U.S. patent application number 10/394896 was filed with the patent office on 2003-12-25 for composition used as a therapeutic agent against chronic viral hepatic diseases.
This patent application is currently assigned to Medeva Holdings B.V.. Invention is credited to Thoma, Hans.
Application Number | 20030235591 10/394896 |
Document ID | / |
Family ID | 8204863 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235591 |
Kind Code |
A1 |
Thoma, Hans |
December 25, 2003 |
Composition used as a therapeutic agent against chronic viral
hepatic diseases
Abstract
A combination, comprising at least one polypeptide sequence,
mediating the antigenicity of one or more epitopes, and a carrier,
capable of presenting this/these polypeptide sequence(s), which are
useful for the production of a medicament for the treatment of
chronic viral hepatitis, is provided.
Inventors: |
Thoma, Hans; (Munchen,
DE) |
Correspondence
Address: |
Thomas E. Popovich
Popovich & Wiles, PA
Suite 1902, IDS Center
80 South 8th Street
Minneapolis
MN
55402
US
|
Assignee: |
Medeva Holdings B.V.
|
Family ID: |
8204863 |
Appl. No.: |
10/394896 |
Filed: |
March 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10394896 |
Mar 21, 2003 |
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09304807 |
May 4, 1999 |
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09304807 |
May 4, 1999 |
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08075520 |
Jan 31, 1994 |
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6020167 |
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08075520 |
Jan 31, 1994 |
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PCT/EP91/02460 |
Dec 19, 1991 |
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Current U.S.
Class: |
424/189.1 ;
530/350 |
Current CPC
Class: |
A61K 2039/6018 20130101;
A61K 39/385 20130101; C12N 2730/10122 20130101; A61K 2039/55505
20130101; Y02A 50/30 20180101; A61P 31/12 20180101; C07K 14/005
20130101; A61P 1/16 20180101; Y02A 50/466 20180101; A61K 39/00
20130101 |
Class at
Publication: |
424/189.1 ;
530/350 |
International
Class: |
A61K 039/29; C07K
014/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 1990 |
EP |
EP 90124775.9 |
Claims
1. Use of a composition for the production of a medicament for the
treatment of chronic viral hepatitis, said composition comprising a
combination of a) at least one polypeptide sequence having one or
more antigenic T cell-activating epitopes and b) a carrier capable
of presenting the epitope sequence(s) a), wherein the polypeptide
sequence(s) a) is bound to carrier b) by covalent or hydrophobic
bonding.
2. Use according to claim 1 wherein said polypeptide sequence(s) a)
is a polypeptide of hepatitis B virus.
3. Use according to claim 2 wherein said polypeptide sequence(s) a)
is the amino acid sequence of one or more members selected from the
group comprising the HB viral pre-S1, pre-S2 and S peptides and the
HB core antigens.
4. Use according to one of claims 1 to 3 wherein said polypeptide
sequence(s) a) may be a native polypeptide sequence modified: i) by
having arbitrary deletions, whereby an epitope comprising at least
six consecutive amino acid residues is preserved, ii) by having
substitutions of one or several amino acids, or iii) by carrying an
additional amino acid sequence either at its N-terminus, at its
C-terminus or as an insertion into the polypeptide sequence(s)
a).
5. Use according to one of claims 1 to 4 wherein said polypeptide
sequence(s) a) is myristylated.
6. Use according to any one of claims 1 to 5 wherein said
polypeptide sequence(s) a) is produced by expression of a
recombinant DNA molecule.
7. Use according to one of claims 1 to 6 wherein said carrier b) is
a polysaccharide, a hydrophobic polymer or an inorganic molecule
having particle form.
8. Use according to one of claims 1 to 7 wherein said carrier b) is
a second polypeptide sequence.
9. Use according to claim 8 wherein said polypeptide sequence b)
upon secretion forms particles having a diameter of at least 10
nm.
10. Use according to claim 8 or 9 wherein said polypeptide sequence
b) is a substantial part of or the complete amino acid sequence of
a polypeptide selected from a group consisting of the HBV
S-peptide, the HBV core-, the HAV core- and the HIV core-antigen as
well as the surface antigens of poliovirus, HAV or HIV.
11. Use according to one of claims 8 to 10 wherein said polypeptide
sequence b) may be modified i) by having arbitary deletions,
whereby the particle forming capacity is preserved, ii) by having
substitutions of one or several amino acids, or iii) by carrying an
additional amino acid sequence either at its N-terminus, at its
C-terminus or as an insertion into the polypeptide sequence b).
12. Use according to one of claims 8 to 11 wherein said polypeptide
sequence b) is myristylated.
13. Use according to any one of claims 8 to 12 wherein said
polypeptide sequence b) is produced by expression of a recombinant
DNA expression.
14. Use according to one of claims 1 to 6 or 8 to 13 wherein said
polypeptide sequences a) and b) are linked via disulfide
bridges.
15. Use according to one of claims 1 to 6 or 8 to 14 wherein said
polypeptide sequences a) and b) are linked via "hydrophobic
anchoring" (mediated by myristic acid).
16. Use according to one of claims 1 to 6 or 8 to 15 wherein said
polypeptide sequences a) and b) are linked by a peptide bond,
wherein optionally a spacer sequence is inserted in between
polypeptide sequence(s) a) and polypeptide sequence b), said
spacer-sequence being linked via peptide bonds to polypeptide
sequences a) and b).
17. Use according to claim 16 wherein said polypeptide sequence(s)
a) and said polypeptide sequence b) are encoded in tandem on the
same recombinant DNA molecule.
18. Use according to claim 17 wherein said polypeptide sequence(s)
a) and said polypeptide sequence b) are expressed from a single
open reading frame on said DNA molecule, and are optionally
separated by a spacer polypeptide sequence also encoded in the
single open reading frame.
19. Use as claimed in any one of claims 1 to 18 wherein said
composition is for administration by intravenous or intramuscular
injection.
20. Method of treatment of viral hepatitis characterised in that a
composition as defined in any one of claims 1 to 19 is administered
in a patient.
21. A method as claimed in claim 20 for the treatment of viral
hepatitis B.
Description
[0001] The present invention relates to a composition comprising a
polypeptide sequence prepared by recombinant DNA techniques and a
carrier to provide a curing agent against chronic viral hepatic
diseases. The invention relates to DNA sequences coding for said
polypeptide sequences and to transfected cells for the expression
of the same.
[0002] At least five different viruses, namely Hepatitis virus A,
B, C, D and E, are able to trigger the clinical aspect of an acute
hepatitis. Hepatitis A and E, which are transferred enterically,
always heal, whereas hepatitis B, C (formerly called parenteral
hepatitis Non-A Non-B), and D can progress into a chronic stage of
inflammation, which in turn can result in liver cirrhosis and
primary hepatocellular carcinoma.
[0003] There is relatively little data available on hepatitis C and
D, on methods for the diagnosis and their treatment and on the
respective viruses. The hepatitis D virus is a RNA virus which is
known to be incomplete. Therefore, it needs a helper virus to
develop in patients and is found only in individuals infected with
HBV. Only very recently the hepatitis C virus has been detected,
and an antibody test (anti-HCV) facilitating the diagnosis of
chronic hepatitis C infections has been developed. However, there
is an increasingly urgent need for a treatment to cure this
disease.
[0004] The same holds true for chronic hepatitis B, a much better
studied disease with respect to its recognition by immunological
methods, its causative virus and the viral life cycle and DNA
sequence. Patients are said to be chronic carriers of the hepatitis
B virus if the viral DNA persists longer than ten weeks, the
HBe-antigen (HBeAg) for more than 12 weeks, or if the hepatitis B
surface-antigen (HBsAg) is persistent longer than six months.
[0005] Roughly three hundred million people are deemed to suffer
from chronic hepatitis B, most of them living in the Far East.
[0006] For these people the main risk to be infected appears to be
during or immediately after birth, since a chronically infected
mother transfers the virus to her newborn. 90 percent of the
children infected this way will become chronically infected, too,
during later life. In the Western World infection occurs more
commonly later in life, during childhood or even adulthood, mainly
by a parenteral or sexual transmission. In these cases of hepatitis
B infection after birth only five to ten percent of the infected
become chronic carriers. The virus transferred, however, is not
responsible for distinct reactions shown by infected people either
to eliminate the virus or to retain it in the body lifelong.
Consequently, it seems to be a matter of the immunological status
that determines the future physical condition.
[0007] The HB-virion (Dane particle) is composed of different
structural proteins, the core proteins and the surface (S)
proteins. The latter are translation products of an open reading
frame encompassing the coding sequence of three S-type domains,
each of which starts with an ATG triplet capable of initiating
translation in vivo. The domains are referred to as preS1, preS2
and S in the order of 5' to the 3'end of the molecule. There are
six protein products derived from this ORF: a glycosylated and a
non-glycosylated form of the major protein (gp27 and p24)
translated from the S domain only (226 amino acids), a middle
protein (281 amino acids) having one or two polysaccharide side
chains (gp33 and gp36, respectively), that is encoded by the preS2-
and S-region, and finally, both a glycosylated (gp42) and a
non-glycosylated (p39) form of the large protein (389-400 amino
acids, depending upon the viral serotype), which is formed by
translation of preS1, preS2 and S. The core proteins are HBcAg and
HBeAg, the latter one conceivably being a processing product of
HBcAg.
[0008] The Dane particle, which is the infectious virion, comprises
both core and surface proteins, whereas the filaments consist of a
mixture of the six surface antigens. The S peptides alone assemble
to form the so-called 20 nm particles, which are completely
uninfectious.
[0009] Patients infected by the HB virus pass through several
stages of the hepatitis, before they are regarded to be chronically
HBV-infected. Immediately after infection an infectious stage will
follow, characterized by the presence of HBeAg in the serum.
Continued HBs antigenaemia in spite of inhibited HBV replication
indicates the presence of viral DNA sequences integrated into the
cellular genome of the patient. The integrated viral sequences do
not enable the host cell to synthesize the complete virus. However,
liver cells having HBV-sequences integrated are capable of
producing HBsAg only, which in turn is detectable in the serum of
the patient and is an indicator for chronic hepatitis B. Most
probably the transformed hepatocytes are not lysed by cytotoxic
T-cells, but proliferate and induce either chronic persistent
hepatitis (CPH) or chronic active hepatitis (CAH), which may then
proceed to cirrhosis of the liver or to primary hepatocellular
carcinoma resulting in premature death of the patient.
[0010] Recently it has been established that patients who are
chronically HBV infected show a defect in endogenous interferon
production (Abb et al., 1985; J. Med. Virol 16. 171-176). This was
the rationale to treat patients suffering from chronical hepatitis
B, as indicated by the presence of HBeAg and HBV-DNA in the serum,
with interferon .alpha. (IFN.alpha.). Controlled trials with large
numbers of patients showed that the administration of interferon
.alpha. resulted in significantly increased elimination of the
hepatitis B-virus, when compared to controls. However, persons
infected at or around time of birth do not appear to seroconvert in
response to this therapy. This phenomenon unfortunately precludes
some 75% of carriers from IFN.alpha. therapy.
[0011] At present, the exact mode of action of interferon .alpha.
on chronic hepatitis B remains unclear. Its antiviral activity
might protect infected cells from infection or reduce viral
transcription, translation and replication in HBV-infected cells.
Interferon further has immunomodulatory effects by activating
T-cells, macrophages and NK-cells and by inducing the expression of
MHC class I proteins.
[0012] Another approach to treat chronic hepatitis B is based on
the idea to inhibit replication of the virus, thus impairing its
defence sufficiently to render the host immune system capable of
eliminating the virus. This led to test antiviral drugs such as
adenine arabinoside and adenine arabinoside monophosphate for
treatment of chronically HBV infected individuals. However, less
than half of the patients responded to this therapy, either by
sustained or transient seroconversion (HBeAg.sup.+ to
anti-HBe.sup.+). A further negative aspect of these antiviral drugs
are their immunosuppressive properties.
[0013] Other drugs that have beeen tested for treatment of chronic
carriers include interferon .beta. and, acycloguanosine
(acyclovir), interleukin 2, steroids, such as prednisolone, and
combinations thereof. But none of them could provide better results
than treatment with interferon .alpha.. Only a combination therapy,
including the initial administration of steroids followed by that
of IFN .alpha. may increase the response rate in selected
patients.
[0014] It is known from the prior art, that chronically HBV
infected chimpanzees can not be cured by treatment with HBsAg
(bound to a tetanus toxoid) nor with anti-HBs antibodies.
Furthermore, it has been attempted to immunize chronically HBV
infected patients by administration of S peptides. This treatment
did not even result in anti-HBs antibody formation in these
persons.
[0015] Additionally, according to the definition, chronic carriers
of hepatitis B virus are characterized in that HBsAg is detectable
in their serum. Therefore, it has been absolutely unforeseeable,
that a combination, comprising a T-cell activating epitope of the
viral S peptide, according to the present invention, is able to
induce an immunization in and a final healing of chronic carriers
of hepatitis virus B.
[0016] Considering the above-discussed state of the art it is the
objective of the present invention to provide an effective
therapeutic agent for the treatment of viral chronic hepatic
diseases which leads to a complete response (i.e. to the sustained
inhibition of HBV-replication, the loss of HBV DNA and DNA
polymerase and to a decrease and finally the disappearance of HBeAg
and HBsAg in the serum of patients).
[0017] According to the present invention this goal is achieved by
a combination of a) at least one polypeptide sequence mediating the
antigenicity of one or more epitopes and b) a carrier, capable of
presenting the epitope sequence(s) a), wherein the polypeptide
sequence(s) a) can be bound to carrier b) by adsorption, any
chemical bonding or secondary valences.
[0018] This invention is furthermore directed to the use of this
combination for the production of a medicament for the treatment of
chronic viral hepatitis.
[0019] The present invention is further directed to a method for
the treatment of chronic viral hepatitis by administering to a
patient the above described combination of a) at least one
polypeptide sequence mediating the antigenicity of one or more
epitopes and b) a carrier capable of presenting the epitope
sequence(s) a), wherein the polypeptide sequence(s) a) can be bound
to carrier b) by adsorption, any chemical bonding or secondary
valences.
[0020] It is important that polypeptide sequence a), which may be
one or more different polypeptides, mediates the antigenicity of a
T cell-activating epitope in a direct or indirect way.
[0021] According to the present invention polypeptide sequence(s)
a) may be a polypeptide or a combination of two or more
polypeptides of hepatitis B virus of any subtype, particularly adw,
ayw, adr and ady.
[0022] These peptides derived from hepatitis B virus may be HBV
peptides preS1, preS2 or S or the HBV core antigens.
[0023] Useful as polypeptide sequence(s) a) are futhermore any of
the above-stated polypeptides or a combination of two or more
polypeptides which are modified either by amino acid deletions,
whereby at least one epitope comprising at least six consecutive
amino acid residues must be preserved, or by adding further amino
acids either at the N-terminus, the C-terminus or as insertions
into the polypeptide sequence(s) a). In each of these cases it is
essential, however, that the biologial activity is maintained.
[0024] Preferably, polypeptide sequence(s) a) is myristylated.
[0025] In order to display the appropriate pharmacological activity
it is necessary that in the combination of the present invention
polypeptide sequences(s) a) is presented on a carrier b). This
carrier consists of a particular substance which for example may
consist of particles of a hydrophobic polymer, of inorganic
particles, or of particles of a polysaccharide. Preferably, carrier
b) is a second polypeptide sequence which forms particles upon
secretion, said particles having preferably a diameter of at least
10 nm.
[0026] It is preferred that the particle forming polypeptide
sequence b) is a substantial part of or the complete amino acid
sequence of a polypeptide which may be selected from HBV S peptide,
HBV core antigen, HAV core antigen, HAV surface antigen, HIV
surface antigen and HIV core antigen as well as the surface antigen
of polio virus. Preferred as the particle-forming carrier b) is HBV
S peptide and/or core peptide.
[0027] When used as the carrier sequence b) the above-stated
polypeptides may be modified by arbitrary deletions of amino acids,
by substitutions of one or more amino acids or by adding one or
more amino acids either at the N-terminus, the C-terminus or by
insertion of one or more amino acids into the polypeptide sequence
b), provided that the particle-forming capacity is maintained.
Preferably, polypeptide sequence b) is myristylated.
[0028] If the carrier b) is a polypeptide sequence, both sequences
a) and b) may be linked via one or more of the following
interactions: hydrophobic anchoring (mediated by myristic acid),
disulfide bridge formation, or both sequences may be connected by a
peptide bond to form a fusion peptide. In the latter case
optionally a spacer sequence may be inserted between polypeptide
sequence(s) a) and polypeptide sequence b), which spacer sequence
is linked to both polypeptides via peptide bonds.
[0029] The present invention furthermore provides a recombinant DNA
molecule coding for a combination, that is useful for production of
a medicament to treat chronic viral hepatic diseases. The
recombinant DNA molecule comprises at least one first DNA sequence,
optionally a second, a third and/or a fourth DNA sequence
wherein
[0030] i) said at least one first DNA sequence codes for at least
one polypeptide sequence a) as defined above,
[0031] ii) said second DNA sequence codes for a polypeptide
sequence b) according to the above definition of the particle
forming peptide,
[0032] iii) said third DNA sequence codes for a spacer sequence,
and
[0033] iv) said fourth DNA sequence codes for a selection
marker,
[0034] and wherein the DNA sequences are controlled by DNA elements
essential for expression, and optionally have a common reading
frame.
[0035] On account of the fact, that many amino acids are designated
by more than one triplet, there exist several DNA sequences
embraced by the present invention, which code for the above-defined
peptide sequences a) and b).
[0036] Apart from this, the invention further embraces recombinant
DNA molecules, which differ from the above-defined recombinant DNA
molecules by the fact, that up to 30% of the nucleotides may be
substituted.
[0037] A further object of the present application is to provide a
host cell transfected with a recombinant DNA molecule coding for
the above combination, which is useful for treatment of chronically
HBV-infected patients. This host cell may be a mammalian, a yeast
or a bacterial cell. For the purpose of this invention it is
preferred, that this cell does not produce any human serum proteins
or any primate serum proteins other than the polypeptide(s) being
comprised within the above combination.
[0038] The term "HBV S peptide" as used herein refers to the
peptide encoded by the entire region of the HBV genome. The term
"HBV pre-S2 peptide" as used herein refers to the peptide encoded
by the entire pre-S2 and S regions of the HBV genome. The term "HBV
pre-S1 peptide" as used herein refers to the polypeptide encoded by
the entire pre-S1, pre-S2 and S regions of the HBV genome. The term
"epitope" as used herein refers to a sequence of at least six
consecutive amino acids encoded by the designated genome region
(e.g. a "HBV pre-S2 epitope" refers to a sequence of at least six
amino acids encoded by the pre-S2 region of the HBV genome). The
term "T-cell epitope" as used herein refers to an epitope that
interacts with receptors on the surface of T-cells to enhance or
otherwise effect an immune response.
[0039] As used herein "antigenicity" means the ability to provoke
an immune response (e.g. acting as an antigen), the ability to
cause the production of antibodies (e.g. acting as an antigen)
and/or the ability to interact with a cell surface receptor so as
to enhance an immune response or production of antibodies.
[0040] The term "HBV" means any subtype of the virus, particularly
adw, ayw, adr and ayr, described in the literature (P. Valenzuela,
Nature Vol. 280, p. 815 (1979), Gerlich, EP-A-85 111 361, Neurath,
EP-A-85 102 250). Examples of peptide sequences thereof,
constituting polypeptide sequence(s) a), which mediate the
antigenicity of one or more epitopes, are shown in the Sequence
Listing (SEQ ID No. 17-20, 22).
[0041] Preferred embodiments of the present invention are the
following combinations:
[0042] HB S-antigen particles with specific epitopes (determinants)
of the pre-S1-, pre-S2-, and/or core peptides;
[0043] HB core-antigen particles with specific epitopes
(determinants) of the pre-S1-, pre-S2-, S-peptide, and/or of the
core antigens;
[0044] Hepatitis A-antigen particles with specific epitopes
(determinants) of the hepatitis B S-,pre-S1-, pre-S2-, and/or
core-peptides.
[0045] Recombinant DNA molecules preferred for the present
invention are characterized by the presence of sequences coding for
polypeptide sequence(s) a), mediating the antigenicity of one or
more T-cell epitopes, and for polypeptide b), which upon secretion
forms particles having a diameter of 10 nm or more, both of which
are under control of a suitable promoter. As examples for sequences
coding for a) there may be mentioned any of the sequences listed
under ID numbers 1 to 24 in the Sequence Listing. Examples for the
DNA sequence coding for polypeptide sequence b) are represented by
any of the ID-sequences 25 to 27 in the Sequence Listing.
[0046] Any of the 24 sequences (ID numbers 1 to 24) may be combined
to any sequence disclosed under ID number 25 to 27 in the Sequence
Listing, therein both orders a-b and b-a are included.
[0047] A particular preferred embodiment of the present invention
consists in a combination of the epitope sequence ID No. 28
(corresponding to amino acids 9 to 28 of the S1 sequence of HBV) in
combination with sequence ID No. 26 and/or 27 as a particle
former.
[0048] Hepatitis virus sequences used in the recombinant DNA
construct of the present invention can be formed or isolated by any
means including isolation and ligation of restriction fragments,
chemical synthesis of oligonucleotides using a synthesizer (Cyclon,
Bio-Search), and synthesis by the PCR method (T. J. White, N.
Arnleim, H. E. Erlich, 1989; The Polymerase Chain Reaction,
Technical Focus 5 (6)).
[0049] Preferred recombinant DNA molecules were formed by the
ligation of synthetic oligonucleotides to a 5' XbaI-BglII 3'
fragment (ID number 27) from the S region of the HBV genome, which
is derived from a BglII-BglII HBV fragment including the entire
pre-S1-pre-S2-S-region, or to the entire S-region. Oligonucleotides
used in making such constructs are summarized in Table I below.
1TABLE I Function Definition SEQ ID No. core (adw) aa* 59-87 6 core
(adw) aa 2-28 7 core (adw) aa -10-28 8 core (adw) aa 29-58 9 core
(adw) aa 1-87 10 core (adw) aa -10-87 11 core (adw) aa 70-110 12
core (adw) aa 80-125 13 core (adw) aa 88-120 15 S1 (ayw) aa 9-28 17
S1 (ayw) aa 83-103 18 S1 (ayw) aa 20-40 19 S1 (ayw) aa 59-94 20 S1
(adw) aa 94-114 21 S1 (adw) aa 70-105 22 S2 (ayw) aa 2-21 23 S2
(ayw) aa 14-33 24 *aa = amino acid
[0050] Other preferred DNA molecules were formed by ligation of
core sequences, which are prepared by the PCR method and which code
for T-cell epitopes, to the core sequence of HBV (SEQ ID NO 25)
functioning as polypeptide sequence b). Oligonucleotides used in
preparing these constructs are given in Table II-1.
2 TABLE II-1 Function Definition SEQ ID No. core complete, bp
1901-2500 1 core C-terminal deletion, 2 bp 1901-2405 core
C-terminal deletion and 3 stop codon inserted, bp 1901-2405
core/precore 10 aa precore, 4 C-terminal deletion, bp 1871-2405
core/precore 10 aa precore, 5 C-terminal deletion and stop codon
inserted, bp 1871-2405 core aa (-10-120) 16 core/precore 10 aa
precore, 35 complete core, bp 1871-2500
[0051] Table II-2 shows several examples, where the T-cell
epitope-coding DNA sequences have been isolated by restriction
fragmentation of the HBV genome and have been ligated to the DNA
sequence coding for polypeptide sequence b) as defined above.
3 TABLE II-2 Function Definition SEQ ID No. core/precore complete,
bp 1403-311 ** S2 ay/ad ** S2 (K) ay/ad S2-S, 7 codons 14 deleted,
start codon ATG changed to ATA **Sequence has been published by
Galibert, F. et al. (1979: Nature 281, 646-650) and by Ono, Y. et
al. (1983: Nucl. Acid Res. 11(6), 1747-1757)
[0052] In Table II-3 specific recombinant DNA molecules are listed.
The procedure for their construction will be described in more
detail in the Examples.
4TABLE II-3 Se- lec- Final T-cell Particle tion construct epitope
Former Gene MT-core(-10-120) + core(aa -10-120) S adw/ayw or neo
SAg + neo S/Xbal/BglII MT-S1(aa 9-28) - S1(aa 9-28)ay S adw/ayw or
egpt# S + egpt S/XbaI/BglII MT-core-neo core/precore core adw neo
bp 1403-31 MT-core(1-87) + core(aa 1-87) S adw/ayw or neo HBsAg -
neo S/XbaI/BglII #egpt = E coli xanthine guanine phosphoribosyl
transferase
[0053] Preferred recombinant DNA molecules according to the present
invention comprise, apart from the regions coding for polypeptides
a) and b), an additional DNA sequence coding for a selection
marker. Furthermore, they comprise all usual elements essential for
the expression, such as promoter sequence, start codon and a
polyadenylation signal.
[0054] Examples of suitable promoters are the methallothionein
(MT), the U2 and the H2K promoter in case of using mammalian cells
as a host cell. If yeast or bacterial cells are to be employed,
appropriate yeast and bacterial promoters, such as the GCN4- and
the GAL 1/10 promoter or the prokaryotic trp- and tac promoters,
respectively, may be used.
[0055] In order to produce the combination of polypeptide(s) a) and
polypeptide b) according to this application the recombinant DNA
molecule is inserted into host cells by transfection (in case of
mammalian cells), by transformation (in case of yeast and bacterial
cells), or by other means. As a host cells of any organism may be
used that are capable of transcribing and translating recombinant
DNA molecules, such as mammalian, bacterial and yeast cells.
[0056] Suitable mammalian cells according to this invention are for
example VERO cells (a monkey kidney cell line), 3T3-, C127 and L
cells (murine fibroblast cell lines), and CHO (Chinese hamster
ovary) cells, which are either positive or negative in
dehydrofolate reductase.
[0057] According to a specific embodiment of the present invention
it is furthermore possible that the above-defined first DNA
sequence and the above-defined second DNA sequence, which code for
polypeptide sequence(s) a) and for a polypeptide sequence b),
respectively, are present in different recombinant DNA molecules,
in which case the host cells are cotransfected with both of these
recombinant DNA molecules.
5TABLE III Possible alternatives of compositions for particles
containing T-cell epitopes for targeting chronic hepatitis carrier
FINAL T-CELL-EPITOPE PARTICLE SELECTION PURIFI- CONSTRUCT.sup.1
PROMOTER.sup.2 SYN PCR GENE FORMER GENE CATION.sup.3 12 MT/112/U2
Core(AA -10-+87) entire S adw/ayw neo/egpt S/XbaI/BglII 13
MT/112/U2 Core(AA 70-110) entire S adw/ayw neo/egpt S/XbaI/BglII 14
MT/112/U2 Core(AA 80-125) entire S adw/ayw neo/egpt S/XbaI/BglII 15
MT/112/U2 Core(AA 88-120) entire S adw/ayw neo/egpt S/XbaI/BglII 16
MT/112/U2 Core(AA -10 + SATG + entire S adw/ayw neo/egpt AA 2-87)
S/XbaI/BglII 17 MT-core MT/112/U2 Core(AA -10-+120) entire S
adw/ayw neo/egpt (-10-+120) + S/XbaI/BglII SAg + neo 18 MT-S1
MT/112/U2 S1-(AA 9-28)(ay) entire S adw/ayw neo/egpt (AA 9-28) -
S/XbaI/BglII S + egpt 19 MT/112/U2 S1-(AA 83-103)(ay) entire S
adw/ayw neo/egpt S/XbaI/BglII 20 MT/112/U2 S1-(AA 20-40)(ay) entire
S adw/ayw neo/egpt Materials S/XbaI/BglII and Methods 21 MT/112/U2
S1-(AA 59-94)(ay) entire S adw/ayw neo/egpt S/XbaI/BglII 22
MT/112/U2 S1-(AA 94-114)(ad) entire S adw/ayw neo/egpt S/XbaI/BglII
23 MT/112/U2 S1-(AA 70-105)(ad) entire S adw/ayw neo/egpt
S/XbaI/BglII 24 MT/112/U2 S1-(AA 9-28)(ay) core adw neo/egpt 25
MT/112/U2 S2-(AA 2-21)(ay) entire S adw/ayw neo/egpt S/XbaI/BglII
26 MT/112/U2 S2-(AA 14-33)(ay) entire S adw/ayw neo/egpt
S/XbaI/BglII 27 MT/112/U2 S2 ayw S ayw/adw neo/egpt 28 MT/112/U2
Adapter S2-(X) S ayw/adw neo/egpt ayw 1 MT/112/U2 Core without
pre-core Core(adw) neo/egpt e.g. bp 1901-2500 2 MT/112/U2 Core
without pre-core; Core(adw) neo/egpt with deletion of the
C-terminus e.g. bp 1901-2405 3 MT/112/U2 Core without pre-Core;
Core(adw) neo/egpt with deletion at the C-terminus + stop signal
e.g. bp 1901-2405 4 MT-core-neo MT/112/U2 Core and pre core Core
with Core(adw) neo/egpt Materials 10 AA pre-core and Methods i.e.
bp 1403-31 5 MT/112/U2 Core and pre-core Core(adw) neo/egpt 10 AA;
with deletion at the C-terminus e.g. bp 1871-2405 6 MT/112/U2 Core
and pre-core with deletion at the C- Core(adw) neo/egpt terminus +
stop signal e.g. bp 1871-2405 7 MT/112/U2 Core(AA 59-87) entire S
adw/ayw neo/egpt S/XbaI/BglII 8 MT/112/U2 Core(AA 2-28) entire S
adw/ayw neo/egpt S/XbaI/BglII 9 MT/112/U2 Core(AA -10-+28) entire S
adw/ayw neo/egpt S/XbaI/BglII 10 MT/112/U2 Core(AA 29-58) entire S
adw/ayw neo/egpt S/XbaI/BglII 11 MT-core MT/112/U2 Core(AA 1-87)
entire S adw/ayw neo/egpt (1-87) + S/XbaI/BglII IIBsAg-neo Notes:
.sup.1: see example 3 .sup.2: any of the stated promoters is
suitable .sup.3: see examples
[0058] Table III gives an overview on how to combine suitable DNA
sequences to get DNA constructs according to the present invention.
It is to be noted that any constituents disclosed in this table may
be combined to provide a DNA sequence which may be taken, if
transfected into a host cell, to produce a combination (comprising
polypeptides(s) a) and b)) as a medicament for the treatment of
chronic viral hepatitis.
[0059] The DNA sequences coding for the T-cell epitope sequences
have been prepared synthetically (SYN) with a Biosearch Cyclon
synthesizer, by PCR procedure (PCR), or by restriction enzyme
fragmentation of the viral genome (GENE).
[0060] For the treatment of patients suffering from chronic viral
hepatitis the combination of polypeptide sequence(s) a) and a
carrier b) may be formulated in any type of a pharmaceutical
composition, which furthermore comprises a suitable diluent or
pharmaceutical carrier material, such as a buffer solution.
[0061] The administration may be effected by any method, i.e. by
parenteral (e.g. intravenous or intramuscular) or oral (e.g. by
using typhoid bacterial cells to encapsulate the active substance)
administration.
[0062] The pharmaceutical preparation comprises the above-described
combination in sufficient concentration to elicit a response upon
administration.
BRIEF DESCRIPTION OF THE FIGURES
[0063] FIG. I shows a DNA construct, coding for a promoter, a
particle former sequence and a selection gene (described in Example
3/4).
[0064] FIG. II shows a DNA-gene construct containing a promoter, an
epitope with the entire HB-S-Ag and a selection gene (described in
Example 3/18).
[0065] FIG. III shows a DNA construct presenting a promoter, a
T-cell epitope with a particle former residue and a selection gene
(described in Example 3/21).
[0066] FIG. IV shows the AST values of chimpanzee 1 during the
Hepa-Care treatment (described in Example 10/1).
[0067] FIG. V shows the antigen values of chimpanzee 1 during the
Hepa-care treatment (described in Example 10/1)
[0068] FIG. VI shows values of liver enzymes ALT and GGT of
chimpanzee 1 booster treated three times with Hepa-Care (described
in Example 10/2).
[0069] FIG. VII shows values of liver enzymes ALT, AST, and GGT and
of antigen of chimpanzee 2 during the Hepa-Care treatment
(described in Example 10/3).
[0070] FIG. VIII shows the liver enzymes as determined for an
untreated control chimpanzee (described in Example 10/3).
[0071] FIGS. IX & X show the antigen and antibody titers of
patient 1 during the Hepa-Care treatment, respectively (described
in Example 11).
[0072] FIGS. XI & XII show the antigen and antibody titers of
patient 2 during the Hepa-Care treatment, respectively (described
in Example 11).
[0073] FIGS. XIII & XIV show the antigen and antibody titers of
patient 2 during the Hepa-Care treatment, respectively (described
in Example 11).
[0074] The invention is more specifically described by the
following examples.
EXAMPLE 1
[0075] 1. Fractionated Precipitation with Polyethylene Glycol
(PEG)
[0076] The supernatant of HBV protein producing cultures was
collected and split into portions of 2,400 ml. To each portion 144
g of PEG 6000 (Serva) were added and dissolved by stirring at room
temperature for 20 minutes and was stirred for another 6 hours at
4.degree. C. The precipitate was separated by centrifugation in 500
ml bottles in a GS 3 rotor at 9,000 rpm (15,000.times.g) for 30
minutes at 10.degree. C. The supernatant was collected and 144 g of
PEG 6000 were added and dissolved as described above. The solution
was stirred at 4.degree. C. for 3 hours. The precipitate from this
solution was harvested as described above except that
centrifugation was continued for 60 minutes.
[0077] 2. Gel Chromatography
[0078] The material obtained after PEG precipitation was
redissolved in 20 ml PBS and submitted to gel chromatography on
A-5m (BioRad). Column dimensions were 25.times.1000 mm and 480 ml
bed volume. In a typical fractionation run 1,000 .mu.g of PEG
precipitated HBV protein in 10 to 15 ml was loaded and eluted with
PBS at a speed of 6 drops/min (18 ml/h). 3 ml fractions were
collected. HBV protein eluted with the first peak. Collected
fractions were submitted to a CsCl gradient.
[0079] 3. Sedimentation in CsCl Gradient
[0080] About 30 fractions covering the first peak in column
chromatography on A-5m and containing prepurified HBV protein were
collected to approximately 100 ml. This solution was adjusted to a
density of 1.30 g/cc with CsCl and subsequently transferred to a
polyallomer tube fitting into a SW 27/28 rotor (Beckman). A
gradient was set by underlaying 4 ml of a CsCl solution of 1.35
g/cc and by overlaying 4 ml of 1.25 g/cc followed by 4 ml of 1.20
g/cc density. This gradient had been run at 28,000 rpm for 50 hours
at 10.degree. C. Thereafter the gradient was fractionated, and
purified HBV protein floating in the 1.20 g/cc density layer was
collected. The solution was desalted by three cycles of dialysis in
bags against water.
EXAMPLE 2
Quantitative Determination of HBV Protein
[0081] 1. With Radioimmunoassay
[0082] In the AUSRIA II-125 "sandwich" radioimmunoassay
(commercially available from Abbot), beads coated with guinea pig
antibody to Hepatitis B surface antigen (Anti-HBs) were incubated
with serum or plasma or purified protein and appropriate controls.
Any HBsAg present was bound to the solid phase antibody. After
aspiration of the unbound material and washing of the bead, human
125T-Anti-HBs was allowed to react with the antibody-antigen
complex on the bead. The beads were then washed to remove unbound
.sup.125I-Anti-HBs.
[0083] )-Anti-HBs HBsAg
[0084] )-Anti-HBs . HBsAg .sup.125I-Anti-HBs
[0085] )-Anti-HBs . HBsAg . .sup.125I-Anti-HBs
[0086] The radioactivity remaining on the beads was counted in a
gamma scintillation counter.
[0087] 2. With ELISA
[0088] In the Enzygnost HBsAg micro "sandwich" assay (commercially
available from Behring), wells-were coated with anti-HBs. Serum
plasma or purified protein and appropriate controls were added to
the wells and incubated. After washing, peroxidase-labelled
antibodies to HBsAg were reacted with the remaining antigenic
determinants. The unbound enzyme-linked antibodies are removed by
washing and the enzyme activity on the solid phase was determined.
The enzymatically catalyzed reaction of hydrogen peroxide and
chromogen was stopped by adding diluted sulfuric acid. The colour
intensity was proportional to the RBsAg concentration of the sample
and was obtained by photometric comparison of the colour intensity
of the unknown samples with the colour intensities of the
accompanying negative and positive control sera.
EXAMPLE 3
Preparation of Gene Constructs of the Present Invention Containing
Promoter, Desired Antigen Sequences and Selection Gene
[0089] 1. Isolation of the MT-Promoter.
[0090] The plasmid pBPV-342-12 (ATCC 37224) was digested with the
endonucleases BglII and BamHI. Three DNA molecules were generated.
The fragment of interest contains the methallothionein promoter and
a pBR322 sequence comprising 4.5 kb and is easily detectable from
the other fragments (2.0 kb and 7.6 kb).
[0091] The reaction was performed in a total volume of 200 .mu.l of
reaction buffer at a final concentration of 0.5 .mu.g/.mu.l DNA
including 100 units of each restriction enzyme. The completion of
the digestion was checked after incubation at 37.degree. C. for
three hours by agarose gel electrophoresis at a 0.8% agarose gel.
The reaction was stopped by adding 4 .mu.l 0.5 M EDTA.
[0092] The 4.5 kb fragment was separated from the other fragments
by preparative 1.2% agarose gel electrophoresis. The DNA was eluted
from the agarose gel on DE-81 Whatman filter paper from which the
DNA was removed in a high salt buffer. The DNA was purified by a
phenol-chloroform extraction and two ethanol precipitations.
[0093] 2. Ligation of a 1.8 kb Fragment Coding for the
HBV-Core-Antigen.
[0094] A 1.8 kb BamHI-BamHI fragment, containing the HBV-core
coding regions was isolated from HBV-containing DNA. This fragment
was ligated together with the 4.5 kb fragment containing the
MT-promoter and the pBR residue (described in 1).
[0095] 2 .mu.l of the 1.8 kb fragment were mixed with 3 .mu.l of
the 4.5 kb fragment and ligated together in a total volume of 10
.mu.l ligation buffer, containing 2 units T4-DNA ligase and 2 mM
ATP at 14.degree. C. overnight.
[0096] The ligation mixture was added to 150 .mu.l competent
bacterial cell suspension for DNA up-take. After the DNA up-take
the bacterial cells were spread on LB agar plates containing 50
.mu.l/ml ampicillin at volumes of 50 to 300 .mu.l cell suspension
per plate. The agar plates were incubated at 37.degree. C.
overnight. Single isolated bacterial colonies were screened for the
presence of a plasmid containing the desired fragments.
[0097] 3. Screening for Desired Plasmid Containing Bacterial
Colonies.
[0098] Single colonies were picked with a toothpick and transferred
to a LB-ampicillin medium containing tube (5 ml). The tubes were
incubated overnight at 37.degree. in a rapidly shaking environment.
A mini-plasmid preparation of each grown bacterial suspension was
made. The different resulting DNAs were proved by digestion with
the restriction endonuclease BglII. Two molecules were expected, a
400 bp fragment and a 5.9 kb fragment. The digestion was analysed
by agarose gel electrophoresis. Plasmid DNA was isolated from the
bacterial cells.
[0099] 4. Insertion of a Neomycin Selection Marker.
[0100] The plasmid resulting from (3) above was linearized by
digestion with the restriction enzyme EcoRI. The reaction was
performed in a total volume of 50 .mu.l and a final concentration
of 1 .mu.g/.mu.l plasmid DNA. 50 units of EcoRI were added and the
digestion was proved after incubation at 37.degree. C. for three
hours by agarose gel electrophoresis. The reaction was stopped by
adding 1 .mu.l of 0.5 M EDTA and the DNA was precipitated with a
final concentration of 0.3 M sodium acetate and 3-4 volumes of
ethanol at -80.degree. C. for 30 minutes. The precipitated DNA was
dissolved in 50 .mu.l distilled water.
[0101] 2 .mu.l of the linearized plasmid was mixed with 3 .mu.l of
the DNA fragment containing the methallothionein promoter and the
neomycin selection gene (isolated from the plasmid pMT-neo-E
(available from ATCC/Exogene) by digestion with the endonuclease
EcoRI as a 3.9 kb fragment), and ligated together. Single bacterial
colonies were screened for the presence of the desired plasmid.
[0102] 5. Isolation of a Fragment Containing the U2 Promoter
Sequence.
[0103] The plasmid pUC-8-42 (available from Exogene) was digested
with the restriction endonucleases EcoRI and ApaI. Two DNA
molecules were generated. The fragment of interest contains the
U2-promoter comprising 340 bp and is easily detectable from the
other fragment (3160 bp). The digestion was performed in a total
volume of 200 .mu.l reaction buffer at a final concentration of 0.5
.mu.g/.mu.l DNA including 100 units of each restriciton enzyme. The
completion of the digest was checked after incubation at 37.degree.
C. for three hours by agarose gel electrophoresis in a 0.7% agarose
gel. The reaction was stopped by adding 4 .mu.l 0.5 M EDTA. The 340
bp fragment was separated from the plasmid DNA by preparative 1.2%
agarose gel electrophoresis. The DNA was eluted from the agarose
gel on DE-81 Whatman filter paper from which the DNA was removed in
a high salt buffer. The DNA was purified by a phenol/chloroform
extraction and two ethanol precipitations.
[0104] 6. Insertion of the Fragment Containing the Promoter
Sequence into a Polylinker Plasmid.
[0105] The plasmid pSP165 (commercially available from Promega
Diotec) containing a polylinker sequence (containing the following
restriction sites: EcoRI, SacI, SmaI, AvaI, BanHI, BglII, SalI,
PstI, HindIII) was linearized with the restriction enzyme EcoRI.
The reaction was performed in a total voume of 50 .mu.l and a final
concentration of 1 .mu.g/.mu.l plasmid DNA. 50 units of EcoRI was
added and the digestion was proved after incubation at 37.degree.
C. for three hours by agarose gel electrophoresis. The reaction was
stopped by adding 1 .mu.l of 0.5 M EDTA and the DNA was
precipitated with a final concentration of 0.3 M sodium acetate and
3-4 volumes of ethanol at -80.degree. C. for 30 minutes. The
precipitated DNA was dissolved in 50 .mu.l distilled water.
[0106] 2 .mu.l of plasmid DNA was mixed with 10 .mu.l of the
fragment DNA containing the U2 promoter sequence, and ligated
together in a total volume of 25 .mu.l of ligation buffer
containing 2 units T4-DNA ligase and 2 mM ATP at 14.degree. C.
overnight. Thereafter, the DNA purified by phenol/chloroform
extractions followed by two ethanol precipitations and dissolved in
10 .mu.l distilled water. The resulting sticky ends of EcoRI and
ApaI had to be converted into blunt ends and ligated. The sticky
ends were converted into blunt ends by reaction with the Mung bean
nuclease as follows: to 25 .mu.l DNA (1 .mu.g/.mu.l concentration)
in reaction buffer 20 units of enzyme were added to give a final
concentration of 1% glycerol and a final reaction volume of 35
.mu.l. After an incubation for 30 minutes at 30.degree. C. the DNA
was purified by phenol-chloroform extractions followed by two
ethanol precipitations. The DNA was dissolved again in 5 .mu.l of
distilled water. The resulting blunt ends were ligated together in
15 .mu.l reaction volume containing 10.times. more T4 ligase than
used above and 2 mM ATP at 14.degree. C. overnight.
[0107] The ligation mixture was added to 150 .mu.l competent
bacterial cell suspension for DNA up-take. After the DNA up-take
the bacterial cells were spread on LB agar plates containing 50
.mu.g/ml ampicillin at volumes of 50 to 300 .mu.l cell suspension
per plate. The agar plates were incubated at 37.degree. C.
overnight. Single isolated bacterial colonies were screened for the
presence of a plasmid containing the desired U2-promoter fragment.
The resulting plasmid was isolated from the bacterial cells and
characterized by restriction enzyme analysis.
[0108] 7. Ligation of Synthetic Oligo-DNA-Nucleotide 89 (SEQ ID
No.: 30) Together with MT-Promoter Fragment (4.5 kb) .
[0109] The 4.5 kb fragment (described in 1) containing the
MT-promoter and a pBR residue were ligated together with the
synthetic oligonucleotide 89 (SEQ ID No.: 30). The ligation mixture
was added to 150 .mu.l competent bacterial cell suspension for DNA
up-take. Single isolated bacterial colonies were screened for the
presence of the desired plasmid. The new plasmid was proved by a
digestion with the restriction endonucleases EcoRI and XbaI. Two
molecules were expected, one 2.0 kb and one 2.6 kb.
[0110] 8. Ligation of the Synthetic Oligonucleotide 101 (SEQ ID
No.: 32) Together with Plasmid (Described in 7).
[0111] The plasmid (described in 7) was digested with BglII and
BamHI and a fragment of 13 nucleotides was removed (described in
1). The resulting fragment containing the first oligonucleotide 89
(SEQ ID No.: 30), was ligated together with oligonucleotide 101
(SEQ ID No.: 32), a BglII-BamHI fragment. After DNA up-take single
cells were screened for the presence of the desired plasmid. The
new plasmid was proved by a digestion with the endonucleases EcoRI
and XbaI, or EcoRI and BglII.
[0112] 9. Ligation of Synthetic DNA-Oligonucleotide 99 (SEQ ID No.:
31) to the 4.5 kb Fragment (Described in 1).
[0113] The 4.5 kb fragment (BglII-BamHI) was ligated together with
the DNA oligonucleotide 99 (SEQ ID No.: 31). After screening of
single bacterial colonies, containing different DNAS, the desired
plasmid was characterized by digestion with EcoRI, resulting in two
fragments, 1.9 kb and 2.7 kb, and by positive linearization with
BglII or BamHI.
[0114] The new plasmid was then digested with PstI and BamHI. Two
molecules were expected, one 2.6 kb fragment, containing a pBR
residue, the MT-promoter and the oligonucleotide and a 2.0 kb pBR
residue. The 2.6 kb fragment was isolated.
[0115] 10. Ligation of the 2.6 kb Fragment of the Plasmid Described
in 9, with a Fragment Isolated from Plasmid (Described in 8).
[0116] The plasmid (described in 8) containing the DNA
oligonucleotides 89 and 101 (SEQ ID No.: 30 and 32, respectively)
was digested with PstI and BglII. Two fragments were expected. A
2.5 kb fragment containing a pBR residue and the MT-promoter and
2.2 kb fragment, containing a pBR residue and both oligos.
[0117] This 2.2 kb fragment was ligated together with the 2.6 kb
fragment, containing the pBR residue, the MT-promoter and oligo 99
(SEQ ID No.: 31) described in 8.
[0118] After screening for the desired plasmid, it was
characterized by restriction endonuclease digestion with
BglII-XbaI. Two fragments were expected, a 270 bp fragment of the
oligo-DNA-nucleotides and a 4.5 kb fragment of the MT-promoter and
the pBR.
[0119] 11. Ligation of the 2.3 kb HBV BglII-BglII Fragment.
[0120] A 2.3 kb BglII-BglII fragment containing the HBV pre-S1,
pre-S2 and S coding regions was isolated from HBV-containing DNA.
The 2.3 kb fragment was ligated together with the 4.5 kb fragment
(obtained as described in 1) containing the methallothionein
promoter.
[0121] 2 .mu.l of the 2.3 kb fragment was mixed with 3 .mu.l of the
4.5 kb fragment and ligated together in a total volume of 10 .mu.l
ligation buffer, containing 2 units T4-DNA ligase and 2 mM ATP at
14.degree. C. overnight.
[0122] The ligation mixture was added to 150 .mu.l competent
bacterial cell suspension for DNA up-take. After the DNA up-take
the bacterial cells were spread on LB agar plate containing 50
.mu.g/ml ampicillin at volumes of 50 to 300 .mu.l cell suspension
per plate. The agar plates were incubated at 37.degree. C.
overnight. Single isolated bacterial colonies were screened for the
presence of a plasmid containing the desired fragment.
[0123] 12. Conversion of a Part of the HBV-Gene Sequence with
HBV-core epitopes.
[0124] The plasmid resulting from 11 above was digested with the
endonucleases BglII and XbaI. Two molecules were expected, one 550
bp fragment and a 6.25 kb fragment which was isolated after agarose
gel electrophoresis.
[0125] The 6.25 kb fragment was ligated together with the 270 bp
fragment (after digestion with BglII and XbaI and fragment
isolation as described above) of the plasmid described in 10,
coding for an epitope part of the HBV-core gene.
[0126] The ligation mixture was added to 150 .mu.l competent
bacterial cell suspension for DNA up-take. Single isolated
bacterial colonies were screened for the presence of the desired
plasmid. The new plasmid was proved by a digestion with BamHI.
Three molecules were expected, a 950 bp, a 450 bp and a 5,150 bp
fragment.
[0127] 13. Preparation of a "Vehicle" Plasmid.
[0128] The plasmid (described in 11) was digested with EcoRI and
XbaI. Two molecules were expected, one 2,450 bp fragment and a
4,350 bp fragment which was isolated after gel electrophoresis.
[0129] This 4,350 bp fragment was ligated together with the
oligo-DNA-nucleotide 39 (SEQ ID No: 29) coding for the entire
DNA-sequence of HBV-S-gene from ATG to the XbaI site, wherein the
ATG was changed into ATA.
[0130] 14. Core-Epitope Upstream of the Entire HBV-S Gene.
[0131] This "vehicle" plasmid was then digested with PstI and XbaI,
two molecules were expected, one 600 bp plasmid residue and a 3,850
bp fragment which was isolated and ligated together with a
PstI-XbaI fragment of 2,800 bp (2,700 bp) isolated after digestion
of the plasmid described in 10.
[0132] After screening for the desired plasmid, it was
characterized by restriction endonuclease digestion with EcoRI and
XbaI, EcoRI and BglII and BamHI.
[0133] 15. Insertion of a Selection Marker.
[0134] The plasmid (described in 14) was linearized with Eco RI.
The reaction was performed in a total volume of 50 .mu.l and a
final concentration of 1 .mu.g/.mu.l plasmid DNA. 50 units of EcoRI
were added and the digestion was proved after incubation at
37.degree. C. for three hours by agarose gel electrophoresis.
[0135] The reaction was stopped by adding 1 .mu.l of 0.5 M EDTA and
DNA was precipitated with a final concentration of 0.3 M sodium
acetate and 3-4 volumes of ethanol at -80.degree. C. for 30
minutes. The precipitated DNA was dissolved in 50 .mu.l distilled
water.
[0136] 2 .mu.l of the linearized plasmid was mixed with 3 .mu.l of
the DNA fragment containing the methallothionein promoter and the
neomycin selection gene (described in 4) and ligated together.
Single bacterial colonies were screened for the desired plasmid
which was isolated, purified and characterized.
[0137] Each gene construct described above can be constructed also
with the U2-promoter whereby the MT-promoter-containing DNA
fragment, after digestion with EcoRI and BglII, is replaced by a
DNA fragment containing the U2-promoter isolated after digestion
with EcoRI and BglII.
[0138] 16. Isolation of the E coli Xanthine Quanine Phosphoribosyl
Transferase (egpt) Selection Gene.
[0139] The fragment containing the egpt selection gene was isolated
after digestion of the plasmid PMSG with BamHI and BglII (1.8 kb)
and ligated together with a 4.5 kb fragment (BglII-BamHI, described
in 1) containing the MT-promoter.
[0140] After screening for the desired plasmid it was isolated,
purified and finalized by a conversion of the BamHI site into an
EcoRI site.
[0141] 17. Isolation of Desired DNA Sequences by PCR-Method.
[0142] One DNA fragment (400 bp) was isolated after gel
electrophoresis. It was generated by PCR-method (described in
Example 5) by using the specific oligonucleotides 131 and 132 (SEQ
ID No.: 33 and 34) as primers.
[0143] The DNA fragemnt was digested with the endonucleases BamHI
and XbaI and then purified by gel electrophoresis. The isolated
PCR-fragment was ligated together with a 6.25 kb fragment which was
isolated from the plasmid (described in 13) after digestion with
BglII and XbaI. After DNA up-take and bacterial transformation the
single bacterial colonies were screened for the desired
plasmid.
[0144] 18. Insertion of a Selection Marker.
[0145] The plasmid (described in 17) was finalized by adding a
selection gene to the plasmid (described in 15).
[0146] 19. Isolation of the H2K Promoter.
[0147] The H2K promoter was isolated as an EcoRI and BglII fragment
(2 kb) from pSP65H2 (available from Exogene).
[0148] In all constructs described all promoters are replaceable as
EcoRI/BglII fragments.
[0149] 20. Conversion of a Part of the HBV-Gene Sequence.
[0150] The plasmid resulting from 11) above was digested with the
endonucleases BglII and XbaI. Two molecules were expected, one of
which is a 6.250 kb fragment which was isolated after agarose gel
electrophoresis.
[0151] The 6.250 kb fragment was ligated together with
oligo-DNA-nucleotide 23 (SEQ ID No.: 28). The ligation mixture was
added to 150 .mu.l competent bacterial cell suspension for DNA
up-take. Single isolated bacterial colonies were screened for the
presence of the desired plasmid. The new plasmid was proven by a
digestion with the endonucleases EcoRI and BglII. Two molecules
were expected, one 1,9 kb and one 4.450 kb.
[0152] 21. Insertion of a egpt Selection Marker.
[0153] The plasmid (described in 20) was linearized with EcoRI. The
reaction was performed in a total volume of 100 .mu.l and a final
concentration of 0.6 .mu.g/.mu.l plasmid DNA. 60 units of EcoRI
were added and the digestion was proved after incubation at
37.degree. C. for three hours by agarose gel electrophoresis. The
reaction was stopped by adding 2 .mu.l of 0.5 M EDTA and the DNA
was precipitated with a final concentration of 0.3 M sodium acetate
and 4 volumes of ethanol at -80.degree. C. for 1 hour. The
precipitated DNA was dissolved in 50 .mu.l distilled water.
[0154] 2 .mu.l of the linearized plasmid was mixed with 3 .mu.l of
the DNA-fragment (3.7 kb) containing the methallothionein promoter
and the egpt selection gene (described in 16) by digestion with
EcoRI and ligated together. Single colonies were screened for the
presence of the desired plasmid. Each of the described gene
constructs in Table III are preparable in the same way as described
above.
EXAMPLE 4
[0155] Transfection of Mammalian Cells with Constructs of the
Present Invention
[0156] In order to achieve secretion of substantial amounts of the
HBV peptides encoded by constructs of the present invention,
mammalian cells must be transfected with a DNA construct of the
present invention. The cotransfection was performed in two steps
(i.e. a separate transfection for each construct) or in a single
step (i.e. one transfection using preparation of both constructs).
Cotransfection was confirmed either by use of different selection
markers on the two constructs or by detection of secretion of
expression products of both constructs by immunoassay.
[0157] Alternatively, a sequence encoding the HBV peptide sequence
of the present invention and a separate sequence encoding the
entire S or core or HAV protein could be combined in a single
construct.
EXAMPLE 5
Polymerase Chain Reaction (PCR).
[0158] The polymerase chain reaction allows to amplify specific DNA
necleotide sequences of a selected region of a known genomic
sequence in vitro by more than a millionfold (Thomas J. White,
Norman Arnleim, Henry A. Erlich 1989: The polymerase chain
reaction. Technical Focus, Vol. 5. No. 6; S. Kwok and R. Higuchi
1989: Avoiding false positives with PCR. Nature, Vol. 339, pp
237-238).
[0159] DNA isolated from cells or plasmid DNA is treated to
separate its complementary strands. These strands are then annealed
with an excess of two DNA oligonucleotides (each 20-25 base pairs
long) that have been chemically synthesized to match sequences
separated by X nucleotides (where X is generally between 50 to
2,000 base pairs).
[0160] The two oligonucleotides serve as specific primers for in
vitro DNA synthesis catalysed by DNA polymerase which copies the
DNA between the sequences corresponding to the two
oligonucleotides. If the two primer oligonucleotides contain the
correct sequence it is possible to create new digestion sites at
the 5' and 3'.
[0161] After multiple cycles of reaction, a large amount of a DNA
fragment of the desired length was obtained, purified by gel
electrophoresis and characterized by restriction enzyme digestion
and agarose gel electrophoresis. The amplified, purified DNA
fragment was then used to ligate it together with other fragments
i.e. plasmid.
[0162] The PCR-DNA fragments were amplified with blunt end. To get
sticky end (for the ligation procedure) the fragment has to be
digested with the desired endonucleases and purified again.
[0163] The PCR-reaction will work for 20 to 30 cycles. One cycle is
separated into three steps with different reaction times and
different reaction temperatures which is controlled by a
PCR-thermo-cycler. The first step is "Denaturation" of the
matrix-DNA (1 min-95.degree. C.), the second step is
"Hybridisation" of matrix DNA and primers. (1 min/55.degree. C.)
followed by "Polymerisation" (2 min/72.degree. C.).
[0164] The final volume for one assay is 30 .mu.l for example,
which contains the following final concentrations: PCR-buffer
(1.times.), nucleotide-mix with 200 .mu.M of each of the four
nucleotides, 200 ng for 30 .mu.l of each of the two primers, 0.5
units Taq-Polymerase per 30 .mu.l aqua bidest.
EXAMPLE 6
Culturing of Transfected Cells to Secrete Protein
[0165] The recipient cells (C127 or CHO-cells available from ATCC)
were seeded in normal growth medium (DMEN+10% Fetal Calf Serum,
Glucose and Glutamine) into petri-dishes (1-2.times.10.sup.6 cells
per dish, .phi. 10 cm) at day 1. The next day the medium was
removed (4 hours before the DNA precipitate was added onto the
cells), and the cells were washed twice with 1.times.PBS. Then 8 ml
DMEM without FCS were added, 4 hours later the DNA precipitate
(prepared as described below) was added to the cells. Again after 4
hours the medium was removed, 3 ml of Glycerol-Mix (50 ml
2.times.TBS buffer, 30 ml glycerol, 120 ml distilled water) were
added. The Glycerol-Mix was immediately removed after an incubation
at 37.degree. C. for 3 minutes and the cells were washed with
1.times.PBS. The cells were cultivated overnight with 8 ml of DMEM
with 10% FCS.
[0166] After 48 hours, the cells were recovered from the dish by
treating with Trypsin-EDTA-Solution (0.025% Trypsin+1 mM EDTA).
Afterwards, to remove the Trypsin-EDTA the cells were washed with
1.times.PBS, suspended in DMEM with 10% FCS and distributed into 24
costar-well-plates (cells from one dish into four
24-well-plates).
[0167] When the cells had grown well, selection medium was added
(concentration 0.5-1 mg/ml of neomycin or: xanthine (250 .mu.g/ml),
hypoxanthine (15 .mu.g/.mu.l) or adenine (25 .mu.g/ml), thymidine
(10 .mu.g/ml), aminopterine (2 .mu.g/ml), mycophenolic acid (25
.mu.g/ml) for eco-gpt, for example). The medium was changed every
week. The first growing cell colonies were seen after 2 weeks.
[0168] To 10 .mu.g of plasmid DNA and 20 .mu.g of carrier-DNA
(salmon sperm DNA, calf-thymus DNA) TE-buffer (10 mM Tris-HCl, 1 mM
EDTA, pH 7.05) was added to a final volume of 440 .mu.l and mixed
together with 60 .mu.l 2 M CaCl.sub.2. Then the same amount of
2.times.TBS (Hepes 50 mM, NaCl 280 mM, Na.sub.2HPO.sub.4 1.5 mM, pH
7.05) was added and mixed well. The precipitation solution was
incubated for 30 minutes at 37.degree. C. and added directly to the
cells which were to be transfected.
EXAMPLE 7
Preparation of the Adjuvant of Purified Particles
[0169] To the desired concentration of antigen suspended in sterile
saline, 1:10,000 volume Thimerosol, 1/10 volume of
filter-sterilized 0.2 M KAl(SO.sub.4).sub.2.phi.12 H.sub.2O were
added. The pH was adjusted to 5.0 with sterile 1 N NaOH and the
suspension was stirred at room temperature for 3 hours. The
alum-precipitated antigen was recovered by centrifugation for 10
minutes at 2,000 rpm, resuspended in sterile normal saline
containing 1:10,000 Thimerosol and aliquoted under sterile
conditions.
EXAMPLE 8
Purification of Hepatitis-B-Core Antigen
[0170] The cell supernatant of HB-core-antigen-secreting cells was
collected and concentrated by ultrafiltration. The concentrate was
cleared by centrifugation at 20,000 rpm for 15 minutes at 4.degree.
C. in a Beckman SW28 rotor.
[0171] Particle formaiton was tested by sucrose density
centrifugation (0-45% sucrose) in a Beckman SW28 rotor for 24 hours
at 28,000 rpm and 4.degree. C. The gradient was fractionated and
the single fractions were analyzed by Elisa.
EXAMPLE 9
[0172] The following tables give some results of Elisa analysis of
immunogenic particles of the present invention as described
below:
[0173] Table IV shows the Elisa data of the purified HBs-antigen
particle produced from any HBV-sequence construct of the present
invention including the pre-S1 epitopes and the S region with the
anti-pre-S1 monoclonal antibody MA 18/7 and the anti-HBs monoclonal
antibody G022.
[0174] Table IV shows the fractions (21) collected after CsCl
density gradient.
6 TABLE IV-1 CsCl-gradient Elisa Measurement (E = 492) Fraction No.
Monoclonal Antibody 18/7 13 0.092 14 0.210 15 0.388 16 1.662 17
2.604 18 0.648 19 0.031
[0175]
7 TABLE IV-2 CsCl-gradient Elisa Measurement (E = 492) Fraction No.
Monoclonal Antibody G022 13 0.136 14 0.426 15 0.822 16 1.970 17
2.954 18 0.967 19 0.076
[0176] Table V shows the Elisa data of the purified HB-core-antigen
particles produced from any HB-core-sequence construct of the
present invention with polyclonal antibodies against HB-core and
with monoclonal antibody G022 against HB-S-Ag.
8 TABLE V-1 Sucrose Gradient Elisa Measurement (E = 492) Fraction
No. Polyclonal Antibodies 6 0.25 7 0.922 8 1.423 9 1.5 10 1.5 11
1.28 12 0.466
[0177]
9 TABLE V-2 Sucrose Gradient Elisa Measurement (E = 492) Fraction
No. Monoclonal Antibody G022 6 0.020 7 0.024 8 0.018 9 0.011 10
0.015 11 0.020 12 0.022
EXAMPLE 10
Studies of Administering Hepa-Care in Chimpanzees
[0178] Hepa-Care are particles presenting hepatitis B surface
antigens (S1 and S) in a specific formulation (ratio 50:50), which
are used for the treatment of chronic carriers of hepatitis
virus.
[0179] Experiment 1
[0180] A Hepatitis-B-carrier chimpanzee 1 was treated
(intramuscularly) with Hepa-Care at time 0, 4, and 8 weeks with a
dosage of 18 .mu.g per injection.
[0181] The liver enzymes were monitored (FIG. IV) as well as the
hepatitis-B antigen level (FIG. V).
[0182] Experiment 2
[0183] Chimpanzee 1 after treatment described above was given a
booster treatment at week 30, 34, and 38. The results are shown in
FIG. VI.
[0184] Experiment 3
[0185] Chimpanzee 2 was treated with Hepa-Care, but contrary to
chimpanzee 1 it was given intravenously. The dosage was 2 mg. The
results are shown in FIG. VII.
[0186] From a control chimpanzee 3 the liver enzymes were also
monitored and shown in FIG. VIII.
EXAMPLE 11
Treatment with Hepa-care
[0187] (For Definition See Example 10)
10 Patient 1 (male, age = 65 years, disease for 2 years):
Hepatitis-B parameters: HBsAg pos. anti-HBs neg. HBeAg neg.
anti-HBe pos. anti-HBc neg.
[0188] was treated (i.m.) with Hepa-Care at month 0, 1, 6, and 7.
The results of the antigen and antibody measurements are given in
FIG. IX and X.
11 Patient 2 (female, age = 48 years, disease for 12 years):
Hepatitis-B parameters: HBsAg pos. HBeAg neg. anti-HBs neg.
anti-HBe pos. anti-HBc pos.
[0189] was treated (i.m.) with Hepa-Care at month 0, 1, and 6.
Results of antigen and antibody measurements are shown in FIG. XI
and XII.
12 Patient 3 (female, age = 41 years, disease for 5 years):
Hepatitis-B parameters: HBsAg pos. HBeAg neg. anti-HBs neg.
anti-HBe pos.
[0190] was treated at month 0, 1, 2, and 5 with Hepa-Care (i.m.).
The measured values of HBs antigen and anti-HBs antibodies are
shown in FIG. XIII and XIV.
Sequence CWU 1
1
* * * * *