U.S. patent application number 10/509249 was filed with the patent office on 2006-04-27 for therapeutic drug using antibody-presenting hollow protein nanoparticles and hollow protein nanoparticles.
Invention is credited to Akihiko Kondo, Shunichi Kuroda, Toshihide Okajima, Masaharu Seno, Katsuyuki Tanizawa, Masakazu Ueda.
Application Number | 20060088536 10/509249 |
Document ID | / |
Family ID | 28677594 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060088536 |
Kind Code |
A1 |
Kuroda; Shunichi ; et
al. |
April 27, 2006 |
Therapeutic drug using antibody-presenting hollow protein
nanoparticles and hollow protein nanoparticles
Abstract
The invention provides a therapeutic drug that uses hollow
protein nanoparticles displaying an antibody against a specific
cell or specific tissue. The effectiveness of the drug has been
proved by animal testing. The invention also provides a therapeutic
method using such a drug. In a drug according to the present
invention, a substance to be transferred into a cell for treating a
disease (for example, a cancer treating gene such as a thymidine
kinase gene derived from simple herpes virus) is encapsulated in
hollow nanoparticles of a particle-forming protein (for example,
hepatitis B virus surface-antigen protein that has been modified to
lack its infectivity to hepatocytes and display an antibody). The
particle surface of the drug displays an antibody, such as a cancer
specific antibody, that recognizes an antigen molecule displayed on
the surface of a specific cancer cell.
Inventors: |
Kuroda; Shunichi; (Suita-shi
Osaka, JP) ; Tanizawa; Katsuyuki; (Toyono-cho,
JP) ; Okajima; Toshihide; (Iguchido Ikeda-shi,
JP) ; Kondo; Akihiko; (Kobe-shi, JP) ; Ueda;
Masakazu; (Shinjuku-ku, JP) ; Seno; Masaharu;
(Kadotabunkamachi Okayama-shi, JP) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O. BOX 8910
RESTON
VA
20195
US
|
Family ID: |
28677594 |
Appl. No.: |
10/509249 |
Filed: |
March 26, 2003 |
PCT Filed: |
March 26, 2003 |
PCT NO: |
PCT/JP03/03694 |
371 Date: |
September 28, 2004 |
Current U.S.
Class: |
424/155.1 ;
424/161.1; 424/489; 977/918 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/2863 20130101; C07K 16/40 20130101; C12N 2730/10123
20130101; A61K 47/6849 20170801; A61K 2039/55555 20130101; B82Y
5/00 20130101; A61K 9/5184 20130101; A61K 47/6925 20170801; C12N
2810/859 20130101; C12N 2730/10122 20130101; C12N 7/00 20130101;
A61K 48/0041 20130101; C12N 15/88 20130101; C07K 2317/622 20130101;
C07K 16/18 20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/155.1 ;
424/489; 424/161.1; 977/918 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/42 20060101 A61K039/42; A61K 9/14 20060101
A61K009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2002 |
JP |
2002-97424 |
Feb 21, 2003 |
JP |
2003-45088 |
Claims
1. A drug that comprises hollow nanoparticles of a particle-forming
protein, the hollow nanoparticles displaying an antibody against a
specific cell or specific tissue, and encapsulating a substance to
be transferred into a cell for treating a disease.
2. The drug as set forth in claim 1, wherein the antibody is a
cancer specific antibody or anti-virus protein antibody.
3. The drug as set forth in claim 1, wherein the antibody is
displayed on a particle surface by binding to a ZZ tag fused with
the particle-forming protein.
4. The drug as set forth in claim 1, wherein the antibody is
biotin-modified and displayed on a particle surface with its biotin
binding to streptavidin or its derivative that is ligated to a
streptag fused with the particle-forming protein.
5. The drug as set forth in claim 1, wherein the antibody is a
single chain antibody fused with the particle-forming protein.
6. The drug as set forth in claim 1, wherein the hollow
nanoparticles of a particle-forming protein are expressed in a
eukaryotic cell.
7. The drug as set forth in claim 6, wherein the eukaryotic cell is
selected from a group consisting of a yeast cell, insect cell, and
animal cell.
8. The drug as set forth in claim 1, wherein the particle-forming
protein comprises a modified hepatitis B virus surface-antigen
protein.
9. The drug as set forth in claim 8, wherein the modified hepatitis
B virus surface-antigen protein is modified to lack some of amino
acids in a pre-S region.
10. The drug as set forth in claim 8, wherein the modified
hepatitis B virus surface-antigen protein is serotype y, and
modified to retain at least N-terminal amino acid residues 1 to 20
in the entire amino acid sequence of the pre-S region.
11. The drug as set forth in claim 10, wherein the modified
hepatitis B virus surface-antigen protein is modified to lack
N-terminal amino acids 50 to 153 in the entire amino acid sequence
of the pre-S region.
12. The drug as set forth in claim 8, wherein the modified
hepatitis B virus surface-antigen protein is serotype d, and
modified to retain at least N-terminal amino acid residues 12 to 31
in the entire amino acid sequence of the pre-S region.
13. The drug as set forth in claim 12, wherein the modified
hepatitis B virus surface-antigen protein is modified to lack
N-terminal amino acids 61 to 164 in the entire amino acid
14. The drug as set forth in claim 1, wherein the disease-treating
substance comprises a gene.
15. The drug as set forth in claim 14, wherein the gene comprises a
thymidine kinase (HSV1tk) gene derived from simple herpes
virus.
16. The drug as set forth in claim 1, wherein the drug is
administered to the human body through intravenous injection.
17. A disease treating method comprising administering the drug of
claims 1.
18. Hollow nanoparticles that comprise a hepatitis B virus
surface-antigen protein of serotype y, the hepatitis B virus
surface-antigen protein forming particles and being modified to
retain at least N-terminal amino acid residues 1 to 20 in the
entire amino acid sequence of a pre-S region.
19. The hollow nanoparticles as set forth in claim 18, wherein the
modified hepatitis B virus surface-antigen protein is modified to
lack N-terminal amino acids 50 to 153 in the entire amino acid
sequence of the pre-S region.
20. Hollow nanoparticles that comprise a hepatitis B virus
surface-antigen protein of serotype d, the hepatitis B virus
surface-antigen protein forming particles and being modified to
retain at least N-terminal amino acid residues 12to 31 in the
entire amino acid sequence of a pre-S region.
21. The hollow nanoparticles as set forth in claim 20, wherein the
modified hepatitis B virus surface-antigen protein is modified to
lack N-terminal amino acids 61 to 164 in the entire amino acid
sequence of the pre-S region.
22. The drug as set forth in claim 2, wherein the antibody is
displayed on a particle surface by binding to a ZZ tag fused with
the particle-forming protein.
23. The drug as set forth in claim 2, wherein the antibody is
biotin-modified and displayed on a particle surface with its biotin
binding to streptavidin or its derivative that is ligated to a
streptag fused with the particle-forming protein.
24. The drug as set forth in claim 2, wherein the antibody is a
single chain antibody fused with the particle-forming protein.
25. The drug as set forth in claim 2, wherein the hollow
nanoparticles of a particle-forming protein are expressed in a
eukaryotic cell.
26. The drug as set forth in claim 9, wherein the modified
hepatitis B virus surface-antigen protein is serotype y, and
modified to retain at least N-terminal amino acid residues 1 to 20
in the entire amino acid sequence of the pre-S region.
27. The drug as set forth in claim 9, wherein the modified
hepatitis B virus surface-antigen protein is serotype d, and
modified to retain at least N-terminal amino acid residues 12 to 31
in the entire amino acid sequence of the pre-S region.
28. A disease treating method comprising administering the drug of
claim 2.
Description
TECHNICAL FIELD
[0001] The present invention relates to therapeutic drugs using
antibody-displaying hollow protein nanoparticles, and to hollow
protein nanoparticles. The invention particularly relates to a drug
whose particle surface displays bio-recognizing molecules such as
an antibody against a specific cell or tissue, and which contains
particles encapsulating a substance to be transferred into a cell
for treating a disease, wherein the drug allows the
disease-treating substance to be specifically incorporated into a
specific cell or tissue. The invention also relates to particles
suitable for the drug.
BACKGROUND ART
[0002] In the field of medicine, there has been active research on
drugs that directly and effectively act on the affected area
without causing serious side effects. One area of active research
is a method known as a drug delivery system (DDS), in which active
ingredients of drugs or other substances are specifically delivered
to a target cell or tissue, where they can exhibit their
effects.
[0003] Another area of active research is a technique of gene
transfer to a specific cell, which is now essential in the field of
molecular cell biology. With the genetic background of various
diseases being revealed by the Human Genome Project, a method of
highly specific gene transfer to a specific cell or tissue holds
great promise because, once the method is established, it is
applicable to the field of gene therapy.
[0004] In one known example of a gene transfer method to cells,
uptake of genes takes place in the form of a giant molecule by
endocytosis (calcium phosphate method, lipofectamin method). In
another method, genes are transferred through cell membrane pores
that are formed by the stimulation of the cell membrane with an
electrical pulse (electroporation method, gene gun method). Both of
these methods are commonly used in molecular biology
experiments.
[0005] Despite the simplicity of these methods, they cannot be
readily applied to cells or tissues of internal body, because the
methods involve direct physical contact with the cells and
surgically expose the site of gene transfer. It is also difficult
to achieve near 100% uptake.
[0006] A transfer method that is safe to use is a liposome method.
The liposome method does not damage the cell and is applicable to
cells or tissues of internal body. A problem of the method,
however, is that the liposome, which is a simple lipid, cannot have
a high level of specificity to the cells or tissues, and uptake of
genes in vivo is far below the required level.
[0007] In a recently developed technique, a therapeutic gene is
inserted in viral DNA, and the gene is transferred by an infectious
virus. The method is innovative in the sense that it does not
expose the site of transfer, is applicable to individuals, and
provides near 100% uptake. However, the method suffers from a
serious drawback in that the virus non-specifically infects a wide
range of cells, transferring the gene to cells other than the
target cell. Further, the method has a potential risk of unexpected
side effect if the viral genome is incorporated in the chromosomes.
In fact, the method is not used in initial stages of disease
treatment. Only the terminal patients can receive the benefit of
the method.
[0008] In sum, none of the conventional gene transfer methods is
sufficient to specifically transfer genes to a target cell and
express the protein therein to produce a drug. To this date, there
has been no effective method of directly delivering a protein as a
drug into a target cell or tissue.
[0009] Under these circumstances, the inventors of the present
invention have previously proposed a method of specifically and
safely delivering and transferring various substances (including
genes, proteins, compounds) into a target cell or tissue, using
hollow nanoparticles of a protein that has the ability to form
particles and has incorporated a bio-recognizing molecule, as
disclosed in International Publication with International
Publication No. WO01/64930 (published on Sep. 7, 2001) (hereinafter
referred to as "International Publication WO01/64930"), and in
Japanese Publication for Unexamined Publication No. 316298/2001
(published on Nov. 13, 2001). However, these publications do not
fully discuss how the method can be used to develop drugs for the
treatment of diseased cells or tissues (cancer, for example).
Specifically, the development of drugs displaying a specific
antibody for specific cancer cells or tissues remains to be one of
the most important goals to be achieved, particularly in view of
the following problems.
[0010] Owning to the difficulty in specifically and safely
delivering and transferring a protein (drug) into a target cell or
tissue, a great burden has been put on the patients receiving
treatment using such a protein drug.
[0011] For example, for the treatment of viral hepatitis (hepatitis
C in particular), an interferon, which is one form of a protein
drug, is administered systemically through intravenous injection
over an extended time period. Though the effectiveness of the
treatment is well recognized, it has many side effects due to the
non-specific action of the interferon, including high fever, loss
of hair, tiredness, and immune response, which occur every time the
drug is administered.
[0012] The hepatocyte growth factor is known to be effective for
the treatment of liver cirrhosis. However, since systemic
administration of the drug through intravenous injection may cause
unexpected side effects, the hepatocyte growth factor is directly
administered to the liver with a catheter. The use of catheter
requires surgery, which puts a burden on the patient if he or she
must receive prolonged treatment.
[0013] The present invention was made in view of the foregoing
problems, and an object of the invention is to provide a
therapeutic drug, proved to be effective by animal testing, that
specifically acts on a target cell or tissue with its hollow
protein nanoparticles displaying bio-recognizing molecules such as
an antibody. The invention also provides a therapeutic method, and
hollow nanoparticles for use in such a therapeutic drug and
therapeutic method.
DISCLOSURE OF INVENTION
[0014] The inventors of the present invention accomplished the
present invention by successfully preparing different types of
hollow protein nanoparticles displaying an antibody, and by finding
that hollow nanoparticles displaying an antibody specific to the
human squamous carcinoma cell was effective in the treatment of
transplanted cancer when a drug encapsulating a cancer treating
gene in the hollow nanoparticles was administered in laboratory
animals through intravenous injection.
[0015] That is, the present invention discloses a drug in which a
substance to be transferred into a cell for treating a disease is
encapsulated in hollow nanoparticles of a protein-forming protein
displaying an antibody against a specific cell or specific
tissue.
[0016] An example of such a protein is a hepatitis B virus
surface-antigen protein that has been modified to lose its
infectivity to the hepatocytes and display an antibody. In
eukaryotic cells, the protein is expressed as a membrane protein on
the endoplasmic reticulum and accumulates thereon before it is
released as particles into the lumen. With the antibody displayed
on the particle surface, the hollow nanoparticles can act as a
carrier, delivering the substance encapsulated in the particles
specifically to a specific cell or specific tissue. As used herein,
"specific cell or specific tissue" refers to cells into which the
substance encapsulated in the particles is introduced by the
binding of the antibody with an antigen displayed on the cell
surface, or tissues as a collection of such cells into which the
substance is introduced.
[0017] The pre-S regions (pre-S1, pre-S2) of the hepatitis B virus
surface-antigen protein have important roles in the binding of HBV
to the hepatocytes. Thus, the hepatitis B virus surface-antigen
protein can be modified to lose its infectivity to the hepatocytes
by deleting some of the amino acids in the pre-S regions. In this
way, the substance in the particles can also be introduced into
cells or organs other than the liver.
[0018] When some of the amino acids in the pre-S region are deleted
to remove the infectivity of the protein to the hepatocytes, the
level of expression of the modified hepatitis B virus
surface-antigen protein in the eukaryotic cell varies depending on
the deleted area of pre-S region. The level of protein expression
in the eukaryotic cell tends to decrease particularly when the
protein is modified to display an antigen.
[0019] It is therefore preferable, in order to maintain a
sufficient level of protein expression in the eukaryotic cell, that
the protein (in the case of serotype y) be modified to retain at
least N-terminal amino acid residues 1 to 20 in the entire amino
acid sequence of the pre-S region (pre-S1, pre-S2 regions), or more
preferably the protein be modified by deleting N-terminal amino
acids 50 to 153 in the entire amino acid sequence of the pre-S
region. For serotype d, the protein is preferably modified to
retain at least N-terminal amino acid residues 12 to 31, or more
preferably the protein is modified by deleting N-terminal amino
acids 61 to 164 in the entire amino acid sequence of the pre-S
region.
[0020] In this way, the hepatitis B virus surface-antigen protein
modified to lose its infectivity to the hepatocytes and display an
antibody is expressed in large amounts in the eukaryotic cell. With
the increased amount of protein, more substance in the protein can
be transported into specific cells or tissues, thereby greatly
enhancing the effectiveness of the substance.
[0021] An example of the antibody is a cancer specific antibody or
an anti-virus protein antibody. For example, a cancer treating
substance (medicament) may be encapsulated in the hollow
nanoparticles displaying a cancer-specific antibody. This provides
an effective therapeutic drug that specifically and effectively
acts on cancer cells. The anti-virus protein antibody is effective
in the removal of virus-infected cells.
[0022] The antibody has a single chain or double chain. Due to its
structure, the double chain antibody cannot readily be displayed on
the particle surface by directly fusing it with the
particle-forming protein. The inventors of the present invention
found ways to successfully display the double chain antibody on the
surface of the hollow nanoparticles by indirectly binding the
double chain antibody to the protein. Specifically, the double
chain antibody was displayed on the particle surface by first
introducing a ZZ tag into the protein (fused with the protein),
wherein the ZZ tag specifically binds to the Fc site of the double
chain antibody, and then by ligating the ZZ tag to the Fc site.
Another way to display the double chain antibody on the particle
surface is to introduce a streptag into the protein (fused with the
protein), wherein the streptag specifically binds to streptavidin
(or its derivative), and bind the streptag to the streptavidin (or
its derivative). The double chain antibody, which has been modified
by biotin that specifically binds to the streptavidin (or its
derivative), can then be displayed on the particle surface by
ligating the streptavidin (or its derivative) to the biotin
attached to the double chain antibody. The single chain antibody
can be displayed on the particle surface by expressing it with the
protein directly fused with the antibody.
[0023] Other than these methods, the antibody may be displayed on
the particle surface by common binding methods involving chemical
modification.
[0024] The hollow protein nanoparticles are preferably the product
of expression in eukaryotic cells. The eukaryotic cell may be
obtained from yeasts, insects, or animals including mammals.
[0025] The target-cell substance encapsulated in the hollow
nanoparticles may be a cancer treating gene, for example. When the
cancer treating gene encapsulated in the drug is a thymidine kinase
(HSV1tk) gene derived from simple herpes virus, ganciclovir is
additionally administered, as will be described in Examples.
[0026] The present invention discloses a drug that can be used by a
convenient method of intravenous injection to effectively treat
specific diseased cells or tissues. The drug is a great leap
forward from conventional disease treatment methods in that it does
not require large dose or any surgical operation in disease
treatment including gene therapy, and that the risk of side effect
is greatly reduced. The drug is therefore usable in clinical
applications in its present form.
[0027] The present invention discloses a treatment method for
treating diseases through administration of the drug disclosed in
the present invention.
[0028] The present invention discloses hollow nanoparticles of a
hepatitis B virus surface-antigen protein of serotype y, the
hepatitis B virus surface-antigen protein forming particles and
being modified to retain at least N-terminal amino acid residues 1
to 20 in the entire amino acid sequence of the pre-S region.
Preferably, the protein is modified by deleting N-terminal amino
acids 50 to 153 in the entire amino acid sequence of the pre-S
region.
[0029] The present invention discloses hollow nanoparticles of a
hepatitis B virus surface-antigen protein of serotype d, the
hepatitis B virus surface-antigen protein forming particles and
being modified to retain at least N-terminal amino acid residues 12
to 31 in the entire amino acid sequence of the pre-S region.
Preferably, the protein is modified by deleting N-terminal amino
acids 61 to 164 in the entire amino acid sequence of the pre-S
region.
[0030] The hollow nanoparticles are expressed in large amounts
particularly in the eukaryotic cell, and are suitable for
displaying bio-recognizing molecules. For example, the hollow
nanoparticles may be used as hollow bio-nanoparticles in gene
therapy or DDS.
[0031] For a fuller understanding of the nature and advantages of
the invention, reference should be made to the ensuing detailed
description taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0032] FIG. 1 is a schematic diagram showing protein regions of
HBsAg gene described in Examples of the present invention, where
the numbers 1 through 8 indicate respective functions of different
sites on a surface antigen, and Pre-S1 indicates 108 amino acid
residues for serotype y, and 119 amino acid residues for serotype
d.
[0033] FIG. 2 is an explanatory drawing schematically showing one
example of expression and purification procedures for HBsAg
particles using recombinant yeasts, as described in Examples of the
present invention, wherein (a) illustrates preparation of
recombinant yeasts, (b) illustrates incubation in High-Pi medium,
(c) illustrates incubation in 8S5N-P400 medium, (d) illustrates
disruption, (e) illustrates density gradient centrifugation, and
(f) illustrates HBsAg particles.
[0034] FIG. 3 is a diagram showing steps of constructing a plasmid
used for preparation of HBsAg-streptag particles with yeasts, as
described in Examples of the present invention.
[0035] FIG. 4 is a diagram showing steps of constructing a plasmid
used for preparation of HBsAg-streptag particles with insect cells,
as described in Examples of the present invention.
[0036] FIG. 5 is a diagram showing steps of constructing a plasmid
used for preparation of HBsAg-streptag particles with animal cells,
as described in Examples of the present invention.
[0037] FIG. 6 is a diagram showing steps of constructing a plasmid
used for preparation of HBsAg-ZZ tag particles with yeasts, as
described in Examples of the present invention.
[0038] FIG. 7 is a diagram representing results of SDS-PAGE and
Western blotting performed on the HBsAg-ZZ tag particles obtained
with yeasts.
[0039] FIG. 8 is a diagram showing steps of constructing a plasmid
used for preparation of HBsAg-ZZ tag particles (or HBsAg-scFv
particles displaying single chain antibody A22 or 3A21) with insect
cells, as described in Examples of the present invention.
[0040] FIG. 9 is a diagram showing steps of constructing a plasmid
used for preparation of HBsAg-ZZ tag particles (or HBsAg-scFv
particles displaying single chain antibody A22 or 3A21) with animal
cells, as described in Examples of the present invention.
[0041] FIG. 10 is a graph showing a result of treatment on
laboratory animals using the HBsAg-ZZ tag particles as a drug of
the present invention.
[0042] FIG. 11 is a diagram representing results of SDS-PAGE and
Western blotting performed on the HBsAg-scFv particles.
[0043] FIG. 12 is a schematic diagram showing deletion HBsAg
protein expression genes, as described in Examples of the present
invention.
[0044] FIG. 13 is a diagram showing reaction compositions of PCR as
described in Examples of the present invention.
[0045] FIG. 14 is a diagram showing a PCR cycle as described in
Examples of the present invention.
[0046] FIG. 15 is a schematic diagram illustrating deletion HBsAg
protein expression genes and a plasmid into which the genes are
transferred, as described in Examples of the present invention.
[0047] FIGS. 16(a) and 16(b) are graphs showing results of enzyme
immunoassay performed on deletion HBsAg protein in animal cells as
described in Examples of the present invention, wherein FIG. 16(a)
is a result in supernatant, and FIG. 16(b) is a result in
cells.
[0048] FIG. 17 is a diagram representing the results of FIGS. 16(a)
and 16(b) in data form.
[0049] FIG. 18 is a diagram showing results of SDS-PAGE performed
on the deletion HBsAg protein expressed in FIGS. 16(a) and 16(b),
wherein (a) is a result in supernatant, and (b) is a result in
cells.
[0050] FIG. 19 is a diagram showing results of Western blotting
performed on the deletion HBsAg protein expressed in FIGS. 16(a)
and 16(b), wherein (a) is a result in supernatant, and (b) is a
result in cells.
[0051] FIG. 20 is a schematic diagram illustrating deletion HBsAg
protein expression genes transferred into yeasts, and a plasmid
into which the genes are transferred, as described in Examples of
the present invention.
[0052] FIG. 21 is a diagram showing a result of enzyme immunoassay
in data form, confirming the expression of deletion HBsAg L protein
using the plasmid of FIG. 20.
[0053] FIG. 22 is a graph representing a result of enzyme
immunoassay, confirming the expression of deletion HBsAg L protein
using the plasmid of FIG. 20.
[0054] FIG. 23 is a diagram listing examples of target-cell
substances according to the present invention.
[0055] FIG. 24 is a diagram listing examples of target-cell
substances according to the present invention.
[0056] FIG. 25 is a diagram listing examples of target-cell
substances according to the present invention.
[0057] FIG. 26 is a diagram listing examples of target-cell
substances according to the present invention.
[0058] FIG. 27 is a table representing a result of treatment on
laboratory animals using a drug according to the present
invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0059] The present invention discloses a drug including hollow
nanoparticles whose particle surface displays an antibody as a
bio-recognizing molecule (molecule that recognizes a specific
cell), and which contains particles encapsulating a substance to be
transferred into a cell for treating a disease, wherein the drug
allows the disease-treating substance to be specifically delivered
to a target cell or tissue. The hollow nanoparticles may be a
protein able to form particles, which may be sub viral particles
obtained from various viruses. Specific examples of such a protein
include hepatitis B virus (HBV) surface-antigen protein.
[0060] Particles of such a protein may be obtained through the
protein expression in the eukaryotic cell. Specifically, in
eukaryotic cells, the particle-forming protein is expressed on the
endoplasmic reticulum as a membrane protein and accumulates thereon
before it is released as particles. The eukaryotic cell may be
obtained from yeasts, insects, or animals including mammals.
[0061] As will be described later in Examples, the inventors of the
present invention have reported that the expression of HBV
surface-antigen L protein in recombinant yeast cells produces
ellipsoidal hollow particles with a minor axis of 20 nm and a major
axis of 150 nm, with a large number of L proteins embedded in the
yeast-derived lipid bilayer membrane (J. Biol. Chem., Vol. 267, No.
3, 1953-1961, 1992). The particles contain no HBV genome and lack
the viral function. Therefore, the particles are very safe to the
human body.
[0062] The HBV surface-antigen L protein may be modified to lack
its infectivity to the hepatocytes and display an antibody (cancer
specific antibody, for example) on the particle surface. With the
antibody on the particle surface of the expressed protein, the
protein can effectively serve as a carrier for specifically
delivering substances to cells or tissues (cancer cell or cancer
tissue in the case of a cancer specific antibody) whose cell
surface has an antigen against the antibody.
[0063] The pre-S regions (pre-S1, pre-S2) of the HBV
surface-antigen L protein have important roles in the binding of
HBV to the hepatocytes (see FIG. 1). Thus, the HBV surface-antigen
L protein can be modified to lose its infectivity to the hepatocyte
by deleting some of the amino acids in the pre-S regions. As used
herein, "deletion of some of the amino acids in the pre-S region"
means deleting some of the amino acids in the preS1 region or preS2
region, or both of these regions. For example, infectivity to the
hepatocytes can be lost by deleting N-terminal amino acids 3 to 66
(serotype y) or N-terminal amino acids 4 to 77 (serotype d), known
as a recognition site for the human hepatocytes, in the pre-S
region (specifically PreS1 region).
[0064] When the protein is modified to lose its infectivity to the
hepatocytes by deleting at least some of the amino acids in the
pre-S region, the level of protein expression in the eukaryotic
cell varies in the modified hepatitis B virus surface-antigen
protein, depending on the region of amino acid deleted. The level
of protein expression is prone to decrease particularly when the
protein is modified to display antigens.
[0065] The modified hepatitis B virus surface-antigen protein can
be expressed in a large amount in the eukaryotic cell when amino
acids in the pre-S region are deleted in domain, as will be
described in Examples. Specifically, as noted above, the level of
protein expression in the eukaryotic cell can be increased by
deleting N-terminal amino acids 3 to 66 (serotype y) or N-terminal
amino acids 4 to 77 (serotype d), known as a recognition site for
the human hepatocytes, in the preS1 region. For serotype y, the
protein may be modified to retain at least N-terminal amino acid
residues 1 to 20. For serotype d, at least N-terminal amino acid
residues 12 to 31 may be retained.
[0066] The level of protein expression can be further increased by
preferably deleting some of the amino acids in the preS2 region, in
addition to some of the amino acids making up the recognition site
for the human hepatocytes in the preS 1 region.
[0067] More specifically, it is preferable in the entire amino acid
sequence in the pre-S region (pre-S1 region, pre-S2 region) that
the protein be modified to delete N-terminal amino acids 50 to 153
and retain at least N-terminal amino acid residues 1 to 20. For
example, for serotype y, it is preferable in the entire amino acid
sequence of the pre-S region that the protein be modified to lack
domains of amino acid in the first 153 amino acids from the
N-terminus, specifically, from amino acids 50 to 153, 33 to 153,
and 21 to 153, as will be described later in Examples. Among these
domains, it is particularly preferable to delete amino acids 50 to
153. Note that, the deleted range of amino acid is not just limited
to this example.
[0068] For serotype d, it is preferable in the entire amino acid
sequence of the pre-S region (preS1 region, preS2 region) that the
protein be modified to lack N-terminal amino acids 61 to 164 and
retain at least N-terminal amino acid residues 12 to 31.
[0069] The hepatitis B virus surface-antigen protein so modified to
lose its infectivity to the hepatocytes and display antibody is
expressed in a large amount in the eukaryotic cell, and therefore
is highly advantageous in terms of productivity. With the increased
amount of protein, more substance in the protein can be transported
into specific cells or tissues, thereby greatly enhancing the
effectiveness of the substance.
[0070] Therefore, forming the protein particles using recombinant
yeasts offers a preferable method of efficiently producing
particles from soluble proteins in the yeasts.
[0071] The insect cell, being a eukaryote closer to some of the
higher animals than the recombinant yeast, is able to form a higher
order structure such as a sugar chain unachievable by yeasts. In
this connection, the insect cell provides a preferable method of
producing heteroproteins in large amounts. The conventional insect
cell line used the baculovirus and involved viral expression. This
has caused a cell death or lysis in the protein expression. A
problem of this method, then, is that the protein expression
proceeds continuously, or the proteins are decomposed by the free
protease separated from the dead cells. Further, in the secretion
and expression of proteins, inclusion of a large amount of fetal
bovine serum contained in the culture medium has made it difficult
to purify proteins even when proteins are secreted in the medium.
In recent years, Invitrogen Corporation has developed and marketed
an insect cell line that can be cultured without a serum and
without being meditated by the baculovirus. Such an insect line can
be used to obtain protein particles that are easy to purify and
form into higher order structures.
[0072] Hollow protein nanoparticles of the present invention are
prepared by binding an antibody to the surface of particles
obtained by the foregoing methods. With various substances (DNA,
RNA, proteins, peptides, drugs, etc.) incorporated into the
particles, the hollow protein nanoparticles can very specifically
deliver and transfer these substances to cells bearing
corresponding antigens on its cell surface.
[0073] The particle-forming protein is not just limited to the
modified hepatitis B virus surface-antigen protein. For example,
animal cells, plant cells, viruses, natural proteins derived from
fungi, and various types of synthetic proteins may be used.
Further, when there is a possibility that, for example,
virus-derived antigen proteins may trigger antibody reaction in a
target organism, a particle-forming protein with suppressed
antigenic action may be used. For example, such a protein may be
the hepatitis B virus surface-antigen protein modified to suppress
its antigenic action, or other types of modified proteins
(hepatitis B virus surface-antigen protein modified by genetic
engineering), as disclosed in International Publication
WO01/64930.
[0074] The type of antibody bound to the particle surface is not
particularly limited as long as it recognizes a surface molecule of
a specific cell as an antigen. For example, the antibody may be a
cancer specific antibody that recognizes a surface molecule of a
specific cancer cell as an antigen. As another example, an antibody
may be used that specifically recognizes an antigen on the surface
of a specific cell as a growth factor receptor or cytokine
receptor. Other than these examples, various types of antibodies
specific to other types of antigens displayed on the cell surface
or tissue surface may be used as well. Specifically, an anti-viral
protein antibody may be used, in addition to the antibodies used in
the Examples below. The antibody should be suitably selected
according to the type of target cell or tissue.
[0075] As described, the present invention provides hollow protein
nanoparticles that encapsulate a substance (target-cell substance)
to be transferred into a target cell or tissue, and thereby
provides a substance carrier (drug) having cell specificity. The
substance carrier may encapsulate any substance including, for
example, genes in the form of DNA or RNA, natural or synthetic
proteins, oligonucleotides, peptides, drugs, and natural or
synthetic compounds.
[0076] For example, human RNase1 or RNase3 may be used, as
previously reported by the inventors of the present invention.
Human RNase1 is documented in Jinno H, Ueda M, Ozawa S, Ikeda T,
Enomoto K, Psarras K, Kitajima M, Yamada H, Seno M Life Sci. 1996;
58(21): 1901-8. Human RNase3 (also known as ECP (eosinophil
cationic protein)) is documented in Mallorqui-Fernandez G, Pous J,
Peracaula R, Aymami J, Maeda T, Tada H, Yamada H, Seno M, de
Llorens R, Gomis-Ruth F X, Coll M; J Mol Boil. 2000 July 28;
300(5): 1297-307.
[0077] The proteins have cytotoxicity, the effects of which are
both intracellular and extracellular. With the RNase encapsulated
in the substance carrier (drug) of the present invention, the
cytotoxicity of the protein can be masked outside the cell, and the
protein exhibits its effect only inside the cell. It is expected
that this will provide a novel cancer treatment method that causes
fewer side effects.
[0078] Note that, the target-cell substance may be proteins shown
in FIG. 23 through 26, or genes that encode these proteins. Other
examples of the substance are various proteins including: cancer
suppressor genes (p53, etc.); interferons; interleukins; cytokines;
colony stimulating factors; tumor necrosis factors; transforming
growth factors .beta.; platelet-derived growth factors;
erythropoietins; and Fas antigens. The target-cell substance may
also be genes that encode these proteins.
[0079] These target-cell substances may be incorporated into the
hollow nanoparticles by various methods commonly used in chemical
or molecular biological experimental techniques. Some of the
preferred examples include an electroporation method, ultrasonic
method, simple diffusion method, and a method using charged
lipids.
[0080] The hollow protein nanoparticles or substance carrier allow
the substance to be specifically transported into cells or tissues
in vivo or in vitro. Specific transport of the substance into a
specific cell or specific tissue with the use of the hollow protein
nanoparticles or substance carrier may be used as a treatment
method of various diseases, or one of the steps in the procedure of
the treatment method.
[0081] In a drug according to the present invention, the antibody
may be displayed on the particle surface by four different methods,
as will be described in the Examples. In the first method, a ZZ tag
that specifically binds to an Fc site of a double chain antibody is
incorporated into a particle-forming protein (in other words,
particles are formed by expressing the protein with the ZZ tag
fused with the protein), and the ZZ tag is bound to the Fc site to
display the double chain antibody on the particle surface. In the
second method, a streptag that specifically binds to streptavidin
is incorporated into a particle-forming protein (in other words,
particles are formed by expressing the protein with the streptag
fused with the protein). The streptag is bound to the streptavidin
(or its derivative), which is then bound to a double chain antibody
that has been modified with biotin that specifically binds to the
streptavidin (or its derivative), thereby displaying the antibody
to the particle surface. In the third method, particles are formed
by expressing the particle-forming protein with a single chain
antibody fused with the protein, thereby displaying the antibody on
the particle surface. The fourth method is chemical binding of the
antibody with particles with the use of common crosslinking agents,
which may be, for example, compounds including the NHS
(N-hydroxysuccinimide) group, maleimide group, or imidoester group
(available from Pierce Biotechnology, Inc.). These methods may be
partly modified by taking advantage of their principles.
[0082] The effectiveness of the treatment using the drug of the
present invention has been confirmed by animal testing, as will be
described later in the Examples. In the Examples, cells derived
from human squamous cell carcinoma were transplanted in nude rats,
and the drug of the present invention and ganciclovir (GCV) were
administered to each rat in separate doses. The drug on its
particle surface had an antibody that recognizes an antigen, the
epidermal growth factor (EGF receptor), expressed by the cancer
cells. Inside the drug, a thymidine kinase (HSV1tk) gene derived
from simple herpes virus was encapsulated. The effectiveness of the
treatment was confirmed by observing the size of grafted cancer
tissue. The drug was administered intravenously. However, oral
administration, intramuscular administration, intraperitoneal
administration, subcutaneous administration, or other
administration routes are also available.
[0083] In the following, the present invention will be described in
more detail by way of Examples with reference to the attached
drawings. It should be appreciated that the present invention is
not limited in any ways by the following Examples, and various
modifications to details of the invention are possible.
[0084] It should also be noted that the techniques described in the
following Examples are all novel and were independently developed
by the inventors of the present invention. The novel techniques
include: incorporating a protein in the preS1 region of the
deletion HBV surface-antigen L protein; producing a deletion HBV
surface-antigen L protein suitable for efficient expression in the
eukaryotic cell; incorporating a bio-recognizing molecule
(antibody) in the deletion HBV surface-antigen L protein for
displaying it on the deletion HBV surface-antigen L protein; and
application of these techniques in gene therapy or DDS.
EXAMPLES
[0085] In the following, HBsAg refers to hepatitis B virus surface
antigen. HBsAg is an envelope protein of HBV, and includes three
kinds of proteins S, M, and L, as schematically illustrated in FIG.
1. S protein is an important envelope protein common to all three
kinds of proteins. M protein includes the entire sequence of the S
protein with additional 55 amino acids (pre-S2 peptide) at the
N-terminus. L protein contains the entire sequence of the M protein
with additional 108 amino acids (serotype y) or 119 amino acids
(serotype d) at the N-terminus. In the following Examples, serotype
y was used.
[0086] The pre-S regions (pre-S1, pre-S2) of HBV have important
roles in the binding of HBV to the hepatocytes. The Pre-S1 region
has a direct binding site for the hepatocytes, and the pre-S2
region has a polymeric albumin receptor that binds to the
hepatocytes via polymeric albumin in the blood.
[0087] Expression of HBsAg in the eukaryotic cell causes the
protein to accumulate as membrane protein on the membrane surface
of the endoplasmic reticulum. The L protein molecules of HBsAg
agglomerate and are released as particles into the ER lumen,
carrying the ER membrane with them as they develop.
[0088] The Examples below used L proteins of HBsAg. FIG. 2 briefly
illustrates procedures of expression and purification of HBsAg
particles described in the following Examples.
Example A
Expression of HBsAg Particles in Recombinant Yeasts
[0089] Recombinant yeasts (Saccharomyces cerevisiae AH22R-strain)
carrying (pGLDLIIP39-RcT) were cultured in synthetic media High-Pi
and 8S5N-P400, and HBsAg L protein particles were expressed (FIG.
2a through 2c). The whole procedure was performed according to the
method described in J. Biol. Chem., Vol. 267, No. 3, 1953-1961,
1992 reported by the inventors of the present invention.
[0090] From the recombinant yeast in stationary growth phase (about
72 hours), the whole cell extract was obtained with the yeast
protein extraction reagent (product of Pierce Chemical Co., Ltd.).
The sample was then separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the
HBsAg in the sample was identified by silver staining.
[0091] The result showed that HBsAg was a protein with a molecular
weight of about 52 kDa.
Example B
Purification of HBsAg Particles from the Recombinant Yeasts
[0092] (1) The recombinant yeast (wet weight of 26 g) cultured in
synthetic medium 8S5N-P400 was suspended in 100 ml of buffer A (7.5
M urea, 0.1 M sodium phosphate, pH 7.2, 15 mM EDTA, 2 mM PMSF, and
0.1% Tween 80), and disrupted with glass beads by using a
BEAD-BEATER. The supernatant was collected by centrifugation (FIG.
2d).
[0093] (2) The supernatant was mixed with a 0.75 volume of PEG 6000
solution (33%, w/w), and cooled on ice for 30 min. The pellets were
collected by centrifugation at 7000 rpm for 30 min, and resuspended
in buffer A without Tween 80.
[0094] (3) The solution was layered onto a 10-40% CsCl gradient,
and ultracentrifuged at 28000 rpm for 16 hours. The centrifuged
sample was divided into 12 fractions, and each fraction was tested
for the presence of HBsAg by Western blotting (the primary antibody
was the anti-HBsAg monoclonal antibody). The HBsAg fractions were
dialyzed against buffer A without Tween 80.
[0095] (4) 12 ml of the dialyzed solution obtained in (3) was
layered onto a 5-50% sucrose gradient, and ultracentrifuged at
28000 rpm for 16 hours. As in (3), the centrifuged sample was
divided into fractions, and each fraction was tested for the
presence of HBsAg. The HBsAg fractions were dialyzed against buffer
A containing 0.85% NaCl, without urea or Tween 80 ((2) through (4):
FIG. 2e).
[0096] (5) By repeating the procedure (4), the dizlyzed sample was
concentrated with the ultrafilter Q2000 (Advantec), and stored at
4.degree. C. for later use (FIG. 2f).
[0097] The result of Western blotting after CsCl equilibrium
centrifugation in (3) revealed that HBsAg was a protein with S
antigenicity with a molecular weight of 52 kDa. At the end of the
procedure, about 24 mg of pure HBsAg particles were obtained from
the yeast (26 g wet weight) derived from 2.5 L medium.
[0098] Each fraction obtained in the purification process was
analyzed by SDS-PAGE. In order to confirm whether the purification
had successfully removed the yeast-derived protease, the HBsAg
particles obtained in (5) were incubated at 37.degree. C. for 12
hours, separated by SDS-PAGE, and identified by silver
staining.
[0099] The result of confirmation showed that the yeast-derived
protease had been completely removed by the purification
process.
[0100] The HBsAg particles specifically infect the human
hepatocytes. The strong infectivity of the HBsAg particles is
rendered by the hepatocyte recognition site displayed on the
particle surface, which has been found on amino acid residues 3 to
77 in the pre-S1 region (Le Seyec J, Chouteau P, Cannie I,
Guguen-Guillouzo C, Gripon P., J. Virol. 1999, March; 73(3):
2052-7).
[0101] In the following, description is made as to a producing
method of the drug in which a cancer-specific antibody is displayed
on the particle surface. In the producing method described below,
the strong infectivity of the HBsAg particles to the hepatocytes
has been removed in order to ensure that the drug of the present
invention only acts on a specific cancer cell whose cell surface
bears a molecule that is recognized as an antigen by the drug
antibody. Further, the drug of the present invention was prepared
in three different forms: (1) HBsAg particles displaying a
cancer-specific antibody with a streptag; (2) HBsAg particles
displaying a cancer-specific antibody with a ZZ tag; and (3) HBsAg
particles displaying a cancer-specific single chain antibody
expressed with the HBsAg protein fused with the antibody.
Example C
Preparation of HBsAg Particles Displaying a Cancer-Specific
Antibody using a Streptag
Example C-1
Preparation of HBsAg-Streptag Particles in Yeast Cells
[0102] In order to delete a gene region that encodes a human
hepatocyte recognition site of the pGLDLIIP39-RcT plasmid discussed
in Example A and at the same time insert a restriction enzyme NotI
site (gcggccgc), PCR was run for the pGLDLIIP39-RcT plasmid using
the oligonucleotides of SEQ ID NOs: 1 and 2 as PCR primers. The PCR
was carried out with the QuickChange.TM. Site-Directed Mutagenesis
Kit (Stratagene).
[0103] Specifically, using Pfu DNA polymerase (Stratagene) as a
heat-resistant DNA polymerase, PCR was run in 30 cycles as follows:
30 second denature at 95.degree. C., 1 minute annealing at
55.degree. C., and 30 minute synthesis at 68.degree. C. The PCR
product was treated with restriction enzyme DpnI and transformed
into E. coli DH5.alpha.. Then, vector DNA was extracted from the
resultant colonies, and the extract was screened for mutant
pGLDLIIP39-RcT plasmid based on the base sequence. In the
following, the resultant plasmid will be called pGLDLIIP39-RcT-Null
plasmid. Note that, in FIG. 3 and the subsequent drawings, a gene
region of plasmid encoding HBsAg L protein that lacks the human
hepatocyte recognition site will be denoted by "Null." For
convenience of explanation, such a gene region will be called a
"Null region."
[0104] In order to add a SacI site and SaII site in the
pGLDLIIP39-RcT-Null plasmid, PCR was run using the oligonucleotides
of SEQ ID NOs: 3 and 4 as PCR primers, as shown in FIG. 3, wherein
the oligonucleotides of SEQ ID NOs: 3 and 4 had a SacI site and a
SalI site, respectively. The PCR amplified the Null region, which
contained a promoter (GLDp) and a terminator (PGKt), and cDNA
fragments including the Null region were obtained.
[0105] Then, a pRS405+2 .mu.m plasmid, which was prepared by
inserting a 2 .mu.m origin into AatII site of a universal yeast
vector pRS405 (Stratagene), was digested with restriction enzymes
SacI and SalI. The DNA fragments including the Null region were
then inserted into the cleaved pRS405+2 .mu.m plasmid, so as to
prepare a pRS405+2 .mu.m-Null plasmid.
[0106] Thereafter, synthetic oligonucleotides (oligonucleotide of
SEQ ID NO: 5, and oligonucleotide of SEQ ID NO: 6 complementary to
SEQ ID NO: 5) that encode a streptag were annealed and inserted
into pRS405+2 .mu.m-Null plasmid digested with NotI. As a result, a
pRS405+2 .mu.m-streptag plasmid was prepared that included a gene
region encoding the streptag. The streptag is a peptide that binds
to streptavidin with strong affinity like biotin, and has the
sequence (1) SAWRHPQFGG (SEQ ID NO: 27) or (2) WSHPQFEK (SEQ ID NO:
28) from the N-terminus. Sequence (1) functions at the C-terminus
of the protein. The present Examples used the streptag of sequence
(2).
[0107] The pRS405+2 .mu.m-streptag plasmid was used to transform
yeasts (Saccharomyces cerevisiae AH22R- strain). The resultant
transformants were cultured, and the cultured cells were purified
to obtain modified HBsAg particles (particles obtained by
expressing the streptag fused with the HBsAg L protein lacking the
human hepatocyte recognition site; hereinafter referred to as
HBsAg-streptag particles) according to the method described in
Example B. At the end of the procedure, about 200 .mu.g of pure
HBsAg-streptag particles were obtained from the yeasts derived from
1.0 L medium.
Example C-2
Preparation of HBsAg-Streptag Particles in Insect Cells in
Serum-Free Medium
[0108] Example below describes a producing method of
HBsAg-strept-tag particles using insect cell lines that can be
cultured serum-free without the mediation of baculovirus. With the
producing method using insect cell lines, a higher order structure
such as a sugar chain can be realized.
[0109] As shown in FIG. 4, PCR was run for the pGLDLIIP39-RcT-Null
plasmid obtained in Example C-1, using the oligonucleotides of SEQ
ID NO: 7 and SEQ ID NO: 8 as PCR primers, wherein the
oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 had a kpni site
(ggtacc) and a SacII site (ccgcgg), respectively. The PCR amplified
the Null region, which contained a coding region for a
lysozym-secreted signal peptide derived from chicks.
[0110] The PCR product was electrophorased on agarose, and gene
fragments of a target band about 1.3 kbp were collected. The gene
fragment was ligated between the kpni site and SacII site of vector
pIZT/V5-His (used for stable expression in insect cells)
(Invitrogen Corporation) to close the ring, using TaKaRa Ligation
kit ver. 2 (TaKaRa). The base sequence was confirmed, and the
plasmid was named pIZT-Null.
[0111] Thereafter, as in Example C-1, synthetic oligonucleotides
(oligonucleotide of SEQ ID NO: 5, and oligonucleotide of SEQ ID NO:
6 complementary to SEQ ID NO: 5) that encodes a streptag were
annealed and inserted into the pIZT-Null plasmid digested with
NotI. As a result, a pIZT-streptag plasmid was prepared that
included a gene region encoding the streptag.
[0112] Meanwhile, the insect cell High Five line (BTI-TN-5B1-4):
(Invitrogen Corporation) was slowly conditioned from the fetal
bovine serum-contained medium to a serum-free medium (Ultimate
Insect Serum-Free Medium: Invitrogen Corporation) over a period of
about 1 month. Then, using the gene transfer lipid Insectin-Plus
(Invitrogen Corporation), the pIZT-streptag plasmid was transferred
for the transformation of the High Five line conditioned to the
serum-free medium. The sample was incubated in the serum-free
medium at 27.degree. C. for 48 hours, followed by further
incubation that extended 4 to 7 days until confluent cells were
obtained on the serum-free medium with the additional 400 .mu.g/mL
antibiotic zeocin (Invitrogen Corporation). As a result,
HBsAg-streptag particles were obtained.
[0113] The sample was centrifuged at 1500.times.g for 5 min, and
the supernatant was collected. The HBsAg-streptag particles in the
medium were measured for the presence or absence of expression,
using the IMx kit (Dainabot Co. Ltd.). The result confirmed the
expression of HBsAg-streptag particles. The HBsAg-streptag
particles obtained from the supernatant were separated by SDS-PAGE
and analyzed by Western blotting using an anti-S antibody (prepared
by the inventors), followed by enzyme immunoassay IMx. The
HBsAg-streptag particles fused with the streptag had a molecular
weight of about 42 kDa.
[0114] 1 L of the supernatant was concentrated with an
ultrafiltration unit (filter UK-200, the product of Advantec,
exclusion molecular weight 200 K), and purified through an anion
exchange column (DEAE-Toyopearl 650 M, Toyo Soda). As a result,
about 1 mg of pure uniform HBsAg-streptag particles were
obtained.
(Example C-3
Preparation of HBsAg-Streptag Particles in Animal Cells
[0115] As shown in FIG. 5, restriction enzyme XhoI was used to
cleave the pGLDLIIP39-RcT-Null plasmid at the Xho site, so as to
obtain fragments containing the Null region with a terminator
(PGKt). After digesting pcDNA3.1 (Invitrogen Corporation) with
restriction enzyme XhoI, the fragments were inserted into the
pcDNA3.1 to prepare a pcDNA3.1-Null plasmid.
[0116] Thereafter, as in Example C-1, synthetic oligonucleotides
(oligonucleotide of SEQ ID NO: 5, and oligonucleotide of SEQ ID NO:
6 complementary to SEQ ID NO: 5) that encodes a streptag gene were
annealed and inserted into the pcDNA3.1-Null plasmid digested with
NotI. As a result, a pcDNA3.1-streptag plasmid was prepared that
included a coding region for the streptag.
[0117] The pcDNA3.1-streptag plasmid so obtained was then
transferred into COS7 cells derived from the monkey kidney, using
the gene transfer device gene pulser (Bio-Rad Laboratories, Inc.).
After the transfer, the sample was incubated overnight in a
Dulbecco-modified medium containing 10% fetal bovine serum. After
further incubation in a serum-free medium CHO-SFMII (Gibco-BRL) for
a week, the medium was purified to obtain HBsAg-streptag
particles.
[0118] As in Example C-2, the HBsAg-streptag particles obtained
from the supernatant were separated by SDS-PAGE and analyzed by
Western blotting using an anti-S antibody, followed by enzyme
immunoassay IMx. The HBsAg-streptag particles fused with the
streptag had a molecular weight of about 42 kDa. The measured
values of IMx were 8.81 (against cut-off value) for the wild-type
HBsAg L particles expressed with the pcDNA3.1 vector, 3.47 for the
HBsAg Null particles, and 2.41 for the HBsAg-streptag particles.
All of these values can be considered to be sufficient.
(Example C-4
Method of Displaying an Antibody on the HBsAg-Streptag Particles
having a Streptag
[0119] The foregoing Examples C-1 through C-3 prepared
HBsAg-streptag particles with a streptag. The streptag specifically
binds to streptavidin, which in turn specifically binds to biotin.
By taking advantage of these specific bindings, the HBsAg-streptag
particles are first bound to streptavidin, which is then ligated to
a biotin-modified antibody. The result is HBsAg-streptag particles
with the antibodies arrayed on the particle surface (such
HBsAg-streptag particles will be referred to as "HBsAg-streptag-Ab
particles" hereinafter).
[0120] Specifically, the anti-human EGFR mouse monoclonal antibody
7G7B6 (purified), which is an antibody against the human epidermal
growth factor receptor (EGFR), is used as an antibody, and the
NHS-biotin (EZ-Link.RTM. NHS-Biotin, the product of Pierce
Biotechnology, Inc.) was tagged according to the protocol described
in the instructions of the Pierce product. The purified
HBsAg-streptag particles were then ligated to the avidin protein
(ImmunoPure Avidin, the product of Pierce Biotechnology, Inc.) by
mixing the two in PBS at a molar ratio of 2:1 and at ordinary
temperature for 30 min (molar calculation was made on a molecular
basis). Thereafter, the biotin-tagged anti-human EGFR mouse
monoclonal antibody was allowed to react with an equimolar amount
of HBsAg-streptag particles bearing the avidin protein. The
reaction was carried out in PBS at ordinary temperature for 30 min.
The result was HBsAg-streptag-Ab particles bearing the antibodies
on the particle surface.
Example C-5
Transfer of Genes into the HBsAg-Streptag-Ab Particles
[0121] According to the method described in International
Publication WO01/64930, the HBsAg-streptag-Ab particles were mixed
with a green fluorescent protein expression plasmid (pEGFP-F
(Clontech)), and the pEGFP-F was sealed in the HBsAg-streptag-Ab
particles by an electroporation method. The result was
HBsAg-streptag-Ab particles that had anti-human EGFR antibodies on
the particle surface and encapsulated GFP expression plasmid inside
the particles.
[0122] Next, there were prepared human squamous cell
carcinoma-derived cells A431 (JCRB9009), along with human hepatic
cancer-derived cells NUE and human colon cancer-derived cells WiDr
as negative controls. The A431 and negative controls (NUE, WiDr)
were each placed on a 3.5 cm glass-bottomed Petri dish, and
incubated for 4 days with 1 .mu.g of HBsAg-streptag-Ab particles
encapsulating the GFP expression vector plasmid. GFP expression in
the cells of the respective samples were observed with a confocal
laser fluorescence microscope.
[0123] The observation found GFP fluorescence in A431 but not in
the negative controls (NUE cells, WiDr).
[0124] Thus, with the HBsAg-streptag-Ab particles that had
anti-human EGFR antibodies on the particle surface and encapsulated
GFP expression plasmid inside the particles, the experiment showed
that the transfer and expression of the gene was very specific and
efficient in the A431 cells on the cultured cell level. The
experiment therefore suggests that the HBsAg-streptag-Ab particles
encupsulating a substance to be transferred into a cell for
treating a disease have a potential use in the effective treatment
of specific diseased cells or tissues.
Example D
Preparation of Antibody-Displaying HBsAg-ZZ Particles using a ZZ
Tag
Example D-1
Preparation of HBsAg-ZZ Particles in Yeast Cells
[0125] As in Example C-1, a pRS405+2 .mu.m-Null plasmid was
prepared as shown in FIG. 6.
[0126] Using NotI site-containing oligonucleotides of SEQ ID NOs: 9
and 10 as PCR primers, PCR was run for a plasmid that contained a
coding region for a ZZ tag (indicated by "ZZ" in the figure;
hereinafter referred to as "ZZ region") (prepared by inserting a ZZ
region based on a Protein A gene derived from Staphyrococcus
aureus). The PCR amplified regions including the ZZ region. The ZZ
tag is defined as an amino acid sequence with the ability to bind
to the Fc region of immunoglobulin G, wherein the amino acid
sequence has the following two repeating units from the N-terminus
(ZZ tag sequence (SEQ ID NO: 29)): TABLE-US-00001
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNDAQAPK
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNDAQAPK
[0127] The pRS405+2 .mu.m-Null plasmid was then digested with
restriction enzyme NotI, and the amplified fragments were inserted
in the cleaved plasmid to prepare a pRS405+2 .mu.m-ZZ plasmid.
[0128] As in Example A, the plasmid gene pRS405+2 .mu.m-ZZ was used
to transform the yeast S. cerevisiae AH22R--by a spheroplast
method. The resulting transformants were incubated in medium
High-Pi (3 ml) at 30.degree. C. for 3 days, and subsequently in
medium 8S5N-P400 (3 ml) at 30.degree. C. for another 3 days, so as
to prepare HBsAg particles displaying a ZZ tag.
[0129] The pRS405+2 .mu.m-ZZ plasmid so obtained was used to
transform the yeast (Saccharomyces cerevisiae AH22R-strain). The
resulting transformants were incubated, and the cultured cells were
purified according to the method described in Example B to obtain
modified HBsAg particles (particles obtained by expressing the ZZ
tag fused with the HBsAg L protein lacking the human hepatocyte
recognition site). The efficiency of particle expression was very
high, producing about 20 mg of pure HBsAg-ZZ tag particles from the
yeasts derived from 1.0 L medium.
[0130] The HBsAg-ZZ tag particles obtained from the supernatant
were separated by SDS-PAGE, and analyzed by Western blotting using
an anti-S antibody, followed by enzyme immunoassay IMx. FIG. 7
shows the results of SDS-PAGE and Western blotting. The measured
values of IMx were 49.43 (against cut-off value, .times.100
diluted) for the wild-type HBsAg L particles expressed with the
pRS405+2 .mu.m vector, 21.87 for the HBsAg Null particles, and
253.64 for the HBsAg-ZZ tag particles. All of these values can be
considered to be sufficient. The HBsAg-ZZ tag particles with the ZZ
tag had a molecular weight of about 56 kDa.
Example D-2
Preparation of HBsAg-ZZ Tag Particles in Insect Cells in Serum-Free
Medium
[0131] As shown in FIG. 8, a pIZT-Null plasmid was obtained
according to the method described in Example C-2.
[0132] A region including the ZZ region was inserted in the
pIZT-Null plasmid according to the method of Example D-1, so as to
prepare a pIZT-ZZ plasmid.
[0133] The pIZT-ZZ plasmid so obtained was inserted in insect
cells, and HBsAg-ZZ tag particles were expressed therein according
to the method of Example C-2.
[0134] After incubating the insect cells, the supernatant was
collected. The HBsAg-ZZ tag particles obtained from the supernatant
were separated by the SDS-PAGE, and analyzed by Western blotting
using an anti-S antibody (prepared by the inventors, mouse
polyclonal antibody). The result showed that the HBsAg-ZZ tag
particles displaying the ZZ tag had a molecular weight of about 56
kDa.
[0135] The amount of HBsAg-ZZ tag particles obtained from 1 L
supernatant was about 1 mg according to the method of Example
C-2.
Example D-3
Preparation of HBsAg-ZZ Tag Particles in Animal Cells
[0136] As shown in FIG. 9, a pcDNA3.1-Null plasmid was obtained
according to the method described in Example C-3.
[0137] Then, a region including the ZZ region was inserted in the
pcDNA3.1-Null plasmid according to the method of Example D-1, so as
to prepare a pcDNA3.1-ZZ plasmid.
[0138] The pcDNA3.1-ZZ plasmid so obtained was inserted in COS7
cells, and HBsAg-ZZ tag particles were expressed therein according
to the method of Example C-3.
[0139] After incubating the COS7 cells, the supernatant was
collected. The HBsAg-ZZ tag particles obtained from the supernatant
were separated by the SDS-PAGE, and analyzed by Western blotting
using an anti-S antibody (prepared by the inventors, mouse
polyclonal antibody), followed by enzyme immunoassay IMx. The
measured values of IMx were 8.81 (against cut-off value) for the
wild-type HBsAg L particles expressed with the pcDNA3.1 vector,
3.47 for the HBsAg Null particles, and 2.41 for the HBsAg-ZZ tag
particles. All of these values can be considered to be sufficient.
The HBsAg-ZZ tag particles with the ZZ tag had a molecular weight
of about 56 kDa.
(Example D-4
Method of Displaying an Antibody on the HBsAg-ZZ Tag Particles
[0140] The ZZ tag has strong affinity to the Fc portion of the
antibody molecule. For example, the ZZ tag can specifically bind to
the cancer-specific mouse monoclonal antibody 7G7B6 against the
human EGF receptor (EGFR). Other examples of antibodies specific to
the ZZ tag include the mouse monoclonal antibody 528 against the
human IL-2 receptor (Tac antigen), and the colon cancer-specific
mouse monoclonal antibody ST-421 against the human colon cancer. By
binding the HBsAg-ZZ tag particles to these antibodies, HBsAg-ZZ
tag particles were prepared that had the antibodies arrayed on the
particle surface (hereinafter, such HBsAg-ZZ tag particles will be
referred to as "HBsAg-ZZ tag-Ab particles").
[0141] Specifically, the HBsAg-ZZ tag particles were mixed with an
equimolar amount of anti-human EGFR mouse monoclonal antibody 7G7B6
(purified) (molar calculation was made on a molecular basis), and
the mixture was allowed to react for one hour in PBS. The result
was HBsAg-ZZ tag-Ab particles with the antibodies displayed on the
particle surface.
Example D-5
Transfer of Genes into the HBsAg-ZZ Tag-Ab Particles
[0142] According to the method disclosed in International
Publication WO01/64930, the HBsAg-ZZ tag-Ab particles were mixed
with a green fluorescent protein expression plasmid (pEGFP-F
(Clontech)), and the pEGFP-F was sealed in the HBsAg-ZZ tag-Ab
particles by an electroporation method. The result was HBsAg-ZZ
tag-Ab particles that had anti-human EGFR antibodies on the
particle surface and encapsulated GFP expression plasmid inside the
particles.
[0143] Next, as in Example C-5, there were prepared human squamous
cell carcinoma-derived cells A431, along with human hepatic
cancer-derived cells NUE and HuH-7 (JCRB0403) and human colon
cancer-derived cells WiDr (ATCC CCL-218) as negative controls. The
A431 and negative controls (NUE, HuH-7, WiDr) were each placed on a
3.5 cm glass-bottomed Petri dish, and incubated for 4 days with 1
pg of HBsAg-ZZtag-Ab-GFP particles encapsulating the GFP expression
vector plasmid. GFP expression in the cells of the respective
samples were observed with a confocal laser fluorescence
microscope.
[0144] The observation found GFP fluorescence in A431 but not in
the other cells (e.g., NUE cells).
[0145] Thus, with the HBsAg-ZZ tag-Ab particles that had anti-human
EGFR antibodies on the particle surface and encapsulated GFP
expression plasmid inside the particles, the experiment showed that
the transfer and expression of the gene was very specific and
efficient in the A431 cells on the cultured cell level.
[0146] Meanwhile, human tumor strains (A431, HuH-7, WiDr) were
injected by hypodermic injection into nude mice (lineage: BALB/c,
nu/nu, microbiological quality: SPF, male, 5 weeks of age). The
injection was made in the bilateral dorsal area of the mouse with
1.times.10.sup.7 cells for each strain. In order to obtain a
carrier mice, the mice were grown for 2 to 4 weeks until the
transplanted tumor developed into a solid cancer tumor of about 2
cm diameter.
[0147] The HBsAg-ZZ tag-Ab particles encapsulating the GFP
expression plasmid were administered into the abdomen of each mouse
with a 26G syringe. The mouse was killed 4 days after the
administration, and the tumor area was removed along with various
organs including liver, spleen, kidney, and intestines. The tissues
were fixed and embedded using the GFP resin embedding kit
(Technovit 7100).
[0148] Specifically, the samples were fixed by immersing them in 4%
neutralized formaldehyde, and were dried in 70% EtOH at room
temperature for 2 hours, 96% EtOH at room temperature for 2 hours,
and 100% EtOH at room temperature for one hour. Pre-fixation was
carried out for 2 hours at room temperature in a mixture containing
equal amounts of 100% EtOH and Technovit 7100. The samples were
further immersed in Technovit 7100 for no longer than 24 hours at
room temperature. Out of the solution, the samples were allowed to
stand for one hour at room temperature and for another one hour at
37.degree. C. for polymerization.
[0149] According to ordinary method, the sample were sliced and
stained with hematoxin-eosin (common method of tissue staining).
GFP fluorescence of each slice was observed with a fluorescent
microscope. The result showed that human squamous cell
carcinoma-derived cells A431 had GFP fluorescence. No fluorescence
was observed in the organs removed from the same mouse, including
liver, spleen, kidney, and intestines. On the other hand, in
carrier mice that have incorporated cells derived from other types
of human cancer (HuH-7, WiDr), no GFP fluorescence was observed in
the tumor area, or in the liver, spleen, kidney, or intestines.
Fluorescence was not observed either in carrier mice to which the
HBsAg-ZZ tag was not administered.
[0150] Thus, with the HBsAg-ZZ tag-Ab particles that had anti-human
EGFR antibodies on the particle surface and encapsulated GFP
expression plasmid inside the particles, the experiment showed that
the transfer and expression of the gene was very specific and
efficient in the A43 1 cells on the laboratory animal level.
Example D-6
Effectiveness of Treatment using the HBsAg-ZZ Tag-Ab Particles
[0151] In order to produce the HBsAg-ZZ tag-Ab particles as a drug
of the present invention encapsulating HSV1tk gene, a
cancer-treating thymidine kinase derived from simple herpes virus
(HSV1tk) was sealed in the HBsAg-ZZ tag-Ab particles that were
prepared in yeasts according to the described method.
[0152] The cancer cells that have incorporated the HSV1tk gene
become ganciclovir (GCV) sensitive when they express the gene.
Administration of ganciclovir therefore kills off the cancer cells
by the strong effect it exhibits on the cancer cells. This is one
reason the HSV1tk gene has been widely used in the gene therapy of
cancer.
[0153] In this Example, the HSV1tk gene was sealed in the HBsAg-ZZ
tag-Ab particles using a vector pGT65-hIFN-.alpha. (the product of
Invitrogen Corporation) that expresses the HSV1tk gene. The
HBsAg-ZZ tag-Ab particles encapsulating the HSV1tk gene were
obtained by transferring the expression vector into the HBsAg-ZZ
tag-Ab particles by an electroporation method. Specifically, 10
.mu.g of expression vector was transferred into 50 .mu.g of L
protein particles in the HBsAg-ZZ tag-Ab particles. The vector was
transferred using a PBS buffer, and the electroporation was carried
out with a 4 mm cuvette under 220 V and 950 .mu.F.
[0154] As the laboratory animal, the present Example used nude rats
purchased from CLEA Japan, Inc. (lineage: F344/NJcl-rnu/rnu,
female). By hypodermic injection, human squamous cell
carcinoma-derived cells A431 were transplanted into the nude rats,
along with the human colon cancer-derived cells WiDr as a negative
control. The injection was made in the bilateral dorsal area of the
rats with 1.times.10.sup.7 cells for each cell type. The rats were
grown for about 3 weeks until the grafted tumor developed into a
solid cancer tumor of about 2 to 3 cm diameter.
[0155] 10 .mu.g of HBsAg-ZZ tag-Ab particles encapsulating the
HSV1tk gene were administered to each nude rat through the tail
vein (intravenous injection). Starting from 5 days after the
intravenous injection, ganciclovir (GCV) was administered to each
rat with the dose of 50 mg/kg/day, using an osmotic pump (alzet
osmotic pump; Cat No. 2ML2). Here, the GCV was administered to the
back of each nude rat subcutaneously. The GCV was administered for
no longer than 14 days. After the administration, the state (size)
of the tumor tissue of the nude rats was observed over time.
Specifically, the major axis and minor axis of the tumor part were
measured with a gauge, and a tumor volume was approximated (major
axis x minor axis x minor axis/2). The rats were measured in
triplet. The results are shown in FIG. 10 and FIG. 27.
[0156] Thus, with the HBsAg-ZZ tag-Ab particles that had anti-human
EGFR antibodies on the particle surface and encapsulated the HSV1tk
gene inside the particles, the experiment showed that the transfer
and expression of the gene was very specific and efficient in the
A431 cells and therefore highly effective in cancer treatment on
the laboratory animal level.
Example E
Preparation of HBsAg-scFv Particles Displaying a Single Chain
Antibody
Example E-1
Preparation of HBsAg-scFv Particles in Yeast Cells
[0157] A region of including a coding region for antibody A22 or
3A21 was amplified by PCR, where the antibody A22 is a single chain
anti-human serum albumin antibody derived from mice, and the
antibody 3A2 1 is a single chain anti-human RNase antibody derived
from mice. PCR was performed according to the procedure of Example
D-1, except that a different plasmid and different PCR primers were
used. As the plasmid that includes a ZZ region, the present Example
used either a plasmid that includes a coding region for the
antibody A22 (generous gift of TOTO LTD.), or a plasmid that
includes a coding region for the antibody 3A2 1. (prepared
according to the method described in Mol Immunol. 1997
August-September; 34(12-13): 887-90 Katakura Y, Kumamoto T, Iwai Y,
Kurokawa Y, Omasa T, Suga K., and Mol Immunol 1997 July; 34(10):
731-4 Katakura Y, Kumamoto T, Iwai Y, Kurokawa Y, Omasa T, Suga K.)
As the PCR primers, the present Example used either
oligonucleotides of SEQ ID NOs: 11 and 12 (in the case of A22), or
oligonucleotides of SEQ ID NOs: 13 and 14 (in the case of 3A21),
where each oligonucleotide had a NotI site. The single chain
antibody (scFv) is a pseudo antibody molecule that has been
restructured to have the antigen recognition site only on a single
chain polypeptide, rather than the normal double chain
polypeptide.
[0158] The amplified fragments obtained by the PCR were inserted in
the pRS405+2 .mu.m-Null plasmid to prepare pRS405+2 .mu.m-A22
plasmid or pRS405+2 .mu.m-3A21 plasmid. The pRS405+2 .mu.m-A22 or
pRS405+2 .mu.m-3A21 plasmid was transferred into yeasts and
expressed therein. The result was particles whose particle surface
had single chain antibody A22 or 3A21 expressed with the HBsAg L
protein fused with the antibody (such particles will be referred to
as HBsAg-scFv particles hereinafter).
[0159] After incubating the yeasts, the supernatant was collected.
The HBsAg-scFv particles obtained from the supernatant were
separated by SDS-PAGE, and analyzed by Western blotting using an
anti-S antibody, followed by enzyme immunoassay IMx. FIG. 11 shows
the results of SDS-PAGE and Western blotting. The measured values
of IMx were 49.43 (against cut-off value, .times.100 diluted) for
the wild-type HBsAg L particles expressed with the pRS405+2 .mu.m
vector, 21.87 for the HBsAg Null particles, 2.41 for the HBsAg-scFv
particles displaying A22, and 4.02 for the HBsAg-scFv particles
displaying 3A21. All of these values can be considered to be
sufficient. The HBsAg-scFv particles with antibody A22 had a
molecular weight of about 76 kDa. The result was the same for the
HBsAg-scFv particles with antibody 3A21. By the method of Example
C-2, about 200 .mu.g of pure HBsAg-scFv particles were obtained
from the yeasts derived from 1.0 L medium.
Example E-2
Preparation of HBsAg-scFv Particles in Insect Cells in Serum-Free
Medium
[0160] As shown in FIG. 8, a region of including a coding region
for antibody A22 or 3A21 was amplified by PCR, where the antibody
A22 is a single chain anti-human serum albumin antibody derived
from mice, and the antibody 3A21 is a single chain anti-human RNase
antibody derived from mice. PCR was performed according to the
procedure of Example D-2, except that a different plasmid and
different PCR primers were used. As the plasmid that includes a ZZ
region, a plasmid that includes a coding region for the antibody
A22 or 3A21 was used. As the PCR primers, the present Example used
either oligonucleotides of SEQ ID NOs: 11 and 12 (in the case of
A22), or oligonucleotides of SEQ ID NOs: 13 and 14 (in the case of
3A21), where each oligonucleotide had a NotI site.
[0161] The amplified fragments obtained by the PCR were inserted in
the pRS405+2 .mu.m-Null plasmid to prepare pIZT-A22 plasmid or
pIZT-3A21 plasmid. The pIZT-A22 or pIZT-3A21 plasmid was
transferred into insect cells and expressed therein. The result was
HBsAg-scFv particles displaying the single chain antibody A22 or
3A2 1.
[0162] After incubating the insect cells, the supernatant was
collected. The HBsAg-scFv particles obtained from the supernatant
were separated by SDS-PAGE, and analyzed by Western blotting using
an anti-S antibody. The HBsAg-scFv particles with antibody A22 had
a molecular weight of about 76 kDa. The result was the same for the
HBsAg-scFv particles with antibody 3A2 1.
[0163] By the method of Example C-2, about 1 mg of pure HBsAg-scFv
particles were obtained from 1.0 L supernatant.
Example E-3
Preparation of HBsAg-scFv Particles in Animal Cells
[0164] As shown in FIG. 9, a region including a coding region for
antibody A22 or 3A2 1 was amplified by PCR, where the antibody A22
is a single chain anti-human serum albumin antibody derived from
mice, and the antibody 3A21 is a single chain anti-human RNase
antibody derived from mice. PCR was performed according to the
procedure of Example D-3, except that a different plasmid and
different PCR primers were used. As the plasmid that includes a ZZ
region, a plasmid that includes a coding region for the antibody
A22 or 3A21 was used. As the PCR primers, the present Example used
either oligonucleotides of SEQ ID NOs: 11 and 12 (in the case of
A22), or oligonucleotides of SEQ ID NOs: 13 and 14 (in the case of
3A21), where each oligonucleotide had a NotI site.
[0165] The amplified fragments obtained by the PCR were inserted in
the pcDNA3. 1 plasmid to prepare pcDNA3. 1-A22 plasmid or pcDNA3.
1-3A21 plasmid. The pcDNA3. 1-A22 or pcDNA3.1-3A21 plasmid was
transferred into animal cells and expressed therein. The result was
HBsAg-scFv particles displaying the single chain antibody A22 or
3A2 1.
[0166] After incubating the COS7 cells, the supernatant was
collected. The HBsAg-scFv particles obtained from the supernatant
were separated by SDS-PAGE, and analyzed by Western blotting using
an anti-S antibody. The result showed that HBsAg-scFv particles
with antibody A22 had a molecular weight of about 76 kDa. The
result was the same for the HBsAg-scFv particles with antibody 3A2
1.
(Example E-4
Transfer of Genes into the HBsAg-scFv Particles
[0167] The HBsAg-scFv particles so prepared were fixed on 96-well
plates, wherein human serum albumin was used for the HBsAg-scFv
particles that had antibody A22, and human RNase 1 was used for the
HBsAg-scFv particles that had 3A2 1 antibody. Binding factors of
the respective samples were measured by an ELISA. The amount of
HBsAg-scFv particles that bound in the stationary phase was
quantified using the HRP tag anti-HBsAg polyclonal antibody
provided in the AUSZYME II of Dainabot Co. Ltd. The result showed
that the HBsAg-scFv particles had binding factors of not more than
100 nM for A22, and not more than 50 nM for 3A2 1. The results are
based on proteins building the HBsAg-scFv particles, not the
HBsAg-scFv particles themselves. The binding factors in these
ranges are sufficient for the HBsAg-scFv particles to serve as a
carrier for delivering a drug or other substances to a specific
site inside the body.
[0168] The experiment showed that the HBsAg-scFv particles
displaying the antibody A22 or 3A2 1 on the particle surface were
highly specific to the A43 1 cells.
Example F
[0169] By expressing various types of deletion HBsAg L proteins
lacking amino acids in the pre-S region (pre-S1, pre-S2), which is
the human hepatocyte recognition site of the HBsAg L protein, the
present Example evaluated the level of expression and antigenicity
in eukaryotic cells among different amino acid deletion
regions.
Example F-1
Preparation of Deletion HBsAg L Protein Expression Genes
[0170] Deletion HBsAg L protein expression genes were prepared by
PCR according to the method described below.
[0171] In order to obtain deletion HBsAg L proteins, there were
prepared deletion HBsAg L protein expression genes that express 5
types of deletion HBsAg L proteins (a) to (e) below in which part
of the pre-S regions (pre-S1 region, pre-S2 region) has been
deleted. Specifically, the deletion HBsAg L proteins prepared in
this Example are (a) a protein lacking N-terminal amino acids 21 to
153 in the pre-S region (.DELTA.21-153 in FIG. 12; the same
notation is used below), (b) a protein lacking N-terminal amino
acids 33 to 153 in the pre-S region (.DELTA.33-153), (c) a protein
lacking N-terminal amino acids 50 to 153 in the pre-S region
(.DELTA.50-153), (d) a protein lacking N-terminal amino acids 108
to 153 in the pre-S region (.DELTA.108-153), and (e) a protein
lacking N-terminal amino acids 127 to 153 in the pre-S region
(.DELTA.127-153).
[0172] In order to amplify deletion HBsAg L protein expression
genes of the respective proteins (a) through (e), PCR was run for
pB0477 (plasmid that has incorporated HbsAg L protein expression
genes, prepared by the inventors) according to the described
method. As the PCR primers, the oligonucleotides of SEQ ID NOs: 15
through 24 were used. The oligonucleotides of SEQ ID NOs: 15 and 16
were for amplifying the deletion HBsAg L protein (a), the
oligonucleotides of SEQ ID NOs: 17 and 18 for (b), the
oligonucleotides of SEQ ID NOs: 19 and 20 for (c), the
oligonucleotides of SEQ ID NOs: 21 and 22 for (d), and the
oligonucleotides of SEQ ID NOs: 23 and 24 for (e). Further, among
the primers of SEQ ID NOs: 15 through 24, the odd-numbered ones are
forward primers, and the even-numbered ones are reverse
primers.
[0173] The reaction compositions of PCR are shown in FIG., 13:
Pyrobest DNA polymerase (TaKaRa) (heat-resistant DNA polymerase)
(0.5 .mu.L), PCR buffer (5 .mu.L .times.10), dNTP mixture (10 mM, 5
.mu.L), template DNA (pB0477; plasmid that has incorporated the
HbsAg L protein expression genes, prepared by the inventors) (5
.mu.g/mL, 2 .mu.L), and a primer set (SEQ ID NOs: 15 to 24) (1
.mu.L each). The total volume was 50 .mu.L with the addition of
distilled water.
[0174] The PCR was run in 30 cycles as follows: 30 second denature
at 98.degree. C., 30 second denature at 98.degree. C., 1 minute
annealing at 55.degree. C., and 30 minute synthesis at 68.degree.
C. The reaction was ended upon cooling to 4.degree. C., as shown in
FIG. 14. In order to cut the template DNA, the restriction enzyme
DpnI (1OU) was added to the PCR product. After incubation at
37.degree. C. for 1 hour, the resulting plasmid was used to
transform E. coli JM109 strain. The plasmid was removed from the
expression colonies, and its base sequence was confirmed.
[0175] Thereafter, restriction enzyme NotI sites were introduced
into the deletion HBsAg L protein. FIG. 15 schematically
illustrates an expression gene that was prepared by introducing
restriction enzyme NotI sites in the deletion HBsAg L protein
expression gene. The schematic diagram of FIG. 15 also illustrates
a plasmid that has incorporated such a gene. In FIG. 15, the
restriction enzyme NotI sites are indicated by 0aa, 25aa, and
.DELTA.PreS, wherein 0aa is an insertion site at an end (5' end) of
the deletion HBsAg L protein expression gene, 25aa is an insertion
site at the 3' end of the first 25 amino acid residues from the 5'
end, and .DELTA.PreS is an insertion site at an end (5' end) of an
S protein expression gene.
[0176] The deletion HBsAg L protein expression gene with the NotI
sites was then inserted in a plasmid pB0477 (plasmid that has
incorporated the HBsAg L protein expression gene, for expression in
animal cells, prepared by the inventors) with XhoI, so as to obtain
a recombinant HBsAg L protein expression gene.
[0177] Note that, in FIG. 15 and subsequent drawings, the notation
A127-153 indicates that the HBsAg L protein expression gene shown
in FIG. 12 lacks a gene that encodes amino acids 127 to 153, for
example. (The same notation is used below.) Similarly, Apre-S
indicates that a gene that encodes all amino acids in the pre-S
regions (pre-S1, pre-S2) is lacking.
Example F-2
Preparation of Deletion HBsAg L Protein in Animal Cells
[0178] The plasmid (2 .mu.g) constructed in Example F-1 was used to
transform Cos7 cells (3 to 8.times.10.sup.4 cells) by
electroporation (300 V, 950 .mu.F). The resulting plasmid was
allowed to stand at 37.degree. C. for 4 days in the presence of 5%
CO.sub.2. The amount of mutant L particles (deletion HBsAg L
protein) in the supernatant and cell extract was measured with an
enzyme immunoassay device (Dainabot Co. Ltd.). The measurement was
made based on the antigenicity of the mutant L particles. The
supernatant used in the measurement had been diluted with the equal
amount of PBS. The cell extract was obtained by causing the cells
to lyse in a lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl,
10 mM 2-mercaptoethanol, 1% (v/v) Triton X-100), followed by
.times.200 dilution of the lysate supernatant with PBS after
centrifugation.
[0179] FIGS. 16(a) and 16(b) and FIG. 17 represent the results of
measurement, showing the produced amount of mutant L particles
(given in numerical values and graph). In these drawings, greater
values of S/N and RATE indicate greater antigenicity. That is,
samples .DELTA.21-153, .DELTA.33-153, and .DELTA.50-153 produced
good results, of which the deletion HBsAg L protein .DELTA.50-153
was particularly desirable.
[0180] The level of expression was also measured by SDS-PAGE and
Western blotting, as shown in FIG. 18 and FIG. 19. As the primary
antibody, a mouse anti-S protein antibody (prepared by the
inventors) was used. The anti-mouse IgG antibody AP tag (Promega)
was used as the secondary antibody. Note that, FIG. 19 shows the
result of Western blotting after enzyme treatment (EndH), which was
performed to remove N-sugar chains. The result is shown along with
the molecular weights. The EndH treatment revealed that the Pre-S
region of the product mutant L particles had N-sugar chains. For
.DELTA.51-66 in FIG. 19, a plasmid prepared with the primers of SEQ
ID NOs: 25 and 26 were used according to the method described in
Example F-1.
[0181] The experiment showed that the level of protein expression
was particularly desirable in the deletion HBsAg L proteins (a)
through (c).
Example F-3
Preparation of Deletion HBsAg L Proteins with Inserted Epithelial
Growth Factor (EGF)
[0182] Using the deletion HBsAg L protein expression genes (a)
through (c) (.DELTA.21-153, .DELTA.33-153, and .DELTA.50-153) which
showed desirable levels of protein expression, the EGF gene was
inserted in these genes at the NotI sites and expressed therein.
The EGF gene was obtained by cleaving the pGLDLIIP39-RcT-EGF
(prepared by the inventors) with the restriction enzyme NotI. The
resulting plasmid was used to transform the Cos7 cells. After 24 hr
incubation in serum media, the samples were further incubated for 3
days on serum-free media. The culture media were collected and
concentrated with an ultrafilter, so as to obtain mutant L
particles (deletion HBsAg L proteins (a) through (c)).
[0183] The green fluorescent protein expression plasmid (pEGFP-F
(Clontech)) was electroporated in the particles of the respective
proteins, and the GFP expression plasmid was encapsulated in the
particles. The resulting particles were used in a gene transfer
experiment using hepatocyte HepG2 and epithelial cell A431.
Observation of the GFP fluorescence showed that specificity to the
hepatocyte HepG2 had been lost, and that binding to the epithelial
cell A431 was highly selective. That is, the experiment
successfully retargeted the epithelial cell A431.
Example F-4
Preparation of Deletion HBsAg L Protein by Transformation in Yeast
Cells
[0184] Genes that express the deletion HBsAg L proteins
.DELTA.21-153, .DELTA.33-153, and .DELTA.50-153 (proteins (a)
through (c)) which showed desirable levels of protein expression in
Cos7 cells were obtained by cleaving the plasmid at the XhoI sites.
The genes so obtained were inserted at the XhoI sites of the yeast
expressed plasmids pGLDLIIP39-RcT (see FIG. 20), which were then
transferred to S. cerevisiae AH22R-strain by a spheroplast method.
The transformants were incubated for 3 days in industrial media
High-Pi and another 3 days in 8S5N-P400 media. The cultured cells
were collected and disrupted with glass beads. Then, the cell
extract was measured to confirm antigenicity and the level of
expression. Antigenicity was measured with a cultured yeast enzyme
immunoassay device IMx (Dainabot Co. Ltd.), and the level of
protein expression was measured by SDS-PAGE and Western blotting
(using anti-S protein antibody as the primary antibody, and AP
tagged anti-mouse IgG antibody as the secondary antibody) (see FIG.
21 and FIG. 22).
[0185] Additionally, two kinds of plasmids were constructed using
the NotI sites: a plasmid for displaying a ZZ domain gene of
protein A; and a plasmid for displaying EGF (see FIG. 20). In sum,
the following plasmids were constructed (expression plasmid for
efficiently expressing deletion HBsAg L protein in yeasts):
pGLDLIIP39-RcT-.alpha.50-153; pGLDLIIP39-RcT-.DELTA.33-153,
pGLDLIIP39-RcT-.DELTA.21-153; pGLDLIIP39-RcT-.DELTA.50-153-ZZ;
pGLDLIIP39-RcT-.DELTA.33-153-ZZ; pGLDLIIP39-RcT-.DELTA.21-153-ZZ;
pGLDLIIP39-RcT-.DELTA.50-153-EGF; pGLDLIIP39-RcT-.DELTA.33-153-EGF;
and pGLDLIIP39-RcT-.DELTA.21-153-EGF. In addition,
pGLDLIIP39-RcT-.DELTA.3-66 was constructed as a control. These
yeast-expressed plasmids were transferred into S. cerevisiae
AH22R-strain by a spheroplast method. The transformants were
incubated for 3 days in industrial media High-Pi and another 3 days
in 8S5N-P400 media. The cultured cells were collected and disrupted
with glass beads, and the cell extract was measured to confirm the
level of protein expression by measuring antigenicity with a
cultured yeast enzyme immunoassay device IMx (Dainabot Co. Ltd.)
(see FIG. 21 and FIG. 22).
[0186] The enzyme immunoassay confirmed formation of deletion
particles. The levels of antigenicity for the deletion HBsAg L
proteins (deletion HBsAg particles) .DELTA.21-153, .DELTA.33-153,
and .DELTA.50-153 compared to that of wild-type particles (LAg in
the drawings) (see FIG. 21 and FIG. 22). The deletion HBsAg
particles A3-66 used as a control produced no transformant (not
shown). The deletion HBsAg particles .DELTA.50-153 are particularly
advantageous since its level of protein expression, combined with a
considerably large amount of deletion HBsAg particles displaying a
ZZ domain (.DELTA.50-153+ZZ), exceeds that of the wild-type.
[0187] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be obvious to one skilled in
the art are intended to be included within the scope of the
following claims.
INDUSTRIAL APPLICABILITY
[0188] As described above, the present invention provides a drug
whose particle surface displays an antibody such as a cancer
specific antibody. The drug can be used by a convenient method of
intravenous injection to specifically and effectively treat
specific diseased cells or tissues. The invention is a great leap
forward from conventional gene therapy in that it does not require
any surgical operation, and that the risk of side effect is greatly
reduced. The drug is therefore usable in clinical applications in
its present form.
Sequence CWU 1
1
29 1 39 DNA Artificial Sequence Synthesized Oligonucleotide 1
cgacaaggca tgggaggcgg ccgcagccct caggctcag 39 2 39 DNA Artificial
Sequence Synthesized Oligonucleotide 2 ctgagcctga gggctgcggc
cgcctcccat gccttgtcg 39 3 36 DNA Artificial Sequence Synthesized
Oligonucleotide 3 ggggacctcg gatccgcgag cttaccagtt ctcaca 36 4 36
DNA Artificial Sequence Synthesized Oligonucleotide 4 gaggtcgacc
agctttaacg aacgcagaat tttcga 36 5 33 DNA Artificial Sequence
Synthesized Oligonucleotide 5 ggccgctgga gccacccgca gttcgaaaaa ggc
33 6 33 DNA Artificial Sequence Synthesized Oligonucleotide 6
ggccgccttt ttcgaactgc gggtggctcc agc 33 7 29 DNA Artificial
Sequence Synthesized Oligonucleotide 7 ggggtaccat gagatctttg
ttgatcttg 29 8 28 DNA Artificial Sequence Synthesized
Oligonucleotide 8 ggccgcggtt aaatgtatac ccaaagac 28 9 36 DNA
Artificial Sequence Synthesized Oligonucleotide 9 gggggcggcc
gcgcgcaaca cgatgaagcc gtagac 36 10 36 DNA Artificial Sequence
Synthesized Oligonucleotide 10 ggttgagata aaagagcttt tggcgcggcc
gccttt 36 11 36 DNA Artificial Sequence Synthesized Oligonucleotide
11 gggggcggcc gcgatattga tatgacccaa tctcca 36 12 36 DNA Artificial
Sequence Synthesized Oligonucleotide 12 cccgcggccg cccgaggaga
cggtgactga ggtccc 36 13 36 DNA Artificial Sequence Synthesized
Oligonucleotide 13 gggggcggcc gcgatgtgca gcttcaggag tcggga 36 14 30
DNA Artificial Sequence Synthesized Oligonucleotide 14 ggggcggccg
ccttttattt ccaactttgt 30 15 31 DNA Artificial Sequence Synthesized
Oligonucleotide 15 ccagttggac ggcggccgcc ctgcaccgaa c 31 16 31 DNA
Artificial Sequence Synthesized Oligonucleotide 16 gttcggtgca
gggcggccgc cgtccaactg g 31 17 34 DNA Artificial Sequence
Synthesized Oligonucleotide 17 caatccagat tggggcggcc gccctgcacc
gaac 34 18 34 DNA Artificial Sequence Synthesized Oligonucleotide
18 gttcggtgca gggcggccgc cccaatctgg attg 34 19 31 DNA Artificial
Sequence Synthesized Oligonucleotide 19 ggtaggagcg ggcggccgcc
ctgcaccgaa c 31 20 31 DNA Artificial Sequence Synthesized
Oligonucleotide 20 gttcggtgca gggcggccgc ccgctcctac c 31 21 30 DNA
Artificial Sequence Synthesized Oligonucleotide 21 cctcaggccg
gcggccgccc tgcaccgaac 30 22 30 DNA Artificial Sequence Synthesized
Oligonucleotide 22 gttcggtgca gggcggccgc cctgaggatg 30 23 31 DNA
Artificial Sequence Synthesized Oligonucleotide 23 cagagtgagg
ggcggccgcc ctgcaccgaa c 31 24 31 DNA Artificial Sequence
Synthesized Oligonucleotide 24 gttcggtgca gggcggccgc ccctcactct g
31 25 30 DNA Artificial Sequence Synthesized Oligonucleotide 25
ggtaggagcg ggcggccgca gccctcaggc 30 26 30 DNA Artificial Sequence
Synthesized Oligonucleotide 26 gcctgagggc tgcggccgcc cgctcctacc 30
27 10 PRT Artificial Sequence Artificially Synthesized Peptide
Sequence 27 Ser Ala Trp Arg His Pro Gln Phe Gly Gly 1 5 10 28 8 PRT
Artificial Sequence Artificially Synthesized Peptide Sequence 28
Trp Ser His Pro Gln Phe Glu Lys 1 5 29 116 PRT Artificial Sequence
Artificially Synthesized Peptide Sequence 29 Val Asp Asn Lys Phe
Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu
Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser
Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40
45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn
50 55 60 Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro
Asn Leu 65 70 75 80 Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu
Lys Asp Asp Pro 85 90 95 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
Lys Lys Leu Asn Asp Ala 100 105 110 Gln Ala Pro Lys 115
* * * * *