U.S. patent application number 09/816391 was filed with the patent office on 2002-05-09 for anaerobic bacterium as a drug for cancer gene therapy.
Invention is credited to Amano, Jun, Fujimori, Minoru, Kano, Yasunobu, Nakamura, Toshiyuki, Sasaki, Takayuki, ichiro Taniguchi, Shun?apos, Yazawa, Kazuyuki.
Application Number | 20020054865 09/816391 |
Document ID | / |
Family ID | 18771403 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020054865 |
Kind Code |
A1 |
Fujimori, Minoru ; et
al. |
May 9, 2002 |
Anaerobic bacterium as a drug for cancer gene therapy
Abstract
The present invention provides a bacterium belonging to the
genus Bifidobacterium, by which DNA coding for a protein having an
antitumor activity or DNA coding for a protein having the activity
of converting a precursor of an antitumor substance into the
antitumor substance is delivered to tumor tissues specifically
under anaerobic conditions thereby expressing the protein encoded
by the DNA, as well as a pharmaceutical composition comprising said
anaerobic bacterium.
Inventors: |
Fujimori, Minoru;
(Matsumoto-shi, JP) ; Taniguchi, Shun?apos;ichiro;
(Matsumoto-shi, JP) ; Amano, Jun; (Matsumoto-shi,
JP) ; Yazawa, Kazuyuki; (Matsumoto-shi, JP) ;
Kano, Yasunobu; (Kyoto, JP) ; Nakamura,
Toshiyuki; (Matsumoto-shi, JP) ; Sasaki,
Takayuki; (Matsumoto-shi, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
18771403 |
Appl. No.: |
09/816391 |
Filed: |
March 26, 2001 |
Current U.S.
Class: |
424/93.21 ;
435/252.3; 435/454 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 48/00 20130101; C12N 1/205 20210501; C07K 14/195 20130101;
C12R 2001/01 20210501 |
Class at
Publication: |
424/93.21 ;
435/454; 435/252.3 |
International
Class: |
A61K 048/00; C12N
015/03; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 21, 2000 |
JP |
2000-287688 |
Claims
What is claimed is:
1. A method for delivering a gene in a system for delivering DNA
specifically to tumor tissues under anaerobic conditions, wherein a
bacterium belonging to the genus Bifidobacterium is used as a gene
delivery vector and then the DNA delivered specifically to tumor
tissues under anaerobic conditions is expressed in said tumor
tissues.
2. A method for delivering a gene in a system for delivering DNA
specifically to tumor tissues under anaerobic conditions, wherein a
bacterium belonging to the genus Bifidobacterium and having the DNA
coding for a protein which has a higher activity than in its parent
strain is used as a gene delivery vector and then the DNA delivered
specifically to tumor tissues under anaerobic conditions is
expressed in said tumor tissues.
3. A method for delivering a gene in a system for delivering DNA
specifically to tumor tissues under anaerobic conditions, wherein a
bacterium belonging to the genus Bifidobacterium transformed with a
recombinant DNA having said DNA is used as a gene delivery vector
and the DNA delivered specifically to tumor tissues under anaerobic
conditions is expressed in the tumor tissues.
4. The method as claimed in any one of claims 1 to 3, wherein the
DNA is selected from the group consisting of: (a) DNA coding for a
protein having an antitumor activity, and (b) DNA coding for a
protein having an activity of converting a precursor of an
antitumor substance into the antitumor substance.
5. The method as claimed in claim 4, wherein the protein having an
antitumor activity is interleukin-2.
6. The method as claimed in claim 4, wherein the precursor of an
antitumor substance is selected from the group consisting of
5-fluorocytosine, 5-aziridino-2,4-dinitrobenzamide, ganciclovir, a
glucuronic acid-conjugated antitumor substance and a
lysine-conjugated antitumor substance.
7. The method as claimed in claim 4, wherein the protein having the
activity of converting a precursor of an antitumor substance into
the antitumor substance is a protein selected from the group
consisting of cytosine deaminase, nitroreductase, herpes simplex
virus type 1 thymidine kinase and .beta.-glucuronidase.
8. The method as claimed in claim 3, wherein the recombinant DNA is
an expression vector.
9. The method as claimed in claim 8, wherein the expression vector
has a promoter and a terminator functioning in a bacterium
belonging to the genus Bifidobacterium.
10. The method as claimed in claim 9, wherein the promoter and
terminator are those involved in expressing a gene coding for
histone-like DNA-binding protein (HU protein) derived from
Bifidobacterium longum.
11. The method as claimed in claim 9, wherein the promoter and
terminator are DNAs located at the 1- to 192-positions and at the
472- to 600-positions respectively in the nucleotide sequence set
forth in SEQ ID NO: 1.
12. The method as claimed in any one of claims 1 to 11, wherein the
bacterium is Bifidobacterium longum.
13. The method as claimed in any one of claims 1 to 4 or 6 to 12,
wherein the bacterium is Bifidobacterium longum
105-A/pBLES100-S-eCD (FERM BP-7274).
14. A method for expressing a gene coding for a protein having an
antitumor activity in tissue tumors specifically, which comprises
use of the bacterium as claimed in any one of claims 1 to 5 or 8 to
12.
15. A method for expressing a gene coding for a protein having the
activity of converting a precursor of an antitumor substance into
the antitumor substance in tissue tumors specifically, which
comprises use of the bacterium as claimed in any one of claims 1 to
4 or 6 to 12.
16. A pharmaceutical composition comprising the bacterium as
claimed in any one of claims 1 to 13.
17. The pharmaceutical composition as claimed in claim 16, wherein
the pharmaceutical composition comprises a combination of the
bacterium as claimed in any one of claims 1 to 4 or 6 to 13 and the
precursor of an antitumor substance.
18. The pharmaceutical composition as claimed in claim 16, wherein
the pharmaceutical composition comprises the bacterium as claimed
in any one of claims 1 to 4 or 6 to 13 and the precursor of an
antitumor substance.
19. The pharmaceutical composition as claimed in any one of claims
16 to 18, wherein the bacterium is Bifidobacterium longum.
20. The pharmaceutical composition as claimed in any one of claims
16 to 19, wherein bacterium is Bifidobacterium longum
105-A/pBLES100-S-eCD (FERM BP-7274).
21. A bacterium belonging to the genus Bifidobacterium, which is
used in the method as claimed in any one of claims 1 to 13.
22. Bifidobacterium longum 105-A/pBLES100-S-eCD (FERM BP-7274.
23. DNA having the nucleotide sequence set forth in SEQ ID NO:
1.
24. A method of treating a solid tumor, which comprises use of the
method as claimed in any one of claims 1 to 15.
25. A method of treating a solid tumor, which comprises
administering the bacterium as claimed in any one of claims 1 to 4
or 6 to 13 in combination with the precursor of an antitumor
substance.
26. An anaerobic bacterium belonging to the genus Bifidobacterium
capable of expressing a gene coding for a protein having an
antitumor activity in only cancer cells under substantially
anaerobic conditions.
27. An anaerobic bacterium belonging to the genus Bifidobacterium
capable of expressing a gene coding for a protein having the
activity of converting a precursor of an antitumor substance with
low toxicity to humans and animals into an antitumor substance in
only cancer cells under substantially anaerobic conditions.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to anaerobic bacteria
belonging to the genus Bifidobacterium useful for gene therapy of
solid tumors, a pharmaceutical composition containing the same, a
method of delivering a gene and a method of treating solid tumors
by use of the same.
[0003] 2. Description of the Prior Art
[0004] Hypoxic regions are characteristic of solid tumors in animal
(Int. J. Radiat. Oncol. Biol. Phys., 10: 695-712(1984)) and occur
with high frequency in many types of human solid tumors
(Fischer-Verlag, stuttgart, 219-232(1994), New York). Tissue oxygen
electrode measurements (i.e. a membrane examination device capable
of measuring dissolved oxygen) taken in cancer patients have shown
a median range of oxygen partial pressure of 10 to 30 mmHg in
tumors, with a significant proportion of readings below 2.5 mmHg,
whereas those in normal tissues range from 24 to 66 mmHg.
[0005] Accordingly, gene therapy in solid tumors that targets gene
expression to hypoxic tumor cells is currently being investigated
(Nat. Med. 3: 515-520 (1997)). As a result, it is known that
certain species of anaerobic bacteria, including the genera
Clostridium and Bifidobacterium, can selectively germinate and grow
in the hypoxic regions of solid tumors after intravenous (i.v.)
injection (Cancer Res. 40: 2061-2068 (1980) & 15: 473-478
(1955)).
[0006] Further, anaerobic bacteria such as Clostridia or Salmonella
have been examined for the availability as gene delivery vectors
(Gene Ther. 4: 791-796 (1997) & 3: 173-178 (1996), FEMS
Microbiol. Rev. 17:357-364(1995), Cancer Biother Radio. 11: 145-153
(1996), Nat. Biotechnol. 17: 37-41 (1999)).
[0007] However, these bacteria have pathogenicity in humans and are
thus not always safe gene delivery vectors in gene therapy of solid
tumors. Actually, some reports have demonstrated febrile adverse
reactions as side effects after injection with Clostridium
butyricum spores or oral intake of Salmonella typhi (Eur. J.
Cancer. 3: 37-41 (1967), J. Clin. Invest. 90: 412-420 (1992),
Infect. Immun. 60: 536-541 (1992)).
[0008] The genera Bifidobacterium and Lactobacillus, on the other
hand, are Gram-positive and are domestic, nonpathogenic bacteria
found in the lower small intestine and large intestine of humans
and other animals.
[0009] In particular, Bifidobacterium strains have widely used for
preparation of fermented dairy products in many Asiatic and Western
countries, and it is now generally accepted that these bacteria are
nonpathogenic. In addition, it is known that these bacteria are not
only nonpathogenic but also have health-promoting properties for
their host. Such useful properties include e.g. an increase of the
immune response (J. Dairy Sci. 74: 1187-1195 (1991)), inhibition of
carcinogenesis (Cancer Res. 53: 3914-3918 (1993))and protection of
the host against viral infection (Lancet. 344: 1046-1049 (1994)),
etc.
[0010] Despite the increasing attention to these bacteria in the
fields of food science, medicine and industry, they have rarely
been used in gene therapy.
[0011] To be able to exploit the potential of these bacteria for
cancer gene therapy, detailed knowledge is required about such
basic biological phenomena as cellular metabolism, gene expression,
protein secretion and genetics. But little is known about genetic
properties of the genus Bifidobacterium, mainly due to the lack of
efficient and reproducible systems for genetic transfer and
adequate selectable markers.
[0012] In recent years, however, a system for the convenient and
reproducible genetic transformation of stains of the genus
Bifidobacterium was developed (Microbiology, 142: 109-114 (1996);
Biosci. Biotechnol. Biocem. 61: 1211-1212 (1997)).
[0013] However, the development of regulatory sequences including a
promoter for highly expressing an introduced gene was still not
satisfactory.
SUMMARY OF THE INVENTION
[0014] An object of the present invention is to provide anaerobic
bacteria belonging to the genus Bifidobacterium which are effective
as gene delivery vectors in gene therapy of solid tumors and safe
to humans and animals, as well as a pharmaceutical composition
comprising the anaerobic bacteria.
[0015] Another object of the present invention is to provide a
method of delivering a gene in which DNA effective in gene therapy
of solid tumors specifically to tumor tissues under aerobic
conditions by use of said anaerobic bacteria as gene delivery
vectors, as well as a method of treating solid tumors by expressing
a protein encoded by said DNA by use of said method.
[0016] The present inventors found that the bacteria of the genus
Bifidobacterium can be used as gene delivery vectors on the basis
of the known facts (a) human and animal solid tumors are in a
hypoxic region, (b) the bacteria of the genus Bifidobacterium are
anaerobes so they hardly grow in normal tissues, but will grow in
tumor tissues under anaerobic conditions, and (c) the bacteria of
the genus Bifidobacterium are less pathogenic than those bacteria
(e.g. Clostridia and Salmonella) used conventionally as gene
delivery vectors.
[0017] Further, the present inventors examined a system of
genetically transforming the bacteria belonging to the genus
Bifidobacterium, and as a result, they found that an introduced
gene can be efficiently expressed by using expression vector
containing a promoter and a terminator involved in expressing a
gene coding for a histone-like DNA-binding protein (abbreviated
herein after to HU protein) (Biochimie, 72: 207-212 (1990))
inherently highly expressed in the bacteria belonging to the genus
Bifidobacterium, particularly in Bifidobacterium longum.
[0018] To achieve the object described above, the present inventors
made further study to complete the present invention.
[0019] That is, the present invention relates to:
[0020] (1) A method for delivering a gene in a system for
delivering DNA specifically to tumor tissues under anaerobic
conditions, wherein a bacterium belonging to the genus
Bifidobacterium is used as a gene delivery vector and then the DNA
delivered specifically to tumor tissues under anaerobic conditions
is expressed in said tumor tissues;
[0021] (2) A method for delivering a gene in a system for
delivering DNA specifically to tumor tissues under anaerobic
conditions, wherein a bacterium belonging to the genus
Bifidobacterium and having the DNA coding for a protein which has a
higher activity than in its parent strain is used as a gene
delivery vector and then the DNA delivered specifically to tumor
tissues under anaerobic conditions is expressed in said tumor
tissues;
[0022] (3) A method for delivering a gene in a system for
delivering DNA specifically to tumor tissues under anaerobic
conditions, wherein a bacterium belonging to the genus
Bifidobacterium transformed with a recombinant DNA having said DNA
is used as a gene delivery vector and the DNA delivered
specifically to tumor tissues under anaerobic conditions is
expressed in the tumor tissues;
[0023] (4) The method as described above in any one of (1) to (3),
wherein the DNA is selected from the group consisting of: (a) DNA
coding for a protein having an antitumor activity, and (b) DNA
coding for a protein having an activity of converting a precursor
of an antitumor substance into the antitumor substance;
[0024] (5) The method as described above in (4), wherein the
protein having an antitumor activity is interleukin-2;
[0025] (6) The method as described above in (4), wherein the
precursor of an antitumor substance is selected from the group
consisting of 5-fluorocytosine, 5-aziridino-2,4-dinitrobenzamide,
ganciclovir, a glucuronic acid-conjugated antitumor substance and a
lysine-conjugated antitumor substance;
[0026] (7) The method as described above in (4), wherein the
protein having the activity of converting a precursor of an
antitumor substance into the antitumor substance is a protein
selected from the group consisting of cytosine deaminase,
nitroreductase, herpes simplex virus type 1 thymidine kinase and
.beta.-glucuronidase;
[0027] (8) The method as described above in (3), wherein the
recombinant DNA is an expression vector;
[0028] (9) The method as described above in (8), wherein the
expression vector has a promoter and a terminator functioning in a
bacterium belonging to the genus Bifidobacterium;
[0029] (10) The method as described above in (9), wherein the
promoter and terminator are those involved in expressing a gene
coding for histone-like DNA-binding protein (HU protein) derived
from Bifidobacterium longum;
[0030] (11) The method as described above in (9), wherein the
promoter and terminator are DNAs located at the 1- to 192-positions
and at the 472- to 600-positions respectively in the nucleotide
sequence set forth in SEQ ID NO: 1;
[0031] (12) The method as described above in any one of (1) to
(11), wherein the bacterium is Bifidobacterium longum;
[0032] (13) The method as described above in any one of (1) to (4)
or (6) to (12), wherein the bacterium is Bifidobacterium longum
105-A/pBLES100-S-eCD (FERM BP-7274);
[0033] (14) A method for expressing a gene coding for a protein
having an antitumor activity in tissue tumors specifically, which
comprises use of the bacterium as described above in any one of (1)
to (5) or (8) to (12);
[0034] (15) A method for expressing a gene coding for a protein
having the activity of converting a precursor of an antitumor
substance into the antitumor substance in tissue tumors
specifically, which comprises use of the bacterium as described
above in any one of (1) to (4) or (6) to (12);
[0035] (16) A pharmaceutical composition comprising the bacterium
as described above in any one of (1) to (13);
[0036] (17) The pharmaceutical composition as described above in
(16), wherein the pharmaceutical composition comprises a
combination of the bacterium as described above in any one of (1)
to (4) or (6) to (13) and the precursor of an antitumor
substance;
[0037] (18) The pharmaceutical composition as described above in
(16), wherein the pharmaceutical composition comprises the
bacterium as described above in any one of (1) to (4) or (6) to
(13) and the precursor of an antitumor substance;
[0038] (19) The pharmaceutical composition as described above in
any one of (16) to (18), wherein the bacterium is Bifidobacterium
longum;
[0039] (20) The pharmaceutical composition as described above in
any one of (16) to (19), wherein bacterium is Bifidobacterium
longum 105-A/pBLES100-S-eCD (FERM BP-7274);
[0040] (21) A bacterium belonging to the genus Bifidobacterium,
which is used in the method as described above in any one of (1) to
(13);
[0041] (22) Bifidobacterium longum 105-A/pBLES100-S-eCD (FERM
BP-7274;
[0042] (23) DNA having the nucleotide sequence set forth in SEQ ID
NO: 1;
[0043] (24) A method of treating a solid tumor, which comprises use
of the method as described above in any one of (1) to (15);
[0044] (25) A method of treating a solid tumor, which comprises
administering the bacterium as described above in any one of (1) to
(4) or (6) to (13) in combination with the precursor of an
antitumor substance;
[0045] (26) An anaerobic bacterium belonging to the genus
Bifidobacterium capable of expressing a gene coding for a protein
having an antitumor activity in only cancer cells under
substantially anaerobic conditions;
[0046] (27) An anaerobic bacterium belonging to the genus
Bifidobacterium capable of expressing a gene coding for a protein
having the activity of converting a precursor of an antitumor
substance with low toxicity to humans and animals into an antitumor
substance in only cancer cells under substantially anaerobic
conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1 shows an illustration of plasmid pBLES100. The solid
line is Escherichia coil vector pBR322, the smeared part is plasmid
pTB6 (3.6 kb) derived from B. longum, and the non-smeared part is a
1.1-kb Hind III-Eco RI fragment derived from Enterococcus faecalis.
Spec.sup.r represents a spectinomycin resistance gene, and Ori
represents an origin of replication.
[0048] FIG. 2 shows an illustration of plasmid pBL595.
[0049] FIG. 3 shows an illustration of plasmid pBLEM100. The
smeared part is plasmid pBL595 derived from B. longum SBT595, the
non-smeared part is an Ava I-Hind III fragment of pBR329 derived
from Escherichia coli, and the solid line is a Hind III-Ava I
fragment of pAM.beta.I derived from Enterococcus faecalis.
[0050] FIG. 4 shows an illustration of plasmid pBL67.
[0051] FIG. 5 shows an illustration of plasmid pBL78.
[0052] FIG. 6 shows an illustration of plasmid pBLES100 having a HU
gene and a cytosine deaminase (abbreviated hereinafter to CD) gene
integrated into it. The solid line is Escherichia coil vector
pBR322, the smeared part is plasmid pTB6 (3.6 kb) derived from B.
longum, the non-smeared part is a 1.1-kb Hind III-Eco RI fragment
derived from Enterococcus faecalis, the dotted part is a Hind
III-treated fragment from a gene in B. longum, the non-smeared part
with the arrow inside is a CD gene derived from Escherichia coli,
the netted part located upstream (toward Ori) from the CD gene is a
region containing a promoter for the HU gene, and the shaded part
located downstream from the CD gene is a region containing a
terminator. Spec.sup.r represents a spectinomycin resistance gene,
and Ori represents an origin of replication.
[0053] FIG. 7 is a graph showing the number of B. longum bacilli
present in each kind of organ tissues and tumor tissues after
intravenous injection of B. longum bacteria into tumor-bearing
mice. The circle shows the result of administration of B. longum
105-A. The square shows the result of administration of B. longum
108-A.
[0054] FIG. 8 is a graph showing the number of B. longum bacilli
present in each kind oforgan tissues and tumor tissues in 168 hours
after intravenous injection of B. longum 105-A (shown in the netted
bar) or B. longum 105-A transformed with pBLES100 (shown in the
white bar) into tumor-bearing rats.
[0055] FIG. 9 is a graph showing the number of B. longum bacilli
present tumor cells after intravenous injection of B. longum 105-A
or B. longum 105-A/pBLES100 and administration of spectinomycin
into tumor-bearing mice. The white bar indicates a group given B.
longum 105-A, and the netted bar indicates a group given B. longum
105-A/pBLES100, and the control shows a group not given
spectinomycin, and spectinomycin shows a group given
spectinomycin.
[0056] FIG. 10 shows an illustration of plasmid pBLHU15 containing
DNA coding for HU protein derived from B. longum. The dotted part
is a Hind III-treated fragment of the gene from B. longum, the
solid line and the non-smeared part indicate plasmid pBR322, and
the smeared part is the HU gene derived from B. longum. Amp.sup.r
represents an ampicillin resistance gene, Ter.sup.r represents a
tetracycline resistance gene and Ori represents an origin of
replication.
[0057] FIG. 11 shows a process for constructing plasmid vector
pBLES100-S-eCD in which the CD gene derived from Escherichia coli
was integrated, which is used as expression vector for B.
longum.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention provides bacteria belonging to the
genus Bifidobacterium (abbreviated hereinafter to the bacteria of
the genus Bifidobacterium) having a gene coding for a substance
having an antitumor activity. Preferably said substance has a
higher antitumor activity than in its parent strain.
[0059] The substance having a higher antitumor activity than in its
parent strain is e.g. the substance which is expressed in a larger
amount than in its parent strain, has an improvement in Km value as
compared with the counterpart (enzyme) expressed in its parent
strain, or is hardly degradable than in the counterpart expressed
in its parent strain, resulting in the higher activity. The parent
strain in the microbiology usually means the wild stain, from which
substains, clones and mutants and the like are derived (Biologic
dictionary, third edition, tokyokagakudoujin 1998).
[0060] Whether the activity of the substance having an antitumor
activity is higher than in its parent strain can be easily
determined by a screening method known in the art. For example, the
bacteria of the genus Bifidobacterium are cultured in a suitable
medium, and the produced substance having an antitumor activity is
measured for its antitumor activity (expression level, enzyme
activity etc.) by a known method.
[0061] The substance having an antitumor activity may be any
substance having an antitumor activity and its mechanism is not
limited. The antitumor activity includes the action of preventing
or inhibiting the development, maturation, multiplication or
diffusion of tumor cells or tissues, or the activity of regressing
tumor cells or tissues. The tumor includes e.g. carcinoma or
sarcoma. However, the substance having an antitumor activity in the
present invention is usually a polypeptide or a protein whose
structure can be encoded by the nucleotide sequence of DNA.
[0062] The substance having an antitumor activity in the present
invention includes e.g. cytokines. The cytokines having an
antitumor activity include e.g. interferon (IFN)-.alpha., .beta.,
.gamma., granulocyte macrophage colony-stimulating factor (GM-CSF),
interleukin (IL)-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-6,
IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, anti-Fas antibody, tumor
necrosis factor (TNF)-.alpha., lymphotoxin (LT)-.beta., granulocyte
colony-stimulating factor (G-CSF), macrophage colony-stimulating
factor (M-CSF), macrophage migration inhibition factor (MIF),
leukocyte inhibitory factor (LIF), co-stimulating factor of
activated T cell B7 (CD80) and B7-2 (CD86), kit ligand, oncostatin
M, etc. In particular, IL-2 is preferable.
[0063] These may be a combination thereof. For example, a
combination of IL-6 and TNF-.alpha., a combination of IFN-.alpha.
and IFN-.beta. or IFN-.gamma., a combination of TNF-.alpha. and
IFN-.gamma., and a combination of anti-Fas antibody and IFN-.gamma.
are preferable.
[0064] The present invention provides those bacteria belonging to
the genus Bifidobacterium having a gene coding for an enzyme
capable of converting a precursor of an antitumor substance with
low toxicity to humans and animals into the antitumor substance
(referred to hereinafter as converting enzyme), said enzyme being
capable of production in only tumor cells under substantially
anaerobic conditions. Preferably the converting enzyme has a higher
activity than in its parent strain. The converting enzyme having a
higher activity than in its parent strain has the almost same
meaning as defined above.
[0065] The antitumor substance may be any known substance having an
antitumor activity. The antitumor activity has the same meaning as
defined above. However, the precursor of an antitumor substance
should be low toxic to humans and animals. The precursor of an
antitumor substance may be in an inactive form. The substance in an
inactive form means the one converted by the converting enzyme into
an active substance expressing an antitumor activity.
[0066] The converting enzyme can be selected as necessary depending
on the combination of the precursor of an antitumor substance and
the antitumor substance. The converting enzyme may be either a
single enzyme or a group of plural enzymes, preferably a single
enzyme.
[0067] The combination of the precursor of an antitumor substance,
the antitumor substance and the converting enzyme used in the
present invention is not particularly limited insofar as they are
known in the art.
[0068] Mention is made of e.g. a combination of 5-fluorocytosine
(5-FC) as the precursor of an antitumor substance, 5-fluorouracil
as the antitumor substance and cytosine deaminase as the converting
enzyme.
[0069] Mention is also made of a combination of
5-aziridino-2,4-dinitroben- zamide (CB1954) as the precursor of an
antitumor substance, an alkylating agent as the antitumor substance
known to form bridge-linkages in double-stranded DNA, and
nitroreductase as the converting enzyme.
[0070] Mention is also made of a combination of ganciclovir as the
precursor of an antitumor substance, its metabolite as the
antitumor substance, and herpes simplex virus type 1 thymidine
kinase (HSV1-TK) as the converting enzyme.
[0071] Further, the antitumor substance may be converted into the
precursor rendered low-toxic (e.g. in an inactivated form) to
humans by modifying it by conjugation with glucuronic acid, glycine
or lysine, and the converting enzyme may be an enzyme for
de-modifying said precursor. The enzyme for de-modifying the
precursor may be any enzyme known in the art, and for example a
combination of a glucuronic acid-conjugated precursor of an
antitumor substance and .beta.-glucuronidase as the converting
enzyme can be mentioned.
[0072] The bacteria of the genus Bifidobacterium used in the
present invention may be any known strains belonging to the
aforementioned genus, which is anaerobic.
[0073] Examples thereof include Bifidobacterium adolescentis, B.
longum, B. bifidum, B. pseudolongum, B. thermophirum, B. breve, B.
infantis etc.
[0074] Particularly preferably used are those bacteria known to be
resident in intestines in humans of any age, such as B.
adolescentis, B. longum, B. bifidum and B. infantis, among which B.
longum is the most preferable. Further, their resistant strains,
mutants etc. may also be used.
[0075] Any of these bacteria are commercially available or easily
obtainable from the depository organizations. For example,
Bifidobacterium longum has been deposited under ATCC-15707, B.
bifidum under ATCC-11863, and B. infantis under ATCC-15697.
[0076] The bacteria of the genus Bifidobacterium include those
strains capable of producing the substance having an antitumor
activity or the converting enzyme, and such strains can be used
preferably as the gene delivery vectors in the present invention.
Such strains include B. longum producing cytosine deaminase capable
of converting 5-FC into 5-FU.
[0077] Whether the bacterium in question is a strain capable of
producing the substance having an antitumor activity or the
converting enzyme can be easily judged by examining whether the
substance having an antitumor activity or the converting enzyme is
detected by a known screening method or whether the antitumor
substance is detected upon culture of the bacterium in a medium
containing a precursor of the antitumor substance.
[0078] The strain not capable of producing the substance having an
antitumor activity or the converting enzyme is transformed in the
following manner with DNA coding for the substance having an
antitumor activity or the converting enzyme, whereby the bacterium
can be preferably used as the gene delivery vector in the present
invention.
[0079] The following fundamental procedures in genetic engineering
or biological engineering can be conducted according to the methods
described commercial books on experiments, such as "Idenshi Manual
(Gene Manual)" published by Kodansha, "Idenshi Sosa Jikkenho
(Experimental Methods in Gene Manipulation)" edited by Y. Takagi
and published by Kodansha, Molecular Cloning, Cold Spring Harbor
Laboratory (1982), Molecular Cloning, 2nd ed., Cold Spring Harbor
Laboratory (1989), Methods in Enzymology, 194 (1991), "Genetic
Experimental Methods by Yeasts", Extra Issue of "Jikken Igaku
(Experimental Medicine)" published by Yodosha (1994), etc.
[0080] First, it is necessary to obtain DNA coding for the
substance having an antitumor activity or the converting
enzyme.
[0081] The DNA described above can be easily obtained on the basis
of the information on the known nucleotide sequence.
[0082] For example, it can be obtained by chemical synthesis using
a known method on the basis of the information on the known
nucleotide sequence. The chemical synthesis method includes e.g. a
chemical synthesis method using a DNA synthesizer such as DNA
synthesizer model 392 (Perkin Elmer) utilizing the phosphoamidite
method.
[0083] Alternatively, the DNA described above can also be obtained
by amplification of the DNA in the PCR method (PCR Protocols,
Academic Press (1990)) where nucleotides prepared on the basis of
the 5'- and 3'-terminal nucleotide sequences of said nucleotide
sequence are used as the primers, while cDNA synthesized from mRNA
contained in tissues or cells in various organisms or cDNA selected
from cDNA library is used as the template.
[0084] Further, the above-described DNA can also be obtained by
colony hybridization or plaque hybridization with cDNA library or
cDNA synthesized from mRNA contained in tissues or cells in various
organisms (Molecular Cloning, 2nd ed.) where full-length or partial
DNA or polynucleotide chemically synthesized on the basis of the
information on the known nucleotide sequence is used as the
probe.
[0085] Alternatively, the above DNA can also be easily obtained
from the information on the known amino acid sequence.
[0086] As the method of obtaining the above-described DNA from the
information on the known amino acid sequence, a method known in the
art may be used. Specifically, there is a method of amplifying the
desired DNA from the cDNA library etc. by the PCR method using
synthetic DNA primers having a partial nucleotide sequence of the
DNA coding for the known amino acid sequence, or a selection method
by hybridizing the DNA integrated in a suitable vector with a
labeled DNA fragment or synthetic DNA (probe) coding for a part or
the whole of the substance having an antitumor activity or the
converting enzyme.
[0087] If the substance or the enzyme is known to have an antitumor
activity or a converting enzyme activity, but neither the amino
acid sequence thereof nor the nucleotide sequence of DNA coding
therefor is known, the method of obtaining the DNA coding for the
substance having an antitumor activity or the converting enzyme
involves e.g. preparing an expression cDNA library from organisms
confirmed to have the antitumor activity or the converting enzyme
activity and then screening individual cells constituting the
library by using the antitumor activity or converting enzyme
activity as the indicator in order to obtain those cells carrying
the DNA coding for the substance having an antitumor activity or
the converting enzyme.
[0088] Further, the substance having an antitumor activity or the
converting enzyme can be purified by a combination of methods known
in the art, then the N-terminal amino acid sequence of the
substance having an antitumor activity or the converting enzyme is
analyzed by a method known in the art, and hybridization with the
cDNA library etc. is conducted where a synthetic DNA having the
nucleotide sequence of DNA coding for said amino acid sequence is
used as the probe, whereby the substance having an antitumor
activity or the converting enzyme can be obtained.
[0089] Specifically, DNA coding for cytosine deaminase is
preferably the one isolated from plasmid pAdexlCSCD (RDB No. 1591,
Gene Bank, Institute of Physical and Chemical Research) containing
DNA coding for cytosine deaminase derived from E. coli, or from
plasmid pMK116 containing DNA coding for cytosine deaminase derived
from E. coli (D. A. Mead et al., Protein Engineering 1: 67-74
(1986)).
[0090] Nitroreductase is preferably the one isolated from E. coil
B. Its amino acid sequence is described in Biochem. Pharmacol, 44:
2289-2295, and on the basis of its amino acid sequence, the DNA
coding for nitroreductase can be easily obtained by the method
described above.
[0091] In addition to the DNA coding for the substance having an
antitumor activity or the converting enzyme, which is obtained on
the basis of the information on the known nucleotide or amino acid
sequence, DNA hybridizing with said DNA under stringent conditions
can also be used in the present invention. That is, a plurality of
genetic codes are generally present for one amino acid, so that
even DNA having a nucleotide sequence different from the nucleotide
sequence based on the known nucleotide or amino acid sequence or
DNA coding for an amino acid sequence different from the known
amino acid sequence due to one or several amino acid residues are
deleted, substituted or added in the known amino acid sequence can
be used in the present invention insofar as it can express the
substance having an antitumor activity or the converting
enzyme.
[0092] The DNA capable of hybridization under stringent conditions
means DNA obtained by colony hybridization, plaque hybridization or
Southern hybridization with the above-described DNA as the
probe.
[0093] Specifically, the DNA includes those DNAs which can be
identified by hybridization conducted in the presence of about 0.7
to 1.0 M sodium chloride at about 65.degree. C. on a filter onto
which DNA derived from colonies or plaques has been immobilized,
and then washing the filter under the condition of about 65.degree.
C. with about 0.1- to 2-fold conc. SSC solution (1-fold conc. SSC
solution consists of 150 mM sodium chloride and 15 mM sodium
citrate). Hybridization can be conducted by a method described in
e.g. Molecular Cloning, Second Edition, Current Protocols in
Molecular Biology, DNA Cloning 1: Core Techniques, A Practical
Approach, Second Edition, Oxford University (1995), etc.
[0094] Specifically, the hybridizable DNA includes those DNAs
having at least 60% or more, preferably about 80% or more and most
preferably about 95% or more homology to the nucleotide sequence of
DNA coding for the substance having an antitumor activity or the
converting enzyme obtained on the basis of the information on the
nucleotide sequence or the information on the amino acid sequence
described above.
[0095] The homology of a nucleotide sequence or an amino acid
sequence can be determined using the algorithm "BLAST" by Karlin
and Altschl (Proc. Natl. Acad. Sci. USA, 90, 5873-5877 (1993)). The
programs called "BLASTN" and "BLASTX" have developed based on the
above algorithm (J. Mol. Biol., 215, 403-410 (1990)). In the case
of analyzing a nucleotide sequence based on BLAST, the parameter
can be set to e.g. score=100, wordlength=12. And in the case of
analyzing an amino acid sequence based on BLASTX, the parameter can
be set to e.g. score=50, wordlength=3. In the case of using BLAST
or Gapped BLAST program, a default parameter of each program can be
used. The specific analysis method of using the above programs are
known in the art (http://www.ncbi.nlm.nih.gov.).
[0096] In the present invention, it is also possible to employ a
protein or a polypeptide having an amino acid sequence wherein one
or several amino acid residues are deleted, substituted or added in
the amino acid sequence coding for the above substance or
converting enzyme.
[0097] Such protein or polypeptide can be obtained by site-specific
mutation of the DNA coding for the substance having an antitumor
activity or the converting enzyme by means of site-specific
mutagenesis described in Molecular Cloning, A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press (1989), Current
Protocols in Molecular Biology, John Wiley & Sons (1987-1997),
Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci.,
USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research,
13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)
etc.
[0098] The number of amino acids deleted, substituted or added is
not particularly limited, but preferably one to dozens,
particularly one to few amino acids are deleted, substituted or
added.
[0099] Secondly, a recombinant DNA containing the DNA coding for
the substance having an antitumor activity or the converting enzyme
obtained as described above is prepared. In the present invention,
the recombinant DNA is preferably an expression vector.
[0100] The expression vector can be produced for example by cutting
out the desired DNA fragment and ligating the DNA fragment to a
region downstream from a promoter in a suitable expression
vector.
[0101] As the DNA fragment inserted into the expression vector, the
DNA coding for the substance having an antitumor activity or the
converting enzyme can be used as such or after digestion with
restriction enzymes if necessary or after addition of a linker. The
DNA fragment may have ATG as the translation initiation codon at
the 5'-terminal or TAA, TGA or TAG as the translation termination
codon at the 3'-terminal. These translation initiation and
termination codons can also be added via a suitable synthetic DNA
adaptor to the DNA coding for the substance having an antitumor
activity or the converting enzyme.
[0102] For expression or advantageous expression of the substance
having an antitumor activity or the converting enzyme according to
the present invention, the expression vector usually has regulatory
sequences added to a cloning vector as described below. Each
regulatory sequence may be endogenous or extraneous to the cloning
vector.
[0103] Such regulatory sequences include, but are not limited to, a
promoter, a leader, a pro-peptide sequence, an enhancer, a signal
sequence, a selective marker and a terminator. In particular, the
regulatory sequences are preferably those containing at least a
promoter and a terminator.
[0104] The regulatory sequences may have a linker (restriction
enzyme cleavage site) to facilitate linkage thereof to the DNA
coding for the substance having an antitumor activity or for the
converting enzyme or to facilitate linkage between the regulatory
sequences described above.
[0105] The promoter and terminator used in the present invention
are particularly preferably those involved in expression of HU gene
(SEQ ID NO: 1) that is expressed inherently highly in B. longum.
Specifically, it is preferable that the DNA containing the DNA
located in the 1- to 192-positions in SEQ ID NO: 1 is used as the
promoter and the DNA in the 472- to 600-positions in SEQ ID NO: 1
as the terminator.
[0106] The expression vector having the promoter and terminator
involved in expressing the HU gene is constructed preferably by
cutting the HU gene out from DNA of B. longum with a restriction
enzyme, integrating it in a cloning vector described below, and
integrating e.g. the DNA coding for the substance having an
antitumor activity or for the converting enzyme in a region
downstream from the promoter involved in expression of the HU gene.
By use of the promoter and terminator involved in expression of the
HU gene, the substance having an antitumor activity or the
converting enzyme can be efficiently expressed.
[0107] The method of isolating the HU gene involves e.g. digesting
the chromosomal DNA of B. longum with a restriction enzyme Hind
III.
[0108] Specifically, the following method can be mentioned. The
chromosomal DNA of B. longum is digested with a restriction enzyme
Hind III and purified by phenol treatment and ethanol
precipitation. Separately, pBR322 (Takara Shuzo Co., Ltd.) is also
digested with Hind III, dephosphorylated, and purified in analogous
manner. The respective DNAs are ligated to give a recombinant
DNA.
[0109] This recombinant DNA is then used to transform E. coli mH3
(Gene, 45, 37 (1986)) in a usual manner, whereby an
ampicillin-resistant and tetracycline-sensitive transformant is
obtained. A plasmid DNA is extracted in a usual manner from the
transformant thus obtained, and the plasmid DNA is introduced in a
usual manner into E. coil YK2741 strain (Gene, 89, 133 (1990))
thereby transforming said strain. The YK2741 strain is deficient in
HU gene and IHF (integration host factor) gene and is thus
sensitive to low temperatures, and the capability of its
low-temperature sensitivity can be utilized for selection of the
transformant containing the DNA encoding for HU by plating it onto
an ampicillin-containing agar medium and culturing it at 27.degree.
C.
[0110] Then, the YK2741 transformant thus obtained is further
cultured, and a plasmid possessed in said strain is extracted in a
usual manner, and the plasmid DNA is introduced in a usual manner
into E. coli YK1340 strain (J. Mol. Biol., 204, 581 (1988)) thereby
transforming said strain. The resulting transformant is subjected
in a usual manner to a test of infection with Mu phage. The YK1340
strain is a strain deficient in HU gene, but Mu phage necessitates
the HU protein for its growth, and thus a transformant infected
with Mu phage and lyzed by growth of Mu phage therein is a
promising candidate for a strain carrying the HU gene derived from
B. longum. Accordingly, plasmid pBLHU15 having the promoter and
terminator involved in expression of the HU gene derived from B.
longum can be obtained by selecting the plasmid possessed in the
strain resistant to ampicillin and infected with Mu phage and lyzed
by growth of Mu phage therein.
[0111] By further integrating a signal sequence therein, the
substance having an antitumor activity or the converting enzyme
produced e.g. in host cells can be positively secreted into the
outside of the host cells. That is, the signal sequence can be used
to express the substance having an antitumor activity or the
converting enzyme in a form having a signal peptide, resulting in
positive secretion of the substance having an antitumor activity or
the converting enzyme into the outside of the host cells.
[0112] The method of adding the signal peptide includes e.g. the
method of Paulson et al. (J. Biol. Chem., 264,17619(1989)), the
method of Low et al. (Proc. Natl. Acad. Sci., USA, 86, 8227 (1989),
Genes Develop., 4, 1288 (1990)), or by the methods described in
JP-A 5-336963, WO94/23021, etc.
[0113] The selective marker is used for specifically selecting the
transformed bacteria of the genus Bifidobacterium. For example,
mention is made of selection by chemical resistance markers with
ampicillin resistance, tetracycline resistance, neomycin resistance
or kanamycin resistance; nutrition requirements; and mediums such
as HAT medium etc.
[0114] If the cloning vector described below has the selective
marker, integration of another additional selective marker is not
necessary.
[0115] The cloning vector that can be used in the present invention
includes a cloning vector (a) capable of easily producing a
recombinant vector in vitro with the DNA coding for the substance
having an antitumor activity or for the converting enzyme, (b)
having the ability to autonomously replicate in the bacteria of the
genus Bifidobacterium or to integrate into genomic DNA of the
bacteria of the genus Bifidobacterium, (c) capable of being
introduced into the bacteria of the genus Bifidobacterium, and (d)
permitting specific detection of the bacteria of the genus
Bifidobacterium transformed by introducing the cloning vector.
[0116] As the cloning vector, plasmid pBLES100 is specifically
mentioned, and this plasmid can be used preferably in the present
invention.
[0117] This plasmid is illustrated in FIG. 1. As can be seen from
FIG. 1, a 1.1-kb Hind III-Eco RI fragment (the non-smeared part in
FIG. 1) derived from Enterococcus faecalis is integrated in a
composite plasmid consisting of Escherichia coli vector pBR322 (the
solid line in FIG. 1) and B. longum-derived pTB6 plasmid (3.6 kb)
(the smeared part in FIG. 1). This fragment contains a region
showing spectinomycin resistance, that is, a region coding for
spectinomycin adenyltransferase.
[0118] Mention is also made of plasmid pBL595 of about 2.9 kb in
size derived from B. longum SBT595 (FERM P-14162 deposited with the
National Institute of Bioscience and Human-Technology, Agency of
Industrial Science and Technology, Japan) and having restriction
enzyme recognition sites shown in FIG. 2.
[0119] Mention is also be made of plasmid pBLEM100 (FIG. 3)
consisting of plasmid pBL595, an Ava I-Hind III fragment from E.
coli-derived pBR329, and a Hind III-Ava I fragment from pAMP1
derived from Enterococcus faecalis. E. coil carrying the plasmid
pBLEM100 has been deposited under FERM P-14102 with the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, Japan.
[0120] Further, a plasmid vector constructed by binding a
conjugated plasmid consisting of a plasmid derived from a bacterium
belonging to the genus Streptococcus and a plasmid from E. coil to
plasmid pBL67 or pBL78 derived from B. longum may also be used
(JP-A 5-130876).
[0121] Plasmid pBL67 is an about 3.7-kb plasmid having restriction
enzyme recognition sites shown in FIG. 4, which was derived from B.
longum M09101 (FERM P-12167) or B. longum M09102 (FERMP-12168).
Plasmid pBL78 is an about 8.5-kb plasmid having restriction enzyme
recognition sites shown in FIG. 5, which was derived from B. longum
MO9103 (FERM P-12169).
[0122] A plasmid in which plasmid pBR322 derived from E. coli is
bound to plasmid pTB4, pTB6 or pTB10 derived from B. longum is also
mentioned. Further, a plasmid in which the whole of pC194 (or a
chloramphenicol resistance gene therein) derived from
Staphylococcus oureus is bound to the above plasmid is also
mentioned. Further, a plasmid in which genes involved in tryptophan
synthetic pathway from B. longum is bound to each of the above two
plasmids may also be used (JP-A 63-123384).
[0123] Those E. coil bacteria carrying these plasmids have been
deposited with the National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology,
Japan (FERM P-9040, 9041, 9042, 9043, 9044, 9045, 9046, 9047 and
9048).
[0124] Plasmid pTB4 or pTB10 is a plasmid derived from B. longum
BK25 (FERM P-9049). Plasmid pTB6 is a plasmid derived from B.
longum BK51 (FERM P-9050).
[0125] As a preferable embodiment of the expression vector in the
present invention, there is an expression vector wherein the
promoter and terminator involved in expression of the HU gene
described above, and a gene (CD gene) coding for cytosine deaminase
(abbreviated hereinafter to CD) capable of converting 5-FC into
5-FU, have been integrated in the vector pBLES100 described
above.
[0126] As a more specific embodiment, there is an expression vector
illustrated in FIG. 6. By way of example, a method of constructing
this expression vector is as follows: A recombinant DNA comprising
the E. coli-derived CD gene inserted into TOPO vector (Funakoshi
Co., Ltd.) is used to transform E. coli JM109, and plasmid DNA is
extracted from the resulting transformant. The desired plasmid
pTOPO-eCD is digested with restriction enzymes Nsp V and Hpa I,
followed by purification of the desired 1.3-kb CD-coding DNA
fragment.
[0127] Similarly, plasmid pBLHU15 carrying the promoter and
terminator involved in expression of the B. longum-derived HU gene
obtained in the manner described above is also digested with Nsp V
and Hpa I, followed by purification of the desired 6.7-kb DNA
fragment.
[0128] The 1.3-kb and 6.7-kb DNA fragments obtained above are
ligated in a usual manner to prepare a recombinant DNA, and this
recombinant DNA is used to transform E. coli JM109 in a usual
manner.
[0129] Then, the plasmid DNA is extracted in a usual manner from
the resulting transformant, then the plasmid DNA is digested with
Hind III, and the promoter and terminator involved in expression of
the HU gene and a 3.6-kb DNA fragment containing the CD gene are
separated and purified by conventional techniques such as a garose
gel electrophoresis. Separately, the Escherichia-Bifidobacterium
shuttle vector pBLES100 described above is also digested with Hind
III and dephosphorylated.
[0130] The 3.6-kb DNA fragment and the above Hind III digest of
pBLES100 are ligated in a usual manner to construct a recombinant
DNA, and this recombinant DNA is used to transform E. coli JM109 in
a usual manner. The transformant can be selected by spectinomycin
resistance. The Escherichia-Bifidobacterium shuttle vector
pBLES100-S-eCD having the CD-coding gene downstream from the
promoter for the HU gene can thus be constructed.
[0131] Thirdly, the recombinant DNA, preferably an expression
vector thereof, is introduced into the bacteria of the genus
Bifidobacterium as the host. For this introduction, any methods
known in the art can be used. Such methods include e.g. the
electroporation method (Cytotechnology, 3, 133 (1990)), the calcium
phosphate method (JP-A 2-227075), the lipofection method (Proc.
Natl. Acad. Sci., USA, 84, 7413 (1987)), the method of using
calcium ion (Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)), the
protoplast method (JP-A 63-2483942), and those methods described in
Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111
(1979), etc.
[0132] In the present invention, the electroporation method is
preferably used. Electroporation is carried out for about 4.1 to
4.5 ms under the conditions of about 10.0 kV/cm, about 200 .OMEGA.
and about 25 .mu.F.
[0133] Although a combination of the recombinant DNA (preferably
its expression vector) introduced and the bacterium of the genus
Bifidobacterium as the host is not particularly limited, plasmid
pBLES100 is introduced preferably into B. longum 105-A or 108-A
(Biosci. Biotech. Biochem. 61(7), 1211-1212 (1997)).
[0134] B. longum 105-A/pBLES100-S-eCD i.e. B. longum 105-A
transformed with plasmid pBLES100-S-eCD in which the promoter and
terminator involved in expression of the HU gene shown in FIG. 6
and the CD gene were integrated has been deposited under FERM
BP-7274 with the National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology.
[0135] The bacteria of the genus Bifidobacterium into which the
above expression vector was introduced are cultured in a known
medium in which only the transformed bacteria are selected. As the
medium, a known medium suitable for the intended strain can be
selected as necessary. Depending on the selective marker used, a
chemical, an amino acid or the like has been added to the medium to
select the transformed bacteria of the genus Bifidobacterium.
[0136] For example, B. longum BK25 or BK51 strain is cultured
preferably in a Briggs medium having the following composition:
1 Briggs medium Tomato juice extract (*1) 400 ml Glucose 20 g
Soluble starch 0.5 g Yeast extract 6 g Peptone 15 g Monosodium
glutamate 2 g Tween 80 1 g Sodium acetate .multidot. 3H.sub.2O 10 g
Potassium dihydrogen phosphate 4 g Sodium chloride 5 g Distilled
water 600 ml pH 6.8 (*1) A product obtained by mixing a commercial
tomato juice with an equal volume of distilled water, keeping it at
60.degree. C. for 1 hour and then at 100.degree. C. for 5 minutes,
and removing residues therefrom.
[0137]
2 B. longum SBT0595 strain is cultured preferably in a TGAM medium
having the following composition: Composition of the TGAM medium
Tomato juice extract 400 ml Peptone 10 g Yeast extract 5 g Liver
extract powder 1.2 g Glucose 3 g Soluble starch 5 g Sodium chloride
3 g Tween 80 1 g L-cysteine-HCl .multidot. H.sub.2O 0.3 g Soybean
peptone 3 g Proteose peptone 10 g Digested serum powder 13.5 g Meat
extract 2.2 g
[0138] For example, B. longum 105-A or 108-A strain is cultured
preferably in the Briggs medium having the same composition as
described above except that glucose is exchanged with 2% lactose,
0.2 g/L L-cysteine and 3.4 g/L sodium ascorbate are added
thereto.
[0139] B. longum MO9101, MO9102 and MO9103 strains are cultured
preferably in GAM bouillon liquid medium (Nissui Seiyaku Co.,
Ltd.).
[0140] The above bacteria belonging to the genus Bifidobacterium
can be cultured in the following manner. If the bacteria are
cultured in the liquid medium described above, a sufficient amount
of the bacteria belonging to the genus Bifidobacterium are
inoculated into the liquid medium, and they are cultured under
anaerobic conditions at about 30 to 40.degree. C., preferably about
37.degree. C. for about 12 hours or more, preferably until their
growth reaches the middle phase of logarithmic growth. The aerobic
conditions are those conditions achieved in a completely airtight
vessel (e.g. an anaerobic chamber or an anaerobic box) capable of
keeping the anaerobic degree at which the bacteria belonging to the
genus Bifidobacterium can grow.
[0141] The above-described transformed bacteria of the genus
Bifidobacterium can grow only in tumor tissues under anaerobic
conditions, to express the substance having an antitumor activity
or the converting enzyme in the tumor tissues. Accordingly, such
transformants of the genus Bifidobacterium can be used as a
pharmaceutical composition effective for treating tumors preferably
solid tumors under anaerobic conditions.
[0142] The administration route of the pharmaceutical composition
of the present invention includes, but is not limited to, oral or
parenteral administration, preferably parenteral administration.
The parenteral administration includes administration into the
respiratory tract or rectum, or subcutaneous, intramuscular or
intravenous administration.
[0143] Examples of the pharmaceutical composition suitable for oral
administration include e.g. tablets, granules, finely divided
agents, powders, syrups, solutions, capsules and suspensions, while
examples of the pharmaceutical composition suitable for parenteral
administration include e.g. injections, drip infusions,
inhalations, sprays, suppositories, and agents absorbed through the
skin or mucous membrane, etc.
[0144] The pharmaceutical composition of the present invention is
used preferably as an injection, particularly as an intravenous
injection.
[0145] The transformed bacteria of the genus Bifidobacterium
described above may be subjected to post-treatment known in the
art.
[0146] The transformed bacteria may be purified in a crude form by
e.g. centrifugation. Also, the transformed bacteria may be purified
in a crude form and then dissolved or suspended in conventionally
used solvent such as physiological saline, PBS (phosphate-buffered
saline) or a Ringer's solution blended with lactic acid.
[0147] If desired, the bacteria may be lyophilized or spray-dried
to form powders or particles.
[0148] As the pharmaceutical composition of the present invention,
the solution, the suspension or the granular or powdery dried
product of the transformed bacteria of the genus Bifidobacterium
may be administered as such. However, it is generally desired to
administer a pharmaceutical composition containing the
above-described substance as the active ingredient and one or more
additives for the pharmaceutical composition.
[0149] Such a pharmaceutical composition can be produced in a known
method or a conventional method in pharmacology.
[0150] For production of the liquid pharmaceutical compositions
suitable for oral administration, it is possible to employ water
and pharmaceutical additives e.g. sugars such as sucrose, sorbitol,
fruit sugar etc.; glycols such as polyethylene glycol, propylene
glycol etc.; oils such as sesame oil, olive oil, soybean oil etc.;
and preservatives such as p-hydroxybenzoates.
[0151] For production of solid pharmaceutical compositions such as
capsules, tablets, powders and granules, it is possible to employ
e.g. fillers such as lactose, glucose, sucrose and mannitol;
disintegrating agents such as starch and sodium alginate;
lubricants such as magnesium stearate and talc; binders such as
polyvinyl alcohol, hydroxypropyl cellulose and gelatin; surfactants
such as fatty esters; and plasticizers such as glycerin.
[0152] Among those pharmaceutical compositions suitable for
parenteral administration, the compositions for administration into
blood vessels, for example injections and drip infusions can be
prepared preferably using an aqueous medium isotonic to human
blood.
[0153] For example, the injections can be prepared in a usual
manner as solution, suspension or dispersion by using an aqueous
medium selected from a salt solution, a glucose solution and a
mixture of a salt solution and a glucose solution, along with
suitable auxiliary agents.
[0154] The suppositories for administration into intestines can be
prepared using carriers such as cacao fat, hydrogenated fats or
hydrogenated carboxylic acids.
[0155] The sprays can be prepared using carriers which do not
irritate the oral cavity or the mucous membrane in the respiratory
tract mucus in humans and can disperse the active ingredient of the
present invention i.e. the bacteria of the genus Bifidobacterium
into fine particles thereby promoting absorption thereof. Such
carriers include e.g. lactose and glycerin. Depending on the
properties of the present bacteria of the genus Bifidobacterium and
the carriers used, the pharmaceutical composition can be prepared
in the form of aerosol or dry powder.
[0156] For production of the parenteral pharmaceutical
compositions, it is possible to use one or more pharmaceutical
additives selected from diluents, perfumes, preservatives, fillers,
disintegrating agents, lubricants, binders, surfactants and
plasticizers.
[0157] The form of the pharmaceutical composition of the present
invention as well as the process for producing the same is not
limited to those exemplified above.
[0158] The dose of the pharmaceutical composition of the present
invention and the frequency of administration thereof are not
particularly limited and can be selected as necessary depending on
various conditions such as the type of the gene possessed by the
bacteria of the genus Bifidobacterium, the type of the morbid state
to be treated, the administration route, the age and body weight of
the patient, the symptoms, and the severeness of the disease. For
e.g. systemic administration thereof by intravenous injection,
about 2.times.10.sup.6 to 2.times.10.sup.7 bacteria/body are
administered daily to an adult, and for topical administration
thereof into tumors, about 5.times.10.sup.8 bacteria are
administered preferably per tumor. However, the dose is not limited
to this specific example.
[0159] The pharmaceutical composition according to the present
invention can be applied to tumors under anaerobic conditions,
preferably various solid tumors. The solid tumors include e.g.
colon (large intestine) cancer, cerebral tumor, head cervical
cancer, breast cancer, lung cancer, esophagus cancer, stomach
cancer, hepatic cancer, cholecystic cancer, bile-duct cancer,
pancreatic cancer, Langerhans islet cancer, chorionic cancer, colon
cancer, renal cell cancer, adrenal cortical cancer, bladder cancer,
testicle cancer, prostate cancer, testicle tumor, ovary cancer,
uterine cancer, chorionic cancer, thyroid cancer, malignant
carcinoid tumor, skin cancer, malignant melanoma, osteosarcoma,
soft-part tissue sarcoma, neuroblastoma, Wilms' tumor,
retinoblastoma, melanoma, cancroid etc.
[0160] The pharmaceutical composition of the present invention may
be used in combination with other pharmaceutical compositions.
[0161] If the bacteria of the genus Bifidobacterium having the gene
coding for the converting enzyme introduced therein are
administered, it is essential to administer a precursor of an
antitumor substance. However, both the precursor of an antitumor
substance and the bacteria of the genus Bifidobacterium having the
gene coding for the converting enzyme introduced therein may
constitute one pharmaceutical composition or may be administered
separately at the same time or after a predetermined period.
[0162] Further, 20% lacturose is preferably used in combination.
Lacturose is a nutrient source for the bacteria of the genus
Bifidobacterium and cannot be metabolized by humans, mice and pigs
so that by administering lacturose, the number of bacteria of the
genus Bifidobacterium is increased specifically in tumor
tissues.
[0163] The dose is preferably about 24 to 48 g/day for an adult,
and the frequency of administration is not particularly
limited.
[0164] Further, the pharmaceutical composition of the present
invention can be used in combination with other antitumor agents.
Generally, it is used preferably in combination with several kinds
of antitumor agents which are different in the working
mechanism.
[0165] The other antitumor agents include alkylating agents,
various antimetabolites, antitumor antibiotics, other antitumor
agents, antitumor plant components, BRM (biological response
metabolite), angiogenesis inhibitors, cell adhesion inhibitors,
matrix metalloprotease inhibitors, hormones, vitamins,
antimicrobial antibiotics and chemotherapeutic agents.
[0166] Specifically, the alkylating agents include e.g. alkylating
agents such as nitrogen mustard, nitrogen mustard N-oxide and
chloram butyl; aziridine-type alkylating agents such as carboqoune
and thio-TEPA; epoxide-type alkylating agents such as
dibromomannitol and dibromodansitol; nitrosourea-type alkylating
agents such as calmstine, romstine, semstine, nimustine
hydrochloride, streptozotocin, chlorozotocin and ranimustine;
busulfan; inprosulfane tocylate and dacarbazine.
[0167] The antimetabolites include e.g. purine antimetabolites such
as 6-mercaptopurine, 6-thioguanine and thioinosine, pyrimidine
antimetabolites such as fluorouracil, tegafur, tegafur uracil,
carmofur, doxifluridine, broxuridine, cytarabine and enocitabine,
folate antimetabolites such as methotrexate and trimethoxalate, as
well as salts or complexes thereof.
[0168] The antitumor antibiotics include e.g. anthracycline-type
antibiotic antitumor agents such as mitomycin C, bleomycin,
peplomycin, daunorubicin, aclarubicin, doxorubicin, pyralbicin,
THP-adriamycin, 4'-epidoxysorbicin and epirbicin, chromomycin
A.sub.3, actinomycin D etc. as well as salts or complexes
thereof.
[0169] The other antitumor agents include e.g. cisplatin,
carboplatin, tamoxifen, camptothecine, ifosfamide,
cyclophosphamide, melphalan, L-asparaginase, acecratone,
schizophyllan, picibanil, Ubenimex, crestine etc. as well as salts
or complexes thereof. Further, procarbazine, pipobroman,
neocarzinostatin, and hydroxyurea can also be mentioned.
[0170] The antitumor plant components include e.g. vinca alkaloids
such as vindesine, vincristine and vinblastine,
epipodophyllotoxines suchasetoposide, teniposide etc., as well as
salts or complexes thereof.
[0171] The BRM includes e.g. tumor necrosis factor, indomethacin
and salts or complexes thereof.
[0172] The angiogenesis inhibitors include e.g. fumadirol
derivatives and salts or complexes thereof.
[0173] The cell adhesion inhibitors include e.g. substances having
the RGD sequence and salts or complexes thereof.
[0174] The matrix matalloprotease inhibitors include e.g.
marimastat, batimastat and salts or complexes thereof.
[0175] The hormones include e.g. hydrocortisone, dexamethasone,
methylprednisolone, prednisolone, plastelone, betamethasone,
triamcinolone, oxymetholone, nandrolone, methenolone, fosfestorol,
ethynylestradiol, chlormadinone, medroxyprogesterone etc. as well
as salts or complexes thereof.
[0176] The vitamins include e.g. vitamin C, vitamin A and salts or
complexes thereof.
[0177] The bacteria of the genus Bifidobacterium according to the
present invention administered to the patient can be easily killed
by antibiotics. This is important for further improvements in the
safety of the gene delivery system of the present invention.
EXAMPLES
[0178] Hereinafter, the present invention is described by reference
to the Examples, which however are not intended to limit the
present invention. Unless otherwise specified, DNAs etc. in the
Examples were handled according to the methods described in
Molecular Cloning, Second Edition.
Example 1
Confirmation of Accumulation and Growth of B. longum in Tumor
Tissues
[0179] (1) Preparation of a Suspension of B. longum for
Administration to Tumor-bearing Animals
[0180] A suspension of B. longum 105-A or 108A (Biosci. Biotech.
Biochem., 61, 1211 (1997)) for administration to tumor-bearing
animals was prepared in the manner described below. B. longum 105-A
can be obtained by culturing FERM BP-7274 under non-selective
conditions (in the absence of spectinomycin) in the manner
described below, then plating it onto an agar-containing modified
Briggs broth (modified Briggs broth containing 1.5% agar) to which
75 .mu.g/ml spectinomycin had been added, and obtaining it as a
strain rendered sensitive to spectinomycin by removal of a plasmid.
B. longum 105-A or 108A was inoculated into a modified Briggs
medium (a mixture of 100 parts of solution A, 10 parts of solution
B and 1 part of solution C wherein the composition of each solution
is as follows: solution A (0.5 g/l soluble starch, 6.0 g/l Bacto
Yeast extract (Difco), 15.0 g/l polypeptone, 2.0 g/l sodium
glutamate, 10.0 g/l sodium acetate.3H.sub.2O, 4.0 g/l potassium
dihydrogen phosphate, 5.0 g/l sodium chloride, 1.0 g/l Tween 80,
400 ml/l tomato juice extract (prepared by adding 400 ml water to
400 ml canned tomato juice (Delmonte), keeping it at 60.degree. C.
for 1 hour and then at 100.degree. C. for 5 minutes, adding a small
amount of High-Flow Super Cell (Wako Pure Chemical Industries,
Ltd.) and filtering it by an aspirator), adjusted to pH 6.8 with
sodium hydroxide and sterilized in an autoclave), solution B (20%
aqueous lactose solution sterilized in an autoclave) and solution C
(20.0 g/l L-cysteine, 340 g/l sodium ascorbate sterilized by
filtration)). In this broth, the bacteria were multiplied by
stationary culture under anaerobic conditions at 37.degree. C.
until their growth reached the middle phase of logarithmic growth
phase.
[0181] The resulting culture liquid was centrifuged to precipitate
the bacteria, and after the supernatant was removed, the bacteria
were suspended in PBS (phosphate buffered saline, 8 g/l sodium
chloride, 0.2 g/l potassium chloride, 1.44 g/l disodium hydrogen
phosphate, 0.24 g/l potassium dihydrogen phosphate, pH 7.4), and
the suspension was centrifuged further twice as described above, to
wash the bacteria. After the second centrifugation, the supernatant
was removed from the bacteria, which were then suspended in a 10-
or {fraction (1/50)}-fold volume of PBS relative to the volume of
the culture liquid subjected to the first centrifugation, whereby
the B. longum suspension was prepared.
[0182] (2) Administration of B. longum into Tumor-bearing
Animals
[0183] The tumor-bearing animals for administration of B. longum
were two kinds of tumor-bearing mice i.e. 6- to 8-week-old male
C57BL/6 mice (purchased from Nippon SLC) transplanted with B16-F10
melanoma cells and Luwis lung cancer cells respectively, as well as
tumor-bearing rats created by administering
7,12-dimethylbenz[a]anthracene (DMBA) to 6-week-old male
Spraque-Dawley rats (purchased from Nippon SLC).
[0184] The B16-F10 melanoma cells and Luwis lung cancer cells used
in inoculation into the mice were prepared by monolayer culture in
a Dulbecco's modified Eagle's medium (Virology, 8, 396 (1959),
Virology, 12, 185 (1960)) supplemented with 10% fetal bovine serum
at 37.degree. C. in an atmosphere of 5% CO.sub.2. These cultured
cancer cells, each 5.times.10.sup.5 cells, were inoculated into the
right thigh muscle of each C57BL/6 mouse, and in 2 weeks after this
inoculation, the mice having the solid tumors in their right thighs
were examined as the tumor-bearing mice in the B. longum injection
test.
[0185] The rats with chemically induced breast cancer were created
by administering 1 ml DABA (10 mg/ml) solution in sesame oil via a
probe into the stomach of each 6-week-old male Spraque-Dawley rat,
and one week later, an equal amount of DABA was administered again
to the rat. In 1.5 to 2 months after the second administration, the
rats having the tumor with a diameter of 5 mm were examined as the
rats with chemically induced breast cancer in the B. longum
injection test.
[0186] In administration of B. longum into the tumor-bearing
animals, 0.5 ml (5 to 6.times.10.sup.6 cells) suspension diluted
10-fold from the suspension of B. longum prepared in item (1) above
were administered once via tail veins into the whole body of mouse,
and 0.5 ml (2.times.10.sup.8 cells) suspension concentrated into a
{fraction (1/50)} volume from the suspension of B. longum prepared
in item (1) above were administered once via tail veins into the
whole body of rat, respectively.
[0187] (3) Observation of Selective Accumulation of B. longum in
Tumor Tissues and Selective Growth Thereof in the Tumor Tissues
[0188] Six to eight tumor-bearing mice to which B. longum had been
administered were sacrificed at 1, 24, 48, 72, 96, and 168 hours
respectively after injection, while 6 tumor-bearing rats were
sacrificed at 168 hours after injection, and the tumor tissues and
normal tissues were excised and each tissue extract was
anaerobically cultured in order to analyze the accumulation and
growth of B. longum in the tumor tissues and normal tissues.
[0189] The normal tissues used were obtained from the lung, liver,
spleen, kidney and heart, and the mouse tumor tissues used were
tumor tissues grown at the right tights, and the rat tumor tissues
used were breast cancer tissues. After the tissues were weighed
under aseptic conditons, the tissue extract was obtained by cutting
the tissues, mashing, homogenizing them with ice-cold PBS in a
10-fold volume relative to the tissues and filtering.
[0190] Distribution of B. longum in each of the tissues was
analyzed in the following manner. 100 .mu.l sample (containing 0.01
g tissues/100 .mu.l) prepared by diluting the tissue homogenate
prepared above was put into two Petri dishes per sample, and a
Briggs agar medium (prepared by adding 1.5% agar to the Briggs
medium) at 55.degree. C. containing 20 mg/l L-cysteine and 340 mg/l
sodium ascorbate was poured into the Petri dish, and the medium was
stirred well and then solidified by leaving it at room temperature.
Each Petri dish was placed in a completely airtight desiccator at
37.degree. C. under anaerobic conditions for 3 days, and the number
of growing B. longum colonies therein was counted to analyze the
distribution of B. longum in each tissue.
[0191] As a result, 60,000 B. longum colonies per gram of the tumor
tissues were observed in tumors of the tumor-bearing mice which
underwent inoculation of the Lewis lung cancer cells and subsequent
systemic administration of B. longum 105-A or 108-A respectively.
On the other hand, no colony was observed in the normal tissues,
that is, the lung, liver, spleen, kidney and heart after 96 hours
with B. longum 108-A and after 168 hours with B. longum 105-A (FIG.
7).
[0192] The same results as above were obtained in the case of the
tumor-bearing mice, which underwent inoculation with B16-F10
melanoma cells and subsequent administration of B. longum 105-A or
108-A.
[0193] 10,000 B. longum colonies per gram of the tumor tissues were
observed in tumors of the rats with chemically induced breast
cancer, which underwent systemic administration of B. longum 105-A,
but no colony was observed in the normal tissues, that is, the
lung, liver, spleen and kidney after 168 hours (FIG. 8).
[0194] From the results described above, it was confirmed that B.
longum was accumulated and multiplied in tumor tissues
specifically.
[0195] (4) Growth of B. longum in Tumor Tissues by Administering
Lacturose (4-0-.beta.-D-galactopyranosyl-D-fructofuranose)
[0196] Lacturose (provided by Nikken Chemicals Co., Ltd.) is a
synthetic saccharide not occurring in nature, and it is known that
lacturose cannot be metabolized by humans, mice and pigs (Biochem.
Biophys. Acta, 110, 635 (1965), Pediatrics, 32, 1033 (1963),
Gastroenterology, 47, 26 (1964), Die Nahrung, 11, 39 (1967)). On
the other hand, B. longum is capable of growing with lacturose as a
carbon source.
[0197] Accordingly, 6 to 8 mice bearing Lewis lung cancer cells to
which B. longum 105-A had been administered was intraperitoneally
given 1 ml of 20% lacturose solution for 8 successive days after
injection of B. longum, and on the 9th day, the mice were
sacrificed to analyze the number of B. longum bacteria present in
each tissue. As a result, the number of B. longum bacteria present
of the tumor tissues in the tumor-bearing mouse group given
lacturose was 200-times as more as that of the control group not
given lacturose.
[0198] From the results described above, it was demonstrated that
B. longum in tumor tissues can be selectively grown by
administration of lacturose.
Example 2
Specific Accumulation and Growth of Recombinant B. longum Having
Plasmid DNA in Tumor Tissues
[0199] (1) Preparation of Recombinant B. longum having Plasmid
DNA
[0200] According to the method described in Example 1 (1), B.
longum 105-A was cultured under anaerobic conditions and then left
at 4.degree. C. Then, the culture liquid was centrifuged to
precipitate the bacteria, and after the supernatant was removed,
the bacteria were suspended in ice-cold 10% glycerol. The operation
described above was repeated 3 times, whereby the B. longum
bacteria were sufficiently washed. After the final washing, the
supernatant was removed, and the bacteria were suspended in
ice-cold 10% glycerol in a {fraction (1/10)} volume relative to the
volume of the culture liquid subjected to the first centrifugation
and used as a bacterial sample to be subjected to transformation by
electroporation (2.times.10.sup.8 to 2.times.10.sup.9 colony
forming unit (CFU)/50 .mu.l).
[0201] The plasmid DNA used in transformation, that is, plasmid
pBLES100 (Biosci. Biotech. Biochem., 61, 1211 (1977)) can be
constructed by the method described in Biosci. Biotech. Biochem.,
61, 1211 (1997) or can be obtained by extracting the plasmid in a
usual manner from FERM BP-7274 deposited with the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, then digesting the DNA with restriction
enzyme Hind III (Takara Shuzo Co., Ltd.), treating the DNA with
phenol, precipitating it with ethanol and dissolving it in e.g.
water, followed by ligation thereof through self-cyclization
reaction with T4 DNA ligase (Takara Shuzo Co., Ltd.) according to
manufacture's instructions.
[0202] Plasmid pBLES100 used for transformation of B. longum was
prepared in the following manner. Plasmid pBLES100 obtained in the
manner as described above was used to transform E. coli JM109, and
the resulting transformant was cultured in the presence of
75.mu./ml spectinomycin. Plasmid pBLES100 was extracted in a usual
manner from the culture obtained in this culture and purified by
cesium chloride density gradient ultracentrifugation (Molecular
Cloning, Second Edition), to give plasmid pBLES100 for use in
transformation of B. longum.
[0203] 50 .mu.l of the B. longum 105-A sample and 4 .mu.l of
plasmid pBLES100 (1 .mu.g DNA/4 .mu.l) prepared above were put to
an electroporation cuvette of 0.2 cm in width (Bio-Rad), mixed and
placed on ice for 5 minutes. The cuvette was set in Gene Pulser
(Bio-Rad) and subjected to transformation by electroporation under
the conditions of 2.0 KV, 25 .mu.F capacitor and 200 .OMEGA.
parallel resistance.
[0204] After electric pulses were applied, 1 ml Briggs medium was
rapidly added to the cuvette, and after the cuvette was left at
37.degree. C. for 3 hours, the sample in the cuvette was plated
onto a Briggs agar medium plate containing 75 .mu.g/ml
spectinomycin. The plate was incubated at 37.degree. C. for 3 to 4
days under anaerobic conditions in Gas Pack Anaerobic Systems
(BBL).
[0205] A few of the resulting colonies were picked up and cultured
in the method described in Example 1 (1), and the plasmid DNA
possessed by the colonies was extracted by using QIAGEN Plasmid
Mini Kit (Qiagen) according to manufacture's instruction provided
that lysozyme was added to a P1 solution obtained by lyzing the
bacteria and then the solution was incubated at 37.degree. C. for
40 minutes.
[0206] The plasmid DNA thus extracted was digested with several
restriction enzymes and its structure was confirmed by agarose gel
electrophoresis, and it was confirmed that the colonies carried
pBLES100.
[0207] The resulting recombinant was designated B. longum
105-A/pBLES100.
[0208] (2) Administration of Recombinant B. longum into
Tumor-bearing Animals
[0209] A suspension of B. longum 105-A/pBLES100 for use in
administration into tumor-bearing animals was prepared in the same
manner as in Example 1 (1) except that 75 .mu.g/ml spectinomycin
was added to the modified Briggs medium.
[0210] The suspension was administered in the same manner as in
Example 1 (2) into tumor-bearing mice transplanted with B16-F10
melanoma cells and into tumor-bearing rats with chemically induced
breast cancer.
[0211] (3) Selective Accumulation and Growth of the Recombinant B.
longum in Tumor Tissues
[0212] Six to eight tumor-bearing mice and 6 tumor-bearing rats to
which the suspension of B. longum 105-A/pBLES100 had been
administered were sacrificed on the fourth day, and the
distribution of B. longum 105-A/pBLES100 in the tumor tissues and
normal tissues was analyzed according to the method described in
Example 1 (3). However, 75 .mu.g/ml spectinomycin was added to the
medium mixed with the tissue extract.
[0213] As a result, the tumor-bearing mice inoculated with B16-F10
melanoma cells or Luwis lung cancer cells did not indicate a
reduction in the number of cells of B. longum 105-A/pBLES100
distributed in the tumor tissues in the tumor-bearing mice given B.
longum 105-A/pBLES100, as compared with the number of cells of B.
longum distributed in the tumor tissues in the control group given
non-recombinant B. longum 105-A (FIG. 9). Further, the rats with
chemically induced breast cancer gave the same results (FIG.
8).
[0214] From the results described above, it was found that in tumor
tissues, B. longum 105-A can stably maintain plasmid pBLES100.
[0215] Those tumor-bearing mice into which non-recombinant B.
longum or B. longum 105-A/pBLES100 had been administered were
intraperitoneally given 200 mg/kg spectinomycin every day from the
next day of administration, and the mice were sacrificed on the
fourth day, and distribution of B. longum in each tissue was
analyzed.
[0216] The number of bacteria of B. longum distributed in the tumor
tissues was reduced to 1% or less by giving spectinomycin to the
tumor-bearing mice into which non-recombinant B. longum had been
administered, as compared with the control group given daily
intraperitoneally PBS in place of spectinomycin. In the
tumor-bearing mice into which B. longum 105-A/pBLES100 had been
administered, the number of bacteria of B. longum 105-A/pBLES100
distributed in the tumor tissues was kept at 81% of the number of
bacteria in the control group (FIG. 9).
[0217] From the results described above, it was confirmed that the
spectinomycin resistance gene is expressed specifically in the
tumor tissues.
Example 3
Antitumor Agent Containing Recombinant B. longum Highly Expressing
Cytosine Deaminase (CD) Gene
[0218] (1) Acquisition of the Gene Highly Expressed in B. longum
Cells
[0219] The HU gene (HU protein: histone-like DNA-binding protein,
Biochimie, 72, 207 (1990)) known as a gene highly expressed in B.
longum cells was obtained in the following manner.
[0220] B. longum ATCC15707 was cultured in the Briggs medium
according to the method described in Example 1 (1), and from the
resulting bacterium, chromosomal DNA was extracted and purified
according to the method described in Molecular Cloning, Second
Edition. 1 .mu.g of the chromosomal DNA was digested with
restriction enzyme Hind III and purified by treatment with phenol
and precipitation with ethanol. Separately, plasmid pBR322
(purchased from Takara Shuzo Co., Ltd.) was also digested with Hind
III, dephosphorylated and purified in analogous manner. 100 ng each
of the DNAs were ligated by use of T4 DNA ligase (Takara Shuzo Co.,
Ltd.) according to manufacture's instructions, to give recombinant
DNA.
[0221] Then, the recombinant DNA was used to transform E. coli mH3
(Gene, 45, 37 (1986)), to give transformants resistant to
ampicillin and sensitive to tetracycline.
[0222] From about 2000 transformants thus obtained, plasmid DNA was
extracted therefrom in a usual manner and transformed into E. coli
YK2741 (Gene, 89, 133 (1990)). The YK2741 strain is a strain
deficient in the HU gene and an IHF (integration host factor) gene
and thus sensitive to low temperatures. Accordingly, a transformant
capable of growing even at low temperatures can be a strain
carrying the HU gene derived from B. longum. Transformation was
carried out in a usual manner, and the transformants were plated
onto an ampicillin-containing agar medium and cultured at
27.degree. C., and the growing transformants were subjected to the
subsequent experiment.
[0223] Then, the transformants of YK2741 strain obtained above were
cultured, and the plasmid possessed by each transformant was
extracted by the method described above and transformed into E.
coli YK1340 (J. Mol. Biol., 204, 581 (1988)). The resulting
transformants were examined in a Mu phage transfection test
according to the method described in Molecular Cloning, Second
Edition. The YK1340 strain is a strain deficient in the HU gene,
but Mu phage necessitates the HU protein for its growth, and thus
its transformant infected with Mu phage and lyzed by growth of Mu
phage is a promising candidate for a strain carrying the HU gene
derived from B. longum.
[0224] One of the plasmids possessed by the transformants resistant
to ampicillin and infected with Mu phage and lyzed by growth of Mu
phage was designated pBLHU15, and its structure and properties were
analyzed, and said plasmid was confirmed to be a plasmid carrying
the HU gene derived from B. longum (FIG. 10).
[0225] (2) Preparation of a Plasmid Highly Expressing Cytosine
Deaminase (CD) Gene
[0226] A gene coding for CD was obtained by PCR where plasmid
pAdex1CSCD (RDB No. 1591, Gene Bank, Institute of Physical and
Chemical Research) containing a gene coding for CD derived from E.
coli was used as the template, while the DNA set forth in SEQ ID
NO: 2 and the DNA in SEQ ID NO: 3 were used as a primer set. In
PCR, 40 .mu.l reaction solution (125 ng/l template DNA, 0.5
.mu.mol/l each primer, 2.5 units Pfu DNA polymerase (Stratagene), 4
.mu.l of .times.10 buffer for Pfu DNA polymerase (Stratagene) and
200 .mu.mol/l each deoxy NTP) was subjected repeatedly 30 times to
the step of reaction at 94.degree. C. for 1 min., 55.degree. C. for
1 min. and 72.degree. C. for 1 min., and then the reaction solution
was kept at 72.degree. C. for 15 minutes.
[0227] After it was confirmed by agarose gel electrophoresis of an
aliquot of the reaction solution that an about 1.3-kb fragment had
been amplified, the remainder of the reaction solution was purified
by treatment with phenol and precipitation with ethanol, and the
fragment was ligated to TOPO vector (Funakoshi) by use of T4 DNA
ligase. The recombinant DNA thus obtained by ligation was used to
transform E. coil JM109, then the plasmid DNA was extracted from
the resulting transformant, and digested with various restriction
enzyme, it was confirmed that the desired plasmid pTOPO-eCD had
been constructed carrying out agarose gel electrophoresis of the
digests. Plasmid pTOPO-eCD was digested with restriction enzymes
Nsp V (Takara Shuzo Co., Ltd.) and Hpa I (Takara Shuzo Co., Ltd.)
and then electrophoresed on agarose gel, and the about 1.3-kb DNA
fragment coding for CD was purified by Gene clean kit (Funakoshi)
according to manufacturer's instructions.
[0228] Separately, plasmid pBLHU15 obtained in Example 3 (1) was
also digested with Nsp V and Hpa I and a 6.7-kb DNA fragment was
purified.
[0229] The 1.3-kb DNA fragment and the 6.7-kb DNA fragment obtained
above were ligated by use of T4 DNA ligase to prepare a recombinant
DNA, and this recombinant DNA was used to transform E. coil JM109
in a usual manner. Some of the resulting transformants were
cultured, and the plasmid was extracted from the culture, then
digested with various restriction enzymes and analyzed by agarose
gel electrophoresis, and it was thus confirmed that the plasmid DNA
having the CD gene integrated downstream from the promoter for the
HU gene had been constructed.
[0230] The plasmid DNA was then digested with Hind III and
electrophoresed on agarose gel to separate a 3.6-kb DNA fragment
containing the HU gene and the CD-coding gene, followed by
purification thereof by the Gene Clean kit. Further, the
Escherichia-Bifidobacterium shuttle vector pBLES100 described above
was also digested with Hind III and dephosphorylated.
[0231] The 3.6-kb DNA fragment and the Hind III digest of pBLES100
obtained above were ligated by use of T4 DNA ligase to construct a
recombinant DNA, and this recombinant DNA was used to transform E.
coli JM109 in a usual manner.
[0232] A few of the transformants having spectinomycin resistance
were picked up, and the plasmid DNA possessed by the transformants
was extracted in a usual manner, then digested with various
restriction enzymes and subjected to agarose gel electrophoresis,
and it was thereby confirmed that the desired plasmid had been
constructed.
[0233] The resulting Escherichia-Bifidobacterium shuttle vector
having the CD-coding gene downstream from the promoter for the HU
gene was designated pBLES100-S-eCD (FIG. 11).
[0234] From E. coil JM109 carrying the plasmid pBLES100-S-eCD
obtained above, the plasmid for use in transformation of B. longum
was prepared by cesium chloride density gradient centrifugation
according to the method described in Example 2 (1). The plasmid
pBLES100-S-eCD thus prepared was used to transform B. longum 105-A
in the method described in Example 2 (1), and the resulting
transformant strain was designated B. longum
105-A/pBLES10-S-eCD.
[0235] The transformant B. longum 105-A/pBLES100-S-eCD has been
deposited under FERM BP-7274 under the Budapest Treaty with the
National Institute of Bioscience and Human-Technology, Agency of
Industrial Science and Technology, the Ministry of International
Trade and Industry, Higashi 1-1-3, Tsukuba City, Ibaraki Pref.
Japan (Zip Code: 305-8566) from Aug. 15, 2000.
Example 4
Antitumor Agent Containing Recombinant B. longum Highly Expressing
CD Gene
[0236] (1) Injection of B. longum 105-A/pBLES100-S-eCD into
Tumor-bearing Animals
[0237] A suspension of B. longum 105-A/pBLES100-S-eCD used for
injection to tumor-bearing mice was prepared according to the
method described in Example 2 (2).
[0238] The suspension containing 1.times.10.sup.7 bacteria was
injected topically into tumors in the thigh of each tumor-bearing
mouse transplanted with B16-F10 melanoma cells in the right
thigh.
[0239] (2) Specific Conversion of 5-fluorocytosine (5-FC) into
5-fluorouracil (5-FU) in Tumor Tissues
[0240] 500 mg/kg 5-FC was administered intraperitoneally every day
into 6 to 8 tumor-bearing mice to which B. longum
105-A/pBLES100-S-eCD had been injected in Example 4 (1), while 1 ml
of 20% lacturose solution was administered intraperitoneally every
day into each mice from the next day of administration of B. longum
105-A/pBLES100-S-eCD. The administration was conducted until the
tumor-bearing mice were sacrificed. Separately, 5-FC was
administered every day in the same manner as above into the control
group of tumor-bearing mice to which B. longum 105-A/pBLES100-S-eCD
was not administered.
[0241] On the 8th day after injection of B. longum
105-A/pBLES100-S-eCD, the tumor-bearing mice were sacrificed, and
the concentration of 5-FU in the tumor tissues in the thighs was
examined. For measurement of the concentration of 5-FU, the tumor
tissues to which the transformant B. longum 105-A/pBLES100-S-eCD
had been topically injected, and the tumor tissues to which the
transformant had not been injected were excised, and the
concentration of 5-FU in the tumor tissues was measured by GC-MS
method (J. Chromatography, 564, 137 (1991)) in Otsuka Assay
Laboratories.
[0242] As a result, only about 10.0 ng/g 5-FU could be detected in
the tumor tissues to which B. longum 105-A/pBLES100-S-eCD had not
injected, while 588.8 ng/g 5-FU was detected in the tumor tissues
to which B. longum 105-A/pBLES100-S-eCD had been topically
injected.
[0243] From the results described above, it was confirmed that
systemically administered 5-FC is converted into 5-FU in tumor
tissues specifically.
Industrial Applicability
[0244] The present invention provides a method of expressing a
substance having an antitumor activity or a converting enzyme in
tumor tissues specifically under anaerobic conditions by using, as
gene delivery vectors, anaerobic bacteria belonging to the genus
Bifidobacterium, some of which are domestic in human intestine and
nonpathogenic bacteria, as well as transformed or mutated bacteria
belonging to the genus Bifidobacterium for use in said method.
[0245] By use of this method in treating solid tumors, there is the
effect that selective treatment of tumors is feasible and the side
effect of a conventional chemotherapeutic agent against tumors is
relieved. Also, there is another effect that those compositons
which were effective against cancer but could not be used due to
their side effects may become usable.
[0246] Further, the present invention provides an expression vector
for high expression of a protein encoded by DNA introduced into
bacteria of the genus Bifidobacterium. Tumor tissues particularly
solid tumors under anaerobic conditions can thereby be efficiently
treated.
Sequence CWU 1
1
3 1 600 DNA Bifidobacterium longum CDS (193)..(471) 1 gctgggcgcg
gcggccatga agtggcttga caagcataat cttgtctgat tcgtctattt 60
tcaatacctt cggggaaata gatgtgaaaa cccttataaa acgcgggttt tcgcagaaac
120 atgcgctagt atcattgatg acaacatgga ctaagcaaaa gtgcttgtcc
cctgacccaa 180 gaaggatgct tt atg gca tac aac aag tct gac ctc gtt
tcg aag atc gcc 231 Met Ala Tyr Asn Lys Ser Asp Leu Val Ser Lys Ile
Ala 5 10 cag aag tcc aac ctg acc aag gct cag gcc gag gct gct gtt
aac gcc 279 Gln Lys Ser Asn Leu Thr Lys Ala Gln Ala Glu Ala Ala Val
Asn Ala 15 20 25 ttc cag gat gtg ttc gtc gag gct atg aag tcc ggc
gaa ggc ctg aag 327 Phe Gln Asp Val Phe Val Glu Ala Met Lys Ser Gly
Glu Gly Leu Lys 30 35 40 45 ctc acc ggc ctg ttc tcc gct gag cgc gtc
aag cgc ccg gct cgc acc 375 Leu Thr Gly Leu Phe Ser Ala Glu Arg Val
Lys Arg Pro Ala Arg Thr 50 55 60 ggc cgc aac ccg cgc act ggc gag
cag att gac att ccg gct tcc tac 423 Gly Arg Asn Pro Arg Thr Gly Glu
Gln Ile Asp Ile Pro Ala Ser Tyr 65 70 75 ggc gtt cgt atc tcc gct
ggc tcc ctg ctg aag aag gcc gtc acc gag 471 Gly Val Arg Ile Ser Ala
Gly Ser Leu Leu Lys Lys Ala Val Thr Glu 80 85 90 tgaccttctg
ctcgtagcga ttacttcgag cattactgac gacaaagacc ccgaccgaga 531
tggtcggggt ctttttgttg tggtgctgtg acgtgttgtc caaccgtatt attccggact
591 agttcagcg 600 2 18 DNA Artificial sequence Description of
Artificial Sequencesynthetic DNA 2 ggttcgaata acgcttta 18 3 23 DNA
Artificial sequence Description of Artificial Sequencesynthetic DNA
3 cggttaactc aacgtttgta atc 23
* * * * *
References