WO2006121232A1 - Adjuvant comprising oligonucleotide and non toxic lps - Google Patents
Adjuvant comprising oligonucleotide and non toxic lps Download PDFInfo
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- WO2006121232A1 WO2006121232A1 PCT/KR2005/002490 KR2005002490W WO2006121232A1 WO 2006121232 A1 WO2006121232 A1 WO 2006121232A1 KR 2005002490 W KR2005002490 W KR 2005002490W WO 2006121232 A1 WO2006121232 A1 WO 2006121232A1
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- lps
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- adjuvant
- adjuvant composition
- oligodeoxynucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
Definitions
- the present invention relates to an adjuvant using a lipopolysaccharide
- LPS low-molecular compound
- ODNs oligodeoxynucleotides
- DNAs is the significant CpG suppression and the selective methylation of CpG
- c-myc mRNA and increase expression of myn, blc2 and bcl-XL mRNAs to protect the
- CpG motif directly activates B cells to facilitate secretion of IL-6 and IL- 12 within a short time.
- Clinical trials of an adjuvant and a therapeutic agent for treatment of asthma using synthetic oligonucleotides including the CpG sequences have been in
- CpG dinucleotides is not associated with an anti-cancer effect, and it is also reported
- LPS is a typical thymus-independent antigen, and known to causes side effects
- LPS has a strong toxicity as a typical endotoxin.
- binding of a general LPS to DNA may cause a serious condition such as
- the present invention is designed to solve the problems of the prior art, and therefore it is an object of the present invention to provide a material which is
- the present invention provides an
- adjuvant including oligodeoxynucleotides (ODNs) and bacterial LPS-derived non-toxic ODNs (ODNs) and bacterial LPS-derived non-toxic ODNs (ODNs)
- the non-toxic compound preferably has a molecular weight
- LPS-derived non-toxic compounds may be used if they are mixed at a minimum amount
- the two components are preferably mixed by shaking.
- the interaction of the CpG motif as described above mainly appears by inducing immunoactivation of T
- helper type 1 cells and activation of NK cells helper type 1 cells and activation of NK cells.
- the bacterium, used in the present invention is preferably Escherichia coli
- mycobacteria or mycobacteria, and more preferably Escherichia coli.
- composition is preferably used as a vaccine adjuvant, and more
- HBV vaccine adjuvant preferably as an HBV vaccine adjuvant.
- Fig. 1 is an electrophoretic diagram showing separated products of lipopolysaccharides from the outer membrane of E. coli cells. The diagram shows the
- Fig. 2 is an electrophoretic diagram showing that Lipid A is degraded by alkaline
- lane 1 represents a marker
- lane 2 represents separated products of lipopolysaccharides (CIA04)
- lane 3 represents
- Fig. 3 is a diagram showing that a molecular weight of CIA04 is measured using
- the CIA04 is dissolved in distilled water at a concentration of 5
- Axima-LNR V 2.3.5 (Mode Liner, Power: 106) from the company Shimadz is used as the MALDI-MASS.
- Fig. 4 is a diagram showing that an amount of TNF- ⁇ secreted in THP-I
- THP-I cells to secrete a large amount of TNF- ⁇ , while the non-toxic CIA05 induces
- FIG. 5 is a diagram showing, from an amount of IL- 12 expressed in human blood
- CIA05 has an effect of stimulating immune reaction regardless of whether or
- ODNs oligodeoxynucleotides
- Fig. 6 is a diagram showing, from an amount of IL- 12 expressed in human blood
- Fig. 7 is a diagram showing that immunity of the ODN having phosphorothioate
- 7909(s) represents the phosphorothioate form ODN
- Fig. 8 is a graph showing an effect of the ODN by CIA05 as a vaccine adjuvant in a mouse model. Here, it shows that binding of CIA05 to the ODN play an important role, particularly in activation of the immune cells.
- Fig. 9 is an electrophoretic diagram showing that major fractions having a low
- M represents a pre-stained marker
- lane 1 represents a treated LPS (Fraction 1)
- lane 2 represents lane 1
- lane 3 represents a treated LPS (Fraction 2)
- lane 3 represents a treated LPS (Fraction 3)
- lane 4 represents untreated LPS (20 kD).
- Fig. 10 is a graph showing, from a level of TNF- ⁇ secreted in human PBMCs,
- Fig. 11 is a graph showing, from a level of TNF- ⁇ secreted in the healthy
- the inventors designed a bacterial LPS-derived non-toxic high-molecular
- the oligonucleotide has the effect as described above if it is used alone, but the oligonucleotide has the effect as described above if it is used alone, but the oligonucleotide has the effect as described above if it is used alone, but the oligonucleotide has the effect as described above if it is used alone, but the oligonucleotide has the effect as described above if it is used alone, but the oligonucleotide has the effect as described above if it is
- Binding of DNA to general lipopolysaccharides allows the lipopolysaccharides to participate in various reactions, for example by functioning as a T cell-independent
- the inventors screened a strain (E. coli EG0021) having a very short sugar chain
- ODNs oligodeoxynucleotides
- ODN 1826 TCCATGACGTTCCTGACGTT (SEQ ID NO: 1 ; 20 mer)
- ODN 7909 TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 2; 24 mer) ODN 7909m TCmGTCmGTTTTGTCmGTTTTGTCmGTT (cytosine methylation)
- Example 1 Screening of Non-toxic Strains Screening and Finding of Mutant Escherichia Coli Strain with Very Short
- the strain E. coli EG0021 having a very short sugar chain of lipopolysaccharides having a very short sugar chain of lipopolysaccharides
- a single colony of the E. coli obtained from the healthy male adults was cultured in a liquid medium, and then a selection procedure was repeated 5 times to obtain 50 E.
- Proteinase K (20 mgM) was added thereto, and then reacted at 37 ° C overnight.
- a human lymphocyte cell line differentiated with GM-CSF was
- TNF- ⁇ was selected (Table 1), and a molecular weight of the lipopolysaccharide was
- cytocine residues of CG sequences were selectively methylated with Sss I methyalse.
- DNA methylation was carried out by mixing 1 unit of CpG methylase (M. Sss I;
- SAM S-adenosylmethionine
- the strain E. coli was prepared in the same manner as in the DNA separation.
- the strain prepared thus was mixed with 2 X volumes of ethanol, centrifuged
- lipopolysaccharides The resultant lipopolysaccharides were dissolved in ethyl ether,
- lipopolysaccharides was measured, normalized as a standard to measure its
- the lipopolysaccharide has a molecular weight of about 2,000 to about 10,000 daltons, which is very smaller than the general E. coli lipopolysaccharides (Fig. 2).
- Fig. 3 is a diagram showing that a molecular weight of CIA04 is
- the CIA04 is dissolved in distilled water at a
- G-5254 is used as a matrix.
- Axima-LNR V 2.3.5 (Mode Liner, Power: 106) from the
- the MALDI-MASS As seen from Fig. 3, it was revealed that the CIA04, as measured using the MALDI-MASS, has a molecular weight of about
- the purified E. coli lipopolysaccharide was adjusted to a concentration of 3
- THP-I acute monocytic leukemia
- thermometer ⁇ g/ 1 mi per 1 kg of a rabbit, and then each thermometer was inserted into their recta
- thermometer which can measure temperature with a 0.1 ° C
- thermometer into a rectum at the constant depth of 60 mm to 90 mm, and checking its
- the sample pre-warmed to about
- control temperature was measured.
- the body temperature was checked very 3 hours, at leased every 1 hour after injection. A difference of the measured temperatures and
- control body temperature was calculated, and the difference was referred to as a
- Venous blood was aseptically taken from healthy adult males, and put into a
- ODN oligodeoxynucleotide
- CG-free ODN (nonCG) showed a similar immune-stimulating effect to that of saline
- ODN That is, the ODN (m7909) methylated at a cytosine residue of a GC sequence
- 7909(s) is a oligodeoxynucleotide in which a diester
- - Yeast recombinant HBs antigen was used at a concentration of 219 as
- Alum hydroxide was used as the adjuvant in the control group, and CIA05 was mixed with DNA methylated at the base C of the CpG (CIA07m) and general bacterial DNA (CIA07) at a mixing ratio of 1:100 and used as the adjuvant in the
- the used animal is an ICR mouse, and each group was grouped into 6 mice, and intramuscularly injected once every a week (3 times).
- the negative control group was injected with 0.1 ml of saline injection per mouse, and the positive control group was
- the experimental group was injected with 2 ⁇ g of HBs Ag and 50 ⁇ g of
- a level of IgG against HBs antigen in the blood serum was measured using
- Example 6 Toxicity and Efficiency of LPS according to its Size LPS Lvsate with Molecular Weight of 2,000 to 10,000 Da Obtained by Lysine
- Fig. 9 is an electrophoretic diagram showing that major fractions having a low
- M represents a pre-stained marker
- lane 1 represents a treated LPS (Fraction 1)
- lane 2 represents a treated LPS (Fraction 2)
- lane 3 represents a treated LPS (Fraction 3)
- lane 4 represents untreated LPS (20 kD). As seen in Fig. 9, the major fractions having
- Human PBMCs obtained from the healthy males were put into a 24 well tissue
- Fig. 10 is a graph showing, from a level of TNF- ⁇ secreted in human PBMCs,
- Fig. 11 is a graph showing, from a level of TNF- ⁇ secreted in the healthy
- Lane 4 of the present invention showed an excellent immune-enhancing effect.
- the bacteria-derived material (CIA05) of the present invention As described abode, the bacteria-derived material (CIA05) of the present invention
- oligodeoxynucleotide is used alone, and also may induce a specific immune reaction.
- the E. co/7-derived adjuvant of the present invention may have a high
Abstract
Disclosed is an adjuvant composition of the present invention including the oligodeoxynucleotides and the LPS-derived non-toxic polysaccharides as the major components.
Description
ADJUVANT COMPRISING OLIGONUCLEOTIDE AND NON-TOXIC LPS
TECHNICAL FIELD
The present invention relates to an adjuvant using a lipopolysaccharide
(LPS)-derived non-toxic high-molecular compounds (CIA05) and oligodeoxynucleotides (ODNs).
BACKGROUND ART
Generally, one of the distinctive differences between mammalian and bacterial
DNAs is the significant CpG suppression and the selective methylation of CpG
dinucleotides at cytosine residues in the mammalian DNA. Recently, the researchers
has proposed that CpG motifs present in the bacterial DNA rapidly activate polyclonal B
cells to facilitate secretion of IgM, and the bacterial CpG motifs inhibit expression of
c-myc mRNA and increase expression of myn, blc2 and bcl-XL mRNAs to protect the
cells from being apoptosed in the B cells in which the cell cycles are stopped by
anti-IgM antibodies and apoptosis is initiated. In another study, it was reported that a
CpG motif directly activates B cells to facilitate secretion of IL-6 and IL- 12 within a short time. Clinical trials of an adjuvant and a therapeutic agent for treatment of asthma using synthetic oligonucleotides including the CpG sequences have been in
progress by the company CPG (U.S), based on the characteristics described above.
In the recent studies, it was, however, reported that cytosine methylation in the
CpG dinucleotides is not associated with an anti-cancer effect, and it is also reported
that an effect of stimulating immune reaction by bacterial DNA depends on its structural
factor, etc.
However, it was reported that such a role of the unmethylated CpG is not
necessary in DNA anti-cancer drugs, and methods which may be in place of this method
remain to be developed.
LPS is a typical thymus-independent antigen, and known to causes side effects
such as an inflammation, etc. by directly acting on B cells to induce non-specific
immune reactions. But, it was seen that LPS can use its toxicity to kill cancer cells,
and its subunit Lipid A especially shows an anti-cancer effect by inducing expression of
the various transcription factors. But, LPS has a strong toxicity as a typical endotoxin.
In addition, binding of a general LPS to DNA may cause a serious condition such as
sepsis.
DISCLOSURE OF INVENTION
Accordingly, the present invention is designed to solve the problems of the prior art, and therefore it is an object of the present invention to provide a material which is
much safer and more effective than the conventional therapeutic agents and induces
more specific immune reactions.
In order to accomplish the above object, the present invention provides an
adjuvant including oligodeoxynucleotides (ODNs) and bacterial LPS-derived non-toxic
high-molecular materials.
In the present invention, it is not important whether or not an unmethylated CG
is present in the ODNs, but the non-toxic compound preferably has a molecular weight
of about 2,000 to 10,000 daltons.
Also in the present invention, a content of the ODNs and the bacterial
LPS-derived non-toxic compounds may be used if they are mixed at a minimum amount
to show the effect of the present invention. Particularly, their efficiency is increased in
the weight ratio of 500:1 to 1 :500 in a dose-dependant manner, and the range is
preferred, considering their non-toxicity, economical efficiency, etc.
Also, the two components are preferably mixed by shaking. The interaction of the CpG motif as described above mainly appears by inducing immunoactivation of T
helper type 1 cells and activation of NK cells.
Also, the bacterium, used in the present invention, is preferably Escherichia coli
or mycobacteria, and more preferably Escherichia coli.
Also, the composition is preferably used as a vaccine adjuvant, and more
preferably as an HBV vaccine adjuvant.
BRIEF DESCRIPTION OF THE DRAWINGS Other objects and aspects of the present invention will become apparent from the
following description of embodiments with reference to the accompanying drawings in
which:
Fig. 1 is an electrophoretic diagram showing separated products of lipopolysaccharides from the outer membrane of E. coli cells. The diagram shows the
separated products of the lipopolysaccharides in 5 batch experiments, respectively.
Fig. 2 is an electrophoretic diagram showing that Lipid A is degraded by alkaline
treatment, and its size is reduced in the separated E. coli lipopolysaccharides, and
therefore their toxicity is removed. In the diagram, lane 1 represents a marker, lane 2
represents separated products of lipopolysaccharides (CIA04), and lane 3 represents
alkaline-treated non-toxic lipopolysaccharides (CIA05).
Fig. 3 is a diagram showing that a molecular weight of CIA04 is measured using
a MALDI-MASS. The CIA04 is dissolved in distilled water at a concentration of 5
mg/ml, and used. Gentisic acid (2,5-dihydroxybenzoic acid, Sigma, G-5254) is used as
a matrix. Axima-LNR V 2.3.5 (Mode Liner, Power: 106) from the company Shimadz is used as the MALDI-MASS.
Fig. 4 is a diagram showing that an amount of TNF- α secreted in THP-I
(Acute monocytic leukemia) is measured. The control lipopolysaccharide induces the
THP-I cells to secrete a large amount of TNF- α , while the non-toxic CIA05 induces
the THP-I cells to secrete an extremely low amount of TNF- α , indicating that
inflammatory reaction by the toxicity of the lipopolysaccharide is reduced significantly. Fig. 5 is a diagram showing, from an amount of IL- 12 expressed in human blood
cells, that CIA05 has an effect of stimulating immune reaction regardless of whether or
not a GC sequence is present in the oligodeoxynucleotides (ODNs).
Fig. 6 is a diagram showing, from an amount of IL- 12 expressed in human blood
cells, that improved DNA anti-cancer efficiency of CIA05 is not associated with the
unmethylated CG by means of the CG methylation. Here, m7909 represents
cytosine-methylated ODN 7909. Fig. 7 is a diagram showing that immunity of the ODN having phosphorothioate
is improved by CIA05. Here, 7909(s) represents the phosphorothioate form ODN
7909.
Fig. 8 is a graph showing an effect of the ODN by CIA05 as a vaccine adjuvant
in a mouse model. Here, it shows that binding of CIA05 to the ODN play an important role, particularly in activation of the immune cells.
Fig. 9 is an electrophoretic diagram showing that major fractions having a low
molecular weight, obtained from LPS lysate by a gel filtration using a sephacryl
S-200HR (Pharmacia), are observed on SDS-PAGE. At this time, 14 % tris-glycine
gel is used and silver-stained. Then, it was confirmed that Fractions 2 and 3 used in
this experiment have a molecular weight of less than 10,000 daltons. The diagram
shows the SDS-PAGE of LPS and its cleaved derivatives from E. coli. In the diagram,
M represents a pre-stained marker, lane 1 represents a treated LPS (Fraction 1), lane 2
represents a treated LPS (Fraction 2), lane 3 represents a treated LPS (Fraction 3), and
lane 4 represents untreated LPS (20 kD).
Fig. 10 is a graph showing, from a level of TNF- α secreted in human PBMCs,
that toxicity of LPS is varied according to its size. In the graph, "1" represents saline,
"2" represents LPS (20 kD Sigma L2880), "3" represents lysed LPS (5 kD to 10 kD), "4"
represents CIA05 (3.5 kD), and "5" represents MPL (2 kD LPS, Sigma L6638).
Fig. 11 is a graph showing, from a level of TNF- α secreted in the healthy
male's venous blood, that an immune-enhancing effect of LPS is varied according to its size. In the graph, "1" represents saline, "2" represents LPS (20 kD Sigma L2880), "3"
represents lysed LPS (5 kD to 10 kD), "4" represents CIA05 (3.5 kD), and "5" represents
MPL (2 kD LPS, Sigma L6638).
BEST MODES FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described in
detail referring to the accompanying drawings.
The inventors designed a bacterial LPS-derived non-toxic high-molecular
material (CIA05) as the adjuvant, and confirmed that the high-molecular material
(CIA05) is effectively used as the adjuvant. Especially, an oligonucleotide shows no
effect if it is used alone, but the oligonucleotide has the effect as described above if it is
used in combination with the high-molecular material (CIA05).
Binding of DNA to general lipopolysaccharides allows the lipopolysaccharides to participate in various reactions, for example by functioning as a T cell-independent
antigen in various sites of the immune system, and therefore their synergic effect may
cause serious conditions such as sepsis. However, CIA05 show no specific toxicity
even though it is used in combination with DNA.
The inventors screened a strain (E. coli EG0021) having a very short sugar chain
of lipopolysaccharide from Escherichia coli living in the bowls of healthy humans and
deposited the strain E. coli EG0021 to the Korean Culture Center of Microorganisms
(KCCM) at 361-221 Hongje-dong, Seodaemun-gu, Seoul, on May 2, 2002, and its accession number was KCCM 10374. And, there was established a method for
purifying lipopolysaccharides from the strain E. coli EG0021. Also, fatty acid was removed from the resultant very small LPS by alkaline treatment to obtain CIA05,
which is very safe and shows an anti-cancer effect.
The following oligodeoxynucleotides (ODNs) were synthesized, which are commercially available from the company Genotech Co. Ltd.
ODN 1826 TCCATGACGTTCCTGACGTT (SEQ ID NO: 1 ; 20 mer)
ODN 7909 TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 2; 24 mer)
ODN 7909m TCmGTCmGTTTTGTCmGTTTTGTCmGTT (cytosine methylation)
ODN 7909s TCGTCGTTTTGTCGTTTTGTCGTT (phosphorothioate)
ODN nonCG CTGGTCTTTCTGGTTTTTTTCTGG (SEQ ID NO: 3; 24 mer)
It was confirmed that a mixture of the ODN and CIA05, prepared by the method,
might show a more improved efficiency.
The present invention has been described in detail with reference to non-limiting
embodiments of the invention.
Example 1: Screening of Non-toxic Strains Screening and Finding of Mutant Escherichia Coli Strain with Very Short
Lipopolysaccharides
The strain E. coli EG0021 having a very short sugar chain of lipopolysaccharides
was found from Escherichia coli living in the bowls of healthy humans, and there was
established a method for purifying the lipopolysaccharides from the strain E. coli
EG0021.
A single colony of the E. coli obtained from the healthy male adults was cultured in a liquid medium, and then a selection procedure was repeated 5 times to obtain 50 E.
coli strains. And, each colony was taken from the 50 selected strains on the plates,
dissolved in 4 m£of 0.9 % saline, and then 1 ml, of the resultant solutions were
transferred into Eppendorf tubes and treated with 2μ£ of DNase 1, and then reacted at
37 °C in an incubator for 1 hours. After treatment with DNase 1, lysates were treated
with 50 μi of RNase (10 mg/ml), and then reacted at 37 °C in an incubator for 1 hours.
Then, 100 μi of Proteinase K (20 mgM) was added thereto, and then reacted at
37 °C overnight. A human lymphocyte cell line differentiated with GM-CSF was
treated with each LPS of the strains obtained by the procedure as described above, and a
level of the secreted TNF- α was measured, and then a strain having the lowest level of
TNF- α was selected (Table 1), and a molecular weight of the lipopolysaccharide was
confirmed on an electrophoresis. It was confirmed that generic characteristics of the
attenuated strain itself or its morphological Characteristics did not changed, but a ladder
of the lipopolysaccharide having a molecular weight of 50,000 to 100,000 daltons is
absent and the lipopolysaccharide having a molecular weight of 2,000 to 10,000 daltons
is produced mainly when its lipopolysaccharides were isolated and electrophoresed on
the SDS-PAGE (Fig. 1 ). Accordingly, this strain was named EG0021.
Table 1
Example 2; CG methylation In order to characterize functions of unmethylated CG in the oligonucleotide, the
cytocine residues of CG sequences were selectively methylated with Sss I methyalse.
DNA methylation was carried out by mixing 1 unit of CpG methylase (M. Sss I;
NEB M0226S) with 10 μg of ODN 7909, and then reacting each other at 37 °C for 12
hours. At this time, 160 μ M S-adenosylmethionine (SAM) was mixed as a methyl
donor and reacted together. After the methylation was completed, the remaining salts
and enzymes were, then, removed off using a DNA clean kit (CPG DPC60050) and a micropure EZ (Amicon 42529).
Example 3: Purification of CIA 04 from Mutant E. coli
Purification of Lipopolysaccharide from Mutant E. coli
The strain E. coli was prepared in the same manner as in the DNA separation.
The strain prepared thus was mixed with 2 X volumes of ethanol, centrifuged
at 4,000 g to precipitate a pellet, and then 1.5 X volumes of acetone was added to the
resultant pellet, mixed throughly and centrifuged at 4,000 g.
The equivalent amount of ethyl ether wad added to the resultant pellet, mixed
throughly and centrifuged at 4,000 g. The cell pellet obtained by centrifugation was
covered with an aluminum foil with holes in it, and dried, and the cell body was weighed, and then an extraction mixture (90 % Phenol : Chloroform : Petroleum ether =
2 : 5 : 8) was added at an amount of 7.5 mi per 1 g of the dried weight. The resultant
mixture was divided into glass centrifuge tubes and centrifuged at 25 °C and 3,000 rpm
(1,200 g) for 20 minutes to obtain supernatant. The resultant supernatant was kept in a hood for 12 hours to precipitate the residues, divided into glass centrifuge tubes and
centrifuged at 25 °C and 3,000 rpm (1,200 g) for 20 minutes to obtain
lipopolysaccharides. The resultant lipopolysaccharides were dissolved in ethyl ether,
and then the lipopolysaccharide solutions were transferred to Eppendorf tubes, dried in a
hood, and their dried weights were measured using a chemical balance, and then ethanol
was added to the dried lipopolysaccharide, which was stored for the future use.
Ethanol was completely removed from the purified E. coli lipopolysaccharide stored in
ethanol, and then an amount of KDO (2-keto-3-deoxyoctonate) in the
lipopolysaccharides was measured, normalized as a standard to measure its
concentration, and separated according to their molecular weight on the SDS-PAGE,
and their molecular weight was confirmed using a silver staining method. It was
confirmed that the lipopolysaccharide has a molecular weight of about 2,000 to about 10,000 daltons, which is very smaller than the general E. coli lipopolysaccharides (Fig. 2).
Meanwhile, Fig. 3 is a diagram showing that a molecular weight of CIA04 is
measured using a MALDI-MASS. The CIA04 is dissolved in distilled water at a
concentraion of 5 rag/ml, and used. Gentisic acid (2,5-dihydroxybenzoic acid, Sigma,
G-5254) is used as a matrix. Axima-LNR V 2.3.5 (Mode Liner, Power: 106) from the
company Shimadz is used as the MALDI-MASS. As seen from Fig. 3, it was revealed that the CIA04, as measured using the MALDI-MASS, has a molecular weight of about
3,500 daltons (Fig. 3).
Example 4: Removal of Toxicity of Lipopolysaceharide Purified from
Mutant E. coli Removal of Toxicity by Degradation of Lipid A in Lipopolysaccharide
The purified E. coli lipopolysaccharide was adjusted to a concentration of 3
mg/ml, and 0.2 N NaOH was mixed with the lipopolysaccharide at a mixing ratio of 1 :1
(by volume), deacylated for 140 minutes while shaking at 60 °C every 10 minutes, and
then 1 N acetic acid was added at about 1/5 amount of the initial 0.2 N NaOH to titrate
to pH 7.0. After titration of pH, the resultant mixture was precipitated by ethanol to obtain non-toxic lipopolysaccharide.
Concentration of the non-toxic lipopolysaccharide was measured using a KDO
method, and the non-toxic lipopolysaccharide was compared with an untreated
lipopolysaccharide on the SDS-PAGE, and then its molecular weight was confirmed
using a silver staining method.
As a result of the staining, it was revealed that Lipid A of the lipopolysaccharide
was degraded by the alkaline treatment, and therefore was smaller than the untreated lipopolysaccharide (CIA04) (Fig. 2).
Confirmation on Removal of Toxicity from Non-toxic Lipopolysaccharide
A secretion test of inflammatory proteins and a pyrogen test were conducted in
order to confirm that toxicity of the LPS is reduced to at least 1/1,000 times in the
method for selecting strains that synthesize smaller LPSs, and that its toxicity is further
reduced using the alkaline treatment.
- Secretion of Inflammatory Protein
A level of TNF- α secreted in THP-I (Acute monocytic leukemia) was
measured. It was seen that a large amount of TNF- α was secreted by the control
lipopolysaccharide, while a very small amount of TNF- α was secreted by the
non-toxic LPS (CIA05), indicating that the inflammatory reactions by its toxicity was
significantly relieved (Fig. 4).
- Pyrogen Test
3 rabbits were vaccinated to check a change of temperature in their recta, as
follows. A vaccine was intravenously injected into the rabbit ears at an amount of 0.2
βg/ 1 mi per 1 kg of a rabbit, and then each thermometer was inserted into their recta
to check their abnormal changes of temperature. Rabbits with body weights of at least
1.5 kg was used in this experiment. The rabbits used in the test should be re-used
after at least 3 days. A thermometer, which can measure temperature with a 0.1 °C
resolution, was used to measure their body temperatures. Syringes and needles,
previously sterilized by heating at 250 °C for at least 30 minutes, were used. Animals
were fed only with water at a period from 16 hours before their use until the experiment
was completed. Fixation of animals was conducted as moderate as it can be. Measurement of the body temperature was carried out by inserting the
thermometer into a rectum at the constant depth of 60 mm to 90 mm, and checking its
temperature after a predetermined time. The temperature measured before injection of
the vaccine was used as a control temperature. The sample pre-warmed to about
37 °C were intravenously injected into the rabbit ears within 15 minutes after the
control temperature was measured. The body temperature was checked very 3 hours, at leased every 1 hour after injection. A difference of the measured temperatures and
the control body temperature was calculated, and the difference was referred to as a
difference of body temperature. And, the maximum difference of body temperature
was considered as an exothermic reaction of the test animal. 3 animals of a specimen were used in this experiment.
If a sum of the temperatures measured in the 3 animals is 1.3 °C or less, a
pyrogen test is considered to be "negative", while if it is 2.5 °C or more, a pyrogen test
is considered to be "positive". This experiment was repeated 3 times, and the vaccine
was suitable for this experiment since the pyrogen test was proven to be negative. The
result is listed in the following Table 2.
Table 2
Lipopolvsaccharide-Derived Polysaccharide (CIA05) and Their Activity
Efficiency Test of Mixture of ODN and CIA05
Venous blood was aseptically taken from healthy adult males, and put into a
vacuum tube including an anti-coagulant heparin. The resultant whole blood was
mixed with an RPMI 1640 medium (2 mM L-glutamine, 1 mM Sodium pyruvate, 80
μglmi of gentamycin) at a mixing ratio of 1 :1. 20 ≠ of CIA07 (50 μg of CIA02 +
\βg or 500 ng of CIA05, 100 ng) or 20 ≠ of HBSS were added to 1 ml of the whole
blood mixed with the medium together, and then incubated at 37 °C in a 5 % CO2
incubator for 24 hours. Then, a culture supernatant was collected to measure levels of
secreted TNF- α (R&D system, DY210) and secreted IL- 12 p40 (R&D system,
DYl 240) using a commercially-available ELISA kit. The results are shown in Figs. 4
to 7.
From the result as described above, it was revealed that CIA05 showed an
immune-stimulating effect regardless of whether or not a GC sequence is present in the
oligodeoxynucleotide (ODN). In particular, it was revealed that the unmethylated
CG-free ODN (nonCG) showed a similar immune-stimulating effect to that of saline
used as the control if it was used alone, but showed a strong immune-stimulating effect
if it was used in combination with CIA05 (nonCG + CIA05) (Fig. 5). Such a synergic
effect was clearly confirmed by the cytosine methylation of the GC sequence in the
ODN. That is, the ODN (m7909) methylated at a cytosine residue of a GC sequence
of 7909 ODN (7909) showed a low immune-stimulating effect if it was used alone, but
showed the nearly same strong immune-stimulating effect as in the case of the mixture of the 7909 ODN and the CIA05 (7909 + CIA05) if it was used in combination with
CIA05 (m7909 +CIA05). Accordingly, it was confirmed that the improved DNA
anti-cancer efficacy of the CIA05 was not correlated with the unmethylated CG (Fig. 6).
Also, it was revealed that the ODN including phosphorothioate also showed an
improved immunoefficiency. 7909(s) is a oligodeoxynucleotide in which a diester
bond is substituted with phosphorothioate in the 7909 ODN (Fig. 7).
Measurement of Adjuvant Effect of CIA using Mouse Model System
the antigen. Alum hydroxide was used as the adjuvant in the control group, and CIA05 was mixed with DNA methylated at the base C of the CpG (CIA07m) and general bacterial DNA (CIA07) at a mixing ratio of 1:100 and used as the adjuvant in the
experimental group.
The used animal is an ICR mouse, and each group was grouped into 6 mice, and intramuscularly injected once every a week (3 times). The negative control group was
injected with 0.1 ml of saline injection per mouse, and the positive control group was
injected with 2 βg of HBs Ag + 50 βg of the alum, dissolved in 0.1 ml of saline for 2
hours. The experimental group was injected with 2 βg of HBs Ag and 50 βg of
CIA07 or CIA07m. Finally, after 7 day of injection, whole blood was collected and centrifuged to obtain blood serum.
A level of IgG against HBs antigen in the blood serum was measured using
ELISA. As a result, it was seen that CIA07 and CIA07m showed an excellent effect on
antibody production, compared to the case where the alum was used alone, and a level
of IgG2 was especially increased, indicating that they may considerably contribute to
improving their cell-mediated immunity, which is important for development of virus or
cancer vaccines (Fig. 8).
Example 6; Toxicity and Efficiency of LPS according to its Size LPS Lvsate with Molecular Weight of 2,000 to 10,000 Da Obtained by Lysine
General LPS
A procedure where E. coli LPS (055 :B5, Sigma) is sonicated at 150 J for 2
minutes, and then kept for 1 minute was repeated 20 times. LPS lysate obtained thus
was gel-filtered using a sephacryl S-200HR (Pharmacia).
Fig. 9 is an electrophoretic diagram showing that major fractions having a low
molecular weight, obtained from LPS lysate by a gel filtration using a sephacryl S-200HR (Pharmacia), are observed on SDS-PAGE. At this time, 14 % tris-glycine gel is used and silver-stained. Then, it was confirmed that Fractions 2 and 3 used in
this experiment have a molecular weight of less than 10,000 daltons. The diagram
shows the SDS-PAGE of LPS and its cleaved derivatives from E. coli. In the diagram,
M represents a pre-stained marker, lane 1 represents a treated LPS (Fraction 1), lane 2 represents a treated LPS (Fraction 2), lane 3 represents a treated LPS (Fraction 3), and
lane 4 represents untreated LPS (20 kD). As seen in Fig. 9, the major fractions having
a low molecular weight were confirmed on SDS-PAGE.
Toxicity Test of LPS Lysate
Human PBMCs obtained from the healthy males were put into a 24 well tissue
culture plate, and RPMI 1640 + 10 % FBS was added at a concentration of 5 X 105 per 1
m£/well. The resultant mixture was treated with BSS or test materials, incubated for 12
hours, treated with 100 μi of BSS (Balanced salt solution) and 1 //g/100 μi of LPS,
and then a level of TNF- α secreted by PBMCs was quantified using ELISA (R&D
system, D Y210).
Fig. 10 is a graph showing, from a level of TNF- α secreted in human PBMCs,
that toxicity of LPS is varied according to its size. In the graph, " 1 " represents saline,
"2" represents LPS (20 kD Sigma L2880), "3" represents lysed LPS (5 kD to 10 kD), "4"
represents CIA05 (3.5 kD), and "5" represents MPL (2 kD LPS, Sigma L6638). As seen in Fig. 10, it was revealed that LPS with the low molecular weight, for example
CIA05 of Lane 3, etc., showed a low toxicity.
Immune-Enhancing Test on LPS Lysate
Whole blood, aseptically taken from healthy adult males and put into a vacuum tube including an anti-coagulant heparin, was mixed with an RPMI 1640 medium at a
mixing ratio of 1 : 1. 1 ml of the whole blood mixed with the medium was added to a
24 well plate, respectively, treated with LPS, incubated at 37 °C in a 5 % CO2
incubator for 12 hours, centrifuged to obtain supernatant, and then a level of IFN- γ
was measured from the resultant supernatant using an ELISA kit (R&D system IFN- Y ;
DY285).
Fig. 11 is a graph showing, from a level of TNF- α secreted in the healthy
male's venous blood, that an immune-enhancing effect of LPS is varied according to its
size. In the graph, " 1 " represents saline, "2" represents LPS (20 kD Sigma L2880), "3 "
represents lysed LPS (5 kD to 10 kD), "4" represents CIA05 (3.5 kD), and "5" represents
MPL (2 kD LPS, Sigma L6638). As seen in Fig. 11, it was revealed that CIA05 of
Lane 4 of the present invention showed an excellent immune-enhancing effect.
INDUSTRIAL APPLICABILITY
As described abode, the bacteria-derived material (CIA05) of the present
invention is significantly effective when compared to the case where the conventional
oligodeoxynucleotide is used alone, and also may induce a specific immune reaction.
Accordingly, the E. co/7-derived adjuvant of the present invention may have a high
industrial value.
Claims
1. An adjuvant composition comprising:
(a) oligodeoxynucleotides (ODNs); and (b) (b) bacterial LPS-derived non-toxic high-molecular materials.
2. The adjuvant composition according to claim 1, wherein the
oligodeoxynucleotides are at least 20-mers in size.
3. The adjuvant composition according to claim 1, wherein the
oligodeoxynucleotides comprise CG motifs.
4. The adjuvant composition according to claim 1, wherein the
oligodeoxynucleotides do not comprise a CG motif.
5. The adjuvant composition according to claim 1 or 3, wherein the
oligodeoxynucleotides are methylated at cytosine residues.
6. The adjuvant composition according to claim 1 or 3, wherein the
oligodeoxynucleotides are not methylated at a cytosine residue.
7. The adjuvant composition according to any of claims 1 to 4, wherein the
oligodeoxynucleotides comprise the nucleotide sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
8. The adjuvant composition according to claim 1, wherein the LPS-derived
non-toxic high-molecular materials have a molecular weight of 2,000 to 10,000
daltons.
9. The adjuvant composition according to claim 1 , wherein a weight ratio of
the oligodeoxynucleotides to the LPS-derived non-toxic high-molecular
materials ranges from 500:1 to 1 :500.
10. The adjuvant composition according to claim 1, wherein the bacteria is
selected from the group consisting of Escherichia coli and mycobacteria.
11. The adjuvant composition according to claim 1, wherein the component
(a) and the component (b) are mixed by shaking.
12. The adjuvant composition according to claim 1, wherein the composition
is used as a vaccine adjuvant.
13. The adjuvant composition according to claim 1, wherein the composition is used as an HBV vaccine adjuvant.
Priority Applications (4)
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AT05774237T ATE506954T1 (en) | 2005-05-10 | 2005-07-29 | ADJUVANT WITH AN OLIGONUCLEOTIDE AND NON-TOXIC LPS |
DE602005027754T DE602005027754D1 (en) | 2005-05-10 | 2005-07-29 | ADJUVANS WITH AN OLIGONUCLEOTIDE AND NON-TOXIC LPS |
EP05774237A EP1879598B1 (en) | 2005-05-10 | 2005-07-29 | Adjuvant comprising oligonucleotide and non toxic lps |
US11/920,070 US20100112013A1 (en) | 2005-05-10 | 2005-07-29 | Adjuvant comprising oligonucleotide and non-toxic lipopolysaccharide |
Applications Claiming Priority (2)
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KR1020050038771A KR100740237B1 (en) | 2005-05-10 | 2005-05-10 | Adjuvant comprising oligonucleotide and non-toxic LPS |
KR10-2005-0038771 | 2005-05-10 |
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US (1) | US20100112013A1 (en) |
EP (1) | EP1879598B1 (en) |
KR (1) | KR100740237B1 (en) |
AT (1) | ATE506954T1 (en) |
DE (1) | DE602005027754D1 (en) |
WO (1) | WO2006121232A1 (en) |
Cited By (5)
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EP1986664A1 (en) * | 2006-02-01 | 2008-11-05 | Eyegene Inc. | Composition for treating cancer comprising oligonucleotide and non-toxic lps |
WO2010147268A1 (en) | 2009-06-19 | 2010-12-23 | 아이진 주식회사 | Vaccine for cervical cancer |
CN103384532A (en) * | 2010-12-18 | 2013-11-06 | 艾金株式会社 | Vaccine for inducing an improved immune reaction |
WO2018080253A1 (en) | 2016-10-31 | 2018-05-03 | 아이진 주식회사 | Immunity enhancing composition including immune response regulator and cationic liposome, and use of same |
EP3533463A4 (en) * | 2016-10-31 | 2020-06-17 | Eyegene Inc. | Immune response regulatory substance and vaccine composition containing same |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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KR101452830B1 (en) | 2012-04-06 | 2014-10-21 | 아이진 주식회사 | E. coli strain for Providing Two Anti-tumor Substances |
KR20140060053A (en) * | 2012-11-09 | 2014-05-19 | 아이진 주식회사 | Immunity reinforcement agent for influenza vaccine |
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WO2004039413A1 (en) * | 2002-05-22 | 2004-05-13 | Eyegene Inc | Immune stimulating and controlling composition comprising bacterial chromosomal dna fragments and non-toxic lipopolysaccharides |
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US7585847B2 (en) * | 2000-02-03 | 2009-09-08 | Coley Pharmaceutical Group, Inc. | Immunostimulatory nucleic acids for the treatment of asthma and allergy |
GB0025577D0 (en) * | 2000-10-18 | 2000-12-06 | Smithkline Beecham Biolog | Vaccine |
CA2388049A1 (en) * | 2002-05-30 | 2003-11-30 | Immunotech S.A. | Immunostimulatory oligonucleotides and uses thereof |
-
2005
- 2005-05-10 KR KR1020050038771A patent/KR100740237B1/en active IP Right Grant
- 2005-07-29 AT AT05774237T patent/ATE506954T1/en not_active IP Right Cessation
- 2005-07-29 EP EP05774237A patent/EP1879598B1/en active Active
- 2005-07-29 US US11/920,070 patent/US20100112013A1/en not_active Abandoned
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Patent Citations (1)
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WO2004039413A1 (en) * | 2002-05-22 | 2004-05-13 | Eyegene Inc | Immune stimulating and controlling composition comprising bacterial chromosomal dna fragments and non-toxic lipopolysaccharides |
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Also Published As
Publication number | Publication date |
---|---|
ATE506954T1 (en) | 2011-05-15 |
EP1879598A1 (en) | 2008-01-23 |
KR20060117387A (en) | 2006-11-17 |
EP1879598A4 (en) | 2009-07-22 |
DE602005027754D1 (en) | 2011-06-09 |
EP1879598B1 (en) | 2011-04-27 |
US20100112013A1 (en) | 2010-05-06 |
KR100740237B1 (en) | 2007-07-18 |
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