WO2007089051A1

WO2007089051A1 - Composition for treating cancer comprising oligonucleotide and non-toxic lps

Info

Publication number: WO2007089051A1
Application number: PCT/KR2006/000360
Authority: WO
Inventors: Bo-Young Ahn; Yang-Je Cho; Won-Il Yoo; Na-Gyong Lee; Doo-Sik Kim
Original assignee: Eyegene Inc.; Daewoong Co., Ltd.
Priority date: 2006-02-01
Filing date: 2006-02-01
Publication date: 2007-08-09
Also published as: EP1986664A4; CN101394857A; EP1986664A1

Abstract

Disclosed is a composition for treating cancer including oligodeoxynucleotides and LPS-derived non-toxic lipopolysaccharides as effective components.

Description

COMPOSITION FOR TREATING CANCER COMPRISING

OLIGONUCLEOTIDE AND NON-TOXIC LPS

TECHNICAL FIELD

The present invention relates to an anti-cancer drug using an LPS-derived

non-toxic high molecular compound (CIA05) and oligodeoxynucleotides (ODNs).

BACKGROUND ART

Methods for treating cancer, which have been progressed since 1960s, are

mainly divided into three groups: surgery, irradiation, and chemotherapy. The success

of the therapeutic methods were told by the fact that cancer mortality that had increased

suddenly up to 1973 was slow in the past upward pattern in the United States.

However, the surgery and the immunotherapy have a limitation that they have a good

prognosis if in situ cancer was interrupted at an early stage since they are limited to

topical therapies. And, the chemotherapy is successful in killing the entire cancer cells.

In this case, old and weak persons may lose their lives since normal tissues,

particularly immune tissues, of hosts, namely patients, were also damaged seriously.

Non-specific immunotherapy is the most spotlighted immunotherapy which has

been used for treating nearly all kinds of tumors alone or in combination with the

chemotherapy. By the non-specific immunotherapy, it is meant that the immunotherapy is irrespective of the kinds of the cancers by expression. And, many theories concerning its intrinsic mechanisms have been proposed until now, but the

theories remain to be studied. However, it is considered that the non-specific immunotherapy stimulates the kinetics of reticuloendothelial cells, in particular

lymphocytes, and there is a point of action in an aspect of the immunological

surveillance. Corynebacterium has been actually used as a leading material in clinical

tests, and Picibanil (OK-432) was recommended in Korea a long time ago and the

material has been already used for treating many patients. The above-mentioned

materials was mainly studied and have been on the market in Japan, and they have been

commercially available in Japan, Korea or some of Southeast Asia. A family of the

materials had been usually applied for treating cancer a very long time ago. Already in

1968, Bush Fehleison et al. (Germany) found that cancer, which was developed in

patients suffering from erysipelas, was stopped or reduced by the use of the family of

the materials. In 1891, the American surgeon Coley (Chicago, USA) reported that

so-called Coley' s mixed toxin was manufactured and used to treating many cancer

patients, and the toxin had an efficiency to the significantly numerous patients. The

Coley' s mixed toxin is composed of substances extracted from the Streptococci medium

obtained by culturing Streptococci.

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

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 as 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 anticancer effect of bacterial DNA depends on its structural factors, etc.

However, it was reported that such a role of the unmethylated CpG is not

necessary in DNA anti-cancer drugs, and therefore various methods remain to be

developed as an alternative of the above method.

It has been known that LPS is a typical thymus-independent antigen which

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, a problem is that 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 QF 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 substance capable

of inducing more stable, effective and specific immune responses than conventional

therapeutic agents. In order to accomplish the above object, the present invention provides an

anti-cancer drug, including oligodeoxynucleotides (ODNs) and bacterial LPS-derived

non-toxic high molecular substances.

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 (Da).

Also in the present invention, the ODNs and the bacterial LPS-derived non-toxic

compound may be used if they are mixed at a minimum content 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.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of preferred embodiments of

the present invention will be more fully described in the following detailed description, taken accompanying drawings. In the drawings:

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 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. 4 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. 5 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, ni7909 represents

cytosine-methylated ODN 7909.

FIG. 6 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. 7 is a graph showing an anticancer effect of the ODN by the CIA05 in a

mouse model. BEST MODES FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in

detail with reference to the accompanying drawings.

The inventors designed a bacterial LPS-derived non-toxic high-molecular

material (CIA05) as the anti-cancer adjuvant, and confirmed that the oligonucleotides

are effectively used as the adjuvant. Especially, they found that the oligonucleotides

show no effect if it is used alone, but the oligonucleotides exhibit the effect as described

above if they are used in combination with the high-molecular material (CIA05).

Binding of DNA to conventional lipopolysaccharides allows the

lipopolysaccharides to participate in various reactions, for example by acting 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, the CIA05

show no specific toxicity even if 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 means of 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 (Korea).

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 enhanced efficiency.

The non-limiting embodiments of the present invention will be described in

more detail. See PCT05-073.

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 2μi 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 ofRNase (10 mgM), and then reacted at 37 ^°C in an incubator for 1 hours.

Then, 100 μJt of Proteinase K (20 mg/ral) 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 ⁰C 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 02 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 ml 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 (Fig. 2). It

was confirmed that the lipopolysaccharide has a molecular weight of about 3,000 to

about 10,000 daltons, which is very smaller than the general E. coli lipopolysaccharides.

Example 4: Removal of Toxicity of Lipopolvsaeeharide Purified from

Mutant E. coli

Removal of Toxicity by Degradation of Lipid A in Lipopolvsaeeharide The purified E. coli lipopolysaccharide was adjusted to a concentration of 3 mg/m£, 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 ⁰C every 10 minutes

- 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.

- A 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 silver staining, it was revealed that Lipid A of the

lipopolysaccharide was degraded by the alkaline treatment, and therefore was smaller

than the untreated lipopolysaccharide (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. 3).

- 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/\ 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 ⁰C resolution, was used to measure their body

temperatures. Syringes and needles, previously sterilized by heating at 250 ⁰C 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 the 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

Example 5; Mix of Oligodeoxynucleotide (ODN) and Non-toxic

Lipopolysaccharide-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

βglnt of gentamycin) at a mixing ratio of 1:1. 20 μl of CIA07 (50 μg of CIA02 + \μg or 500 ng of CIA05, 100 ng) or 20 μi of HBSS were added to 1 mi 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,

DY1240) using a commercially-available ELISA kit. The results are shown in Figs. 3

to 6.

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. 4). 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. 5). 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. 6). Measurement of Anti-cancer Effect of CIA using Mouse Model System

- A MBT-2 cell line was used to determine an effect of ODN + CIA05 on

treatment of bladder cancer in C3H/HeJ mice (Female, 4 weeks old, 19-2Ig) which

suffer from a bladder cancer. The C3H/HeJ mice were treated with the ODN + CIA05

from the day after adminstration of the tumor cell line. The tumor cell line was

administered (totally 13 times) daily for one week and every 2 days for the next 2 weeks.

The tumor cell line was administered into right flanks of the C3H/HeJ mice, and a

subcutaneous injection was used for the route of administration. The effect of ODN +

CIA05 on the treatment of the bladder cancer was determined by measuring a

perpendicular diameter of a cancerous lesion and comparing mortalities of the

experimental groups.

The detailed experiment was described, as follows. The C3H/HeJ mice were

selected and adapted to an experimental environment for one week, and then the MBT-2

bladder cancer cell line was administered subcutaneously into right flanks of the

C3H/HeJ mice at a density of 5 X 10⁵ cell/100 ul at the time point of Day 0. The

CIA07 was administered subcutaneously into the same site that the tumor cell line was

administered, and the administration was performed daily for one week from the time

point of Day 1, and then every 2 days for the next 2 weeks. The CIA07 was

administered every 2 days for a period from Day 10 to Day 45 to measure perpendicular

diameters of lesions (FIG. 7).

As shown in FIG. 7, it was revealed that the CIA05 and the ODN exhibits a more strong anticancer effect when they are used together than when they are used alone.

In particular, it was revealed that the presence of the unmethlated CG in the ODN is not important when the CIA05 and the ODN are mixed together.

INDUSTRIAL APPLICABILITY

The bacteria-derived substance (CIA05) of the present invention may induce

more effective and specific anti-cancer functions than the conventional

oligodeoxynucleotide used alone as the therapeutic agent.

Accordingly, the E. co//-derived anti-cancer drug of the present invention may be

very useful for the industrial aspects.

Claims

What is claimed is;

1. A composition for treating cancer comprising

(a) oligodeoxynucleotides (ODNs); and

(b) bacterial LPS-derived non-toxic high molecular substances.

2. The composition for treating cancer according to claim 1, wherein the

oligodeoxynucleotides are at least 20-mers in size.

3. The composition for treating cancer according to claim 1, wherein the

oligodeoxynucleotides contain CG motifs.

4. The composition for treating cancer according to claim 1, wherein the

oligodeoxynucleotides are free from a CG motif.

5. The composition for treating cancer according to claim 1 or 3, wherein

the oligodeoxynucleotides are methylated at cytosine residues.

6. The composition for treating cancer according to claim 1 or 3, wherein

the oligodeoxynucleotides are unmethylated at cytosine residues.

7. The composition for treating cancer according to any of claims 1 to 4,

wherein the oligodeoxynucleotides include a sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and

SEQ ID NO: 3.

8. The composition for treating cancer according to claim 1, wherein the

LPS-derived non-toxic high molecular substances have a molecular weight of 2,000 to

10,000 dalton (Da).

9. The composition for treating cancer according to claim 1, wherein a

weight ratio of the oligodeoxynucleotide to the LPS-derived non-toxic high molecular

substances ranges from 500:1 tol:500.

10. The composition for treating cancer according to claim 1, wherein the

bacterium is selected from the group consisting of Escherichia coli and Mycobacterium.

11. The composition for treating cancer according to claim 1, wherein the

components (a) and (b) were mixed with shaking.

12. The composition for treating cancer according to claim 1, wherein the

composition immunostimulates T helper type 1.

13. The composition for treating cancer according to claim 1, wherein the

composition stimulates NK cell activity.