KR20140107954A - Composition for preventing and treating cancer comprising attenuated bacteria and IL-1? As an active ingredient - Google Patents
Composition for preventing and treating cancer comprising attenuated bacteria and IL-1? As an active ingredient Download PDFInfo
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- KR20140107954A KR20140107954A KR1020130022219A KR20130022219A KR20140107954A KR 20140107954 A KR20140107954 A KR 20140107954A KR 1020130022219 A KR1020130022219 A KR 1020130022219A KR 20130022219 A KR20130022219 A KR 20130022219A KR 20140107954 A KR20140107954 A KR 20140107954A
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Abstract
본 발명은 치료용 약독화 혐기성 박테리아의 항암 효과를 증가시키기 위해, 약독화된 살모넬라 속 균주 및 IL-1β(interleukin-1β)를 포함하는, 암 예방 및 치료용 조성물을 제공한다.The present invention provides a composition for preventing and treating cancer, which comprises attenuated Salmonella spp. And IL-1? (Interleukin-1?) To increase the anticancer effect of attenuated anaerobic bacteria for treatment.
Description
본 발명은 암 예방 및 치료용 조성물에 관한 것으로서, 더 상세하게는 약독화된 혐기성 박테리아와 IL-1β(interleukin 1-beta)를 유효성분으로 포함하는 암 예방 및 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing and treating cancer, and more particularly, to a composition for preventing and treating cancer comprising an attenuated anaerobic bacterium and IL-1? (Interleukin 1-beta) as an active ingredient.
일부 세균 종은 암 내에서 증식하고 축척되는 것을 선호하는 것으로 보고된 바 있다. 더구나, 세균은 다중의 치료용 단백질을 동시에 운반하고 발현할 수 있는 능력을 포함하는 유리한 특성들을 가지고 있고, 항생제에 의해 용이하게 제거될 수 있기 때문에, 이러한 점은 세균 치료법이 암 치료에 있어서 희망적인 새로운 전략이 될 수 있음을 시사한다(Nauts et al., Acta. Med. Scand. Suppl. 276: 1-103, 1953). 뿐만 아니라, 살아있는 약독화 또는 유전학적으로 변형된 비병원성 세균의 직접적인 암살해 효과 또는 상기 세균을 이용하여 암살해 분자를 암 조직에 전달함으로써 항암제로 사용하려는 시도가 있으며, 이러한 접근 방법은 세균이 약물이 효과적으로 도달하지 못하는 암 조직까지 이동할 수 있다는 점에서 장점을 가지고 있다(Forbes, Nat. Biotechnol., 24: 1484-1485, 2006).Some bacterial species have been reported to prefer proliferation and accumulation in cancer. Moreover, since bacteria have advantageous properties including the ability to simultaneously carry and express multiple therapeutic proteins, and because they can be easily removed by antibiotics, this suggests that bacterial therapy is promising for cancer treatment (Nauts et al., Acta. Med. Scand. Suppl . 276: 1-103, 1953). In addition, there is a direct assassination effect of living attenuated or genetically modified non-pathogenic bacteria, or an attempt to use the bacteria as an anticancer agent by assassinating and transferring molecules to cancer tissue, (Forbes, Nat. Biotechnol. , 24: 1484-1485, 2006).
그 중에서도 살모넬라 티피무리움(Salmonella typhimurium)은 운동성을 가진 침습성 그람음성 세균으로서 고형 암에서 군집화할 수 있다(Pawelek et al., Cancer Res., 57: 4537-4544, 1997). 시험관 내 조건에서는 휴지기 암세포에 의해 생성되는 주화성 화합물이 이러한 효과를 유발하는 것으로 나타났다(Kasinskas and Forbes, Biotechnol. Bioeng., 94: 710-721, 2006). 생체 내 조건에서 살모넬라는 일차적으로 영양성분이 풍부한 괴사 지역에서 군집화하는 것으로 나타났다(Forbes et al., Cancer Res., 63: 5188-93, 2003). 아울러 살모넬라는 프로드러그-변환 효소와 항원과 같은 항암 치료용 분자를 암으로 전달하는데 성공적으로 이용되어 왔다(King et al., Hum. Gene Ther., 13: 1225-1233, 2002).As Salmonella typhimurium (Salmonella typhimurium) Among them may be clustering in solid cancer as invasive Gram-negative bacteria with mobility (Pawelek et al, Cancer Res, 57:.. 4537-4544, 1997). In vitro conditions, the chemotactic compounds produced by the pausing cancer cells have been shown to induce this effect (Kasinskas and Forbes, Biotechnol. Bioeng. , 94: 710-721, 2006). In vivo, salmonella has been shown to clusters primarily in nourishment-rich necrotic areas (Forbes et al., Cancer Res. , 63: 5188-93, 2003). Salmonella has also been successfully used to deliver cancer-mediated molecules such as prodrug-converting enzymes and antigens to cancer (King et al., Hum. Gene Ther. , 13: 1225-1233, 2002).
본 발명은 약독화된 박테리아의 항암 효과가 나타나는 기저 작용을 통하여, 약독화된 박테리아 균주의 항암 효과를 현저하게 증진시키는 방법을 최초로 규명한 것으로서, 약독화된 살모넬라 균주와 IL-1β를 유효성분으로 함유하는 암 예방 및 치료용 조성물을 제공하는 것을 목적으로 한다. 그러나 이러한 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 한정되는 것은 아니다.The present invention firstly identifies a method for remarkably enhancing the anticancer effect of an attenuated bacterial strain through the basal action in which an antitumor effect of attenuated bacteria is exhibited. The attenuated Salmonella strain and IL-1? And to provide a composition for preventing and treating cancer. However, these problems are exemplary and do not limit the scope of the present invention.
본 발명의 일 관점에 따르면, 약독화된 혐기성 박테리아 및 IL-1β(interleukin-1β)를 포함하는, 암 예방 및 치료용 조성물이 제공된다.According to one aspect of the present invention, there is provided a composition for preventing and treating cancer, which comprises attenuated anaerobic bacteria and IL-1? (Interleukin-1?).
본 발명의 다른 일 관점에 따르면, 포유류의 IL-1β를 발현하도록 형질전환된 약독화 혐기성 박테리아를 유효성분으로 포함하는 암 예방 및 치료용 조성물이 제공된다.According to another aspect of the present invention, there is provided a composition for preventing and treating cancer comprising an attenuated anaerobic bacterium transformed to express a mammalian IL-1? As an active ingredient.
상기 약독화된 혐기성 박테리아는 ppGpp 합성능이 결여된 변이체일 수 있으며, ppGpp 합성을 위한 ppGpp 씬세타아제를 코딩하는 불활성화된 relA 유전자 또는 spoT 유전자를 포함할 수 있다. The attenuated anaerobic bacterium may be a mutant lacking the ability to synthesize ppGpp, and may contain an inactivated relA gene or spoT gene encoding ppGpp cytosetase for ppGpp synthesis.
상기 약독화된 혐기성 박테리아는 에스케리치아(Escherichia) 속, 살모넬라(Salmonella) 속, 클로스트리듐(Clostridium) 속, 비피도박테리움(Bifidobacterium) 속, 여시니아(Yersinia) 속, 리스테리아(Listeria) 속, 마이코플라스마(Mycoplasma) 속 또는 스트렙토코코스(Streptococcus)속 일 수 있으며, 상기 살모넬라 속 박테리아는 Salmonella typhimurium, Salmonella choleraesuis 또는 Salmonella enteritidis일 수 있다. The attenuated anaerobic bacteria Escherichia (Escherichia) genus, Salmonella (Salmonella) genus, Clostridium (Clostridium) genus Bifidobacterium (Bifidobacterium), A yeosi California (Yersinia) genus Listeria (Listeria) in , mycoplasma (mycoplasma), or in may be a genus Streptomyces Cocos (Streptococcus), the genus Salmonella bacteria may be a Salmonella typhimurium, Salmonella choleraesuis, or Salmonella enteritidis.
상기 암은 간암, 대장암, 자궁경부암, 신장암, 위암, 전립선암, 유방암, 뇌암, 폐암, 자궁암, 결장암, 방광암, 혈액암 및 췌장암으로 구성되는 군으로부터 선택되는 어느 하나일 수 있다.The cancer may be any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain cancer, lung cancer, cervical cancer, bladder cancer, blood cancer and pancreatic cancer.
또한, 상기 포유류는 인간일 수 있다.The mammal may also be a human.
이때, 본 발명의 일실시예 따른 조성물은 바람직하게는 조성물 총 중량에 대하여 상기 암 예방 및 치료용 조성물을 0.1 내지 50 중량% 로 포함할 수 있다. 또한, 추가로 암 예방 및 치료용 조성물과 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상을 함유할 수 있으나, 상기 유효성분의 함량을 초과하지 않는 것이 바람직하다. At this time, the composition according to an embodiment of the present invention may preferably contain 0.1 to 50% by weight of the composition for preventing and treating cancer, based on the total weight of the composition. In addition, the composition may further contain at least one kind of active ingredient which exhibits the same or similar function as the composition for cancer prevention and treatment, but preferably does not exceed the content of the effective ingredient.
상기 조성물은 임상 투여 시에 경구 또는 비경구로 투여가 가능하며 비경구 투여 시 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내주사, 자궁내 경막주사, 뇌혈관내 주사 또는 흉부내 주사에 의해 투여될 수 있고, 일반적인 의약품 제제의 형태로 사용될 수 있다.The composition can be administered orally or parenterally at the time of clinical administration and can be administered orally or parenterally in the case of parenteral administration by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, intracerebral injection, And may be used in the form of a general pharmaceutical preparation.
본 발명의 일 실시예에 따른 조성물은 제약상 허용되는 담체를 비롯한 불활성 성분을 추가로 포함한다. 본 명세서에서 사용된 "제약상 허용된 담체"란 조성물, 구체적으로 의약 조성물의 활성 물질을 제외한 성분을 지칭하는 용어이다. 제약상 허용되는 담체의 예로는 결합제, 붕해제, 희석제, 충진제, 활택제, 가용화제 또는 유화제 및 염이 포함된다.Compositions in accordance with one embodiment of the present invention further comprise an inert ingredient, including a pharmaceutically acceptable carrier. As used herein, the term " pharmaceutically acceptable carrier "refers to a composition, specifically a component other than the active ingredient of a pharmaceutical composition. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, lubricants, solubilizers or emulsifiers and salts.
실제 사용에 있어서, 본 발명의 일실시예에 따른 조성물은 통상적인 제약 조제 기술에 따른 제약 담체와 조합될 수 있다. 담체는, 예를 들어 경구 또는 (정맥내 투여를 비롯한) 비경구 투여에 바람직한 제조에 따라 광범위하게 다양한 형태를 지닐 수 있다. In practical use, the composition according to one embodiment of the present invention may be combined with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier may take a wide variety of forms depending upon the preparation desired, for example, for oral or parenteral administration (including intravenous administration).
아울러, 본 발명의 일 실시예에 따른 조성물은 0.1 mg/kg 내지 1 g/kg의 용량으로 투여될 수 있으며, 더 바람직하게는 0.1 mg/kg 내지 500 mg/kg의 투여량으로 투여된다. 한편, 상기 투여량은 환자의 나이, 성별 및 상태에 따라 적절히 조절될 수 있다.In addition, the composition according to one embodiment of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably 0.1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.
본 발명의 다른 관점에 따르면, 상기 조성물을 암에 걸린 개체에 투여하는 단계를 포함하는, 암 치료방법이 제공된다.According to another aspect of the present invention, there is provided a method for treating cancer, comprising administering the composition to a subject having cancer.
상기한 바와 같이 이루어진 본 발명의 일 실시예에 따르면, 약독화된 살모넬라 균주의 항암효과를 증진시키는, 암 예방 및 치료용 조성물 및 이를 이용한 치료방법을 구현할 수 있다. 물론 이러한 효과에 의해 본 발명의 범위가 한정되는 것은 아니다.According to an embodiment of the present invention as described above, a composition for preventing and treating cancer, which improves the anticancer effect of attenuated Salmonella strains, and a method of treating using the composition can be implemented. Of course, the scope of the present invention is not limited by these effects.
도 1은 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 암 동물모델에 투여한 후, 암 조직의 부피 변화(도 1a), 및 FACS 분석을 이용한 암 세포 사멸 수준의 변화(도 1b, 1c) 결과를 나타낸다.
도 2는 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 암 동물모델에 투여한 후, 생체 내(in vivo) 사이토카인 mRNA 수준의 변화(도 2a), 단백질 수준의 변화(도 2b) 및 사이토카인 분비량 변화(도 2c)를 나타낸 그래프이다.
도 3은 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 암 동물모델에 투여한 후, 조직면역염색 방법을 이용하여 암 조직을 염색한 사진(도 3a), 및 FACS 분석을 이용한 암 조직 내의 사이토카인 수준의 변화(도 1b, 1c)를 나타낸다.
도 4는 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 암 동물모델에 투여한 후, 조직면역염색 방법을 이용하여 CD68(Mac)(도 4a), CD68(Neu)(도 4b) 및 CD11c(DC)(도 4c)와 IL-1β의 이중 염색된 사진이다.
도 5는 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 암 동물모델에 투여한 후, 암성장이 억제되는 첫 10 일간의 ‘억제시기(suppression)’와 이후 암 부피가 1,500 mm3 까지 성장하는 시기를 ‘재발시기(reccurrence)’의 IL-1β와 TNF-α mRNA 수준의 변화(도 5a) 및 단백질 수준변화(도 5b)를 나타낸 결과이다.
도 6은 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주와 함께 IL-1β를 암 동물모델에 투여하여 항암효과를 확인을 위한 실험 과정(도 6a), 암 조직 부피의 변화(도 6b), 및 암 조직을 촬영한 사진(도 6c)이다. FIG. 1 is a graph showing changes in the volume of cancer tissues (FIG. 1A) and the degree of cancer cell death (FACS analysis) after administration of an attenuated StΔppGpp / lux strain according to an embodiment of the present invention 1b, 1c).
FIG. 2 is a graph showing changes in the level of cytokine mRNA in vivo (FIG. 2A), changes in protein level (FIG. 2b) and cytokine secretion amount change (Fig. 2C).
FIG. 3 is a photograph (FIG. 3A) of staining cancer tissue using a tissue immunostaining method after administration of an attenuated StΔppGpp / lux strain according to an embodiment of the present invention to a cancer animal model 1b < / RTI > and 1c) in cancer tissues.
4 (a) and 4 (b) are graphs showing the expression of CD68 (Mac) (Fig. 4A), CD68 (Neu) (Fig. 4B) using tissue immunostaining method after administering the attenuated St.DELTA.ppGpp / lux strain according to an embodiment of the present invention to a cancer animal model. ) And CD11c (DC) (Figure 4c) and IL-1 [beta].
FIG. 5 is a graph showing the results of inhibition of cancer growth after the administration of an attenuated St.DELTA.ppGpp / lux strain according to an embodiment of the present invention to a cancer animal model, 3 shows changes in IL-1β and TNF-α mRNA levels (FIG. 5A) and protein levels (FIG. 5B) of 'reccurrence'.
FIG. 6 is a graph showing an experimental procedure (FIG. 6A) for confirming the anti-cancer effect by administering IL-1β to a cancer animal model together with an attenuated StΔppGpp / lux strain according to an embodiment of the present invention 6b), and a photograph of cancer tissue (Fig. 6c).
본 문서에서 사용되는 용어를 정의하면 하기와 같다.The terms used in this document are defined as follows.
본 문서에서 사용되는 “약독화”는 미생물의 병원성을 감소시키도록 변형시키는 것을 의미한다. 약독화는 미생물을 환자에게 투여한 경우 독성 및 다른 부작용을 감소시킬 목적으로 이루어진다. 약독화 박테리아는 당업계에 공지된 다양한 방법을 통하여 제조될 수 있다. 예를 들어, 약독화는 박테리아가 숙주세포에서 생존하도록 하는 독성인자(virulence factor)를 결실시키거나 또는 파괴하여 달성된다. 상기 결실 및 파괴는 당업계에 잘 알려져 있으며, 예컨대, 상동성 재조합, 화학적 변이유발, 조사 변이유발 또는 트랜스포존 변이유발 등과 같은 방법에 의해 실시된다. 결실된 경우 약독화를 초래할 수 있는 Salmonella의 독성인자의 예는 다음과 같다: 5'-아데노신 모노포스페이트(Biochenko et al., Bull. Eksp. Biol. Med. 103: 190-192, 1987), 사이토라이신(Libby et al., Proc. Nati. Acad. Sci. USA 91: 489-493, 1994), 디펜신 내성 loci(Fields et al., Science, 243: 1059-62, 1989), DNAK(Buchmeier et al., Science 248: 730-732, 1990), 핌브리애(Ernst et al., Infect. Immun., 58: 2014-2016, 1990), GroEL(Buchmeier et al., Science 248: 730-732, 1990), Inv loci(Ginocchio et al., Proc. Natl. Acad. Sci. USA 89: 5976-5980, 1992), 리포단백질(Stone et al., J. Bacteriol. 174: 3945-3952, 1992), LPS(Gianella et al., J. Infect. Dis. 128: 69-75, 1973), PhoP 및 PhoQ(Miller et al., Proc. Natl. Acad. Sci. USA 86: 5054-5058, 1989), Pho 활성화 유전자(Abshiro et al., J. Bacteriol., 175: 3734-3743, 1993), PhoP 및 PhoQ 조절 유전자(Behlau et al., J. Bacteriol. 175: 4475-4484, 1993), 포린(Tufano et al., Eur. J. Epiderniol. 4: 110- 114, 1988), 독성인자(Loos et al., Immun. Infekt. 22: 14-19, 1994; Sansonetti, Rev. Prat. 42: 2263-2267, 1992). 약독화 박테리아에 대한 내용은 WO 96/40238에 상세하게 개시되어 있으며, 이 특허문헌은 본 명세서에 참조로서 삽입된다.As used herein, " attenuation " means modifying the pathogenicity of a microorganism to reduce it. Attenuation is done for the purpose of reducing toxicity and other side effects when the microorganism is administered to the patient. The attenuated bacteria may be produced through a variety of methods known in the art. For example, attenuation is achieved by destroying or destroying the virulence factor that causes the bacteria to survive in the host cell. Such deletions and deletions are well known in the art and are carried out by methods such as homologous recombination, chemical mutagenesis, irradiation mutagenesis or transposon mutagenesis. Examples of toxic agents of Salmonella that can lead to attenuation if deleted include: 5'-adenosine monophosphate (Biochenko et al. , Bull. Eksp. Biol. Med. 103: 190-192, 1987) (Fields et al., Science, 243: 1059-62, 1989), DNAK (Buchmeier et al., Proc. Nat . Acad. Sci . USA 91: 489-493, 1994) al., Science 248: 730-732, 1990), Pimbria (Ernst et al., Infect. Immun., 58: 2014-2016, 1990), GroEL (Buchmeier et al., Science 248: 1990), Inv loci (Ginocchio et al., Proc. Natl. Acad Sci . USA 89: 5976-5980, 1992), lipoprotein (Stone et al., J. Bacteriol. 174: 3945-3952, LPS (Gianella et al, J. Infect Dis 128:... 69-75, 1973), PhoP and PhoQ (Miller et al, Proc Natl Acad Sci USA 86:..... 5054-5058, 1989), Pho active genes (Abshiro et al, J. Bacteriol, 175:.. 3734-3743, 1993), PhoP and PhoQ regulatory genes (Behlau et al, J. Bacteriol 175 :.. 4475-4484, 1993), morpholine (Tufano et al., Eur. J. Ep 4: 110-114 , 1988), toxic factors (Loos et al., Immun. Infekt. 22: 14-19, 1994; Sansonetti, Rev. Prat . 42: 2263-2267, 1992). The contents of attenuated bacteria are described in detail in WO 96/40238, which is incorporated herein by reference.
본 문서에서 사용되는 “불활성화된 relA 유전자 및/또는 spoT 유전자”는 유전자의 전사 또는 해독, 유전자 산물의 활성의 손상(impairment)을 초래하는 relA 유전자 및/또는 spoT 유전자의 변형(modifications)을 의미한다. 이런 유전자 변형은 ppGpp 씬세타아제 코딩 서열(CDS)의 불활성화뿐만 아니라 이의 프로모터의 불활성화도 포함될 수 있다. 박테리아 지놈 상에서 목적한 유전자만의 특이적 불활성화는 코딩하는 유전자의 전체 또는 하나 이상의 부분 영역에서 치환, 삽입, 결실 또는 이들의 조합을 통하여 돌연변이시킴으로써 가능하다. 예들 들어, 유전자의 결실 및 유전자로의 이형 서열(heterogenous sequence)의 삽입은 유전자의 절단(truncation), 넌센스 돌연변이(nonsense mutation), 프레임쉬프트 돌연변이(frameshift mutation), 미스센스 돌연변이(missense mutation) 등을 초래할 수 있다. 이러한 유전자의 특이적 불활성화는 당업계에서 확립된 방법을 통하여 수행할 수 있다. 한편 유전자의 결실은 당업계에 공지된 다양한 변이유발(mutagenesis) 방법을 통하여 실시할 수 있다. 예컨대, relA 유전자 또는 spoT 유전자의 결실은 PCR 돌연변이유발법 및 카세트 돌연변이유발법으로 실시할 수 있다(Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press(2001)). ppGpp 합성 결여는 본 발명의 박테리아가 암 조직으로 타겟팅하는 데에 있어서 결정적인 기여를 한다.As used herein, the term " inactivated relA gene and / or spOT gene " refers to a modification of the relA gene and / or the spoT gene that results in transcription or translation of the gene or impairment of activity of the gene product do. Such genetic modification may include inactivation of the ppGpp cytoset sequencing (CDS) as well as inactivation of its promoter. The specific inactivation of only the gene of interest on the bacterial genome is possible by mutation through substitution, insertion, deletion, or a combination thereof in all or at least one subdomain of the coding gene. For example, deletion of a gene and insertion of a heterogenous sequence into a gene can be accomplished by truncation of a gene, nonsense mutation, frameshift mutation, missense mutation, etc. . The specific inactivation of these genes can be accomplished through methods established in the art. On the other hand, gene deletion can be carried out through various mutagenesis methods known in the art. For example, deletion of the relA gene or spoT gene can be performed by PCR mutagenesis and cassette mutagenesis (Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001) ). ppGpp synthesis deficiencies make a crucial contribution in targeting the bacteria of the present invention to cancer tissue.
이하, 실시예 및 실험예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예 및 실험예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예 및 실험예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to fully inform the owner of the scope of the invention.
실시예 1: 마우스 CT26 세포주의 배양Example 1: Culture of mouse CT26 cell line
마우스 대장암 세포주(colon adenocarcinoma) CT26은 단층으로 배양하였으며, 37℃, 5% 이산화탄소 조건하에서 10% FBS(Gibco, Invitrogen) 및 1% P/S(penicillin and streptomycin, Gibco, Invitrogen)가 포함된 cDMEM (endotoxin-free DMEM high glucose medium, Hyclone, Thermal scientific) 배지에서 배양하였다.Colon adenocarcinoma CT26 was cultured as a monolayer and cultured in a cDMEM (Invitrogen) containing 10% FBS (Gibco, Invitrogen) and 1% P / S (penicillin and streptomycin, Gibco, Invitrogen) at 37 ° C under 5% and cultured in endotoxin-free DMEM high glucose medium (Hyclone, Thermal scientific).
실시예 2: 약독화된 살모넬라 균주의 제조Example 2: Preparation of attenuated Salmonella strains
약독화된 Salmonella typhimurium 균주(StΔppGpp/lux, SHJ2168)와 lux 유전자를 미발현하는 E. coli MG1655(E. coli, not E coli-lux)를 하기 실험에 이용하였다(10. Min, J.J. et al., Mol. Imaging Biol., 10, 54-61, 2008; Yoshimura, K. et al., Cancer Res., 66, 1096-1104, 2006). 상기 E. coli MG1655는 암피실린 내성을 가지고 있어, 이를 이용하여 박테리아 위치파악 및 계수에 이용하였다. To an attenuated Salmonella typhimurium strain (StΔppGpp / lux, SHJ2168) and E. coli MG1655 (E. coli, not E coli -lux) to non-expression of the lux genes it was used in the experiment (10. Min, JJ et al. , Mol. Imaging Biol. , 10, 54-61, 2008; Yoshimura, K. et al., Cancer Res. , 66, 1096-1104, 2006). The E. coli MG1655 was resistant to ampicillin and was used for bacterial localization and counting.
상기 약독화된 Salmonella typhimurium (StΔppGpp/lux)는 relA 유전자 및 spoT 유전자를 결손시켜 ppGpp(Guanosine 5'-diphosphate 3'-diphosphate) 합성능이 결여된 것을 특징으로 하며, 구체적으로 50 μg/ml 카나마이신(Sigma)을 포함하는 LB 배지(Difco Laboratories)에서 격렬하게 공기공급을 하면서 37℃에서 S. typhimurium ΔppGpp 균주(SHJ2037, 기탁번호 KCTC10787BP)를 배양하였다. 생발광 이미징을 위하여, 상기 ΔppGpp 균주를 생발광 리포터 유전자로 형질전환시켰다. S. typhimurium-Xen26(Xenogen-Caliper)의 박테리아 루시퍼라아제 유전자(lux)를 P22HT int 트랜스덕션 방법으로 SHJ 2037 균주에 도입시킨 후, 50 μg/ml 카나마이신을 포함하는 LB 배지에서 배양하였다. 그 후, 상기 균주에 pBAD-pelB-RLuc8 플라스미드를 전기-형질전환 방식으로 도입시켰다. 암피실린(20 μg/ml)을 포함하는 LB 아가 플레이트 상에서 성장하는 콜로니를 선별하였다(S. typhimurium 균주(StΔppGpp/lux, SHJ2168)).The attenuated Salmonella typhimurium (StΔppGpp / lux) lacks the ability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) due to deletion of the relA gene and spoT gene. Specifically, 50 μg / ml kanamycin (Difco Laboratories) with 37 ° C, and incubated at 37 ° C with S. typhimurium ΔppGpp strain (SHJ2037, Accession No. KCTC10787BP) with vigorous air supply. For bioluminescence imaging, the? PpGpp strain was transformed with a bioluminescent reporter gene. The bacterial luciferase gene (lux) of S. typhimurium- Xen26 (Xenogen-Caliper) was introduced into strain SHJ 2037 by the P22HT intransfection method and then cultured in LB medium containing 50 μg / ml kanamycin. The pBAD-pelB-RLuc8 plasmid was then introduced into the strain by electro-transformation. Colonies growing on LB agar plates containing ampicillin (20 μg / ml) were selected ( S. typhimurium strain (StΔppGpp / lux, SHJ2168)).
실시예 3: 암 동물 모델의 제조Example 3: Preparation of female animal model
5-6주령 웅성 Balb/c 마우스(20 g, Samtaco, 오리엔트-찰스 리버 랩, 대한민국)를 실험에 이용하였다. 전남대학교 동물연구회에서 승인된 프로토콜 및 NIH(National Institute of Health)에서 발표한 실험동물 사육 및 이용에 대한 가이드(발표 85-23, 개정 1985)에 따라 모든 동물 사육, 실험 및 안락사를 실시하였다. 5-6 week old male Balb / c mice (20 g, Samtaco, Orient-Charles River Lab., Korea) were used for the experiments. All animal breeding, experiments and euthanasia were conducted in accordance with the protocol approved by the Animal Research Association of Chonnam National University and the Guidance on the Rehabilitation and Use of Laboratory Animals (published by the National Institute of Health) (Announcement 85-23, Rev. 1985).
상기 실시예 1에서 배양한 대장암 세포 CT26 세포 1 ×106을 마우스의 오른쪽 허벅지 피하에 이식하였으며, 이식 후 10일 경과한 후 하기 실험에서 약독화된 박테리아 또는 IL-β를 투여하는 실험을 수행하였다.1x10 < 6 > of the colon cancer cell CT26 cells cultured in Example 1 was implanted subcutaneously in the right thigh of the mouse. Ten days after the transplantation, experiments were carried out in which the attenuated bacteria or IL- Respectively.
실험예 1: 약독화된 살모넬라 균주의 항암 효과 분석Experimental Example 1: Antitumor effect analysis of attenuated Salmonella strains
1-1: 암 부피 변화1-1: Cancer volume change
약독화된 살모넬라 균주의 항암효과를 분석하기 위하여, 상기 실시예 2에서 제조한 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168) 및 lux 유전자를 발현하지 않는 E. coli MG1655를 실시예 3의 암 동물모델에 투여한 후, 이식된 암의 크기 변화를 관찰하였다. 상기 S. typhimurium 균주(StΔppGpp/lux, SHJ2168)는 4.5 × 107 세포수/100 μl PBS 용액을 정맥으로 투여하였으며, E. coli MG1655(not E coli-lux)는 5 × 107 세포수/100 μl PBS 용액을, 대조군은 100 μl PBS 용액을 1회 투여하였다. 그 후, 18일 동안 암의 부피(mm3) 변화를 캘리퍼스(caliper)를 이용하여 측정하였다. In order to analyze the anticancer effect of the attenuated Salmonella strain, the attenuated Salmonella strain (St? PpGpp / lux, SHJ2168) prepared in Example 2 and E. coli MG1655 not expressing the lux gene were cultured in the same manner as in Example 3 After administration to the model, changes in the size of the implanted cancers were observed. The S. typhimurium strain (StΔppGpp / lux, SHJ2168) is 4.5 × 10 7 cells / 100 μl PBS was administered intravenously and the solution, E. coli MG1655 (not E-coli lux) is 5 × 10 7 cells / 100 μl PBS solution, and the control group was administered with 100 μl PBS solution once. Thereafter, changes in the volume (mm < 3 >) of the cancer for 18 days were measured using a caliper.
그 결과, 도 1a에 나타난 바와 같이, 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 투여한 군은 대조군 및 E. coli MG1655 (not E coli-lux)를 투여한 군에 비하여 암의 부피가 현저하게 감소하였다(도 1a 참조).As a result, as shown in FIG. 1A, the group to which the attenuated StΔppGpp / lux strain according to one embodiment of the present invention was administered showed a significantly higher incidence of cancer than the control group and E. coli MG1655 (not E coli-lux) (See Fig. 1A).
1-2: FACS 분석1-2: FACS analysis
이어, 본 발명자는 암세포가 주입된 림프절을 분리한 후, FACS 분석을 수행하였다.Next, the present inventors performed FACS analysis after separating lymph nodes injected with cancer cells.
상기 실험예 1-1에서 박테리아를 주입한 마우스를 이용하였으며, 박테리아 주입 후 2일이 경과한 후 암을 추출하였다. 암세포가 주입된 림프절을 분리한 후, 40 μm 멸균된 스테인레스 스틸 메쉬(BD Falcon, USA)를 이용하여 균질화한 후, T 세포용 배지인 RPMI1640에 현탁하였다. 이때, 상기 RPMI1640 배지는 10% FBS, 1% 페니시린/스트렙토마이신, L-글루타민(glutamine), 피루브산나트륨(sodium pyruvate), 비필수 아미노산 및 2-메르캅토에탄올(2-mercaptoethanol)이 첨가되어 있으며, 상기 현탄액을 annexin V(early stage apoptosis marker) 및 7-ADD (late stage apoptosis marker) 마커를 이용하여 FACS 분석을 수행하였다. In Experimental Example 1-1, a mouse injected with bacteria was used. After 2 days from the injection of the bacteria, the cancer was extracted. The lymph nodes injected with cancer cells were separated, homogenized using a 40 μm sterilized stainless steel mesh (BD Falcon, USA), and suspended in RPMI 1640 medium for T cells. At this time, the RPMI 1640 medium was supplemented with 10% FBS, 1% penicillin / streptomycin, L-glutamine, sodium pyruvate, non-essential amino acid and 2-mercaptoethanol , And the FACS analysis was performed using annexin V (early stage apoptosis marker) and 7-ADD (late stage apoptosis marker) markers.
그 결과, 도 1b에 나타난 바와 같이, 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 투여한 군은 대조군 및 E. coli MG1655를 투여한 군에 비하여 암 세포 사멸이 현저하게 증가하였다. As a result, as shown in FIG. 1B, the group to which the attenuated StΔppGpp / lux strain according to one embodiment of the present invention was administered showed a significantly increased cancer cell death compared with the control group and the group administered with E. coli MG1655 .
1-3: 사멸된 암 세포 분석1-3: Analysis of killed cancer cells
이어, 본 발명자는 상기 실험예 1-2의 FACS 분석 결과를 정량화였다. Next, the present inventor quantified the FACS analysis result of Experimental Example 1-2.
그 결과, 도 1c에 나타난 바와 같이 본 발명의 일 실시예에 따른 약독화된 약독화된 StΔppGpp/lux 균주를 투여한 군에서 사멸된 암 세포가 대조군 및 대조군 및 E. coli MG1655를 투여한 군에 비하여 현저하게 높은 비율을 보였으며, 약 35% 정도 암 세포가 사멸된 것으로 나타났으며, 도 1a의 결과와 일치하였다. 즉, 상기 결과는 본 발명의 일 실시예에 따른 약독화된 살모넬라 균주의 항암효과를 입증하는 것이며, 이는 생체 내(in vivo) 암 조직의 부피 감소 및 사멸된 암 세포수의 증가에 의하여 뒷받침된다. 이하 실험예에서는 암성장이 억제되는 첫 10 일간의 ‘억제시기(suppression)’와 이후 암 부피가 1,500 mm3 까지 성장하는 시기를 ‘재발시기(reccurrence)’로 나누고 각 시기별, 균주별 cytokine을 측정하여 서로 비교하였다.As a result, as shown in FIG. 1C, in the group administered with the attenuated attenuated StΔppGpp / lux strain according to one embodiment of the present invention, the killed cancer cells were treated with the control and the control group and the group administered with E. coli MG1655 And the cancer cells were killed by about 35%, which is consistent with the result of FIG. 1A. That is, the above results demonstrate the anticancer effect of the attenuated Salmonella strain according to one embodiment of the present invention, which is supported by the reduction of the volume of cancer tissue in vivo and the increase in the number of killed cancer cells . In the following examples, the 'suppression' during the first 10 days in which cancer growth is inhibited and the period of growth of cancer volume up to 1,500 mm 3 are divided into 'reccurrence' And compared with each other.
실험예 2: 사이토카인 분석Experimental Example 2: Cytokine analysis
본 발명의 일 실시예에 따른 약독화된 박테리아에 의하여 생체 내 사이토카인(cytokine) 변화를 분석을 통하여, 암 세포를 억제할 수 있는 특이적인 사이토카인의 분비가 증가하는지의 여부를 확인하였다. Through analysis of cytokine changes in vivo by attenuated bacteria according to an embodiment of the present invention, it was confirmed whether the secretion of specific cytokines capable of inhibiting cancer cells is increased.
2-1: RT-PCR 분석을 이용한 사이토카인 수준 분석2-1: Analysis of cytokine levels by RT-PCR analysis
상기 실시예 3의 암 동물모델에 살모넬라 균주(StΔppGpp/lux, SHJ2168) 및 E. coli MG1655를 감염시킨 이후, 2일이 경과한 시점(억제 단계(suppression stage))에 암 조직을 멸균된 도구를 이용하여 마우스로부터 분리하였다. 피부와 이를 둘러싼 지질을 제거하여 감염과 암 이외의 조직이 혼합되는 것을 억제하였다. 조직을 분리한 즉시, 조직을 분해(dissociation)였다. 상기 조직의 분해는 효소를 이용하여 수행하였으며, 이는 Invitrogen Laboratories의 ‘Dissociation of cells from primary tissue’에 따라서 수행하였다. 간략하게 정리하면, 암 조직을 멸균된 PBS를 이용하여 세척한 후, 2~3 mm3 부피로 다졌다. 그 후, 상기 조직을 25% 트립신(Gibco, Invitrogen, USA)과 반응시켰으며, 조직 1g당 효소 10 ml의 비율로 반응시켰으며, 효소가 암 조직 내에 침투하는 것을 용이하게 하며 효소의 활성을 극대화하기 위해서 4℃에서 2시간 동안 반응시켰다. 그 후, 상기 효소처리된 암 조직을 세포 배양기(37℃, 5% 이산화탄소)에서 20분 동안 반응시킨 후, 상층액을 제거하고 cDMEM을 첨가하여 효소반응을 중단시켰다. 그 후, 상기 암 조직은 조심스럽게 분쇄한 후, 멸균된 스테인레스 스틸 메쉬(BD Falcon, USA) 100 μm를 이용하여 여과하여 단일 세포 혼합물을 분리하였다.The cancer tissues of Example 3 were infected with Salmonella strains (StΔppGpp / lux, SHJ2168) and E. coli MG1655, and the cancer tissues were sterilized by a sterilized tool at the point in time two days passed (suppression stage) Lt; / RTI > The skin and surrounding lipids were removed to inhibit the mixing of tissues other than the infection and cancer. As soon as the tissue was separated, the tissue was dissociated. The degradation of the tissue was performed using an enzyme, which was performed according to the 'Dissociation of cells from primary tissue' of Invitrogen Laboratories. To summarize briefly, cancer tissues were washed with sterile PBS and then diluted to 2-3 mm 3 volumes. The tissue was then reacted with 25% trypsin (Gibco, Invitrogen, USA) and reacted at a ratio of 10 ml of enzyme per gram of tissue, facilitating penetration of the enzyme into cancer tissue and maximizing enzyme activity For 2 hours at < RTI ID = 0.0 > 4 C. < / RTI > Then, the enzyme-treated cancer tissues were reacted in a cell culture incubator (37 ° C, 5% carbon dioxide) for 20 minutes, the supernatant was removed, and the enzyme reaction was stopped by adding cDMEM. Thereafter, the cancerous tissue was carefully pulverized and then filtered using a 100 占 퐉 sterilized stainless steel mesh (BD Falcon, USA) to isolate a single cell mixture.
상기 과정을 통하여 분리된 암 세포 1×106 에 TRI 시약(Molecular Research Center, USA)을 이용하여 mRNA를 추출하였다. 역전사 반응을 수행하기 위하여, 상기 추출된 mRNA 1 μg, oligo (dT) 프라이머 (Promega, Madison, WI, USA) 및 Improm-II Reverse Transcriptase (Promega)의 전체 부피가 20 μl로 맞추어 반응시켜 cDNA를 합성하였다. 상기 합성한 cDNA 1 μg을 하기 프라이머를 이용하여 증폭하였다:
MRNA was extracted from 1 x 10 < 6 > of isolated cancer cells using the TRI reagent (Molecular Research Center, USA). To perform the reverse transcription reaction, cDNA was synthesized by reacting 1 μg of the extracted mRNA, 20 μl of oligo (dT) primer (Promega, Madison, Wis. USA) and Improm-II Reverse Transcriptase (Promega) Respectively. 1 μg of the synthesized cDNA was amplified using the following primers:
그 결과, 도 2a에 나타난 바와 같이, 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주는 E.coli에 비하여 사이토카인의 mRNA 수준을 현저하게 증가시켰으며, 특히, IL-1β와 TNF-α는 약 200 배 및 20 배 각각 증가시켰다. 이러한 결과는 상기 약독화된 StΔppGpp/lux 균주를 이용하는 박테리아 치료방법에 있어서, IL-1β와 TNF-α가 암 억제에 중요한 역할을 수해할 수 있음을 시사한다(도 2a 참조). As a result, as shown in FIG. 2A, the attenuated StΔppGpp / lux strain according to an embodiment of the present invention significantly increased cytokine mRNA levels compared to E. coli , and particularly IL-1β and TNF -α was increased about 200 times and 20 times, respectively. These results suggest that IL-1 [beta] and TNF- [alpha] may play an important role in tumor suppression in the bacterial treatment method using the attenuated St [Delta] ppGpp / lux strains (see FIG.
2-2: 웨스턴 블랏 분석을 이용한 사이토카인 수준 분석2-2: Analysis of cytokine levels using Western blot analysis
세포에서 합성되는 pro IL-1β 및 케스페이즈-1의 양을 측정하기 위하여, 상기 실험예 2-1에서 분리된 암 세포 1×104를 단백질 추출 용액(Intron Biotechnology, Korea)을 이용하여 용해시켰다. 그 후, 15% SDS-page(선형 구배 gel)에 로딩하여 단백질을 분리한 후, 나이트로셀룰로오스 멤브레인(Bio-rad, USA)에 이동시켰다. 상기 멤브레인은 다클론성 염소 항-마우스 IL-1β 항체(catalog# AF-401-NA, R&D Systems, 1:2000 dilution) 또는 다클론성 토끼 항-마우스 케스페이즈-1(sc-514, Santa Cruz Biotechnology, 1:1000 dilution)를 이용하여 반응시킨 후, 이어서 2차 항체로 HRP 당나귀 항-염소 IgG(Santa Cruz Biotechnology) 및 다클론성 염소 항-토끼 IgG(P0448, Dako)를 각각 이용하여 반응시켰다. 그 후, 생물발광(bioluminescence)은 루미노(lumino) 시약을 이용하여 촉매반응을 시킨 후, LAS3000 기기를 이용하여 밴드를 검출하였다. In order to measure the amount of pro IL-1 beta and caspase-1 synthesized in the cells, 1 x 10 4 cancer cells isolated in Experimental Example 2-1 were dissolved using a protein extraction solution (Intron Biotechnology, Korea) . The protein was then loaded on a 15% SDS-PAGE (linear gradient gel) and transferred to a nitrocellulose membrane (Bio-rad, USA). The membranes were incubated with polyclonal goat anti-mouse IL-1β antibody (catalog # AF-401-NA, R & D Systems, 1: 2000 dilution) or polyclonal rabbit anti- mouse kesephase- Biotechnology, 1: 1000 dilution), followed by reaction with HRP donkey anti-goat IgG (Santa Cruz Biotechnology) and polyclonal goat anti-rabbit IgG (P0448, Dako) as secondary antibodies . After that, the bioluminescence was catalyzed by using a lumino reagent and then the band was detected using a LAS3000 instrument.
그 결과, 도 2b와 같이 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 감염시킨 암 동물 모델에서 Pro IL-1β이 IL-1β에 비하여 단백질 발현 수준이 높게 나타났으며, 이는 pro IL-1β가 성숙한 IL-1β의 형태로 분리되지 않았음을 의미한다(도 2b 참조).As a result, as shown in FIG. 2B, the expression level of pro IL-1β was higher than that of IL-1β in a cancer animal model infected with an attenuated StΔppGpp / lux strain according to an embodiment of the present invention, It means that IL-1 [beta] has not been isolated in the form of mature IL-1 [beta] (see FIG. 2b).
2-3: ELISA 키트를 이용한 사이토카인 분비량 분석2-3: Analysis of cytokine secretion using ELISA kit
상기 실시예 3의 암 동물모델에 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168) 및 E. coli MG1655를 감염시킨 이후, 2일이 경과한 시점(억제 단계(suppression stage))에 암 조직을 멸균된 도구를 이용하여 마우스로부터 분리한 후, 조직과 단백질 추출 용액(Intron Biotechnology, Korea)을 1:2(w/v)의 비율이 되도록 혼합한 후 균질화하였다. 그 후, 상층액을 분리하고 4℃, 12,000 rpm 조건에서 15분씩 3회 원심분리를 수행하고, IL-1β와 TNFα ELISA test kit (eBioscience, USA)를 이용하여 사이토카인의 수준을 측정하였다. 기질의 정색반응은 450 nm 파장에서 ELISA 리더기(SpectraMax, Molecular dervices, US)를 이용하여 측정하였다.After infecting Salmonella strains (StΔppGpp / lux, SHJ2168) and E. coli MG1655 attenuated in the animal model of Example 3, the cancer tissues were sterilized after 2 days (suppression stage) The mixture was homogenized by mixing the tissue and protein extraction solution (Intron Biotechnology, Korea) at a ratio of 1: 2 (w / v). Subsequently, the supernatant was separated and centrifuged three times for 15 minutes at 4 ° C and 12,000 rpm. IL-1β and TNFα ELISA test kit (eBioscience, USA) was used to measure the levels of cytokines. The color reaction of the substrate was measured at 450 nm wavelength using an ELISA reader (SpectraMax, Molecular dervices, US).
그 결과, 도 2c에 나타난 바와 같이 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주는 대조군 및 E.coli에 비하여 IL-1β와 TNF-α의 분비를 약 4배 및 1,000 배씩 증가시켰다(도 2c 참조). 이러한 결과는 상기 도 2a의 결과와 일치하였다. As a result, as shown in FIG. 2C, the attenuated StΔppGpp / lux strain according to an embodiment of the present invention increased the secretion of IL-1β and TNF-α by about 4-fold and 1,000-fold compared to the control and E. coli (See FIG. 2C). These results were consistent with the results of FIG. 2A.
실험예 3: 면역 반응 분석Experimental Example 3: Analysis of immune response
3-1: 조직면역염색 방법을 이용한 암 조직내 면역 반응 분석3-1: Analysis of Immune Response in Cancer Tissue Using Tissue Immunostaining
상기 실시예 3의 암 동물 모델에 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168) 및 E. coli MG1655를 감염시킨 이후, 2일이 경과한 시점(억제 단계(suppression stage))에 4% 파라포름알데하이드로 관류(perfusion)시켜 조직을 고정시켰다. 그 후, 암 조직을 적출하여 30% 수크로오스를 포함하는 PBS에 넣어 탈수시킨 후, -80℃에서 조직을 냉동시켰다. 이어, 상기 조직을 냉동절편기(Cryomicrotome)을 이용하여 5 μm 두께로 자른 후, CD 45(BD science, USA), Gr-1(BD science, USA), CD68(BD science, USA), CD11c(BD science, USA) 항체를 이용하여 염색하였다. 조직염색결과는 광학 현미경(Olympus)을 이용하여 확인하였다. After infestation with Salmonella strains (StΔppGpp / lux, SHJ2168) and E. coli MG1655 attenuated in the animal model of Example 3, 2 days later (suppression stage), 4% paraformaldehyde The tissues were fixed by perfusion with aldehyde. Then, the cancer tissue was removed, dehydrated in PBS containing 30% sucrose, and the tissue was frozen at -80 ° C. The tissues were cut to a thickness of 5 탆 using a cryomicrotome and then seeded on CD 45 (BD science, USA), Gr-1 (BD science, USA), CD68 (BD science, USA) BD science, USA) antibody. Tissue staining results were confirmed using an optical microscope (Olympus).
그 결과, 도 3a에 나타난 바와 같이, 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 감염시킨 암 동물 모델에서 심각한 염증 반응이 유도되는 것을 CD45, Gr-1 CD68, 및 CD11c의 발현 증가를 통하여 확인할 수 있었다(도 3a 참조).As a result, as shown in FIG. 3A, in a cancer animal model infected with an attenuated StΔppGpp / lux strain according to an embodiment of the present invention, the induction of a severe inflammatory reaction was confirmed by the expression of CD45, Gr-1 CD68, and CD11c (See FIG. 3A).
3-2: FACS 분석을 이용한 암 조직내 면역 반응 분석3-2: Analysis of immune response in cancer tissues using FACS analysis
상기 실시예 3의 암 동물모델에 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168) 및 E. coli MG1655를 감염시킨 이후, 2일이 경과한 시점(억제 단계(suppression stage))에 암세포가 주입된 림프절을 분리한 후, 40 μm 멸균된 스테인레스 스틸 메쉬(BD Falcon, USA)를 이용하여 균질화한 후, T 세포용 배지인 RPMI1640에 현탁하였다. 이때, 상기 RPMI1640 배지는 10% FBS, 1% 페니시린/스트렙토마이신, L-글루타민(glutamine), 피루브산나트륨(sodium pyruvate), 비필수 아미노산 및 2-메르캅토에탄올(2-mercaptoethanol)이 첨가되어 있으며, 상기 현탄액을 FACS 분석에 이용하였다. 마커로는 CD45(Leukocyte antigen), Ly-6G(Neutrophil), F4/80(macrophage), CD11c(DC)를 사용하였다. After the infection of Salmonella strains (StΔppGpp / lux, SHJ2168) and E. coli MG1655 attenuated in the animal model of Example 3, cancer cells were injected at the time of 2 days (suppression stage) The lymph nodes were separated, homogenized using a 40 μm sterilized stainless steel mesh (BD Falcon, USA) and suspended in RPMI 1640 medium for T cells. At this time, the RPMI 1640 medium was supplemented with 10% FBS, 1% penicillin / streptomycin, L-glutamine, sodium pyruvate, non-essential amino acid and 2-mercaptoethanol , And the present tan solution was used for FACS analysis. Leukocyte antigen (CD45), Ly-6G (Neutrophil), F4 / 80 (macrophage) and CD11c (DC) were used as markers.
그 결과, 도 3b에 나타난 바와 같이, 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 감염시킨 암 동물 모델에서 상기 사이토카인이 대조군 및 E. coli MG1655를 감염시킨 모델에 비하여 현저하게 증가된 것을 확인할 수 있었다(도 3b 참조). As a result, as shown in FIG. 3B, in a cancer animal model infected with an attenuated strain StΔppGpp / lux according to an embodiment of the present invention, the cytokine significantly inhibited the expression of E. coli MG1655 (See FIG. 3B).
이어, 본 발명자는 도3b의 실험을 3회 이상 반복하였으며, 그 결과를 정량화하여 도 3c에 정량화하여 나타냈다. 도 3c의 x축에 기재된 M①은 macrophage를 의미하며 F4/80 마커를 이용하여 분석한 결과를 정량화한 것이며, DC는 수지상세포(dendritic cell)를 의미하며, CD11c 마커를 이용하여 분석한 결과를 정량화한 것이다. Next, the inventor repeated the experiment of FIG. 3B three times or more, and the results were quantified and quantified in FIG. 3c. 3 (c) represents the macrophage, which is a quantitative result of analysis using F4 / 80 marker. DC means dendritic cell, and the result of analysis using CD11c marker is quantified It is.
실험예 4: 조직 염색 방법을 이용한 IL-1β 사이토카인 분비 세포의 분석Experimental Example 4: Analysis of IL-1β cytokine-secreting cells using a tissue staining method
상기 실시예 3의 암 동물모델에 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168) 및 E. coli MG1655를 감염시킨 이후, 5일이 경과한 시점(억제 단계(suppression stage))에 4% 파라포름알데하이드로 관류(perfusion)시켜 조직을 고정시켰다. 그 후, 암 조직을 적출하여 30% 수크로오스(sucrose)를 포함하는 PBS에 밤새 넣고, 조직이 가라앉을 때까지탈수시킨 후, PBS 용액으로 1회 세척하고 OCT compound(Leika microsystem, German)에 포매하였다. 조직절편 이전까지 -80℃에서 보관하였다. . 이어, 상기 조직을 냉동절편기(Cryomicrotome, Microm HM 525 (Thermo Scientific)를 이용하여 5 μm 두께로 자른 후, 상기 OCT compound에 포매된 조직에 0.3% Triton X-100, 5% BSA 및 TBS-T 혼합용액을 2시간 동안 반응시키며 투과(permeable) 및 블로킹(blocking)을 함께 수행하였다. 그 후, DAPI/Antifade (1:200; Invitrogen, USA)를 이용하여 암 조직의 세포핵(청색)을, CD68(1:500; ABD serotech, USA)을 이용하여 대식세포 및 호중구(neutrophil)를, CD11c(1:100; BD Pharmingen, USA)을 이용하여 수지상 세포를 염색하였으며, 이와 함께 IL-1β(1:100; Santa cruz, USA)를 항체를 이용하여 4℃ 염색을 수행하였다. 이어, Alexa Fluor 488 chicken anti-rabbit (1:100, Invitrogen, USA) 항체를 2차 항체로 이용하여 반응시키고, Pro-long®Gold antifade(P36930, Invitrogen) 시약을 그 위에 첨가하였다. 염색된 조직은 공초점 광학 현미경(Fluoview-1000 (FV-1000) laser scanning confocal microscope, Olympus)을 이용하여 분석하였다. After infestation with Salmonella strains (StΔppGpp / lux, SHJ2168) and E. coli MG1655 attenuated in the animal model of Example 3 above, 4 days after the passage of 5 days (suppression stage) The tissues were fixed by perfusion with aldehyde. Then, cancer tissues were extracted and placed in PBS containing 30% sucrose overnight, dehydrated until the tissue was submerged, washed once with PBS solution and embedded in OCT compound (Leica microsystem, German) . And stored at -80 ° C until the tissue section. . Next, the tissue was cut into a thickness of 5 μm using a cryomicrotome (Microm HM 525 (Thermo Scientific)), and the tissues embedded in the OCT compound were coated with 0.3% Triton X-100, 5% BSA and TBS-T (Blue) of cancer tissues was stained with CD68 (Invitrogen, USA) using DAPI / Antifade (1: 200; Invitrogen, USA). Then, the mixed solution was reacted for 2 hours and permeable and blocking were performed together. (1: 100; BD Pharmingen, USA) were used to stain dendritic cells with macrophages and neutrophils using 1: 500 (USA serotech, USA) 100, Invitrogen, USA) was reacted with Alexa Fluor 488 chicken anti-rabbit (1: 100, Invitrogen, USA) as a secondary antibody, and Pro- (P36930, Invitrogen) reagent was added thereto. The stained tissue was examined using a confocal optical microscope (Fluoview-1000 (FV-1000) laser scanner ng confocal microscope, Olympus).
그 결과, 도 4a 내지 도 4c에 나타난 바와 같이, 사이토카인 IL-1β는 수지상세포와 일부 호중구 세포에서 생산되는 것을 확인할 수 있었다(도 4a 내지 도 4c 참조). As a result, as shown in Figs. 4A to 4C, it was confirmed that cytokine IL-1 beta is produced in dendritic cells and some neutrophil cells (Figs. 4A to 4C).
실험예 5: 암 시기에 따른 사이토카인 발현량 분석Experimental Example 5: Analysis of cytokine expression level by cancer stage
박테리아 감염 이후 2일이 경과한 시점(억제 시기(suppression stage)), 또는 StΔppGpp/lux가 감염된 암 부피가 1,500 mm3(재발 시기(recurred stage))일 때 암 조직을 멸균된 도구를 이용하여 마우스로부터 분리하였다. 피부와 이를 둘러싼 지질을 제거하여 감염과 암 이외의 조직이 혼합되는 것을 억제하였다. 조직을 분리한 즉시, 조직을 분해(dissociation)였다. 그 후, 상기 실험예 2-1에 기재된 바와 동일한 방법으로 조직을 분해한 후, 이로부터 mRNA를 추출하였다. 역전사 반응을 수행하기 위하여, 상기 추출된 mRNA 1 μg, oligo (dT) 프라이머 (Promega, Madison, WI, USA) 및 Improm-II Reverse Transcriptase (Promega)의 전체 부피가 20 μl로 맞추어 반응시켜 cDNA를 합성하였다. 상기 합성한 cDNA 1 μg을 하기 프라이머를 이용하여 증폭하였다:At the time of 2 days after the bacterial infection (suppression stage), or when the cancer volume infected with StΔppGpp / lux was 1,500 mm 3 (recurred stage), the cancer tissues were treated with a sterilized tool . The skin and surrounding lipids were removed to inhibit the mixing of tissues other than the infection and cancer. As soon as the tissue was separated, the tissue was dissociated. Thereafter, the tissue was digested in the same manner as described in Experimental Example 2-1, and mRNA was extracted therefrom. To perform the reverse transcription reaction, cDNA was synthesized by reacting 1 μg of the extracted mRNA, 20 μl of oligo (dT) primer (Promega, Madison, Wis. USA) and Improm-II Reverse Transcriptase (Promega) Respectively. 1 μg of the synthesized cDNA was amplified using the following primers:
IL- 1β 정방향: 5‘-GCAACTGTTCCTGAACTCAACT-3’ (서열번호 3);IL-1? Forward: 5'-GCAACTGTTCCTGAACTCAACT-3 '(SEQ ID NO: 3);
IL- 1β 역방향: 5‘-ATCTTTTGGGGTCCGTCAACT-3’ (서열번호 4);IL-1? Reverse direction: 5'-ATCTTTTGGGGTCCGTCAACT-3 '(SEQ ID NO: 4);
TNFα 정방향: 5‘-CATCTTCTCAAAATTCGAGTGACAA-3’(서열번호 9); 및TNF? Forward: 5'-CATCTTCTCAAAATTCGAGTGACAA-3 '(SEQ ID NO: 9); And
TNFα 역방향: 5‘-TGGGAGTAGACAAGGTACAACCC-3’(서열번호 10).TNF? Reverse direction: 5'-TGGGAGTAGACAAGGTACAACCC-3 '(SEQ ID NO: 10).
그 결과, 도 5a에 나타난 바와 같이 재발시기(recurrence) 시기에 IL- 1β 와 TNFα의 mRNA 수준이 본 발명의 일 실시예에 따른 약독화된 StΔppGpp/lux 균주를 감염시킨 암 동물 모델의 암조직에서 현저하게 감소하는 것을 관찰할 수 있었다.As a result, as shown in FIG. 5A, the levels of IL-1β and TNFα mRNA levels at the recurrence time were significantly higher in the cancer tissues of the cancer animal model infected with the attenuated StΔppGpp / lux strain according to one embodiment of the present invention It was observed that it was remarkably decreased.
이어, 본 발명자는 세포내에서 합성되는 pro IL-1β 및 케스페이즈-1의 양을 측정하기 위하여, 박테리아 감염 이후 2일이 경과한 시점(억제 단계(suppression stage)), 또는 StΔppGpp/lux가 감염된 암 부피가 1,500 mm3(재발 단계(recurred stage))일 때 암 조직을 상기 실험예 2-1에 기재된 바와 동일한 방법으로 분해하였다. 그 후, 1×104 살아있는 암 세포 현탁액을 단백질 추출 용액(Intron Biotechnology, Korea)을 이용하여 용해시키고, 15% SDS-page(선형 구배 gel)에 로딩하여 단백질을 분리한 후, 나이트로셀룰로오스 멤브레인(Bio-rad, USA)에 이동시켰다. 상기 멤브레인은 다클론성 염소 항-마우스 IL-1β 항체(catalog# AF-401-NA, R&D Systems, 1:2,000 dilution) 또는 다클론성 토끼 항-마우스 케스페이즈-1(sc-514, Santa Cruz Biotechnology, 1:1,000 dilution)를 이용하여 반응시킨 후, 이어서 2차 항체로 HRP 당나귀 항-염소 IgG(Santa Cruz Biotechnology, USA) 및 다클론성 염소 항-토끼 IgG(P0448, Dako, USA)를 각각 이용하여 반응시켰다. 그 후, 생물발광(bioluminescence)은 루미노(lumino) 시약을 이용하여 촉매반응을 시킨 후, LAS3000 기기를 이용하여 밴드를 검출하였다. The present inventors then investigated whether pro-IL-1? And caspase-1 synthesized intracellularly are effective in inhibiting the proliferation of cells infected at the time point two days after the bacterial infection (suppression stage) When the cancer volume was 1,500 mm 3 (recurred stage), cancer tissues were digested in the same manner as described in Experimental Example 2-1. Thereafter, the 1 × 10 4 live cancer cell suspension was dissolved using a protein extraction solution (Intron Biotechnology, Korea) and loaded on a 15% SDS-PAGE (linear gradient gel) to separate the proteins. The proteins were separated on a nitrocellulose membrane (Bio-Rad, USA). The membranes were incubated with polyclonal goat anti-mouse IL-1β antibody (catalog # AF-401-NA, R & D Systems, 1: 2,000 dilution) or polyclonal rabbit anti- mouse kesephase- (Santa Cruz Biotechnology, USA) and polyclonal goat anti-rabbit IgG (P0448, Dako, USA) were used as secondary antibodies, respectively. . After that, the bioluminescence was catalyzed by using a lumino reagent and then the band was detected using a LAS3000 instrument.
그 결과, 도 5b에 나타난 바와 같이 재발시기(recurrence) 시기에 본 발명의 일 실시예에 따른 StΔppGpp/lux 균주를 감염시킨 암 동물 모델의 암 조직에서 pro IL-1β, IL-1β 및 케스페이즈-1의 발현이 현저하게 감소하는 것을 관찰할 수 있었다. As a result, as shown in FIG. 5B, at the recurrence time, pro IL-1β, IL-1β and caspase-1β in cancer tissues of a cancer animal model infected with StΔppGpp / lux strain according to an embodiment of the present invention, 1 expression was markedly decreased.
실험예 6: 약독화된 살모넬라 균주와 IL-1β 병용투여에 의한 항암효과 분석Experimental Example 6: Antitumor effect analysis by the combination of attenuated Salmonella strains and IL-1β
IL-1β 생산을 억제하기 위하여, IL-1β 특이적 항체(AF401-NA, R&D system) 5 μg을 1×PBS 100 μl에 현탁시킨 후, 상기 실시예 3의 암 동물모델에 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168)를 감염시키기 하루 전날 체내에 투여하였으며, 감염 후에는 상기 IL-1β 특이적 항체를 2주 동안 1주에 2회 투여하였다. 또한, 대조군으로 햄스터 면역글로불린(EQUITECH Bio)을 투여하였다. 또한, 살모넬라 및 IL-1β 병용 투여는 감염 5일째 날로부터 2일 마다 재조합 IL-1β(rIL-1β, 401-ML/CF, R&D systems, USA)를 투여하였으며, 이때 투여량은 재조합 IL-1β 0.5 μg을 1×PBS 5 μl에 현탁시킨 후, PrecisionGlide Needle (BDM011455-1, BD bioscience)을 구비하고 있는 마이크로필터 주사기(Hamilton company)를 이용하여 암 내로 투여하였다. 또한, 실시예 3의 암 동물모델에 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168)를 감염시키지 않은 상태에서 실험 0일 째에 재조합 IL-1β만을 투여한 군을 비교를 위하여 이용하였다(도 6a 참조). In order to inhibit IL-1? Production, 5 占 퐂 of IL-1? Specific antibody (AF401-NA, R & D system) was suspended in 100 占 퐇 of 1 占 PBS, and Salmonella strain attenuated in the animal model of Example 3 (StΔppGpp / lux, SHJ2168), and the IL-1β-specific antibody was administered twice a week for 2 weeks after the infection. In addition, a hamster immunoglobulin (EQUITECH Bio) was administered as a control. In addition, recombinant IL-1β (rIL-1β, 401-ML / CF, R & D systems, USA) was administered every 2 days from the 5th day after infection with salmonella and IL- 0.5 μg was suspended in 5 μl of 1 × PBS, and then administered into a cancer using a microfilter syringe (Hamilton company) equipped with a PrecisionGlide needle (BDM011455-1, BD bioscience). In addition, a group to which only recombinant IL-1? Was administered on the 0th day of the experiment without infecting the Salmonella strain (St? PpGpp / lux, SHJ2168) attenuated in the animal model of Example 3 was used for comparison Reference).
박테리아 감염 시기를 0일로 정하고, 20일 동안 암 동물모델의 암 부피를 측정한 결과, 재조합 IL-1β 단백질과 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168)를 병용 투여한 군에서 암 조직의 부피가 가장 현저하게 감소하였으며, 그 뒤를 이어 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168) 또는 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168)와 IgG를 투여한 군에서 암 조직의 부피가 감소하였다. 반면, IL-1β 항체를 살모넬라 균주 투여 1일 전에 투여한 군이나 IL-1β 단독으로 투여한 군은 대조군에 비해서 약간의 항암효과는 있었으나 본 발명의 일 실시예에 따른 방법에 비하면 현저하게 항암 효과가 감소하였다(도 6b 참조).The bacterial infection time was determined as 0 day, and the cancer volume of the cancer animal model was measured for 20 days. As a result, the volume of cancer tissue in the combination of recombinant IL-1β protein and attenuated salmonella strain (StΔppGpp / lux, SHJ2168) Followed by a decrease in the volume of cancer tissues in the group treated with attenuated Salmonella strains (StΔppGpp / lux, SHJ2168) or attenuated Salmonella strains (StΔppGpp / lux, SHJ2168) and IgG. On the other hand, in the group administered with IL-1 beta antibody one day before the administration of Salmonella strain or in the group administered with IL-1 beta alone, the anti-cancer effect was slight compared to the control group, (See FIG. 6B).
또한, 실험 0, 5 및 21일 째 되는 날, 암 동물 모델의 허벅지 부위를 절개하여 암 조직을 관찰한 결과에서도 약독화된 살모넬라 균주와 재조합 IL-1β를 병용투여한 군에서 암이 현저하게 감소한 것을 관찰할 수 있었다(도 6c 참조).In addition, on the 0th, 5th, and 21st days after the experiment, the cancer tissue was observed by incising the thigh region of the animal model, and the cancer was remarkably decreased in the group treated with the Salmonella strain which was attenuated and the recombinant IL-1β (See FIG. 6C).
상기 결과를 종합하면, 본 발명자는 본 발명의 일 실시예에 따른 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168)의 항암작용에 있어서, IL-1β가 중요한 작용을 한다는 것을 입증하였으며, 약독화된 살모넬라 균주(StΔppGpp/lux, SHJ2168) 또는 IL-1β 단일 투여에 비하여 이를 병용투여하였을 때, 항암 효과가 현저하게 상승한다는 생체 내(in vivo) 실험 결과를 통하여 약독화된 혐기성 박테리아와 IL-1β를 포함하는 조성물을 암 치료에 유용하게 적용할 수 있음을 밝혔다.
The present inventors have demonstrated that IL-1β plays an important role in the anticancer activity of the attenuated Salmonella strain (StΔppGpp / lux, SHJ2168) according to one embodiment of the present invention, In vivo experiments were carried out to compare the effects of attenuated anaerobic bacteria and IL-1β on the anticancer efficacy of co-administration of Salminella (StΔppGpp / lux, SHJ2168) or IL-1β Can be usefully applied in the treatment of cancer.
본 발명은 도면에 도시된 실시예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.
<110> Industry Foundation of Chonnam National University <120> Pharmaceutical composition comprising attenuated facultative bacteria and Il-1 beta for preventing or treating cancer <130> PD12-0526 <160> 16 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HPRT Forward primer <400> 1 ttatggacag gactgaaaga c 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HPRT Reverse primer <400> 2 gctttaatgt aatccagcag gt 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-1 beta Forward primer <400> 3 gcaactgttc ctgaactcaa ct 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-1 beta Reverse primer <400> 4 atcttttggg gtccgtcaac t 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Forward primer <400> 5 gaggatacca ctcccaacag acc 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Reverse primer <400> 6 aagtgcatca tcatcgttgt tca 23 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IFN-gamma Forward primer <400> 7 tcaagtggca tagatgtgga agaa 24 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IFN-gamma Reverse primer <400> 8 tggctctgca ggattttcat g 21 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Forward primer <400> 9 catcttctca aaattcgagt gacaa 25 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Reverse primer <400> 10 tgggagtaga caaggtacaa ccc 23 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta Forward primer <400> 11 gaaggcagag ttcagggtct t 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta Reverse primer <400> 12 ggttcctgtc tttgtggtga a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S Forward primer <400> 13 gtaacccgtt gaaccccatt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S Reverse primer <400> 14 ccatccaatc ggtagtagcg 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 28S Forward primer <400> 15 ctaataggga acgtgagctg gg 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 28S Reverse primer <400> 16 gcggttcctc tcgtactgag 20 <110> Industry Foundation of Chonnam National University <120> Pharmaceutical composition comprising attenuated facultative bacteria and Il-1 beta for preventing or treating cancer <130> PD12-0526 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HPRT Forward primer <400> 1 ttatggacag gactgaaaga c 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HPRT Reverse primer <400> 2 gctttaatgt aatccagcag gt 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-1 beta Forward primer <400> 3 gcaactgttc ctgaactcaa ct 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> IL-1 beta reverse primer <400> 4 atcttttggg gtccgtcaac t 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Forward primer <400> 5 gaggatacca ctcccaacag acc 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Reverse primer <400> 6 aagtgcatca tcatcgttgt tca 23 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IFN-gamma Forward primer <400> 7 tcaagtggca tagatgtgga agaa 24 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> IFN-gamma Reverse primer <400> 8 tggctctgca ggattttcat g 21 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Forward primer <400> 9 catcttctca aaattcgagt gacaa 25 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Reverse primer <400> 10 tgggagtaga caaggtacaa ccc 23 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta Forward primer <400> 11 gaaggcagag ttcagggtct t 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta Reverse primer <400> 12 ggttcctgtc tttgtggtga a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S Forward primer <400> 13 gtaacccgtt gaaccccatt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S Reverse primer <400> 14 ccatccaatc ggtagtagcg 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 28S Forward primer <400> 15 ctaataggga acgtgagctg gg 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 28S Reverse primer <400> 16 gcggttcctc tcgtactgag 20
Claims (10)
상기 약독화된 혐기성 박테리아는 ppGpp 합성능이 결여된 변이체인, 암 예방 및 치료용 조성물.3. The method according to claim 1 or 2,
Wherein the attenuated anaerobic bacteria is a mutant lacking the ability to synthesize ppGpp.
상기 약독화된 혐기성 박테리아는 ppGpp 합성을 위한 ppGpp 씬세타아제를 코딩하는 불활성화된 relA 유전자 또는 spoT 유전자를 포함하는, 암 예방 및 치료용 조성물.3. The method according to claim 1 or 2,
Wherein the attenuated anaerobic bacterium comprises an inactivated relA gene or a spoT gene encoding ppGpp < RTI ID = 0.0 > cytosetase < / RTI > for ppGpp synthesis.
상기 약독화된 혐기성 박테리아는 에스케리치아(Escherichia) 속, 살모넬라(Salmonella) 속, 클로스트리듐(Clostridium) 속, 비피도박테리움(Bifidobacterium) 속, 여시니아(Yersinia) 속, 리스테리아(Listeria) 속, 마이코플라스마(Mycoplasma) 속 또는 스트렙토코코스(Streptococcus)속 인, 암 예방 및 치료용 조성물.3. The method according to claim 1 or 2,
The attenuated anaerobic bacteria Escherichia (Escherichia) genus, Salmonella (Salmonella) genus, Clostridium (Clostridium) genus Bifidobacterium (Bifidobacterium), A yeosi California (Yersinia) genus Listeria (Listeria) in , Mycoplasma genus or Streptococcus genus.
상기 살모넬라 속 균주는 Salmonella typhimurium, Salmonella choleraesuis 또는 Salmonella enteritidis인, 암 예방 및 치료용 조성물.6. The method of claim 5,
Wherein said Salmonella genus is Salmonella typhimurium , Salmonella choleraesuis or Salmonella enteritidis .
상기 살모넬라 속 균주는 Salmonella typhimurium인, 암 예방 및 치료용 조성물.The method according to claim 6,
Wherein the Salmonella genus is Salmonella typhimurium .
상기 암은 간암, 대장암, 자궁경부암, 신장암, 위암, 전립선암, 유방암, 뇌암, 폐암, 자궁암, 결장암, 방광암, 혈액암 및 췌장암으로 구성되는 군으로부터 선택되는 어느 하나인, 암 예방 및 치료용 조성물.3. The method according to claim 1 or 2,
Wherein said cancer is any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain cancer, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer. / RTI >
상기 포유류는 인간인, 암 예방 및 치료용 조성물.3. The method of claim 2,
Wherein said mammal is a human.
A method for treating cancer, comprising administering to a subject a composition of any one of claims 1 to 9
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021086085A1 (en) * | 2019-10-31 | 2021-05-06 | (주)최현일바이오랩 | Tumor-targeting salmonella gallinarum strain and use thereof |
| WO2022231043A1 (en) * | 2021-04-30 | 2022-11-03 | 전남대학교산학협력단 | Attenuated salmonella gallinarum strain and use thereof |
| KR20220149313A (en) * | 2021-04-30 | 2022-11-08 | 전남대학교산학협력단 | Attenuated salmonella gallinarum and use thereof |
| WO2023080703A1 (en) * | 2021-11-05 | 2023-05-11 | 전남대학교 산학협력단 | Pharmaceutical composition comprising salmonella strain and immune checkpoint inhibitor as active ingredient for prevention or treatment of cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20240114350A (en) * | 2023-01-16 | 2024-07-24 | 주식회사 오디세우스바이오 | Attenuated Salmonella Gallinarum expressing FliC or FliC-hIL2 and uses thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021086085A1 (en) * | 2019-10-31 | 2021-05-06 | (주)최현일바이오랩 | Tumor-targeting salmonella gallinarum strain and use thereof |
| KR20210052337A (en) * | 2019-10-31 | 2021-05-10 | (주)최현일바이오랩 | Tumor Targeting Salmonella gallinarum and Use Thereof |
| WO2022231043A1 (en) * | 2021-04-30 | 2022-11-03 | 전남대학교산학협력단 | Attenuated salmonella gallinarum strain and use thereof |
| KR20220149313A (en) * | 2021-04-30 | 2022-11-08 | 전남대학교산학협력단 | Attenuated salmonella gallinarum and use thereof |
| WO2023080703A1 (en) * | 2021-11-05 | 2023-05-11 | 전남대학교 산학협력단 | Pharmaceutical composition comprising salmonella strain and immune checkpoint inhibitor as active ingredient for prevention or treatment of cancer |
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