KR20190094106A - 신규 다가 hpv 백신 조성물 - Google Patents
신규 다가 hpv 백신 조성물 Download PDFInfo
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Abstract
본 발명은 신규 다가 HPV DNA 백신 및 그에 사용되는 융합단백질 및 이를 암호화하는 폴리뉴클레오타이드에 관한 것으로서, 보다 구체적으로는 6, 11, 16, 18, 39, 45 및 56형 인유두종바이러스(HPV)의 조기 단백질 항원 6(E6) 또는 그의 면역원성 단편, 및 조기 단백질 항원 7(E7) 또는 그의 면역원성 단편을 각각 암호화하는 폴리뉴클레오타이드를 포함하는, 다가 HPV DNA 백신 조성물에 관한 것이다.
Description
본 발명은 신규 다가 HPV DNA 백신 및 그에 사용되는 융합단백질 및 이를 암호화하는 폴리뉴클레오타이드에 관한 것이다.
자궁경부암은 전세계적으로 여성에서 암 사망의 주된 원인들 중 하나이며(Einstein et al., Lancet Infect. Dis., 9: 347-356, 2009; Parkin and Bray, Vaccine 24(3S): 11-25, 2007), 그 경우의 약 75%가 가장 흔한 고위험 인유두종바이러스(HPV) 유형, 즉 HPV16 및 HPV18에 의한 지속적인 감염에 의해 야기된다(Schiffman et al., Lancet, 370:890-907, 2007; Forman et al., Vaccine 30(5S): F12-23, 2012). HPV 감염 지속성은 통상적으로 명백한 HPV-특이적 T-세포 면역의 결여와 연관되며, 전-악성 및 악성종양 환자에서 발견되는 바이러스-특이적 T 세포는 일반적으로 기능장애성이고 때때로 심지어 억제성인 것으로 보고되어 있다(Trimble, Cancer Immunol. Immunother. CII 59:799-803, 2010). 이러한 결과들은 바이러스-특이적 T 세포의 기능 손상이 HPV-유도된 자궁경부암의 발생과 연관될 수 있음을 시사한다.
자궁경부암은 고위험 HPV 감염, 바이러스 지속성, 지속적으로 감염된 세포의 전-악성 병변으로의 클론 확장 및 분화, 및 침습성 암으로의 그의 점진적 형질전환의 과정을 통해 발생한다(Schiffman et al., Lancet 370: 890-907, 2007). 전-악성 자궁경부 상피내 종양 2 및 3(CIN2 및 3), 특히 HPV16에 양성인 상기 종양들은, 침습성 암으로 발전할 가능성이 약 30%인 고-등급 병변으로 간주된다(Moscicki et al., Vaccine 30(5S): F24-33, 2012). 그러므로, 지속적인 HPV 감염의 심각한 합병증을 예방하고 HPV-관련 종양을 뿌리뽑을 수 있는 효과적인 치료 백신이 시급하게 필요하다.
현재 한국에서 시판되고 있는 두 가지 HPV 백신은 4가 백신(가다실)과 2가 백신(서바릭스)가 있는데 4가 백신의 경우 HPV6, 11, 16, 18형의 L1 VLP를 함유하고 있고 2가 백신의 경우 HPV 16, 18형의 L1 VLP를 함유하고 있다. 그러나 HPV6, 11형은 생식기 사마귀의 주요원인이지만 자궁경부암과는 관련이 없는 저위험군 HPV 이므로 자궁경부암을 예방하는 차원에서는 두 백신 모두 HPV 16, 18형을 예방하는 2가 백신에 해당한다.
한편, HPV E6 및 E7은 종양 억제 단백질 p53 및 망막아세포종(pRb) 각각을 결합시키고 분해를 촉진함으로써 바이러스 종양단백질(oncoprotein)로 작용한다(Yugawa and Kiyono, Rev. Med. Virol., 19: 97-113, 2009). 상기 바이러스 종양단백질들은, 이들 단백질이 종양형성을 유도할 뿐만 아니라 이들이 또한 HPV-감염된 전-악성 및 악성 세포에서 구성적으로 발현되기 때문에 CIN2/3 및 자궁경부암에 대한 치료 백신에 이상적인 표적들로 여겨지고 있다Yugawa and Kiyono, Rev. Med. Virol., 19: 97-113, 2009). 자궁경부 병변의 퇴행은 세포성 면역 반응과 연관되지만 체액성 면역 반응과는 연관되지 않으므로(Deligeoroglou et al., Infect. Dis. Obstet. Gynecol., 2013: 540850, 2013; Woo et al., Int. J. Cancer, 126: 133-141, 2010), 강력한 E6/E7-특이적 T-세포 면역을 선택적으로 유도할 수 있는 치료 백신이 매우 바람직하다.
현재 HPV E6/E7 항원을 이용하여 개발되고 있는 백신으로는 한국공개특허 제2017-0045254호에 개시된 HPV 16/18 E6/E7 항원을 이용한 DNA 백신 조성물이 존재한다. 그러나, 상기 시판 백신 조성물 및 상기 HPV 16/18 E6/E7 항원을 이용한 DNA 백신 조성물 모두 가장 고위험군인 16형 및 18형 HPV만을 표적으로 하고 있기 때문에, 전체 자궁경부암의 70% 정도만을 커버하고 있을 뿐이다.
따라서, 거의 모든 자궁경부암 유발 위험성을 갖고 있는 다양한 HPV 타입에 대한 면역반응을 유발할 수 있는 다가 백신의 개발이 절실한 상황이다.
이에 본 발명은 보다 다양한 타입의 HPV에 대한 면역반응을 유도함으로써 자궁경부암 및 기타 HPV 감염에 의해 유발되는 질환의 예방 및 치료에 효율적인 다가 HPV DNA 백신을 제공하는 것을 목적으로 한다. 그러나, 본 발명의 범위가 상기 목적에 의해 제한되는 것은 아니다.
본 발명의 일 측면에 따르면, 6, 11, 16, 18, 39, 45 및 56형 인유두종바이러스(HPV)의 조기 단백질 항원 6(E6) 또는 그의 면역원성 단편, 및 조기 단백질 항원 7(E7) 또는 그의 면역원성 단편을 각각 암호화하는 폴리뉴클레오타이드를 포함하는, 다가 HPV DNA 백신 조성물이 제공된다.
본 발명의 일 관점에 따르면, 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 및 59형으로 구성되는 군으로부터 선택되는 인유두종바이러스(human papilloma virus)의 조기 단백질 6(E6) 및 조기 단백질 항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(E6N)-E7의 C-말단 단편(E7C)-E7의 N-말단 단편(E7N)-E6의 C-말단 단편(E6C)의 순으로 연결된 폴리펩타이드를 E6/E7 항원단위체로 하며, 상기 HPV 중 적어도 4 타입의 HPV의 항원단위체가 링커에 의해 연결된 항원 연결체 융합단백질이 제공된다.
본 발명의 일 관점에 따른 상기 융합단백질을 암호화하는 폴리뉴클레오타이드가 제공된다.
본 발명의 다른 일 관점에 따르면 상기 폴리뉴클레오타이드가 발현조절부위에 작동가능하게 연결된 발현벡터가 제공된다.
본 발명의 다른 일 관점에 따르면, 상기 융합단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동 가능하게 연결된 발현벡터를 포함하며, 상기 14타입의 모든 E6/E7 항원 단위체를 암호화하는 폴리뉴클레오타이드를 포함하도록 복수의 발현벡터를 포함하는 14가 HPV DNA 백신 조성물이 제공된다.
본 발명의 다른 일 관점에 따르면, 상기 다가 HPV DNA 백신 조성물 또는 상기 14가 HPV DNA 백신 조성물을 개체에 투여하는 단계를 포함하는 HPV 감염에 의해 유발되는 질환의 치료방법이 제공된다.
도 1은 본 발명의 일 실시예에 따른 다가 HPV DNA 백신에 포함되는 세 가지 융합단백질(BD-14A, BD-14B 및 BD-14C)의 구조를 나타낸 개요도이다.
도 2a는 본 발명의 일 실시예에 따른 백신 면역보조제 BD-121의 구조를 나타내는 개요도이고, 도 2b는 본 발명의 일 실시예에 따른 백신 면역보조제 BD-121A의 구조를 나타내는 개요도이다.
도 3a는 본 발명의 일 실시예에 따른 BD-14A, BD-14B 및 BD-14C 플라스미드로 각각 COS-7 세포를 형질감염시킨 후 Flt3L의 발현정도를 ELISA로 분석한 결과를 나타내는 그래프이고, 도 3b는 상기 도 3a의 세포를 파쇄한 세포파쇄액을 대상으로 항-Flt3L 항체를 이용하여 웨스턴블랏 분석을 수행한 결과를 나타내는 사진이다.
도 4a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14)의 면역반응을 분석하기 위한 실험의 백신 접종 스케쥴을 나타낸다.
도 4b는 공벡터(Mock), 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 및 종래 2가 HPV DNA 백신으로 접종된 마우스로부터 적출된 비장세포 중 각 타입의 HPV의 E6/E7에 특이적으로 반응한 비장세포의 수를 분석한 ELISPOT 분석결과를 나타내는 그래프이다.
도 5a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14)의 HPV 유발 암에 대한 항암효과를 분석하기 위한 실험의 백신 접종 스케쥴을 나타낸다.
도 5b는 공벡터(pGX27), 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 및 종래 2가 HPV DNA 백신으로 접종된 이종종양 이식 마우스의 시간의 경과에 따른 종양조직의 부피의 변화를 기록한 그래프이다.
도 5c는 공벡터(pGX27), 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 및 종래 2가 HPV DNA 백신으로 접종된 이종종양 이식 마우스의 시간의 경과에 따른 생존률을 기록한 그래프이다.
도 6a는 본 발명의 일 실시예에 따른 백신 면역보조제 hBD-121 컨스트럭트로 형질감염된 COS-7 세포의 배양 상등액 내의 IL-12 및 IL-21의 농도를 ELISA로 분석한 결과를 나타내는 그래프이다.
도 6b는 본 발명의 일 실시예에 따른 백신 면역보조제 mBD-121 컨스트럭트로 형질감염된 COS-7 세포의 배양 상등액 내의 IL-12 및 IL-21의 농도를 ELISA로 분석한 결과를 나타내는 그래프이다.
도 6c는 본 발명의 일 실시예에 따른 백신 면역보조제 hBD-121 컨스트럭트 및 mBD-121로 형질감염된 COS-7 세포의 세포파쇄액 내의 IL-12 및 IL-21의 발현 정도를 웨스턴블랏 분석을 통해 확인한 결과를 나타낸다.
도 7a는 본 발명의 일 실시예에 따른 DNA 백신 조성물의 항암효과를 확인하기 위한 백신 접종 스케쥴을 나타낸다.
도 7b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 단독 투여 또는 상기 BD-14와 백신 면역보조제 BD-121A의 병용투여시 각 타입의 HPV의 E6/E7 특이적 면역반응 비장세포의 수를 ELISPOT 분석으로 분석한 결과를 나타내는 그래프이다.
도 8a는 본 발명의 일 실시예에 따른 DNA 백신 조성물과 종래 2가 백신 조성물과의 항암효과의 비교를 위한 백신 접종 스케쥴을 나타낸다.
도 8b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물 투여시 종양조직의 시간의 경과에 따른 부피의 변화를 종래 2가 백신 조성물과 비교하여 나타낸 그래프이다.
도 8c는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물 투여시 실험 동물의 시간의 경과에 따른 생존율을 종래 2가 백신 조성물과 비교하여 나타낸 그래프이다.
도 9a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물의 작용기전을 확인하기 위한 동물실험의 백신 접종 스케쥴을 나타낸다.
도 9b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물을 항-CD4 항체 또는 항-CD8 항체를 투여하여 각각 CD4 T 세포 및 CD8 T 세포를 결손시킨 실험동물에 투여하였을 때의 시간의 경과에 따른 종양의 부피를 나타낸 그래프이다.
도 10a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물을 IL-7과 병용시 항암활성을 조사하기 위한 동물실험의 백신 접종 스케쥴을 나타낸다.
도 10b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물을 단독 또는 IL-7과 병용 투여시 시간의 경과에 따른 암세포의 부피를 조사한 결과를 나타내는 그래프이다.
도 2a는 본 발명의 일 실시예에 따른 백신 면역보조제 BD-121의 구조를 나타내는 개요도이고, 도 2b는 본 발명의 일 실시예에 따른 백신 면역보조제 BD-121A의 구조를 나타내는 개요도이다.
도 3a는 본 발명의 일 실시예에 따른 BD-14A, BD-14B 및 BD-14C 플라스미드로 각각 COS-7 세포를 형질감염시킨 후 Flt3L의 발현정도를 ELISA로 분석한 결과를 나타내는 그래프이고, 도 3b는 상기 도 3a의 세포를 파쇄한 세포파쇄액을 대상으로 항-Flt3L 항체를 이용하여 웨스턴블랏 분석을 수행한 결과를 나타내는 사진이다.
도 4a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14)의 면역반응을 분석하기 위한 실험의 백신 접종 스케쥴을 나타낸다.
도 4b는 공벡터(Mock), 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 및 종래 2가 HPV DNA 백신으로 접종된 마우스로부터 적출된 비장세포 중 각 타입의 HPV의 E6/E7에 특이적으로 반응한 비장세포의 수를 분석한 ELISPOT 분석결과를 나타내는 그래프이다.
도 5a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14)의 HPV 유발 암에 대한 항암효과를 분석하기 위한 실험의 백신 접종 스케쥴을 나타낸다.
도 5b는 공벡터(pGX27), 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 및 종래 2가 HPV DNA 백신으로 접종된 이종종양 이식 마우스의 시간의 경과에 따른 종양조직의 부피의 변화를 기록한 그래프이다.
도 5c는 공벡터(pGX27), 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 및 종래 2가 HPV DNA 백신으로 접종된 이종종양 이식 마우스의 시간의 경과에 따른 생존률을 기록한 그래프이다.
도 6a는 본 발명의 일 실시예에 따른 백신 면역보조제 hBD-121 컨스트럭트로 형질감염된 COS-7 세포의 배양 상등액 내의 IL-12 및 IL-21의 농도를 ELISA로 분석한 결과를 나타내는 그래프이다.
도 6b는 본 발명의 일 실시예에 따른 백신 면역보조제 mBD-121 컨스트럭트로 형질감염된 COS-7 세포의 배양 상등액 내의 IL-12 및 IL-21의 농도를 ELISA로 분석한 결과를 나타내는 그래프이다.
도 6c는 본 발명의 일 실시예에 따른 백신 면역보조제 hBD-121 컨스트럭트 및 mBD-121로 형질감염된 COS-7 세포의 세포파쇄액 내의 IL-12 및 IL-21의 발현 정도를 웨스턴블랏 분석을 통해 확인한 결과를 나타낸다.
도 7a는 본 발명의 일 실시예에 따른 DNA 백신 조성물의 항암효과를 확인하기 위한 백신 접종 스케쥴을 나타낸다.
도 7b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신(BD-14) 단독 투여 또는 상기 BD-14와 백신 면역보조제 BD-121A의 병용투여시 각 타입의 HPV의 E6/E7 특이적 면역반응 비장세포의 수를 ELISPOT 분석으로 분석한 결과를 나타내는 그래프이다.
도 8a는 본 발명의 일 실시예에 따른 DNA 백신 조성물과 종래 2가 백신 조성물과의 항암효과의 비교를 위한 백신 접종 스케쥴을 나타낸다.
도 8b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물 투여시 종양조직의 시간의 경과에 따른 부피의 변화를 종래 2가 백신 조성물과 비교하여 나타낸 그래프이다.
도 8c는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물 투여시 실험 동물의 시간의 경과에 따른 생존율을 종래 2가 백신 조성물과 비교하여 나타낸 그래프이다.
도 9a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물의 작용기전을 확인하기 위한 동물실험의 백신 접종 스케쥴을 나타낸다.
도 9b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물을 항-CD4 항체 또는 항-CD8 항체를 투여하여 각각 CD4 T 세포 및 CD8 T 세포를 결손시킨 실험동물에 투여하였을 때의 시간의 경과에 따른 종양의 부피를 나타낸 그래프이다.
도 10a는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물을 IL-7과 병용시 항암활성을 조사하기 위한 동물실험의 백신 접종 스케쥴을 나타낸다.
도 10b는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 조성물을 단독 또는 IL-7과 병용 투여시 시간의 경과에 따른 암세포의 부피를 조사한 결과를 나타내는 그래프이다.
본 발명의 일 측면에 따르면, 6, 11, 16, 18, 39, 45 및 56형 인유두종바이러스(HPV)의 조기 단백질 항원 6(E6) 또는 그의 면역원성 단편, 및 조기 단백질 항원 7(E7) 또는 그의 면역원성 단편을 각각 암호화하는 폴리뉴클레오타이드를 포함하며, 상기 E6 및 E7은 야생형의 기능을 갖지 않는, 다가 HPV DNA 백신 조성물이 제공된다.
상기 다가 HPV DNA 백신 조성물은, 31, 33, 35, 51, 52, 58 및 59형 인유두종바이러스(HPV)로 구성되는 군으로부터 선택되는 1종 또는 그 이상의 HPV의 조기 단백질 항원 6(E6) 또는 그의 면역원성 단편, 및 조기 단백질 항원 7(E7) 또는 그의 면역원성 단편을 각각 암호화하는 폴리뉴클레오타이드를 추가로 포함할 수 있는데, 상기 추가된 E6 및 E7 역시 야생형의 기능을 갖지 않는다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 E6 및 E7은 각각 N-말단 단편 및 C-말단 단편으로 나뉘어 무작위적으로 뒤섞인 E6/E7 셔플드 항원단위체의 형태로 발현될 수 있고, 상기 E6/E7 셔플드 항원단위체는 상기 E6 및 E7의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(E6N)-E7의 C-말단 단편(E7C)-E7의 N-말단 단편(E7N)-E6의 C-말단 단편(E6C)의 순으로 연결된 폴리펩타이드일 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 적어도 2개 이상, 3개 이상, 또는 4개 이상의 인유두종바이러스의 E6/E7 셔플드 항원단위체가 융합단백질 형태로 연결되어 발현될 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 E6/E7 셔플드 항원단위체 또는 상기 융합단백질은 신호서열을 추가로 포함할 수 있고, 상기 E6/E7 셔플드 항원단위체 또는 상기 융합단백질은 Flt3L을 추가로 포함할 수 있다.
상기 다가 HPV DNA 백신 조성물은, 추가로 IL-7을 포함할 수 있다. 본 발명자들은 본 발명의 일 실시예에 따른 다가 HPV DNA 백신 투여시 IL-7을 병용투여할 경우 항암 효과가 현저하게 상승됨을 확인하였다(도 10b 참조).
본 발명의 다가 HPV DNA 백신 조성물은 하나 이상의 약학적으로 허용가능한 백신보조제를 추가로 포함할 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 백신 면역보조제는 IL-12 단백질 및 IL-21 단백질을 유효성분으로 포함하거나 상기 IL-12 단백질을 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 암호화하는 폴리뉴클레오타이드를 유효성분으로 포함하는 T 림프구 특이적 면역반응 촉진용 백신 면역보조제일 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 T 림프구 특이적 면역반응 촉진용 백신 면역보조제는 하기로 구성되는 군으로부터 선택되는 하나 이상을 포함할 수 있다:
p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)로 구성된 IL-12 단백질 및 IL-21 단백질;
상기 IL-12를 구성하는 p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)를 각각 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드를 포함하는 하나 내지 세 개의 벡터; 및
상기 IL-12p35, IL-12p40 및 IL-21 단백질을 각각 암호화하는 mRNA 분자.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 IL-12p35 단백질은 서열번호 1로 기재되는 아미노산 서열로 구성되는 인간 IL-12p35와 서열 상동성 90% 이상인 것일 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 IL-12p40 단백질은 서열번호 2로 기재되는 아미노산 서열로 구성되는 인간 IL-12p40와 서열 상동성 90% 이상인 것일 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 IL-21 단백질은 서열번호 3으로 기재되는 아미노산 서열로 구성되는 인간 IL-21과 서열 상동성 90% 이상인 것일 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 백신 면역보조제는 하기로 구성되는 군으로부터 선택되는 하나 이상을 추가로 포함할 수 있다:
ⅰ) MIP-1α 단백질;
ⅱ) 상기 MIP-1α 단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동 가능하게 연결된 MIP-1α 유전자컨스트럭트; 및
ⅲ) 상기 MIP-1α 단백질을 암호화하는 폴리뉴클레오타이드가 상기 IL-12p35, IL-12p40 및 IL-21 단백질 중 어느 하나 이상에 IRES 또는 링커 펩타이드를 암호화하는 폴리뉴클레오타이드에 의해 작동가능하게 연결된 복합 유전자컨스트럭트; 및
ⅳ) MIP-1α 단백질을 암호화하는 mRNA 분자.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 MIP-1α 유전자컨스트럭트는 별도의 발현벡터에 포함되거나, 상기 IL-12를 구성하는 p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)를 각각 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드를 포함하는 하나 내지 세 개의 벡터 중 어느 하나 이상의 벡터 내에 포함될 수 있다.
상기 다가 HPV DNA 백신 조성물에 있어서, 상기 MIP-1α 단백질은 서열번호 10으로 기재되는 아미노산 서열로 구성되는 인간 MIP-1α 단백질과 서열 상동성 90% 이상인 것일 수 있다.
본 발명의 일 관점에 따르면, 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 및 59형으로 구성되는 군으로부터 선택되는 인유두종바이러스(human papilloma virus)의 조기 단백질 6(E6) 및 조기 단백질 항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(E6N)-E7의 C-말단 단편(E7C)-E7의 N-말단 단편(E7N)-E6의 C-말단 단편(E6C)의 순으로 연결된 폴리펩타이드를 E6/E7 항원단위체로 하며, 상기 HPV 중 적어도 4 타입의 HPV의 항원단위체가 링커에 의해 연결된 항원 연결체 융합단백질이 제공된다.
본 문서에서 사용되는 용어 "HPV"는 인유두종바이러스(human papilloma virus)로 직경이 52~55 nm인 DNA 기반의 바이러스이며, 사람을 비롯한 여러 동물의 피부나 피하에 감염된다. 현재까지 130 여종의 HPV가 발견되었는데(zur Hausen H., Vaccine 24 Suppl 3: S3, 2006), HPV는 피부의 각질세포(keratinocyte)나 점막을 통하여 감염된다. 알려진 HPV 중 대부분은 사람들에게 어떠한 징후를 나타내지 않으나, 일부의 인유두종바이러스(HPV)의 경우, 사람에게 유두종(papilloma)을 발생시킬 수 있다. 또한 소수의 인유두종바이러스의 경우, 자궁경부암, 고환암과 같은 암을 발생시킨다. 인유두종바이러스 16(HPV 16)과 인유두종바이러스 18(HPV 18)의 경우, 전 세계 자궁경부암 환자의 70%에서 발견되고 있다. 이와 같은 인유두종바이러스는 고위험군으로 분류된다. HPV의 DNA는 8,000개의 염기쌍을 포함하고 있고 지질막이 아닌 오량체의 캡시드 단백질에 둘러싸여 존재하는데, 캡시드 단백질은 2개의 구조 단백질인 L1과 L2로 이루어 졌고, 이 단백질은 바이러스 복제 사이클의 후기에서 발현된다. 모든 HPV의 게놈에서는 8개의 개방판독틀(ORF)이 존재하고 각 ORF는 3가지의 기능적 부위로 구분된다. 바이러스의 복제에 필요한 유전자인 조기 단백질 E1-E7, 비리온(virion)을 구성하는 구조 단백질을 발현하는 유전자 L1-L2, 마지막으로 바이러스의 복제와 전사를 조절하는 LCR로 구성된다.
본 문서에서 사용되는 용어 "E6"는 HPV의 복제에 필요한 조기 발현 단백질 중 하나로, p53에 결합하여 p53의 유비퀴틴화를 촉진시킴으로서 암종양 억제 유전자로서의 p53의 기능을 저해시킨다. 또한 세포자살 단백질(pro-apoptotic protein)인 BAK의 분해를 유도한다. 아울러, 텔로머레이스(telomerase)의 활성화를 통하여 숙주세포의 세포주기를 활성화시키는 역할을 수행한다.
본 문서에서 사용되는 용어 "E7"은 HPV의 복제에 필요한 조기 발현 단백질 중 하나로, RB(retinoblastoma)와 상호작용하여 RB를 분해한다. 이를 통하여 RB에 의해 저해되고 있던 전사촉진 인자인 E2F를 방출시킨다. 더구나, 세포주기 S기에 작용하는 사이클린 E(cycilin E)와 사이클린 A(cycilin A)를 활성화시켜 숙주세포의 세포주기를 활성화시킨다.
본 문서에서 사용되는 용어 "면역원성 단편"은 전장 길이 항원단백질의 단편 중 항원으로 기능을 발휘할 수 있는, 즉, 항원-특이적인 면역반응을 유발할 수 있는 단편을 의미한다.
상기 융합단백질에 있어서, 상기 N-말단 단편 및 C-말단 단편은 10 내지 30 a.a.가 중복이 될 수 있고, 상기 항원단위체는 셔플드 단백질(shuffled protein)이기 때문에 항원으로서의 기능을 보유하고 있으나, 원래의 야생형 E6 및 E7 단백질의 고유의 기능(p53 및 pRb 결합 기능)이 결여되어 있다.
상기 융합단백질에 있어서, 상기 항원 연결체 단백질은 N-말단에 Flt3(fms-like tyrosine kinase-3) 리간드(Flt3L)가 부가될 수 있고, 분비 신호서열이 부가될 수 있다. 상기 분비 신호서열은 세포내에서 발현되는 재조합 단백질의 세포 밖으로의 분비를 유도하며, tPA(tissue plasminogen activator) 신호서열, HSV gDs(단순포진 바이러스 당단백질 Ds) 신호서열 또는 성장호르몬 신호서열일 수 있다.
상기 융합단백질은 하나 또는 둘 이상의 면역 증진 펩타이드를 암호화하는 폴리뉴클레오타이드를 추가로 포함할 수 있고, 상기 면역 증진 펩타이드는 CD28, ICOS(inducible costimulator), CTLA4(cytotoxic T lymphocyte associated protein 4), PD1(programmed cell death protein 1), BTLA(B and T lymphocyte associated protein), DR3(death receptor 3), 4-1BB, CD2, CD40, CD30, CD27, SLAM(signaling lymphocyte activation molecule), 2B4(CD244), NKG2D(natural-killer group 2, member D)/DAP12(DNAX-activating protein 12), TIM1(T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3(lymphocyte activation gene 3), B7-1, B7-H1, GITR(glucocorticoid-induced TNFR family related protein), Flt3 리간드(fms-like tyrosine kinase 3 ligand), 플라젤린(flagellin), HVEM(herpesvirus entry mediator) 또는 OX40L[ligand for CD134(OX40), CD252]의 세포질 도메인 또는 이들 중 둘 이상의 연결체일 수 있다.
상기 융합단백질에 있어서, 상기 링커는 링커 펩타이드인 것이 바람직한데, 이러한 링커 펩타이드에는 (G4S)n(단위체: 서열번호 32, n은 1 내지 10의 정수), (GS)n(n은 1 내지 10의 정수), (GSSGGS)n(단위체: 서열번호 33, n은 1 내지 10의 정수), KESGSVSSEQLAQFRSLD(서열번호 34), EGKSSGSGSESKST(서열번호 35), GSAGSAAGSGEF(서열번호 36), (EAAAK)n(단위체: 서열번호 37, n은 1 내지 10의 정수), CRRRRRREAEAC(서열번호 38), A(EAAAK)4ALEA(EAAAK)4A(서열번호 39), GGGGGGGG(서열번호 40), GGGGGG(서열번호 41), AEAAAKEAAAAKA(서열번호 42), PAPAP(서열번호 43), (Ala-Pro)n(n은 1 내지 10의 정수), VSQTSKLTRAETVFPDV(서열번호 44), PLGLWA(서열번호 45), TRHRQPRGWE(서열번호 46), AGNRVRRSVG(서열번호 47), RRRRRRRR(서열번호 48), GFLG(서열번호 49), 및 GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE(서열번호 50) 등이 포함될 수 있다.
본 발명에서 사용되는 용어 "융합단백질"은 둘 이상의 단백질 또는 단백질 내 특정 기능을 담당하는 도메인이 연결된 재조합 단백질(recombinant protein)을 의미한다. 상기 둘 이상의 단백질 또는 도메인 사이에는 통상적으로 유연한 구조를 갖는 링커(linker) 펩타이드가 삽입될 수 있는데, 연결되는 폴리펩타이드의 본래의 기능을 제한하지 않고, 융합단백질의 발현을 저해하지 않는 유연성 펩타이드 링커라면 어느 것이라도 사용이 가능하며, 구체적인 예시는 상술한 바와 같다.
본 발명의 일 관점에 따른 상기 융합단백질을 암호화하는 폴리뉴클레오타이드가 제공된다.
상기 폴리뉴클레오타이드는 디옥시리보핵산(DNA) 또는 리보핵산(RNA)일 수 있다.
본 발명의 다른 일 관점에 따르면 상기 폴리뉴클레오타이드가 조절서열에 작동가능하게 연결된 발현벡터가 제공된다.
본 문서에서 사용되는 용어 "작동 가능하게 연결된(operably linked to)"이란 목적으로 하는 핵산서열(예컨대, 시험관내 전사/번역 시스템에서 또는 숙주세포에서)이 그의 발현이 이루어질 수 있도록 하는 방식으로 상기 조절서열에 연결되어 있다는 것을 의미한다.
상기 "조절서열"이란 용어는 프로모터, 인핸서 및 다른 조절 요소(예, 폴리아데닐화 신호)를 포함하는 의미이다. 조절서열에는 많은 숙주세포에서 목적으로 하는 핵산이 항상적으로 발현될 수 있도록 지시하는 것, 특정한 조직세포에서만 목적으로 하는 핵산이 발현될 수 있도록 지시하는 것(예, 조직특이적 조절서열), 그리고 특정 신호에 의해 발현이 유도되도록 지시하는 것(예, 유도성 조절서열)이 포함된다. 발현벡터의 설계는 형질전환될 숙주세포의 선택 및 원하는 단백질 발현의 수준 등과 같은 인자에 따라 달라질 수 있다는 것은 당업자라면 이해할 수 있다. 본 발명의 발현벡터는 숙주 세포에 도입되어 상기 융합 단백질을 발현할 수 있다. 상기 진핵세포 및 원핵세포에서 발현을 가능하게 하는 조절서열들은 당업자에게 잘 알려져 있다. 상술한 바와 같이, 이들은 보통 전사개시를 담당하는 조절서열들 및, 선택적으로 전사물의 전사종결 및 안정화를 담당하는 폴리-A 신호를 포함한다. 추가적인 조절서열들은 전사조절인자 외에도 번역 증진인자 및/또는 천연-조합 또는 이종성 프로모터 영역을 포함할 수 있다. 예를 들어 포유류 숙주 세포에서 발현을 가능하게 하는 가능한 조절서열들은 CMV-HSV 티미딘 키나아제 프로모터, SV40, RSV-프로모터(로우스 육종 바이러스), 인간 신장 요소 1α-프로모터, 글루코코르티코이드-유도성 MMTV-프로모터(몰로니 마우스 종양 바이러스), 메탈로티오네인-유도성 또는 테트라사이클린-유도성 프로모터 또는, CMV 증폭제 또는 SV40-증폭제와 같은 증폭제를 포함한다. 신경 세포 내 발현을 위해, 신경미세섬유-프로모터(neurofilament-promoter), PGDF-프로모터, NSE-프로모터, PrP-프로모터 또는 thy-1-프로모터들이 사용될 수 있다는 것이 고려되고 있다. 상기 프로모터들은 당 분야에 알려져 있으며, 문헌(Charron, J. Biol. Chem. 270: 25739-25745, 1995)에 기술되어 있다. 원핵세포내 발현을 위해, lac-프로모터, tac-프로모터 또는 trp 프로모터를 포함하는 다수의 프로모터들이 개시되어 있다. 전사를 개시할 수 있는 인자들 외에, 상기 조절서열들은 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 하류(downstream)에 SV40-폴리-A 부위 또는 TK-폴리-A 부위와 같은 전사 종결 신호를 포함할 수도 있다. 본 문서에서, 적당한 발현 벡터들은 당 분야에 알려져 있으며, 그 예로는 오카야마-베르그(Okayama-Berg) cDNA 발현 벡터 pcDV1(Parmacia), pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1(GIBCO BRL), pGX27(특허 제1442254호), pX(Pagano (1992) Science 255, 1144-1147), 효모 2-혼성(two-hybrid) 벡터, 가령 pEG202 및 dpJG4-5(Gyuris (1995) Cell 75, 791-803) 또는 원핵 발현 벡터, 가령 람다 gt11 또는 pGEX(Amersham-Pharmacia)가 있다. 본 발명의 핵산 분자들 외에, 벡터는 분비신호 펩타이드를 암호화하는 폴리뉴클레오타이드를 추가로 포함할 수 있다. 상기 분비신호 펩타이드들은 당업자에게 잘 알려져 있다. 그리고, 사용된 발현 시스템에 따라, 융합단백질을 세포 구획으로 이끌 수 있는 리더서열(leader sequence)이 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 코딩 서열에 조합되며, 바람직하게는 해독된 단백질 또는 이의 단백질을 세포질 주변 또는 세포외 매질로 직접 분비할 수 있는 리더 서열이다.
또한, 본 발명의 벡터는 예를 들면, 표준 재조합 DNA 기술에 의하여 제조될 수 있으며, 표준 재조합 DNA 기술에는 예를 들면, 평활말단 및 접착말단 라이게이션, 적절한 말단을 제공하기 위한 제한 효소 처리, 부적합한 결합을 방지하기 위하여 알칼리 포스테이즈 처리에 의한 인산기 제거 및 T4 DNA 라이게이즈에 의한 효소적 연결 등이 포함된다. 화학적 합성 또는 유전자 재조합 기술에 의하여 얻어진 신호 펩타이드를 코딩하는 DNA, 본 발명의 일 실시예에 따른 융합단백질을 암호화하는 DNA를 적절한 조절서열이 포함되어 있는 벡터에 재조합함으로써 본 발명의 벡터가 제조될 수 있다. 상기 조절 서열이 포함되어 있는 벡터는 상업적으로 구입 또는 제조할 수 있으며, 본 발명의 일 실시예에서는 DNA 백신 제조용 벡터인 pGX27(한국 등록특허 제1442254호)을 사용하였다.
본 발명의 일 실시예에 따른 상기 발현벡터는 숙주세포에서 상기 융합단백질을 발현하도록 할 수 있는 발현벡터일 수 있으며, 상기 발현벡터는 플라스미드 벡터, 바이러스 벡터, 코스미드 벡터, 파지미드 벡터, 인공 인간 염색체 등 그 어떠한 형태를 나타내더라도 무방하다.
본 발명의 다른 일 관점에 따르면, 상기 융합단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동 가능하게 연결된 발현벡터를 포함하며, 상기 14 타입의 모든 E6/E7 항원 단위체를 각각 암호화하는 폴리뉴클레오타이드를 포함하는 복수의 발현벡터를 포함하는 14가 HPV DNA 백신 조성물이 제공된다.
상기 14가 HPV DNA 백신 조성물은, 상기 14 타입의 HPV의 E6/E7 항원 단위체 4 내지 5개가 연결된 세 개의 융합단백질을 각각 암호화하는 폴리뉴클레오타이드가 발현벡터에 각각 클로닝되어 구축된 세 개의 발현벡터를 포함할 수 있다.
상기 세 개의 발현벡터는 하기와 같이 구성될 수 있으나 이로 제한되는 것이 아니라 다양한 HPV 타입의 조합이 가능하다:
ⅰ) 16형 인유두종바이러스(HPV16)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(16E6N)-E7의 C-말단 단편(16E7C)-E7의 N-말단 단편(16E7N)-E6의 C-말단 단편(16E6C)의 순으로 연결된 HPV16 E6/E7 항원단위체, 18형 인유두종바이러스(HPV18)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(18E6N)-E7의 C-말단 단편(18E7C)-E7의 N-말단 단편(18E7N)-E6의 C-말단 단편(18E6C)의 순으로 연결된 HPV18 E6/E7 항원단위체, 35형 인유두종바이러스(HPV35)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(35E6N)-E7의 C-말단 단편(35E7C)-E7의 N-말단 단편(35E7N)-E6의 C-말단 단편(35E6C)의 순으로 연결된 HPV35 E6/E7 항원단위체, 45형 인유두종바이러스(HPV45)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(45E6N)-E7의 C-말단 단편(45E7C)-E7의 N-말단 단편(45E7N)-E6의 C-말단 단편(45E6C)의 순으로 연결된 HPV45 E6/E7 항원단위체, 및 58형 인유두종바이러스(HPV58)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(58E6N)-E7의 C-말단 단편(58E7C)-E7의 N-말단 단편(58E7N)-E6의 C-말단 단편(58E6C)의 순으로 연결된 HPV58 E6/E7 항원단위체가 링커 펩타이드에 의해 연결된 제1융합단백질을 암호화하는 제1핵산분자가 프로모터에 작동가능하게 연결된 제1유전자컨스트럭트를 포함하는 제1발현벡터;
ⅱ) 31형 인유두종바이러스(HPV31)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(31E6N)-E7의 C-말단 단편(31E7C)-E7의 N-말단 단편(31E7N)-E6의 C-말단 단편(31E6C)의 순으로 연결된 HPV31 E6/E7 항원단위체, 33형 인유두종바이러스(HPV33)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(33E6N)-E7의 C-말단 단편(33E7C)-E7의 N-말단 단편(33E7N)-E6의 C-말단 단편(33E6C)의 순으로 연결된 HPV33 E6/E7 항원단위체, 6형 인유두종바이러스(HPV6)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(6E6N)-E7의 C-말단 단편(6E7C)-E7의 N-말단 단편(6E7N)-E6의 C-말단 단편(6E6C)의 순으로 연결된 HPV6 E6/E7 항원단위체, 11형 인유두종바이러스(HPV11)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(11E6N)-E7의 C-말단 단편(11E7C)-E7의 N-말단 단편(11E7N)-E6의 C-말단 단편(11E6C)의 순으로 연결된 HPV11 E6/E7 항원단위체, 및 52형 인유두종바이러스(HPV52)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(52E6N)-E7의 C-말단 단편(52E7C)-E7의 N-말단 단편(52E7N)-E6의 C-말단 단편(52E6C)의 순으로 연결된 HPV52 E6/E7 항원단위체가 링커 펩타이드에 의해 연결된 제2융합단백질을 암호화하는 제2핵산분자가 프로모터에 작동가능하게 연결된 제2유전자컨스트럭트를 포함하는 제2발현벡터; 및
ⅲ) 39형 인유두종바이러스(HPV39)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(39E6N)-E7의 C-말단 단편(39E7C)-E7의 N-말단 단편(39E7N)-E6의 C-말단 단편(39E6C)의 순으로 연결된 HPV39 E6/E7 항원단위체, 51형 인유두종바이러스(HPV51)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(51E6N)-E7의 C-말단 단편(51E7C)-E7의 N-말단 단편(51E7N)-E6의 C-말단 단편(51E6C)의 순으로 연결된 HPV51 E6/E7 항원단위체, 56형 인유두종바이러스(HPV56)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(56E6N)-E7의 C-말단 단편(56E7C)-E7의 N-말단 단편(56E7N)-E6의 C-말단 단편(56E6C)의 순으로 연결된 HPV56 E6/E7 항원단위체, 및 59형 인유두종바이러스(HPV59)의 조기항원 6(E6) 및 조기항원 7(E7) 각각의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(59E6N)-E7의 C-말단 단편(59E7C)-E7의 N-말단 단편(59E7N)-E6의 C-말단 단편(59E6C)의 순으로 연결된 HPV59 E6/E7 항원단위체가 링커 펩타이드에 의해 연결된 제3융합단백질을 암호화하는 제3핵산분자가 프로모터에 작동가능하게 연결된 제3유전자컨스트럭트를 포함하는 제3발현벡터.
상기 백신 조성물에 있어서, 상기 제1융합단백질 내지 제3융합단백질은 N-말단에 분비 신호서열 및 Flt3L이 부가된 것일 수 있다. 상기 분비 신호서열은 상술한 바와 같다.
상기 백신 조성물은 하나 이상의 약학적으로 허용 가능한 백신 면역보조제를 포함할 수 있다. 상기 백신 면역보조제로는 알루미늄 하이드록사이드, 알루미늄 포스페이트, 알룸(포타슘 알루미늄 설페이트), MF59, virosome, AS04[알루미늄 하이드록사이드 및 모노포스포릴 리피드 A(MPL)의 혼합물], AS03(DL-α-tocopherol, squalene 및 유화제인 polysorbate 80의 혼합물), CpG, Flagellin, Poly I:C, AS01,AS02, ISCOMs 및 ISCOMMATRIX 등이 사용될 수 있다.
본 문서에서 사용되는 용어 "보조제(adjuvant)" 또는 "백신 면역보조제(vaccine adjuvant)"는 백신의 면역반응을 향상시킬 목적으로 투여되는 약학적 또는 면역학적 제제를 의미한다.
아울러 상기 백신 면역보조제는 IL-12 단백질 및 IL-21 단백질을 유효성분으로 포함하거나 상기 IL-12 단백질을 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 암호화하는 폴리뉴클레오타이드를 유효성분으로 포함하는 T 림프구 특이적 면역반응 촉진용 백신 면역보조제일 수 있다.
이 때 상기 백신 면역보조제는 하기로 구성되는 군으로부터 선택되는 하나 이상을 포함할 수 있다:
p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)로 구성된 IL-12 단백질 및 IL-21 단백질;
상기 IL-12를 구성하는 p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)를 각각 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드를 포함하는 하나 내지 세 개의 벡터; 및
상기 IL-12p35, IL-12p40 및 IL-21 단백질을 각각 암호화하는 mRNA 분자.
아울러, 상술한 백신 면역보조제는 상기 IL-21p35, IL-12p40 및 IL-21 중 일부는 단백질로 포함되고, 나머지는 발현벡터 및/또는 mRNA 분자와 같이 이종분자가 혼용되어 사용되는 것도 가능하다.
이때 상기 하나 내지 세 개의 벡터는 상기 IL-12p35, IL-12p40 및 IL-21을 발현시킬 수 있도록 상기 폴리뉴클레오타이드가 프로모터와 같은 조절서열에 작동가능하게 연결된 유전자 컨스트럭트를 포함할 수 있다. 상기 백신 면역보조제는 상기 IL-12p35, IL-12p40 및 IL-21을 각각 암호화하는 폴리뉴클레오타이드들이 개별적인 발현벡터 내에 삽입되거나(3 벡터 시스템) 하나 또는 두 개의 발현벡터 내에 삽입됨으로써(단일벡터 또는 이중벡터 시스템) 하나 내지 세 개의 벡터로 구성될 수 있다. 이러한 단일벡터 내지 삼중벡터 시스템의 구체적인 구현예는 하기와 같다:
ⅰ) 상기 IL-12p35, IL-12p40 및 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드가 프로모터에 각각 작동가능하게 연결된 제4 내지 제6유전자컨스트럭트를 포함하는 제4발현벡터;
ⅱ) 상기 제4 내지 제6유전자컨스트럭트를 각각 포함하는 제5 내지 제7발현벡터
ⅲ) 상기 제4 내지 제6유전자컨스트럭트 중 둘 및 나머지 하나가 각각 포함된 제8발현벡터 및 제9발현벡터;
ⅳ) 상기 IL-12p35 및 IL-12p40 중 어느 하나에 IL-21이 연결된 융합단백질 및 상기 IL-12p35 및 IL-12p40 중 상기 융합단백질에 포함되지 않은 펩타이드;
ⅴ) 상기 ⅳ의 융합단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동 가능하게 연결된 제7유전자컨스트럭트 및 상기 ⅳ의 펩타이드를 암호화하는 폴리뉴클레오타이드가 프로모터에 작동가능하게 연결된 제8유전자컨스트럭트를 포함하는 제10발현벡터;
ⅵ) 상기 제7유전자컨스트럭트 및 제8유전자컨스트럭트를 각각 포함하는 제11발현벡터 및 제12발현벡터;
ⅶ) 상기 IL-12p35, IL-12p40 및 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드 중 적어도 둘 이상이 내부 리보솜 진입 부위(IRES)로 연결된 폴리뉴클레오타이드가 프로모터에 작동가능하게 연결된 제9유전자컨스트럭트; 및 선택적으로 상기 세 폴리뉴클레오타이드 중 상기 제9유전자컨스트럭트에 포함되지 않은 폴리뉴클레오타이드가 프로모터에 작동가능하게 연결된 제10유전자컨스트럭트를 포함하는 제13발현벡터; 및
ⅷ) 상기 제9유전자컨스트럭트를 포함하는 제14발현벡터 및 선택적으로 상기 제10유전자컨스트럭트를 포함하는 제15발현벡터.
상기 백신 조성물에 있어서, 상기 IL-12p35 단백질은 서열번호 1로 기재되는 아미노산 서열로 구성되는 인간 IL-12p35와 서열 상동성 90% 이상, 바람직하게는 95% 이상의 아미노산 서열로 구성될 수 있으며, 인체 내에서 면역반응을 유도하지 않을 수준의 높은 상동성을 가진 비인간 예컨대 영장류나 유인원 유래의 IL-12p35의 사용도 가능하다. 상기 IL-12p40 단백질은 서열번호 2로 기재되는 아미노산 서열로 구성되는 인간 IL-12p40과 서열 상동성 90% 이상, 바람직하게는 95% 이상의 아미노산 서열로 구성될 수 있으며, 인체 내에서 면역반응을 유도하지 않을 수준의 높은 상동성을 가진 비인간 예컨대 영장류나 유인원 유래의 IL-12p40의 사용도 가능하다. 상기 IL-12p35 및 IL-12p40은 한국 등록특허 제0399728호에 기재된 서열 또한 사용 가능하다. 상기 IL-21 단백질은 서열번호 3으로 기재되는 아미노산 서열로 구성되는 인간 IL-21과 서열 상동성 90% 이상, 바람직하게는 95% 이상의 아미노산 서열로 구성될 수 있으며, 인체 내에서 면역반응을 유도하지 않을 수준의 높은 상동성을 가진 비인간 예컨대 영장류나 유인원 유래의 IL-21의 사용도 가능하다.
상기 백신 조성물에 있어서, 상기 백신 보조제는 하기로 구성되는 군으로부터 선택되는 하나 이상을 추가로 포함할 수 있다:
ⅰ) MIP-1α 단백질;
ⅱ) 상기 MIP-1α 단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동 가능하게 연결된 MIP-1α 유전자컨스트럭트; 및
ⅲ) 상기 MIP-1α 단백질을 암호화하는 폴리뉴클레오타이드가 상기 IL-12p35, IL-12p40 및 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드 중 어느 하나 이상에 IRES 또는 링커 펩타이드를 암호화하는 폴리뉴클레오타이드에 의해 작동가능하게 연결된 복합 유전자컨스트럭트; 및
ⅳ) MIP-1α 단백질을 암호화하는 mRNA 분자.
상기 MIP-1α 단백질을 암호화하는 폴리뉴클레오타이드는 상기 IL-12p35, IL-12p40 및 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드에 펩타이드 링커를 암호화하는 폴리뉴클레오타이드 또는 IRES를 통해 작동가능하게 연결되거나 별도의 유전자컨스트럭트 형태로 제공될 수 있다. 아울러 상기 MIP-1α 유전자컨스트럭트는 상기 IL-12를 구성하는 p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)를 각각 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드를 포함하는 하나 내지 세 개의 벡터 중 어느 하나 이상의 벡터 내에 포함될 수 있다. 즉, 상기 MIP-1α 유전자컨스트럭트는 상기 백신 면역보조제의 구현예에 기재된 제4발현벡터 내지 제15발현벡터로 구성되는 군으로부터 선택되는 어느 하나 이상의 발현벡터 내에 포함될 수 있다.
상기 백신 조성물에 있어서, 상기 MIP-1α 단백질은 서열번호 10으로 기재되는 아미노산 서열로 구성되는 MIP-1α 단백질과 서열 상동성 90% 이상, 바람직하게는 95% 이상의 아미노산 서열로 구성될 수 있으며, 인체 내에서 면역반응을 유도하지 않을 수준의 높은 상동성을 가진 비인간 예컨대 영장류나 유인원 유래의 MIP-1α 단백질의 사용도 가능하다.
상기 백신 조성물은 추가로 IL-7을 포함할 수 있다.
상기 백신 조성물은 상기 담체 외에 약학적으로 허용가능한 보조제, 부형제 또는 희석제를 추가적으로 포함할 수 있다.
본 문서에서 사용되는 용어 "약학적으로 허용가능한"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.
상기 백신 조성물은 상기 백신 면역보조제 외에 통상적으로 사용되는 백신 면역보조제를 추가로 포함할 수 있는데 이러한 백신 면역보조제로는 알루미늄 하이드록사이드, 알루미늄 포스페이트, 알룸(포타슘 알루미늄 설페이트), MF59, virosome, AS04[알루미늄 하이드록사이드 및 모노포스포릴 리피드 A(MPL)의 혼합물], AS03(DL-α-tocopherol, squalene 및 유화제인 polysorbate 80의 혼합물), CpG, Flagellin, Poly I:C, AS01,AS02, ISCOMs 및 ISCOMMATRIX 등이 사용될 수 있다.
또한, 본 발명에 일 실시예에 따른 백신 조성물은 포유동물에 투여시, 활성 성분의 신속한 방출, 또는 지속 또는 지연된 방출이 가능하도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말 형태를 포함한다.
본 발명의 일 실시예에 따른 백신 조성물은 다양한 경로로 투여될 수 있으며, 예를 들면, 경구, 비경구, 예를 들면 좌제, 경피, 정맥, 복강, 근육내, 병변내, 비강, 척추관내 투여로 투여될 수 있으며, 또한 서방형 또는 연속적 또는 반복적 방출을 위한 이식장치를 사용하여 투여될 수 있다. 투여횟수는 원하는 범위 내에서 하루에 1회, 또는 수회로 나누어 투여할 수 있으며, 투여 기간도 특별히 한정되지 않는다.
본 발명의 일 실시예에 따른 백신 조성물은 일반적인 전신성 투여 또는 국소성 투여, 예컨대, 근육내 주사 또는 정맥 주사 방식으로 투여될 수 있으나, DNA 백신 조성물로 제공되는 경우, 가장 바람직하게는 전기천공기(electroporator)를 이용하여 주입될 수 있다. 상기 전기천공기는 시판 중인 DNA 약물 체내 주입용 전기천공기, 예컨대, 이탈리아의 IGEA 사의 GlinporatorTM, 한국의 JCBIO사의 CUY21EDIT, 스위스의 Supertech사의 SP-4a, 한국의 SLVAXiGEN사의 OrbiJector 등이 사용될 수 있다.
본 발명에 일 실시예에 따른 백신 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 이와 같은 투여경로는 비경구 투여, 예를 들어, 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 활막강 내 투여될 수 있으나, 이에 제한되지는 않는다.
아울러, 본 발명의 일 실시예에 따른 다가 HPV DNA 백신조성물은 IL-7 단백질 또는 그를 암호화하는 폴리뉴클레오타이드와 병용 투여될 수 있는데, 통상적으로 DNA 약물과 단백질 약물은 투여경로가 다를 수 있기 때문에, 하나의 제형(preparation)으로 투여될 수도 있으나, 별도의 제형으로 포장되어 다른 경로를 통해 투여될 수 있다. 예컨대 DNA 백신 조성물은 전기천공법에 의해 근육내 주입에 의해 투여될 수 있고, IL-7 단백질은 일반적인 근육내 주사 또는 정맥내 투여, 복강내 투여 등 일반적인 단백질 약물의 투여방법에 따라 투여될 수 있다.
본 발명에 일 실시예에 따른 백신 조성물은 일반적으로 사용되는 약학적으로 허용가능한 담체와 함께 적합한 형태로 제형화될 수 있다. 약학적으로 허용되는 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 또한 본 발명에 따른 조성물은 그 투여방법이나 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 산화방지제 등을 적절히 포함할 수 있다. 상기에 예시된 것들을 비롯하여 본 발명에 적합한 약학적으로 허용되는 담체 및 제제는 문헌[Remington's Pharmaceutical Sciences, 최신판]에 상세히 기재되어 있다.
상기 백신 조성물의 환자에 대한 투여량은 환자의 신장, 체표면적, 연령, 투여되는 특정 화합물, 성별, 투여 시간 및 경로, 일반적인 건강, 및 동시에 투여되는 다른 약물들을 포함하는 많은 요소들에 따라 다르다. 약학적으로 활성인 DNA는 100 ng/체중(kg) - 10 ㎎/체중(㎏)의 양으로 투여될 수 있고, 더 바람직하게는 1 내지 500 ㎍/kg(체중)으로 투여될 수 있으며, 가장 바람직하게는 5 내지 50 ㎍/kg(체중)으로 투여될 수 있는데, 상기 요소들을 고려하여 투여량이 조절될 수 있다.
아울러 본 발명의 백신 조성물은 치료적으로 유효한 양으로 투여된다.
본 문서에서 사용되는 용어 "치료적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 백신 조성물은 0.1 mg/kg 내지 1 g/kg의 용량으로 투여될 수 있으며, 더 바람직하게는 1 mg/kg 내지 500 mg/kg의 투여량으로 투여될 수 있으며 단위 용량으로 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg 등으로 투여될 수 있다 한편, 상기 투여량은 환자의 나이, 성별 및 상태에 따라 적절히 조절될 수 있다.
본 발명의 다른 일 관점에 따르면, 상기 다가 HPV DNA 백신 조성물 또는 상기 14가 HPV DNA 백신 조성물을 개체에 투여하는 단계를 포함하는 HPV 감염에 의해 유발되는 질환의 치료방법이 제공된다.
상기 치료방법에 있어서, 상기 HPV 감염에 의해 유발되는 질환은 편평세포암종(SCC), 선암, 선편평세포암종, 소세포암종, 신경내분비 종양(NET), 유리 세포 암종, 융모샘 선암(VGA), 비-암종 악성종양, 흑색종, 림프종, 또는 자궁경부 상피내 종양(CIN)일 수 있다.
상기 치료방법에 있어서, 상기 백신 조성물은 생체내 전기천공법에 의해 투여될 수 있다.
본 발명자들은 종래 HPV 백신들이 자궁경부암 등의 HPV 감염 질환에 범용적으로 적용하지 못한다는 문제점을 해결하기 위해 자궁경부전암 (Cervical intraepithelial neoplasia, CIN), 자궁경부암 (Cervical cancer), 외음부전암 (Vulvar intraepithelial neoplasia, VIN), 외음부암 (Vulvar cancer), 질전암 (Viginal intraepithelial neoplasia), 질암 (Viginal cancer), 사마귀 (Anogenital warts, genital warts)에서 주요 유병률을 나타내어 고위험군에 속하는 6형, 11형, 16형, 18형, 31형, 33형, 35형, 39형, 45형, 51형, 52형, 56형, 58형 및 59형 인간유두종 바이러스(human papilloma virus, HPV)를 모두 커버하기 위한 다가 DNA 백신을 각 HPV의 E6/E7 셔플드 단백질이 4개 또는 5개의 타입당 하나의 거대 융합단백질 형태로 발현될 수 있는 3개의 플라스미드로 구성된 다가 HPV DNA 백신을 제조하였으며, 이를 이용한 동물실험 결과, 대표적인 항원인 HPV16 및 HPV18은 물론 다른 타입의 HPV에 대하여 모두 T 세포 특이적인 면역반응을 유도함을 실험적으로 입증하였다. 이러한 결과는, 원래의 항원 단백질을 사용한 것도 아닌 셔플드 항원단백질을 그것도 여러 개의 셔플드 단백질이 단지 링커에 의해 연결된 다중 셔플드 항원단백질을 발현하는 발현벡터를 이용하여 달성된 것이라는 점에서 매우 고무적인 것이다. 더구나, 놀랍게도 본 발명의 일 실시예에 따른 다가 HPV DNA 백신은 종래 2가 백신에 비해 1/4이라는 매우 낮은 투여량으로 투여되더라도 종래 2가 백신과 유사한 HPV16 및 HPV18에 대한 T 세포 특이적 면역반응을 유도하였을 뿐만 아니라, 6, 11, 39, 45 및 56형의 경우 HPV16이나 HPV18에 대한 것보다도 더 높은 T 세포 특이적 면역반응을 유도하였다. 따라서, 본 발명의 일 실시예에 따른 다가 HPV DNA 백신은 상기 7 타입의 HPV에 대한 감염을 동시에 예방할 수 있는 예방 백신으로서 매우 효율적으로 사용될 수 있을 뿐 아니라, 그 외에 31, 33, 35, 51, 52, 58 및 59형으로 구성되는 군으로부터 선택되는 1종 이상의 고위험성 HPV의 감염 예방 및 감염증 치료를 위한 백신으로 효율적으로 사용될 수 있다.
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, 본 발명이 속한 기술분야의 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다.
실시예 1: 다가 HPV DNA 백신의 제조
본 발명자들은 다가 HPV DNA 백신을 제조하기 위해, 고위험군에 속하는 6형, 11형, 16형, 18형, 31형, 33형, 35형, 39형, 45형, 51형, 52형, 56형, 58형 및 59형 인간유두종 바이러스(human papilloma virus, HPV)의 조기발현 단백질 E6 및 E7 항원을 야생형 E6 및 E7의 기능을 나타내지 않도록 뒤섞은 셔플드 단백질의 형태로 발현시키기 위해 각 타입의 E6 항원 및 E7 항원의 N-말단 단편 및 C-말단 단편을 암호화하는 폴리뉴클레오타이드를 PCR 반응을 통해 수득한 후, 도 1 및 표 1에 개시된 순서와 같이 연결하여 세 개의 유전자컨스트럭트를 제조한 후, 이를 각각 pGX-27 벡터에 삽입함으로써 HPV DNA 백신 컨스트럭트를 제조하였고, 이를 각각 BD-14A, BD-14B 및 BD-14C로 명명하였으며, 상기 세 벡터를 포함하는 조성물을 BD-14로 명명하였다. 상기와 같이 3 벡터 시스템으로 다가 HPV DNA 백신 컨스트럭트를 제조한 이유는 삽입되는 유전자컨스트럭트의 크기가 너무 클 경우 pGX-27 벡터의 용량상 비효율적이기 때문이다.
도 1 및 하기 표 1에서 나타난 바와 같이, 각 타입의 HPV E6 및 E7 항원은 각각 일부 서열(20 a.a)이 중복이 되는 N-말단 단편과 C-말단 단편으로 나뉜 후 E6의 N-말단 단편(E6N) 뒤에 E7의 C-말단 단편(E7C)이 연결된 융합 폴리펩타이드가 (GS)5 링커 펩타이드로 E7의 N-말단 단편(E7N) 뒤에 E6의 C-말단 단편(E6C)이 연결된 융합 폴리펩타이드가 다시 연결된 구조를 가지며, 이러한 각 타입의 항원 단위체 4개 내지 5개가 상기 (GS)5 링커에 연결되어 하나의 벡터에 포함되도록 작제를 하였다. 하나의 발현벡터에 삽입되는 각 서브타입의 종류는 표 1에 예시적으로 기재되어 있으나, 이는 예시적인 것일 뿐 그 어떠한 다른 순서로 제조하더라도 무방하다.
| 구성요소 | 구체적인 구조 | 서열번호 | 기원 | ||
| 단백질 | 핵산 | ||||
| 공통 요소 |
링커 | (GS)5 | 20 | 21 | N/A |
| tPA | tPA1-22 | 22 | 23 | Uniprot: P00750 | |
| Flt3L | Flt3L27-182, △1-26 | 24 | 25 | Uniprot: P49771 | |
| BD-14A | 16 E6E7 | 16E6N1-85-16E7C41-105-(GS)5-16E7N1-60-16E6C66-158 | 26 | 27 | GenBank: K02718.1 |
| 18 E6E7 | 18E6N1-85-18E7C41-98-(GS)5-18E7N1-60-18E6C66-158 | GenBank: X05015.1 | |||
| 35 E6E7 | 35E6N1-78-35E7C42-99-(GS)5-35E7N1-61-35E6C59-149 | GenBank: X74477.1 | |||
| 45 E6E7 | 45E6N1-85-45E7C41-106-(GS)5-45E7N1-60-45E6C66-158 | GenBank: X74479.1 | |||
| 58 E6E7 | 58E6N1-85-58E7C41-98-(GS)5-58E7N1-60-58E6C66-149 | GenBank: D90400.1 | |||
| BD-14B | 31 E6E7 | 31E6N1-85-31E7C42-98-(GS)5-31E7N1-61-31E6C66-149 | 28 | 29 | GenBank: J04353.1 |
| 33 E6E7 | 33E6N1-85-33E7C42-96-(GS)5-33E7N1-61-33E6C66-149 | GenBank: M12732.1 | |||
| 06 E6E7 | 6E6N1-85-6E7C42-98-(GS)5-6E7N1-61-6E6C66-150 | GenBank: X00203.1 | |||
| 11 E6E7 | 11E6N1-85-11E7C42-98-(GS)5-11E7N1-61-11E6C66-150 | GenBank: M14119.1 | |||
| 52 E6E7 | 52E6N1-85-52E7C41-99-(GS)5-52E7N1-60-52E6C66-148 | GenBank: X74481.1 | |||
| BD-14C | 39 E6E7 | 39E6N1-85-39E7C44-109-(GS)5-39E7N1-63-51E6C66-158 | 30 | 31 | GenBank: M62849.1 |
| 51 E6E7 | 51E6N1-83-51E7C45-101-(GS)5-51E7N1-64-51E6C64-151 | Uniprot : P26554(E6), P26558(E7) |
|||
| 56 E6E7 | 56E6N1-86-56E7C48-105-(GS)5-56E7N1-67-56E6C67-155 | Uniprot : P24836(E6), P36833(E7) | |||
| 59 E6E7 | 59E6N1-85-59E7C50-107-(GS)5-59E7N1-69-59E6C66-160 | GenBank: CAA54849.1(E6), CAA54850.1(E7) | |||
실시예 2: 인간 IL-12 및 IL-21 발현 벡터의 제조
2-1: 단일벡터 시스템
본 발명자들은 IL-12 및 IL-21이 하나의 벡터를 통해 발현되도록 단일벡터 시스템을 고안하였다.
이를 위해 구체적으로, 본 발명자들은 인간 IL-12 단백질의 두 소단위체인 서열번호 1로 기재되는 아미노산 서열로 구성되는 hIL-12p35 및 서열번호 2로 기재되는 아미노산 서열로 구성되는 hIL-12p40 폴리펩타이드를 각각 암호화하는 폴리뉴클레오타이드(서열번호 4 및 5)를 서열번호 6으로 기재되는 핵산서열을 갖는 EMCV-유래 내부 리보솜 진입부위(internal ribosome entry site, IRES)로 연결하였고, 상기 hIL-12p40 폴리펩타이드를 암호화하는 폴리뉴클레오타이드의 3'-말단에 서열번호 7로 기재되는 RSV 프로모터(pRSV), 그리고 서열번호 3으로 기재되는 아미노산 서열로 구성되는 인간 IL-21 단백질(hIL-21)을 암호화하는 폴리뉴클레오타이드(서열번호 8)를 순차적으로 연결한 유전자컨스트럭트를 제조한 후, 상기 유전자컨스트럭트를 pGX-27 벡터(한국 등록특허 제1442254호)의 다중클로닝 부위에 삽입하여 본 발명의 일 실시예에 따른 벡터를 제조하고 이를 'hBD-121'으로 명명하였다(도 2a).
1-2: 이중벡터 시스템
본 발명자들은 상기 IL-12 및 IL-21이 별도의 벡터에 삽입되어 발현되도록 이중벡터 시스템을 고안하였다.
상기 이중벡터 시스템은 하기와 같이 제조된다. 상기 hIL-12p35 폴리펩타이드를 암호화하는 폴리뉴클레오타이드(서열번호 1) 및 hIL-12p40 폴리펩타이드를 암호화하는 폴리뉴클레오타이드(서열번호 2)를 서열번호 6으로 기재되는 핵산서열을 갖는 EMCV-IRES에 연결하고 이를 pGX-27 벡터의 다중클로닝 부위에 삽입하고, 마찬가지로 서열번호 3으로 기재되는 아미노산 서열로 구성되는 인간 IL-21 단백질(hIL-21)을 암호화하는 폴리뉴클레오타이드(서열번호 8) 역시 pGX-27 벡터의 다중클로닝 부위에 삽입하여, IL-12 및 IL-21을 각각 발현하는 벡터를 제조한다. 이를 'hBD-12 및 hBD-21'로 명명하였다.
1-3: 삼중벡터 시스템
IL-12의 경우 hIL-12p35 폴리펩티드 및 hIL-12p40 폴리펩티드로 이루어진 이량체 단백질이기 때문에, 상기 hIL-12p35 폴리펩티드 및 hIL-12p40 폴리펩티드는 독립적인 벡터로부터 발현될 수 있다. 이와 같이 본 발명의 일 실시예에 따르면 상기 hIL-12p35 폴리펩티드, hIL-12p40 폴리펩티드 및 IL-21은 각각 독립적으로 구성된 세 개의 벡터를 통해 발현될 수 있다. 이를 본 발명자들은 편의상 '삼중벡터 시스템'으로 명명하였다.
상기 삼중벡터 시스템은 하기와 같이 제조될 수 있다:
상기 hIL-12p35 폴리펩타이드, hIL-12p40 폴리펩타이드 및 hIL-21을 각각 암호화하는 폴리뉴클레오타이드(서열번호 4, 5 및 8)을 pGX-27 벡터의 다중클로닝 부위에 삽입하여 삼중벡터 시스템을 제조한다.
실시예 3: 인간 IL-12, IL-21 및 MIP-α 발현 벡터의 제조
서열번호 1로 기재되는 아미노산 서열로 구성되는 hIL-12p35 폴리펩타이드 및 서열번호 2로 기재되는 아미노산 서열로 구성되는 hIL-12p40 폴리펩타이드를 각각 암호화하는 폴리뉴클레오타이드(서열번호 4 및 5)를 서열번호 6으로 기재되는 핵산서열을 갖는 EMCV-유래 내부 리보솜 진입부위(internal ribosome entry site, IRES)로 연결하였고, 상기 hIL-12p40 폴리펩타이드를 암호화하는 폴리뉴클레오타이드의 3'-말단에 서열번호 7로 기재되는 RSV 프로모터(pRSV), 그리고 서열번호 3으로 기재되는 아미노산 서열로 구성되는 인간 IL-21 단백질(hIL-21)을 암호화하는 폴리뉴클레오타이드(서열번호 8)이 순차적으로 연결된 폴리뉴클레오타이드에 서열번호 9로 기재되는 핵산서열로 구성되는 인간 EF-1α 프로모터(pEF-1α) 및 서열번호 10으로 기재되는 아미노산 서열로 구성되는 인간 MIP-1α 단백질(hMIP-1α)을 암호화하는 폴리뉴클레오타이드(서열번호 11)를 순차적으로 연결한 유전자컨스트럭트를 제조하여 pGX-27 벡터의 다중클로닝 부위에 삽입하였으며, 이를 hBD-121A로 명명하였다(도 2b).
실시예 4: 마우스 IL-12 및 IL-21 발현 벡터의 제조
마우스 IL-12 단백질의 두 소단위체인 서열번호 12로 기재되는 아미노산 서열로 구성되는 mIL-12p35 폴리펩타이드 및 서열번호 13로 기재되는 아미노산 서열로 구성되는 mIL-12p40 폴리펩타이드를 각각 암호화하는 폴리뉴클레오타이드(서열번호 14 및 15)를 서열번호 6으로 기재되는 핵산서열을 갖는 EMCV-유래 내부 리보솜 진입부위(internal ribosome entry site, IRES)로 연결하였고, 상기 mIL-12p40 폴리펩타이드를 암호화하는 폴리뉴클레오타이드의 3'-말단에 서열번호 7로 기재되는 RSV 프로모터(pRSV), 그리고 서열번호 16으로 기재되는 아미노산 서열로 구성되는 마우스 IL-21 단백질(mIL-21)을 암호화하는 폴리뉴클레오타이드(서열번호 17)을 순차적으로 연결한 유전자컨스트럭트를 제조한 후, 상기 유전자컨스트럭트를 pGX-27 벡터의 다중클로닝 부위에 삽입하여 본 발명의 일 실시예에 따른 벡터를 제조하고 이를 'mBD-121'으로 명명하였다(도 2a).
실시예 5: 마우스 IL-12, IL-21 및 MIP-1α 발현 벡터의 제조
서열번호 12로 기재되는 아미노산 서열로 구성되는 mIL-12p35 폴리펩타이드 및 서열번호 13으로 기재되는 아미노산 서열로 구성되는 mIL-12p40 폴리펩타이드를 각각 암호화하는 폴리뉴클레오타이드(서열번호 14 및 15)를 서열번호 6으로 기재되는 핵산서열을 갖는 EMCV-유래 내부 리보솜 진입부위(internal ribosome entry site, IRES)로 연결하였고, 상기 mIL-12p40 폴리펩타이드를 암호화하는 폴리뉴클레오타이드의 3'-말단에 서열번호 7로 기재되는 RSV 프로모터(pRSV), 그리고 서열번호 16으로 기재되는 아미노산 서열로 구성되는 마우스 IL-21 단백질(mIL-21)을 암호화하는 폴리뉴클레오타이드(서열번호 17)이 순차적으로 연결된 폴리뉴클레오타이드에 서열번호 9로 기재되는 핵산서열로 구성되는 인간 EF-1α 프로모터 (pEF-1α) 및 서열번호 18로 기재되는 아미노산 서열로 구성되는 마우스 MIP-1α 단백질(mMIP-1α)을 암호화하는 폴리뉴클레오타이드(서열번호 19)를 순차적으로 연결한 유전자컨스트럭트를 제조하여 pGX-27 벡터의 다중클로닝 부위에 삽입하였으며, 이를 mBD-121A로 명명하였다(도 2b).
실험예 1: BD-14의 발현 분석
1-1: ELISA 분석
본 발명자들은 상기 실시예 1에서 제조된 본 발명의 일 실시예에 따른 다가 HPV DNA 백신인 BD-14가 포유동물 세포에 도입시 정상적으로 발현되는지 여부를 확인하기 위해, 포유동물 세포에 상기 BD-14에 포함되는 세 가지 발현벡터 BD-14A, BD-14B 및 BD-14C를 각각 형질도입한 후 단백질 발현 여부를 각 컨스트럭트에 포함된 Flt3L에 특이적인 항원을 이용하여 분석하였다.
구체적으로 COS-7 세포주를 100 mm culture dish에 접종하여 16시간 배양 후 공벡터(mock plasmid DNA) 및 실시예 1에서 제조된 BD-14A, BD14-B 및 BD-14C plasmid DNA를 Lipofectamine 2000을 이용하여 각각 형질감염시키고, 37℃ CO2 배양기에서 3일 동안 배양 후에 각 조건의 COS-7 세포의 배양 상등액을 회수하여 검체로 사용하였다. 검체 내 존재하는 단백질들은 Flt3L가 결합되어 있는 형태이므로 Flt3L ELISA kit를 이용하여 정량 하였다(도 3a).
그 결과, 도 3a에서 나타난 바와 같이, 상기 BD-14A, BD-14B 및 BD-14C는 모두 잘 발현이 됨을 확인하였다.
1-2: 웨스턴 블랏 분석
본 발명자들은 상기 실험예 1-1에서 수득한 세포의 세포파쇄액(cell lysate)을 대상으로 SDS-PAGE 전기영동을 수행하고, nylon 막으로 전사한 후, 항-Flt3L 항체(Abcam, Cat# ab52648)를 이용하여 웨스턴블랏 분석을 수행하였다(도 3b).
그 결과 도 3b에서 확인되는 바와 같이, BD-14A, BD-14B 및 BD-14C 모두 예상되는 크기의 융합단백질을 정상적으로 발현시킬 수 있는 것으로 확인되었다.
실험예 2: BD-14의 생체내 면역반응 분석
2-1: T 세포 특이적 면역반응 분석
본 발명자들은 본 발명의 일 실시예에 따른 BD-14를 실험동물에 투여한 후 T 세포 특이적인 면역반응을 유발하는지 여부를 각 항원에 대하여 면역반응을 보이는 비장 면역세포의 수를 계수하는 방법을 이용하여 조사하였다. 구체적으로 실험동물인 C57BL/6 마우스를 대조군으로 공 벡터 투여군(n=5), 종래 2가 HPV DNA 백신(대한민국 공개특허 제10-2017-0045254호) 투여군(n=5) 및 본 발명의 일 실시예에 따른 BD-14 투여군(n=5)으로 나누어, 각각 2 ㎍의 플라스미드 DNA(BD-14A 내지 BD-14C의 경우 각각 0.67 ㎍)를 대퇴근육에 OrbiJector (SLVAXiGEN, Korea) 생체내 전기천공기를 이용하여 2주 간격으로 두 차례 투여하였고, 마지막 투여 2주 경과 후 실험동물을 희생시킨 다음 비장을 적출하여 각 타입의 E6/E7 항원에 반응하는 비장 면역세포를 ELISPOT 분석을 이용하여 계수하였다(도 4a 및 4b).
그 결과, 도 4b에 나타난 바와 같이, 본 발명의 일 실시예에 따른 BD-14의 경우 비록 35형, 52형 및 59형 HPV에 대하여는 상대적으로 약하긴 하였으나 모든 타입의 HPV E6/E7 항원에 대한 T 세포 특이적인 면역반응을 유도하는데 성공하였다. 한편, 16형 및 18형 HPV E6/E7 항원에 대한 반응성에 있어서 종래의 2가 DNA 백신과의 큰 차이는 없었다. 이는 본 발명의 일 실시예에 따른 BD-14 내에 16형 및 18형 HPV E6/E7 항원의 비율이 종래 2가 HPV DNA 백신의 1/3 정도에 불과함에도 불구하고 동등한 효력이 가능함을 입증하는 것이다. 특히, 6, 11, 39, 45, 및 56형 HPV에 대해서는 16, 18형 HPV에 의해 유도된 면역반응보다 훨씬 강한 반응이 유도됨을 확인하였다.
2-2: 항-종양 효과 분석
본 발명자들은 본 발명의 일 실시예에 따른 BD-14가 인유두종바이러스의 감염에 의해 유발되는 암의 예방 및 치료에 효과적인지 확인하기 위해 종양모델 동물을 이용한 생체내 항암활성 분석을 수행하였다.
구체적으로, 실험동물인 C57BL/6 마우스를 대조군으로 공 벡터 투여군(n=8), 종래 2가 HPV DNA 백신(대한민국 공개특허 제2017-0045254호) 투여군(n=8) 및 본 발명의 일 실시예에 따른 BD-14 투여군(n=8)으로 나누어, C57BL/6 마우스의 폐상피 세포(lung epithelial cell)에서 유래하였고, HPV16의 E6/E7 항원을 발현하도록 형질전환된 TC-1 세포 5x105 개를 등쪽에 피하주사로 주입하여 종양을 유발시킨 후, 상기 암세포 투여 3일 후에 각각 2 ㎍의 플라스미드 DNA(BD-14A 내지 BD-14C의 경우 각각 0.67 ㎍)를 대퇴근육에 OrbiJector(SLVAXiGEN, Korea) 생체내 전기천공기를 이용하여 2주 간격으로 두 차례 투여하였고, 종양 주입 7일째부터 3일 내지 4일 간격으로 종양의 크기 및 실험동물의 생존률을 조사하였다(도 5a 내지 도 5c).
그 결과, 도 5b에서 나타난 바와 같이, 본 발명의 일 실시예에 따른 BD-14는 대조군에 비해서 종양 크기를 현저하게 감소시켰을 뿐만 아니라, 종래 2가 HPV DNA 백신과 유사한 항종양효능을 보였다. 아울러, 도 5c에서 나타난 바와 같이, 생존률 면에서는 상기 종양 크기 분석결과와 상반되게 본 발명의 일 실시예에 따른 BD-14 투여군의 생존률이 종래 2가 HPV DNA 백신 투여군에 비해 더 높은 것으로 나타났다. 이러한 결과를 종합적으로 검토해보면, 본 발명의 일 실시예에 따른 BD-14 백신 조성물은 HPV16이나 HPV18과 같은 고위험군에 대하여 투여량은 1/3임에도 불구하고 종래의 2가 DNA 백신과 동등하거나 더 나은 항암 활성을 나타낼 뿐만 아니라 다른 타입의 HPV에 대하여도 T 세포 특이적인 면역반응을 유발함으로써 자궁경부암에 대한 예방 범위를 90% 이상으로 높일 수 있는 매우 획기적인 백신 조성물임이 입증된 것이다.
실험예 3: 백신 보조제로서의 BD-121의 효과 분석
3-1: ELISA 분석
본 발명자들은 상기 실시예 2 및 4에서 각각 제조된 본 발명의 일 실시예에 따른 hBD-121 컨스트럭트 및 mBD-121 컨스트럭트를 세포에 형질도입한 후 이들 형질전환 세포에서 IL-12 및 IL-21가 정상적으로 발현되는지 여부를 조사하였다. 구체적으로 COS-7 세포주를 100 mm culture dish에 접종하여 16시간 배양 후 공벡터(mock plasmid DNA) 및 실시예 2-1에서 제조된 hBD-121 plasmid DNA 및 실시예 4에서 제조된 mBD-121 plasmid DNA로 Lipofectamine 2000을 이용하여 각각 형질감염시키고, 37℃ CO2 배양기에서 3일 동안 배양 후에 각 조건의 COS-7 세포의 배양 상등액을 회수하여 검체로 사용하였다. 검체 내 존재하는 IL-12 및 IL-21 단백질들은 각각 IL-12 및 IL-21을 특이적으로 인식하는 항체(IL-12: R&D Systems, Cat# D1200, IL-21: BioLegend, Cat# 433808)를 이용한 ELISA 분석방법을 이용하여 정량하였다(도 6a 및 6b).
그 결과, 도 6a에서 나타난 바와 같이, hBD-121 plasmid DNA를 도입한 검체 내 단백질 발현량은 hIL-12의 경우 4 ㎍ DNA 도입시 4,000 pg/ml을 상회하여 정상적으로 발현됨을 확인하였고, hIL-21 역시 4 ㎍ DNA 도입시 무려 200 ng/ml에 가까운 수치를 나타내 매우 고발현되고 있음을 확인할 수 있었다. 아울러, 도 6b에서 나타난 바와 같이, 마우스 컨스트럭트 역시 인간 컨스트럭트와 유사한 결과를 나타냈다. 한편 공벡터를 도입한 대조군의 경우 양 단백질 모두 전혀 발현되지 않아, 본 발명의 백신 면역보조제 발현 시스템이 정상적으로 작동함을 확인할 수 있었다.
3-2: 웨스턴블랏 분석
본 발명자들은 상기 실험예 3-1에서 수득한 세포의 세포파쇄액을 대상으로 SDS-PAGE 전기영동을 수행한 후, nylon 막으로 전사한 후, 항-IL-12A 항체, 항-IL-12B 항체 및 항-IL-21 항체를 이용하여 웨스턴블랏 분석을 수행하였다(도 6c).
그 결과 도 6c에서 확인되는 바와 같이, IL-12 및 IL-21 모두 본 발명의 일 실시예에 따른 BD-121 플라스미드 DNA의 형질도입에 의해 정상적으로 발현됨을 확인하였다.
실험예 4: 백신 보조제로서의 BD-121A의 효과 분석
4-1: T 세포 특이적 면역반응 분석
본 발명자들은 백신 보조제로서 BD-121A가 BD-14의 백신 성능을 향상시키는지 여부에 대하여 분석을 수행하였다.
이를 위해 본 발명자들은 본 발명의 일 실시예에 따른 BD-14 및 상기 실시예 5에서 제조된 mBD-121A를 실험동물에 투여한 후 T 세포 특이적인 면역반응을 유발하는지 여부를 각 항원에 대하여 면역반응을 보이는 비장 면역세포의 수를 계수하는 방법을 이용하여 조사하였다. 구체적으로 실험동물인 C57BL/6 마우스를 대조군으로 공벡터 투여군(n=3), BD-14 단독투여군(n=5) 및 본 발명의 일 실시예에 따른 BD-14 및 mBD-121A 투여군(n=5)으로 나누어, BD-14 단독투여군의 경우 각 플라스미드 DNA(BD-14A, BD-14B 및 BD-14C) 1.3 ㎍를 그리고 BD-14 및 mBD-121A 복합 투여군의 경우를 각각의 플라스미드 DNA 1 ㎍를 대퇴근육에 OrbiJector(SLVAXiGEN, Korea) 생체내 전기천공기를 이용하여 1회 투여하였고, 투여 2주 경과 후 실험동물을 희생시킨 다음 비장을 적출하여 각 타입의 E6/E7 항원에 반응하는 비장 면역세포를 ELISPOT 분석을 이용하여 계수하였다(도 7a 및 도 7b).
그 결과, 도 7b에서 나타난 바와 같이, 본 발명의 일 실시예에 따른 mBD-121A는 BD-14 단독 투여시보다 다양한 HPV 타입의 E6/E7-특이적인 T 세포반응을 강화시키는 것으로 나타났다. 특히 고위험군인 16형 HPV의 경우 T 세포 면역반응이 두 배 이상 증가하는 것으로 나타났고, 31형, 33형, 51형 및 58형의 경우 현저한 면역반응 상승을 나타냈다. 이는 본 발명의 일 실시예에 따른 BD-121A가 다가 HPV DNA 백신에 대한 매우 효율적인 백신 면역보조제임을 입증하는 결과이다.
4-2: 항-종양 활성분석
구체적으로, 실험동물인 C57BL/6 마우스를 대조군으로 공 벡터 투여군(n=10), 종래 2가 HPV DNA 백신(대한민국 공개특허 제2017-0045254호) 투여군(n=13) 및 본 발명의 일 실시예에 따른 BD-14 및 BD-121 투여군(n=13)으로 나누어, 상기 실험예 2-2에서 사용된 TC-1 세포 5x105 개를 등쪽에 피하주사로 주입하여 종양을 유발시킨 후, 상기 TC-1 세포 접종 7일 후에 총량 기준으로 4 ㎍의 플라스미드 DNA(BD-14A 내지 BD-14C 및 BD-121의 경우 각각 1 ㎍씩)를 대퇴근육에 OrbiJector(SLVAXiGEN, Korea) 생체내 전기천공기를 이용하여 2주 간격으로 두 차례 투여하였고, 종양 접종 7일째부터 3일 내지 4일 간격으로 종양의 크기 및 실험동물의 생존률을 조사하였다(도 8a 내지 도 8c).
그 결과, 도 8b 및 8c에서 확인되는 바와 같이, 본 발명의 일 실시예에 따른 다가 백신은 음성대조군(PBS 투여군)에 비해서 유의미한 항암 효능을 보여 주었으며, 양성 대조군인 종래 2가 백신과 비교하였을 때 동등 또는 동등 이상의 항암 효능을 가짐을 확인할 수 있었다. 이는 투여되는 항원(HPV16의 E6/E7)의 양이 종래 2가 백신보다 1/4 수준이라는 점을 감암할 때, 유사한 항암 효능을 보인 것을 의미함과 동시에 많은 종류(수)의 항원이 투여됨에도 불구하고 다양한 면역반응을 유도할 수 있음은 물론이고 특정 타입(HPV16)에 대한 항암효능이 훼손되지 않음을 의미한다.
실험예 5: 백신의 작용 기전 분석
본 발명자들은 본 발명의 일 실시예에 따른 다가 HPV DNA 백신이 어떤 기전을 통해 작용하는 것인지 조사하기 위해, 항-CD4 또는 항-CD8 항체를 투여하여 각각 CD-4 T 세포 및 CD-8 T 세포가 제거된 실험동물을 제조하여 본 발명의 일 실시예에 따른 HPV DNA 백신의 항암 활성을 비교분석하였다.
구체적으로, 실험동물인 C57BL/6 마우스를 네 그룹으로 나누어 실험을 수행하였는데, 대조군으로 공 벡터 투여군(n=9)을 사용하였고, 본 발명의 일 실시예에 따른 백신 및 백신 보조제(BD14A+BD121A) 투여군은 대조군으로 동형항체(isotype) 투여군(n=9), 항-CD4 항체 투여군(n=9) 및 항-CD8 항체 투여군(n=9)으로 나누어 실험을 수행하였다. 실험스케쥴은 다음과 같다: 상기 실험예 2-2의 TC-1 암세포를 두당 5x105 세포로 피하접종한 후, 항체(isotype, 항-CD4 및 항-CD8 항체)는 암세포 접종일로부터 1일째부터 7일 간격으로 200 ㎍/injection/mouse의 투여량으로 7회 복강투여하였고, 본 발명의 일 실시예에 따른 백신 조성물(BD-14A 및 BD-121)은 암세포 접종일로부터 3일째부터 7일 간격으로 3회 전기천공법을 이용하여 8 ㎍/injection/mouse의 투여량으로 뒷다리 대퇴근에 근육내 투여하였다(도 9a). 종양 접종 9일째부터 3일 내지 4일 간격으로 종양의 크기 및 실험동물의 생존률을 조사하였다(도 9b 내지 도 9c).
그 결과, 도 9b 및 9c에서 확인되는 바와 같이, CD4 T 세포를 제거한 마우스에서는 항암 효능이 유지되었으나, CD8 T 세포가 제거된 마우스에서는 항암 효능이 대조군(mock 및 동형항체)보다 더욱 좋지 않는 결과를 관찰할 수 있었다. 이는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신이 CD8 T 세포를 통해서 항암 면역작용을 나타냄을 보여주는 것으로서, 이는 기존에 알려진대로 항암 치료 백신의 효능에는 CD8 T 세포가 매우 중요하는 것과 일치하는 결과이다.
실험예 6: IL-7과의 병용 투여 효과 분석
IL-7(Interleukin 7)은 다능성 조혈모세포의 림프구성 선조세포로의 분화를 촉진시키는 사이토카인으로 B 세포 및 T 세포의 발달에 중요한 역할을 하는 것으로 알려지고 있다. IL-7은 급성 림프구성 백혈병이나 T 세포 림프종과 같은 혈액암의 악성화를 촉진하는 것으로 알려져 있으나, 일반 고형암에 있어서는 CD8 및 CD4 세포의 항상성을 붕괴시켜 CD4+CD25+Foxp3+ 조절 T 세포의 비율을 감소시키는 것으로 알려지고 있어, 현재 몇몇 암에 대하여 임상 1상 및 2상 시험이 진행중이다. 본 발명자들은 상기 실험예 5의 결과로부터 IL-7이 CD4 T 세포 및 CD8 T 세포 사이의 균형에 영향을 미치고 CD8 T 세포의 증가를 야기하는 경향이 있기 때문에, 본 발명의 일 실시예에 따른 다가 HPV DNA 백신과 병용투여시 상승효과를 기대할 수 있을 것이라는 가설을 수립하였다. 이러한 가설이 맞는지 확인하기 위해 본 발명자들은 본 발명의 일 실시예에 따른 백신 조성물을 단독 또는 IL-7과 병용하여 투여한 후 항암 활성을 분석하였다.
구체적으로, 본 발명자들은 실험동물인 C57BL/6 마우스를 세 그룹으로 나누어서 실험을 수행하였는데, 대조군으로 공 벡터 투여군(n=8), 본 발명의 일 실시예에 따른 백신(BD14) 단독 투여군(n=8), 및 본 발명의 일 실시예에 따른 백신 및 IL-7 투여군(BD14 + IL-7)을 사용하였으며, 상기 실험예 2-2의 TC-1 암세포를 두당 1x105 세포로 질내 주입시켜(i.va) 종양을 유발한 정위적 종양 모델을 제조한 후, 백신 4 ㎍ 또는 상기 백신 4㎍ 및 IL-7 50 ㎍은 암세포 접종일로부터 7일째, 14일째 및 28일째 3회 근육내 투여하였고, 암세포 접종일로부터 7일 간격으로 종양의 부피를 측정하였다(도 10a).
그 결과, 도 10b에서 확인되는 바와 같이, 정위적 종양(orthotopic tumor) 모델을 이용하여 수행한 결과, PBS 투여군과 비교시 BD14 투여에 의해서 항암효능이 유의미하게 향상됨을 확인 할 수가 있었다. 이는 피하주입에 따른 적위적 종양 모델(ectopic tumor model)을 이용한 실험에서 확인된 결과와 일치하는 결과임을 알 수 있었다. 더욱 흥미로운 점은 IL-7과 병용 투여하였을 때 항암 효능은 BD14 단독 투여군보다 유의미하게 향상되었다는 점이다. 이는 본 발명의 일 실시예에 따른 다가 HPV DNA 백신과 T 세포에 대한 작용기전을 갖는 다른 사이토카인과의 병용 가능성을 보여 주는 것이라고 할 수 있다.
상술한 바와 같이, 본 발명의 일 실시예에 따른 다가 HPV DNA 백신인 BD-14는 복잡한 구조에도 불구하고 내부에 포함된 항원 단백질인 E6/E7 셔플드 단백질을 정상적으로 발현하였고, 실제 다양한 타입의 HPV E6/E7 항원에 대한 T 세포 특이적 면역반응을 성공적으로 유도하였으며, 고위험군인 HPV16 E6/E7 항원을 발현하는 암 모델에 대한 항암 활성 분석 결과 종래의 2가 DNA 백신과 동등하거나 더 나은 항암효과를 나타냈다. 더 나아가, 본 발명의 일 실시예에 따른 다가 HPV DNA 백신은 백신 면역 백신보조제로서 본 발명의 일 실시예에 따른 BD-121A와 병용투여시 HPV E6/E7 항원에 대한 T 세포-특이적 면역반응을 현저하게 증가시켰을 뿐만 아니라, 항암효과에 있어서도 더욱 현저한 효과를 나타냈다.
따라서, 본 발명의 일 실시예에 따른 다가 HPV DNA 백신, 그리고 상기 DNA 백신과 BD-121 또는 BD-121A 백신 면역보조제를 포함한 백신조성물은 자궁경부암 등 치명적인 질환을 유발할 위험성을 갖는 다양한 HPV 감염의 예방 및 HPV 감염증의 치료에 매우 효과적으로 사용될 수 있다.
본 발명은 상술한 실시예 및 실험예를 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.
<110> SL VAXIGEN, Inc.
<120> A novel polyvalent HPV vaccine compostion
<130> PD18-5742
<150> KR 10-2018-0013328
<151> 2018-02-02
<160> 50
<170> KoPatentIn 3.0
<210> 1
<211> 253
<212> PRT
<213> Homo sapiens
<400> 1
Met Trp Pro Pro Gly Ser Ala Ser Gln Pro Pro Pro Ser Pro Ala Ala
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Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val
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Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro
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Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
65 70 75 80
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
85 90 95
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
100 105 110
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
115 120 125
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
130 135 140
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
145 150 155 160
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
165 170 175
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
180 185 190
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
195 200 205
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
210 215 220
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
225 230 235 240
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
245 250
<210> 2
<211> 328
<212> PRT
<213> Homo sapiens
<400> 2
Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu
1 5 10 15
Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val
20 25 30
Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu
35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln
50 55 60
Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys
65 70 75 80
Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val
85 90 95
Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
100 105 110
Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe
115 120 125
Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp
130 135 140
Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg
145 150 155 160
Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser
165 170 175
Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
180 185 190
Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile
195 200 205
Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr
210 215 220
Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
225 230 235 240
Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp
245 250 255
Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr
260 265 270
Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg
275 280 285
Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala
290 295 300
Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser
305 310 315 320
Glu Trp Ala Ser Val Pro Cys Ser
325
<210> 3
<211> 155
<212> PRT
<213> Homo sapiens
<400> 3
Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe Leu Gly Thr Leu
1 5 10 15
Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met
20 25 30
Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp
35 40 45
Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys
50 55 60
Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala
65 70 75 80
Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu
85 90 95
Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg
100 105 110
Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu
115 120 125
Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His
130 135 140
Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
145 150 155
<210> 4
<211> 759
<212> DNA
<213> Homo sapiens
<400> 4
atgtggcccc ctgggtcagc ctcccagcca ccgccctcac ctgccgcggc cacaggtctg 60
catccagcgg ctcgccctgt gtccctgcag tgccggctca gcatgtgtcc agcgcgcagc 120
ctcctccttg tggctaccct ggtcctcctg gaccacctca gtttggccag aaacctcccc 180
gtggccactc cagacccagg aatgttccca tgccttcacc actcccaaaa cctgctgagg 240
gccgtcagca acatgctcca gaaggccaga caaactctgg aattttaccc ttgcacttct 300
gaagagattg atcatgaaga tatcacaaaa gataaaacga gcacagtgga ggcctgttta 360
ccattggaat taaccaagaa tgagagttgc ctaaattcca gagagacctc tttcataact 420
aatgggagtt gcctggcctc cagaaagacc tcttttatga tggccctgtg ccttagtagt 480
atttatgaag acttgaagat gtaccaggtg gagttcaaga ccatgaatgc aaagcttctg 540
atggatccta agaggcagat ctttctggat caaaacatgc tggcagttat tgatgagctg 600
atgcaggccc tgaatttcaa cagtgagact gtgccacaaa aatcctccct tgaagaaccg 660
gatttttata aaactaaaat caagctctgc atacttcttc atgctttcag gattcgggca 720
gtgactattg atagagtgat gagctatctg aatgcttcc 759
<210> 5
<211> 987
<212> DNA
<213> Homo sapiens
<400> 5
atgtgccacc agcagctggt catcagctgg ttctccctgg tctttctggc ttctcctctg 60
gtggcaattt gggagctgaa gaaagacgtg tacgtggtcg aactggactg gtatccagat 120
gcccccggag agatggtggt cctgacctgc gacacaccag aggaagatgg catcacttgg 180
accctggacc agagctccga ggtcctggga agcggcaaga cactgactat tcaggtgaaa 240
gaattcgggg atgctggaca gtacacatgt cataagggcg gggaggtgct gtcccactct 300
ctgctgctgc tgcataagaa agaagatggc atctggtcta ctgacattct gaaggatcag 360
aaagagccca agaacaaaac cttcctgaga tgcgaagcca agaattatag cgggaggttt 420
acctgttggt ggctgaccac aatctctact gacctgacct ttagtgtgaa gtctagtagg 480
gggtcaagcg atcctcaggg agtgacctgc ggagcagcta cactgagcgc agagcgggtc 540
agaggagaca acaaggagta cgaatattcc gtggagtgcc aggaagattc tgcatgtccc 600
gcagccgagg aatccctgcc tatcgaagtg atggtggacg ccgtgcacaa gctgaaatac 660
gaaaactaca catcctcttt ctttatccgg gacatcatta agccagatcc ccctaaaaac 720
ctgcagctga agcccctgaa aaattcacga caggtggagg tcagctggga ataccctgat 780
acatggagca ctccacattc ttatttcagt ctgacttttt gcgtgcaggt ccagggcaag 840
agtaaacgag agaagaaaga ccgggtcttc accgataaga catccgctac tgtgatctgt 900
cggaaaaacg ccagtatttc agtgagggct caggaccgct actatagttc aagctggtca 960
gagtgggcaa gcgtgccctg ttcctag 987
<210> 6
<211> 579
<212> DNA
<213> Artificial Sequence
<220>
<223> ECMV IRES
<400> 6
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataatatgg 579
<210> 7
<211> 527
<212> DNA
<213> Artificial Sequence
<220>
<223> RSV promoter
<400> 7
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca 60
acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg 120
ctgcttcgcg atgtacgggc cagatatacg cgtatctgag gggactaggg tgtgtttagg 180
cgaaaagcgg ggcttcggtt gtacgcggtt aggagtcccc tcaggatata gtagtttcgc 240
ttttgcatag ggagggggaa atgtagtctt atgcaatact cttgtagtct tgcaacatgg 300
taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg 360
tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga 420
ttggacgaac cactgaattc cgcattgcag agatattgta tttaagtgcc tagctcgata 480
caataaacgc catttgacca ttcaccacat tggtgtgcac ctccaag 527
<210> 8
<211> 468
<212> DNA
<213> Homo sapiens
<400> 8
atggaacgga ttgtcatttg cctgatggtc atttttctgg gaaccctggt ccacaagtca 60
agcagtcagg gccaggatag gcacatgatt aggatgcgcc agctgatcga cattgtggat 120
cagctgaaga actacgtgaa tgacctggtc cctgagtttc tgcctgcacc agaggatgtc 180
gaaacaaact gcgaatggag cgccttctcc tgttttcaga aggcccagct gaaatccgct 240
aacaccggca acaatgagcg aatcatcaac gtgagcatca agaagctgaa gcggaaaccc 300
cctagcacta atgctgggcg gagacagaaa catagactga cctgcccctc ttgtgacagt 360
tatgaaaaga aaccacccaa ggagttcctg gaacgcttta aaagtctgct gcagaaaatg 420
attcaccagc acctgtcctc cagaactcac gggtccgaag attcctaa 468
<210> 9
<211> 1189
<212> DNA
<213> Artificial Sequence
<220>
<223> hEF-1alpha promoter
<400> 9
cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60
tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120
aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180
gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240
gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300
gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 360
agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 420
gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 480
ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 540
tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 600
tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 660
ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct 720
ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 780
tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 840
aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg 900
gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 960
cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1020
tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1080
ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1140
cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaa 1189
<210> 10
<211> 92
<212> PRT
<213> Homo sapiens
<400> 10
Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala
1 5 10 15
Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala
20 25 30
Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala
35 40 45
Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val Ile Phe
50 55 60
Leu Thr Lys Arg Ser Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp
65 70 75 80
Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala
85 90
<210> 11
<211> 279
<212> DNA
<213> Homo sapiens
<400> 11
atgcaggtgt caaccgccgc cctggctgtc ctgctgtgca ctatggctct gtgcaatcag 60
ttttccgcaa gtctggccgc tgatactccc accgcctgct gtttctctta cacaagtagg 120
cagatccctc agaacttcat tgctgactat tttgagacta gctcccagtg cagcaagccc 180
ggcgtgatct ttctgaccaa gcggagccgg caggtctgtg ccgatccctc cgaagaatgg 240
gtgcagaagt atgtctccga cctggaactg tcagcataa 279
<210> 12
<211> 215
<212> PRT
<213> Mus musculus
<400> 12
Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu
1 5 10 15
Asn His Leu Ser Leu Ala Arg Val Ile Pro Val Ser Gly Pro Ala Arg
20 25 30
Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp Met Val
35 40 45
Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp
50 55 60
Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr
65 70 75 80
Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg
85 90 95
Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr
100 105 110
Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys
115 120 125
Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His
130 135 140
Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp
145 150 155 160
Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys
165 170 175
Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys
180 185 190
Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val
195 200 205
Met Gly Tyr Leu Ser Ser Ala
210 215
<210> 13
<211> 335
<212> PRT
<213> Mus musculus
<400> 13
Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu
1 5 10 15
Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val
20 25 30
Val Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu
35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln
50 55 60
Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys
65 70 75 80
Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr
85 90 95
Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp
100 105 110
Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys
115 120 125
Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln
130 135 140
Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro
145 150 155 160
Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys
165 170 175
Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln
180 185 190
Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu
195 200 205
Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser
210 215 220
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln
225 230 235 240
Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro
245 250 255
Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val
260 265 270
Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys
275 280 285
Asn Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln
290 295 300
Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn
305 310 315 320
Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser
325 330 335
<210> 14
<211> 648
<212> DNA
<213> Mus musculus
<400> 14
atgtgccaga gcagatacct gctgttcctg gccaccctgg ccctgctgaa ccacctgagc 60
ctggccagag tgatccccgt gagcggcccc gccagatgcc tgagccagag cagaaacctg 120
ctgaagacaa ccgacgacat ggtgaagacc gccagagaga agctgaagca ctacagctgc 180
accgccgagg acatcgacca cgaggacatc accagagacc agaccagcac cctgaagacc 240
tgcctgcccc tggagctgca caagaacgag agctgcctgg ccacaagaga gaccagcagc 300
accacaagag gcagctgcct gcctccccag aagaccagcc tgatgatgac cctgtgcctg 360
ggcagcatct acgaggacct gaagatgtac cagaccgagt tccaggccat caacgctgcc 420
ctgcagaacc acaatcacca gcagatcatc ctggacaagg gcatgctggt ggccatcgac 480
gagctgatgc agagcctgaa ccacaacggc gagaccctga gacagaagcc ccctgtgggc 540
gaggccgatc cctacagagt gaagatgaag ctgtgcatcc tgctgcacgc cttcagcacc 600
agagtggtga ccatcaacag agtgatgggc tacctgagca gcgcctga 648
<210> 15
<211> 1008
<212> DNA
<213> Mus musculus
<400> 15
atgtgccccc agaagctgac catcagctgg ttcgccatcg tgctgctggt gagccccctg 60
atggccatgt gggagctgga gaaggacgtg tacgtggtgg aggtggactg gacccccgac 120
gcccccggcg agaccgtgaa cctgacctgc gacacccccg aggaggacga catcacctgg 180
accagcgacc agaggcacgg cgtgatcggc agcggcaaga ccctgaccat caccgtgaag 240
gagttcctgg acgccggcca gtacacctgc cacaagggcg gcgagaccct gagccacagc 300
cacctgctgc tgcacaagaa ggagaacggc atctggagca ccgagatcct gaagaacttc 360
aagaacaaga ccttcctgaa gtgcgaggcc cccaactaca gcggcaggtt cacctgcagc 420
tggctggtgc agaggaacat ggacctgaag ttcaacatca agagcagcag cagcagcccc 480
gacagcaggg ccgtgacctg cggcatggcc agcctgagcg ccgagaaggt gaccctggac 540
cagagggact acgagaagta cagcgtgagc tgccaggagg acgtgacctg ccccaccgcc 600
gaggagaccc tgcccatcga gctggccctg gaggccaggc agcagaacaa gtacgagaac 660
tacagcacca gcttcttcat cagggacatc atcaagcccg acccccccaa gaacctgcag 720
atgaagcccc tgaagaacag ccaggtggag gtgagctggg agtaccccga cagctggagc 780
accccccaca gctacttcag cctgaagttc ttcgtgagaa tccagaggaa gaaggagaag 840
atgaaggaga ccgaggaggg ctgcaaccag aagggcgcct tcctggtgga gaagaccagc 900
accgaggtgc agtgcaaggg cggcaacgtg tgcgtgcagg cccaggacag gtactacaac 960
agcagctgca gcaagtgggc ctgcgtgccc tgcagggtga ggagctaa 1008
<210> 16
<211> 146
<212> PRT
<213> Mus musculus
<400> 16
Met Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val
1 5 10 15
Ala His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu
20 25 30
Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp
35 40 45
Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys
50 55 60
Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser
65 70 75 80
Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu
85 90 95
Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile
100 105 110
Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu
115 120 125
Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His
130 135 140
Leu Ser
145
<210> 17
<211> 441
<212> DNA
<213> Mus musculus
<400> 17
atggagagaa cactggtctg cctcgtggtc atcttcctgg gtactgtggc tcataaatcc 60
tcacctcagg gtcccgatag actgctgatc aggctgcggc acctcatcga cattgtggag 120
cagctcaaaa tctacgaaaa cgacctggac cccgagctgc tctctgcccc ccaggatgtc 180
aaggggcact gcgaacatgc cgctttcgca tgttttcaga aggccaaact gaagcccagc 240
aatcctggca acaataagac cttcatcatt gacctggtgg ctcagctcag gagacggctg 300
ccagcacgac gaggaggaaa gaaacagaaa catatcgcta agtgccctag ctgtgattcc 360
tatgagaaaa gaacaccaaa ggagttcctc gaaaggctca aatggctcct ccagaagatg 420
attcaccagc acctctccta a 441
<210> 18
<211> 92
<212> PRT
<213> Mus musculus
<400> 18
Met Lys Val Ser Thr Thr Ala Leu Ala Val Leu Leu Cys Thr Met Thr
1 5 10 15
Leu Cys Asn Gln Val Phe Ser Ala Pro Tyr Gly Ala Asp Thr Pro Thr
20 25 30
Ala Cys Cys Phe Ser Tyr Ser Arg Lys Ile Pro Arg Gln Phe Ile Val
35 40 45
Asp Tyr Phe Glu Thr Ser Ser Leu Cys Ser Gln Pro Gly Val Ile Phe
50 55 60
Leu Thr Lys Arg Asn Arg Gln Ile Cys Ala Asp Ser Lys Glu Thr Trp
65 70 75 80
Val Gln Glu Tyr Ile Thr Asp Leu Glu Leu Asn Ala
85 90
<210> 19
<211> 279
<212> DNA
<213> Mus musculus
<400> 19
atgaaggtga gcaccaccgc tctggctgtg ctgctctgca ccatgaccct ctgcaaccag 60
gtgttctcag ctccctacgg cgctgatacc cccaccgcct gctgcttcag ctacagccgg 120
aagatccccc ggcagttcat cgtggactac ttcgaaacca gcagcctgtg cagccagccc 180
ggcgtgatct tcctgaccaa acggaaccgg cagatctgcg ctgacagcaa agagacctgg 240
gtgcaggaat acatcaccga cctggaactg aacgcctaa 279
<210> 20
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> (GS)5 linker
<400> 20
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
1 5 10
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding (GS)5 linker
<400> 21
ggatcaggca gtggttcagg atcaggtagt 30
<210> 22
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> tPA signal sequence
<400> 22
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala
20 25
<210> 23
<211> 75
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding tPA signal sequence
<400> 23
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgcc 75
<210> 24
<211> 156
<212> PRT
<213> Artificial Sequence
<220>
<223> Flt3L_27-182
<400> 24
Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala
1 5 10 15
Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val
20 25 30
Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp
35 40 45
Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala
50 55 60
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His
65 70 75 80
Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95
Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu
100 105 110
Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125
Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140
Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
145 150 155
<210> 25
<211> 468
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding Flt3L_27-182
<400> 25
acccaggact gcagcttcca gcacagcccc atcagcagcg acttcgccgt gaagatcaga 60
gagctgagcg actacctgct gcaggactac cccgtgaccg tggccagcaa cctgcaggac 120
gaggagctgt gcggcggcct gtggagactg gtgctggccc agagatggat ggagagactg 180
aagaccgtgg ccggcagcaa gatgcagggc ctgctggaga gagtgaacac cgagatccac 240
ttcgtgacca agtgcgcctt ccagcctccc cccagctgcc tgaggttcgt gcagaccaac 300
atcagcagac tgctgcagga gaccagcgag cagctggtgg ccctgaagcc ctggatcacc 360
agacagaact tcagcagatg cctggagctg cagtgccagc ccgacagcag caccctgccc 420
cctccctgga gccccagacc cctggaggcc accgctccca cagcccct 468
<210> 26
<211> 1957
<212> PRT
<213> Artificial Sequence
<220>
<223> BD-14A polypeptide
<400> 26
Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala
1 5 10 15
Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His
20 25 30
Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp
35 40 45
Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp
50 55 60
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp
65 70 75 80
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu
85 90 95
Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln
100 105 110
Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu
115 120 125
Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr
130 135 140
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser
145 150 155 160
Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala
165 170 175
Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Asp
180 185 190
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val
195 200 205
Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His Ser
210 215 220
Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr
225 230 235 240
Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu
245 250 255
Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met
260 265 270
Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu
275 280 285
Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro
290 295 300
Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu
305 310 315 320
Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg
325 330 335
Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser
340 345 350
Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro
355 360 365
Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met His Gln
370 375 380
Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu
385 390 395 400
Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu
405 410 415
Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp
420 425 430
Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr
435 440 445
Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr
450 455 460
Arg His Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile
465 470 475 480
Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln
485 490 495
Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr
500 505 510
Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Gly Ser Gly Ser
515 520 525
Gly Ser Gly Ser Gly Ser Met His Gly Asp Thr Pro Thr Leu His Glu
530 535 540
Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu
545 550 555 560
Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala
565 570 575
Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys
580 585 590
Cys Lys Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys
595 600 605
Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Val Tyr Gly Thr Thr Leu
610 615 620
Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile
625 630 635 640
Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp
645 650 655
Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys
660 665 670
Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Gly
675 680 685
Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala Arg Phe Glu Asp Pro
690 695 700
Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu Cys Thr Glu Leu Asn Thr
705 710 715 720
Ser Leu Gln Asp Ile Glu Ile Thr Cys Val Tyr Cys Lys Thr Val Leu
725 730 735
Glu Leu Thr Glu Val Phe Glu Phe Ala Phe Lys Asp Leu Phe Val Val
740 745 750
Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His Lys Cys Ile Asp Phe
755 760 765
Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr Ser Asp Ser Val Ile Asp
770 775 780
Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg
785 790 795 800
His Thr Met Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu
805 810 815
Val Val Glu Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe
820 825 830
Leu Ser Thr Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Gly
835 840 845
Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Tyr Gly Pro Lys Ala Thr
850 855 860
Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn Glu Ile Pro Val
865 870 875 880
Asp Leu Leu Cys His Glu Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp
885 890 895
Glu Ile Asp Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu
900 905 910
Pro Gln Arg His Thr His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg
915 920 925
Glu Leu Arg His Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys
930 935 940
Leu Thr Asn Thr Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys
945 950 955 960
Gln Lys Pro Leu Asn Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys
965 970 975
Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser
980 985 990
Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr
995 1000 1005
Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Leu Glu Met Phe
1010 1015 1020
Gln Asp Pro Ala Glu Arg Pro Tyr Lys Leu His Asp Leu Cys Asn Glu
1025 1030 1035 1040
Val Glu Glu Ser Ile His Glu Ile Cys Leu Asn Cys Val Tyr Cys Lys
1045 1050 1055
Gln Glu Leu Gln Arg Ser Glu Val Tyr Asp Phe Ala Cys Tyr Asp Leu
1060 1065 1070
Cys Ile Val Tyr Arg Glu Gly Gln Pro Tyr Gly Val Cys Met Lys Cys
1075 1080 1085
Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg Trp Pro Ala Gly Gln
1090 1095 1100
Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile Val Thr Ser Cys Cys Lys
1105 1110 1115 1120
Cys Glu Ala Thr Leu Arg Leu Cys Val Gln Ser Thr His Ile Asp Ile
1125 1130 1135
Arg Lys Leu Glu Asp Leu Leu Met Gly Thr Phe Gly Ile Val Cys Pro
1140 1145 1150
Gly Cys Ser Gln Arg Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
1155 1160 1165
Met His Gly Glu Ile Thr Thr Leu Gln Asp Tyr Val Leu Asp Leu Glu
1170 1175 1180
Pro Glu Ala Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Cys Asp Ser Ser
1185 1190 1195 1200
Glu Glu Glu Glu Asp Thr Ile Asp Gly Pro Ala Gly Gln Ala Lys Pro
1205 1210 1215
Asp Thr Ser Asn Tyr Asn Ile Val Thr Ser Cys Cys Lys Pro Tyr Gly
1220 1225 1230
Val Cys Met Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg
1235 1240 1245
Trp Tyr Arg Tyr Ser Val Tyr Gly Glu Thr Leu Glu Lys Gln Cys Asn
1250 1255 1260
Lys Gln Leu Cys His Leu Leu Ile Arg Cys Ile Thr Cys Gln Lys Pro
1265 1270 1275 1280
Leu Cys Pro Val Glu Lys Gln Arg His Leu Glu Glu Lys Lys Arg Phe
1285 1290 1295
His Asn Ile Gly Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Trp Lys
1300 1305 1310
Pro Thr Arg Arg Glu Thr Glu Val Gly Ser Gly Ser Gly Ser Gly Ser
1315 1320 1325
Gly Ser Val Glu Gly Ser Met Ala Arg Phe Asp Asp Pro Lys Gln Arg
1330 1335 1340
Pro Tyr Lys Leu Pro Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln
1345 1350 1355 1360
Asp Val Ser Ile Ala Cys Val Tyr Cys Lys Ala Thr Leu Glu Arg Thr
1365 1370 1375
Glu Val Tyr Gln Phe Ala Phe Lys Asp Leu Cys Ile Val Tyr Arg Asp
1380 1385 1390
Cys Ile Ala Tyr Ala Ala Cys His Lys Cys Ile Asp Phe Tyr Ser Arg
1395 1400 1405
Ile Arg Glu Leu Arg Tyr Tyr Ser Asn Ser Val Glu Ala Asp Gly Val
1410 1415 1420
Ser His Ala Gln Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Lys
1425 1430 1435 1440
Ile Leu Cys Val Cys Cys Lys Cys Asp Gly Arg Ile Asp Leu Thr Val
1445 1450 1455
Glu Ser Ser Ala Asp Asp Leu Arg Thr Leu Gln Gln Leu Phe Leu Ser
1460 1465 1470
Thr Leu Ser Phe Val Cys Pro Trp Cys Ala Thr Asn Gln Gly Ser Gly
1475 1480 1485
Ser Gly Ser Gly Ser Gly Ser Met His Gly Pro Arg Ala Thr Leu Gln
1490 1495 1500
Glu Ile Val Leu His Leu Glu Pro Gln Asn Glu Leu Asp Pro Val Asp
1505 1510 1515 1520
Leu Leu Cys Tyr Glu Gln Leu Ser Glu Ser Glu Glu Glu Asn Asp Glu
1525 1530 1535
Ala Asp Gly Val Ser His Ala Gln Leu Pro Ala Arg Arg Ala Glu Pro
1540 1545 1550
Gln Arg His His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu
1555 1560 1565
Arg Tyr Tyr Ser Asn Ser Val Tyr Gly Glu Thr Leu Glu Lys Ile Thr
1570 1575 1580
Asn Thr Glu Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys
1585 1590 1595 1600
Pro Leu Asn Pro Ala Glu Lys Arg Arg His Leu Lys Asp Lys Arg Arg
1605 1610 1615
Phe His Ser Ile Ala Gly Gln Tyr Arg Gly Gln Cys Asn Thr Cys Cys
1620 1625 1630
Asp Gln Ala Arg Gln Glu Arg Leu Arg Arg Arg Arg Glu Thr Gln Val
1635 1640 1645
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Phe Gln Asp Ala Glu
1650 1655 1660
Glu Lys Pro Arg Thr Leu His Asp Leu Cys Gln Ala Leu Glu Thr Ser
1665 1670 1675 1680
Val His Glu Ile Glu Leu Lys Cys Val Glu Cys Lys Lys Thr Leu Gln
1685 1690 1695
Arg Ser Glu Val Tyr Asp Phe Val Phe Ala Asp Leu Arg Ile Val Tyr
1700 1705 1710
Arg Asp Gly Asn Pro Phe Ala Val Cys Lys Val Cys Leu Arg Leu Leu
1715 1720 1725
Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser Leu Tyr Gly Arg
1730 1735 1740
Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile Val Thr
1745 1750 1755 1760
Cys Cys Tyr Thr Cys Asp Thr Thr Val Arg Leu Cys Ile Asn Ser Thr
1765 1770 1775
Thr Thr Asp Val Arg Thr Leu Gln Gln Leu Leu Met Gly Thr Cys Thr
1780 1785 1790
Ile Val Cys Pro Ser Cys Ala Gln Gln Gly Ser Gly Ser Gly Ser Gly
1795 1800 1805
Ser Gly Ser Met Arg Gly Asn Asn Pro Thr Leu Arg Glu Tyr Ile Leu
1810 1815 1820
Asp Leu His Pro Glu Pro Thr Asp Leu Phe Cys Tyr Glu Gln Leu Cys
1825 1830 1835 1840
Asp Ser Ser Asp Glu Asp Glu Ile Gly Leu Asp Arg Pro Asp Gly Gln
1845 1850 1855
Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile Val Thr Cys Cys Tyr Cys
1860 1865 1870
Leu Arg Leu Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser
1875 1880 1885
Leu Tyr Gly Asp Thr Leu Glu Gln Thr Leu Lys Lys Cys Leu Asn Glu
1890 1895 1900
Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro Leu Cys Pro Gln Glu
1905 1910 1915 1920
Lys Lys Arg His Val Asp Leu Asn Lys Arg Phe His Asn Ile Ser Gly
1925 1930 1935
Arg Trp Thr Gly Arg Cys Ala Val Cys Trp Arg Pro Arg Arg Arg Gln
1940 1945 1950
Thr Gln Val Gly Ser
1955
<210> 27
<211> 5304
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding BD-14A polypeptide
<400> 27
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120
gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180
agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240
tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300
aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420
aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480
agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540
cctggcagcg ggtccggaag tgggtctgga tctatgcacc agaagagaac cgccatgttc 600
caggaccccc aggagagacc cagaaagctg ccccagctgt gcaccgagct gcagaccaca 660
atccacgaca tcatcctgga gtgcgtgtac tgcaagcagc agctgctgag aagagaggtg 720
tacgacttcg ccttcagaga cctgtgcatc gtgtacagag atggcaaccc ttatgctgtc 780
tgtgataaat gtctcaaatt ttattccaaa attagtgaat ataggcatcc agcaggacag 840
gctgaaccag atagggctca ttataatatt gtcacatttt gttgtaaatg cgacagcacc 900
ctgagactgt gcgtgcagag cacccacgtg gacatcagaa ccctggagga cctgctgatg 960
ggcaccctgg gcatcgtgtg ccccatctgc agccagaagc ctggcagcgg ctctggctcc 1020
ggcagtggct caatgcacgg cgacacaccc accctgcacg agtacatgct ggacctgcag 1080
cccgagacta ccgacctgta ctgctacgag cagctgaacg acagcagcga ggaagaggac 1140
gagatcgacg gccctgctgg ccaggccgag cccgacagag cccactacaa catcgtgacc 1200
ttctgctgca agccctacgc cgtgtgcgac aagtgcctga agttctacag caagatcagc 1260
gagtacagac actactgcta cagcgtgtac ggcaccaccc tggagcagca gtacaacaag 1320
cccctgtgcg acctgctgat cagatgcatc aactgccaga agcccctgtg ccccgaggag 1380
aagcagagac acctggacaa gaagcagaga ttccacaaca tcagaggcag atggaccggc 1440
agatgcatga gctgctgcag aagcagcaga accagaagag agacccagct gggatctggc 1500
agtggatctg gaagcggctc tatggccaga ttcgaagatc ccaccagaag accctacaag 1560
ctgcccgacc tgtgcaccga gctgaacacc agcctgcagg acatcgagat cacctgcgtg 1620
tactgcaaga ccgtgctgga gctgaccgag gtgttcgagt tcgccttcaa ggacctgttc 1680
gtggtgtaca gagacagcat cccccacgct gcctgccata aatgtattga tttttattcc 1740
aggattaggg aactcaggca ttatagtgat tctgtcattg atggtgtcaa tcatcagcat 1800
ctcccagcta ggagggctga acctcagagg cataccatgc tgtgcatgtg ctgcaagtgc 1860
gaggccagaa tcgagctggt ggtggagagc agcgccgacg acctgagagc cttccagcag 1920
ctgttcctga gcaccctgag cttcgtgtgc ccctggtgcg ccagccagca gggctcagga 1980
tctggcagcg gaagtggatc tatgtacggc cccaaggcta ccctgcagga catcgtgctg 2040
cacctggagc cccagaacga gatccccgtg gacctgctgt gccacgagca gctgagcgac 2100
agcgaggaag aaaacgacga gatcgacggc gtgaaccacc agcacctgcc tgccagaaga 2160
gccgagcccc agagacacac ccacaagtgc atcgacttct acagcagaat cagagagctg 2220
agacactaca gcgacagcgt gtacggcgac accctggaga agctgaccaa caccggcctg 2280
tacaacctgc tgatcagatg cctgagatgc cagaagcccc tgaaccctgc cgagaagctg 2340
agacacctga acgagaagag aagattccac aacatcgccg gccactacag aggccagtgc 2400
cacagctgct gcaacagagc cagacaggag agactgcaga gaagaagaga gacccaggtg 2460
ggatctggca gcggctctgg ctccggctca ctcgagatgt tccaggaccc tgccgaaaga 2520
ccctacaagc tgcatgatct gtgcaatgaa gtcgaagaga gtatccatga aatctgtctg 2580
aattgcgtgt actgtaagca ggagctgcag cgcagtgaag tctacgactt cgcctgctat 2640
gacctgtgca tcgtgtaccg agagggacag ccatatggcg tctgcatgaa gtgtctgaag 2700
ttctactcta agatcagtga atataggtgg ccagccggcc aggctaaacc cgacacttcc 2760
aactataata ttgtgacctc ttgctgtaaa tgcgaggcta ccctgagact gtgcgtgcag 2820
agcacacaca tcgacattag gaagctggag gacctgctga tggggacctt cggaatcgtg 2880
tgcccaggat gttcccagcg agctggatct ggcagtgggt caggaagcgg ctccatgcat 2940
ggagagatta ccacactgca ggactacgtc ctggatctgg agcctgaagc aactgacctg 3000
tactgctatg aacagctgtg cgatagctcc gaggaagagg aagacaccat cgatggccct 3060
gcagggcagg ccaagccaga tacaagtaac tacaacatcg tgacttcatg ctgtaaaccc 3120
tacggcgtct gcatgaaatg tctgaaattc tactcaaaga tcagcgagta tcggtggtac 3180
agatatagcg tgtacgggga gacactggaa aagcagtgca acaaacagct gtgccacctg 3240
ctgatccggt gcattacttg tcagaagccc ctgtgccctg tggagaaaca gcgacacctg 3300
gaggaaaaga aacggtttca taatattggc gggaggtgga caggccgctg catgagctgt 3360
tggaagccta ccagacggga gaccgaagtg ggcagcggca gtgggagcgg aagcgggagt 3420
gtcgagggat ctatggccag attcgacgac cccaagcaga gaccctacaa gctgcccgac 3480
ctgtgcaccg agctgaacac cagcctgcag gacgtgagca tcgcctgcgt gtactgcaag 3540
gccaccctgg agagaaccga ggtgtaccag ttcgccttca aggacctgtg catcgtgtac 3600
agagactgca tcgcctacgc cgcctgccat aaatgtattg atttttattc caggattcgg 3660
gagctccgct attattctaa tagtgtcgaa gctgatggag tcagtcatgc tcagctccct 3720
gctcggaggg cagaacctca gaggcataag atcctgtgcg tgtgctgcaa gtgcgacggc 3780
agaatcgacc tgaccgtgga gagcagcgcc gacgacctga gaaccctgca gcagctgttc 3840
ctgagcaccc tgagcttcgt gtgcccctgg tgcgccacca accagggcag cggaagcgga 3900
agcggcagcg gcagcatgca cggccccaga gccaccctgc aggagatcgt gctgcacctg 3960
gagccccaga acgagctgga ccccgtggac ctgttgtgct acgagcagct gagcgaaagc 4020
gaggaagaga acgacgaggc cgacggcgtg agccacgccc agctgcccgc cagaagagcc 4080
gagccccaga gacaccacaa gtgcatcgac ttctacagca gaatcagaga gctgagatac 4140
tacagcaaca gcgtgtacgg cgagaccctg gagaagatca ccaacaccga gctgtacaac 4200
ctgttgatca gatgcctgag atgccagaag cccctgaacc ccgccgagaa gagaagacac 4260
ctgaaggaca agagaagatt ccacagcatc gccggccagt acagaggcca gtgcaacacc 4320
tgctgcgacc aggccagaca ggagagactg agaaggagga gagagaccca ggtgggatca 4380
ggaagtggat ctgggtccgg cagcatgttc caggacgccg aggagaagcc cagaaccctg 4440
cacgacctgt gccaggccct ggagaccagc gtgcacgaga tcgagctgaa gtgcgtggag 4500
tgcaagaaga ccctgcagag aagcgaggtg tatgacttcg tgttcgccga cctgagaatc 4560
gtgtatagag acggcaaccc cttcgccgtg tgcaaggtgt gtttgaggct cctctccaaa 4620
atttctgaat atcggcatta taactattcc ctctatggaa ggcctgatgg acaggctcag 4680
ccagctacag caaattatta tattgtcaca tgttgctata cctgcgacac caccgtgaga 4740
ctgtgcatca acagcaccac aaccgacgtg agaaccctgc agcagctgct gatgggcacc 4800
tgcaccatcg tgtgccccag ctgcgcccag cagggctcag gcagcggctc cggcagcgga 4860
tctatgagag gcaacaaccc caccctgaga gagtacatcc tggacctgca ccccgagccc 4920
accgacctgt tctgctacga gcagctgtgc gacagcagcg acgaggacga gatcggcctg 4980
gacagacccg acggccaggc ccagcccgcc accgccaact actacatcgt gacctgctgc 5040
tactgcctga gactgctgag caagatcagc gagtacagac actacaacta cagcctgtac 5100
ggcgacaccc tggagcagac cctgaagaag tgcctgaacg agatcctgat cagatgcatc 5160
atctgccaga gacccctgtg cccccaggag aagaagagac acgtggacct gaacaagaga 5220
ttccacaaca tcagcggcag atggaccggc agatgcgccg tgtgctggag acccagaagg 5280
agacagaccc aggtgggatc ctaa 5304
<210> 28
<211> 1909
<212> PRT
<213> Artificial Sequence
<220>
<223> BD-14B polypeptide
<400> 28
Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala
1 5 10 15
Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His
20 25 30
Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp
35 40 45
Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp
50 55 60
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp
65 70 75 80
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu
85 90 95
Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln
100 105 110
Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu
115 120 125
Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr
130 135 140
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser
145 150 155 160
Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala
165 170 175
Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Asp
180 185 190
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val
195 200 205
Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His Ser
210 215 220
Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr
225 230 235 240
Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu
245 250 255
Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met
260 265 270
Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu
275 280 285
Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro
290 295 300
Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu
305 310 315 320
Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg
325 330 335
Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser
340 345 350
Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro
355 360 365
Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Phe Lys
370 375 380
Asn Pro Ala Glu Arg Pro Arg Lys Leu His Glu Leu Ser Ser Ala Leu
385 390 395 400
Glu Ile Pro Tyr Asp Glu Leu Arg Leu Asn Cys Val Tyr Cys Lys Gly
405 410 415
Gln Leu Thr Glu Thr Glu Val Leu Asp Phe Ala Phe Thr Asp Leu Thr
420 425 430
Ile Val Tyr Arg Asp Asp Thr Pro Tyr Gly Val Cys Thr Lys Cys Leu
435 440 445
Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr Arg Tyr Ser Val
450 455 460
Tyr Gly Pro Ala Gly Gln Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile
465 470 475 480
Val Thr Phe Cys Cys Gln Cys Glu Ser Thr Leu Arg Leu Cys Val Gln
485 490 495
Ser Thr Gln Val Asp Ile Arg Ile Leu Gln Glu Leu Leu Met Gly Ser
500 505 510
Phe Gly Ile Val Cys Pro Asn Cys Ser Thr Arg Leu Gly Ser Gly Ser
515 520 525
Gly Ser Gly Ser Gly Ser Met Arg Gly Glu Thr Pro Thr Leu Gln Asp
530 535 540
Tyr Val Leu Asp Leu Gln Pro Glu Ala Thr Asp Leu His Cys Tyr Glu
545 550 555 560
Gln Leu Pro Asp Ser Ser Asp Glu Glu Asp Val Ile Asp Ser Pro Ala
565 570 575
Gly Gln Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile Val Thr Phe Cys
580 585 590
Cys Gln Cys Leu Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr
595 600 605
Arg Tyr Ser Val Tyr Gly Thr Thr Leu Glu Lys Leu Thr Asn Lys Gly
610 615 620
Ile Cys Asp Leu Leu Ile Arg Cys Ile Thr Cys Gln Arg Pro Leu Cys
625 630 635 640
Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Lys Arg Phe His Asn
645 650 655
Ile Gly Gly Arg Trp Thr Gly Arg Cys Ile Val Cys Trp Arg Arg Pro
660 665 670
Arg Thr Glu Thr Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
675 680 685
Met Phe Gln Asp Thr Glu Glu Lys Pro Arg Thr Leu His Asp Leu Cys
690 695 700
Gln Ala Leu Glu Thr Thr Ile His Asn Ile Glu Leu Gln Cys Val Glu
705 710 715 720
Cys Lys Asn Pro Leu Gln Arg Ser Glu Val Tyr Asp Phe Ala Phe Ala
725 730 735
Asp Leu Thr Val Val Tyr Arg Glu Gly Asn Pro Phe Gly Ile Cys Lys
740 745 750
Leu Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn
755 760 765
Tyr Ser Val Tyr Gly Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asp
770 775 780
Tyr Tyr Ile Val Thr Cys Cys His Thr Cys Asn Thr Thr Val Arg Leu
785 790 795 800
Cys Val Asn Ser Thr Ala Ser Asp Leu Arg Thr Ile Gln Gln Leu Leu
805 810 815
Met Gly Thr Val Asn Ile Val Cys Pro Thr Cys Ala Gln Leu Gly Ser
820 825 830
Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly His Lys Pro Thr Leu
835 840 845
Lys Glu Tyr Val Leu Asp Leu Tyr Pro Glu Pro Thr Asp Leu Tyr Cys
850 855 860
Tyr Glu Gln Leu Ser Asp Ser Ser Asp Glu Asp Glu Gly Leu Asp Arg
865 870 875 880
Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asp Tyr Tyr Ile Val Thr
885 890 895
Cys Cys His Thr Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg
900 905 910
His Tyr Asn Tyr Ser Val Tyr Gly His Thr Leu Glu Gln Thr Val Lys
915 920 925
Lys Pro Leu Asn Glu Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro
930 935 940
Leu Cys Pro Gln Glu Lys Lys Arg His Val Asp Leu Asn Lys Arg Phe
945 950 955 960
His Asn Ile Ser Gly Arg Trp Ala Gly Arg Cys Ala Ala Cys Trp Arg
965 970 975
Ser Arg Arg Arg Glu Thr Ala Leu Gly Ser Gly Ser Gly Ser Gly Ser
980 985 990
Gly Ser Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp
995 1000 1005
Gln Leu Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn
1010 1015 1020
Cys Val Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr
1025 1030 1035 1040
Ala Tyr Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala
1045 1050 1055
Ala Cys Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg
1060 1065 1070
His Phe Asp Tyr Ala Gly Tyr Asp Gly Gln Asp Ser Gln Pro Leu Lys
1075 1080 1085
Gln His Tyr Gln Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn Val
1090 1095 1100
Arg Leu Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln Gln
1105 1110 1115 1120
Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys
1125 1130 1135
Thr Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met His Gly Arg His
1140 1145 1150
Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val
1155 1160 1165
Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val
1170 1175 1180
Asp Glu Val Asp Gly Gln Asp Ser Gln Pro Leu Lys Gln His Tyr Gln
1185 1190 1195 1200
Ile Val Thr Cys Cys Cys Gly Cys Cys Leu Glu Phe His Gly Lys Ile
1205 1210 1215
Asn Gln Tyr Arg His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu
1220 1225 1230
Glu Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu
1235 1240 1245
Cys His Lys Pro Leu Cys Glu Val Glu Lys Val Lys His Ile Leu Thr
1250 1255 1260
Lys Ala Arg Phe Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu
1265 1270 1275 1280
His Cys Trp Thr Thr Cys Met Glu Asp Met Leu Pro Gly Ser Gly Ser
1285 1290 1295
Gly Ser Gly Ser Gly Ser Met Glu Ser Lys Asp Ala Ser Thr Ser Ala
1300 1305 1310
Thr Ser Ile Asp Gln Leu Cys Lys Thr Phe Asn Leu Ser Leu His Thr
1315 1320 1325
Leu Gln Ile Gln Cys Val Phe Cys Arg Asn Ala Leu Thr Thr Ala Glu
1330 1335 1340
Ile Tyr Ala Tyr Ala Tyr Lys Asn Leu Lys Val Val Trp Arg Asp Asn
1345 1350 1355 1360
Phe Pro Phe Ala Ala Cys Ala Cys Cys Leu Glu Leu Gln Gly Lys Ile
1365 1370 1375
Asn Gln Tyr Arg His Phe Asn Tyr Ala Ala Tyr Asp Lys Gln Asp Ser
1380 1385 1390
Gln Pro Leu Thr Gln His Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys
1395 1400 1405
Asp Ser Asn Val Arg Leu Val Val Glu Cys Thr Asp Gly Asp Ile Arg
1410 1415 1420
Gln Leu Gln Asp Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile
1425 1430 1435 1440
Cys Ala Pro Lys Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met
1445 1450 1455
His Gly Arg Leu Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln Pro
1460 1465 1470
Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu Glu Asp Ser Ser
1475 1480 1485
Glu Asp Glu Val Asp Lys Val Asp Lys Gln Asp Ser Gln Pro Leu Thr
1490 1495 1500
Gln His Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys Cys Leu Glu Leu
1505 1510 1515 1520
Gln Gly Lys Ile Asn Gln Tyr Arg His Phe Asn Tyr Ala Ala Tyr Ala
1525 1530 1535
Pro Thr Val Glu Glu Glu Thr Asn Glu Asp Ile Leu Lys Val Leu Ile
1540 1545 1550
Arg Cys Tyr Leu Cys His Lys Pro Leu Cys Glu Ile Glu Lys Leu Lys
1555 1560 1565
His Ile Leu Gly Lys Ala Arg Phe Ile Lys Leu Asn Asn Gln Trp Lys
1570 1575 1580
Gly Arg Cys Leu His Cys Trp Thr Thr Cys Met Glu Asp Leu Leu Pro
1585 1590 1595 1600
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Phe Glu Asp Pro Ala
1605 1610 1615
Thr Arg Pro Arg Thr Leu His Glu Leu Cys Glu Val Leu Glu Glu Ser
1620 1625 1630
Val His Glu Ile Arg Leu Gln Cys Val Gln Cys Lys Lys Glu Leu Gln
1635 1640 1645
Arg Arg Glu Val Tyr Lys Phe Leu Phe Thr Asp Leu Arg Ile Val Tyr
1650 1655 1660
Arg Asp Asn Asn Pro Tyr Gly Val Cys Ile Met Cys Leu Arg Phe Leu
1665 1670 1675 1680
Ser Lys Ile Ser Glu Tyr Arg His Tyr Gln Tyr Ser Leu Tyr Gly Asp
1685 1690 1695
Arg Pro Asp Gly Gln Ala Glu Gln Ala Thr Ser Asn Tyr Tyr Ile Val
1700 1705 1710
Thr Tyr Cys His Ser Cys Asp Ser Thr Leu Arg Leu Cys Ile His Ser
1715 1720 1725
Thr Ala Thr Asp Leu Arg Thr Leu Gln Gln Met Leu Leu Gly Thr Leu
1730 1735 1740
Gln Val Val Cys Pro Gly Cys Ala Arg Leu Gly Ser Gly Ser Gly Ser
1745 1750 1755 1760
Gly Ser Gly Ser Met Arg Gly Asp Lys Ala Thr Ile Lys Asp Tyr Ile
1765 1770 1775
Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu His Cys Tyr Glu Gln Leu
1780 1785 1790
Gly Asp Ser Ser Asp Glu Glu Asp Thr Asp Gly Val Asp Arg Pro Asp
1795 1800 1805
Gly Gln Ala Glu Gln Ala Thr Ser Asn Tyr Tyr Ile Val Thr Tyr Cys
1810 1815 1820
Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Gln Tyr
1825 1830 1835 1840
Ser Leu Tyr Gly Lys Thr Leu Glu Glu Arg Val Lys Lys Pro Leu Ser
1845 1850 1855
Glu Ile Thr Ile Arg Cys Ile Ile Cys Gln Thr Pro Leu Cys Pro Glu
1860 1865 1870
Glu Lys Glu Arg His Val Asn Ala Asn Lys Arg Phe His Asn Ile Met
1875 1880 1885
Gly Arg Trp Thr Gly Arg Cys Ser Glu Cys Trp Arg Pro Arg Pro Val
1890 1895 1900
Thr Gln Val Gly Ser
1905
<210> 29
<211> 5160
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding BD-14B polypeptide
<400> 29
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120
gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180
agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240
tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300
aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420
aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480
agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540
cctggcagcg ggtccggaag tgggtctgga tctatgttca agaaccccgc cgagagaccc 600
agaaagctgc acgagctgag cagcgccctg gagatcccct acgacgagct gagactgaac 660
tgcgtgtact gcaagggcca gctgaccgag accgaggtgc tggacttcgc cttcaccgac 720
ctgaccatcg tgtacagaga cgacaccccc tacggcgtgt gcaccaagtg tctcaggttt 780
tatagtaaag tctctgaatt taggtggtat aggtattccg tctatggtcc tgcaggacag 840
gctaaacctg atacaagtaa ttataatatt gtcacatttt gttgtcagtg tgagagcacc 900
ctgagactgt gcgtgcagag cacccaggtg gacatcagaa tcctgcagga gctgctgatg 960
ggcagcttcg gcatcgtgtg ccccaactgc agcaccagac tgggcagtgg aagcggctca 1020
ggaagcggca gcatgagagg cgagaccccc accctgcagg actacgtgct ggacctgcag 1080
cccgaggcca ccgacctgca ctgctacgag cagctgcccg acagcagcga cgaggaggat 1140
gtgatcgaca gccccgccgg ccaggccaag cccgacacca gcaactacaa catcgtgacc 1200
ttctgctgcc agtgcctgag attctacagc aaggtgagcg agttcagatg gtacagatac 1260
agcgtgtacg gcaccaccct ggagaagctg accaacaagg gcatctgcga cctgctgatc 1320
agatgcatca cctgccagag acccctgtgc cccgaggaga agcagagaca cctggacaag 1380
aagaaaagat tccacaacat cggcggcaga tggaccggca gatgcatcgt gtgctggaga 1440
agacccagaa ccgagaccca ggtgggcagc ggctccggat caggcagcgg aagtatgttc 1500
caggacaccg aggagaagcc cagaaccctg cacgacctgt gccaggccct ggagaccacc 1560
atccacaaca tcgagctgca gtgcgtggag tgcaagaacc ccctgcagag aagcgaggtg 1620
tacgacttcg ccttcgccga cctgaccgtg gtgtacagag agggcaaccc cttcggcatc 1680
tgcaagctgt gtctcaggtt tctcagtaaa atttctgaat ataggcatta taattattcc 1740
gtctatggac ctgatggaca ggctcagcct gctacagcag attattatat tgtcacatgt 1800
tgtcatacat gtaacaccac cgtgagactg tgcgtgaaca gcaccgccag cgatctgaga 1860
accatccagc agctgctgat gggcaccgtg aacatcgtgt gccccacctg cgcccagctg 1920
ggctcaggaa gtggaagcgg ctctggatct atgagaggcc acaagcccac cctgaaggag 1980
tacgtgctgg acctgtaccc cgagcccacc gacctgtact gctacgagca gctgagcgat 2040
agcagcgacg aggacgaggg cctggacaga cccgatggcc aggcccagcc cgccaccgcc 2100
gactactaca tcgtgacctg ctgccacacc tgcctgagat tcctgagcaa gatcagcgag 2160
tacagacact acaactacag cgtgtacggc cacaccctgg agcagaccgt gaagaagccc 2220
ctgaacgaga tcctgatcag atgcatcatc tgccagagac ccctgtgccc ccaggagaag 2280
aagagacacg tggacctgaa caagagattc cacaacatca gcggcagatg ggccggcaga 2340
tgcgccgcct gctggagaag cagaagaaga gagaccgccc tgggcagcgg ctctggctcc 2400
ggctcaggat ctatggagtc tgctaacgct tccacatccg ctacaactat cgaccagctg 2460
tgcaagactt tcaacctcag catgcacacc ttgcagatca actgtgtgtt ttgcaaaaac 2520
gccctgacca cagcagaaat ttacagttac gcctacaaac atctgaaggt gctctttcgg 2580
gggggctatc cctacgccgc atgcgcttgt tgcttggaat ttcatggaaa aatcaaccag 2640
tatcggcatt tcgattatgc cggatacgat gggcaggata gtcagcctct gaaacagcac 2700
tatcagattg tgacctgttg ctgtggatgt gacagcaacg tgaggctggt cgtgcagtgt 2760
acagaaacag acatcagaga ggtgcagcag cttcttctgg gcactctcaa catcgtgtgt 2820
cccatctgcg ctccaaaaac cgggtccggc agcggatctg gaagcggctc catgcacggg 2880
cggcacgtga cacttaaaga catcgtcctg gaccttcagc cccctgatcc tgtcggcttg 2940
cactgttacg agcagctggt ggactcatct gaggatgagg tggacgaagt ggacggacag 3000
gattcacagc ctctgaaaca gcattaccag attgtgacct gctgctgcgg ctgttgtctt 3060
gagttccatg gaaaaatcaa ccagtacaga catttcgatt atgccggata cgcaacaacc 3120
gtcgaagagg agactaaaca ggacatcctc gacgtcctga ttcgctgcta cctgtgtcac 3180
aaaccactgt gtgaggtcga aaaggtgaaa cacattctta ccaaggcaag attcatcaaa 3240
ctcaattgta cctggaaggg acggtgcctg cactgttgga ctacatgcat ggaagatatg 3300
cttccaggaa gtgggagcgg ctcaggaagc gggagcatgg aaagtaaaga cgcttccaca 3360
agtgccactt caatcgacca gctctgtaag acattcaact tgagtctgca caccctgcag 3420
atccagtgcg tgttttgcag aaacgcactc acaaccgctg agatttacgc ctatgcttac 3480
aagaacctca aggtcgtgtg gagggataat ttccccttcg ctgcctgcgc ctgttgcctg 3540
gaactgcagg ggaaaatcaa tcagtatcgg catttcaact atgctgctta cgacaaacag 3600
gattctcagc ctctgaccca gcactaccag attctcacct gctgctgcgg ctgcgatagt 3660
aatgtgaggc tcgtggtcga gtgtaccgac ggcgacatta ggcagctcca ggatcttctc 3720
cttggcacac tgaatatcgt gtgtcctatt tgtgccccaa aacccgggtc tggaagtggc 3780
tccggatctg ggagtatgca tggacgcctc gtgacactga aggatattgt gctcgatctg 3840
cagccacctg atcccgtggg cctccactgt tatgagcagc tggaggattc ctcagaagat 3900
gaggtggata aagtggacaa acaggactcc cagcctctta cccagcatta tcagatcctg 3960
acctgctgct gcggatgttg tctggaattg cagggcaaaa tcaaccagta tagacatttc 4020
aattatgctg catacgcccc tacagtcgag gaggaaacca atgaagacat cctcaaggtg 4080
ctgatcagat gttacctctg tcacaagcct ctttgcgaaa tcgagaaact gaagcatatc 4140
ctgggaaagg ctcgctttat caagcttaac aatcagtgga aaggcaggtg cctgcactgc 4200
tggaccacct gtatggaaga cctgctgccc gggtccggct caggaagcgg ctccggctct 4260
atgtttgaag acccagccac caggccaaga acattgcacg agctttgcga agtcctcgaa 4320
gagagtgtgc atgagattag gctccagtgt gtgcagtgca agaaggaact tcagcgcaga 4380
gaggtctaca agttcttgtt tacagacctg cggatcgtgt acagggataa taatccctat 4440
ggcgtctgca ttatgtgtct taggttcctg tccaagattt cagagtacag acattaccag 4500
tattcactgt atggggacag gccagatggc caggccgagc aggctactag taactactac 4560
attgtgacct actgtcactc ctgcgactca accctccggc tgtgcattca cagcaccgcc 4620
accgaccttc gcactctgca gcagatgctg ctcggcacct tgcaggtggt gtgtcccgga 4680
tgcgccaggt tgggcagcgg gagtgggtcc ggaagcggca gtatgagagg cgataaggca 4740
accatcaagg actacatcct ggacctgcag cctgagacca ctgatttgca ttgctacgaa 4800
cagctgggag actcaagcga tgaagaagac actgatggcg tggacaggcc cgacggacag 4860
gccgaacagg ccaccagtaa ctattatatc gtcacctatt gctgcctgag gtttctcagt 4920
aaaatttctg agtacagaca ctatcagtac tcactttacg gcaagacatt ggaggagagg 4980
gtgaagaagc ctctgtccga gatcactatt aggtgcatca tctgtcagac tcccctgtgt 5040
cctgaggaaa aggagcggca tgtcaatgct aacaagagat tccacaacat catgggacgg 5100
tggacaggcc gctgctctga atgctggcgc cccaggccag tgactcaggt gggatcctaa 5160
5160
<210> 30
<211> 1467
<212> PRT
<213> Artificial Sequence
<220>
<223> BD-14C polypeptide
<400> 30
Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala
1 5 10 15
Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His
20 25 30
Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp
35 40 45
Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp
50 55 60
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp
65 70 75 80
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu
85 90 95
Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln
100 105 110
Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu
115 120 125
Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr
130 135 140
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser
145 150 155 160
Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala
165 170 175
Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala
180 185 190
Arg Phe His Asn Pro Ala Glu Arg Pro Tyr Lys Leu Pro Asp Leu Cys
195 200 205
Thr Thr Leu Asp Thr Thr Leu Gln Asp Ile Thr Ile Ala Cys Val Tyr
210 215 220
Cys Arg Arg Pro Leu Gln Gln Thr Glu Val Tyr Glu Phe Ala Phe Ser
225 230 235 240
Asp Leu Tyr Val Val Tyr Arg Asp Gly Glu Pro Leu Ala Ala Cys Gln
245 250 255
Ser Cys Ile Lys Phe Tyr Ala Lys Ile Arg Glu Leu Arg Tyr Tyr Ser
260 265 270
Asp Ser Val Gln Leu Leu Ala Arg Arg Asp Glu Pro Gln Arg His Thr
275 280 285
Ile Gln Cys Ser Cys Cys Lys Cys Asn Asn Thr Leu Gln Leu Val Val
290 295 300
Glu Ala Ser Arg Asp Thr Leu Arg Gln Leu Gln Gln Leu Phe Met Asp
305 310 315 320
Ser Leu Gly Phe Val Cys Pro Trp Cys Ala Thr Ala Asn Gln Gly Ser
325 330 335
Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly Pro Lys Pro Thr Leu
340 345 350
Gln Glu Ile Val Leu Asp Leu Cys Pro Tyr Asn Glu Ile Gln Pro Val
355 360 365
Asp Leu Val Cys His Glu Gln Leu Gly Glu Ser Glu Asp Glu Ile Asp
370 375 380
Glu Pro Asp His Ala Val Asn His Gln His Gln Leu Leu Ala Arg Arg
385 390 395 400
Asp Glu Pro Gln Arg His Thr Ile Gln Cys Ser Cys Cys Lys Gln Ser
405 410 415
Cys Ile Lys Phe Tyr Ala Lys Ile Arg Glu Leu Arg Tyr Tyr Ser Asp
420 425 430
Ser Val Tyr Ala Thr Thr Leu Glu Asn Ile Thr Asn Thr Lys Leu Tyr
435 440 445
Asn Leu Leu Ile Arg Cys Met Cys Cys Leu Lys Pro Leu Cys Pro Ala
450 455 460
Glu Lys Leu Arg His Leu Asn Ser Lys Arg Arg Phe His Lys Ile Ala
465 470 475 480
Gly Ser Tyr Thr Gly Gln Cys Arg Arg Cys Trp Thr Thr Lys Arg Glu
485 490 495
Asp Arg Arg Leu Thr Arg Arg Glu Thr Gln Val Gly Ser Gly Ser Gly
500 505 510
Ser Gly Ser Gly Ser Met Phe Glu Asp Lys Arg Glu Arg Pro Arg Thr
515 520 525
Leu His Glu Leu Cys Glu Ala Leu Asn Val Ser Met His Asn Ile Gln
530 535 540
Val Val Cys Val Tyr Cys Lys Lys Glu Leu Cys Arg Ala Asp Val Tyr
545 550 555 560
Asn Val Ala Phe Thr Glu Ile Lys Ile Val Tyr Arg Asp Asn Asn Pro
565 570 575
Tyr Ala Val Cys Lys Gln Cys Leu Leu Phe Tyr Ser Lys Ile Arg Glu
580 585 590
Tyr Arg Arg Tyr Ser Arg Ser Val Leu Pro Glu Arg Arg Ala Gly Gln
595 600 605
Ala Thr Cys Tyr Arg Ile Glu Ala Pro Cys Cys Arg Cys Ser Ser Val
610 615 620
Val Gln Leu Ala Val Glu Ser Ser Gly Asp Thr Leu Arg Val Val Gln
625 630 635 640
Gln Met Leu Met Gly Glu Leu Ser Leu Val Cys Pro Cys Cys Ala Asn
645 650 655
Asn Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly Asn Val
660 665 670
Pro Gln Leu Lys Asp Val Val Leu His Leu Thr Pro Gln Thr Glu Ile
675 680 685
Asp Leu Gln Cys Tyr Glu Gln Phe Asp Ser Ser Glu Glu Glu Asp Glu
690 695 700
Val Asp Asn Met Arg Asp Gln Leu Pro Glu Arg Arg Ala Gly Gln Ala
705 710 715 720
Thr Cys Tyr Arg Ile Glu Ala Pro Cys Cys Arg Lys Gln Cys Leu Leu
725 730 735
Phe Tyr Ser Lys Ile Arg Glu Tyr Arg Arg Tyr Ser Arg Ser Val Tyr
740 745 750
Gly Thr Thr Leu Glu Ala Ile Thr Lys Lys Ser Leu Tyr Asp Leu Ser
755 760 765
Ile Arg Cys His Arg Cys Gln Arg Pro Leu Gly Pro Glu Glu Lys Gln
770 775 780
Lys Leu Val Asp Glu Lys Lys Arg Phe His Glu Ile Ala Gly Arg Trp
785 790 795 800
Thr Gly Gln Cys Ala Asn Cys Trp Gln Arg Thr Arg Gln Arg Asn Glu
805 810 815
Thr Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Glu Pro
820 825 830
Gln Phe Asn Asn Pro Gln Glu Arg Pro Arg Ser Leu His His Leu Ser
835 840 845
Glu Val Leu Glu Ile Pro Leu Ile Asp Leu Arg Leu Ser Cys Val Tyr
850 855 860
Cys Lys Lys Glu Leu Thr Arg Ala Glu Val Tyr Asn Phe Ala Cys Thr
865 870 875 880
Glu Leu Lys Leu Val Tyr Arg Asp Asp Phe Pro Tyr Ala Val Cys Arg
885 890 895
Val Cys Leu Leu Phe Tyr Ser Lys Val Arg Lys Tyr Arg Tyr Tyr Asp
900 905 910
Tyr Ser Val Gln Ala Arg Gln Ala Lys Gln His Thr Cys Tyr Leu Ile
915 920 925
His Val Pro Cys Cys Glu Cys Lys Phe Val Val Gln Leu Asp Ile Gln
930 935 940
Ser Thr Lys Glu Asp Leu Arg Val Val Gln Gln Leu Leu Met Gly Ala
945 950 955 960
Leu Thr Val Thr Cys Pro Leu Cys Ala Ser Ser Asn Gly Ser Gly Ser
965 970 975
Gly Ser Gly Ser Gly Ser Met His Gly Lys Val Pro Thr Leu Gln Asp
980 985 990
Val Val Leu Glu Leu Thr Pro Gln Thr Glu Ile Asp Leu Gln Cys Asn
995 1000 1005
Glu Gln Leu Asp Ser Ser Glu Asp Glu Asp Glu Asp Glu Val Asp His
1010 1015 1020
Leu Gln Glu Arg Pro Gln Gln Ala Arg Gln Ala Lys Gln His Thr Cys
1025 1030 1035 1040
Tyr Leu Ile His Val Pro Cys Cys Glu Arg Val Cys Leu Leu Phe Tyr
1045 1050 1055
Ser Lys Val Arg Lys Tyr Arg Tyr Tyr Asp Tyr Ser Val Tyr Gly Ala
1060 1065 1070
Thr Leu Glu Ser Ile Thr Lys Lys Gln Leu Cys Asp Leu Leu Ile Arg
1075 1080 1085
Cys Tyr Arg Cys Gln Ser Pro Leu Thr Pro Glu Glu Lys Gln Leu His
1090 1095 1100
Cys Asp Arg Lys Arg Arg Phe His Leu Ile Ala His Gly Trp Thr Gly
1105 1110 1115 1120
Ser Cys Leu Gly Cys Trp Arg Gln Thr Ser Arg Glu Pro Arg Glu Ser
1125 1130 1135
Thr Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala Arg Phe
1140 1145 1150
Glu Asp Pro Thr Gln Arg Pro Tyr Lys Leu Pro Asp Leu Ser Thr Thr
1155 1160 1165
Leu Asn Ile Pro Leu His Asp Ile Arg Ile Asn Cys Val Phe Cys Lys
1170 1175 1180
Gly Glu Leu Gln Glu Arg Glu Val Phe Glu Phe Ala Phe Asn Asp Leu
1185 1190 1195 1200
Phe Ile Val Tyr Arg Asp Cys Thr Pro Tyr Ala Ala Cys Leu Lys Cys
1205 1210 1215
Ile Ser Phe Tyr Ala Arg Val Arg Glu Leu Arg Tyr Tyr Arg Asp Ser
1220 1225 1230
Val Leu Leu Leu Ala Arg Arg Ala Glu Pro Gln Arg His Asn Ile Val
1235 1240 1245
Cys Val Cys Cys Lys Cys Asn Asn Gln Leu Gln Leu Val Val Glu Thr
1250 1255 1260
Ser Gln Asp Gly Leu Arg Ala Leu Gln Gln Leu Phe Met Asp Thr Leu
1265 1270 1275 1280
Ser Phe Val Cys Pro Leu Cys Ala Ala Asn Gln Gly Ser Gly Ser Gly
1285 1290 1295
Ser Gly Ser Gly Ser Met His Gly Pro Lys Ala Thr Leu Cys Asp Ile
1300 1305 1310
Val Leu Asp Leu Glu Pro Gln Asn Tyr Glu Glu Val Asp Leu Val Cys
1315 1320 1325
Tyr Glu Gln Leu Pro Asp Ser Asp Ser Glu Asn Glu Lys Asp Glu Pro
1330 1335 1340
Asp Gly Val Asn His Pro Leu Leu Leu Ala Arg Arg Ala Glu Pro Gln
1345 1350 1355 1360
Arg His Asn Ile Val Cys Val Cys Cys Lys Leu Lys Cys Ile Ser Phe
1365 1370 1375
Tyr Ala Arg Val Arg Glu Leu Arg Tyr Tyr Arg Asp Ser Val Tyr Gly
1380 1385 1390
Glu Thr Leu Glu Ala Glu Thr Lys Thr Pro Leu His Glu Leu Leu Ile
1395 1400 1405
Arg Cys Tyr Arg Cys Leu Lys Pro Leu Cys Pro Thr Asp Lys Leu Lys
1410 1415 1420
His Ile Thr Glu Lys Arg Arg Phe His Asn Ile Ala Gly Ile Tyr Thr
1425 1430 1435 1440
Gly Gln Cys Arg Gly Cys Arg Thr Arg Ala Arg His Leu Arg Gln Gln
1445 1450 1455
Arg Gln Ala Arg Ser Glu Thr Leu Val Gly Ser
1460 1465
<210> 31
<211> 4407
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding BD-14C polypeptide
<400> 31
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120
gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180
agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240
tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300
aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420
aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480
agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540
cctggcagcg ggtccggaag tgggtctgga tctatggcta gatttcataa ccccgccgag 600
cgcccttaca aactgcccga cctgtgcacc actctggata ccactctgca ggacatcact 660
atcgcatgcg tgtactgtcg gagaccactg cagcagaccg aggtctatga gttcgccttt 720
tccgacctgt acgtggtcta tagagatggc gagcccctgg ccgcttgcca gtcttgtatc 780
aagttttacg ctaagatcag ggagctgcgc tactatagcg actccgtgca gctgctggca 840
aggcgcgatg aaccccagag gcacaccatc cagtgctcct gctgtaagtg taacaataca 900
ctgcagctgg tggtcgaggc ctcacgcgac actctgcgac agctgcagca gctgtttatg 960
gatagcctgg ggttcgtgtg cccttggtgt gccactgcta accagggctc tgggagtgga 1020
tcaggcagcg ggtccatgcg aggaccaaag cctaccctgc aggagatcgt gctggacctg 1080
tgcccctaca acgaaattca gcctgtggat ctggtctgtc acgagcagct gggcgaaagc 1140
gaggatgaaa tcgacgagcc agatcatgca gtgaatcacc agcatcagct gctggcccga 1200
cgggacgaac cacagcggca cacaattcag tgcagctgct gtaagcagtc ctgtatcaag 1260
ttctacgcaa aaattcgaga gctgcggtac tattctgata gcgtgtacgc caccacactg 1320
gaaaacatca ctaataccaa actgtataac ctgctgatta gatgcatgtg ctgtctgaag 1380
ccactgtgcc ccgccgagaa actgaggcac ctgaatagca agagaaggtt tcataaaatc 1440
gctgggtcct acaccggaca gtgccgccga tgttggacta ccaagagaga ggaccggaga 1500
ctgaccaggc gcgaaacaca agtgggatca ggcagcgggt ccggatctgg cagtatgttc 1560
gaggataaac gggaaagacc aaggacactg cacgagctgt gcgaagccct gaacgtgtcc 1620
atgcataata ttcaggtggt ctgcgtctac tgtaagaaag aactgtgccg cgcagacgtg 1680
tataatgtcg cctttactga gatcaagatc gtgtaccggg ataacaatcc ctatgccgtc 1740
tgcaagcagt gtctgctgtt ctactctaaa atccgcgaat accgacggta ttcacggagc 1800
gtgctgcctg agagaagggc aggccaggcc acttgctata gaattgaggc cccatgctgt 1860
aggtgtagct ccgtggtcca gctggctgtg gaatctagtg gagacaccct gagagtggtc 1920
cagcagatgc tgatgggaga gctgagcctg gtgtgcccat gctgtgccaa caatgggtcc 1980
ggatctggca gtgggtcagg aagcatgagg ggcaacgtgc cacagctgaa ggacgtggtc 2040
ctgcacctga ctccacagac cgagatcgac ctgcagtgct acgaacagtt tgattcaagc 2100
gaggaagagg acgaagtgga taatatgcga gatcagctgc cagagcgccg agctggacag 2160
gcaacctgct accgcatcga ggcaccttgc tgtcggaaac agtgcctgct gttctattcc 2220
aaaattagag agtaccggcg gtacagccgg agcgtgtacg gcacaactct ggaagctatc 2280
acaaagaaat ctctgtatga cctgagtatt agatgccaca ggtgtcagcg ccctctggga 2340
ccagaagaga agcagaaact ggtggatgag aagaaacgct ttcatgaaat cgcaggccgg 2400
tggaccggac agtgcgctaa ctgttggcag cgcacacgac agcggaatga gactcaagtg 2460
ggcagtgggt caggaagcgg ctccgggtct atggagcccc agttcaacaa tcctcaggaa 2520
agaccaaggt cactgcacca tctgagcgag gtgctggaaa tccctctgat tgacctgaga 2580
ctgagctgcg tgtactgtaa gaaagagctg acaagggctg aagtctataa ctttgcatgc 2640
actgagctga agctggtgta ccgcgacgat tttccctatg ccgtgtgccg ggtctgtctg 2700
ctgttctact ccaaggtgcg aaaataccgg tactatgatt atagtgtcca ggcccgccag 2760
gctaaacagc acacatgcta tctgatccat gtgccatgct gtgagtgtaa gttcgtggtc 2820
cagctggaca ttcagagcac taaagaggac ctgcgggtgg tccagcagct gctgatggga 2880
gctctgacag tgacttgccc cctgtgcgca tcctctaacg gaagtggctc agggagcggc 2940
agcggctcta tgcacggcaa ggtgcccaca ctgcaggacg tggtcctgga gctgacacct 3000
cagactgaaa tcgacctgca gtgcaatgag cagctggata gttcagagga cgaagatgag 3060
gacgaagtgg atcatctgca ggaaagacct cagcaggcaa ggcaggccaa gcagcacacc 3120
tgctacctga ttcacgtccc atgctgtgag cgcgtctgtc tgctgtttta cagcaaggtg 3180
agaaaatata ggtactatga ctacagtgtc tatggcgcca ctctggagtc aatcaccaag 3240
aaacagctgt gcgatctgct gattcgatgc taccggtgcc agagcccact gacccctgaa 3300
gagaagcagc tgcactgcga cagaaaaagg cgcttccacc tgatcgccca tggatggaca 3360
ggcagctgcc tgggctgttg gaggcagact tcccgggagc ctagagaatc taccgtgggg 3420
agtggatcag gcagcgggtc cggatctatg gctagatttg aggaccccac acagaggcct 3480
tacaagctgc ccgacctgag caccaccctg aacattccac tgcatgacat ccgcattaat 3540
tgcgtcttct gtaaaggcga gctgcaggag cgggaagtgt tcgaatttgc cttcaacgac 3600
ctgtttatcg tgtacaggga ttgcaccccc tatgcagcct gcctgaagtg tatttccttc 3660
tacgctcgcg tgcgagagct gaggtactat cgcgattctg tcctgctgct ggctcgacgg 3720
gcagaacctc agcgccacaa tatcgtgtgc gtctgctgta aatgtaacaa tcagctgcag 3780
ctggtcgtgg agaccagcca ggacggactg cgggccctgc aacaactgtt tatggataca 3840
ctgagcttcg tgtgccctct gtgcgctgca aaccaaggca gtgggtcagg aagcggctcc 3900
gggtctatgc atggaccaaa ggccaccctg tgcgacatcg tgctggatct ggaaccccag 3960
aattacgaag aggtggacct ggtctgttat gagcagctgc ctgatagtga ctcagagaac 4020
gaaaaagacg aaccagatgg cgtgaatcac ccactgctgc tggccagaag ggctgagcca 4080
cagagacata acatcgtgtg cgtctgctgc aagctgaaat gtattagttt ttacgctcgg 4140
gtgagagaac tgcgatacta tcgggactct gtctatgggg agactctgga ggcagaaacc 4200
aagacacccc tgcacgagct gctgatcaga tgctacaggt gtctgaaacc tctgtgcccc 4260
accgataagc tgaaacacat tacagagaaa cgccgattcc ataatatcgc cggaatctac 4320
accggccagt gcagggggtg tagaacacga gcaaggcatc tgaggcagca gcggcaggca 4380
aggtccgaga ctctggtggg atcctaa 4407
<210> 32
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> (GGGGS)n linker peptide unit
<400> 32
Gly Gly Gly Gly Ser
1 5
<210> 33
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> (GSSGGS)n linker peptide unit
<400> 33
Gly Ser Ser Gly Gly Ser
1 5
<210> 34
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 34
Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser
1 5 10 15
Leu Asp
<210> 35
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 35
Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr
1 5 10
<210> 36
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 36
Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe
1 5 10
<210> 37
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> (EAAAK)n linker peptide unit
<400> 37
Glu Ala Ala Ala Lys
1 5
<210> 38
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 38
Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys
1 5 10
<210> 39
<211> 46
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 39
Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
1 5 10 15
Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala
20 25 30
Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala
35 40 45
<210> 40
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 40
Gly Gly Gly Gly Gly Gly Gly Gly
1 5
<210> 41
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 41
Gly Gly Gly Gly Gly Gly
1 5
<210> 42
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 42
Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala
1 5 10
<210> 43
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 43
Pro Ala Pro Ala Pro
1 5
<210> 44
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 44
Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp
1 5 10 15
Val
<210> 45
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 45
Pro Leu Gly Leu Trp Ala
1 5
<210> 46
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 46
Thr Arg His Arg Gln Pro Arg Gly Trp Glu
1 5 10
<210> 47
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 47
Ala Gly Asn Arg Val Arg Arg Ser Val Gly
1 5 10
<210> 48
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 48
Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 49
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 49
Gly Phe Leu Gly
1
<210> 50
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 50
Gly Ser Ser Gly Gly Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly Asp
1 5 10 15
Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu
20 25 30
Claims (20)
- 6, 11, 16, 18, 39, 45 및 56형 인유두종바이러스(HPV)의 조기 단백질 항원 6(E6) 또는 그의 면역원성 단편, 및 조기 단백질 항원 7(E7) 또는 그의 면역원성 단편을 각각 암호화하는 폴리뉴클레오타이드를 포함하며, 상기 E6 및 E7은 야생형의 기능을 갖지 않는, 다가 HPV DNA 백신 조성물.
- 제1항에 있어서,
31, 33, 35, 51, 52, 58 및 59형 인유두종바이러스(HPV)로 구성되는 군으로부터 선택되는 1종 또는 그 이상의 HPV의 조기 단백질 항원 6(E6) 또는 그의 면역원성 단편 및 조기 단백질 항원 7(E7) 또는 그의 면역원성 단편을 각각 암호화하는 폴리뉴클레오타이드를 추가로 포함하며, 상기 E6 및 E7는 야생형의 기능을 갖지 않는, 다가 HPV DNA 백신 조성물. - 제1항 또는 제2항에 있어서,
상기 E6 및 E7은 각각 N-말단 단편 및 C-말단 단편으로 나뉘어 무작위적으로 뒤섞인 E6/E7 셔플드 항원단위체의 형태로 발현되는, 다가 HPV DNA 백신 조성물. - 제3항에 있어서,
상기 E6/E7 셔플드 항원단위체는 상기 E6 및 E7의 N-말단 단편 및 C-말단 단편이 E6의 N-말단 단편(E6N)-E7의 C-말단 단편(E7C)-E7의 N-말단 단편(E7N)-E6의 C-말단 단편(E6C)의 순으로 연결된 폴리펩타이드인, 다가 HPV DNA 백신 조성물. - 제3항에 있어서,
적어도 2개 이상의 인유두종바이러스의 E6/E7 셔플드 항원단위체가 융합단백질 형태로 연결되어 발현되는, 다가 HPV DNA 백신 조성물. - 제5항에 있어서,
상기 E6/E7 셔플드 항원단위체 또는 상기 융합단백질은 신호서열을 추가로 포함하는, 다가 HPV DNA 백신 조성물. - 제5항에 있어서,
상기 E6/E7 셔플드 항원단위체 또는 상기 융합단백질은 Flt3L을 추가로 포함하는, 다가 HPV DNA 백신 조성물. - 제1항에 있어서,
IL-7을 추가로 포함하는, 다가 HPV DNA 백신 조성물. - 제1항에 있어서,
하나 이상의 약학적으로 허용가능한 백신보조제를 추가로 포함하는, 다가 HPV DNA 백신 조성물. - 제9항에 있어서,
상기 백신 면역보조제는 IL-12 단백질 및 IL-21 단백질을 유효성분으로 포함하거나 상기 IL-12 단백질을 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 암호화하는 폴리뉴클레오타이드를 유효성분으로 포함하는 T 림프구 특이적 면역반응 촉진용 백신 면역보조제인, 다가 HPV DNA 백신 조성물. - 제10항에 있어서,
상기 T 림프구 특이적 면역반응 촉진용 백신 면역보조제는 하기로 구성되는 군으로부터 선택되는 하나 이상을 포함하는, 다가 HPV DNA 백신 조성물:
p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)로 구성된 IL-12 단백질 및 IL-21 단백질;
상기 IL-12를 구성하는 p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)를 각각 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드를 포함하는 하나 내지 세 개의 벡터; 및
상기 IL-12p35, IL-12p40 및 IL-21 단백질을 각각 암호화하는 mRNA 분자. - 제10항에 있어서,
상기 IL-12p35 단백질은 서열번호 1로 기재되는 아미노산 서열로 구성되는 인간 IL-12p35인, 다가 HPV DNA 백신 조성물. - 제10항에 있어서,
상기 IL-12p40 단백질은 서열번호 2로 기재되는 아미노산 서열로 구성되는 인간 IL-12p40인, 다가 HPV DNA 백신 조성물. - 제10항에 있어서,
상기 IL-21 단백질은 서열번호 3으로 기재되는 아미노산 서열로 구성되는 인간 IL-21인, 다가 HPV DNA 백신 조성물. - 제10항에 있어서,
상기 백신 면역보조제는 하기로 구성되는 군으로부터 선택되는 하나 이상을 추가로 포함하는, 다가 HPV DNA 백신 조성물:
ⅰ) MIP-1α 단백질;
ⅱ) 상기 MIP-1α 단백질을 암호화하는 폴리뉴클레오타이드가 프로모터에 작동 가능하게 연결된 MIP-1α 유전자컨스트럭트; 및
ⅲ) 상기 MIP-1α 단백질을 암호화하는 폴리뉴클레오타이드가 상기 IL-12p35, IL-12p40 및 IL-21 단백질 중 어느 하나 이상에 IRES 또는 링커 펩타이드를 암호화하는 폴리뉴클레오타이드에 의해 작동가능하게 연결된 복합 유전자컨스트럭트; 및
ⅳ) MIP-1α 단백질을 암호화하는 mRNA 분자. - 제15항에 있어서,
상기 MIP-1α 유전자컨스트럭트는 별도의 발현벡터에 포함되거나, 상기 IL-12를 구성하는 p35 사슬(IL-12p35) 및 p40 사슬(IL-12p40)를 각각 암호화하는 폴리뉴클레오타이드 및 상기 IL-21 단백질을 각각 암호화하는 폴리뉴클레오타이드를 포함하는 하나 내지 세 개의 벡터 중 어느 하나 이상의 벡터 내에 포함되는, 다가 HPV DNA 백신 조성물. - 제16항에 있어서,
상기 MIP-1α 단백질은 서열번호 10으로 기재되는 아미노산 서열로 구성되는 인간 MIP-1α 단백질인, 다가 HPV DNA 백신 조성물. - 제1항 내지 제17항 중 어느 한 항의 다가 HPV DNA 백신을 개체에 투여하는 단계를 포함하는 HPV 감염에 의해 유발되는 질환의 치료방법.
- 제18항에 있어서,
상기 HPV 감염에 의해 유발되는 질환은 편평세포암종(SCC), 선암, 선편평세포암종, 소세포암종, 신경내분비 종양(NET), 유리 세포 암종, 융모샘 선암(VGA), 비-암종 악성종양, 흑색종, 림프종, 또는 자궁경부 상피내 종양(CIN)인, 치료방법. - 제18항에 있어서,
상기 백신 조성물은 생체내 전기천공법에 의해 투여되는, 치료방법.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20180013328 | 2018-02-02 | ||
| KR1020180013328 | 2018-02-02 |
Publications (2)
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| TW (1) | TWI731300B (ko) |
| WO (1) | WO2019151760A1 (ko) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021241928A1 (ko) * | 2020-05-25 | 2021-12-02 | (주)진매트릭스 | 사람 파필로마바이러스의 구조적으로 변형된 키메라 폴리펩타이드 및 그 폴리펩타이드를 포함하는 재조합 단백질, 그 단백질의 용도 |
| KR20210145672A (ko) * | 2020-05-25 | 2021-12-02 | (주)진매트릭스 | 사람 파필로마바이러스의 구조적으로 변형된 키메라 폴리펩타이드 및 그 폴리펩타이드를 포함하는 재조합 단백질 및 그 단백질의 용도 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111918666A (zh) * | 2018-02-02 | 2020-11-10 | Sl瓦西基因公司 | 一种新型疫苗佐剂 |
| AU2021271860A1 (en) * | 2020-05-14 | 2022-12-15 | Inovio Pharmaceuticals, Inc. | Vaccines for recurrent respiratory papillomatosis and methods of using the same |
| KR20230104175A (ko) * | 2020-11-05 | 2023-07-07 | 네오이뮨텍, 인코퍼레이티드 | Il-7 단백질과 뉴클레오타이드 백신의 조합물을 사용한 종양의 치료 방법 |
| CN115724951B (zh) * | 2022-11-15 | 2023-10-03 | 怡道生物科技(苏州)有限公司 | 与11型hpv结合的抗体或其抗原结合片段及其应用 |
| WO2024140767A1 (zh) * | 2022-12-29 | 2024-07-04 | 仁景(苏州)生物科技有限公司 | 用于预防或治疗hpv感染相关疾病的多核苷酸分子 |
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| WO2016198531A2 (en) * | 2015-06-10 | 2016-12-15 | Hookipa Biotech Ag | Hpv vaccines |
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| ATE518958T1 (de) * | 2007-01-30 | 2011-08-15 | Transgene Sa | Zur impfung verwendetes papillomavirus-e2- polypeptid |
| KR101361416B1 (ko) * | 2008-10-08 | 2014-02-21 | 인트렉손 코포레이션 | 다수의 면역조절자를 발현하는 조작된 세포 및 그 용도 |
| WO2014165291A1 (en) * | 2013-03-12 | 2014-10-09 | The Trustees Of The University Of Pennsylvania | Improved vaccines for human papilloma virus and methods for using the same |
| WO2016024255A1 (en) * | 2014-08-15 | 2016-02-18 | Genexine, Inc. | Methods of treating cervical cancer |
| AU2015323944B2 (en) * | 2014-10-01 | 2018-11-29 | Inovio Pharmaceuticals, Inc. | Vaccines having an antigen and interleukin-21 as an adjuvant |
| CA2995740A1 (en) * | 2015-08-20 | 2017-02-23 | Janssen Vaccines & Prevention B.V. | Therapeutic hpv18 vaccines |
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| WO2016198531A2 (en) * | 2015-06-10 | 2016-12-15 | Hookipa Biotech Ag | Hpv vaccines |
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| Human vaccines & Immunotherapeutics, 2017, 13(12):2837-2848.* * |
| Nature Communications, 2014, 5:5317.* * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021241928A1 (ko) * | 2020-05-25 | 2021-12-02 | (주)진매트릭스 | 사람 파필로마바이러스의 구조적으로 변형된 키메라 폴리펩타이드 및 그 폴리펩타이드를 포함하는 재조합 단백질, 그 단백질의 용도 |
| KR20210145672A (ko) * | 2020-05-25 | 2021-12-02 | (주)진매트릭스 | 사람 파필로마바이러스의 구조적으로 변형된 키메라 폴리펩타이드 및 그 폴리펩타이드를 포함하는 재조합 단백질 및 그 단백질의 용도 |
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| Publication number | Publication date |
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| TW201941786A (zh) | 2019-11-01 |
| KR102222582B1 (ko) | 2021-03-04 |
| WO2019151760A1 (ko) | 2019-08-08 |
| TWI731300B (zh) | 2021-06-21 |
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