KR20190082227A - 병용 요법용 wt1 표적화 dna 백신 - Google Patents
병용 요법용 wt1 표적화 dna 백신 Download PDFInfo
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Abstract
빌름 종양 단백질(WT1)을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주에 관한 것으로서, 본 치료법은 관문 억제제, 특히 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택되는 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함한다. 본 발명은 추가로 WT1을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주를 포함하는 약학 조성물에 관한 것으로서, 본 치료법은 관문 억제제, 특히 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택되는 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함한다.
Description
본 발명은 빌름 종양 단백질(Wilm's Tumor Protein; WT1)을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주에 관한 것으로서, 본 치료법은 관문 억제제, 특히 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택되는 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함한다. 본 발명은 추가로 WT1을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주를 포함하는 약학 조성물에 관한 것으로서, 본 치료법은 관문 억제제, 특히 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택되는 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함한다.
빌름 종양 유전자 1(WT1)은 세포 증식과 분화에 수반되는 아연 핑거 전사 인자(zinc finger transcription factor)를 암호화한다. 이는, 혈액학적 악성종양 몇 가지 유형과 다양한 고형 종양을 비롯한 다양한 악성 종양에서 많이 발현된다. 이와는 대조적으로, 성체에서 WT1의 정상적인 조직내 발현은 다양한 유형의 조직 내 CD34+ 전구체 세포, 중피, 신장, 자궁 및 생식선에 제한된다. WT1은 원래 종양 억제제 유전자로서 제안되었다. 그러나 더욱 최근의 증거는 이 전사 인자의 발암 역할을 시사하고 있는데; 즉 Wt-1은 전구체 세포의 분화에 부정적인 영향을 미치고 이의 증식을 촉진한다고 한다. 게다가 과발현된 WT1은 면역원성으로서; WT1 특이적 T 세포뿐만 아니라 IgG 항WT1 항체가 암 환자에서 관찰된 바 있다. 그러므로 WT-1은 암 백신 개발에 있어서 촉망받는 후보물질이다.
HLA(인간 백혈구 항원) 제한 WT1 펩티드 단편을 기반으로 한 WT1 백신의 인간 대상 임상 시험이 보고된 바 있다. Osada 외 다수는, WT1 암호화 아데노바이러스 백신을 개시하였다[Clin Cancer Res 2009;15:2789-2796].
WO 2014/173542는 암 면역요법에 사용되는, WT1 암호화 재조합 DNA 분자를 포함하는 살모넬라 약독화 균주를 개시하고 있다. 이 WT1 암호화 살모넬라 약독화 균주는 마우스 백혈병 세포로 면역공격된 마우스 모델에서 항종양 활성을 나타내는 것으로 보인다. 그러므로 WT1 암호화 살모넬라 약독화 균주는 이러한 적응증 치료를 위한 암 백신으로서의 잠재성이 크다.
WO 2013/09189는, 갈락토스 에피머라아제 활성이 결여되고, 재조합 DNA 분자를 보유하는 약독화 돌연변이 살모넬라 타이피(Salmonella typhi) 균주를 생육하기 위한 방법을 개시하고 있다.
종양은 면역원성일 수 있다는 발견은, 정상 세포는 보존하면서 악성 세포를 선택적으로 제거하는데 면역계를 사용하도록 디자인된 암 면역요법 다수의 개발을 이끌었다. 그러나 종양 항원에 대한 백신화만으로부터 얻어지는 생존상 이득은 그다지 크지 않다. 항암 백신은 다수의 난관에 봉착하고 있는데, 그중 하나는 면역억제성 미세환경이다. 비정상적 종양 혈관조직은 저과산소성 미세환경을 형성하여, 염증 세포를 면역 억제쪽으로 치우치게 만든다. 게다가 종양은 성장 인자 및 시토카인의 분비를 통해 면역 세포의 증식, 분화 및 기능을 조직적으로 변경시킨다.
암 치유를 위해서는 암 줄기 세포를 완전히 제거하는 것이 매우 중요하다. 인간 종양의 다수 면역 도피 기작은 암 면역요법에 있어서 주요 난관으로 남아있다. 그러므로 지금까지 보지 못했던 개선된 암의 치료적 접근법이 절실히 필요한 실정이다.
선행기술을 고려하였을 때, 본 발명의 목적은 신규 암 요법을 제공하는 것이다. 이러한 신규 요법은 암 환자들의 치료 선택권(treatment option)을 개선한다는 주요 이점을 제공할 것이다.
일 양태에서, 본 발명은 빌름 종양 단백질(WT1)을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주에 관한 것으로서, 이 치료법은 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함한다.
WT1을 암호화하는 살모넬라 약독화 균주 및 적어도 하나의 관문 억제제의 병용 치료법은 놀랍게도 강력하고 지속적인 항종양 효과를 보이는 것으로 파악되었다. 놀랍게도 WT1을 암호화하는 살모넬라 약독화 균주와, 관문 억제제인 항PD-L1 및 항CTLA-4 중 어느 하나의 병용 투여는 전반적인 생존률에 상승적 효과를 나타내는 것이 관찰되었다.
이론에 국한되기를 바라지 않을 때, VXM06은 종양 미세환경에 의해 비활성화될 수 있는 WT-1 특이적 효과기 T 세포를 생성할 수 있는 것으로 생각된다. 관문 억제제 모노클로날 항체는 면역 세포 또는 종양 세포 각각에 있는 구조를 표적화하므로, T 세포 억제 효과를 막거나 상쇄시키는 것으로 보고되고 있다.
특정 구현예에서, 적어도 하나의 관문 억제제는 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택된다.
특정 구현예에서, 살모넬라 약독화 균주는 살모넬라 엔테리카(Salmonella enterica) 종이다. 구체적으로 살모넬라 약독화 균주는 살모넬라 타이피 Ty21a이다.
특정 구현예에서, 발현 카세트는 진핵생물 발현 카세트이다. 구체적으로 발현 카세트는 CMV 프로모터를 포함한다.
특정 구현예에서, WT1은 서열 번호 4에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택된다. 구체적으로 WT1은 절단된 것이고, 더욱 구체적으로 WT1의 아연 핑거 도메인은 결실되었다. 이러한 특정 구현예에서, WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 WT1과, 이것과 적어도 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택된다.
특정 구현예에서, DNA 분자는 카나마이신 내성 유전자, pMB1 ori 및 CMV 프로모터를 포함한다. 구체적으로 DNA 분자는 서열 번호 2에서 발견되는 바와 같은 DNA 서열을 포함한다.
특정 구현예에서, 살모넬라 약독화 균주는 상기 관문 억제제 적어도 하나와 동시에 투여되거나, 이 관문 억제제 적어도 하나가 투여되기 전 또는 투여된 후에 투여된다.
특정 구현예에서, 치료법은 화학요법, 방사선요법 또는 암 생물요법이 동반되는데, 구체적으로 살모넬라 약독화 균주는 이 화학요법 또는 방사선요법 치료 주기 또는 암 생물요법이 수행되기 전, 수행되는 도중 또는 수행된 후에 투여되거나, 아니면 화학요법 또는 방사선요법 치료 주기 또는 암 생물요법이 수행되기 전 및 수행되는 도중에 투여된다.
특정 구현예에서, 암 생물요법은 종양 항원 및/또는 종양 기질 항원을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 살모넬라의 추가 약독화 균주(들) 하나 이상을 투여하는 단계를 포함한다. 이러한 특정 구현예에서, 상기 살모넬라의 추가 약독화 균주(들) 하나 이상은 진핵생물 발현 카세트를 포함하는 살모넬라 타이피 Ty21a이다. 구체적으로 상기 종양 항원은 메소텔린(MSLN), CEA, CMV pp65로 이루어진 군으로부터 선택되고, 바람직하게 상기 종양 항원은 (a) 메소텔린(MSLN), 구체적으로 서열 번호 5에서 발견되는 바와 같은 아미노산 서열을 가지는 MSLN과, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, (b) CEA, 구체적으로 서열 번호 6에서 발견되는 바와 같은 아미노산 서열을 가지는 CEA와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 그리고 (c) CMV pp65, 구체적으로 서열 번호 7에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 서열 번호 8에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 그리고 서열 번호 9에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질 군으로부터 선택된다. 구체적으로 상기 종양 기질 항원은 VEGF 수용체 단백질 및 인간 섬유아세포 활성화 단백질(FAP)로 이루어진 군으로부터 선택되는데, 바람직하게 이 VEGF 수용체 단백질은 VEGFR-2, 더욱 바람직하게는 인간 VEGFR-2, 더욱더 바람직하게는 서열 번호 10에서 발견되는 바와 같은 아미노산 서열을 가지는 VEGFR-2 또는 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질이다.
특정 구현예에서, 살모넬라 약독화 균주는 경구 투여된다.
특정 구현예에서, 암은 백혈병, 구체적으로 급성 골수성 백혈병(AML) 및 급성 림프성 백혈병(ALL), 다발성 골수종으로부터 선택되고, 고형 종양, 구체적으로 폐암, 유방암, 식도암, 결장암, 결장직장암, 위암, 융모막암, 췌장암, 교모세포종, 두경부암, 활막육종, 혈관육종, 골육종, 감상샘암, 자궁경부암, 자궁내막암, 난소암, 신경모세포종, 횡문근육종 및 전립선암으로부터 선택된다.
특정 구현예에서, 살모넬라 약독화 균주의 1회 용량은 약 105 내지 약 1011, 구체적으로 약 106 내지 약 1010, 더욱 구체적으로 약 106 내지 약 109, 더욱 구체적으로 약 106 내지 약 108, 가장 구체적으로 약 106 내지 약 107 콜로니 형성 단위(Colony Forming Unit; CFU)를 포함한다.
특정 구현예에서, 치료법은 환자에서의 WT1에 대한 전 면역 반응 및/또는 WT1 발현을 평가하는 단계를 포함하는 개별맞춤(individualized) 암 면역요법이다.
추가의 양태에서, 본 발명은 WT1을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는 살모넬라 약독화 균주를 포함하는, 암 치료법에 사용하기 위한 약학 조성물에 관한 것으로서, 이 치료법은 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함한다. 구체적으로 적어도 하나의 관문 억제제는 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택된다.
특정 구현예들에서, 살모넬라 약독화 균주는 살모넬라 타이피 Ty21a이고, 발현 카세트는 진핵생물 발현 카세트이며, WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단벡질로 이루어진 군으로부터 선택된다.
구체적으로 진핵생물 발현 카세트는 CMV 프로모터를 포함한다. 구체적으로 인간 WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가진다.
본 발명은 하기 발명의 상세한 설명과 이에 포함된 실시예를 참고하였을 때 더욱 용이하게 이해될 수 있다.
일 양태에서, 본 발명은 빌름 종양 단백질(WT1)을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주에 관한 것으로서, 이 치료법은 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함한다.
아연 핑거 전사 인자 빌름 종양 단백질 1은 WT1 유전자에 의해 암호화된다. 이는, C 말단에 4개의 아연 핑거 모티프를 함유하고, N 말단에 프롤린/글루타민 풍부 DNA 결합 도메인을 함유한다. 암호화 엑손 2군데에서 이루어진 대안적 스플라이싱(alternative splicing)으로부터 초래된 다수의 전사 변이체는 특성규명이 잘 되어 있다. WT1은 비뇨생식계 발달에 필수적인 역할을 하며, 세포 증식과 분화에 수반된다. WT1 유전자는 유년기 신장 신생물형성, 즉 빌름 종양 발생에 관여하는 유전자로서 분리되었다. 이는 혈액학적 악성종양 몇 가지 유형과 다양한 고형 종양을 비롯한 다양한 악성 종양에서 많이 발현된다. 이와는 대조적으로, 성체에서의 WT1의 정상적인 조직내 발현은 다양한 유형의 조직 내 전구체 세포, 중피, 신장, 자궁 및 생식선에 제한된다. 종양 항원 WT1의 발현 프로필, 발암 작용 및 면역원 잠재성으로 말미암아, 이 WT1은 암 백신 개발에 있어서 촉망받는 후보물질이 된다.
본 발명에 따르면, 살모넬라 약독화 균주는 재조합 DNA 분자의 표적 세포로의 전달을 위한 운반체, 즉 WT1을 암호화하는 발현 카세트를 포함하는 상기 재조합 DNA 분자의 세균 운반체로서의 역할을 한다. 이종 항원, 예컨대 WT1(종양 항원)을 암호화하는 DNA 분자를 포함하는 이러한 전달 벡터는 DNA 백신이라 칭하여진다.
본 발명의 내용 중 "백신"이란 용어는 투여시 대상체에서 면역 반응을 유도할 수 있는 제제를 지칭한다. 백신은, 바람직하게 질환을 예방, 완화 또는 치료할 수 있다.
본 발명에 따른 약독화 살모넬라 생균주는 WT1을 암호화하는 재조합 DNA 분자를 안정적으로 운반한다. 이는, 이 제조합 DNA 분자의 경구 전달을 위한 비이클로서 사용될 수 있다.
유전자 면역화는 종래의 백신화에 비하여 유리할 수 있다. 표적 DNA는 상당한 기간 동안 검출될 수 있으므로, 항원의 데포(depot)로서의 역할을 한다. 몇몇 플라스미드 내 서열 모티프, 예컨대 GpC 섬(GpC island)은 면역자극성으로서, LPS 및 기타 세균 성분으로 밀미암는 면역자극에 의해 촉진되는 아주반트(adjuvant)로서의 역할을 할 수 있다.
오로지 WT1 단백질의 소형 단편에 대한 면역성만을 매개할 수 있는 펩티드 백신과는 대조적으로, 유전자 백신화는 암호화된 WT1 단백질의 전체 길이에 걸쳐 존재하는 다양한 에피토프에 대한 면역성을 초래할 수 있다.
이와는 별도로, 대개 임상 시험에서 사용되어 온 WT1 펩티드 백신은 펩 티드의 HLA 제한, 즉 항원 제시 세포(APC) 상 HLA 분자에 대한 펩티드의 결합능으로 말미암아 제한된 응용예를 보인다. 이와는 대조적으로, 본 발명의 DNA 백신은 HLA 제한적이지 않다. 더욱이 암호화된 펩티드 단편은 WT-1에 대한 양성반응이 보임에도 불구하고 환자의 종양 내에 존재하지 않을 수도 있다. VXM06은 WT1의 아연 핑거 도메인을 제외한 전장 단백질 WT-1을 암호화하므로, 면역계에 제시되는 펩티드 단편은 환자에 의해 생산된다.
약독화 생 살모넬라 벡터는 다른 투여형, 예컨대 마이트로캡슐(microencapsulation)에 비하여 이점을 구성할 수 있는 자체의 면역조정 인자, 예컨대 리포다당체(LPS)를 현장에서 생산한다. 더욱이 본 발명에 따른 점막 백신은 유리한 것으로 판명된 림프내 작용 양상을 보인다. 본 발명에 따른 약독화 백신이 섭취되면, 소장의 페이어 패치(Peyer's patch) 내 대식세포 및 기타 세포에 변형 세균이 침투한다. 이 세균은 이러한 식세포에 의해 취하여진다. 세균의 약독화 돌연변이로 말미암아, 에스. 타이피 Ty21 균주 세균은 이러한 식세포 내부에서 견딜 수 없게 되고, 이 시점에서 사멸하게 된다. 재조합 DNA 분자는 방출된 후, 특이적 운반 시스템 또는 엔도좀 유출(endosomal leakage) 중 어느 하나를 통해 면역 식세포의 세포기질로 이동하게 된다. 마지막으로 재조합 DNA 분자는 핵으로 들어가서 전사되고, 그 결과 식세포의 세포기질 내에서 WT1 대량 발현이 초래된다. 감염된 세포는 WT1 항원을 품은 채 세포자살을 진행하게 되고, 소장의 면역계에 의해 흡수 및 처리된다. 이 과정에서 세균 감염의 위험 신호는 전신 및 점막 구획 둘 다의 차원에서 강력한 표적 항원 특이적 CD8+T 세포 및 항체 반응을 초래하는 강력한 아주반트로서 사용된다. 면역 반응은 백신화 이후 대략 10일경에 최고에 이른다. 항 운반체 반응의 결여는, 동일 백신에 의한 수회에 걸친 면역증강을 허용한다.
본 발명의 내용 중, "약독화"란 용어는, 세균 균주가 약독화 돌연변이를 보유하지 않으면서 부모 세균 균주에 비하여 병독성(virulence)이 감소한 경우를 지칭한다. 약독화 세균 균주는, 바람직하게 자체의 병독성을 잃었지만, 방어 면역성을 유도하는 능력은 보유하고 있다. 약독화는 병독성 유전자, 조절 유전자 및 대사 유전자를 비롯한 다양한 유전자의 결실에 의해 달성될 수 있다. 약독화 세균은 자연에서 발견될 수 있거나, 예를 들어 새로운 배지나 세포 배양액에의 적응을 통해 실험실에서 인공적으로 제조될 수 있거나, 아니면 재조합 DNA 기법에 의해 제조될 수 있다. 본 발명에 따른 살모넬라 약독화 균주 약 1011 CFU만큼의 투여는, 바람직하게 5% 미만, 더욱 바람직하게 1% 미만, 가장 바람직하게 1‰ 미만의 대상체에서 살모넬라증을 일으킨다.
본 발명의 내용 중 "~를 포함하다" 또는 "~를 포함하는"이란 용어는, "~를 포함하되, 그에 한정되는 것은 아니다(아닌)"경우를 의미한다. 이 용어는 진술된 임의의 특징, 요소, 정수, 단계 또는 성분의 존재를 특정하는데 제약을 두지 않되, 하나 이상의 다른 특징, 요소, 정수, 단계, 성분 또는 이것들의 군의 존재 또는 부가를 금지하지 않도록 의도된다. 그러므로 "~를 포함하는"이란 용어는, 더욱 제한적인 용어 "~로 이루어진"과 "본질적으로 ~로 이루어진"을 포함한다. 일 구현예에서, 본원 전체에 걸쳐, 구체적으로는 특허청구범위 내에서 사용된 바와 같은 "~를 포함하는"이란 용어는 "~로 이루어진"이란 용어로 대체될 수 있다.
WT1을 암호화하는 발현 카세트를 포함하는 DNA 분자는 적합한 재조합 DNA 분자, 즉 조작된 DNA 구조체, 바람직하게는 상이한 기원의 DNA 조각들로 구성된, 조작된 DNA 구조체이다. DNA 분자는 선형 핵산일 수 있거나, 바람직하게 WT1을 암호화하는 개방 해독틀을 발현 벡터 플라스미드에 도입함으로써 제조된 환형 DNA 플라스미드일 수 있다.
본 발명의 내용에 있어서, "발현 카세트"란 용어는, 자체의 발현을 제어하는 조절 서열의 제어 하에 있는 개방 해독틀(ORF)을 적어도 하나 포함하는 핵산 단위를 지칭한다. 발현 카세트는, 바람직하게 이에 포함된 개방 해독틀로서, 종양 항원, 예컨대 WT1을 암호화하는 개방 해독틀의 표적 세포 내 전사를 매개할 수 있다. 발현 카세트는, 통상 프로모터, 개방 해독틀 적어도 하나, 그리고 전사 종결 신호를 포함한다.
특정 구현예에서, 적어도 하나의 관문 억제제는 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택된다. 바람직하게 적어도 하나의 관문 억제제는 PD-1, PD-L1 또는 CTLA-4에 대한 항체 적어도 하나, 또는 이의 조합이고, 더욱 바람직하게 적어도 하나의 관문 억제제는 PD-L1 또는 CTLA-4에 대한 항체 적어도 하나, 또는 이의 조합이다.
면역계의 중요 부분은, 체내 정상 세포와 이 면역계가 "외래의 것"으로 보는 것을 구분하는 자체의 능력이다. 이 능력은, 면역계가 정상 세포만은 남겨두고, 외래 세포를 공격하도록 만든다. 이를 위해 면역계는 "관문", 즉 면역 반응을 개시하기 위해 활성화(또는 비활성화)될 필요가 있는 임의의 면역 세포상 분자를 이용한다.
암세포는 종종 면역계에 의해 공격당하는 것을 피하기 위해 이 관문을 이용할 방법을 찾는다. 하지만 이 관문을 표적화하는 약물은 암 치료수단으로서의 역할을 할 가능성이 많다. 예를 들어 PD-1은 T 세포라 칭하여지는 면역 세포상 관문 단백질이다. 이는 보통 T 세포가 체내 다른 세포의 공격을 받지 않도록 도와주는 일종의 "오프 스위치(off switch)"로서의 역할을 한다. PD-1은 몇몇 정상 세포(및 암 세포) 상에 존재하는 단백질인 PD-L1에 부착될 때 이러한 역할을 한다. PD-1이 PD-L1에 결합하면, 이는 기본적으로 T 세포를 구별하여 다른 세포와 격리시킨다. 몇몇 암 세포는 면역 공격으로부터 자신이 도피하는 것을 도와주는 PD-L1을 다량으로 가진다.
PD-1 또는 PD-L1 중 어느 하나를 표적화하는 모노클로날 항체는 임의의 암을 치료하기 위한, 촉망받는 후보물질인 것으로 보인다. 놀랍게도, 관문 억제제는 WT1을 암호화하는 살모넬라 약독화 균주에 의해 매개되는, WT-1 발현 암 세포에 대한 면역 반응을 증강시킬 수 있음이 발견되었다.
특정 구현예에서, 살모넬라 약독화 균주는 살모넬라 엔테리카 종이다. 살모넬라 엔테리카의 약독화된 유도체는 이종 항원의 포유동물 면역계로의 전달을 위한 매력적인 비이클인데, 그 이유는 에스. 엔테리카 균주는 비경구 투여에 비하여 간편성과 안전성이라는 이점을 제공하는 점막 면역화 경로, 즉 경구 또는 비내 경로를 통해 잠재적으로 전달될 수 있기 때문이다. 더욱이 살모넬라 균주는 전신 및 점막 구획 둘 다의 차원에서 강력한 체액성 면역 반응 및 세포성 면역 반응을 유도한다. 회분식 제조에 드는 비용은 저렴하고, 생 세균 백신 제제는 매우 안정적이다. 약독화는 병독성 유전자, 조절 유전자 및 대사 유전자를 비롯하여 다양한 유전자의 결실에 의해 달성될 수 있다.
aro 돌연변이에 의해 약독화된 몇몇 살모넬라 타이피뮤리움 균주는 동물 모델에서 이종 항원에 대한 안전하고 유효한 전달 비이클인 것으로 보였다.
특정 구현예들에서, 살모넬라 약독화 균주와 살모넬라의 추가 약독화 균주 적어도 하나는 살모넬라 타이피 Ty21a이다. 약독화된 생 에스. 타이피 Ty21a 균주는 Vivotif®라고도 공지된 Typhoral L®(제조사: 스위스 소재 Berna Biotech Ltd., Crucell Company)의 활성 성분이다. 이는, 현재 유일하게 장티푸스에 대해 허가받은 경구 생백신이다. 이 백신은 광범위하게 평가되어 왔으며, 그 결과 환자 독성뿐만 아니라 제3자에의 감염에 대해 안전한 것으로 판명되었다(Wahdan et al., J. Infectious Diseases 1982, 145:292-295). 이 백신은 40개국이 넘는 국가에서 허가되었으며, 장티푸스에 대한 예방적 백신화를 위해 수천명의 어린이들을 비롯한 수백만명의 개체들에게 사용되어 왔다. 이는 비할데 없는 안전성 실적을 보인다. 에스. 타이피 Ty21a가 혈류를 통해 전신에 도입될 수 있음을 나타내는 데이터는 얻을 수 없다. 그러므로 약독화 살모넬라 타이피 Ty21a 생백신 균주는 안전성을 보이고 널리 관용되면서 소장 내 면역계의 특이적 표적화를 허용한다. Typhoral L®의 마케팅 허가 번호는 PL 15747/0001(1996년 12월 16일)이다. 백신 1회 용량은 적어도 2 x 109개의 생 에스. 타이피 Ty21a 콜로니 형성 단위와, 적어도 5 x 109개의 무생 에스. 타이피 Ty21a 세포를 함유한다.
이처럼 널리 관용되고 있는, 장티푸스에 대한 경구 생 백신은 야생형 병독성 세균 분리주인 에스. 타이피 Ty2의 화학 돌연변이유발법에 의해 유도되었고, gal/E 유전자에 기능상실돌연변이(loss-of-function mutation)를 보유하는 관계로, 갈락토스를 대사하는 능력을 가지지 못한다. 약독화 세균 균주는 또한 황산염을 황화물로 환원시킬 수 없는데, 이 점은 이 약독화 세균 균주와 야생형 살모넬라 타이피 Ty2 균주가 구별되게 만드는 특징이다. 살모넬라 타이피 Ty21a 균주의 혈청학적 특징과 관련하여, 이 살모넬라 타이피 Ty21a 균주는 세균 외막의 다당체인 O9-항원을 함유하고, 결국 살모넬라 타이피뮤리움의 특징을 이루는 성분인 O5-항원은 결실되어 있다. 이러한 혈청학적 특징은 회분 방출에 대한 동일성 시험의 한 패널에서의 각 시험을 포함하는 것에 대한 근거를 뒷받침해준다.
특정 구현예에서, 발현 카세트는 진핵생물 발현 카세트이다. 구체적으로 발현 카세트는 CMV 프로모터를 포함한다. 본 발명의 내용 중 "진핵생물 발현 카세트"란 용어는, 진핵생물 세포 내 개방 해독틀의 발현을 허용하는 발현 카세트를 지칭한다. 적절한 면역 반응을 유도하는데 필요한 이종 항원의 양은 세균에 독성일 수 있고, 세포 사멸, 과 약독화 또는 이종 항원의 발현 불능을 초래할 수 있다는 사실이 확인되었다. 세균 벡터 내에서 발현되지 않고 오로지 표적 세포에서만 발현되는 진핵생물 발현 카세트가 사용되면, 이와 같은 독성에 관한 문제가 극복될 수 있으며, 발현된 단백질은 통상 진핵생물 당화 패턴을 보인다.
진핵생물 발현 카세트는 진핵생물 세포 내에서 개방 해독틀의 발현을 제어할 수 있는 조절 서열, 바람직하게 프로모터 및 폴리아데닐화 신호를 포함한다. 본 발명의 살모넬라 약독화 균주에 의해 포함된 재조합 DNA 분자에 포함된 프로모터 및 폴리아데닐화 신호는, 바람직하게 면역화될 대상체의 세포 내에서 기능성인 것으로 선택된다. 적합한 프로모터, 특히 인간용 DNA 백신을 생산하는데 적합한 프로모터의 예로서는 거대세포바이러스(CMV) 유래 프로모터, 예컨대 강력 CMV 즉시 초기 프로모터, 유인원 바이러스 40(Simian Virus 40; SV40) 유래 프로모터, 마우스유방종양바이러스(Mouse Mammary Tumor Virus; MMTV) 유래 프로모터, 인간면역결핍증바이러스(HIV) 유래 프로모터, 예컨대 HIV 장 말단 반복부(LTR) 프로모터, 몰로니(Moloney) 바이러스 유래 프로모터, 엡스타인 바 바이러스(Epstein Barr Virus; EBV) 유래 프로모터, 그리고 루 육종 바이러스(Rous Sarcoma Virus;RSV) 유래 프로모터, CMV 초기 인핸서 요소, 프로모터, 닭 베타-액틴 유전자의 제1 엑손 및 제1 인트론, 그리고 토끼 베타 글로빈 유전자의 스플라이싱 수용체로 이루어진 합성 CAG 프로모터뿐만 아니라, 인간 유전자, 예컨대 인간 액틴, 인간 미오신, 인간 헤모글로빈, 인간 근육 크레아틴 및 인간 메탈로티오네인 유래 프로모터를 포함하나, 이에 한정되는 것은 아니다. 특정 구현예에서, 진핵생물 발현 카세트는 CMV 프로모터를 함유한다. 본 발명의 내용 중 "CMV 프로모터"란 용어는 강력 즉시 초기 거대세포바이러스 프로모터를 지칭한다.
특히 인간용 DNA 백신 제조에 적합한 폴리아데닐화 신호의 예로서는 소 성장 호르몬(BGH) 폴리아데닐화 부위, SV40 폴리아데닐화 신호 및 LTR 폴리아데닐화 신호를 포함하나, 이에 한정되는 것은 아니다. 특정 구현예에서, 본 발명의 살모넬라 약독화 균주에 의해 포함되는 재조합 DNA 분자 중에 포함된 진핵생물 발현 카세트는 BGH 폴리아데닐화 부위를 포함한다.
WT1의 발현에 필요한 조절 요소, 예컨대 프로모터 및 폴리아데닐화 신호에 더하여, 기타 요소도 또한 재조합 DNA 분자에 포함될 수 있다. 이러한 추가의 요소는 인핸서를 포함한다. 인핸서는, 예를 들어 인간 액틴, 인간 미오신, 인간 헤모글로빈, 인간 근육 크레아틴의 인핸서와, 바이러스 인핸서, 예컨대 CMV, RSV 및 EBV 유래 인핸서일 수 있다.
조절 서열과 코돈은 일반적으로 종에 의존적이어서, 조절 서열과 코돈은 단백질 생산을 최대화하기 위해, 바람직하게 면역화될 종 내에서 유효한 것으로 선택된다. 당 업자는 주어진 대상 종에서 기능성인 재조합 DNA 분자를 제조할 수 있다.
특정 구현예에서, WT1은 서열 번호 4에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과, 이와 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택된다. 구체적으로 WT1은 절단되고, 더욱 구체적으로 WT1의 아연 핑거 도메인은 결실된다. 이러한 특정 구현예에서, WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과, 이와 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택된다. 구체적으로 WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가진다.
WT1의 C 말단에 있는 아연 핑거 도메인은 4개의 아연 핑거 모티프를 포함한다. 서열 번호 1에서 발견되는 바와 같은 아미노산 서열의 절단된 WT1은 UniProt ref P19544-7의 74번 내지 444번 아미노산을 나타낸다. 아연 핑거 도메인의 절단은, 전사 인자를 함유하는 기타 아연 핑거와의 면역학적 교차 반응성이 발생할 위험을 최소화한다. 게다가 아연 핑거 도메인이 결실된 절단 WT1은 전장 WT1보다 더 큰 면역원 잠재성을 가진다. 게다가 DNA 결합에 필수인 아연 핑거 모티프의 결실은 WT1의 발암 잠재성을 없애주므로, 발암 위험이 최소화된다.
본 발명의 내용 중 "약" 또는 "대략"이란 용어는, 주어진 값이나 범위의 80% 내지 120% 이내, 대안적으로는 90% 내지 110% 이내, 예컨대 95% 내지 105% 이내를 의미한다.
본 발명의 내용 중 "주어진 단백질과 적어도 약 80%의 서열 동일성을 공유하는 단백질", 예컨대 서열 번호 1 또는 서열 번호 4에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1이란 용어는, 상기 기준 단백질, 예컨대 서열 번호 1 또는 서열 번호 4 각각의 아미노산 서열을 가지는 인간 WT1의 아미노산 서열을 암호화하는 핵산 서열 및/또는 이 아미노산 서열에 있어서 차이가 날 수 있는 단백질을 지칭한다. 단백질은 천연 기원의 것, 예컨대 야생형 단백질의 돌연변이체 형태, 예컨대 야생형 WT1의 돌연변이체 형태, 또는 상이한 종의 동족체(homolog), 또는 조작된 단백질, 예컨대 조작된 WT1일 수 있다. 코돈의 선호도는 종들 간에 상이한 것으로 공지되어 있다. 그러므로 표적 세포 내에서 이종 단백질이 발현되면, 핵산 서열을 표적 세포의 코돈 선호도에 적응시키는 것이 필요할 수 있거나, 아니면 최소한 도움이 될 수 있다. 주어진 단백질의 유도체를 디자인 및 구성하기 위한 방법은 임의의 당업자에게 널리 공지되어 있다.
주어진 단백질, 예컨대 서열 번호 1 또는 서열 번호 4에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과 적어도 약 80%의 서열 동일성을 공유하는 단백질은 기준 단백질, 예컨대 서열 번호 1 또는 서열 번호 4 각각의 아미노산 서열을 가지는 WT1에 비하여 하나 이상의 아미노산의 부가, 결실 및/또는 치환을 포함하는 돌연변이 하나 이상을 함유할 수 있다. 본 발명의 교시내용에 따르면, 상기 결실, 부가 및/또는 치환된 아미노산은 연속 아미노산일 수 있거나, 또는 기준 단백질, 예컨대 서열 번호 1 또는 서열 번호 4 각각에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과 적어도 약 80%의 서열 동일성을 공유하는 단백질의 아미노산 서열 길이에 걸쳐 산재될 수 있다. 본 발명의 교시내용에 따르면, 기준 단백질과의 아미노산 서열 동일성이 적어도 약 80%이고, 돌연변이 단백질이 면역원성인 한, 임의의 수의 아미노산이 부가, 결실 및/또는 치환될 수 있다. 바람직하게 주어진 기준 단백질, 예컨대 서열 번호 1 또는 서열 번호 4에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과 적어도 약 80%의 서열 동일성을 공유하는 단백질의 면역원성은, ELISA에 의해 측정되는 바에 따르면 상기 기준 단백질, 예컨대 서열 번호 1 또는 서열 번호 4 각각에서 발견되는 바와 같은 아미노산 서열을 가지는 WT1의 면역원성에 비하여 50% 미만, 40% 미만, 30% 미만, 20% 미만, 10% 미만, 5% 미만 또는 1% 미만까지 감소한다. 단백질 동족체를 디자인 및 구성하기 위한 방법과, 이러한 동족체를 자체의 면역원성 잠재력에 대해 시험하기 위한 방법은 임의의 당 업자에게 널리 공지되어 있다. 특정 구현예에서, 기준 단백질, 예컨대 서열 번호 1 또는 서열 번호 4의 아미노산 서열을 가지는 WT1과의 서열 동일성은 적어도 약 80%, 적어도 약 85%, 적어도 약 90%이거나, 가장 구체적으로는 적어도 약 95%이다. 부모 단백질과, 결실, 부가 및/또는 치환을 가지는 이의 유도체의 부모 서열을 기준으로 하는 비교를 비롯하여 서열 동일성을 측정하기 위한 방법 및 알고리즘은 당 업계의 통상의 지식을 가지는 실무자에게 널리 공지되어 있다. DNA 수준에서 주어진 기준 단백질, 예컨대 서열 번호 1 또는 서열 번호 4에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1과 적어도 약 80%의 서열 동일성을 공유하는 단백질을 암호화하는 핵산 서열은, 유전자 암호의 축퇴성으로 말미암아 매우 상이할 수 있다.
본 발명의 내용 중 "절단된 WT1"이란 용어는, 하나의 아미노산 또는 하나를 초과하는 연속적 아미노산 각각의 결실 하나 이상을 보유하는 WT1을 지칭한다. 본 발명의 교시내용에 따르면, 돌연변이된 단백질이 면역원성인 한 임의의 수의 아미노산이 결실될 수 있다. 바람직하게 절단된 WT1 단백질의 면역원성은 ELISA에 의해 측정되는 바에 따르면 서열 번호 4(UniProt ref P19544-7)에서 발견되는 바와 같은 아미노산 서열을 가지는 전장 WT1의 면역원성에 비하여 50% 미만, 40% 미만, 30% 미만, 20% 미만, 10% 미만, 5% 미만 또는 1% 미만까지 감소한다. 단백질 동족체를 디자인 및 구성하기 위한 방법과, 이러한 동족체를 자체의 면역원 잠재성에 대해 시험하기 위한 방법은 임의의 당업자에게 널리 공지되어 있다. 특정 구현예에서 500개 미만, 400개 미만, 350개 미만, 300개 미만, 250개 미만, 200개 미만, 175개 미만, 150개 미만, 125개 미만, 100개 미만, 75개 미만, 50개 미만 또는 25개 미만의 아미노산이, 서열 번호 4(UniProt ref P19544-7)에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 전장 WT1 또는 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로부터 결실된다. 구체적으로 절단된 WT1은 아연 핑거 도메인이 결실된 WT1이고, 바람직하게는 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 WT1과, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택된다.
특정 구현예에서, DNA 분자는 카나마이신 항생제 내성 유전자, pMB1 ori 및 CMV 프로모터를 포함한다. 특정 구현예에서, 재조합 DNA 분자는 상업적으로 이용 가능한 pVAX1TM 발현 플라스미드(Invitrogen, San Diego, California)로부터 유래한다. 이 발현 벡터는, pBR322의 저 복사체 pMB1 복제 기원으로 고 복사체 pUC 복제 기원을 치환함으로써 변형되었다. 저 복사체 변형은 구조체를 더욱 안정적으로 만들고 대사 부담(metabolic burden)을 줄이기 위해 이루어졌다. 제조된 발현 벡터 백본(backbone)은 pVAX10이라 명명하였다.
특정 구현예에서, DNA 분자는 서열 번호 2에서 발견되는 바와 같은 DNA 서열을 포함한다(벡터 백본 pVAX10).
서열 번호 3에서 발견되는 바와 같은 핵산 서열을 가지는 ORF는 아연 핑거 도메인이 결실된 인간 WT1을 암호화한다. 이 ORF가 NheI/ XhoI를 통해 발현 벡터 백본(pVAX10)에 삽입되어, 발현 플라스미드 pVAX10.hWT1이 수득되었다. 이 발현 플라스미드 pVAX10.hWT1은 도 11에 개략적으로 도시되어 있다. 발현 플라스미드 pVAX10.hWT1을 보유하는 약독화 살모넬라 균주 Ty21a를 포함하는 DNA 백신은 VXM06이라 명명된다.
특정 구현예에서, 살모넬라 약독화 균주는 상기 적어도 하나의 관문 억제제 투여와 동시에, 이의 투여 이전에 또는 투여 이후에 투여된다.
본 발명의 내용 중 "동시에"라는 용어는 WT1을 암호화하는 살모넬라 약독화 균주 및 적어도 하나의 관문 억제제의 투여가 같은 날에, 더욱 구체적으로는 12시간 이내의 간격을 두고, 더욱 구체적으로는 2시간 이내의 간격을 두고 이루어지는 것을 의미한다.
특정 구현예에서, WT1을 암호화하는 살모넬라 약독화 균주 및 적어도 하나의 관문 억제제의 투여는, 8 연속 주, 더욱 구체적으로는 3 내지 6 연속 주 이내에 이루어진다. WT1을 암호화하는 살모넬라 약독화 균주 및 적어도 하나의 관문 억제제는 동일한 경로를 통하거나 상이한 경로를 통해 투여될 수 있다.
특정 구현예에서, 치료법은 화학요법, 방사선요법 또는 암 생물요법이 동반된다. 암 치유를 위해 암 줄기 세포를 완전히 제거하는 것이 필수적일 수 있다. 최대의 효능을 달성하기 위해 상이한 치료적 접근법을 병행하는 것이 유리할 수 있다.
본 발명의 내용 중 "암 생물요법"이란 용어는, 바이러스를 포함하는 살아있는 유기체, 살아있는 유기체로부터 유래하는 성분 또는 이러한 성분의 실험실에서 제조된 것을 이용하는 것을 수반하는 암 요법을 지칭한다. 몇몇 암 생물요법은 몸의 면역계를 자극하여 암 세포에 대항하게 만드는 것을 목표로 한다(소위 암 생물면역요법). 암 생물치료적접근법은, 예컨대 살모넬라 기반 DNA 백신, 구체적으로 에스. 타이피 Ty21a 기반 DNA 백신에 의한 종양 항원 및 종양 기질 항원의 전달, 치료적 항체의 약물로서의 전달, 면역자극성 시토카인의 투여, 그리고 조작된 T 세포를 비롯한 면역 세포의 투여를 포함한다. 치료적 항체는 종양 항원 또는 종양 기질 항원을 표적화하는 항체를 포함한다.
특정 구현예에서, 암 생물요법은 종양 항원 및/또는 종양 기질 항원을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는 살모넬라의 추가 약독화 균주(들) 하나 이상을 투여하는 단계를 포함한다. 이러한 특정 구현예에서, 상기 살모넬라의 추가 약독화 균주(들) 하나 이상은 진핵생물 발현 카세트를 포함하는 살모넬라 타이피 Ty21a이다. 구체적으로 상기 종양 항원은 메소텔린(MSLN), CEA 및 CMV pp65로 이루어진 군으로부터 선택되고, 바람직하게 상기 종양 항원은 (a) 메소텔린(MSLN), 구체적으로 서열 번호 5에서 발견되는 바와 같은 아미노산 서열을 가지는 MSLN과, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, (b) CEA, 구체적으로 서열 번호 6에서 발견되는 바와 같은 아미노산 서열을 가지는 CEA와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 그리고 (c) CMV pp65, 구체적으로 서열 번호 7에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 서열 번호 8에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 그리고 서열 번호 9에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택된다. 구체적으로 상기 종양 기질 항원은 VEGF 수용체 단백질 및 인간 섬유아세포 활성화 단백질(FAP)로 이루어진 군으로부터 선택되는데, 바람직하게 이 VEGF 수용체 단백질은 VEGFR-2, 더욱 바람직하게는 인간 VEGFR-2, 더욱더 바람직하게는 서열 번호 10에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 VEGFR-2와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질이다.
본 발명의 살모넬라 약독화 돌연변이 균주와 함께 사용될 수 있는 화학요법제는, 예를 들어 겜시타빈, 아미포스틴(에티올), 카바지탁셀, 시스플라틴, 다카바진(DTIC), 닥티노마이신, 도세탁셀, 메클로레타민, 스트렙토조신, 사이클로포스파미드, 카머스틴(BCNU), 로머스틴(CCNU), 독소루비신(아드리아마이신), 독소루비신 리포(독실), 폴리닌산, 겜시타빈(겜자), 도노루비신, 도노루비신 리포(도녹솜), 프로카바진, 케토코나졸, 미토마이신, 시타라빈, 에토포시드, 메토트렉세이트, 5-플루오로우라실(5-FU), 빈블라스틴, 빈크리스틴, 블레오마이신, 파클리탁셀(탁솔), 도세탁셀(탁소텔), 알데스루킨, 아스파라기나아제, 부설판, 카보플라틴, 클라드리빈, 캄프토테신, CPT-11, 10-하이드록시-7-에틸-캄프토테신(SN38), 다카바진, 플록수리딘, 플루다라빈, 하이드록시우레아, 이포스파미드, 이다루비신, 메스나, 인터페론 알파, 인터페론 베타, 이리노테칸, 미토잔트론, 토포테칸, 루프롤리드, 메게스트롤, 멜파란, 머캅토퓨린, 옥살플라틴, 플리카마이신, 미토탄, 페가스파르가아제, 펜토스타틴, 피포브로만, 플리카마이신, 스트렙토조신, 타목시펜, 테니포시드, 테스토락톤, 티오구아닌, 티오테파, 우라실 머스타드, 비노렐빈, 클로람부실, 보르테조밉, 탈리도마이드, 레날리도마이드 및 이것들의 조합일 수 있다.
본 발명에 따라 VXM06 및 적어도 하나의 관문 억제제와 함께 사용되기에 가장 바람직한 화학요법제는 카바지탁셀, 카보플라틴, 옥살리플라틴, 시스플라틴, 사이클로포스파미드, 도노루비신, 이다루비신, 에피루비신, 에토포시드, 도세탁셀, 겜시타빈, 독소루비신, 파클리탁셀(탁솔), 이리노테칸, 빈크리스틴, 빈블라스킨, 비노렐빈, 폴리닌산, 5-플루오로우라실, 이포스파미드 및 블레오마이신, 특히 겜시타빈이다.
구체적으로 살모넬라 약독화 균주는 화학요법 또는 방사선요법 치료 주기 또는 암 생물요법이 수행되기 전, 수행되는 도중 또는 수행된 후에 투여된다. 다른 특정 구현예에서, 살모넬라 약독화 균주는 화학요법 또는 방사선요법 치료 주기 또는 암 생물요법이 수행되기 전 및 수행되는 도중에 투여된다.
특정 구현예에서, 살모넬라 약독화 균주는 경구 투여된다. 경구 투여는 비경구 투여보다 더 간단하고, 더 안전하며, 더 편안하다. 그러나 WT1을 암호화하는 살모넬라 약독화 균주는 또한 임의의 다른 적합한 경로에 의해 투여될 수 있음도 주목되어야 한다. 바람직하게 치료적 유효 용량이 대상체에 투여되고, 이 용량은 구체적인 응용예, 악성종양의 유형, 대상체의 체중, 나이, 성별 및 건강 상태, 투여 방식 및 제형 등에 따라 달라진다. 투여는 필요에 따라 1회 투여 또는 다수회 투여일 수 있다.
WT1을 암호화하는 살모넬라 약독화 균주는 용액, 현탁액, 동결건조물, 장용외피캡슐 또는 임의의 다른 적합한 형태로서 제공될 수 있다. 통상 살모넬라 약독화 균주는 음용 용액으로서 제형된다. 이 구현예는, 환자의 수용상태(patient compliance) 개선이라는 이점을 제공한다. 바람직하게 음용 용액은 위산을 적어도 어느 정도 중화하는, 즉 위액의 pH를 pH 7에 더 가깝게 만드는 수단을 포함한다. 바람직하게 음용 용액은 WT1을 암호화하는 살모넬라 약독화 균주를 포함하는 완충 현탁액이다. 특정 구현예에서, 완충 현탁액은 살모넬라 약독화 균주를 적합한 완충제, 바람직하게는 탄산수소나트륨 2.6 g, L-아스코르브산 1.7 g, 락토스 일수화물 0.2 g 및 음용수 100 ml를 함유하는 완충제에 현탁함으로써 수득된다.
적어도 하나의 관문 억제제는, 바람직하게 시판제품의 승인된 생약 제제중에 포함되어 투여된다.
특정 구현예에서, 암은 백혈병, 구체적으로 급성 골수성 백혈병(AML) 및 급성 림프성 백혈병(ALL), 다발성 골수종으로부터 선택되고, 고형 종양, 구체적으로 폐암, 유방암, 식도암, 결장암, 결장직장암, 위암, 융모막암, 췌장암, 교모세포종, 두경부암, 활막육종, 혈관육종, 골육종, 감상샘암, 자궁경부암, 자궁내막암, 난소암, 신경모세포종, 횡문근육종 및 전립선암으로부터 선택된다.
WT1을 암호화하는 살모넬라 약독화 균주는 놀랍게도 비교적 저용량에서 유효하다. 게다가 WT1을 암호화하는 살모넬라 약독화 균주와, 적어도 하나의 관문 억제제의 병용 투여는 놀랍게도 WT1을 암호화하는 살모넬라 약독화 균주가 비교적 저용량으로 투여될 때 WT1 특이적 T 세포 반응, 종양 성장 및/또는 전체 생존율에 상승 효과를 보인다. 생 세균 백신의 저용량 투여는 배출설의 위험도 최소화하고, 이에 따라 제3자에의 전염도 최소화한다.
특정 구현예에서, 살모넬라 약독화 균주의 1회 용량은 약 105 내지 약 1011, 구체적으로 약 106 내지 약 1010, 더욱 구체적으로 약 106 내지 약 109, 더욱 구체적으로 약 106 내지 약 108, 가장 구체적으로 약 106 내지 약 107 콜로니 형성 단위(CFU)를 포함한다.
본 발명의 내용 중 "약" 또는 "대략"이란 용어는, 주어진 값이나 범위의 3 팩터(factor) 이내, 대안적으로 2 팩터 이내, 예컨대 1.5 팩터 이내를 의미한다.
특정 구현예에서, 치료법은 환자에서 WT1의 발현 및/또는 WT1에 대한 전 면역 반응을 평가하는 단계를 포함하는 개별맞춤 암 면역요법이다. 환자의 WT1 발현 및/또는 환자의 WT1에 대한 전 면역 반응은, 예컨대 동반진단(companion diagnostic)에 의하여 제1 단계에서 평가될 수 있다. 표적 유전자, 예컨대 wt1의 mRNA 수준 또는 단백질 수준 중 어느 하나의 수준에서의 발현을 평가하기 위한 방법은 임의의 당업자에게 널리 공지되어 있다. 예를 들어 면역조직화학적 염색법, 유세포분석 방법, RNA 서열결정법, 또는 표지화를 이용하는 대안적 방법이 종양내 표적 발현 수준을 확인하는데 사용될 수 있다. 유사하게, 주어진 단백질, 예컨대 WT1에 대한 환자의 전 면역 반응을 평가하기 위한 방법은 임의의 당업자에게 널리 공지되어 있다. 환자의 기존 WT-1 특이적 T 세포 풀(pool)은, 예컨대 ELISpot 또는 다량체 FACS 분석에 의해 검출될 수 있다. 고 WT1 발현 및/또는 WT1에 대한 전 면역의 유도는, WT1을 암호화하는 살모넬라 약독화 균주가 단독으로, 또는 적어도 하나의 관문 억제제와 함께 처리될 때, 이 처리에 유리하게 반응하는 환자의 소인에 대한 진단 지표이다.
유리하게, 일어날 수 있는 부작용의 발생에 따라서 항생제 또는 소염제를 사용하는 치료법이 포함되는 것이 유리할 수 있다.
만일 히스타민, 루코트리엔 또는 시토카인에 의해 매개되는 과민 반응과 유사한 부작용이 일어난다면, 발열, 아나필락시스, 혈압 불안정, 기관지경련 및 호흡곤란에 대한 치료 선택권이 적용될 수 있다. 원치않는 T 세포 유도성 자기공격(auto-aggression)의 경우 치료 선택권은 줄기 세포 이식 후에 적용되는 급성 및 만성 이식편대 숙주병의 표준 치료 계획으로부터 도출된다. 사이클로스포린 및 글루코코르티코이드가 치료 선택권으로서 제안된다.
전신 살모넬라 타이피 Ty21a형 감염의 경우와는 다른 경우, 예컨대 시프로플록사신 또는 오플록사신을 비롯한 플루오로퀴놀론이 사용되는 적당한 항생제요법이 권장된다. 위장관의 세균 감염은 각각의 제제, 예컨대 리팍시민으로 치료될 것이다.
추가의 양태에서, 본 발명은 WT1을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는 살모넬라 약독화 균주를 포함하는, 암 치료법에 사용하기 위한 약학 조성물에 관한 것으로서, 이 치료법은 적어도 하나의 관문 억제제를 투여하는 단계를 추가로 포함한다. 구체적으로 적어도 하나의 관문 억제제는 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택된다. 바람직하게 적어도 하나의 관문 억제제는 PD-1, PD-L1 또는 CTLA-4에 대한 항체 적어도 하나, 또는 이의 조합이고, 더욱 바람직하게 적어도 하나의 관문 억제제는 PD-L1 또는 CTLA-4에 대한 항체 적어도 하나, 또는 이의 조합이다.
본 약학 조성물은 용액, 현탁액, 장용외피캡슐, 동결건조분말의 형태, 또는 의도로 하는 용도에 적합한 임의의 다른 형태로 존재할 수 있다.
본 약학 조성물은 하나 이상의 약학적으로 허용 가능한 부형제를 추가로 포함할 수 있다.
본 발명의 내용 중 "부형제"란 용어는, 의약의 활성 성분과 함께 제제화되는 천연 또는 합성 성분을 지칭한다. 적합한 부형제로서는 접착방지제, 결합제, 코팅, 붕해제, 향미제, 착색제, 윤활제, 활강제, 흡수제, 보존제 및 감미제를 포함한다.
본 발명의 내용 중 "약학적으로 허용 가능한"이란 용어는, 약학 조성물의 분자적 실체 및 기타 성분이 생리학적으로 관용될 수 있고, 포유동물(예컨대 인간)에 투여될 때 원치않는 방향의 반응을 통상 일으키지 않는 경우를 의미한다. "약학적으로 허용 가능한"이란 용어는 또한 포유동물, 더욱 구체적으로 인간에서 사용될 수 있다고 연방 또는 주 정부 규제국에 의해 승인되었거나, 미국 약전 또는 일반적으로 인지되는 다른 약전에 나열된 경우를 의미한다.
특정 구현예에서, 약학 조성물은 음용 용액으로서 제공된다. 이 구현예는 환자 수용상태를 개선한다는 이점을 제공하고, 신속하고, 실현가능하며, 감당할 수 있는 집단 백신화 프로그램을 허용한다.
구체적으로 적합한 음용 용액은 위산을 적어도 어느 정도 중화하는, 즉 위액의 pH를 pH 7에 더 가깝게 만드는 수단을 포함한다. 특정 구현예에서, 음용 용액은 본 발명에 따른 살모넬라 약독화 균주를 적합한 완충제, 바람직하게는 위산을 적어도 어느 정도 중화하는 완충제, 바람직하게 탄산수소나트륨 2.6 g, L-아스코르브산 1.7 g, 락토스 일수화물 0.2 g 및 음용수 100 ml를 함유하는 완충제에 현탁함으로써 수득된 완충 현탁액이다.
특정 구현예에서, 살모넬라 약독화 균주는 살모넬라 타이피 Ty21a이다.
특정 구현예에서, 발현 카세트는 진핵생물 발현 카세트이다.
특정 구현예에서, WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 WT1과, 이 서열과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택된다.
구체적으로 진핵생물 발현 카세트는 CMV 프로모터를 포함한다.
구체적으로 WT1은 서열 번호 1에서 발견된 바와 같은 아미노산 서열을 가진다.
특정 구현예에서, 치료법은 WT1을 암호화하는 살모넬라 약독화 균주, 또는 이것 및/또는 적어도 하나의 관문 억제제를 포함하는 약학 조성물을 1회 또는 다수회 투여하는 것을 포함한다. 투여의 1회 용량은 동일하거나 상이할 수 있다. 구체적으로 치료법은 WT1을 암호화하는 살모넬라 약독화 균주 및/또는 적어도 하나의 관문 억제제를 1, 2, 3, 4, 5 또는 6 회 투여하는 것을 포함하는데, 다만 다수회 투여는, 바람직하게 3 내지 6 연속 월 이내에 이루어진다.
도면 및 표에 대한 간단한 설명
도 1: 플라스미드 pVAX10.hWT1 내에 함유된 WT1 cDNA에 의해 암호화되고, 아연 핑거 도메인이 결실된 인간 WT1의 아미노산 서열(서열 번호 1).
도 2: 공 발현 벡터(empty expression vector) pVAX10에 포함된 핵산 서열(제한 부위 NheI 및 XhoI 사이에 위치하는 다중 클로닝 부위(multiple cloning site)의 일부가 없는 발현 벡터 pVAX10의 서열(서열 번호 2)).
도 3: 서열 번호 1의 인간 WT1을 암호화하고, 플라스미드 pVAX10.hWT1에 함유된 핵산 서열.
도 4: 인간 전장 WT1의 아미노산 서열(UniProt ref P19544-7; 서열 번호 4).
도 5: 플라스미드 pVAX10.hMSLN에 함유된 MSLN cDNA에 의해 암호화되는 인간 MSLN의 아미노산 서열(서열 번호 5).
도 6: 플라스미드 pVAX10.hCEA에 함유된 CEA cDNA에 의해 암호화되는 인간 CEA의 아미노산 서열(서열 번호 6).
도 7: 플라스미드 pVAX10.CMVpp65_1에 함유된 CMV pp65 cDNA에 의해 암호화되는 CMV pp65의 아미노산 서열(서열 번호 7).
도 8: 플라스미드 pVAX10.CMVpp65_2에 함유된 CMV pp65 cDNA에 의해 암호화되는 CMV pp65의 아미노산 서열(서열 번호 8).
도 9: 플라스미드 pVAX10.CMVpp65_3에 함유된 CMV pp65 cDNA에 의해 암호화되는 CMV pp65의 아미노산 서열(서열 번호 9).
도 10: 플라스미드 pVAX10.VR2-1에 함유된 VEGFR-2 cDNA에 의해 암호화되는 VEGFR-2의 아미노산 서열(서열 번호 10).
도 11: pVAX10.hWT1의 플라스미드 맵
도 12: VXM06m과 항 CTLA-4의 병용 투여가, 산재성 FBL-3을 보유하는 C57BL/6 마우스의 생존률에 미치는 효과.
도 13: VXM06m과 항 PD-L1의 병용 투여가, 산재성 FBL-3을 보유하는 C57BL/6 마우스의 생존률에 미치는 효과.
실시예
PD-L1 및
CTLA
-4 관문 억제제 봉쇄가 병행되었을 때
VXM06m의
항종양
활성에 대한 평가
본 연구의 목적은, C57BL/6 마우스에 마우스 세포주 FBL-3가 복막내 이식되어 유도된 백혈병의 동계유전자 종양 모델에 있어서 PD-L1 및 CTLA-4 관문 억제제 봉쇄가 병행되었을 때 VXM06m의 항종양 활성을 관찰하는 것이었다.
60마리의 수컷 C57/BL6 마우스(4 ~ 6주령, 마우스 1마리의 평균 무게 약 20 g)를 각 군당 15마리의 동물로 이루어진 군 4개에 무작위로 할당하였다.
1군(대조군): 1, 3, 5, 7, 14 및 22 일에 마우스(n = 15)를 공 벡터 VXM0m_empty(외부 발현 플라스미드를 보유하지 않는 에스. 타이피뮤리움 세균 벡터 대조군)로 처리하였다. 20일째 되는 날에, FBL-3 종양 세포를 마우스에 복막내 경로로 이식하였다.
2군: 1, 3, 5, 7, 14 및 22 일에 마우스(n = 15)에 VXM06m(절단된 마우스 WT1을 암호화하는 pVAX10.mWT1 함유 에스. 타이피뮤리움)을, 적용당 108 CFU의 용량으로 처리하였다. 한편, 24일 및 29일에는 마우스에 항 PD-L1 모노클로날 항체(BP0101, 생체 내 플러스(In Vivo Plus) 항 마우스 PD-L1, 클론 10F.9G2, BioXCell)를, 적용당 100 μg으로 복막내 경로로 처리하였다. 20일에 FBL-3 종양 세포를 마우스에 복막내 경로로 이식하였다.
3군: 1, 3, 5, 7, 14 및 22 일에 마우스(n = 15)에 VXM06m을, 적용당 108 CFU의 용량으로 처리하였다. 한편, 11일 및 18일에는 마우스에 항 CTLA-4 모노클로날 항체(항 CTLA-4 항체, 클론 UC10-4F10, BioXCell)를, 적용당 100 μg으로 복막내 경로로 처리하였다. 20일에 FBL-3 종양 세포를 마우스에 복막내 경로로 이식하였다.
4군(대조군): 1, 3, 5, 7, 14 및 22 일에 마우스(n = 15)에 공 벡터 VXM0m_empty(1회 적용당 108 CFU)를 처리하되, 11일과 18일에는 항 CTLA-4(100 μg/용량)도 처리하였다. 20일에 FBL-3 종양 세포를 마우스에 복막내 경로로 이식하였다.
FBL-3은 C57BL/6 마우스로부터 기원하는 프렌드(Friend) 백혈병 바이러스 유도성 적백혈병 세포주이다. 이 세포주는 면역계에 의해 인지될 수 있는 독특한 TSTA(종양 특이적 이식 항원: Tumor Specific Transplantation Antigens)를 발현한다. 동계유전자 마우스를 FBL-3 종양 세포로 프라이밍(priming)한 결과, 미래의 생 종양 면역공격에 따르는 거부현상이 초래되었다. 비록 FBL-3는 면역원성이지만, 생 FBL-3 종양 세포를 처녀 동계유전자 마우스에 주입할 경우 종양 성장이 초래되었는데, 이 점은 FBL-3 종양 세포가, 면역 인지 및 파괴를 도피하는 기구를 가짐을 암시한다. 중요한 점은, FBL-3가 빌름 종양 1(WT1)을 과발현하는 것으로 보였다는 점이다.
마우스 생존 시간을 전체 연구 기간 내내 신중히 모니터링하였으며. 그 결과를 도 12 및 도 13에 도시하였다.
7일에, 공 벡터로 처리된 1군 마우스 중 한마리가 폐사한 것이 관찰되었는데, 이는 아마도 백신 투여일에 식도에 사고로 천공이 발생하였기 때문인 것으로 생각된다.
- 23일에 4군의 마우스 중 한마리가 폐사하였다.
- 25일에 4군의 마우스 중 한마리가 폐사하였다.
- 29일에 각 군의 마우스 다섯마리를 죽이고 나서, 비장을 수집하였다.
- 30일에 2군의 마우스 중 한마리가 폐사하였다.
- 39일에 1군의 마우스 중 한마리가 폐사하였다.
- 58일에 1군의 마우스 중 세마리가 폐사하였다.
- 65일에 1군의 마우스 중 두마리가 폐사하였다.
- 74일에 1군의 마우스 중 두마리가 폐사하였다.
- 83일에 1군 및 4군의 마우스 중 두마리가 폐사하였다.
- 90일에 4군의 마우스 중 한마리가 폐사하였다
즉 20일에 행해진 1차 종양 세포 면역공격 이후, 오로지 1군, 2군 및 4군에서만 동물의 폐사가 관찰되었고, 다른 군의 경우 모든 동물은 건강한 상태로서 종양 발달도 관찰되지 않았다.
이러한 결과는, 관문 억제제 항 CTLA-4 및 항 PD-L1 중 어느 하나와 VXM06m의 조합이 FBL-3 모델에서 신속하고도 지속적인 항 종양 효과를 발휘하며 매우 유효하였음을 명백히 보여준다. VXM06m 및 항 CTLA-4의 조합은, 연구의 막바지(196일)에 가서는 100% 생존률을 달성하였다.
이와는 대조적으로, 공 벡터 처리시에는 항 종양 효과가 보이지 않았는데, 종양 면역공격후 45일 경과시 평균 생존률을 보였고, 회복율은 0%였다. 항 CTLA-4 처리는 연구의 막바지에 가서는 40% 폐사율을 보였다.
SEQUENCE LISTING
<110> VAXIMM AG
<120> WT1 TARGETING DNA VACCINE FOR COMBINATION THERAPY
<130> 113021P855PC
<150> EP16197322.7
<151> 2016-11-04
<160> 10
<170> BiSSAP 1.3.6
<210> 1
<211> 371
<212> PRT
<213> Homo sapiens
<400> 1
Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro
1 5 10 15
Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala
20 25 30
Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr
35 40 45
Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro
50 55 60
Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly
65 70 75 80
Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe
85 90 95
Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe
100 105 110
Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe
115 120 125
Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile
130 135 140
Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr
145 150 155 160
Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe
165 170 175
Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln
180 185 190
Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser
195 200 205
Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp
210 215 220
Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln
225 230 235 240
Met Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser
245 250 255
Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu
260 265 270
Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile
275 280 285
His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro
290 295 300
Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys
305 310 315 320
Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys
325 330 335
Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro
340 345 350
Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp
355 360 365
Gln Leu Lys
370
<210> 2
<211> 3500
<212> DNA
<213> Artificial Sequence
<220>
<223> expression plasmid
<400> 2
tgggcttttg ctggcctttt gctcacatgt tcttgactct tcgcgatgta cgggccagat 60
atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 120
ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 180
gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 240
caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 300
cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 360
ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 420
tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 480
gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 540
gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 600
tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 660
taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 720
cccaagctgg ctagcctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact 780
gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 840
gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 900
agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 960
gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctac tgggcggttt 1020
tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc 1080
cctgcaaagt aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa 1140
gctctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 1200
caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 1260
tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 1320
tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt 1380
ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 1440
gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 1500
ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 1560
ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 1620
aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 1680
aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg 1740
gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 1800
gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 1860
ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 1920
ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg 1980
cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 2040
tacaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 2100
acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca 2160
cgtgctaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 2220
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc cccatcagtg 2280
accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg 2340
cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt 2400
cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa 2460
aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 2520
agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 2580
acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 2640
ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 2700
accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 2760
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 2820
ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 2880
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 2940
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3000
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3060
ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 3120
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3180
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3240
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3300
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 3360
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3420
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 3480
ctcaagaaga tcctttgatc 3500
<210> 3
<211> 1116
<212> DNA
<213> Homo sapiens
<400> 3
atggacttcc tcttgctgca ggacccggct tccacgtgtg tcccggagcc ggcgtctcag 60
cacacgctcc gctccgggcc tgggtgccta cagcagccag agcagcaggg agtccgggac 120
ccgggcggca tctgggccaa gttaggcgcc gccgaggcca gcgctgaacg tctccagggc 180
cggaggagcc gcggggcgtc cgggtctgag ccgcagcaaa tgggctccga cgtgcgggac 240
ctgaacgcgc tgctgcccgc cgtcccctcc ctgggtggcg gcggcggctg tgccctgcct 300
gtgagcggcg cggcgcagtg ggcgccggtg ctggactttg cgcccccggg cgcttcggct 360
tacgggtcgt tgggcggccc cgcgccgcca ccggctccgc cgccaccccc gccgccgccg 420
cctcactcct tcatcaaaca ggagccgagc tggggcggcg cggagccgca cgaggagcag 480
tgcctgagcg ccttcactgt ccacttttcc ggccagttca ctggcacagc cggagcctgt 540
cgctacgggc ccttcggtcc tcctccgccc agccaggcgt catccggcca ggccaggatg 600
tttcctaacg cgccctacct gcccagctgc ctggagagcc agcccgctat tcgcaatcag 660
ggttacagca cggtcacctt cgacgggacg cccagctacg gtcacacgcc ctcgcaccat 720
gcggcgcagt tccccaacca ctcattcaag catgaggatc ccatgggcca gcagggctcg 780
ctgggtgagc agcagtactc ggtgccgccc ccggtctatg gctgccacac ccccaccgac 840
agctgcaccg gcagccaggc tttgctgctg aggacgccct acagcagtga caatttatac 900
caaatgacat cccagcttga atgcatgacc tggaatcaga tgaacttagg agccacctta 960
aagggagttg ctgctgggag ctccagctca gtgaaatgga cagaagggca gagcaaccac 1020
agcacagggt acgagagcga taaccacaca acgcccatcc tctgcggagc ccaatacaga 1080
atacacacgc acggtgtctt cagaggcatt cagtga 1116
<210> 4
<211> 522
<212> PRT
<213> Homo sapiens
<400> 4
Met Asp Phe Leu Leu Leu Gln Asp Pro Ala Ser Thr Cys Val Pro Glu
1 5 10 15
Pro Ala Ser Gln His Thr Leu Arg Ser Gly Pro Gly Cys Leu Gln Gln
20 25 30
Pro Glu Gln Gln Gly Val Arg Asp Pro Gly Gly Ile Trp Ala Lys Leu
35 40 45
Gly Ala Ala Glu Ala Ser Ala Glu Arg Leu Gln Gly Arg Arg Ser Arg
50 55 60
Gly Ala Ser Gly Ser Glu Pro Gln Gln Met Gly Ser Asp Val Arg Asp
65 70 75 80
Leu Asn Ala Leu Leu Pro Ala Val Pro Ser Leu Gly Gly Gly Gly Gly
85 90 95
Cys Ala Leu Pro Val Ser Gly Ala Ala Gln Trp Ala Pro Val Leu Asp
100 105 110
Phe Ala Pro Pro Gly Ala Ser Ala Tyr Gly Ser Leu Gly Gly Pro Ala
115 120 125
Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro Pro His Ser Phe
130 135 140
Ile Lys Gln Glu Pro Ser Trp Gly Gly Ala Glu Pro His Glu Glu Gln
145 150 155 160
Cys Leu Ser Ala Phe Thr Val His Phe Ser Gly Gln Phe Thr Gly Thr
165 170 175
Ala Gly Ala Cys Arg Tyr Gly Pro Phe Gly Pro Pro Pro Pro Ser Gln
180 185 190
Ala Ser Ser Gly Gln Ala Arg Met Phe Pro Asn Ala Pro Tyr Leu Pro
195 200 205
Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg Asn Gln Gly Tyr Ser Thr
210 215 220
Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly His Thr Pro Ser His His
225 230 235 240
Ala Ala Gln Phe Pro Asn His Ser Phe Lys His Glu Asp Pro Met Gly
245 250 255
Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr Ser Val Pro Pro Pro Val
260 265 270
Tyr Gly Cys His Thr Pro Thr Asp Ser Cys Thr Gly Ser Gln Ala Leu
275 280 285
Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn Leu Tyr Gln Met Thr Ser
290 295 300
Gln Leu Glu Cys Met Thr Trp Asn Gln Met Asn Leu Gly Ala Thr Leu
305 310 315 320
Lys Gly Val Ala Ala Gly Ser Ser Ser Ser Val Lys Trp Thr Glu Gly
325 330 335
Gln Ser Asn His Ser Thr Gly Tyr Glu Ser Asp Asn His Thr Thr Pro
340 345 350
Ile Leu Cys Gly Ala Gln Tyr Arg Ile His Thr His Gly Val Phe Arg
355 360 365
Gly Ile Gln Asp Val Arg Arg Val Pro Gly Val Ala Pro Thr Leu Val
370 375 380
Arg Ser Ala Ser Glu Thr Ser Glu Lys Arg Pro Phe Met Cys Ala Tyr
385 390 395 400
Pro Gly Cys Asn Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His
405 410 415
Ser Arg Lys His Thr Gly Glu Lys Pro Tyr Gln Cys Asp Phe Lys Asp
420 425 430
Cys Glu Arg Arg Phe Ser Arg Ser Asp Gln Leu Lys Arg His Gln Arg
435 440 445
Arg His Thr Gly Val Lys Pro Phe Gln Cys Lys Thr Cys Gln Arg Lys
450 455 460
Phe Ser Arg Ser Asp His Leu Lys Thr His Thr Arg Thr His Thr Gly
465 470 475 480
Lys Thr Ser Glu Lys Pro Phe Ser Cys Arg Trp Pro Ser Cys Gln Lys
485 490 495
Lys Phe Ala Arg Ser Asp Glu Leu Val Arg His His Asn Met His Gln
500 505 510
Arg Asn Met Thr Lys Leu Gln Leu Ala Leu
515 520
<210> 5
<211> 630
<212> PRT
<213> Homo sapiens
<400> 5
Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro
1 5 10 15
Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln
20 25 30
Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu
35 40 45
Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg
50 55 60
Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu
65 70 75 80
Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu
85 90 95
Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro
100 105 110
Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro
115 120 125
Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile
130 135 140
Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln
145 150 155 160
Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu
165 170 175
Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu
180 185 190
Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu
195 200 205
Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg
210 215 220
Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp
225 230 235 240
Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly
245 250 255
Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg
260 265 270
Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile
275 280 285
Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser
290 295 300
Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys
305 310 315 320
Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met
325 330 335
Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu
340 345 350
Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val
355 360 365
Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile
370 375 380
Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu
385 390 395 400
Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu
405 410 415
Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln
420 425 430
Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr
435 440 445
Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser
450 455 460
Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln
465 470 475 480
Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn
485 490 495
Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro
500 505 510
Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu
515 520 525
Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val
530 535 540
Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala
545 550 555 560
Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln
565 570 575
Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn
580 585 590
Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr
595 600 605
Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu
610 615 620
Leu Ala Ser Thr Leu Ala
625 630
<210> 6
<211> 702
<212> PRT
<213> Homo sapiens
<400> 6
Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln
1 5 10 15
Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr
20 25 30
Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly
35 40 45
Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly
50 55 60
Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile
65 70 75 80
Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser
85 90 95
Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile
100 105 110
Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp
115 120 125
Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu
130 135 140
Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys
145 150 155 160
Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr
165 170 175
Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln
180 185 190
Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn
195 200 205
Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg
210 215 220
Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro
225 230 235 240
Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn
245 250 255
Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe
260 265 270
Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn
275 280 285
Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser
290 295 300
Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala
305 310 315 320
Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu
325 330 335
Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr
340 345 350
Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg
355 360 365
Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr
370 375 380
Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser
385 390 395 400
Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp
405 410 415
Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn
420 425 430
Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser
435 440 445
Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile
450 455 460
Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn
465 470 475 480
Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val
485 490 495
Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro
500 505 510
Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln
515 520 525
Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser
530 535 540
Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn
545 550 555 560
Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser
565 570 575
Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly
580 585 590
Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly
595 600 605
Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln
610 615 620
Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu
625 630 635 640
Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe
645 650 655
Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile
660 665 670
Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr
675 680 685
Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile
690 695 700
<210> 7
<211> 561
<212> PRT
<213> Cytomegalovirus
<400> 7
Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly
1 5 10 15
Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr
20 25 30
Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val
35 40 45
Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp
50 55 60
Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr
65 70 75 80
Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn
85 90 95
Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr
100 105 110
Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val
115 120 125
His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val
130 135 140
Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg
145 150 155 160
Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys
165 170 175
Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp
180 185 190
Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser Met
195 200 205
Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val
210 215 220
Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu
225 230 235 240
Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met
245 250 255
Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe
260 265 270
Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser
275 280 285
His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu
290 295 300
Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu
305 310 315 320
Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr
325 330 335
Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp
340 345 350
Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr
355 360 365
Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr
370 375 380
Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp
385 390 395 400
Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys
405 410 415
Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser
420 425 430
Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ala
435 440 445
Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro
450 455 460
Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala
465 470 475 480
Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu
485 490 495
Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu
500 505 510
Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu
515 520 525
Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln
530 535 540
Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg
545 550 555 560
Gly
<210> 8
<211> 561
<212> PRT
<213> Cytomegalovirus
<400> 8
Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly
1 5 10 15
Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr
20 25 30
Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val
35 40 45
Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp
50 55 60
Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr
65 70 75 80
Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn
85 90 95
Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr
100 105 110
Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val
115 120 125
His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val
130 135 140
Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg
145 150 155 160
Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys
165 170 175
Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp
180 185 190
Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser Met
195 200 205
Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val
210 215 220
Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu
225 230 235 240
Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met
245 250 255
Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe
260 265 270
Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser
275 280 285
His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu
290 295 300
Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu
305 310 315 320
Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr
325 330 335
Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp
340 345 350
Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr
355 360 365
Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr
370 375 380
Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp
385 390 395 400
Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys
405 410 415
Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser
420 425 430
Ala Gly Arg Asn Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ala
435 440 445
Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro
450 455 460
Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala
465 470 475 480
Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu
485 490 495
Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu
500 505 510
Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu
515 520 525
Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln
530 535 540
Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg
545 550 555 560
Gly
<210> 9
<211> 536
<212> PRT
<213> Cytomegalovirus
<400> 9
Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly
1 5 10 15
Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr
20 25 30
Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val
35 40 45
Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp
50 55 60
Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr
65 70 75 80
Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn
85 90 95
Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr
100 105 110
Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val
115 120 125
His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val
130 135 140
Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg
145 150 155 160
Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys
165 170 175
Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp
180 185 190
Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser Met
195 200 205
Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val
210 215 220
Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu
225 230 235 240
Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met
245 250 255
Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe
260 265 270
Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser
275 280 285
His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu
290 295 300
Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu
305 310 315 320
Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr
325 330 335
Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp
340 345 350
Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr
355 360 365
Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr
370 375 380
Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp
385 390 395 400
Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys
405 410 415
Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser
420 425 430
Ala Gly Arg Asn Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ala
435 440 445
Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro
450 455 460
Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala
465 470 475 480
Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu
485 490 495
Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu
500 505 510
Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu
515 520 525
Gly Val Trp Gln Pro Ala Ala Gln
530 535
<210> 10
<211> 1356
<212> PRT
<213> Homo sapiens
<400> 10
Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu
1 5 10 15
Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro
20 25 30
Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr
35 40 45
Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro
50 55 60
Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser
65 70 75 80
Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn
85 90 95
Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser
100 105 110
Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser
115 120 125
Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys
130 135 140
Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser
145 150 155 160
Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg
165 170 175
Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile
180 185 190
Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser
195 200 205
Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr
210 215 220
Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu
225 230 235 240
Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile
245 250 255
Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu
260 265 270
Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe
275 280 285
Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu
290 295 300
Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr
305 310 315 320
Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met
325 330 335
Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala
340 345 350
Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly
355 360 365
Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr
370 375 380
Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu
385 390 395 400
Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val
405 410 415
Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val
420 425 430
Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr
435 440 445
Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu
450 455 460
Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr
465 470 475 480
Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys
485 490 495
Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys
500 505 510
Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr
515 520 525
Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser
530 535 540
Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln
545 550 555 560
Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser
565 570 575
Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro
580 585 590
Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr
595 600 605
Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile
610 615 620
Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr
625 630 635 640
Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val
645 650 655
Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn
660 665 670
Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys
675 680 685
Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn
690 695 700
Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg
705 710 715 720
Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr
725 730 735
Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe
740 745 750
Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu
755 760 765
Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile
770 775 780
Ile Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly
785 790 795 800
Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His
805 810 815
Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp
820 825 830
Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val
835 840 845
Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr
850 855 860
Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg
865 870 875 880
Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu
885 890 895
Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu
900 905 910
Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu
915 920 925
Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg
930 935 940
Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys
945 950 955 960
Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly
965 970 975
Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Pro
980 985 990
Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr
995 1000 1005
Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys
1010 1015 1020
Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Lys Asn
1025 1030 1035 1040
Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp
1045 1050 1055
Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met
1060 1065 1070
Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln Ser Asp Val
1075 1080 1085
Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser
1090 1095 1100
Pro Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys
1105 1110 1115 1120
Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met Tyr
1125 1130 1135
Gln Thr Met Leu Asp Cys Trp His Gly Glu Pro Ser Gln Arg Pro Thr
1140 1145 1150
Phe Ser Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala Asn Ala
1155 1160 1165
Gln Gln Asp Gly Lys Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr Leu
1170 1175 1180
Ser Met Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser
1185 1190 1195 1200
Cys Met Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn
1205 1210 1215
Thr Ala Gly Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg
1220 1225 1230
Pro Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu
1235 1240 1245
Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val Leu
1250 1255 1260
Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys Leu Ser Pro
1265 1270 1275 1280
Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser Val Ala Ser
1285 1290 1295
Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp
1300 1305 1310
Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu Ala Glu Leu Leu Lys
1315 1320 1325
Leu Ile Glu Ile Gly Val Gln Thr Gly Ser Thr Ala Gln Ile Leu Gln
1330 1335 1340
Pro Asp Ser Gly Thr Thr Leu Ser Ser Pro Pro Val
1345 1350 1355
Claims (17)
- 빌름 종양 단백질(WT1)을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주로서, 이 치료법은 적어도 하나의 관문 억제제를 투여하는 단계를 추가로 포함하는 살모넬라 약독화 균주.
- 제1항에 있어서, 상기 관문 억제제는 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체 적어도 하나로부터 선택되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 또는 제2항에 있어서, 상기 살모넬라 약독화 균주는 살모넬라 엔테리카 종이고, 구체적으로 상기 살모넬라 약독화 균주는 살모넬라 타이피 Ty21a인, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 발현 카세트는 진핵생물 발현 카세트이고, 구체적으로 상기 발현 카세트는 CMV 프로모터를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 내지 제4항 중 어느 한 항에 있어서,
(a) WT1은 서열 번호 4에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1 및 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택되거나,
(b) WT1은 절단형이고, 더욱 구체적으로 WT1의 아연 핑거 도메인은 결실되거나, 또는
(c) WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 WT1 및 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주. - 제1항 내지 제5항 중 어느 한 항에 있어서, 상기 DNA 분자는 카나마이신 항생제 내성 유전자, pMB1 ori 및 CMV 프로모터를 포함하고, 구체적으로 DNA 분자는 서열 번호 2에서 발견되는 바와 같은 DNA 서열을 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 내지 제6항 중 어느 한 항에 있어서, 상기 살모넬라 약독화 균주는 적어도 하나의 관문 억제제 투여와 동시에, 투여 이전 또는 투여 이후에 투여되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 내지 제7항 중 어느 한 항에 있어서, 상기 치료법은 화학요법, 방사선요법 또는 암 생물요법이 동반되고, 구체적으로 살모넬라 약독화 균주는 이 화학요법 또는 방사선요법 치료 주기 또는 암 생물요법이 수행되기 전, 수행되는 도중 또는 수행된 후에 투여되거나, 아니면 화학요법 또는 방사선요법 치료 주기 또는 암 생물요법이 수행되기 전 및 수행되는 도중에 투여되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제8항에 있어서, 암 생물요법은 종양 항원 및/또는 종양 기질 항원을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는, 살모넬라의 추가 약독화 균주(들) 하나 이상을 투여하는 단계를 포함하고, 구체적으로 상기 살모넬라의 추가 약독화 균주(들) 하나 이상은 진핵생물 발현 카세트를 포함하는 살모넬라 타이피 Ty21a인, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제9항에 있어서, 상기 종양 항원은 메소텔린(MSLN), CEA 및 CMV pp65로 이루어진 군으로부터 선택되고/선택되거나, 상기 종양 기질 항원은 VEGF 수용체 단백질 및 인간 섬유아세포 활성화 단백질(FAP)로 이루어진 군으로부터 선택되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제10항에 있어서, 상기 종양 항원은 서열 번호 5에서 발견되는 바와 같은 아미노산 서열을 가지는 MSLN과, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 서열 번호 6에서 발견되는 바와 같은 아미노산 서열을 가지는 CEA와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 서열 번호 7에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 서열 번호 8에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 그리고 서열 번호 9에서 발견되는 바와 같은 아미노산 서열을 가지는 CMV pp65와, 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택되고/선택되거나,
상기 종양 기질 항원은 서열 번호 10에서 발견되는 바와 같은 아미노산 서열을 가지는 VEGFR-2 및 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질, 그리고 인간 섬유아세포 활성화 단백질(FAP)로 이루어진 군으로부터 선택되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주. - 제1항 내지 제11항 중 어느 한 항에 있어서, 상기 살모넬라 약독화 균주는 경구 투여되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 내지 제12항 중 어느 한 항에 있어서, 상기 암은 백혈병, 구체적으로 급성 골수성 백혈병(AML) 및 급성 림프성 백혈병(ALL), 다발성 골수종으로부터 선택되고, 고형 종양, 구체적으로 폐암, 유방암, 식도암, 결장암, 결장직장암, 위암, 융모막암, 췌장암, 교모세포종, 두경부암, 활막육종, 혈관육종, 골육종, 감상샘암, 자궁경부암, 자궁내막암, 난소암, 신경모세포종, 횡문근육종 및 전립선암으로부터 선택되는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 내지 제13항 중 어느 한 항에 있어서, 살모넬라 약독화 균주의 1회 용량은 약 105 내지 약 1011, 구체적으로 약 106 내지 약 1010, 더욱 구체적으로 약 106 내지 약 109, 더욱 구체적으로 약 106 내지 약 108, 가장 구체적으로 약 106 내지 약 107 콜로니 형성 단위(CFU)를 포함하는, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- 제1항 내지 제14항 중 어느 한 항에 있어서, 상기 치료법은 환자에서의 WT1에 대한 전 면역 반응 및/또는 WT1 발현을 평가하는 단계를 포함하는 개별맞춤 암 면역요법인, 암 치료법에 사용하기 위한 살모넬라 약독화 균주.
- WT1을 암호화하는 발현 카세트를 포함하는 DNA 분자 복사체 적어도 하나를 포함하는 살모넬라 약독화 균주를 포함하는, 암 치료법에 사용하기 위한 약학 조성물로서, 상기 치료법은 적어도 하나의 관문 억제제, 구체적으로 PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3 및 LAG-3에 대한 항체로부터 선택되는 관문 억제제 적어도 하나를 투여하는 단계를 추가로 포함하는 약학 조성물.
- 제16항에 있어서, 상기 살모넬라 약독화 균주는 살모넬라 타이피 Ty21a이고, 상기 발현 카세트는 진핵생물 발현 카세트, 구체적으로 CMV 프로모터를 포함하는 진핵생물 발현 카세트이며, 상기 WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 인간 WT1 및 이것과 적어도 약 80%의 서열 동일성을 공유하는 단백질로 이루어진 군으로부터 선택되고, 구체적으로 인간 WT1은 서열 번호 1에서 발견되는 바와 같은 아미노산 서열을 가지는 약학 조성물.
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EP (1) | EP3534935A1 (ko) |
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WO2023080703A1 (ko) * | 2021-11-05 | 2023-05-11 | 전남대학교 산학협력단 | 살모넬라 균주 및 면역관문억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약학조성물 |
WO2024106866A1 (ko) * | 2022-11-14 | 2024-05-23 | 전남대학교 산학협력단 | 암의 치료용 면역증강 살모넬라 균주 및 이의 용도 |
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WO2012149364A1 (en) * | 2011-04-28 | 2012-11-01 | Diamond Don J | Tumor associated vaccines and compositions for disrupting tumor-derived immunosuppression for use in combination cancer immunotherapy |
NO337687B1 (no) | 2011-07-08 | 2016-06-06 | Norsk Elektro Optikk As | Hyperspektralt kamera og metode for å ta opp hyperspektrale data |
KR102090612B1 (ko) * | 2012-07-05 | 2020-03-19 | 백심 아게 | 췌장암 환자용 dna 백신 |
CA2899577C (en) * | 2013-04-03 | 2023-10-17 | Ibc Pharmaceuticals, Inc. | Combination therapy for inducing immune response to disease |
LT2988762T (lt) * | 2013-04-25 | 2018-09-25 | Vaximm Ag | Vektoriai, sukonstruoti salmonella pagrindu, skirti vėžio imunoterapijai, nukreiptai į vilmso naviko geną wt1 |
HUE045567T2 (hu) * | 2013-12-18 | 2020-01-28 | Vaximm Gmbh | MSLN-t célzó DNS vakcina rák immunterápiához |
US10975112B2 (en) * | 2015-06-16 | 2021-04-13 | Hangzhou Dac Biotech Co., Ltd. | Linkers for conjugation of cell-binding molecules |
US20180153976A1 (en) * | 2015-06-18 | 2018-06-07 | Vaximm Ag | Novel cmv pp65 targeting dna vaccine for cancer immunotherapy |
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- 2017-11-03 CN CN201780068135.3A patent/CN109906087A/zh active Pending
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WO2023080703A1 (ko) * | 2021-11-05 | 2023-05-11 | 전남대학교 산학협력단 | 살모넬라 균주 및 면역관문억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약학조성물 |
WO2024106866A1 (ko) * | 2022-11-14 | 2024-05-23 | 전남대학교 산학협력단 | 암의 치료용 면역증강 살모넬라 균주 및 이의 용도 |
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EP3534935A1 (en) | 2019-09-11 |
SG11201903099QA (en) | 2019-05-30 |
US20230158133A1 (en) | 2023-05-25 |
CN109906087A (zh) | 2019-06-18 |
IL265684A (en) | 2019-05-30 |
CA3041375A1 (en) | 2018-05-11 |
JP2019533698A (ja) | 2019-11-21 |
AU2017353432A1 (en) | 2019-04-18 |
RU2019112977A (ru) | 2020-12-04 |
BR112019009003A2 (pt) | 2019-07-16 |
MX2019005212A (es) | 2019-06-20 |
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