KR20230008162A - 암 및 감염성 질환에 대한 면역요법을 위한 융합 단백질 - Google Patents
암 및 감염성 질환에 대한 면역요법을 위한 융합 단백질 Download PDFInfo
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Abstract
암 및 감염성 질환에 대한 면역요법을 위한 융합 단백질이 개시된다. 본 발명에 따른 융합 단백질은 CD40-결합 도메인; 항원; 및 CD40-결합 도메인과 항원 사이에 위치한 전위 도메인을 포함하며, 여기서 퓨린 및/또는 카텝신 L 절단 부위가 융합 단백질의 CD40-결합 도메인과 전위 도메인 사이에 존재한다. 항원은 병원체의 항원 또는 종양 항원이다. 퓨린 및/또는 카텝신 L 절단 부위는 퓨린 및/또는 카텝신 L 절단을 통해 융합 단백질로부터 CD40-결합 도메인이 제거될 수 있게 한다. 또한 항원-특이적 세포-매개 면역 반응을 유도하기 위한, 치료를 필요로 하는 대상체에서 종양 및/또는 병원체에 의해 유발되는 질환을 치료하기 위한 본 발명의 융합 단백질의 약학적 조성물, 발현 벡터 및 용도가 개시된다.
Description
본 발명은 일반적으로 융합 단백질에 관한 것으로, 보다 구체적으로는 암 및 감염성 질환에 대한 T 세포-매개 면역 반응을 유도하기 위한 융합 단백질에 관한 것이다.
적응성 면역계는 체액성 면역 성분 및 세포-매개 면역 성분을 포함하고 침입하는 병원체를 파괴한다. 적응성 면역 반응을 수행하는 세포는 림프구로 알려진 백혈구이다. 두 가지의 서로 다른 유형의 림프구인 B 세포 및 T 세포는 항체 반응 및 세포-매개 면역 반응과 같은 주요 활동을 수행한다. 적응성 면역은 병원체 노출로 활성화되며 향후 이 병원체와의 접촉에 대한 향상된 면역 반응을 유도한다. 백신은 표적 병원체에 대한 보호를 가능하게 하는 적응성 면역 세포에서 항원-특이적 기억을 유도한다. 암과 병원체에 의해 유발되는 감염성 질환을 포함하는 질환들을 치료하기 위한 새로운 치료용 백신에 대한 요구가 여전히 존재한다.
일 양상에서, 본 발명은 융합 단백질로서 (a) CD40-결합 도메인; (b) 항원; 및 (c) 상기 CD40-결합 도메인과 상기 항원 사이에 위치한 전위 도메인을 포함하고, 퓨린 및/또는 카텝신 L 절단 부위가 상기 융합 단백질의 상기 CD40-결합 도메인과 상기 전위 도메인 사이에 존재하는, 융합 단백질에 관한 것이다.
다른 양상에서, 본 발명은 본 발명에 따른 융합 단백질을 암호화하는 DNA 단편에 관한 것이다. 또한 본 발명은 본 발명의 융합 단백질을 암호화하는 DNA 단편을 포함하는 발현 벡터에 관한 것이다.
또 다른 양상에서, 본 발명은 본 발명의 융합 단백질 및 약학적으로 허용가능한 담체 및/또는 아쥬반트를 포함하는 약학적 조성물에 관한 것이다.
또 다른 양상에서, 본 발명은 항원-특이적 세포-매개 면역 반응을 유도하여 치료를 필요로 하는 대상체에서 종양 및/또는 병원체에 의해 유발되는 질환을 치료하기 위한 약제의 제조에서의 본 발명의 융합 단백질 또는 약학적 조성물의 용도에 관한 것이다. 또한 본 발명은 항원-특이적 세포-매개 면역 반응을 유도하여 치료를 필요로 하는 대상체에서 종양 및/또는 병원체에 의해 유발되는 질환을 치료하는 용도를 위한 본 발명의 융합 단백질 또는 약학적 조성물에 관한 것이다.
대안적으로, 본 발명은 항원-특이적 세포-매개 면역 반응을 유도하기 위한, 치료를 필요로 하는 대상체에서 종양 및/또는 병원체에 의해 유발되는 질환을 치료하기 위한 방법으로서, 유효량의 본 발명의 융합 단백질 또는 약학적 조성물을 이를 필요로 하는 대상체에게 투여하는 단계를 포함하는, 방법에 관한 것이다.
도 1은 벡터 지도이다.
도 2는 벡터 지도이다. MCS, 다중 클로닝 부위.
도 3은 벡터 지도이다.
도 4는 벡터 지도이다.
도 5a 내지 도 5e는 본 발명의 다양한 실시형태를 예시하는 개략도이다.
도 6은 각 동물군에서 상대적 사이토카인 유도를 보여주는 그래프이다.
도 7은 각 동물군 비장세포의 IFN-γ+ 면역스팟을 보여주는 그래프이다.
도 8은 각 동물군에서 혈청 HPV16 E7-특이 항체 수준을 보여주는 그래프이다.
도 9는 각 동물군에서 혈청 HPV18 E7-특이 항체 수준을 보여주는 그래프이다.
도 10은 면역화 일정 및 각각 표시된 융합 단백질을 처리하거나 처리하지 않은 동물군을 보여준다.
도 11은 표시된 융합 단백질을 처리하거나 처리하지 않은 각 동물군에서의 종양 크기를 보여주는 그래프이다.
도 12는 표시된 융합 단백질을 처리하거나 처리하지 않은 각 동물군에서의 생존율을 보여주는 그래프이다.
도 13은 표시된 융합 단백질을 처리하거나 처리하지 않은 각 동물군에서의 무종양률(tumor free rate)을 보여주는 그래프이다.
도 14는 표시된 다양한 용량으로 융합 단백질 18sCD40L-TPE-E7을 처리하거나 처리하지 않은 각 동물군에서의 종양 크기를 보여주는 그래프이다.
도 15는 표시된 다양한 용량으로 융합 단백질 E7-TStx-18sCD40L을 처리하거나 처리하지 않은 각 동물군에서의 종양 크기를 보여주는 그래프이다.
도 16은 면역화 계획(상부), 동물군 및 융합 단백질 HBx-preS1-TStx-18sCD40L의 각 투여 일정(하부)을 보여준다.
도 17은 도 16의 각 동물군 비장세포의 IFN-γ+ 면역스팟을 보여주는 그래프이다.
도 18은 도 16의 각 동물군에서의 혈청 HBx-특이 항체 수준을 보여주는 그래프이다.
도 2는 벡터 지도이다. MCS, 다중 클로닝 부위.
도 3은 벡터 지도이다.
도 4는 벡터 지도이다.
도 5a 내지 도 5e는 본 발명의 다양한 실시형태를 예시하는 개략도이다.
도 6은 각 동물군에서 상대적 사이토카인 유도를 보여주는 그래프이다.
도 7은 각 동물군 비장세포의 IFN-γ+ 면역스팟을 보여주는 그래프이다.
도 8은 각 동물군에서 혈청 HPV16 E7-특이 항체 수준을 보여주는 그래프이다.
도 9는 각 동물군에서 혈청 HPV18 E7-특이 항체 수준을 보여주는 그래프이다.
도 10은 면역화 일정 및 각각 표시된 융합 단백질을 처리하거나 처리하지 않은 동물군을 보여준다.
도 11은 표시된 융합 단백질을 처리하거나 처리하지 않은 각 동물군에서의 종양 크기를 보여주는 그래프이다.
도 12는 표시된 융합 단백질을 처리하거나 처리하지 않은 각 동물군에서의 생존율을 보여주는 그래프이다.
도 13은 표시된 융합 단백질을 처리하거나 처리하지 않은 각 동물군에서의 무종양률(tumor free rate)을 보여주는 그래프이다.
도 14는 표시된 다양한 용량으로 융합 단백질 18sCD40L-TPE-E7을 처리하거나 처리하지 않은 각 동물군에서의 종양 크기를 보여주는 그래프이다.
도 15는 표시된 다양한 용량으로 융합 단백질 E7-TStx-18sCD40L을 처리하거나 처리하지 않은 각 동물군에서의 종양 크기를 보여주는 그래프이다.
도 16은 면역화 계획(상부), 동물군 및 융합 단백질 HBx-preS1-TStx-18sCD40L의 각 투여 일정(하부)을 보여준다.
도 17은 도 16의 각 동물군 비장세포의 IFN-γ+ 면역스팟을 보여주는 그래프이다.
도 18은 도 16의 각 동물군에서의 혈청 HBx-특이 항체 수준을 보여주는 그래프이다.
달리 정의하지 않는 한, 본 명세서에 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 분야의 통상의 지식을 가진 사람에 의해 일반적으로 이해되는 것과 같은 의미를 갖는다. 또한, 다음의 정의는 본 발명을 기술하기 위해 사용된 다양한 용어들의 의미 및 범위를 예시 및 정의하기 위해 제시된다.
정의
항원-제시 세포(APC) 또는 보조 세포는 표면에 주요 조직적합성 복합체(MHC)와 복합체를 형성한 항원을 표시하는 세포이며; 이 과정은 항원 제시로 알려져 있다. T 세포는 T 세포 수용체(TCR)를 사용하여 이 복합체를 인식할 수 있다. APC는 항원을 처리하여 이를 T 세포에 제시한다.
항원-제시 세포는 전문성과 비-전문성의 두 가지 범주로 나뉜다. 공동-자극 분자 및 패턴 인식 수용체와 함께 MHC 클래스 II 분자를 발현하는 것들을 전문성 항원-제시 세포라고 부른다. 전문성 항원-제시 세포의 주 유형은 수지상 세포(DC), 대식세포 및 B 세포이다. 비-전문성 APC는 MHC 클래스 I 분자를 발현하며, 신체의 모든 유핵 세포 유형을 포함한다.
전문성 APC는 T 세포에 항원을 제시하는 데 특화되어 있다. 이들은 식균작용 또는 수용체-매개 세포내이입에 의해 항원을 내재화하는 데 매우 효율적이며, 항원을 펩타이드 단편으로 처리한 후 이들 펩타이드(클래스 II MHC 분자와 결함됨)를 막 상에 표시한다. T 세포는 항원-제시 세포의 막 상에 존재하는 항원-클래스 II MHC 분자 복합체를 인식하고 이와 상호작용한다. 이후 항원-제시 세포에 의해 추가 공동-자극 신호가 생성되어 T 세포의 활성화를 유도한다. 또한 모든 전문성 APC는 MHC 클래스 I 분자를 발현한다.
전문성 APC 및 비-전문성 APC는 MHC 클래스 I 분자를 사용하여 세포막에 내인성 펩타이드를 표시한다. 이들 펩타이드는 MHC 클래스 II 분자를 사용하는 전문성 APC에 의해 표시되는 외인성 항원과 달리 세포 자체 내에서 기원된다. 세포독성 T 세포는 MHC 클래스 I 분자에 의해 제시된 항원과 상호작용할 수 있다.
CD40은 항원-제시 세포(예를 들어, 수지상 세포, 대식세포 및 B 세포)에서 발현되는 공동자극 단백질이다. CD40에 CD40L이 결합하면 항원-제시 세포가 활성화되며 다양한 하류 효과를 유도한다. CD40은 암 면역요법의 약물 표적이다.
용어 "CD40-결합 도메인"은 CD40을 인식하고 이에 결합할 수 있는 단백질을 의미한다. CD40-결합 도메인은 "CD40 리간드(CD40L) 또는 이의 기능적 단편", "항-CD40 항체 또는 이의 기능적 단편" 중 하나에서 선택될 수 있다.
용어 "CD40L", "CD40 리간드" 및 "CD154"는 호환가능하다. CD40L은 항원-제시 세포(APC) 상의 CD40(단백질)에 결합하며, 이는 표적 세포 유형에 따른 많은 효과를 유도한다. CD40L은 CD40-발현 면역 세포의 T 세포 프라이밍 및 활성화를 통해 면역 반응의 공동-자극 및 조절에서 중심적 역할을 한다. US 5,962,406에 CD40L의 뉴클레오타이드 및 아미노산 서열이 개시되어 있다.
용어 "항-CD40 항체" 및 "CD40-특이적 항체"는 호환가능하다.
용어 "실질적으로 이루어진다" 또는 "실질적으로 이루어지는"이 폴리펩타이드의 아미노산 서열을 기술하는 데 사용되는 경우, 이는 폴리펩타이드가 단백질 번역 요구사항에 따라 N-말단 부분에 시작 아미노산 "M"(시작 코돈 AUG에서 번역됨)을 갖을 수 있거나 그렇지 않을 수 있음을 의미한다. 예를 들어, 항원 HPV18 E7 단백질(서열번호 39)이 다른 폴리펩타이드(예를 들어, 다른 항원)에 융합된 경우, 시작 아미노산 "M"은 생략되거나 유지될 수 있다.
본 명세서에 사용된 "전위 도메인"은 융합 단백질 내의 항원을 엔도솜의 막을 가로질러 CD40-발현 세포의 세포질로 전위시키는 생물학적 활성을 갖는 폴리펩타이드이다. 전위 도메인은 항원을 항원 제시를 위한 클래스 I 주요 조직적합성 복합체(MHC-1) 경로(즉, 세포독성 T 세포 경로)로 안내하거나 촉진한다.
용어 "슈도모나스 외독소 A(PE) 전위 펩타이드(TPE)"는 PE 도메인 II 펩타이드 또는 전위의 생물학적 활성을 갖는 이의 기능적 단편을 의미한다.
용어 "시가 독소(Stx) 전위 펩타이드(TStx)"는 Stx 전위 도메인 또는 전위의 생물학적 활성을 갖는 이의 기능적 단편을 의미한다.
용어 "퓨린 및/또는 카텝신 L" 또는 "퓨린/카텝신 L"은 호환가능하다. 퓨린 및/또는 카텝신 L 절단 부위는 프로테아제(퓨린 및/또는 카텝신 L) 민감성 부위를 의미한다. 이는 퓨린 또는 카텝신 L 또는 퓨린 및 카텝신 L 모두에 의해 절단될 수 있는 짧은 펩타이드 서열이다. 이는 융합 단백질에 도입되는 상기 절단 부위를 포함하는 펩타이드 링커 또는 융합 단백질의 전위 도메인에 존재하는 고유 프로테아제 절단 부위일 수 있다.
용어 "항원" 및 "면역원"은 호환가능하다. 항원은 항원성 단백질을 의미하며, 종양 항원(암으로부터의 항원 또는 항과 관련된 항원) 또는 병원체의 항원(병원체로부터의 항원)일 수 있다.
용어 "종양" 및 "암"은 호환가능하다.
용어 "암세포의 항원" 및 "종양 항원"은 호환가능하다.
용어 "종양 항원"은 종양-특이적 항원 및/또는 종양-관련 항원을 의미한다. 종양-관련 항원은 종양 세포의 표면 상에 발현되는 단백질 또는 폴리펩타이드일 수 있다.
분화 클러스터 28(CD28)은 T-세포-특이적 표면 당단백질이다. CD28 수용체는 T 세포가 항원-제시 세포와 접촉하는 동안 자극된다. 이의 기능은 T 세포 활성화, 세포 증식 유도 및 사이토카인 생산 및 T 세포 생존 촉진과 관련된다.
용어 "유효량"은 치료하는 대상체에 치료 효과를 부여하는 데 필요한 활성 융합 단백질의 양을 의미한다. 유효 용량은 이 분야의 기술자에게 인식되는 바와 같이 투여 경로, 부형제 사용 및 다른 치료와의 공동-사용 가능성에 따라 다양할 것이다.
"치료하는" 또는 "치료"는 암 또는 감염이 있거나, 이러한 질환에 대한 증상 또는 소인이 있는 치료를 필요로 하는 대상체에게 질환, 이의 증상 또는 이에 대한 소인을 치유, 완화, 경감, 치료, 개선 또는 예방하는 목적으로 유효량의 융합 단백질을 투여하는 것을 의미한다. 이러한 대상체는 임의의 적합한 진단 방법의 결과를 기초로 건강 관리 전문적으로 식별될 수 있다.
"0 내지 12반복" 또는 "2 내지 6반복"은 "0 내지 12" 또는 "2 내지 6" 범위 내의 모든 정수 단위량이 본 발명의 일부로서 구체적으로 개시됨을 의미한다. 따라서, "0, 1, 2, 3, 4, . . . 10, 11 및 12" 또는 "2, 3, 4, 5 및 6" 단위량이 본 발명의 실시형태로서 포함된다.
일 양상에서, 본 발명은 융합 단백질로서, (a) CD40-결합 도메인; (b) 항원; 및 (c) 상기 CD40-결합 도메인과 상기 항원 사이에 위치한 전위 도메인을 포함하고, 퓨린 및/또는 카텝신 L 절단 부위가 상기 융합 단백질의 상기 CD40-결합 도메인과 상기 전위 도메인 사이에 존재하는, 융합 단백질에 관한 것이다.
본 발명의 융합 단백질은 MHC 클래스 I 항원 제시 경로를 통해 항원-특이적 T 세포 면역 반응을 유도할 수 있다. 이들은 공통 작용 기작을 공유한다. 예를 들어 융합 단백질 18sCD40L-TPE-E7을 사용하는 경우, 작용 기작은 다음과 같다:
(1) 융합 단백질이 CD40-발현 세포(예를 들어, 수지상 세포 또는 대식세포)에 결합하고 CD40-매개 세포내이입을 통해 내재화된다;
(2) 융합 단백질이 엔도솜 내의 퓨린 프로테아제 및/또는 카텝신 L 프로테아제에 의해 절단되어 TPE-E7 단편으로부터 18sCD40L 단편이 제거된다;
(3) TPE-E7 단편이 엔도솜의 엔도솜막을 가로질러 세포질로 전위된다;
(4) TPE-E7 단편이 세포액의 프로테아솜에 의해 소화되어 에피토프를 갖는 작은 E7 항원이 생성된다;
(5) E7 항원이 항원 제시를 위한 MHC 클래스 I 경로로 전달된다;
(6) 이들 제시된 항원을 인식하는 T 세포에 의해 CD8+ T 세포 특이적 면역 반응이 유도되거나 향상된다.
상기 작용 기작은 융합 단백질 E7-TStx-18sCD40L에 적용가능하며, 이 경우 퓨린 및/또는 카텝신 L 프로테아제 절단이 18sCD40L 단편으로부터 E7-TStx 단편을 제거한다. 따라서, E7-TStx 단편은 엔도솜의 엔도솜막을 가로질러 세포액으로 전위되고, 세포액 프로테아솜에 의해 소화되어 에피토프를 갖는 작은 E7 항원이 생성되며; E7 항원이 항원 제시를 위한 MHC 클래스 I 경로에 전달되고; CD8+ T 세포 특이적 면역 반응이 이들 제시된 항원을 인식하는 T 세포에 의해 유도 또는 향상된다.
본 발명에 따르면, 퓨린 및/또는 카텝신 L 절단 부위가 융합 단백질의 항원과 전위 도메인 사이에 존재하지 않는다.
퓨린 및/또는 카텝신 L 절단 부위의 존재 및 융합 단백질에서 이의 위치는 퓨린 및/또는 카테킨 L 절단 후 융합 단백질로부터 CD40-결합 도메인의 제거를 허용한다.
일 실시형태에서, 본 발명의 융합 단백질은 펩타이드 링커를 더 포함하며, 상기 링커는 융합 단백질의 CD40-결합 도메인과 전위 도메인 사이에 존재하는 퓨린 및/또는 카텝신 L 절단 부위를 포함한다.
전위 도메인 및 항원은 전위 도메인이 엔도솜의 막을 가로질러 항원을 전위시켜 세포액에 진입시키고, 이후 CD40-발현 세포에서 항원 제시를 위한 MHC 클래스 I 경로로 항원을 촉진시킬 수 있는 방향 및/또는 관계로 융합 단백질 내에 위치한다.
일 실시형태에서, 전위 도메인은 슈도모나스 외독소 A(PE)에서 유래된다. 다른 실시형태에서, 전위 도메인은 시가 독소(Stx)에서 유래된다.
일 실시형태에서, 전위 도메인은 CD40-결합 도메인이 융합 단백질의 N-말단에 위치한다는 조건 하에서 슈도모나스 외독소 A(PE) 전위 펩타이드(TPE)를 포함하거나 이 펩타이드이다.
다른 실시형태에서, 전위 도메인은 항원이 융합 단백질의 N-말단에 위치한다는 조건 하에서 시가 독소(Stx) 전위 펩타이드(TStx)를 포함하거나 이 펩타이드이다.
다른 실시형태에서, 본 발명의 융합 단백질은 다음을 순차적으로 포함한다: (i) 융합 단백질의 N-말단에 위치한 CD40-결합 도메인; (ii) PE 전위 펩타이드(TPE)를 포함하는 전위 도메인; 및 (iii) 융합 단백질의 C-말단에 위치한 항원; 여기서 퓨린 및/또는 카텝신 L 절단 부위는 융합 단백질의 CD40-결합 도메인과 전위 도메인 사이에 존재한다.
다른 실시형태에서, 전위 도메인은 TPE의 기능적 부분(moiety)이며 퓨린 및/또는 카텝신 L 절단 부위는 PE로부터의 고유 퓨린 절단 부위이다.
다른 실시형태에서, 본 발명의 융합 단백질은 다음을 순차적으로 포함한다: (i) 융합 단백질의 N-말단에 위치한 CD40-결합 도메인; (ii) 퓨린 및/또는 카텝신 L 절단 부위를 포함하는 펩타이드 링커; (iii) PE 전위 펩타이드(TPE)를 포함하는 전위 도메인; 및 (iv) 병원체의 항원 또는 종양 항원.
다른 실시형태에서, 본 발명의 융합 단백질은 다음을 순차적으로 포함한다: (i) 융합 단백질의 N-말단에 위치한 항원; (ii) Stx 전위 펩타이드(TStx)를 포함하는 전위 도메인; 및 (iii) CD40-결합 도메인; 여기서 퓨린 및/또는 카텝신 L 절단 부위는 융합 단백질의 CD40-결합 도메인과 전위 도메인 사이에 존재한다.
일 실시형태에서, 전위 도메인은 TStx의 기능적 부분이며 상기 퓨린 및/또는 카텝신 L 절단 부위는 Stx로부터의 고유 퓨린 절단 부위이다.
또 다른 실시형태에서, 본 발명의 융합 단백질은 다음을 순차적으로 포함한다: (i) 융합 단백질의 N-말단에 위치한 항원; (ii) Stx 전위 펩타이드(TStx)를 포함하는 전위 도메인; (iii) 퓨린 및/또는 카텝신 L 절단 부위를 포함하는 절단가능한 링커; 및 (iv) CD40-결합 도메인.
일 실시형태에서, 퓨린 및/또는 카텝신 L 절단 부위는 서열번호 1 또는 2의 아미노산 서열을 포함하거나, 이 서열이거나, 이 서열로 이루어진다.
다른 실시형태에서, PE 전위 펩타이드(TPE)는 슈도모나스 외독소 A 단백질(전장 PE, 서열번호 4)의 도메인 II(253 내지 364번 아미노산 잔기; 서열번호 9)이다.
다른 실시형태에서, PE 전위 펩타이드(TPE)는 최소화 기능적 단편 GWEQLEQCGYPVQRLVALYLAARLSW(서열번호 5)를 포함한다.
다른 실시형태에서, PE 전위 펩타이드(TPE)는 26 내지 112개 길이의 아미노산 잔기로 이루어지며, 상기 PE 전위 펩타이드는 GWEQLEQCGYPVQRLVALYLAARLSW(서열번호 5)의 최소화 기능적 단편을 포함한다.
다른 실시형태에서, PE 전위 펩타이드(TPE)는 서열번호 5, 6, 7, 8 또는 9와 적어도 90%, 95% 또는 99% 동일한 아미노산 서열을 포함한다.
다른 실시형태에서, PE 전위 펩타이드(TPE)는 PE280-305(서열번호 5), PE280-313(서열번호 6), PE268-313(서열번호 7), PE253-313(서열번호 8) 및 PE253-364(서열번호 9; 전장 PE 도메인 II)로 이루어진 군에서 선택된다.
일 실시형태에서, Stx 전위 펩타이드(TStx)는 시가 독소(Stx) 서브유닛 A(서열번호 10) 또는 시가-유사 독소 I(Slt-I) 서브유닛 A(서열번호 11)의 기능적 단편이다. 본 발명에 따르면, Stx 전위 펩타이드는 전위 기능을 갖지만 서브유닛 A의 세포독성 효과는 없다. 시가 독소(Stx) 서브유닛 A와 Slt-I 서브유닛 A 사이의 서열 동일성은 99%이며 두 단백질은 하나의 아미노산 만이 다르다.
다른 실시형태에서, Stx 전위 펩타이드(TStx)는 8 내지 84개 길이의 아미노산 잔기로 이루어진다.
다른 실시형태에서, Stx 전위 펩타이드(TStx)는 LNCHHHAS(서열번호 12)의 최소화 기능적 단편을 포함한다.
다른 실시형태에서, Stx 전위 펩타이드(TStx)는 8 내지 84개 길이의 아미노산 잔기로 이루어지며, 상기 TStx는 LNCHHHAS(서열번호 12)의 최소화 단편을 포함한다.
다른 실시형태에서, Stx 전위 펩타이드(TStx)는 서열번호 12, 13, 14, 15 및 16으로 이루어진 군에서 선택된 아미노산 서열과 적어도 90%, 95% 또는 99% 동일한 아미노산 서열을 포함한다.
다른 실시형태에서, Stx 전위 펩타이드는 서열번호 12, 13, 14, 15 및 16으로 이루어진 군에서 선택된 아미노산 서열을 포함한다.
다른 실시형태에서, Stx 전위 펩타이드(TStx)는 Stx 서브유닛 A의 Stx240-247(서열번호 12), Stx240-251(서열번호 13), Stx211-247(서열번호 14), Stx211-251(서열번호 15) 및 Stx168-251(서열번호 16)으로 이루어진 군에서 선택된다.
CD40-결합 도메인은 CD40-발현 세포의 CD40 단배질에 결합하는 생물학적 활성을 갖는 폴리펩타이드이다. CD40-결합 도메인은 본 발명의 융합 단백질이 CD40-발현 세포(즉, 수지상 세포 또는 대식세포)의 CD40 수용체에 결합하는 것을 허용한다.
일 실시형태에서, CD40-결합 도메인은 (i) CD40 리간드(CD40L) 또는 이의 기능적 단편; 및 (ii) 항-CD40 항체 또는 이의 기능적 단편으로 이루어진 군에서 선택된다.
CD40L, 항-CD40 항체 및 이들 각각의 기능적 단편은 모두 CD40-발현 세포의 CD40 단백질에 결합하는 생물학적 활성을 갖는다.
CD40L의 기능적 단편은 전장 CD40L1-261 단백질(서열번호 17)의 막관통 및 세포질 영역이 실질적으로 결여된 생물학적 활성을 갖는 잘린 CD40L이다.
다른 실시형태에서, CD40L 또는 이의 기능적 단편은 154 내지 261개 길이의 아미노산 잔기로 이루어진다.
다른 실시형태에서, 기능적 활성을 갖는 잘린 CD40L은 CD40L47-261(서열번호 18) 및 CD40L108-261(18sCD40L로 명명됨; 서열번호 19)로 이루어진 군에서 선택된다.
다른 실시형태에서, CD40 리간드(CD40L) 또는 이의 기능적 단편은 154 내지 261개 길이의 아미노산 잔기로 이루어지며, 상기 이의 기능적 단편은 CD40L108-261(서열번호 19)를 포함한다.
다른 실시형태에서, CD40L은 서열번호 17, 18 또는 19와 적어도 90%, 95% 또는 99% 동일한 아미노산 서열을 포함하거나 이로 이루어지며, 상기 CD40L은 CD40-발현 세포의 CD40 단백질에 결합하는 생물학적 활성을 갖는다.
다른 실시형태에서, CD40 리간드(CD40L)는 CD40L47-261(서열번호 18), CD40L108-261(서열번호 19; 18sCD40L로 지칭됨) 및 CD40L1-261(서열번호 17)로 이루어진 군에서 선택된다.
다른 실시형태에서, CD40-결합 도메인은 CD40-특이적 항체(또는 항-CD40 항체)이다. CD40-특이적 항체는 CD40 단백질에 특이적인 항체이다. CD40-특이적 항체는 CD40-발현 세포의 CD40 단백질에 결합할 수 있다.
일 실시형태에서, CD40-특이적 항체는 중쇄 가변 도메인(VH) 및 경쇄 가변 도메인(VL)을 포함하고, 여기서 VH는 서열번호 22의 아미노산 서열을 포함하고, VL은 서열번호 23의 아미노산 서열을 포함한다.
다른 실시형태에서, 본 발명에 따른 CD40-특이적 항체는 단일 사슬 가변 단편(scFv), 디아바디(dscFv), 트리아바디, 테트라바디, 이중특이적-scFv, scFv-Fc, scFc-CH3, 단일 사슬 항원-결합 단편(scFab), 항원-결합 단편(Fab), Fab2, 미니바디 및 완전 항체로 이루어진 군에서 선택된다.
다른 실시형태에서, 본 발명에 따른 CD40-결합 도메인은 중쇄 가변 도메인(VH), 경쇄 가변 도메인(VL) 및 VH 및 VL을 연결하는 유연 링커(L)를 포함하는 CD40-특이적 scFv(항-CD40 scFv)이다.
일 실시형태에서, CD40-특이적 scFv는 서열번호 20 또는 21의 아미노산 서열을 포함한다.
다른 실시형태에서, 본 발명에 따른 CD40-결합 도메인은 (i) CD40-특이적 항체 또는 이의 결합 단편 또는 (ii) CD40-특이적 단일 사슬 가변 단편(scFv) 또는 이의 결합 단편이고; 상기 CD40-특이적 항체 또는 상기 CD40-특이적 scFv는 VH 및 VL을 포함하고, 여기서 (a) VH는 서열번호 22의 아미노산 서열을 포함하고; (b) VL은 서열번호 23의 아미노산 서열을 포함한다.
다른 실시형태에서, CD40-특이적 항체 또는 CD40-특이적 scFv는 VH 및 VL을 포함하고, VH는 VH CDR1, VH CDR2 및 VH CDR3을 포함하고; VL은 VL CDR1, VL CDR2 및 VL CDR3을 포함하고, 여기서 (i) VH CDR1, VH CDR2 및 VH CDR3은 각각 서열번호 24, 25 및 26의 아미노산 서열을 포함하고, (ii) VL CDR1, VL CDR2 및 VL CDR3은 각각 서열번호 27, 28 및 29의 아미노산 서열을 포함한다.
다른 실시형태에서, CD40-결합 도메인은 VH 및 VL을 포함하는 CD40-특이적 scFv이며, 여기서 (a) VH는 서열번호 22의 아미노산 서열을 포함하고; (b) VL은 서열번호 23의 아미노산 서열을 포함한다.
다른 실시형태에서, 본 발명의 융합 단백질은 전위 도메인이 PE 전위 펩타이드(TPE)를 포함한다는 조건 하에서 항원의 C-말단에 위치한 소포체(ER) 잔류 서열을 더 포함한다.
다른 실시형태에서, ER 잔류 서열은 서열번호 30, 31, 32, 33 및 34로 이루어진 군에서 선택된 아미노산 서열을 포함한다.
다른 실시형태에서, 본 발명의 융합 단백질은 CD40-결합 도메인과 퓨린 및/또는 카텝신 L 절단 부위 사이에 위치한 CD28-활성화 펩타이드를 더 포함한다.
다른 실시형태에서, CD28-활성화 펩타이드는 28 내지 53개 길이의 아미노산 잔기로 이루어진다.
다른 실시형태에서, CD28-활성화 펩타이드는 28 내지 53개 길이의 아미노산 잔기를 가지며, 상기 CD28-활성화 펩타이드는 서열번호 35, 36 및 37로 이루어진 군에서 선택된 아미노산 서열을 포함한다.
다른 실시형태에서, CD28-활성화 펩타이드는 28 내지 53개 길이의 아미노산 잔기를 가지며, 상기 CD28-활성화 펩타이드는 서열번호 35의 아미노산 서열을 포함한다.
다른 실시형태에서, CD28-활성화 펩타이드는 서열번호 35, 36 및 37로 이루어진 군에서 선택된 아미노산 서열을 포함한다.
다른 실시형태에서, CD28-활성화 펩타이드는 서열번호 35, 36 또는 37과 적어도 90%, 95% 또는 99% 동일한 아미노산 서열을 포함한다.
본 발명의 융합 단백질의 항원은 병원체의 항원 또는 종양 항원이다.
병원체는 인간 파필로마바이러스(Human Papillomavirus: HPV), 인간 면역결핍 바이러스-1(Human Immunodeficiency Virus-1: HIV-1), 인플루엔자 바이러스(Influenza Virus), 뎅기 바이러스(Dengue Virus), A형 간염 바이러스(Hepatitis A Virus: HAV), B형 간염 바이러스(Hepatitis B Virus: HBV), C형 간염 바이러스(Hepatitis C Virus: HCV), D형 간염 바이러스(Hepatitis D Virus: HDV), E형 간염 바이러스(Hepatitis E Virus: HEV), 중증 급성 호흡기 증후군-관련 코로나바이러스(Severe acute respiratory syndrome-associated coronavirus: SARS-CoV), 중증 급성 호흡기 증후군 코로나바이러스 2(Severe acute respiratory syndrome-associated coronavirus: SARS-CoV-2), 중동 호흡기 증후군 코로나바이러스(Middle East respiratory syndrome coronavirus: MERS-CoV), 엡스타인-바 바이러스(Epstein-Barr virus: EBV), 지카 바이러스(Zika Virus), 광견병 바이러스(Rabies Virus), 두창 바이러스(Variola virus), 치쿤군야 바이러스(Chikungunya Virus), 웨스트 나일 바이러스(West Nile virus), 폴리오바이러스(Poliovirus), 홍역 바이러스(Measles virus), 풍진 바이러스(Rubella virus), 한타바이러스(Hantavirus), 일본 뇌염 바이러스(Japanese encephalitis virus), 콕삭키바이러스(Coxsackievirus), 에코바이러스(Echovirus), 엔테로바이러스(Enterovirus), 멈프스 바이러스(Mumps virus), 수두-대상포진 바이러스(Varicella-zoster virus: VZV), 헤르페스 B 바이러스(Cercopithecine herpesvirus-1: CHV-1), 황열 바이러스(Yellow fever virus: YFV), 리프트 발리열 바이러스(Rift Valley Fever Virus), 라싸 바이러스(Lassa virus), 마르부르그 바이러스(Marburg virus), 에볼라바이러스(Ebolavirus), 노로바이러스(Norovirus), 로타바이러스(Rotavirus), 아데노바이러스(Adenovirus), 사포바이러스(Sapovirus), 아스트로바이러스(Astrovirus), 리케차 프로바제키이(Rickettsia prowazekii), 리케차 타이피(Rickettsia typhi), 오리엔티아 쯔쯔가무시(Orientia tsutsugamushi), 보렐리아 부르그도르페리(Borrelia burgdorferi), 페스트균(Yersinia pestis), 삼일열말라리아원충(Plasmodium vivax), 사일열말라리아원충(Plasmodium malariae), 열대열말라리아원충(Plasmodium falciparum), 난형말라리아원충(Plasmodium ovale), 탄저균(Bacillus anthracis), 클로스트리듐 디피실리(Clostridium Difficile), 클로스트리듐 보톨리늄(Clostridium Botulinum), 디프테리아균(Corynebacterium diphtheriae), 살모넬라 엔테리카 혈청형 타이피(Salmonella enterica serovar Typhi), 살모넬라 엔테리카 혈청형 파라타이피 A(Salmonella enterica serovar Paratyphi A), 시가 독소-생산 대장균(E. coli: STEC), 시겔라 다이센테리에(Shigella dysenteriae), 시겔라 플렉스네리(Shigella flexneri), 시겔라 보이디이(Shigella boydii), 시겔라 소네이(Shigella sonnei), 이질아메바(Entamoeba histolytica), 콜레라균(Vibrio cholerae), 결핵균(Mycobacterium tuberculosis), 수막염균(Neisseria meningitidis), 백일해균(Bordetella pertusis), B형 헤모필루스 인플루엔자(Haemophilus influenzae type B: HiB), 파상풍균(Clostridium tetani), 리스테리아 모노사이토제네스(Listeria monocytogenes) 및 폐렴구균(Streptococcus pneumoniae)으로 이루어진 군에서 선택된 것일 수 있다.
다른 실시형태에서, 병원체는 HPV, HIV-1, 인플루엔자 바이러스, 뎅기 바이러스, HAV, HBV, HCV, SARS-CoV, SARS-CoV-2로 이루어진 군에서 선택된 것이다. 보다 바람직하게는, 병원체는 HPV, HBV, HCV 및 SARS-CoV-2로 이루어진 군에서 선택된 것이다.
다른 실시형태에서, 항원은 HPV16 E7 단백질, HPV18 E7 단백질, HBV X 단백질(HBx), HBV preS1 단백질, HCV 코어 단백질(HCVcore) 및 SARS-CoV-2 스파이크 단백질(CoV2S)로 이루어진 군에서 선택된 병원성 항원이다.
다른 실시형태에서, 상기 항원은 원하는 면역 반응을 유도하기 위한 적어도 하나의 에피토프를 포함하며, 바람직하게는 1 내지 30개의 에피토프를 함유하고, 보다 바람직하게는 1 내지 15개의 에피토프를 함유한다.
다른 실시형태에서, 항원은 서열번호 38, 39, 40, 41, 42 또는 43과 적어도 70%, 80%, 90%, 95% 또는 99% 동일한 아미노산 서열을 포함하거나 실질적으로 이 서열로 이루어지는 병원성 항원이다.
다른 실시형태에서, 항원은 서열번호 38, 39, 40, 41, 42 또는 43과 적어도 80% 동일한 아미노산 서열을 포함하거나 실질적으로 이 서열로 이루어지는 병원성 항원이다.
다른 실시형태에서, 항원은 서열번호 38, 39, 40, 41, 42 및 43으로 이루어진 군에서 선택된 아미노산 서열을 포함한다.
다른 실시형태에서, 항원은 종양 항원이다. 종양 항원은 종양-관련 항원(TAA) 또는 종양-특이적 항원(TSA)이다.
일 실시형태에서, 종양 또는 암은 유방암, 결장암, 직장암, 방광암, 자궁내막암, 신장암, 위암, 교모세포종, 간세포 암종, 담관암, 소세포 폐암, 비-소세포 폐암(NSCLC), 흑색종, 난소암, 자궁경부암, 췌장암, 전립선암, 급성 골수성 백혈병(AML), 만성 골수성 백혈병(CML), 비-호지킨 림프종 및 갑상선암으로 이루어진 군에서 선택된 것이다.
다른 실시형태에서, 종양-관련 항원은 SSX2, MAGE-A3, NY-ESO-1, iLRP, WT12-281, RNF43, CEA-NE3, AFP, ALK, AGR2(Anterior gradient 2), BAGE 단백질, β-카테닌, brc-abl, BRCA1, BORIS, CA9, 탄산무수화효소 IX, 카스파제-8, CD40, CDK4, CEA, CTLA4, 사이클린-B1, CYP1B1, EGFR, EGFRvIll, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EphA2, Fra-1, FOLR1, GAGE 단백질(예를 들어, GAGE-1, -2), GD2, GD3, GloboH, 글리피칸-3, GM3, gp100, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE 단백질(예를 들어, MAGE-1, -2, -3, -4, -6 및 -12), MART-1, 메소텔린, ML-IAP, Muc1, Muc16(CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ES01, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA(FOLH1), RAGE 단백질, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, 서비빈(survivin), TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, 티로시나제 및 유로플라킨(uroplakin)-3으로 이루어진 군에서 선택된 것이다.
다른 실시형태에서, 항원은 SSX2, MAGE-A3, NY-ESO-1, iLRP, WT12-281, RNF43 및 CEA-NE3으로 이루어진 군에서 선택된 종양-관련 항원이다.
다른 실시형태에서, 항원은 서열번호 44, 45, 46, 47, 48, 49 또는 50과 적어도 70%, 80%, 90%, 95% 또는 99% 동일한 아미노산 서열을 포함하는 종양-관련 항원이다.
다른 실시형태에서, 항원은 서열번호 44, 45, 46, 47, 48, 49 및 50으로 이루어진 군에서 선택된 아미노산 서열을 포함하는 종양-관련 항원이다.
항원은 단일 항원 또는 이의 항원성 단편 또는 서로 융합된 적어도 두 개의 항원을 포함하는 융합 항원일 수 있다. 예를 들어, 항원은 HPV16 E7 단백질의 단일 항원 또는 HPV16 E7 및 HPV18 E7 단백질을 포함하는 융합 항원일 수 있다. 융합 항원은 서로 다른 항원을 연결하는 링커를 갖거나 갖지 않을 수 있다.
다른 실시형태에서, 항원은 서로 다른 항원을 연결하는 적어도 하나의 링커를 갖는 융합 항원이다.
다른 실시형태에서, 항원은 서로 다른 항원을 연결하는 경식(rigid) 링커 (EAAAAK)n을 갖는 융합 항원이며, 여기서 n은 0 내지 12, 바람직하게는 2 내지 6, 보다 바람직하게는 3 내지 4의 정수이다. 다시 말해, 경식 링커는 서열 EAAAAK(서열번호 56)의 0 내지 12반복체, 2 내지 6반복체 또는 3 내지 4반복체를 포함한다.
다른 실시형태에서, 본 발명의 융합 단백질은 CD40-결합 도메인과 퓨린 및/또는 카텝신 L 절단 부위 사이에 경식 링커를 더 포함한다. 경식 링커는 아미노산 서열 EAAAAK(서열번호 56)의 0 내지 12반복체를 포함하는 펩타이드 라이너일 수 있다.
경식 링커는 (EAAAAK)n 또는 (서열번호 56)n일 수 있으며, 여기서 n은 0 내지 12, 바람직하게는 2 내지 6, 보다 바람직하게는 3 내지 4의 정수이다.
다른 실시형태에서, 경식 링커는 서열번호 56의 2 내지 6반복체 또는 3 내지 4반복체를 포함한다.
다른 실시형태에서, 본 발명의 융합 단백질은 서열번호 51, 52, 53, 54 또는 55와 적어도 90%, 95% 또는 99% 동일한 아미노산 서열을 포함하거나 실질적으로 이로 이루어진다.
또 다른 실시형태에서, 본 발명의 융합 단백질은 서열번호 51, 52, 53, 54 및 55로 이루어진 군에서 선택된 아미노산 서열을 포함하거나 실질적으로 이로 이루어진다.
다른 양상에서, 본 발명은 본 발명의 융합 단백질을 암호화하는 DNA 단편에 관한 것이다. 또한 본 발명은 본 발명의 융합 단백질을 암호화하는 DNA 단편을 포함하는 발현 벡터에 관한 것이다. 또한 본 발명은 본 발명의 융합 단백질 및 약학적으로 허용가능한 담체 및/또는 아쥬반트를 포함하는 약학적 조성물에 관한 것이다.
약학적 조성물은 경피적, 경점막적, 비인두적, 폐 또는 직접적 주사에 적합한 또는 전신적(예를 들어, 비경구적) 또는 국소적(예를 들어, 종양내 또는 병변내 주사) 투여에 적합한 장관 또는 비경구적 투여 형태일 수 있다. 비경구적 주사는 정맥내, 복강내, 근육내, 피하 또는 피내 경로를 통한 것일 수 있다.
적합한 아쥬반트에는 사포닌-기반 아쥬반트 또는 톨-유사 수용체(TLR) 작용제 아쥬반트가 포함되지만 이로 제한되지 않는다. 사포닌-기반 아쥬반트는 GPI-0100, Quil A 또는 QS-21일 수 있다. TLR 작용제 아쥬반트는 Poly I:C, 모노포스포릴 지질 A(MPL) 또는 CpG 올리고뉴클레오타이드(예를 들어, 클래스 A CpG: CpG1585, CpG2216 또는 CpG2336; 클래스 B CpG: CpG1668, CpG1826, CpG2006, CpG2007, CpG BW006 또는 CpG D-SL01; 클래스 C CpG: CpG2395, CpG M362 또는 CpG D-SL03)일 수 있다. 일 실시형태에서, 아쥬반트는 CpG 올리고뉴클레오타이드이다.
또한 약학적 조성물은 경구적으로, 예를 들어 정제, 코팅된 정제, 당의정, 경질 및 연질 젤라틴 캡슐의 형태로 투여될 수 있다.
융합 단백질의 투여량은 제어할 질환, 환자의 나이 및 개별 상태 및 투여 방식에 따라 달라질 수 있다. 투여량은 원하는 치료 반응을 달성하기 위한 본 발명의 융합 단백질의 치료적 유효량의 얻기 위해 각각의 특정한 경우의 개별 요건에 맞추어질 수 있다.
성인 환자의 경우, 약 0.1 내지 50㎎, 특히 약 0.1 내지 5㎎의 단일 용량이 고려된다. 질환의 중증도 및 정확한 약동학적 프로파일에 따라, 융합 단백질은 주당, 2주당 또는 1개월당 하나의 용량 단위로 투여될 수 있고, 이러한 치료를 만족시키기 위해 주기당 총 1 내지 6회 용량 단위가 제공될 수 있다.
다른 양상에서, 본 발명은 항원-특이적 T 세포 면역 반응을 유도하여 이를 필요로 하는 대상체에서 감염성 질환 또는 종양으로부터 보호하고/보호하거나 이를 치료하기 위한 약제의 제조에서의 본 발명의 융합 단백질 또는 약학적 조성물의 용도에 관한 것이다.
약어: Rap1, Ras-proximate-1 또는 Ras-관련 단백질 1; CD40, 분화 클러스터(Cluster of differentiation) 40; CDR, 상보성 결정 영역.
실시예
동물 종양 모델
C57BL/6 생쥐 폐 상피 세포의 HPV16 E6- 및 E7-발현 종양 세포주를 사용하여 실시예 6 내지 8에서의 생체내 효능 분석을 위한 생쥐 HPV16 종양 모델을 확립하였다. 종양 세포는 FBS(10%) 및 페니실린/스트렙토마이신/암포테리신 B(50유닛/㎖)을 함유하는 RPMI 1640 배지에서 37℃, 5% CO2 조건으로 배양하였다.
서열번호 및 구성 요소
표 1은 서열번호 및 해당 펩타이드, 폴리펩타이드 및 융합 단백질을 보여준다.
유세포 분석. 비장 세포를 37℃에서 2시간 동안 항원성 자극제로 자극한 후, 37℃에서 2시간 동안 50㎍/㎖의 프레펠딘 A 및 모넨신으로 처리하였다. 세포를 수확하고, 0.5% BSA 함유 PBS로 세척하고, APC/Cy7-접합 항-CD3 항체, PerCP/Cy5.5-접합 항-CD4 항체, FITC-접합 항-CD8 항체, PE-접합 항-CD44 항체 및 APC-접합 항-CD62L 항체로 동시에 염색하였다. 세척 후, 세포를 투과화시키고, 고정하고 PE-접합 항-IFN-γ 항체, PE/Cy7-접합 항-IL-2 항체 및 eFluor450-접합 항-TNF-α 항체로 동시에 세포내 염색하였다. 또한 Gallios 유세포 분석기 및 Kaluza 소프트웨어를 사용하여 CD8+ 또는 CD4+ 기억 T 세포 표현형(CD3+CD44hiCD62Llo)을 갖는 비장 세포의 세포내 사이토카인 특성(IFN-γ, IL-2 또는 TNF-α)을 분석하였다.
효소-결합 면역스팟(ELISpot) 분석. 비장 세포를 사전처리한 쥐 IFN-γ 포획 96-웰 플레이트(CTL IMMUNOSPOT®)에 항원성 자극제의 존재 또는 부재 하에서 2Х105 세포/웰의 세포 밀도로 3반복 접종하였다. 37℃에서 24시간 동안 배양한 후 세포를 제거하였다. 세척 후, 상온에서 2시간 동안 비오틴-접합 항-쥐 IFN-γ 항체를 사용하여 포획된 IFN-γ를 검출하고 제조사의 지침에 따라 IFN-γ 면역스팟을 전개하였다. IMMUNOSPOT® S5 Micro 분석기(CTL)를 사용하여 IFN-γ 면역스팟의 스캐닝 및 계수를 수행하였다.
간접적 효소-결합 면역흡착 분석(ELISA). 수집한 전혈 시료를 4℃에서 30 내지 60분 동안 그래도 둔 후 5,000g에서 10분 동안 원심분리하여 혈병을 펠릿화하였다. 혈청 시료를 -20℃에 보관하였다. 항원-특이적 항체 결합을 위한 정제된 코팅 단백질을 구아니딘 코팅 완충액(2M 구아니딘 염산, 500mM Na2HPO4, 25mM 구연산염, pH 4.0 내지 4.4)에 희석하고 96-웰 플레이트에 1㎍/웰로 분배하였다. 4℃에서 밤새 항온처리한 후, 96-웰 플레이트를 37℃에서 1시간 동안 PBS에서 1% BSA로 블로킹하였다. 혈청 시료를 녹이고, 1% BSA 함유 PBS에 10배 연속 희석하였다. 코팅된 단백질을 37℃에서 2시간 동안 100㎕의 1000배 희석 혈청 시료와 함께 항온처리하였다. 인산염 완충 염수 TWEEN®-20(PBST)으로 4회 세척한 후, 37℃에서 30분 동안 겨자무 페록시다제(HRP)-접합 염소 항-쥐 IgG(1:10,000 희석, Cat#31430, Thermo Fisher Science)를 사용하여 항원-특이적 항체를 검출하였다. PBST로 4회 세척한 후, 100㎕ TMB 기질의 존재 하에서 HRP-매개 색 발현을 촉매화하고 100㎕ 1N HCl로 퀀칭하였다. 450㎚에서의 흡광도로 혈청 시료의 항원-특이적 항체의 상대적 역가를 결정하였다.
통계적 분석. 모든 비교의 유의성을 t-검정을 사용하여 계산하였고, p<0.05일 때 결과를 유의한 것으로 간주하였다.
실시예 1
CD40L47-261-TPE-E7 및 18sCD40L-TPE-E7을 위한 발현 벡터 구축
도 5a 내지 도 5e는 본 발명에 따른 융합 단백질의 다양한 실시형태를 나타낸다.
융합 단백질 CD40L47-261-TPE-E7(서열번호 51; 도 5a)은 (a) 잘린 CD40 리간드 CD40L47-261(서열번호 18); (b) (EAAAAK)3(서열번호 3) 및 RX1RX2X3R(서열번호 2)(여기서 X1은 A, X2는 Y, X3은 K)을 포함하는 절단가능 링커; (c) 서열번호 5의 PE 전위 펩타이드(PE280-305); 및 (d) 서열번호 38의 HPV16 E7 단백질 및 서열번호 39의 HPV18 E7 단백질을 포함하는 융합 항원 HPV16/18 E7을 포함한다.
CD40L47-261-TPE-E7을 위한 발현 벡터(도 1)는 다음과 같이 구축된다: CD40L47-261을 포함하는 Hin dIIICD40L-Linker-PE Nco I, Xho I, Sal I 암호화 DNA 단편, 절단가능 링커 및 PE 전위 펩타이드(PE280-305)를 PCR 합성하고, HindIII/SalI로 소화시킨 후, HindIII/XhoI 절단 부위를 갖는 플라스미드 pTAC-MAT-Tag-2에 결찰하여 플라스미드 P07-His-pNC(도 2)를 얻었다. 이후, His 태그를 갖는 융합 항원 HPV16/18 E7 암호화 DNA 단편을 제한 효소 NcoI/XhoI를 통해 플라스미드 P07-His-pNC(도 2)에 삽입하여 융합 단백질 CD40L47-261-TPE-E7(도 1)을 위한 발현 벡터를 생성하였다.
절단가능 링커는 퓨린 및/또는 카텝신 L 프로테아제가 융합 단백질을 절단하여 융합 단백질로부터 TPE-E7 단편이 방출되는 것을 허용한다.
위에 기술된 것과 유사한 방법을 적용하면, 임의의 다른 관심있는 항원(들)으로 E7을 대체하고 도 2의 플라스미드에 삽입하여 본 발명에 따른 관심있는 항원을 포함하는 융합 단백질을 위한 도 1의 플라스미드와 유사한 발현 벡터를 생성할 수 있다.
위에 기술된 유사한 방법을 사용하여 융합 단백질 18sCD40L-TPE-E7(서열번호 52; 도 5b)을 위한 발현 벡터를 구축하였으며, 여기서 잘린 CD40 리간드: CD40L47-261(서열번호 18)을 18sCD40L, 다른 잘린 CD40 리간드: CD40L108-261(서열번호 19)로 대체하였다.
실시예 2
E7-TStx-CD40L47-261 및 E7-TStx-18sCD40L을 위한 발현 벡터의 구축
융합 단백질 E7-TStx-CD40L47-261(서열번호 53; 도 5c)은 (a) 융합 항원 HPV16/18 E7(HPV16 E7 단백질(서열번호 38) 및 HPV18 E7 단백질(서열번호 39)를 포함함), (b) 서열번호 14의 Stx 전위 펩타이드(Stx211-247), (c) 서열번호 1의 RX1X2R(여기서 X1은 V, X2는 A) 및 서열번호 3의 (EAAAAK)3을 포함하는 절단가능 링커 및 (d) 서열번호 18의 잘린 CD40 리간드(CD40L47-261)를 포함한다.
E7-TStx-CD40L47-261을 위한 발현 벡터(도 3)는 다음과 같이 구축된다:
Stx 전위 펩타이드(Stx211-247)를 포함하는 Hin dIII, Xho IStx-Linker-CD40L Sal I을 암호화하는 DNA 단편, 절단가능 링커 및 CD40L47-261을 PCR 합성하고, HindIII/SalI으로 소화시킨 후, HindIII/XhoI 절단 부위를 갖는 플라스미드 pTAC-MAT-Tag-2 백본에 결찰하여 플라스미드 P08(RP)-His-pNC(도 4)를 얻었다. 이후, His 태그를 갖는 융합 항원 HPV16/18 E7 암호화 DNA 단편을 제한 효소 HindIII/XhoI를 통해 플라스미드 P08(RP)-His-pNC(도 4)에 삽입하여 발현 벡터 E7-TStx-CD40L47-261(도 3)을 생성하였다.
절단가능 링커는 융합 단백질이 퓨린 및/또는 카텝신 L 프로테아제에 의해 잘려 융합 단백질로부터 E7-TStx 단편이 방출되는 것을 허용하기 때문에 본 발명의 융합 단백질을 위해 필수적이다. 예를 들어, 도 5c를 참고할 수 있다.
위에 기술된 것과 유사한 방법을 적용하면, 다양한 병원체 또는 암으로부터의 임의의 다른 관심있는 항원(들)으로 E7을 대체하고 도 4의 플라스미드에 삽입하여 본 발명에 따른 관심있는 항원을 포함하는 융합 단백질을 위한 도 3의 플라스미드와 유사한 발현 벡터를 생성할 수 있다.
위에 기술된 유사한 방법을 사용하여, 융합 단백질 E7-TStx-18sCD40L(서열번호 54; 도 5d)을 위한 발현 벡터를 구축하였으며, 여기서 잘린 CD40 리간드: CD40L47-261(서열번호 18)을 18sCD40L, 다른 잘린 CD40 리간드: CD40L108-261(서열번호 19)로 대체하였다.
비교 목적을 위해, 미국 특허 제9,481,714 B2호의 실시예 1에 개시된 작제물과 거의 동일한 융합 단백질 RAP1-CD28convPEt-E7-K3(본 출원에서 "RAP1-E7"로 지칭됨)을 구축하였다. RAP1-CD28convPEt-E7-K3(본 출원에서 "RAP1-E7"로 지칭됨)은 RAP1 도메인 III, CD28 서열, 링커, PE 전위 도메인 II(PE268-313), 항원 E7 단백질 및 소포체 잔류 서열을 포함한다. 여기서 사용된 항원 E7 단백질은 HPV16 E7 단백질(서열번호 38) 및 HPV18 E7 단백질(서열번호 39)을 포함하는 융합 항원 HPV16/18 E7이며, 선행기술에 사용된 항원 E7 단백질은 HPV16 E7 단백질이다.
실시예 3
HBx-preS1-TStx-18sCD40L을 위한 발현 벡터의 구축
융합 단백질 HBx-preS1-TStx-18sCD40L(서열번호 55; 도 5e)은 (a) 서열번호 40의 HBx 단백질 및 서열번호 41의 HBV preS1 단백질을 포함하는 융합 항원 HBx-preS1, (b) 서열번호 14의 Stx 전위 펩타이드(Stx211-247), (c) 서열번호 1의 RX1X2R(여기서 X1은 V, X2는 A) 및 서열번호 3의 (EAAAAK)3을 포함하는 절단가능 링커 및 (d) 서열번호 19의 잘린 CD40 리간드(18sCD40L)를 포함한다.
실시예 2에 기술된 유사한 방법을 사용하여, 융합 단백질 HBx-preS1-TStx-18sCD40L을 위한 발현 벡터를 구축하였으며, 여기서 사용된 잘린 CD40 리간드는 18sCD40L이었고, 사용된 항원은 위에 기술된 바와 같은 융합 항원 HBx-preS1이었다.
실시예 4
단백질 발현
단백질 발현 벡터 CD40L47-261-TPE-E7를 품은 대장균 BL21 세포를 37℃에서 선택된 항생제를 적절한 농도로 함유하는 ZY 배지(10g/ℓ 트립톤 및 5g/ℓ 효모 추출물)에 접종하였다. 배양이 초기 로그 단계(OD600=2 내지 5)에 도달했을 때, 아이소프로필-1-티오-β-D-갈락토파이란노사이드(IPTG)(0.5 내지 2mM)를 사용하여 융합 단백질의 발현을 유도하였다. IPTG 유도 4시간 후 세포를 수확하고 음파처리로 파쇄하였다. 과발현된 융합 단백질을 회수하기 위해 봉입체를 추출하고 가용화 완충액(6M 구아니딘 염산, 20mM 인산칼륨, 500mM NaCl, 20mM 이미다졸, 1mM DTT, pH 7.4)에서 가용화시켰다. 정제 후, 4℃에서 밤새도록 20- 내지 50-배 부피의 투석 완충액(10mM PBS)으로 투석하여 융합 단백질을 리폴딩을 수행하였다. 리폴딩된 융합 단백질을 환원(다이티오트레이톨과 함께; +DTT) 및 비-환원(다이티오트레이톨 없이; -DTT) 조건 하에서 SDS-PAGE로 분석하여 이들이 제대로 리폴딩되었는지를 평가하였다.
또한 다음의 융합 단백질들을 발현시키고 위에 기술된 것과 유사한 방법을 사용하여 리폴딩하였다: (1) 18sCD40L-TPE-E7; (2) E7-TStx-CD40L47-261; (3) E7-TStx-18sCD40L; (4) RAP1-E7; (5) CD40L47-261-TPE-HBx-preS1; (6) 18sCD40L-TPE-HBx-preS1; (7) HBx-preS1-TStx-CD40L47-261; (8) HBx-preS1-TStx-18sCD40L; (9) CD40L47-261-TPE-HCVcore; (10) 18sCD40L-TPE-HCVcore; (11) HCVcore-TStx-CD40L47-261; (12) HCVcore-TStx-18sCD40L; (13) CD40L47-261-TPE-CoV2S; (14) 18sCD40L-TPE-CoV2S; (15) CoV2S-TStx-CD40L47-261; (16) CoV2S-TStx-18sCD40L; (17) CD40L47-261-TPE-SSX2; (18) 18sCD40L-TPE-SSX2; (19) SSX2-TStx-CD40L47-261; (20) SSX2-TStx-18sCD40L. 다음 실험에서 융합 단백질 CD40L47-261-TPE-E7, 18sCD40L-TPE-E7, E7-TStx-18sCD40L, RAP1-E7 및 HBx-preS1-TStx-18sCD40L의 면역원성 분석 또는 효능 분석을 추가로 수행하였다.
실시예 5
융합 단백질의 면역원성 분석
암컷 C57BL/6NCrlBltw 생쥐(5 내지 6주령)를 무작위로 다음의 5개의 군(n=5)으로 나누었다: (A) 위약(즉, PBS); (B) 융합 단백질 CD40L47-261-TPE-E7(100㎍); (C) 융합 단백질 18sCD40L-TPE-E7(100㎍); (D) 융합 단백질 E7-TStx-18sCD40L(100㎍); 및 (E) 융합 단백질 RAP1-E7(100㎍). 융합 단백질을 PBS로 투석하였다. CpG1826(50㎍)을 동물군 B 내지 E에 대한 아쥬반트로 사용하였다. 각 군은 0일부터 7일 간격으로 3회 피하(s.c.)로 면역 접종을 제공받았다. 0, 7 및 14일에 혈액 시료를 수집하였다. 21일에 혈액 시료를 수집하고 비장 세포를 FBS(10%) 및 PSA 함유 RPMI 1640 배지에 재현탁하였다.
항원 자극의 존재 또는 부재 하에서 CD8+ 및 CD4+ 기억 T 세포의 세포내 사이토카인 유도(IFN-γ, IL-2 및 TNF-α)를 분석하기 위해 비장 세포를 사용하였다. 간략하게, 각 동물군의 비장 세포를 항원 E7 단백질(2㎍/㎖의 HPV16 E7 펩타이드 풀)로 처리하거나 처리하지 않고 유세포 분석으로 분석하였다. 항원 E7 자극의 존재 하에서의 사이토카인+/CD8+ 및 사이토카인+/CD4+ 비장 세포의 빈도를 자극하지 않은(비처리) 대조군의 것에 대해 정규화하여 얻은 상대적인 사이토카인 유도로 각 생쥐군의 세포내 사이토카인 유도의 정도 또는 수준을 표시하였다.
또한 비장 세포를 사용하고 효소-결합 면역스팟(ELISpot) 분석법을 사용하여 항원 자극(2㎍/㎖의 HPV16 E7 펩타이드 풀)의 존재 또는 부재 하에서 IFN-γ-분비 비장 세포의 빈도를 분석하였다. 이의 결과를 백만개의 비장 세포 당 IFN-γ+ 면역스팟으로 나타내었다.
혈액 시료를 사용하고 ELISA를 사용하여 혈청 HPV16 E7-특이적 및 HPV18 E7-특이적 항체의 수준을 분석하였으며, 여기서 각각의 코팅 단백질로 정제된 HPV16 E7 및 HPV18 E7 재조합 단백질을 사용하였다.
도 6은 HPV16 E7 펩타이드 풀로 비장 세포를 항원 자극한 후의 사이토카인 유도 결과를 보여준다. 융합 단백질 CD40L47-261-TPE-E7, 18sCD40L-TPE-E7 또는 E7-TStx-18sCD40L(B 내지 D군)로 면역화한 동물의 CD8+ 기억 T 세포에서 IFN-γ 및 TNF-α의 상대적 사이토카인 유도가 모두 RAP1-E7-처리군(E군) 또는 위약군(A군)의 것에 비해 유의적으로 증가하였고, IL2는 그렇지 않았다. CD8+ 기억 T 세포에서 IL-2의 상대적 사이토카인 유도 및 B 내지 E 동물군의 CD4+ 기억 T 세포에서 사이토카인 IFN-γ, IL-2 또는 TNF-α는 약간 증가하였지만, 위약군(A군)에 비해 유의적인 차이를 보이지 않았다.
그럼에도 불구하고, 본 발명의 융합 단백질은 항원 HPV16 E7의 자극에 반응하여 CD8+ 기억 T 세포에서 IFN-γ 및 TNF-α의 발현을 유도하는 데 있어서 선행기술의 융합 단백질보다 우수하다는 결론을 내릴 수 있다.
도 7은 시험관 내에서 HPV16 E7 펩타이드 풀로 자극한 비장 세포의 IFN-γ+ 면역스팟을 보여준다. 각각 CD40L47-261-TPE-E7, 18sCD40L-TPE-E7, E7-TStx-18sCD40L 및 RAP1-E7(B 내지 E군)로 면역화한 동물군의 IFN-γ-분비 비장 세포의 빈도는 위약군에 비해 유의적으로 증가하였다. 특히, E7-TStx-18sCD40L은 CD40L47-261-TPE-E7보다 유의적으로 높은 IFN-γ-분비 세포 빈도를 유도하였다(p=0.035).
이러한 결과는 본 발명의 융합 단백질이 항원성 HPV16 E7 펩타이드 풀로 자극 시 또는 자극 후 IFN-γ-분비 T 세포 집단을 유의적으로 증가시킬 수 있음을 나타낸다.
도 8은 0, 7 및 14일에 다양한 융합 단백질로 면역화한 동물에서 혈청 HPV16 E7-특이적 항체 수준을 보여준다. CD40L47-261-TPE-E7, 18sCD40L-TPE-E7 또는 E7-TStx-18sCD40L로 백신 접종한 동물(각각 B 내지 D군)에서 HPV16 E7-특이적 항체 수준은 7일째의 2차 백신 접종 후 증가하기 시작하여, 14일째의 3차 백신 접종 후 더 증가하였다. 21일째에, B 내지 D 동물군에서 혈청 HPV16 E7-특이적 항체 수준은 위약군 및 RAP1-E7(즉, RAP1-CD28convPEt- E7-K3)으로 백신 접종한 동물군보다 높았다.
융합 단백질 RAP1-E7(RAP1-CD28convPEt- E7-K3)은 2회의 백신 접종(0 및 7일째) 후 HPV16 E7-특이적 항체 수준을 유도하지 못했다. 14일째의 3차 백신 접종 후 HPV16 E7-특이적 항체를 유도하기 시작하였고, 혈청 항체 수준은 21일째에 B 내지 D군에 비해 적게 나타났다. 반면, 본 발명의 융합 단백질은 0 및 7일째의 2회의 백신 접종 후 혈청 HPV16 E7-특이적 항체 수준을 유도하였다.
또한 위에 기술된 것과 같은 요법 및 면역화 일정을 사용하여 동물을 RAP1-CD28convPEt-HBx-K3("RAP1-HBx"로 지침됨)로 백신 접종한 경우 HBx-특이적 항체를 유도하는 유사한 효과가 관찰되었다. RAP1-E7(RAP1-CD28convPEt- E7-K3)에서 HBx 항원으로 E7 항원을 대체하여 융합 단백질 RAP1-HBx를 생성하였다. 융합 단백질 RAP1-HBx는 14일째의 3차 백신 접종 후 혈청 HBx-특이적 항체 수준을 유도하였고, 21일째의 혈청 항체 수준은 본 발명의 융합 단백질로 백신 접종한 동물에 비해 적게 나타났다(데이터 표시 없음).
도 9는 0, 7 및 14일에 다양한 융합 단백질로 면역화한 동물에서 혈청 HPV18 E7-특이적 항체 수준을 보여준다. 융합 단백질 CD40L47-261-TPE-E7, 18sCD40L-TPE-E7 및 E7-TStx-18sCD40L(각각 B 내지 D군)은 위약군에 비해 혈청 HPV18 E7-특이적 항체 수준을 유의적으로 증가시켰다.
따라서, 본 발명의 융합 단백질은 항원-특이적 항체를 유도하는 데 효과적이며 항체 유도는 2회의 백신 접종 후에 발생한다.
요약하면, 본 발명의 융합 단백질은 항원-특이적 T 세포 반응을 유도할 수 있고, 전염증성 사이토카인, 예를 들어 IFN-γ 및 TNF-α의 발현을 증가시킬 수 있고, 항원-특이적 항체 반응을 생성할 수 있다.
실시예 6
융합 단백질의 생체내 효능 분석
암컷 C57BL/6NCrlBltw 생쥐(5 내지 6주령)를 무작위로 5개의 군으로 나누고 PBS(A군, 위약, n=4); 또는 다음의 융합 단백질들 중 하나로 처리하였다: B군, CD40L47-261-TPE-E7(25㎍; n=5); C군, 18sCD40L-TPE-E7(25㎍; n=4); D군, E7-TStx-18sCD40L(25㎍; n=5); 및 E군, RAP1-E7(25㎍; n=5). 융합 단백질을 PBS에 용해시켰으며 B 내지 E군의 동물 백신 접종에서 CpG1826(50㎍)을 아쥬반트로 사용하였다. 도 10은 면역화 일정, 융합 단백질 및 투여량을 보여준다.
생쥐를 실험하기 위해, 0일째에 각 생쥐의 좌측 옆구리에 종양 세포(0.1㎖ 중 1Х105)를 s.c.로 주사하였다. 3회의 면역 접종을 7, 14 및 21일째에 s.c.로 제공하였다. 변형된 타원체 식을 기반으로 한 캘리퍼 측정값의 곱셈으로 주 2회 종양 크기를 결정하였다: 종양 부피 = 1/2 (길이 × 폭 2 ). 생존율 및 무종양률을 계산하였다. 2㎝ 초과의 종양 길이를 갖는 생쥐를 죽은 것으로 간주하였고 종양 덩어리를 측정할 수 없거나 감지할 수 없는 생쥐를 무-종양으로 간주하였다.
접종한 종양이 위약군에서 빠르게 발달하여 25일째에 2마리가 죽었고 따라서 위약군의 데이터는 21일까지만 나타내었다(도 11). C 및 D 동물군(각각 18sCD40L-TPE-E7 및 E7-TStx-18sCD40L로 면역화함)의 종양 덩어리는 적어도 전체 실험 기간(마지막 날은 39일임) 동안 거의 완전하게 억제되었다. B 및 E 동물군(각각 CD40L47-261-TPE-E7 및 RAP1-E7로 면역화함)의 종양은 초기에는 잘 제어되었지만, 면역화를 중단한 후 점차 성장하였다.
이러한 결과는 본 발명의 융합 단백질, 특히 융합 단백질 18sCD40L-TPE-E7 및 E7-TStx-18sCD40L이 종양의 성장을 효과적으로 억제할 수 있음을 나타낸다.
B 내지 E 동물군(각각 CD40L47-261-TPE-E7, 18sCD40L-TPE-E7, E7-TStx-18sCD40L 및 RAP1-E7로 면역화함)의 생존율은 35일째에 0%로 감소한 위약군에 비해 35일째에 100%를 유지하였다(도 12).
이러한 결과는 본 발명의 융합 단백질이 동물 종양 모델에서 생존율을 효과적으로 유지할 수 있음을 나타낸다.
전체 실험 기간(39일) 동안 A, B 및 E 동물군에서 무-종양 동물은 발견할 수 없었다(도 13). C군에서 1마리(25%) 및 D군에서 3마리(60%)(각각 18sCD40L-TPE-E7 및 E7-TStx-18sCD40L로 면역화함)가 측정가능하거나 감지가능한 종양 없이 생존하는 것을 발견하였다. 특히, 이들 무종양 생쥐에서 종양 덩어리는 18sCD40L-TPE-E7 또는 E7-TStx-18sCD40L로 3회 면역화를 완료한 직후 모두 제거되었다.
이러한 결과는 종양을 갖는 동물에서 무종양률을 증가시키는 데 있어서 본 발명의 융합 단백질이 선행기술의 융합 단백질 RAP1-E7보다 강력함을 나타낸다.
실시예 7
18sCD40L-TPE-E7의 용량 차이에 따른 생체 내 효능 분석
암컷 C57BL/6NCrlBltw 생쥐(4 내지 6주령)를 무작위로 다음의 5개의 군(군당 n=5)으로 나누었다: (A) 위약(PBS); (B) 18sCD40L-TPE(100㎍; 융합 항원 E7 없음); (C) 18sCD40L-TPE-E7(100㎍); (D) 18sCD40L-TPE-E7(50㎍); (E) 18sCD40L-TPE-E7(25㎍). 융합 단백질을 PBS에 용해시켰으며 B 내지 E군에서 CpG1826(50㎍)을 아쥬반트로 사용하였다. 0일째에 각 생쥐의 좌측 옆구리에 종양 세포(0.1㎖ 중 1Х106)를 s.c.로 주사하였다. 종양 세포 주사 2주 후, 14, 21 및 28일째에 3회 s.c.로 종양 생쥐에 백신 접종하였다.
종양 부피를 결정하였다. 융합 단백질 18sCD40L-TPE-E7의 모든 투여량(25㎍, 50㎍ 또는 100㎍)에서 종양의 성장을 억제하는 강한 효과가 나타났다. 융합 단백질에 의한 종양 크기의 억제는 14일째의 첫 번째 주사 후에 나타났고, 34일째의 마지막 관찰일까지 전체 실험 기간 동안 지속되었다. 항원 E7이 결여된 위약 및 18sCD40L-TPE는 종양의 성장을 억제하는 효과가 없었다(도 14).
실시예 8
E7-TStx-18sCD40L의 용량 차이에 따른 생체 내 효능 분석
B 내지 E군의 생쥐를 각각 (B) TStx-18sCD40L(100㎍; 융합 단백질 E7 없음); (C) E7-TStx-18sCD40L(100㎍); (D) E7-TStx-18sCD40L(50㎍); 및 (E) E7-TStx-18sCD40L(25㎍)로 백신 접종한 것을 제외하고는 실시예 7과 유사한 방법을 사용하여 생쥐를 군으로 나누고, 종양 세포를 주사하고, 14, 21, 28일째에 융합 단백질을 투여하고, 종양 크기를 측정하였다. 융합 단백질 E7-TStx-18sCD40L의 모든 투여량(25㎍, 50㎍ 또는 100㎍)에서 종양의 성장을 억제하는 강한 효과가 나타났다(도 15). 융합 단백질에 의한 종양 크기의 억제는 첫 번째 주사 후에 나타났고, 34일째의 마지막 관찰일까지 전체 실험 기간 동안 지속되었다. 항원 E7이 결여된 위약 및 TStx-18sCD40L은 종양의 성장을 억제하는 효과가 없었다(도 15).
따라서, 본 발명의 융합 단백질은 우수한 치료 효능으로 종양의 성장을 억제하는 강한 효과를 갖는다.
실시예 9
백신 투여 횟수: HBx-preS1-TStx-18sCD40L의 면역원성 분석
도 16은 각 동물군의 투여 일정을 보여준다. C57BL/6JNarl 암컷 생쥐(5주령)를 무작위로 다음의 4개의 군(군당 n=5)으로 나누었다: (1) 위약군; (2) D0-D7-D14군(3회 투여, 0, 7, 14일째에 백신 접종); (3) D7-D14(2회 투여, 7, 14일째에 백신 접종); 및 (4) D14군(1회 투여, 14일째에 백신 접종). 위약군은 0, 7 및 14일째에 s.c.로 PBS를 제공받았다. 다른 군의 생쥐들은 도 16의 투여 일정에 따라 s.c.로 CpG1826 ODN(50㎍)으로 보강된 HBx-preS1-TStx-18sCD40L(100㎍)을 제공받았다. 혈액 시료는 0, 7, 14 및 21일째에 수집하였다. 21일째에, 동물을 희생시키고, 비장 세포를 수집하고 배양하였다. 각각 ELISpot 분석법을 사용하여 항원성 자극제(HBx-특이적 펩타이드 풀, 즉, HBV 32aa 중첩 9 펩타이드)의 존재 및 부재 하에서 IFN-γ-분비 비장 세포의 빈도를 분석하였다. 혈청 HBx-특이적 항체의 수준을 ELISA로 분석하였으며, 여기서 정제된 HBx 재조합 단백질을 코팅 단백질로 사용하였다.
도 17은 각 동물군에서 시험관 내 HBV 32aa 중첩 9 펩타이드 풀로 자극한 비장 세포에서 IFN-γ+ 면역스팟을 보여준다. 이러한 결과는 3회 투여, 2회 투여 및 1회 투여로 면역화한 동물군(각각 D0-D7-D14, D7-D14 및 D14군)의 비장 세포 모두가 위약군에 비해 IFN-γ-분비 비장 세포의 빈도에서 유의적인 증가를 보임을 나타낸다. IFN-γ-분비 비장 세포의 빈도는 면역화의 횟수와 양의 상관관계가 있었다. D0-D7-D14군(3회 백신 접종)은 가장 높은 IFN-γ-분비 비장 세포 유도를 보였다.
반면, HBx-preS1-TStx-18sCD40L의 단일 프라이밍 투여(D14군)는 HBx-특이적 항체 반응을 명확하게 유도하지는 않았다. 그러나, 2차 면역화는 항체 수준을 상당히 높였고(D7-D14군) 3차 투여는 D0-D7-D14 동물군에 나타난 바와 같이 항체 수준을 훨씬 더 높였다(도 18). 체액성 반응을 유도하는 투여-횟수-의존적 효과는 세포-매개 면역 반응을 유도하는 것과 일치한다. 융합 단백질은 투여 횟수 의존적 방식에서 IFN-γ 생성을 유도하는 데 효과적이다(도 17).
따라서, 융합 단백질 HBx-preS1-TStx-18sCD40L은 2회의 면역화 이후 HBx-특이적 T 세포-매개 면역 반응 및 HBx-특이적 체액성 면역 반응을 효과적으로 유도할 수 있었으며, 이는 복수의 백신 접종에 의해 더욱 증가될 수 있었다.
전술한 예시적인 실시형태의 설명은 예시 및 설명의 목적을 위해서만 제공되었으며 개시된 정확한 형태로 본 발명을 제한하기 위해 철저하게 의도되는 것은 아니다. 본 명세서에서 인용 및 논의된 모든 참고문헌은 각 참고문헌이 개별적으로 참조에 의해 원용되는 경우와 같은 정도로 그 전체가 참조에 의해 본 명세서에 원용된다.
SEQUENCE LISTING
<110> NAVICURE BIOPHARMACEUTICALS LIMITED
<120> FUSION PROTEINS FOR IMMUNOTHERAPY AGAINST CANCER AND INFECTIOUS DISEASES
<130> WO/2021/225820
<140> PCT/US2021/029203
<141> 2021-04-26
<150> US 63/020,545
<151> 2020-05-06
<160> 56
<170> PatentIn version 3.5
<210> 1
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Cleavable linker 1
<220>
<221> MISC_FEATURE
<222> (2)..(3)
<223> Xaa can be any naturally occurring amino acid
<400> 1
Arg Xaa Xaa Arg
1
<210> 2
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Cleavable linker 2
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa is K, F or R.
<400> 2
Arg Xaa Arg Xaa Xaa Arg
1 5
<210> 3
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Rigid linker 1
<400> 3
Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala
1 5 10 15
Ala Lys
<210> 4
<211> 613
<212> PRT
<213> Pseudomonas aeruginosa
<400> 4
Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val
1 5 10 15
Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro
20 25 30
Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val
35 40 45
Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu
50 55 60
Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu
65 70 75 80
Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser
85 90 95
Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn
100 105 110
Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His
115 120 125
Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys
130 135 140
Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu
145 150 155 160
Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met
165 170 175
Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser
180 185 190
Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr
195 200 205
Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile
210 215 220
Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys
225 230 235 240
Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu
245 250 255
Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe
260 265 270
Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly
275 280 285
Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser
290 295 300
Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly
305 310 315 320
Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala
325 330 335
Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg
340 345 350
Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val
355 360 365
Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp
370 375 380
Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe
385 390 395 400
Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn
405 410 415
Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg
420 425 430
Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln
435 440 445
Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala
450 455 460
Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly
465 470 475 480
Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly
485 490 495
Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr
500 505 510
Arg Thr Gly Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu
515 520 525
Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly
530 535 540
Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu
545 550 555 560
Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg
565 570 575
Asn Val Gly Gly Asp Leu Ala Pro Ser Ser Ile Pro Asp Gln Glu Gln
580 585 590
Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Ser
595 600 605
Arg Glu Asp Leu Lys
610
<210> 5
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> PE translocation peptide (PE280-305, minimal)
<400> 5
Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val
1 5 10 15
Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp
20 25
<210> 6
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> PE translocation peptide (PE280-313)
<400> 6
Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val
1 5 10 15
Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
20 25 30
Ile Arg
<210> 7
<211> 46
<212> PRT
<213> Artificial Sequence
<220>
<223> PE translocation peptide (PE268-313)
<400> 7
Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
1 5 10 15
Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu
20 25 30
Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
35 40 45
<210> 8
<211> 61
<212> PRT
<213> Artificial Sequence
<220>
<223> PE translocation peptide (PE253-313)
<400> 8
Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro
1 5 10 15
Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu
20 25 30
Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala
35 40 45
Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
50 55 60
<210> 9
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> PE translocation peptide (PE253-364)
<400> 9
Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro
1 5 10 15
Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu
20 25 30
Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala
35 40 45
Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu
50 55 60
Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln
65 70 75 80
Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu
85 90 95
Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn
100 105 110
<210> 10
<211> 293
<212> PRT
<213> Shigella dysenteriae
<400> 10
Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser
1 5 10 15
Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser
20 25 30
Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Thr Gly Asp Asn
35 40 45
Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly
65 70 75 80
Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser
85 90 95
His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser
100 105 110
Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125
Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser
130 135 140
His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg
145 150 155 160
Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg
165 170 175
Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190
Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205
Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile
210 215 220
Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu
225 230 235 240
Asn Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu
245 250 255
Phe Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile Thr His
260 265 270
Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu Met Arg
275 280 285
Arg Thr Ile Ser Ser
290
<210> 11
<211> 293
<212> PRT
<213> Escherichia coli
<400> 11
Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser
1 5 10 15
Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser
20 25 30
Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn
35 40 45
Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly
65 70 75 80
Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser
85 90 95
His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser
100 105 110
Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125
Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser
130 135 140
His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg
145 150 155 160
Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg
165 170 175
Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190
Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205
Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile
210 215 220
Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu
225 230 235 240
Asn Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu
245 250 255
Phe Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile Thr His
260 265 270
Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu Met Arg
275 280 285
Arg Thr Ile Ser Ser
290
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Stx translocation peptide (Stx240-247, minimal)
<400> 12
Leu Asn Cys His His His Ala Ser
1 5
<210> 13
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Stx translocation peptide (Stx240-251)
<400> 13
Leu Asn Cys His His His Ala Ser Arg Val Ala Arg
1 5 10
<210> 14
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> Stx translocation peptide (Stx211-247)
<400> 14
Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe
1 5 10 15
Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys
20 25 30
His His His Ala Ser
35
<210> 15
<211> 41
<212> PRT
<213> Artificial Sequence
<220>
<223> Stx translocation peptide (Stx211-251)
<400> 15
Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe
1 5 10 15
Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys
20 25 30
His His His Ala Ser Arg Val Ala Arg
35 40
<210> 16
<211> 84
<212> PRT
<213> Artificial Sequence
<220>
<223> Stx translocation peptide (Stx168-251)
<400> 16
Ala Leu Arg Phe Arg Gln Ile Gln Arg Gly Phe Arg Thr Thr Leu Asp
1 5 10 15
Asp Leu Ser Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu
20 25 30
Thr Leu Asn Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His Gly
35 40 45
Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe Gly Ser Ile Asn Ala
50 55 60
Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys His His His Ala Ser
65 70 75 80
Arg Val Ala Arg
<210> 17
<211> 261
<212> PRT
<213> Homo sapiens
<400> 17
Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly
1 5 10 15
Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu
20 25 30
Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg
35 40 45
Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val
50 55 60
Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser
65 70 75 80
Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys
85 90 95
Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu
100 105 110
Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser
115 120 125
Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
130 135 140
Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
145 150 155 160
Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
165 170 175
Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
180 185 190
Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
195 200 205
Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
210 215 220
Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
225 230 235 240
Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
245 250 255
Gly Leu Leu Lys Leu
260
<210> 18
<211> 215
<212> PRT
<213> Artificial Sequence
<220>
<223> Truncated CD40 ligand (CD40L47-261)
<400> 18
His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp
1 5 10 15
Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser
20 25 30
Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe
35 40 45
Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser
50 55 60
Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val
65 70 75 80
Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
85 90 95
Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
100 105 110
Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
115 120 125
Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
130 135 140
Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
145 150 155 160
Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
165 170 175
Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
180 185 190
Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
195 200 205
Ser Phe Gly Leu Leu Lys Leu
210 215
<210> 19
<211> 154
<212> PRT
<213> Artificial Sequence
<220>
<223> Truncated CD40 ligand (CD40L108-261, also referred to as
18sCD40L)
<400> 19
Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala
1 5 10 15
Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
20 25 30
Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu
35 40 45
Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
50 55 60
Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala
65 70 75 80
Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
85 90 95
Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
100 105 110
Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
115 120 125
Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
130 135 140
Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
145 150
<210> 20
<211> 246
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-CD40 scFv (VH-L-VL)
<400> 20
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Gly Met His Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
130 135 140
Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser
145 150 155 160
Ser Asn Ile Gly Ala Gly Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro
165 170 175
Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser
180 185 190
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser
195 200 205
Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
210 215 220
Ala Ala Trp Asp Lys Ser Ile Ser Gly Leu Val Phe Gly Gly Gly Thr
225 230 235 240
Lys Leu Thr Val Leu Gly
245
<210> 21
<211> 246
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-CD40 scFv (VL-L-VH)
<400> 21
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
65 70 75 80
Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Lys Ser
85 90 95
Ile Ser Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
130 135 140
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Gly
145 150 155 160
Met His Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ser
165 170 175
Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val Arg
180 185 190
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr Leu
195 200 205
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly Thr
225 230 235 240
Leu Val Thr Val Ser Ser
245
<210> 22
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> VH of the anti-CD40 scFv
<400> 22
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Gly Met His Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Ala Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 23
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> VL of the anti-CD40 scFv
<400> 23
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
65 70 75 80
Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Lys Ser
85 90 95
Ile Ser Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
<210> 24
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> VH CDR1
<400> 24
Gly Phe Thr Phe Ser Thr Tyr Gly Met His
1 5 10
<210> 25
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> VH CDR2
<400> 25
Gly Lys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Gly Gly Ser Ser Tyr
1 5 10 15
Ile Phe Tyr Ala Asp Ser Val Arg Gly Arg
20 25
<210> 26
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> VH CDR3
<400> 26
Cys Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu
1 5 10
<210> 27
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> VL CDR1
<400> 27
Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asn Val Tyr
1 5 10 15
<210> 28
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> VL CDR2
<400> 28
Gly Asn Ile Asn Arg Pro Ser
1 5
<210> 29
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> VL CDR3
<400> 29
Cys Ala Ala Trp Asp Lys Ser Ile Ser Gly Leu Val
1 5 10
<210> 30
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> ER retention sequence
<400> 30
Lys Asp Glu Leu
1
<210> 31
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> ER retention sequence
<400> 31
Lys Lys Asp Leu Arg Asp Glu Leu Lys Asp Glu Leu
1 5 10
<210> 32
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> ER retention sequence
<400> 32
Lys Lys Asp Glu Leu Arg Asp Glu Leu Lys Asp Glu Leu
1 5 10
<210> 33
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> ER retention sequence
<400> 33
Lys Lys Asp Glu Leu Arg Val Glu Leu Lys Asp Glu Leu
1 5 10
<210> 34
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> ER retention sequence
<400> 34
Lys Asp Glu Leu Lys Asp Glu Leu Lys Asp Glu Leu
1 5 10
<210> 35
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> CD28 consensus sequence
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa is I or L
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa is V, F or A
<220>
<221> MISC_FEATURE
<222> (11)..(11)
<223> Xaa is M or L
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> Xaa is L or I
<400> 35
Thr Asp Ile Tyr Phe Cys Lys Xaa Glu Xaa Xaa Tyr Pro Pro Pro Tyr
1 5 10 15
Xaa Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
20 25
<210> 36
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> CD28-activating peptide (minimal)
<400> 36
Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr
1 5 10 15
Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His
20 25
<210> 37
<211> 53
<212> PRT
<213> Artificial Sequence
<220>
<223> CD28-activating peptide
<400> 37
Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr Leu Gln
1 5 10 15
Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val
20 25 30
Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile
35 40 45
Ile His Val Lys Gly
50
<210> 38
<211> 98
<212> PRT
<213> Human papillomavirus type 16
<400> 38
Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln
1 5 10 15
Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser
20 25 30
Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp
35 40 45
Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr
50 55 60
Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu
65 70 75 80
Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln
85 90 95
Lys Pro
<210> 39
<211> 104
<212> PRT
<213> Human papillomavirus type 18
<400> 39
His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro
1 5 10 15
Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser Asp
20 25 30
Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His Leu
35 40 45
Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys
50 55 60
Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu Ser Ser Ala Asp
65 70 75 80
Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val
85 90 95
Cys Pro Trp Cys Ala Ser Gln Gln
100
<210> 40
<211> 154
<212> PRT
<213> Hepatitis B virus
<400> 40
Met Ala Ala Arg Met Cys Cys Gln Leu Asp Pro Ala Arg Asp Val Leu
1 5 10 15
Cys Leu Arg Pro Val Gly Ala Glu Ser Arg Gly Arg Pro Leu Pro Gly
20 25 30
Pro Leu Gly Ala Leu Pro Pro Ser Ser Ala Ser Ala Val Pro Ala Asp
35 40 45
His Gly Ser His Leu Ser Leu Arg Gly Leu Pro Val Cys Ser Phe Ser
50 55 60
Ser Ala Gly Pro Cys Ala Leu Arg Phe Thr Ser Ala Arg Arg Met Glu
65 70 75 80
Thr Thr Val Asn Ala Pro Trp Ser Leu Pro Thr Val Leu His Lys Arg
85 90 95
Thr Ile Gly Leu Ser Gly Arg Ser Met Thr Trp Ile Glu Glu Tyr Ile
100 105 110
Lys Asp Cys Val Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu Ile Arg
115 120 125
Leu Lys Val Phe Val Leu Gly Gly Cys Arg His Lys Leu Val Cys Ser
130 135 140
Pro Ala Pro Cys Asn Phe Phe Thr Ser Ala
145 150
<210> 41
<211> 108
<212> PRT
<213> Hepatitis B virus
<400> 41
Met Gly Gly Trp Ser Ser Lys Pro Arg Gln Gly Met Gly Thr Asn Leu
1 5 10 15
Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro
20 25 30
Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Asn
35 40 45
Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly Ala Gly Ala Phe Gly
50 55 60
Pro Gly Phe Thr Pro Pro His Gly Gly Leu Leu Gly Trp Ser Pro Gln
65 70 75 80
Ala Gln Gly Ile Leu Thr Thr Val Pro Ala Ala Pro Pro Pro Ala Ser
85 90 95
Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile
100 105
<210> 42
<211> 190
<212> PRT
<213> Hepatitis C virus
<400> 42
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
35 40 45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
50 55 60
Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly
65 70 75 80
Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp
85 90 95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Asn Trp Gly Pro Thr Asp Pro
100 105 110
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys
115 120 125
Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu
130 135 140
Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp
145 150 155 160
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile
165 170 175
Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Thr Pro Ala Ser
180 185 190
<210> 43
<211> 1273
<212> PRT
<213> Artificial Sequence
<220>
<223> Antigen SARS-CoV-2 spike protein
<400> 43
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1205 1210 1215
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met
1220 1225 1230
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro
1250 1255 1260
Val Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270
<210> 44
<211> 187
<212> PRT
<213> Homo sapiens
<400> 44
Met Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Thr Val Gly Ala Gln
1 5 10 15
Ile Pro Glu Lys Ile Gln Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe
20 25 30
Ser Lys Glu Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe Tyr
35 40 45
Val Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe Lys
50 55 60
Ala Thr Leu Pro Pro Phe Met Cys Asn Lys Arg Ala Glu Asp Phe Gln
65 70 75 80
Gly Asn Asp Leu Asp Asn Asp Pro Asn Arg Gly Asn Gln Val Glu Arg
85 90 95
Pro Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Ser Pro Lys Ile Met
100 105 110
Pro Lys Lys Pro Ala Glu Glu Gly Asn Asp Ser Glu Glu Val Pro Glu
115 120 125
Ala Ser Gly Pro Gln Asn Asp Gly Lys Glu Leu Cys Pro Pro Gly Lys
130 135 140
Pro Thr Thr Ser Glu Lys Ile His Glu Arg Ser Gly Pro Lys Arg Gly
145 150 155 160
Glu His Ala Trp Thr His Arg Leu Arg Glu Arg Lys Gln Leu Val Ile
165 170 175
Tyr Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp
180 185
<210> 45
<211> 314
<212> PRT
<213> Homo sapiens
<400> 45
Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu
1 5 10 15
Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala
20 25 30
Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val
35 40 45
Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser
50 55 60
Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp
65 70 75 80
Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser
85 90 95
Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys
100 105 110
Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu
115 120 125
Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln
130 135 140
Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu
145 150 155 160
Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr
165 170 175
Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp
180 185 190
Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile
195 200 205
Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu
210 215 220
Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly
225 230 235 240
Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu
245 250 255
Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu
260 265 270
Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His
275 280 285
His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu
290 295 300
His Glu Trp Val Leu Arg Glu Gly Glu Glu
305 310
<210> 46
<211> 180
<212> PRT
<213> Homo sapiens
<400> 46
Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp
1 5 10 15
Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly
20 25 30
Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala
35 40 45
Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro
50 55 60
His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala
65 70 75 80
Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe
85 90 95
Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp
100 105 110
Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val
115 120 125
Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln
130 135 140
Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met
145 150 155 160
Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser
165 170 175
Gly Gln Arg Arg
180
<210> 47
<211> 296
<212> PRT
<213> Homo sapiens
<400> 47
Phe Ser Gly Ala Leu Asp Val Leu Gln Met Lys Glu Glu Asp Val Leu
1 5 10 15
Lys Phe Leu Ala Ala Gly Thr His Leu Gly Gly Thr Asn Leu Asp Phe
20 25 30
Gln Met Glu Gln Tyr Ile Tyr Lys Arg Lys Ser Asp Gly Ile Tyr Ile
35 40 45
Ile Asn Leu Lys Arg Thr Trp Glu Lys Leu Leu Leu Ala Ala Arg Ala
50 55 60
Ile Val Ala Ile Glu Asn Pro Ala Asp Val Ser Val Ile Ser Ser Arg
65 70 75 80
Asn Thr Gly Gln Arg Ala Val Leu Lys Phe Ala Ala Ala Thr Gly Ala
85 90 95
Thr Pro Ile Ala Gly Arg Phe Thr Pro Gly Thr Phe Thr Asn Gln Ile
100 105 110
Gln Ala Ala Phe Arg Glu Pro Arg Leu Leu Val Val Thr Asp Pro Arg
115 120 125
Ala Asp His Gln Pro Leu Thr Glu Ala Ser Tyr Val Asn Leu Pro Thr
130 135 140
Ile Ala Leu Cys Asn Thr Asp Ser Pro Leu Arg Tyr Val Asp Ile Ala
145 150 155 160
Ile Pro Cys Asn Asn Lys Gly Ala Ala His Ser Val Gly Leu Met Trp
165 170 175
Trp Met Leu Ala Arg Glu Val Leu Arg Met Arg Gly Thr Ile Ser Arg
180 185 190
Glu His Pro Trp Glu Val Met Pro Asp Leu Tyr Phe Tyr Arg Asp Pro
195 200 205
Glu Glu Ile Glu Lys Glu Glu Gln Ala Ala Ala Glu Lys Ala Val Thr
210 215 220
Lys Glu Glu Phe Gln Gly Glu Trp Thr Ala Pro Ala Pro Glu Phe Thr
225 230 235 240
Ala Thr Gln Pro Glu Val Ala Asp Trp Ser Glu Gly Val Gln Val Pro
245 250 255
Ser Val Pro Ile Gln Gln Phe Pro Thr Glu Asp Trp Ser Ala Gln Pro
260 265 270
Ala Thr Glu Asp Trp Ser Ala Ala Pro Thr Ala Gln Ala Thr Glu Trp
275 280 285
Val Gly Ala Thr Thr Asp Trp Ser
290 295
<210> 48
<211> 279
<212> PRT
<213> Homo sapiens
<400> 48
Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro Ser
1 5 10 15
Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala Gln
20 25 30
Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr Gly
35 40 45
Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro
50 55 60
Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly Ala
65 70 75 80
Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe Ser
85 90 95
Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe Gly
100 105 110
Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro
115 120 125
Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg
130 135 140
Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly
145 150 155 160
His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys
165 170 175
His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr
180 185 190
Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys
195 200 205
Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn
210 215 220
Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln Met
225 230 235 240
Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser Ser
245 250 255
Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu Ser
260 265 270
Asp Asn His Thr Thr Pro Ile
275
<210> 49
<211> 406
<212> PRT
<213> Homo sapiens
<400> 49
Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu Leu
1 5 10 15
Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu Val Leu Ala
20 25 30
Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile Arg
35 40 45
Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys Leu Asn Leu Thr Leu
50 55 60
Glu Gly Val Phe Ala Gly Val Ala Glu Ile Thr Pro Ala Glu Gly Lys
65 70 75 80
Leu Met Gln Ser His Pro Leu Tyr Leu Cys Asn Ala Ser Asp Asp Asp
85 90 95
Asn Leu Glu Pro Gly Phe Ile Ser Ile Val Lys Leu Glu Ser Pro Arg
100 105 110
Arg Ala Pro Ala His Pro Leu Ile Cys Gly Pro Pro Gly Leu Asp Lys
115 120 125
Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser Asn Ser Gln Pro
130 135 140
Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu Glu Pro His Pro Pro
145 150 155 160
Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp Thr Ala Glu Gly Arg Pro
165 170 175
Cys Pro Tyr Pro His Cys Gln Val Leu Ser Ala Gln Pro Gly Ser Glu
180 185 190
Glu Glu Leu Glu Glu Leu Cys Glu Gln Ala Val Ser Gly Gly His Gln
195 200 205
Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu Leu Met Ala Thr Leu Gln
210 215 220
Ala Gly Phe Gly Arg Thr Gly Leu Val Leu Ala Ala Ala Val Glu Ser
225 230 235 240
Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile Arg Val Ile Pro Leu Lys
245 250 255
Met Asp Pro Thr Gly Lys Leu Asn Leu Thr Leu Glu Gly Val Phe Ala
260 265 270
Gly Val Ala Glu Ile Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His
275 280 285
Pro Leu Tyr Leu Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly
290 295 300
Phe Ile Ser Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro Ala His
305 310 315 320
Pro Leu Ile Cys Gly Pro Pro Gly Leu Asp Lys Arg Leu Leu Pro Glu
325 330 335
Thr Pro Gly Pro Cys Tyr Ser Asn Ser Gln Pro Val Trp Leu Cys Leu
340 345 350
Thr Pro Arg Gln Pro Leu Glu Pro His Pro Pro Gly Glu Gly Pro Ser
355 360 365
Glu Trp Ser Ser Asp Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His
370 375 380
Cys Gln Val Leu Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu
385 390 395 400
Leu Cys Glu Gln Ala Val
405
<210> 50
<211> 284
<212> PRT
<213> Homo sapiens
<400> 50
Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu
1 5 10 15
Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly Tyr Ser
20 25 30
Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile Gly Tyr
35 40 45
Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg
50 55 60
Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile Ile Gln
65 70 75 80
Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp Leu Val
85 90 95
Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu Pro Lys
100 105 110
Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys Asp Ala
115 120 125
Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr Leu Trp
130 135 140
Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser
145 150 155 160
Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn Asp Thr
165 170 175
Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg Arg Ser
180 185 190
Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Thr Pro Ile Ile
195 200 205
Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn Leu Asn Leu Ser
210 215 220
Cys His Ser Ala Ser Asn Pro Ser Pro Gln Tyr Ser Trp Phe Val Asn
225 230 235 240
Gly Thr Phe Gln Gln His Thr Gln Val Leu Leu Ile Ala Lys Ile Gln
245 250 255
Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr
260 265 270
Gly Arg Asn Asn Ser Ile Val Lys Ser Ile Thr Val
275 280
<210> 51
<211> 528
<212> PRT
<213> Artificial Sequence
<220>
<223> Fusion protein CD40L47-261-TPE-E7
<400> 51
Met His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu
1 5 10 15
Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg
20 25 30
Ser Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly
35 40 45
Phe Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn
50 55 60
Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His
65 70 75 80
Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
85 90 95
Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
100 105 110
Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
115 120 125
Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
130 135 140
Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
145 150 155 160
Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
165 170 175
Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
180 185 190
Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
195 200 205
Thr Ser Phe Gly Leu Leu Lys Leu Glu Phe Gly Ser Glu Leu Glu Ala
210 215 220
Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys
225 230 235 240
Arg Ala Arg Tyr Lys Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr
245 250 255
Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp
260 265 270
Thr Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val
275 280 285
Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu His Glu Tyr
290 295 300
Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln
305 310 315 320
Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly
325 330 335
Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys
340 345 350
Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp
355 360 365
Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys
370 375 380
Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala Lys Glu Ala
385 390 395 400
Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys
405 410 415
Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu
420 425 430
Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser
435 440 445
Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His
450 455 460
Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met
465 470 475 480
Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu Ser Ser Ala
485 490 495
Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe
500 505 510
Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Lys Asp Glu Leu
515 520 525
<210> 52
<211> 467
<212> PRT
<213> Artificial Sequence
<220>
<223> Fusion protein 18sCD40L-TPE-E7
<400> 52
Met Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile
1 5 10 15
Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu
20 25 30
Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr
35 40 45
Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr
50 55 60
Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln
65 70 75 80
Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu
85 90 95
Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys
100 105 110
Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly
115 120 125
Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly
130 135 140
Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu Phe Gly Ser Glu
145 150 155 160
Leu Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala
165 170 175
Ala Ala Lys Arg Ala Arg Tyr Lys Arg Gly Trp Glu Gln Leu Glu Gln
180 185 190
Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg
195 200 205
Leu Ser Trp Thr Met Gly Ser Ser His His His His His His Ser Ser
210 215 220
Gly Leu Val Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu
225 230 235 240
His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys
245 250 255
Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly
260 265 270
Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr
275 280 285
Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr
290 295 300
His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly
305 310 315 320
Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala
325 330 335
Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala
340 345 350
Ala Ala Lys Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu
355 360 365
His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu
370 375 380
Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn
385 390 395 400
His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met
405 410 415
Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu
420 425 430
Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr
435 440 445
Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Lys
450 455 460
Asp Glu Leu
465
<210> 53
<211> 535
<212> PRT
<213> Artificial Sequence
<220>
<223> Fusion protein E7-TStx-CD40L47-261
<400> 53
Met Lys Leu Thr Met Gly Ser Ser His His His His His His Ser Ser
1 5 10 15
Gly Leu Val Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu
20 25 30
His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys
35 40 45
Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly
50 55 60
Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr
65 70 75 80
Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr
85 90 95
His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly
100 105 110
Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala
115 120 125
Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala
130 135 140
Ala Ala Lys Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu
145 150 155 160
His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu
165 170 175
Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn
180 185 190
His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met
195 200 205
Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu
210 215 220
Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr
225 230 235 240
Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Pro
245 250 255
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe Gly
260 265 270
Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys His
275 280 285
His His Ala Ser Arg Val Ala Arg Glu Phe Gly Ser Glu Leu Glu Ala
290 295 300
Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys
305 310 315 320
His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp
325 330 335
Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser
340 345 350
Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe
355 360 365
Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser
370 375 380
Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val
385 390 395 400
Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
405 410 415
Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
420 425 430
Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
435 440 445
Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
450 455 460
Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
465 470 475 480
Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
485 490 495
Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
500 505 510
Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
515 520 525
Ser Phe Gly Leu Leu Lys Leu
530 535
<210> 54
<211> 474
<212> PRT
<213> Artificial Sequence
<220>
<223> Fusion protein E7-TStx-18sCD40L
<400> 54
Met Lys Leu Thr Met Gly Ser Ser His His His His His His Ser Ser
1 5 10 15
Gly Leu Val Pro Arg Gly Ser His Met His Gly Asp Thr Pro Thr Leu
20 25 30
His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys
35 40 45
Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly
50 55 60
Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr
65 70 75 80
Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr
85 90 95
His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly
100 105 110
Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Ala Glu Ala Ala Ala Ala
115 120 125
Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala
130 135 140
Ala Ala Lys Ala His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu
145 150 155 160
His Leu Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu
165 170 175
Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn
180 185 190
His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met
195 200 205
Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val Glu
210 215 220
Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr
225 230 235 240
Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Thr Arg Ala Pro
245 250 255
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile Ser Phe Gly
260 265 270
Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys His
275 280 285
His His Ala Ser Arg Val Ala Arg Glu Phe Gly Ser Glu Leu Glu Ala
290 295 300
Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys
305 310 315 320
Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala
325 330 335
Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
340 345 350
Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu
355 360 365
Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
370 375 380
Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala
385 390 395 400
Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
405 410 415
Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
420 425 430
Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
435 440 445
Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
450 455 460
Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
465 470
<210> 55
<211> 541
<212> PRT
<213> Artificial Sequence
<220>
<223> Fusion protein HBx-preS1-TStx-18sCD40L
<400> 55
Met Lys Leu Thr Met Gly Ser Ser His His His His His His Ser Ser
1 5 10 15
Gly Leu Val Pro Arg Gly Ser His Met Ala Ala Arg Met Cys Cys Gln
20 25 30
Leu Asp Pro Ala Arg Asp Val Leu Cys Leu Arg Pro Val Gly Ala Glu
35 40 45
Ser Arg Gly Arg Pro Leu Pro Gly Pro Leu Gly Ala Leu Pro Pro Ser
50 55 60
Ser Ala Ser Ala Val Pro Ala Asp His Gly Ser His Leu Ser Leu Arg
65 70 75 80
Gly Leu Pro Val Cys Ser Phe Ser Ser Ala Gly Pro Cys Ala Leu Arg
85 90 95
Phe Thr Ser Ala Arg Arg Met Glu Thr Thr Val Asn Ala Pro Trp Ser
100 105 110
Leu Pro Thr Val Leu His Lys Arg Thr Ile Gly Leu Ser Gly Arg Ser
115 120 125
Met Thr Trp Ile Glu Glu Tyr Ile Lys Asp Cys Val Phe Lys Asp Trp
130 135 140
Glu Glu Leu Gly Glu Glu Ile Arg Leu Lys Val Phe Val Leu Gly Gly
145 150 155 160
Cys Arg His Lys Leu Val Cys Ser Pro Ala Pro Cys Asn Phe Phe Thr
165 170 175
Ser Ala Arg Gly Arg Ala Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala
180 185 190
Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala Glu
195 200 205
Phe Met Gly Gly Trp Ser Ser Lys Pro Arg Gln Gly Met Gly Thr Asn
210 215 220
Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp
225 230 235 240
Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro
245 250 255
Asn Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly Ala Gly Ala Phe
260 265 270
Gly Pro Gly Phe Thr Pro Pro His Gly Gly Leu Leu Gly Trp Ser Pro
275 280 285
Gln Ala Gln Gly Ile Leu Thr Thr Val Pro Ala Ala Pro Pro Pro Ala
290 295 300
Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Glu Leu Thr
305 310 315 320
Arg Ala Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile
325 330 335
Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu
340 345 350
Asn Cys His His His Ala Ser Arg Val Ala Arg Glu Phe Gly Ser Glu
355 360 365
Leu Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Glu Ala Ala
370 375 380
Ala Ala Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro
385 390 395 400
Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser
405 410 415
Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu
420 425 430
Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu
435 440 445
Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser
450 455 460
Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg
465 470 475 480
Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys
485 490 495
Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln
500 505 510
Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser
515 520 525
His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
530 535 540
<210> 56
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Rigid linker 1 with n=1
<400> 56
Glu Ala Ala Ala Ala Lys
1 5
Claims (20)
- 융합 단백질로서,
(a) CD40-결합 도메인;
(b) 항원; 및
(c) 상기 CD40-결합 도메인과 상기 항원 사이에 위치한 전위 도메인
을 포함하되, 퓨린 및/또는 카텝신 L 절단 부위가 상기 융합 단백질의 상기 CD40-결합 도메인과 상기 전위 도메인 사이에 존재하는, 융합 단백질. - 제1항에 있어서, 상기 전위 도메인은 슈도모나스(Pseudomonas) 외독소 A(PE) 전위 펩타이드이고, 상기 CD40-결합 도메인은 상기 융합 단백질의 N-말단에 위치하는, 융합 단백질.
- 제1항에 있어서, 상기 전위 도메인은 26 내지 112개 길이의 아미노산 잔기로 이루어진 PE 전위 펩타이드이고, 상기 PE 전위 펩타이드는 서열번호 5의 아미노산 서열을 포함하는, 융합 단백질.
- 제1항에 있어서, 상기 전위 도메인은 시가(Shiga) 독소(Stx) 전위 펩타이드이고, 상기 항원은 상기 융합 단백질의 N-말단에 위치하는, 융합 단백질.
- 제1항에 있어서, 상기 전위 도메인은 8 내지 84개 길이의 아미노산 잔기로 이루어진 Stx 전위 펩타이드이고, 상기 Stx 전위 펩타이드는 서열번호 12의 아미노산 서열을 포함하는, 융합 단백질.
- 제1항에 있어서, 상기 퓨린 및/또는 카텝신 L 절단 부위는 퓨린 및/또는 카텝신 L 절단을 통해 상기 융합 단백질로부터 상기 CD40-결합 도메인의 제거를 허용하는, 융합 단백질.
- 제1항에 있어서, 상기 퓨린 및/또는 카텝신 L 절단 부위는 서열번호 1 또는 2의 아미노산 서열을 포함하는, 융합 단백질.
- 제1항에 있어서, 상기 CD40-결합 도메인 및 상기 전위 도메인 사이에 위치한 상기 퓨린 및/또는 카텝신 L 절단 부위를 포함하는 펩타이드 링커를 더 포함하는, 융합 단백질.
- 제1항에 있어서, 상기 CD40-결합 도메인은 CD40 리간드(CD40L) 또는 이의 기능적 단편인, 융합 단백질.
- 제1항에 있어서, 상기 CD40-결합 도메인은 서열번호 19의 아미노산 서열을 포함하는 CD40 리간드(CD40L) 또는 이의 기능적 단편이고, 상기 CD40L 또는 이의 기능적 단편은 154 내지 261개 길이의 아미노산 잔기를 갖는, 융합 단백질.
- 제1항에 있어서, 상기 CD40-결합 도메인은 CD40-특이적 항체 또는 이의 결합 단편 또는 단일 사슬 가변 단편(scFv)이고,
상기 CD40-특이적 항체 또는 scFv는 VH 및 VL을 포함하고,
(a) 상기 VH는 서열번호 22의 아미노산 서열을 포함하고,
(b) 상기 VL은 서열번호 23의 아미노산 서열을 포함하는, 융합 단백질. - 제1항에 있어서, 상기 CD40-결합 도메인은 CD40-특이적 항체 또는 이의 결합 단편이고, 상기 CD40-특이적 항체는 VH 및 VL을 포함하고, 상기 VH는 VH CDR1, VH CDR2 및 VH CDR3을 포함하고, 상기 VL은 VL CDR1, VL CDR2 및 VL CDR3을 포함하고,
(i) 상기 VH CDR1, VH CDR2 및 VH CDR3은 각각 서열번호 24, 25 및 26의 아미노산 서열을 포함하고,
(ii) 상기 VL CDR1, VL CDR2 및 VL CDR3은 각각 서열번호 27, 28 및 29의 아미노산 서열을 포함하는, 융합 단백질. - 제1항에 있어서, 상기 항원은 종양 항원이고, 상기 종양은 유방암, 결장암, 직장암, 방광암, 자궁내막암, 신장암, 위암, 교모세포종, 간세포 암종, 담관암, 소세포 폐암, 비-소세포 폐암(NSCLC), 흑색종, 난소암, 자궁경부암, 췌장암, 전립선암, 급성 골수성 백혈병(AML), 만성 골수성 백혈병(CML), 비-호지킨 림프종 및 갑상선암으로 이루어진 군에서 선택된 것인, 융합 단백질.
- 제1항에 있어서, 상기 항원은 인간 파필로마바이러스(Human Papillomavirus: HPV), 인간 면역결핍 바이러스-1(Human Immunodeficiency Virus-1: HIV-1), 인플루엔자 바이러스(Influenza Virus), 뎅기 바이러스(Dengue Virus), A형 간염 바이러스(Hepatitis A Virus: HAV), B형 간염 바이러스(Hepatitis B Virus: HBV), C형 간염 바이러스(Hepatitis C Virus: HCV), D형 간염 바이러스(Hepatitis D Virus: HDV), E형 간염 바이러스(Hepatitis E Virus: HEV), 중증 급성 호흡기 증후군-관련 코로나바이러스(Severe acute respiratory syndrome-associated coronavirus: SARS-CoV), 중증 급성 호흡기 증후군 코로나바이러스 2(Severe acute respiratory syndrome-associated coronavirus: SARS-CoV-2), 중동 호흡기 증후군 코로나바이러스(Middle East respiratory syndrome coronavirus: MERS-CoV), 엡스타인-바 바이러스(Epstein-Barr virus: EBV), 지카 바이러스(Zika Virus), 광견병 바이러스(Rabies Virus), 두창 바이러스(Variola virus), 치쿤군야 바이러스(Chikungunya Virus), 웨스트 나일 바이러스(West Nile virus), 폴리오바이러스(Poliovirus), 홍역 바이러스(Measles virus), 풍진 바이러스(Rubella virus), 한타바이러스(Hantavirus), 일본 뇌염 바이러스(Japanese encephalitis virus), 콕삭키바이러스(Coxsackievirus), 에코바이러스(Echovirus), 엔테로바이러스(Enterovirus), 멈프스 바이러스(Mumps virus), 수두-대상포진 바이러스(Varicella-zoster virus: VZV), 헤르페스 B 바이러스(Cercopithecine herpesvirus-1: CHV-1), 황열 바이러스(Yellow fever virus: YFV), 리프트 발리열 바이러스(Rift Valley Fever Virus), 라싸 바이러스(Lassa virus), 마르부르그 바이러스(Marburg virus), 에볼라바이러스(Ebolavirus), 노로바이러스(Norovirus), 로타바이러스(Rotavirus), 아데노바이러스(Adenovirus), 사포바이러스(Sapovirus), 아스트로바이러스(Astrovirus), 리케차 프로바제키이(Rickettsia prowazekii), 리케차 타이피(Rickettsia typhi), 오리엔티아 쯔쯔가무시(Orientia tsutsugamushi), 보렐리아 부르그도르페리(Borrelia burgdorferi), 페스트균(Yersinia pestis), 삼일열말라리아원충(Plasmodium vivax), 사일열말라리아원충(Plasmodium malariae), 열대열말라리아원충(Plasmodium falciparum), 난형말라리아원충(Plasmodium ovale), 탄저균(Bacillus anthracis), 클로스트리듐 디피실리(Clostridium Difficile), 클로스트리듐 보톨리늄(Clostridium Botulinum), 디프테리아균(Corynebacterium diphtheriae), 살모넬라 엔테리카 혈청형 타이피(Salmonella enterica serovar Typhi), 살모넬라 엔테리카 혈청형 파라타이피 A(Salmonella enterica serovar Paratyphi A), 시가 독소-생산 대장균(E. coli: STEC), 시겔라 다이센테리에(Shigella dysenteriae), 시겔라 플렉스네리(Shigella flexneri), 시겔라 보이디이(Shigella boydii), 시겔라 소네이(Shigella sonnei), 이질아메바(Entamoeba histolytica), 콜레라균(Vibrio cholerae), 결핵균(Mycobacterium tuberculosis), 수막염균(Neisseria meningitidis), 백일해균(Bordetella pertusis), B형 헤모필루스 인플루엔자(Haemophilus influenzae type B: HiB), 파상풍균(Clostridium tetani), 리스테리아 모노사이토제네스(Listeria monocytogenes) 및 폐렴구균(Streptococcus pneumoniae)으로 이루어진 군에서 선택된 병원체의 항원인, 융합 단백질.
- 제1항 내지 제14항 중 어느 한 항의 융합 단백질의 용도로서, 항원-특이적 세포-매개 면역 반응을 유도하여 치료를 필요로 하는 대상체에서 종양 및/또는 병원체에 의해 유발되는 질환을 치료하기 위한 약제의 제조에서의 용도.
- 제2항에 있어서, 상기 항원의 C-말단에 위치한 소포체(ER) 잔류 서열을 더 포함하는, 융합 단백질.
- 제2항 또는 제4항에 있어서, 상기 CD40-결합 도메인과 상기 퓨린 및/또는 카텝신 L 절단 부위 사이에 위치한 CD28-활성화 펩타이드를 더 포함하고, 상기 CD28-활성화 펩타이드는 28 내지 53개 아미노산 잔기의 길이를 갖고 서열번호 35, 36 및 37로 이루어진 군에서 선택된 아미노산 서열을 포함하는, 융합 단백질.
- 제1항의 융합 단백질을 암호화하는 DNA 단편을 포함하는 발현 벡터.
- 제2항에 있어서, 상기 PE 전위 펩타이드는 서열번호 5, 6, 7, 8 및 9로 이루어진 군에서 선택된 아미노산 서열을 포함하는, 융합 단백질.
- 제4항에 있어서, 상기 Stx 전위 펩타이드는 서열번호 12, 13, 14, 15 및 16으로 이루어진 군에서 선택된 아미노산 서열을 포함하는, 융합 단백질.
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