TWI772282B - 樹突細胞組成物 - Google Patents
樹突細胞組成物 Download PDFInfo
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- TWI772282B TWI772282B TW105142799A TW105142799A TWI772282B TW I772282 B TWI772282 B TW I772282B TW 105142799 A TW105142799 A TW 105142799A TW 105142799 A TW105142799 A TW 105142799A TW I772282 B TWI772282 B TW I772282B
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Abstract
本發明關注樹突細胞組成物。該樹突細胞組成物利用與抗原或其片段融合之第II類MHC靶向信號以達到MHC II呈遞該抗原或其片段。
尤其，本發明關於樹突細胞疫苗，其包含表現與抗原或其片段融合之第II類MHC靶向信號的樹突細胞。本發明亦描述用於刺激針對與黑色素瘤相關之抗原的免疫反應之樹突細胞疫苗。
Description
本發明關注樹突細胞組成物。該樹突細胞組成物利用與抗原或其片段融合之第II類MHC靶向信號以達到MHC II呈遞該抗原或其片段。
尤其，本發明關於樹突細胞疫苗，其包含表現與抗原或其片段融合之第II類MHC靶向信號的樹突細胞。本發明亦描述用於刺激針對與黑色素瘤相關之抗原的免疫反應之樹突細胞疫苗。
樹突細胞代表免疫療法中非常有效的藥劑，因為它們可在發展由T細胞介導之免疫的過程中有效地起動初始T細胞並刺激適應性免疫反應。樹突細胞具有活化不僅針對病原體，而且針對惡性細胞之免疫反應的能力。在體內，未成熟或中間階段之樹突細胞守衛外周組織以捕捉和處理抗原。在局部細胞因子和危險信號之影響下，樹突細胞經歷複雜之成熟過程並移行至局部淋巴結，它們在那裡與T細胞形成免疫突觸並在第I類或第II類MHC分子之背景
下呈遞源自收集之抗原的肽類。CD4+ T細胞活化係依賴MHC-II複合物結合，而CD8+交互作用係依賴MHC I結合。
樹突細胞授權模型描述CD4+ T細胞藉由交互作用介導之活化來間接協助CD8+ T細胞，該由交互作用介導之活化能使樹突細胞提供共刺激信號。因此，在針對腫瘤之有效免疫反應方面，CD4+ T細胞協助已隨著越來越多關於它們在特異性抗原CD8+ T細胞之擴增和記憶型生成方面的重要性被知道而成為必不可少的。此外，由於腫瘤抗原大部分為自身抗原，其不像致病抗原提供“危險信號”(例如PAMP：病原相關之分子模式)，因而CD4+ T細胞協助對誘導CD8+ T細胞記憶具有關鍵性。
因此，經改善之促進患者之免疫系統可攻擊其自身之腫瘤細胞並建立持久之免疫力的樹突細胞疫苗是有需要的。誘導T細胞增殖更多、增進由活化誘導之T細胞分泌IFN γ及增強T細胞之腫瘤殺傷力的疫苗是令人期待的。
因此，本發明之目的係提供可允許抗原呈遞在該MHC II複合物上之先進的樹突細胞疫苗。
因此，本發明之第一態樣關注包含表現至少一種融合蛋白之樹突細胞的樹突細胞組成物，該融合蛋白包含- 至少一種抗原或其片段，- 在該抗原N端之前的內質網(ER)-轉位信號序
列，及- 包含在該抗原C端之後的核內體(endosomal)/溶酶體(lysosomal)靶向序列的跨膜和胞質結構域。
此外，額外刺激抗原特異性CD8+ T細胞以改善免疫反應是令人期待的。
因此，於一較佳之實施態樣中，該樹突細胞組成物進一步包含表現至少一種抗原或其片段之樹突細胞，其中該抗原未與促進該MHC II呈遞該抗原或其片段之靶向信號序列融合。
通常，促進該MHC II呈遞之靶向信號序列為選自下列群組之至少一者- 在該抗原N端之前的內質網(ER)-轉位信號序列，及- 包含在該抗原C端之後的核內體/溶酶體靶向序列的跨膜和胞質結構域。
通常，該融合蛋白和該抗原(其未與靶向信號序列融合)係經瞬時或穩定地表現，較佳為穩定地表現。例如，該瞬時表現可藉由引入ivt-RNA進行。
於一些實施態樣中，該核內體/溶酶體靶向序列係源自DC-LAMP。較佳地，該核內體/溶酶體靶向序列為人。一種實施態樣係關於如本文所描述之樹突細胞組成物，其中核內體/溶酶體靶向序列包含序列SEQ ID NO：3或其片段。於一特定之實施態樣中，該核內體/溶酶體靶向序列包含序列SEQ ID NO：14或其片段。
該ER轉位信號序列可源自核內體/溶酶體相關蛋白。較佳地，該ER轉位信號序列係源自LAMP1。更佳地，該ER轉位信號序列包含序列SEQ ID NO：1或其片段。
於一些實施態樣中，該樹突細胞為成熟樹突細胞，其係藉由包含下列步驟之方法產生：(i)提供單核細胞；(ii)將步驟i)之單核細胞經IL-4和GM-CSF培育；(iii)將步驟ii)之單核細胞經IL-4、GM-CSF及熟化混合物之組合培育。
例如，該熟化混合物包含IL-β、TNF-α、IFN-γ、TLR7/8激動劑、PGE2和TLR3激動劑之組合。步驟ii)之培育可持續至少2天。步驟iii)之培育可持續至少12小時，較佳為24小時。較佳地，該TLR 7/8激動劑為R848且該TLR3激動劑為聚(I：C)。
於特定之實施態樣中，該抗原為MELAN-A。
本發明之另一態樣關於包含如本文所描述之樹突細胞組成物的樹突細胞疫苗。較佳地，該樹突細胞為自體細胞。
通常，該樹突細胞組成物和樹突細胞疫苗為醫藥上可接受之流體組成物。
本發明之另一態樣係關於根據本發明之樹突細胞組成物或根據本發明之作為藥物的樹突細胞疫苗。
本發明之一實施態樣係關於用於治療癌症之如本文所
描述之樹突細胞疫苗。
特定之實施態樣係關於用於刺激針對與黑色素瘤相關之抗原的免疫反應之根據本發明的樹突細胞組成物或樹突細胞疫苗。於一特定之實施態樣中，該與黑色素瘤相關之抗原為MELAN-A。
在參考本發明之一些較佳實施態樣詳細描述本發明之前提供下列一般定義。
如下文中說明性描述之本發明可在無本文未具體揭示之任何元件、限制之存在下適當地實行。
本發明將參考特定實施態樣並參考某些附圖描述，但本發明並不限於此，而僅受申請專利範圍之限制。
當在本說明書和申請專利範圍中使用術語“包含”時，其不排除其它元件。為了本發明的目的，術語“由......組成”被認為是術語“包含(comprising of)”的較佳實施態樣。若下文中一個群組被定義為包含至少某些數量之實施態樣，其亦被理解為揭示較佳為僅由這些實施態樣組成之群組。
除非另有明確說明，當提及單數名詞時使用不定冠詞或定冠詞(例如“一(a、an)”或“該(the)”)時係包括該名詞之複數。
如本文所使用之術語“表現”係指基於基因之核酸序列產生多肽之過程。因此，術語“經表現”之蛋白質或多肽包含，但不限於細胞內、跨膜和分泌之蛋白質或多肽。
技術術語係按照其通常意義使用。若特定含義被傳達至某些術語，則術語之定義將描述於接續在使用該術語之背景下的下文中。
本發明之一態樣係指包含表現抗原或其片段之樹突細胞的樹突細胞組成物，其中該抗原或其片段係與促進MHC II呈遞該抗原或其片段之靶向信號序列融合。
更具體地，本發明係指包含表現至少一種融合蛋白之樹突細胞的樹突細胞組成物，該融合蛋白包含- 至少一種抗原或其片段，- 在該抗原N端之前的內質網(ER)-轉位信號序列，及- 包含在該抗原或其片段C端之後的核內體/溶酶體靶向序列的跨膜和胞質結構域。
該片段可為該抗原所專有之抗原序列，即不存在於哺乳動物，尤其人的另一種蛋白質或肽中。該片段可能較該抗原之序列短，諸如較該抗原短至少5%、至少10%、至少30%、至少50%、至少70%、至少90%。該片段之長度可為至少9、至少10、至少11、至少12、至少13、至少
14、至少15或更多個胺基酸。
與投予包含表現未與靶向信號序列融合之常規抗原的樹突細胞之樹突細胞組成物相比較，投予包含表現與靶向信號序列融合之抗原的樹突細胞之樹突細胞組成物可導致抗原特異性CD8+ T細胞增加。因此，相較於僅表現未與靶向信號序列融合之常規抗原的樹突細胞，表現與靶向信號序列融合之抗原的樹突細胞提供優越之誘導能力、在刺激時改善之分泌IFN-γ的能力和高滅殺能力。
於一特定之實施態樣中，該樹突細胞組成物進一步包含表現至少一種抗原或其片段之樹突細胞，其中該抗原未與促進MHC II呈遞該抗原或其片段的靶向信號序列融合。
此意味該樹突細胞組成物包含(i)表現至少一種與促進MHC II呈遞該抗原或其片段的靶向信號序列融合的抗原之樹突細胞，和(ii)表現至少一種未與促進MHC II呈遞該抗原或其片段的靶向信號序列的抗原或其片段之樹突細胞融合。
換句話說，本發明關於樹突細胞組成物，其包含(i)表現至少一種融合蛋白之樹突細胞，該融合蛋白包含- 至少一種抗原或其片段，- 在該抗原或其片段N端之前的內質網(ER)-轉位信號序列，及- 包含在該抗原或其片段C端之後的核內體/溶
酶體靶向序列的跨膜和胞質結構域，和(ii)表現至少一種抗原或其片段之樹突細胞，該抗原或其片段不包含- 在該抗原或其片段N端之前的內質網(ER)-轉位信號序列，及- 包含在該抗原或其片段C端之後的核內體/溶酶體靶向序列的跨膜和胞質結構域。
於一較佳之實施態樣中，(i)和(ii)之抗原為相同抗原。例如，此意味本發明之一特定實施態樣中係關於包含下列群組之樹突細胞組成物(i)表現至少一種融合蛋白之樹突細胞，該融合蛋白包含- MELAN-A抗原或其片段，- 在MELAN-A抗原或其片段N端之前的內質網(ER)-轉位信號序列，及- 包含在該MELAN-A抗原或其片段C端之後的核內體/溶酶體靶向序列的跨膜和胞質結構域跨膜和胞質結構域，及(ii)表現MELAN-A抗原或其片段之樹突細胞，其不包含- 在MELAN-A抗原或其片段N端之前的內質網(ER)-轉位信號序列，及- 包含在該MELAN-A抗原或其片段C端之後的核內體/溶酶體靶向序列的跨膜和胞質結構域。
投予表現與靶向信號序列融合之抗原的樹突細胞及表現未與靶向信號序列融合之抗原的樹突細胞之混合物可導致抗原特異性CD8+ T細胞增加。因此，相較於僅表現未與靶向信號序列融合之抗原的樹突細胞或僅表現與靶向信號序列融合之抗原的樹突細胞，該混合物提供優越之誘導能力。該混合物亦顯示在刺激時具有高的分泌IFN-γ之能力和高滅殺能力。
與促進MHC II呈遞該抗原之靶向信號序列融合或未與促進MHC II呈遞該抗原之靶向信號序列融合之抗原可藉由，例如瞬時表現或穩定表現之方式被引入樹突細胞中。換言之，表現該與促進MHC II呈遞該抗原之靶向信號序列融合或未與促進MHC II呈遞該抗原之靶向信號序列融合之抗原為瞬時表現或穩定表現。於較佳之實施態樣中，該表現為瞬時表現，例如藉由引入編碼至少一種融合蛋白之ivt-RNA。由於經控制品質之ivt-RNA可迅速地產生且不攜帶免疫原性蛋白污染物，ivt-RNA表現可具有優勢。
該ER轉位信號序列可源自核內體/溶酶體相關蛋白。
該用於所揭示之方法中之ER-轉位信號序列可為核內體/溶酶體定位蛋白的分選序列。如本文所使用之核內體/溶酶體定位蛋白係指位於細胞之核內體及/或溶酶體的膜或內腔中的蛋白質。
核內體或溶酶體定位蛋白之實例有糖苷酶
(glycosidase)，諸如α-半乳糖苷酶A/GLA、內-β-N-乙醯葡糖胺酶H/Endo H、α-N-乙醯半乳糖胺酶/NAGA、半乳糖基神經醯胺酶/GALC、α-N-乙醯葡糖胺酶/NAGLU、葡糖基神經醯胺酶/GBA、α-半乳糖苷酶/a-Gal、肝素酶/HPSE、α-L-岩藻糖苷酶、肝素酶I、組織α-L-岩藻糖苷酶/FUCA1、肝素酶II、β-半乳糖苷酶-1/GLB1、肝素酶III、β-葡萄醣醛酸酶/GUSB、己糖胺酶A/HEXA、β(1-3)-半乳糖苷酶、透明質酸裂解酶(hyaluronan Lyase)、β(1-4)-半乳糖苷酶、透明質酸酶1/HYAL1、幾丁質酶3樣1(chitinase 3-like 1)、透明質酸酶4/HYAL4、幾丁質酶3樣2、α-L-艾杜糖醛酸酶(iduronidase)/IDUA、幾丁質酶3樣3/ECF-L、幾丁質二糖酶(chitobiase)/CTBS、幾丁質三糖苷酶(chitotriosidase)/CHIT1、乳糖酶樣蛋白/LCTL、軟骨素B裂解酶/軟骨素酶B、溶酶體α-葡糖苷酶、軟骨素酶ABC、MBD4、軟骨素酶AC、NEU-1/唾液酸酶-1、胞質β-葡糖苷酶/GBA3、O-GlcNAcase/OGA、內-β-N-乙醯葡糖胺酶F1/Endo F1、PNGase F、內-β-N-乙醯葡糖胺酶F3/Endo F3、SPAM1；溶酶體蛋白酶，諸如AMSH/STAMBP、組織蛋白酶(cathepsin)H、組織蛋白酶3、組織蛋白酶K、組織蛋白酶6、組織蛋白酶L、組織蛋白酶7/組織蛋白酶1、組織蛋白酶O、組織蛋白酶A/溶酶體羧肽酶A、組織蛋白酶S、組織蛋白酶B、組織蛋白酶V、組織蛋白酶C/DPPI、組織蛋白酶X/Z/P、組織蛋白酶D、半乳糖基神經醯胺酶/GALC、組織蛋白酶F、
öegumain/天門冬醯胺醯內肽酶；硫酸酯酶，諸如芳基硫酸酯酶A/ARSA、艾杜糖酸2-硫酸酯酶/IDS、芳基硫酸酯酶B/ARSB、N-乙醯半乳糖胺-6-硫酸酯酶/GALNSv、芳基硫酸酯酶G/ARSG、磺醯胺酶/SGSH、葡糖胺(N-乙醯基)-6-硫酸酯酶/GNS、硫酸酯酶-2/SULF2；或其他溶酶體蛋白，諸如BAD-LAMP/LAMP5；透明質酸酶1/HYAL1；CD63；LAMP1/CD107a；CD-M6PR；LAMP2/CD107b；網格蛋白(clathrin)重鏈1/CHC17；Rab27a；網格蛋白重鏈2/CHC22；UNC13D、CD68、CD1b或DC-LAMP。
ER轉位信號序列可源自核內體/溶酶體相關蛋白。該核內體/溶酶體相關蛋白可為LAMP1、LAMP2、DC-LAMP、CD68或CD1b，較佳為LAMP1。較佳地，該ER轉位信號為人序列。ER轉位信號序列可包含下列序列至少一者：SEQ ID NO：1、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10和SEQ ID NO：12。於一些實施態樣中，該ER轉位信號序列可包含至少一種選自由下列序列所組成之群組的序列：SEQ ID NO：2、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13。於一些實施態樣中，該ER轉位信號序列可由選自由下列序列所組成之群組的序列之一組成：SEQ ID NO：2、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13。於具體之實施態樣中，該ER轉位信號序列包含序列SEQ ID NO：1或其片段。於更具體之實施態樣中，該ER
轉位信號序列係由序列SEQ ID NO：2所組成。
該核內體/溶酶體靶向序列可源自LAMP1或DC-LAMP，較佳為DC-LAMP。該核內體/溶酶體靶向序列通常為跨膜和細胞質結構域之一部分。因此，包含核內體/溶酶體靶向序列之跨膜和胞質結構域可源自LAMP1或DC-LAMP，較佳為DC-LAMP。較佳地，該包含核內體/溶酶體靶向序列之跨膜和細胞質結構域為人結構域。通常，該核內體/溶酶體靶向序列包含基序(motif)Y-XX，隨後為疏水性胺基酸(SEQ ID NO：4)。較佳地，該核內體/溶酶體靶向信號序列為YQRI(SEQ ID NO：5)。例如，包含核內體/溶酶體靶向序列之跨膜和細胞質結構域可包含序列SEQ ID NO：14或其片段。
術語疏水性胺基酸為本技藝之技術熟習人士所熟知。疏水性胺基酸之實例為Ala、Ile、Leu、Phe、Val、Pro、Gly、Met、Trp、Tyr、Pro、Cys。
該樹突細胞可包含不同群體之抗原呈遞細胞，各群體表現不同的抗原融合蛋白。
通常，該樹突細胞為成熟樹突細胞，其係藉由包含下列步驟之方法產生：i)提供單核細胞；(ii)將步驟i)之單核細胞經IL-4和GM-CSF培育；(iii)將步驟ii)之單核細胞經IL-4、GM-CSF及熟化混合物之組合培育。
該熟化混合物可包含至少一種選自由下列所組成之群組的組分：IL-β、TNF-α、IFN-G、TLR7/8激動劑、PGE2和TLR3激動劑或彼等之組合。TLR7/8激動劑可為R848
或CL075。該TLR3激動劑可為聚(I：C)。例如，該熟化混合物可包含IFN-γ、TLR7/8激動劑、PGE2之組合，諸如IFN-γ、TLR7/8激動劑、PGE2和TLR3激動劑之組合。於一具體之實施態樣中，該熟化混合物可包含IL-β、TNF-α、IFN-γ、TLR7/8激動劑和PGE2之組合。於另一具體之實施態樣中，該熟化混合物可包含IL-β，TNF-α、IFN-γ、TLR7/8激動劑、PGE2和TLR3激動劑之組合。本發明亦關於如本文所描述之熟化混合物。此外，本發明亦關於在玻管內熟化至少一個未成熟之樹突細胞，其包含以如本文所描述之熟化混合物刺激至少一個未成熟的樹突細胞。
步驟ii)之培育可持續至少2天。步驟iii)之培育可持續至少12小時，較佳為24小時。
通常，該抗原為腫瘤抗原或病毒抗原。該腫瘤抗原可選自由下列所組成之群組：病毒腫瘤抗原、腫瘤特異性抗原、腫瘤相關抗原及攜帶患者特異性突變並表現在患者之腫瘤細胞中的抗原。較佳地，該攜帶患者特異性突變並表現在患者之腫瘤細胞中的抗原未表現在患者之非癌性細胞中。
亦稱為致癌病毒抗原之病毒腫瘤抗原為致癌病毒之抗原，諸如致癌DNA病毒類(例如，諸如B型肝炎病毒、皰疹病毒和乳頭狀瘤病毒)及致癌RNA病毒類。腫瘤特異性抗原係指專門由腫瘤細胞表現之腫瘤相關突變。腫瘤相關抗原之群組包含，例如組織特異性癌/睾丸抗原或組
織分化抗原，諸如MART-1(MELAN-A)、酪胺酸酶或CD20。該腫瘤抗原可為腫瘤相關抗原，可選擇地該腫瘤相關抗原為癌/睾丸抗原(C/T抗原)。該C/T抗原可選自由下列所組成之群組：MAGE族成員，例如MAGE-A1、MAGE-A3、MAGE-A4，但不限於此，包含單點突變之腫瘤抗原、NY-ESO1、腫瘤/睾丸抗原1B、GAGE-1、SSX-4、XAGE-1、BAGE、GAGE、SCP-1、SSX-2、SSX-4、CTZ9、CT10、SAGE和CAGE。較佳地，該C/T抗原可選自由下列所組成之群組：GAGE-1、SSX-4和XAGE-1。較佳地，該腫瘤抗原為組織分化抗原，諸如MART-1、酪胺酸酶或CD20。
更佳地，該腫瘤抗原為MART-1，其亦稱為MELAN-A。
因此，於具體之實施態樣中，本發明關於包含表現至少一種融合蛋白之樹突細胞的樹突細胞組成物，該融合蛋白包含- 至少一種抗原或其片段，- 在該抗原或其片段之N端前的內質網(ER)-轉位信號序列，及- 包含在該抗原或其片段C端之後的核內體/溶酶體靶向序列的跨膜和胞質結構域。
其中該融合蛋白為MELAN-A。
本發明之具體實施態樣關於包含樹突細胞的樹突細胞組成物，該樹突細胞表現至少一種其中該抗原未與促進
MHC II呈遞該抗原或其片段之靶向信號序列融合的抗原或其片段，其中該樹突細胞不表現其中該抗原與促進MHC II呈遞該抗原或其片段之靶向信號序列融合之抗原或其片段，其中該抗原為MELAN-A。
於具體之實施態樣中，該樹突細胞組成物包含(i)表現至少一種與促進MHC II呈遞該抗原或其片段之靶向信號序列融合之抗原的樹突細胞及(ii)表現至少一種未與促進MHC II呈遞該抗原或其片段之靶向信號序列融合之抗原的樹突細胞，其中該抗原為MELAN-A。
因此，本發明亦關於作為藥物之樹突細胞組成物。本發明亦關注作為藥物之樹突細胞疫苗。
本發明之另一態樣關於用於治療癌症之樹突細胞組成物。具體之實施態樣關於用於刺激針對與黑色素瘤相關之抗原的免疫反應之樹突細胞組成物。
本發明之進一步的態樣關於用於治療癌症之樹突細胞疫苗。具體之實施態樣關於用於刺激針對與黑色素瘤相關之抗原的免疫反應之樹突細胞疫苗。
以本發明之組成物進行之治療的活化變化形廓可藉由，例如測量從經投予本發明之樹突細胞組成物的生物體分離出之T細胞的由活化誘導之細胞因子釋出或抗原定向滅殺能力來測定。
為了測量由活化誘導之細胞因子分泌，可將T細胞與負載抗原之樹突細胞共同培養。可使用不同的效應細胞與靶細胞(E：T)比。與呈遞對照抗原之刺激細胞(即，模
擬轉染之APC)一起培育或在無刺激細胞之存在下培育之T細胞可作為陰性對照組。藉由標準酶聯免疫吸附分析(ELISA)評估培養上清液。標記物之實例有，但不限於粒細胞-巨噬細胞集落刺激因子(GM-CSF)、干擾素-γ(IFN-γ)、IL-2和TNF-α分泌。當遭遇抗原時分泌IFN-γ、IL-2和TNF-α係與增強之抗腫瘤作用有關，因此，當測量CD8+細胞毒性T細胞之由抗原誘導的細胞因子分泌時特別有用。此外，IFN-γ和粒細胞-巨噬細胞集落刺激因子(GM-CSF)為定義明確之用於評估抗原特異性CD4+ T輔助細胞-1(Th1)極化之T細胞株的細胞因子。
藉由本發明之樹突細胞群活化的T細胞之細胞毒性活性可藉由，例如鉻釋出分析測量。在該等分析中，靶細胞係以放射性鉻標記並暴露於T細胞。當滅殺時，放射性鉻釋入上清液中並可在共同培養開始後4小時內檢測到。將特異性鉻釋出相對於在無效應細胞存在下培育之靶細胞所評估之自發性釋出標準化。因此，上清液中之高含量的鉻與優良之溶細胞性T細胞活性相關聯。較佳地，進行鉻釋出分析以篩選腫瘤抗原特異性CD8+ T細胞。
源自供體之抗原呈遞細胞可為，例如熟化成樹突細胞之分離的單核細胞。熟化之樹突細胞顯現出最佳之活化能力。
通常，該樹突細胞為自體細胞，即，從根據本發明之教示治療的患者取得細胞，再投予同一患者。例如，從患
者分離出單核細胞，熟化成樹突細胞並依本文之描述處理以表現所需抗原，然後再投予同一患者。
本發明亦關於包含如本文所描述之樹突細胞組成物的樹突細胞疫苗。
較佳地，本發明之活性組分(諸如樹突細胞組成物)劑量與可接受之載體或載體物質混合用於醫藥組成物中，以治療或至少緩解該疾病。該等組成物可(除了該活性組分和載體外)包含填充物質、鹽、緩衝劑、穩定劑、增溶劑和其他本技藝已知之物質。
術語“醫藥上可接受的”係定義不會干擾該活性組分(即，本發明之樹突細胞)之生物活性效力的非毒性物質。該載體之選擇係取決於施用方式。
該醫藥組成物可含有增強該活性組分之活性或增補該治療的額外組分。該等額外之組分及/或因子可為該醫藥組成物之一部分，以取得協同效應或將不利的或不欲有之效果最小化。
用於配製或製備及施用/藥物處理本發明之活性組分的技術發表於“Remington’s Pharmaceutical Sciences”，Mack出版公司，賓州Easton，最新版。適當之施用方式為腸胃道外施用，例如皮內、肌內、皮下、髓內注射及鞘內、直接心室內、靜脈內、結節內、腹膜內或腫瘤內注射。靜脈內注射為患者之較佳治療。
該醫藥組成物可為可注射之組成物，即，醫藥上可接受之流體組成物，其包含至少一種活性成分，例如本發明
之樹突細胞組成物。該活性成分通常溶解或懸浮於生理上可接受之載體中，且該組成物可另外包含少量之一或多種無毒性輔助物質，諸如乳化劑、防腐劑和pH緩衝劑，等。該等可與本發明之樹突細胞一起使用之注射組成物為本技藝之一般技術人士所熟知之常規；合適之調製劑。
適合用於腸胃道外投予(諸如藉由關節內(在關節中)、靜脈內、肌肉內、真皮內(intradermal)、皮內(intracutan)、腹膜內和皮下途徑，較佳為真皮內(intradermal)、結節內、皮內(intracutan)或皮下途徑)之調製劑包括水性等張無菌注射溶液(其可含有抗氧化劑、緩衝劑、抑菌劑和使該調製劑與所欲接受者之血液等張之溶質)及水性和非水性無菌懸浮液(其可包括懸浮劑、增溶劑、增稠劑、穩定劑和防腐劑)。真皮內、皮內、皮下或結節內投予為用於本發明之樹突細胞的較佳投予方法。
在本發明之背景下，投予患者之樹突細胞的劑量應足以在該患者體內隨時間使有益之治療反應生效、或抑制癌細胞生長、或抑制感染。因此，投予患者之細胞量係以足以引發有效之對抗病毒或腫瘤抗原的CTL反應及/或減輕、減少、治癒或至少部分地遏止來自該疾病或感染之症狀及/或併發症。足以達成該目的之量定義為“治療有效劑量”。該劑量將由所產生之樹突細胞的活性和患者的狀況決定。劑量之大小亦將由投予特定患者特定細胞時所伴隨存在之任何不良副作用、性質和程度決定。在測定治療或
預防疾病(諸如癌症)時欲投予之細胞的有效量時，醫生需要評估CTL毒性、疾病之進展及是否誘導出針對任何引入之細胞類型的免疫反應。
投予前，取得血液樣本並保存之以用於分析。一般而言，約將104至106，更佳為106至1010個細胞以單劑量或多劑量之形式經由皮內、結節內、皮下或真皮內注射投予70kg之患者。較佳地，每次疫苗接種係使用至少2*106至107個細胞數。注射可每週投予一次，為期4週，接著每個月投予/注射1次，且較佳地，應在接近淋巴結、直接進入淋巴結或藉由真皮內、皮內或皮下注射給藥。可額外執行加強注射。如所述，較佳為在一年之期間內每個月重複進行細胞再輸注，共10至12次治療。第一次治療後，可由臨床醫師決定在門診進行輸注。若在門診給予再輸注，則在該次治療後監測該參與者至少4小時。
樹突細胞組成物/樹突細胞疫苗可投予至少一次、至少二次、至少3次、至少4次、至少5次、至少6次、至少7次、至少8次、至少9次、至少10次。該疫苗可投予不超過15次、18次、20次、25次、30次。投予之間的時間間隔為至少3天、至少7天、至少14天或至少4週。較佳地，每週投予該疫苗一次，為期4週，接著為每月投予1次，共治療10至12次。
在投予方面，本發明之細胞可藉由該細胞類型之LD-50(或毒性之其他測量值)及不同濃度之該細胞類型在投予時對患者之質量和整體健康的副作用來決定投予率。投
予可經由單一或分割劑量完成。本發明之細胞可藉由已知之常規療法(包括細胞毒性劑、核苷酸類似物和生物反應修飾劑)補充其他治療法之條件。類似地，可選擇地添加生物反應修飾劑以用於藉由樹突細胞進行之治療。
本發明亦關於包含a)和b)之組成物：a)表現載體，其包含- 人ER-轉位信號序列，- 包含核內體/溶酶體靶向序列之人跨膜和胞質結構域，和- 至少一種抗原或其片段；及b)表現載體，其包含至少一種抗原或其片段，但缺乏ER轉位信號序列及包含核內體/溶酶體靶向序列之人跨膜和胞質結構域。
本發明進一步關於包含a)和b)之套組：a)表現載體，其包含- 人ER-轉位信號序列，- 包含核內體/溶酶體靶向序列之人跨膜和胞質結構域，和- 至少一種抗原或其片段；及b)表現載體，其包含至少一種抗原或其片段，但缺乏ER轉位信號序列及包含核內體/溶酶體靶向序列之人跨膜和胞質結構域。
“載體”為有能力攜帶核酸序列進入合適之宿主細胞的任何分子或組成物，該宿主細胞為能夠合成該經編碼之多
肽的處所。通常且較佳地，載體為已使用本技藝已知之重組DNA技術處理以合併期望之核酸序列(例如本發明之核酸)的核酸。該載體可包含DNA或RNA及/或包含脂質體。該載體可為質粒、穿梭載體、噬菌粒、黏粒、表現載體、逆轉錄病毒載體、腺病毒載體或顆粒及/或在基因治療中所使用之載體。載體可包括允許其在宿主細胞中複製之核酸序列，諸如複製起點。載體亦可包括一或多個可選擇之標記基因及本技藝之一般技術人士已知的其他遺傳元件。較佳地，載體為包括根據本發明之核酸的表現載體，該根據本發明之核酸係可操作地連接允許該核酸表現之序列。
第1圖：疫苗接種程序之實驗綜述。
第一天，為分成四個不同組之16隻小鼠各自植入10×106百萬個來自健康HLA-A *02：01陽性供體的人PBMC。在接下去的14天內重建外周T細胞貯庫。在第14天和第21天施行疫苗接種，投予1×106個以常規ivt RNA(2)或CrossTAg-ivt RNA(3)轉染之成熟樹突細胞。此外，一組接受以CrossTAg-或常規ivt RNA轉染之成熟樹突細胞的1：1混合物(4)。該對照組未接種疫苗(1)。第28天，分離出脾細胞並使用HLA-A*02：01-Melan-A特異性多聚體藉由多聚體染色篩選Melan-A特異性CD8+ T細胞。在玻管內以適當之樹突細胞再刺激剩餘之脾細胞並再擴增10天。從對照組分離出之脾細胞僅接受人IL-2以供進一步在玻管內培養。第38天定量Melan-A特異性CD8+ T細胞並進行功能測試，像IFN-γ分泌分析及細胞毒性分析。
第2圖：樹突細胞之熟化和電穿孔。
經由下列步驟產生用於第14天(1.疫苗)和第21天(2.疫苗)進行疫苗接種的成熟樹突細胞：從健康HLA-A*02：01捐血者分離出單核細胞，然後依照3d DC實驗計劃(包含IL-β、TNF-α、IFN-γ、TLR7/8激動劑R848、PGE2和TLR3)將該單核細胞熟化。(A)為了驗證成熟狀態，以結合特異於任一成熟(CD80、CD83、CD86、CD40、CCR7、CD209及HLA-DR)或不成熟(CD14)細胞之不同表面標記的單株抗體將樹突細胞染色並藉由FACS分析。使用同種型對照抗體作為陰性對照組。(B)在投予前，以CrossTAg-Melan-A或常規Melan-A-ivt-RNA轉染成熟之樹突細胞。培育6小時後，藉由Melan-A蛋白之細胞內染色(APC)驗證Melan-A表現。
第3圖：經分離之在玻管內擴增之脾細胞的多聚體染色。
(A)多聚體染色之示意圖：藉由螢光標記(藻紅蛋白，PE)互連並被標記MHC分子(HLA-A*02：01)。為了比較不同DC疫苗之誘導效力，考慮CD8-和多聚體-雙陽性細胞。在(B)體外測量脾細胞群中之Melan-A特異性CD8+ T細胞數及之後(C)在玻管內與對應之樹突細胞和IL-2(對照組僅以IL-2處理)再擴增10天的細胞數。以HLA-A *02：01-Melan-A多聚體和用於CD8之單株抗體將細胞染色。各圖形總結在示例性實驗中來自各NSG接受者之Melan-A特異性CD8+ T細胞的百分比。分
析3個個別實驗，每個實驗共16隻小鼠。
第4圖：Melan-A特異性CD8+ T細胞分泌細胞因子之能力。
(A)在玻管內再刺激脾細胞後10天藉由IFN-γ分泌來調查Melan-A特異性CD8+ T細胞之反應性。將擴增之脾細胞與靶細胞以1：1之比率共同培養24小時。施用之靶細胞為K562細胞(MHC-I和-II陰性)、與Melan-A-肽或不與Melan-A-肽一起培育2小時之K562-A2(HLA-A02：01+)及Mel624.38(HLA-A02：01+，Melan-A+)。以ELISpot分析儀(C.T.L.)檢測IFN-γ斑點之數目。圖形中所顯示的每一個數據點代表來自源自一隻小鼠之活化的T細胞之FN-g點的數目。分析三個個別實驗，每個實驗中共16隻小鼠(顯示一個示例性實驗)。(B)使用單株panMHC-I或panMHC-II抗體分析靶細胞株Mel624.38上之MHC-I和MHC-II表現。使用經小Epstein-Barr病毒(EBV)轉形之類淋巴母細胞株(mLCL)作為陽性對照組，未經染色之細胞作為陰性對照組。
第5圖：經擴增之脾細胞的滅殺能力。
以放射性鉻標記靶細胞，若該細胞裂解，則可在上清液中檢測到該放射性鉻。使用不同之腫瘤細胞株作為靶細胞。將經擴增之脾細胞與靶細胞以不同比例(脾細胞：
APC=80：1；40：1；20：1；10：1)共同培養4小時，再測量釋入該上清液中之放射性鉻。施用各種靶細胞以預想所選擇之脾細胞群K562(MHC-I-/-II-)內之NK細胞活性。使用裝載Melan-A肽或Mel624.38(Melan A+，HLA-A*02：01+)之K562-A2(Melan-A-，HLA-A*02：01+)作為陽性對照組。使用MelA375(Melan A-，HLA-A*02：01+)作為陰性對照組。圖形中所顯示之每個數據點代表從二個測量值計算之平均值與標準偏差。分析三個個別實驗，每個實驗中共有16隻小鼠(顯示一個示例性實驗)。
1.1 在NSG小鼠模型中活化Melan-A特異性CD8 +T細胞
為了研發人化小鼠模型以在體內研究我們的DC疫苗接種，我們使用異種移植人PBMC的NOD-scid/IL2r γ -/-(NSG)小鼠。由於NSG小鼠為免疫缺陷的(缺乏NK、T和B細胞)(SPRANGER,S.et al.2012.NOD/scid IL-2Rg(null)mice：a preclinical model system to evaluate human dendritic cell-based vaccine strategies in vivo.J Transl Med,10,30.)，在免疫細胞群中所產生之缺口允許非常有效的植入人PBMC(SHULTZ,et al.2007.Humanized mice in translational biomedical research.Nat Rev Immunol,7,118-30)。
將16隻NSG接受耆小鼠分成四組並在14天之期間內接受異種移植入人HLA-A*02：01 PBMC。為不同組中之小鼠免疫接種疫苗，該疫苗係由二種新鮮製備之自體成熟樹突細胞的靜脈注射液所組成，二次注射之間相隔一週，該新鮮製備之成熟樹突細胞係經CrossTAg-Melan-A-ivt-RNA、常規Melan-A-ivt-RNA或混合之ivt-RNA的其中任一者電穿孔處理。再過七天及在玻管內再刺激之後，藉由FACS分析脾細胞群以計算Melan-A特異性CD8+ T細胞。亦藉由鉻釋出和ELISpot分析來分析全部4組之Melan-A特異性T細胞的細胞毒性和分泌IFN-γ之能力(第1圖)。
1.2 經轉染之成熟樹突細胞的成熟狀態和Melan-A表現
將源自健康HLA-A*02：01-供體之單核細胞分離出並依Spranger等人(2010.Generation of Th1-polarizing dendritic cells using the TLR7/8 agonist CL075.J Immunol,185,738-47)之描述在3天內，在玻管內將其熟化。為了驗證樹突細胞在玻管內之成熟狀態以供稍後體內投予人化小鼠，我們藉由FACS分析測定通常表現在未成熟之樹突細胞和成熟樹突細胞上的細胞表面分子之表現(Burdek et al.2010.Three-day dendritic cells for vaccine development：antigen uptake,processing and presentation.J Transl Med,8,90)。分析之樹突細胞表現成熟表型(第2A圖)。在成熟3d樹突細胞之電穿孔處理後以
CrossTAg-Melan-A-或常規Melan-A-ivt-RNA轉染成熟樹突細胞(Burdek等人，2010)。以單株抗體藉由Melan-A蛋白細胞內染色來檢查轉染效率(電穿孔後6小時)(第2B圖)。
1.3 定量Melan-A特異性CD8+ T細胞
在分離並分析脾細胞之前為經重建之小鼠接種疫苗二次，二次接種之間相隔一週。為了比較不同DC疫苗之誘導效率，藉由經螢光標記之裝載Melan-A抗原決定部位的HLA-A* 02：01多聚體檢測Melan-A特異性CD8+ T細胞之數量(第3A圖)。
脾細胞之體外分析已透露出相較於常規DC組(其與對照組相比較僅表現出稍多之特定細胞數)，CrossTAg-DC-組或混合組中具有較多之Melan-A特異性CD8+ T細胞數。此外，我們檢測到相較於常規組，CrossTAg組中之CD8+ T細胞的百分比明顯較高(第3B圖)。隨後，將剩餘的脾細胞在玻管內藉由經Melan-A轉染之樹突細胞和hIL-2進一步擴增，而對照組僅以hIL-2處理。重新分析經擴增之細胞證明含CrossTAg-Melan-A之樹突細胞與常規Melan-A樹突細胞之間在Melan-A特異性CD8+ T細胞之平均數方面有更明確的差異。相較於常規組，CrossTAg組中之Melan-A-特異性CD8+ T細胞的數目明顯較多(第3C圖)。因此，以裝載CrossTAg-Melan-A-ivt-RNA之樹突細胞進行疫苗接種可導致優異之誘導能
力，此可由Melan-A特異性CD8+ T細胞增殖更多證明。以裝載CrossTAg-Melan-A-ivt-RNA之樹突細胞和裝載Melan-A-ivt-RNA(未與CrossTAg融合)之樹突細胞的組合進行疫苗接種顯示出更強之誘導能力。
1.4 經誘導之Melan-A特異性CD8+ T細胞的功能分析
我們證實經CrossTAg轉染之樹突細胞具有優越之誘導抗原特異性CD8+ T細胞之效率，這指明包含鄰接該靶抗原之CrossTAg序列的RNA構建體的明確益處。為了建立CD8+ T細胞記憶及成功使腫瘤消退，先前已證明CD4+ T細胞之關鍵角色(Mortenson,et al.2013.Effective anti-neu-initiated antitumor responses require the complex role of CD4+ T cells.Clin Cancer Res,19,1476-86；Rosenberg et al.Cancer immunotherapy：moving beyond current vaccines.Nat Med,10,909-15)。因此，以含有CrossTAg-Melan-A之樹突細胞進行疫苗接種可在NSG小鼠中誘導出增強之免疫反應的解釋可能為CrossTAg序列啟動CD4+ T細胞協助，導致Melan-A呈遞在MHC-I和MHC-II上。
為了進一步檢查經誘導之T細胞的功能性，下一個要澄清的重要議題為該經誘導之Melan-A特異性CD8+ T細胞是否亦能夠分泌IFN-γ(其對適當之免疫反應而言具關鍵性)。因此，分析當刺激時Melan-A特異性CD8+ T細胞分泌IFN-γ的能力。將在玻管內擴增之脾細胞與各
種作為刺激細胞之腫瘤細胞株共同培養，以評估IFN-γ分泌。未受刺激之脾細胞被認為是完全無反應的。NK細胞活性(在脾細胞群內)係使用HLA-陰性腫瘤細胞株K562測定，因為NK細胞係被缺乏任何MHC分子的靶細胞活化。未檢測到NK細胞活性。僅表現Melan-A或裝載特異性肽之刺激細胞導致脾細胞強烈活化，然而Melan-A陰性細胞未刺激脾細胞。平均而言，與常規組或對照組相比較，從CrossTAg-組或混合組分離出之CD8+ T細胞可檢測到更多IFN-γ點(第4A圖)。
為了解決在ELISpot分析中所觀察到之反應性是否可以溯回到CD8+ T細胞，而非CD4+ T細胞這個問題，檢查該刺激細胞上之MHC-II表現。該刺激細胞株為MHC-I陽性，但MHC-II則不(第4B圖)。即使在IFN-γ處理刺激細胞一整夜後也未誘導出MHC-II分子之表現(數據未顯示)。因此，僅MHC-I限制性T細胞可被活化，這表明所觀察到之反應性係源自活化之CD8+ T細胞，因為CD4+ T細胞將需要抗原呈遞在欲被活化之MHC-II上。
CD8+ T細胞具細胞毒性以能夠殺死腫瘤細胞也很重要。因此，藉由鉻釋出分析評估Melan-A-特異性T細胞之細胞毒性能力(第5圖)。細胞毒性結果證明經擴增之脾細胞僅特異溶解Melan-A-肽呈遞細胞，而不會溶解靶抗原陰性之對照細胞株。來自對照組之細胞未顯示出任何滅殺活性。所有組中之脾細胞內的NK細胞活性亦非常低。值得注意的是，來自CrossTAg-DC-組和混合組之細
胞顯示出較來自常規DC-組之脾細胞高出許多的減殺能力。
方法
基因構建體
使用pGEM-EGFP-A120載體作為CrossTAg載體之起始構建體。此原始pGEM載體之polyA120變異體使經轉錄之RNA具有較高之穩定性，並導致改善之蛋白質表現。該質粒在eGFP cDNA之5’端進一步含有獨特之AgeI位點，並在3’端具有獨特之EcoRI位點。聚A尾之後為SpeI位點，其允許用於產生ivt-RNA之質粒內化。
該GEM-CrossTAg-A120質粒係藉由以編碼CrossTAg靶向信號之cDNA替換eGFP來選殖。該CrossTAg序列包含5’與DC-LAMP(登錄編號：NP_055213，aa 376-416)之跨膜和胞質結構域融合的人溶酶體膜相關蛋白-1(LAMP-1，登錄編號：NP_005552，aa 1-28)之ER-轉位信號。為了插入編碼抗原之cDNA，藉由含有NheI、KpnI和PstI限制性位點之18-bp間隔子分開不同的CrossTAg序列，而不破壞LAMP1開放閱讀框(ORF)。使用計算選殖軟體虛擬設計密碼子優化之CrossTAg序列並由(GeneArt，Regensbu,rg，德國)合成。隨後，使用AgeI(5’端)和EcoRI(3’端)限制性位點從質粒DNA切出完整的CrossTAg序列，並連接入經同樣分解之pGEM-A120載體的MCS。為了選殖各種C/T抗原-CrossTAg構建體
(pGEM-GAGE-1-CrossTAg-A120、pGEM-MAGE-A4-CrossTAg-A120、pGEM-NY-ESO-1-CrossTAg-A120、pGEM-SSX-4-CrossTAg-A120、pGEM-XAGE-1-CrossTAg-A120)，藉由PCR(登錄編號：GAGE-1，U19142；MAGE-A4，NM_001011550；NY-ESO1，AJ003149；SSX-4，U90841；XAGE-1，AF251237)，使用正向和反向基因特異性引物從質粒擴增抗原cDNA並經由NheI和PstI/NotI限制性位點連接。將所有抗原序列插入pGEM-CrossTAg-A120之分裂CrossTAg信號，不破壞初始ORF。
為了驗證CD4+ T細胞抗原決定部位，合成互補之寡核苷酸(Metabion，Planegg，德國)並使之退火(annealed)。使用在退火時產生的黏端將這些短抗原序列直接連接入CrossTAg載體。
產生ivt-RNA
SpeI線性化之後，使用mMESSAGE mMACHINE T7套組(ThermoFisher Scientific，美國麻薩諸塞州)，根據製造商之說明，使用pGEM-質粒作為玻管內轉錄(ivt)產生RNA之單一物種的模板。為了品管，藉由瓊脂糖凝膠電泳分析ivt-RNA產物長度。藉由Nanodrop ND-1000分光光度計(ThermoFisher Scientific，麻薩諸塞州)測定濃度和純度。
細胞培養
依Bürdek等人(Journal of Translational Medicine 2010,8：90)之描述產生並轉染源自單核細胞之3d成熟樹突細胞。RNA轉染成熟樹突細胞：藉由電穿孔取得經迷你Epstein-Barr病毒(EBV)轉化之類淋巴母細胞株(mLCL)。
樹突細胞之表型分型
以下列抗體檢測由樹突細胞表現之表面標記：PE-軛合之特異性CCR7抗體(3D12)(eBioscience公司，德國法蘭克福)、Hz450軛合之CD4特異性抗體(RPA-T4)、Hz500軛合之CD8特異性抗體(RPA-T8)、FITC軛合之CD14特異性抗體(M5E2)、PE軛合之CD40特異性抗體(5C3)、PE軛合之CD40L特異性抗體(TRAP1)、PE軛合之CD80特異性抗體(L307.4)、FITC軛合之CD83特異性抗體(HB15e)、FITC軛合之CD86特異性抗體(2331)、APC軛合之CD137特異性抗體(4B4-1)、FITC軛合之DC-SIGN特異性抗體(DCN46)、PE軛合之HLA-DR特異性抗體(G46-6)(均來自BD Biosciences公司，德國海德堡)。清洗後，將細胞在4℃染色30分鐘並加入碘化丙啶(2μg/ml)以排除死細胞。藉由流式細胞術(LSRII，BD)分析所有表面標記之表現。使用FlowJo 8軟體(TreeStar)完成採集後數據分析。依(Frentsch,M.et al.(2005)Direct access
to CD4+ T cells specific for defined antigens according to CD154 expression.Nat Med 11(10)：1118-1124)之描述，使用2μg/ml αCD40抗體(選殖株G28.5，由M.Frentsch，柏林-勃蘭登堡再生療法中心提供)分析T細胞上CD40L表面表現並在T細胞：APC共同培養開始後6小時評估。
以RNA轉染之樹突細胞重新啟動PBL
以編碼Melan-A之CrossTAg-RNA轉染健康供體之3d成熟樹突細胞。電穿孔後，收穫該經轉染之成熟樹突細胞，並將該混合物之混合的成熟樹突細胞與外周血淋巴細胞(PBL)(其在成熟樹突細胞產生過程中之PBMC塑料黏附期間為非黏附性)在1：2之比例內共同培養。將細胞培養在37℃，加濕之大氣中。1天後加入介白素2(IL-2，20U/ml；Chiron Behring公司，德國馬爾堡)及5ng IL-7/ml(Promokine，德國海德堡)，之後每隔一天添加。
細胞因子釋出試驗
為了測量活化誘導之細胞因子分泌，將5*104個T細胞與1*105個裝載ivt-RNA之抗原呈遞細胞DC在37℃，加濕之大氣下共同培養在圓底96孔盤中之200μl T細胞培養基中。使用T細胞與模擬轉染之APC或無刺激細胞存在來作為陰性對照組。共同培養16小時後，收穫上清
液並使用OptEIA Human IFN-γ或GM-CSF Set(均來自BD Biosciences，德國海德堡)，藉由酶聯免疫吸附分析(ELISA)評估。
<110> 梅迪基因免疫治療公司(Medigene Immunotherapies GmbH) 德國亥姆霍茲慕尼黑中心德國研究環境衛生(公司)(Helmholtz Zentrum Muenchen Deutsches)
<120> 樹突細胞組成物
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Claims (23)
- 一種包含表現至少一種融合蛋白之樹突細胞之樹突細胞組成物，該融合蛋白包含- MELAN-A抗原或其抗原性片段，- 在該MELAN-A抗原或其抗原性片段的N端之前的源自LAMP1之內質網(ER)-轉位信號序列，及- 包含在該MELAN-A抗原或其抗原性片段的C端之後的源自DC-LAMP之核內體/溶酶體靶向序列的跨膜和胞質結構域，其中該樹突細胞組成物進一步包含表現至少一種抗原或其抗原性片段之樹突細胞，其中該抗原或其抗原性片段並未與促進MHC II呈遞該抗原或其抗原性片段之靶向信號序列融合，其中促進該MHC II呈遞之該靶向信號序列為選自由下列所組成之群組的至少一者：- 在該抗原的N端之前的源自LAMP1之內質網(ER)-轉位信號序列，及- 包含在該抗原的C端之後的源自DC-LAMP之核內體/溶酶體靶向序列的跨膜和胞質結構域。
- 如申請專利範圍第1項之樹突細胞組成物，其中該抗原係MELAN-A。
- 如申請專利範圍第1項之樹突細胞組成物，其中該融合蛋白和該MELAN-A抗原係經瞬時或穩定地表現。
- 如申請專利範圍第3項之樹突細胞組成物，其中該瞬時表現係藉由引入ivt-RNA進行。
- 如申請專利範圍第1至4項中任一項之樹突細胞組成物，其中該源自DC-LAMP之核內體/溶酶體靶向序列為人序列。
- 如申請專利範圍第1至4項中任一項之樹突細胞組成物，其中該源自DC-LAMP之核內體/溶酶體靶向序列包含序列SEQ ID NO：3。
- 如申請專利範圍第1至4項中任一項之樹突細胞組成物，其中該源自DC-LAMP之核內體/溶酶體靶向序列包含序列SEQ ID NO：14。
- 如申請專利範圍第1至4項中任一項之樹突細胞組成物，其中該源自LAMP1之ER-轉位信號序列包含序列SEQ ID NO：1。
- 如申請專利範圍第1至4項中任一項之樹突細胞組成物，其中該樹突細胞為藉由包含下列步驟之方法產生的成熟樹突細胞：(i)提供單核細胞；(ii)將步驟(i)之單核細胞經IL-4和GM-CSF培育；(iii)將步驟(ii)之單核細胞經IL-4、GM-CSF及成熟混合物之組合培育。
- 如申請專利範圍第9項之樹突細胞組成物，其中該成熟混合物包含IFN-α、TLR7/8激動劑、PGE2及可選擇地TLR 3激動劑之組合。
- 如申請專利範圍第10項之樹突細胞組成物，其中 該成熟混合物包含IL-β、TNF-α、IFN-γ、TLR7/8激動劑、PGE2和TLR3激動劑之組合。
- 如申請專利範圍第9項之樹突細胞組成物，其中步驟(ii)之培育持續至少2天。
- 如申請專利範圍第9項之樹突細胞組成物，其中步驟(iii)之培育持續至少12小時。
- 如申請專利範圍第11項之樹突細胞組成物，其中該TLR7/8激動劑為R848且其中該TLR3激動劑為聚(I：C)。
- 一種樹突細胞疫苗，其包含如申請專利範圍第1至14項中任一項之樹突細胞組成物。
- 如申請專利範圍第15項之樹突細胞疫苗，其中該樹突細胞為自體細胞。
- 如申請專利範圍第16項之樹突細胞疫苗，其中該樹突細胞疫苗為醫藥上可接受之流體組成物。
- 如申請專利範圍第15至17項中任一項之樹突細胞疫苗，其係作為藥物。
- 如申請專利範圍第15至17項中任一項之樹突細胞疫苗，其係用於治療黑色素瘤。
- 一種組成物，其包含a)表現載體，其包含- 源自LAMP1之人ER-轉位信號序列，- 包含源自DC-LAMP之核內體/溶酶體靶向序列之人跨膜和胞質結構域，和 - MELAN-A抗原或其抗原性片段；及b)表現載體，其包含至少一種抗原或其抗原性片段，但缺乏源自LAMP1之該人ER-轉位信號序列，及包含源自DC-LAMP之核內體/溶酶體靶向序列之該人跨膜和胞質結構域。
- 如申請專利範圍第20項之組成物，其中該至少一種抗原係MELAN-A。
- 一種套組，其包含a)表現載體，其包含- 源自LAMP1之人ER-轉位信號序列，- 包含源自DC-LAMP之核內體/溶酶體靶向序列之人跨膜和胞質結構域，和- MELAN-A抗原或其抗原性片段；及b)表現載體，其包含至少一種抗原或其抗原性片段，但缺乏源自LAMP1之該人ER-轉位信號序列，及包含源自DC-LAMP之核內體/溶酶體靶向序列之該人跨膜和胞質結構域。
- 如申請專利範圍第22項之套組，其中該至少一種抗原係MELAN-A。
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| JP2022535045A (ja) * | 2019-06-03 | 2022-08-04 | ザ スクリプス リサーチ インスティテュート | 細胞間近接相互作用の検出および免疫療法用の腫瘍特異的抗原反応性ｔ細胞の単離のための化学酵素的方法 |
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| CN112133372B (zh) * | 2020-08-18 | 2022-06-03 | 北京臻知医学科技有限责任公司 | 抗原特异性tcr数据库的建立方法及抗原特异性tcr的评估方法 |
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