TW201929868A - 含核酸之脂質奈米粒子及其用途 - Google Patents
含核酸之脂質奈米粒子及其用途 Download PDFInfo
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- TW201929868A TW201929868A TW107147275A TW107147275A TW201929868A TW 201929868 A TW201929868 A TW 201929868A TW 107147275 A TW107147275 A TW 107147275A TW 107147275 A TW107147275 A TW 107147275A TW 201929868 A TW201929868 A TW 201929868A
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Abstract
本發明提供一種脂質奈米粒子,其包含以下之(a)~(c): (a)編碼嵌合抗原受體(CAR)或外源性T細胞受體(TCR)之核酸; (b)陽離子性脂質;及 (c)非陽離子性脂質。 又,本發明提供一種向活體內(in vivo)或活體外(ex vivo)T細胞導入該脂質奈米粒子而獲得之CAR或外源性TCR表現免疫細胞、以及使用該免疫細胞之活體內或活體外之癌等疾病之治療手段。
Description
本發明係關於一種含有編碼嵌合抗原受體或T細胞受體之核酸之脂質奈米粒子、使用該脂質奈米粒子使對象之免疫細胞表現嵌合抗原受體或外源性T細胞受體之方法、以及該等之醫藥用途等。
利用基因導入嵌合抗原受體(CAR)或源自癌抗原特異性殺手T細胞之T細胞受體(TCR)之CAR-T細胞或TCR-T細胞的癌免疫療法之相關研究、開發進展急速。當前之CAR-T細胞療法一般為如於美國批准上市之Kymriah(商品名)或Yescarta(商品名),使用慢病毒載體等病毒載體於活體外(ex vivo)對采自患者之T細胞導入CAR基因而製造CAR-T細胞,對患者投予該CAR-T細胞之方法。然而,該方法因細胞培養或病毒載體等之製備所需費用而存在製造成本高昂之課題。若可於活體內(in vivo)選擇性地對T細胞等免疫細胞導入CAR或外源性TCR,則無須於活體外進行製備,從而能夠提供製造成本較低之CAR-或TCR-免疫細胞療法。又,即便是活體外,若可不使用製造成本高之病毒載體而選擇性地對T細胞等免疫細胞導入CAR或外源性TCR,則可免去病毒殘存試驗等所需費用,藉此能夠提供製造成本較低之CAR-或TCR-免疫細胞療法。
目前,業界報告有使用奈米粒子(專利文獻1、非專利文獻1)或奈米載體(專利文獻2)進行之CAR向T細胞之活體外或活體內轉染,上述奈米粒子係使編碼CAR之質粒DNA與陽離子性聚合物凝集,並利用與抗CD3抗體片段複合(conjugate)之非陽離子性聚合物被覆該凝集體而獲得,上述奈米載體係利用經抗CD3抗體進行表面修飾之脂質被覆其小孔內封入有編碼CAR之DNA之中孔二氧化矽而獲得。
有別於該等技術,業界報告有於內部無小孔構造之包含陽離子性脂質與非陽離子性輔助脂質及用以向靶細胞進行傳遞之配體的「脂質奈米粒子(LNP)」內封入目標siRNA(Small interfering RNA,小干擾RNA),而向靶細胞傳遞該siRNA之技術。例如報告有將抗CD4抗體片段作為靶向配體,向T細胞活體外或活體內轉染針對CD45之siRNA(專利文獻3、非專利文獻2)。
然而,迄今為止,業界尚無使用LNP選擇性地對T細胞等免疫細胞導入編碼CAR或外源性TCR之核酸(例如mRNA(Messenger RNA,信使RNA)、DNA)之報告。
[先前技術文獻] [專利文獻]
[先前技術文獻] [專利文獻]
專利文獻1:US 2017/0296676 專利文獻2:US 2016/0145348 專利文獻3:WO 2016/189532 [非專利文獻]
非專利文獻1:Nature Nanotechnology 12, 813-820 (2017) 非專利文獻2:ACS Nano, 2015, 9(7), 6706-6716
[發明所欲解決之課題]
本發明之目的在於提供一種能夠效率良好地於活體內或活體外選擇性地對T細胞等免疫細胞導入CAR或外源性TCR之新穎之轉染技術,以提供製造成本較低之CAR-或TCR-免疫細胞療法。又,本發明之另一目的在於提供一種避免因病毒蛋白引起之抗原性問題而安全性更高之CAR-或TCR-免疫細胞療法。
[解決問題之技術手段]
[解決問題之技術手段]
本發明者等人為了達成上述目的,經過不斷之努力研究,結果成功地使用LNP,於活體內及活體外效率良好地將編碼CAR或外源性TCR之核酸選擇性地導入至T細胞等免疫細胞,從而完成本發明。
即,本發明提供如下所述者。
[1]一種脂質奈米粒子,其包含以下之(a)~(c): (a)編碼嵌合抗原受體或外源性T細胞受體之核酸; (b)陽離子性脂質;及 (c)非陽離子性脂質。 [2]如[1]記載之脂質奈米粒子,其中上述陽離子性脂質為
式(I)所表示之化合物或其鹽:
[1]一種脂質奈米粒子,其包含以下之(a)~(c): (a)編碼嵌合抗原受體或外源性T細胞受體之核酸; (b)陽離子性脂質;及 (c)非陽離子性脂質。 [2]如[1]記載之脂質奈米粒子,其中上述陽離子性脂質為
式(I)所表示之化合物或其鹽:
[化1]
[式中,
L1 為C1-22 伸烷基、C2-22 伸烯基或C3-22 伸二烯基(alkadienylene),
n為0或1之整數,
R1 為
(1)氫原子、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(4)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R2 為-CH2 -O-CO-R5 、-CH2 -CO-O-R5 或-R5 ,
R3 為-CH2 -O-CO-R6 、-CH2 -CO-O-R6 或-R6 ,
R4 為氫原子、-CH2 -O-CO-R7 、-CH2 -CO-O-R7 或-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R8 及R9 分別獨立表示C1-6 烷基]。
[3]如[1]或[2]記載之脂質奈米粒子,其中上述核酸為mRNA或DNA。 [4]如[1]至[3]中任一項記載之脂質奈米粒子,其中上述非陽離子性脂質為磷脂質、膽固醇及/或PEG脂質。 [5]如[1]至[4]中任一項記載之脂質奈米粒子,其中上述脂質奈米粒子於表面具有能夠靶向T細胞之配體。 [6]如[5]記載之脂質奈米粒子,其中上述配體為包含選自由針對CD3之抗體、針對CD4之抗體、針對CD8之抗體及針對CD28之抗體所組成之群中之1種以上之抗體之抗原結合區的配體。 [7]如[5]記載之脂質奈米粒子,其中上述配體為包含針對CD3之抗體及/或針對CD28之抗體之抗原結合區的配體。 [8]如[5]記載之脂質奈米粒子,其中上述配體為包含針對CD3之抗體及針對CD28之抗體之抗原結合區的配體。 [9]一種醫藥,其含有如[1]至[8]中任一項記載之脂質奈米粒子而成。 [10]如[9]記載之醫藥,其係癌之預防或治療藥。 [11]如[9]記載之醫藥,其向活體內免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並誘導其之表現。 [12]如[9]記載之醫藥,其向活體內T細胞中基因導入嵌合抗原受體或外源性T細胞受體並誘導其之表現。 [13]一種向哺乳動物之活體內免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:對該哺乳動物投予如[1]至[8]中任一項記載之脂質奈米粒子。 [14]一種向哺乳動物之活體內T細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:對該哺乳動物投予如[1]至[8]中任一項記載之脂質奈米粒子。 [15]一種針對哺乳動物之癌之預防或治療方法,其特徵在於:對該哺乳動物投予如[1]至[8]中任一項記載之脂質奈米粒子。 [16]如[1]至[8]中任一項記載之脂質奈米粒子,其用於癌之預防、治療。 [17]一種如[1]至[8]中任一項記載之脂質奈米粒子之用途,其用於製造癌之預防、治療劑。 [18]一種嵌合抗原受體或外源性T細胞受體表現誘導用組合物,其含有如[1]至[8]中任一項記載之脂質奈米粒子而成。 [19]一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,其係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [20]一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,其係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [21]一種醫藥,其含有表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞而成,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [22]一種醫藥,其含有表現嵌合抗原受體或外源性T細胞受體之活體外T細胞而成,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [23]如[21]或[22]記載之醫藥,其係癌之預防或治療藥。 [24]如[21]或[22]記載之醫藥,其係細胞凋亡誘導藥。 [25]一種向活體外免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [26]一種使活體外T細胞表現嵌合抗原受體或外源性T細胞受體之方法,其特徵在於:於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [27]一種癌之預防或治療方法,其特徵在於:對哺乳動物投予表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [28]一種癌之預防或治療方法,其特徵在於:對哺乳動物投予表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [29]一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,其用於癌之預防、治療,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [30]一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,其用於癌之預防、治療,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [31]一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞之用途,其用於製造癌之預防、治療劑,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [32]一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞之用途,其用於製造癌之預防、治療劑,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [33]一種含有表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞而成之醫藥之製造方法,其包括如下步驟:於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [34]一種含有表現嵌合抗原受體或外源性T細胞受體之活體外T細胞而成之醫藥之製造方法,其包括如下步驟:於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [發明之效果]
[式中,
L1 為C1-22 伸烷基、C2-22 伸烯基或C3-22 伸二烯基(alkadienylene),
n為0或1之整數,
R1 為
(1)氫原子、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(4)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R2 為-CH2 -O-CO-R5 、-CH2 -CO-O-R5 或-R5 ,
R3 為-CH2 -O-CO-R6 、-CH2 -CO-O-R6 或-R6 ,
R4 為氫原子、-CH2 -O-CO-R7 、-CH2 -CO-O-R7 或-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R8 及R9 分別獨立表示C1-6 烷基]。
[3]如[1]或[2]記載之脂質奈米粒子,其中上述核酸為mRNA或DNA。 [4]如[1]至[3]中任一項記載之脂質奈米粒子,其中上述非陽離子性脂質為磷脂質、膽固醇及/或PEG脂質。 [5]如[1]至[4]中任一項記載之脂質奈米粒子,其中上述脂質奈米粒子於表面具有能夠靶向T細胞之配體。 [6]如[5]記載之脂質奈米粒子,其中上述配體為包含選自由針對CD3之抗體、針對CD4之抗體、針對CD8之抗體及針對CD28之抗體所組成之群中之1種以上之抗體之抗原結合區的配體。 [7]如[5]記載之脂質奈米粒子,其中上述配體為包含針對CD3之抗體及/或針對CD28之抗體之抗原結合區的配體。 [8]如[5]記載之脂質奈米粒子,其中上述配體為包含針對CD3之抗體及針對CD28之抗體之抗原結合區的配體。 [9]一種醫藥,其含有如[1]至[8]中任一項記載之脂質奈米粒子而成。 [10]如[9]記載之醫藥,其係癌之預防或治療藥。 [11]如[9]記載之醫藥,其向活體內免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並誘導其之表現。 [12]如[9]記載之醫藥,其向活體內T細胞中基因導入嵌合抗原受體或外源性T細胞受體並誘導其之表現。 [13]一種向哺乳動物之活體內免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:對該哺乳動物投予如[1]至[8]中任一項記載之脂質奈米粒子。 [14]一種向哺乳動物之活體內T細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:對該哺乳動物投予如[1]至[8]中任一項記載之脂質奈米粒子。 [15]一種針對哺乳動物之癌之預防或治療方法,其特徵在於:對該哺乳動物投予如[1]至[8]中任一項記載之脂質奈米粒子。 [16]如[1]至[8]中任一項記載之脂質奈米粒子,其用於癌之預防、治療。 [17]一種如[1]至[8]中任一項記載之脂質奈米粒子之用途,其用於製造癌之預防、治療劑。 [18]一種嵌合抗原受體或外源性T細胞受體表現誘導用組合物,其含有如[1]至[8]中任一項記載之脂質奈米粒子而成。 [19]一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,其係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [20]一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,其係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [21]一種醫藥,其含有表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞而成,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [22]一種醫藥,其含有表現嵌合抗原受體或外源性T細胞受體之活體外T細胞而成,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [23]如[21]或[22]記載之醫藥,其係癌之預防或治療藥。 [24]如[21]或[22]記載之醫藥,其係細胞凋亡誘導藥。 [25]一種向活體外免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [26]一種使活體外T細胞表現嵌合抗原受體或外源性T細胞受體之方法,其特徵在於:於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [27]一種癌之預防或治療方法,其特徵在於:對哺乳動物投予表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [28]一種癌之預防或治療方法,其特徵在於:對哺乳動物投予表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [29]一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,其用於癌之預防、治療,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [30]一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,其用於癌之預防、治療,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [31]一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞之用途,其用於製造癌之預防、治療劑,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [32]一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞之用途,其用於製造癌之預防、治療劑,該活體外T細胞係於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子而獲得。 [33]一種含有表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞而成之醫藥之製造方法,其包括如下步驟:於包含活體外免疫細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [34]一種含有表現嵌合抗原受體或外源性T細胞受體之活體外T細胞而成之醫藥之製造方法,其包括如下步驟:於包含活體外T細胞之培養液中添加如[1]至[8]中任一項記載之脂質奈米粒子。 [發明之效果]
根據本發明,可提供一種由於不僅於活體外、且於活體內亦能夠效率良好地選擇性地對T細胞等免疫細胞導入CAR或外源性TCR,故而製造成本較低之CAR-或TCR-免疫細胞療法。又,由於不使用病毒載體,故而能夠避免因病毒蛋白引起之抗原性問題。
1. 本發明之脂質奈米粒子 (LNP)
本發明提供一種脂質奈米粒子(以下亦稱為「本發明之脂質奈米粒子」、「本發明之LNP」),其包含以下之(a)~(c): (a)編碼嵌合抗原受體(CAR)或外源性T細胞受體(TCR)之核酸; (b)陽離子性脂質;及 (c)非陽離子性脂質。 於本說明書中,所謂「脂質奈米粒子(LNP)」意指於由上述(b)及(c)構成之分子集合體之內部不具有小孔構造(例如中孔材料)之平均粒徑未達1 μm之粒子。 以下,對本發明之脂質奈米粒子之構成要素(a)~(c)進行說明。
(a)編碼嵌合抗原受體(CAR)或外源性T細胞受體(TCR)之核酸 (a-1)編碼CAR之核酸 CAR係包含與T細胞訊號傳遞區連結之抗體之抗原結合區(例如scFv)的人工構建之雜交蛋白(hybrid protein)。關於CAR之特徵,可列舉:利用單株抗體之抗原結合特性,以非MHC限制之樣式對應於所選擇之標靶而轉換T細胞之特異性及反應性之能力。非MHC限制性抗原識別係對表現CAR之T細胞賦予與抗原處理無關地識別抗原之能力,藉此避開腫瘤免疫逃逸之主要機制。進而,若於T細胞中表現,則CAR有利的是不會與內源性TCR α鏈及β鏈進行二聚化。
本發明之脂質奈米粒子中使用之CAR包括:能夠特異性識別應被標靶免疫細胞(例如T細胞、NK細胞、NKT細胞、單核球、巨噬細胞、樹狀細胞等)識別之表面抗原(例如癌抗原肽、於癌細胞中表現亢進之表面受體等)之抗體之抗原結合區、細胞外鉸鏈區、跨膜區及細胞內T細胞訊號傳遞區。
作為抗原結合區所特異性識別之表面抗原,例如可列舉:於各種癌(例如急性淋巴細胞性癌、腺泡狀橫紋肌肉瘤、膀胱癌、骨癌、腦癌(例如神經管胚細胞瘤)、乳癌、肛門、肛門廔管或肛門直腸之癌、眼癌、肝內膽管之癌、關節癌、頸部、膽囊或胸膜之癌、鼻、鼻腔或中耳之癌、口腔癌、外陰癌、慢性骨髄性癌、結腸癌、食道癌、子宮頸癌、纖維肉瘤、消化道類癌腫瘤、頭頸部癌(例如頭頸部扁平上皮癌)、下咽頭癌、腎癌、喉頭癌、白血病(例如急性淋巴母細胞性白血病、急性淋巴細胞性白血病、慢性淋巴細胞性白血病、急性骨髄性白血病)、液性腫瘤、肝癌、肺癌(例如非小細胞肺癌)、淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤、彌漫性大細胞型B細胞淋巴瘤、濾泡性淋巴瘤)、惡性間皮瘤、肥胖細胞瘤、黑色素瘤、多發性骨髓瘤、上咽頭癌、卵巢癌、胰腺癌、腹膜、網膜及腸間膜之癌、咽頭癌、前列腺癌、直腸癌、腎癌、皮膚癌、小腸癌、軟組織癌、實體腫瘤、胃癌、睾丸癌、甲狀腺癌、尿管癌等)中表現亢進之表面受體,例如CD19、EGF受體、BCMA、CD30、Her2、ROR1、MUC16、CD20、間皮素(mesothelin)、B細胞突變抗原(BCMA,B-cell mutation antigen)、CD123、CD3、前列腺特異性膜抗原(PSMA,prostate specific membrane antigen)、CD33、MUC-1、CD138、CD22、GD2、PD-L1、CEA(Carcinoembryonic antigen,癌胚抗原)、硫酸軟骨蛋白多糖(chondroitin sulfate proteoglycan)-4、IL-13受體α鏈、IgGκ輕鏈等;或者癌抗原肽(例如源自WT1、GPC3、MART-1、gp100、NY-ESO-1、MAGE-A4等之肽)等,但並不限定於該等。
作為本發明中使用之抗原結合區,只要為能夠特異性識別靶抗原之抗體片段,則並無特別限制,若考慮到CAR之易製作性,較理想為將輕鏈可變區與重鏈可變區經由連接肽連結而成之單鏈抗體(scFv)。關於單鏈抗體中之輕鏈可變區與重鏈可變區之配置,只要兩者能夠再構成功能性抗原結合區,則並無特別限定,通常可按照自N末端側起依序為輕鏈可變區-連接肽-重鏈可變區之順序設計。作為連接肽,可使用製作單鏈抗體時通常使用之自身公知之連接肽。編碼輕鏈可變區之DNA、編碼重鏈可變區之DNA例如可分別自抗體產生細胞選殖輕鏈基因、重鏈基因,以該等作為模板進行PCR(polymerase chain reaction,聚合酶鏈反應)等而製備,或者可根據既有之抗體序列資訊進行化學合成。將所獲得之各DNA片段與編碼連接肽之DNA利用適宜方法進行接合(ligation),藉此可取得編碼單鏈抗體之DNA。為了使CAR呈現於免疫細胞表面,較佳為於抗原結合區之N末端側進而附加前導序列(leader sequence)。
作為細胞外鉸鏈區及跨膜區,可適當使用該技術領域中通常使用之源自T細胞表面分子之區域,例如可列舉源自CD8α或CD28之各區,但並不限定於該等。
作為細胞內訊號傳遞區,例如可列舉:具有CD3ζ鏈者,於跨膜區與CD3ζ鏈之間進而具有CD28、CD134、CD137、Lck、DAP10、ICOS、4-1BB等共刺激傳遞基元(motif)者,具有2個以上之共刺激傳遞基元者等,但並不限定於該等,可將該技術領域中通常使用之任意區域組合使用。
編碼細胞外鉸鏈區、跨膜區及細胞內訊號傳遞區之核酸序列資訊於該技術領域中為周知,只要是業者,則可基於該等資訊,容易地自T細胞取得編碼各區之DNA片段。
將如此獲得之分別編碼抗原結合區、細胞外鉸鏈區、跨膜區及細胞內訊號傳遞區之DNA片段利用常規方法進行連結,藉此可取得編碼CAR之DNA。
將如此獲得之分別編碼抗原結合區、細胞外鉸鏈區、跨膜區及細胞內訊號傳遞區之DNA片段利用常規方法進行連結,藉此可取得編碼CAR之DNA。
將所獲得之編碼CAR之DNA可直接、或附加適宜之連接基及/或核轉移訊號等後,插入至包含於T細胞內發揮功能之啟動子之表現載體、較佳為質粒載體。作為於T細胞內發揮功能之啟動子,可使用:哺乳動物細胞中構成性SRα啟動子、SV40啟動子、LTR啟動子、CMV(巨細胞病毒)啟動子、RSV(勞斯肉瘤病毒)啟動子、MoMuLV(莫洛尼氏鼠白血病病毒)LTR、HSV-TK(單純疱疹病毒胸苷激酶)啟動子等,但並不限定於該等。又,亦可使用T細胞特異性表現之CD3、CD4、CD8等基因啟動子。
編碼CAR之RNA、較佳為mRNA可藉由以包含上述編碼CAR之DNA之表現載體作為模板,利用自身公知之試管內(in vitro)轉錄系統轉錄成mRNA而製備。
(a-2)編碼外源性TCR之核酸 於本說明書中,所謂「T細胞受體(TCR)」,意指由TCR鏈(α鏈、β鏈)之二聚體所構成,識別抗原或該抗原-HLA(人白血球型抗原)(MHC:主要組織相容性複合體)複合體並向T細胞傳遞刺激訊號之受體。各TCR鏈係由可變區與恆定區構成,可變區存在3個互補決定區(CDR1、CDR2、CDR3)。又,本發明中使用之TCR不僅指TCR之α鏈與β鏈構成雜二聚體(Heterodimer)而成者,亦包括構成同二聚體(Homodimer)而成者。進而,該TCR亦包括:恆定區局部或整體缺損者、或者胺基酸序列經重組者、變為可溶性TCR(soluble TCR)者等。 再者,所謂「外源性TCR」意指對作為本發明之脂質奈米粒子之靶細胞的T細胞而言為外源性。外源性TCR之胺基酸序列與作為本發明之脂質奈米粒子之靶細胞的T細胞所表現之內因性TCR可相同亦可不同。
本發明之脂質奈米粒子中使用之編碼TCR之核酸係編碼能夠特異性識別應被靶T細胞識別之表面抗原(例如癌抗原肽等)之TCR之α鏈及β鏈的核酸。
該核酸可藉由自身公知之方法製備。於目標TCR之胺基酸序列或核酸序列為公知之情形時,可基於該序列,例如化學合成DNA鏈或RNA鏈,或者將所合成之部分重疊之寡DNA短鏈利用PCR法或Gibson Assembly法進行連接,藉此構建編碼本發明之TCR之全長或部分長之DNA。
該核酸可藉由自身公知之方法製備。於目標TCR之胺基酸序列或核酸序列為公知之情形時,可基於該序列,例如化學合成DNA鏈或RNA鏈,或者將所合成之部分重疊之寡DNA短鏈利用PCR法或Gibson Assembly法進行連接,藉此構建編碼本發明之TCR之全長或部分長之DNA。
於目標TCR之序列非公知之情形時,例如可從包含表現目標TCR之T細胞之細胞群中單離目標T細胞,自該T細胞獲得碼TCR之核酸。具體而言,可自生物體(例如:人)採集含有T細胞之細胞群(例如:PBMC),於目標TCR所識別之細胞表面抗原之表位之存在下,一面刺激該等細胞群一面進行培養,將針對表現該細胞表面抗原之細胞之特異性、及CD8或CD4等細胞表面抗原設為指標,利用公知方法從該細胞群中選擇特異性識別表現該細胞表面抗原之細胞之T細胞。T細胞針對表現該表面抗原之細胞之特異性可利用例如Dextramer分析、ELISPOT分析或細胞損傷性分析等測定。上述含有T細胞之細胞群較佳為自例如包含大量表現被目標TCR識別之細胞表面抗原之細胞之生物體(例如:癌等之疾病患者、或含有與該抗原之表位或於該表位經脈衝之樹狀細胞接觸之T細胞之群)中採集。
利用常規方法自上述單離之T細胞萃取DNA,以該DNA作為模板,基於TCR之恆定區之核酸序列而擴增TCR基因,進行選殖,藉此可獲得本發明之核酸。又,亦可藉由如下方式製備:利用常規方法自細胞萃取RNA而合成cDNA,以此為模板,使用與分別編碼TCR之α鏈及β鏈之恆定區之核酸互補之反義引子,進行5'-RACE(Rapid amplification of cDNA ends,快速擴增cDNA末端)。5'-RACE利用公知方法進行即可,例如可使用如SMART PCR cDNA合成套組(SMART PCR cDNA Synthesis Kit)(Clontech公司製造)之市售套組進行。與上述編碼CAR之DNA同樣地,可將所獲得之分別編碼TCR之α鏈及β鏈之DNA插入至適宜之表現載體。編碼α鏈之DNA與編碼β鏈之DNA可插入至同一個載體,亦可插入至分開之載體。於插入至同一個載體之情形時,該表現載體可多順反子(polycistronic)表現兩鏈,亦可單順反子(monocistronic)表現。於前者之情形時,於編碼兩鏈之DNA之間插入如IRES(internal ribosome entry site,內部核糖體進入位點)或FMV(Foot-And-Mouth-Disease Virus,口蹄疫病毒)2A之容允多順反子表現之中介序列。
又,編碼TCR之各鏈之RNA、較佳為mRNA例如可以該表現載體作為模板,與上述編碼CAR之RNA同樣地製備。
又,編碼TCR之各鏈之RNA、較佳為mRNA例如可以該表現載體作為模板,與上述編碼CAR之RNA同樣地製備。
(b)陽離子性脂質 於本說明書中,所謂「陽離子性脂質」意指於生理學pH等所選擇之pH下帶有純淨正電荷之脂質。本發明之脂質奈米粒子中使用之陽離子性脂質並無特別限定,例如可列舉WO 2015/011633、WO 2016/021683、WO 2011/153493、WO 2013/126803、WO 2010/054401、WO 2010/042877、WO 2016/104580、WO 2015/005253、WO 2014/007398、WO 2017/117528、WO 2017/075531、WO 2017/00414、WO 2015/199952、US 2015/0239834等中記載之陽離子性脂質等。
較佳為列舉WO 2015/011633中記載之下述結構式所表示之陽離子性脂質。
[化2]
[化3]
[化4]
[化5]
[化6]
[化7]
[化8]
[化9]
以及該等之鹽。
作為上述陽離子性脂質中之更佳者,可列舉下述結構式所表示之陽離子性脂質。
[化10]
以及該等之鹽。
較佳為列舉WO 2016/021683中記載之下述結構式所表示之陽離子性脂質。
[化11]
[式中,
W表示式-NR1 R2 或式-N+ R3 R4 R5 (Z- ),
R1 及R2 分別獨立表示C1-4 烷基或氫原子,
R3 、R4 及R5 分別獨立表示C1-4 烷基,
Z- 表示陰離子,
X表示可經取代之C1-6 伸烷基,
YA 、YB 及YC 分別獨立表示可經取代之次甲基,
LA 、LB 及LC 分別獨立表示可經取代之亞甲基或鍵結鍵,
RA1 、RA2 、RB1 、RB2 、RC1 及RC2 分別獨立表示可經取代之C4-10 烷基]
所表示之化合物或其鹽。
W表示式-NR1 R2 或式-N+ R3 R4 R5 (Z- ),
R1 及R2 分別獨立表示C1-4 烷基或氫原子,
R3 、R4 及R5 分別獨立表示C1-4 烷基,
Z- 表示陰離子,
X表示可經取代之C1-6 伸烷基,
YA 、YB 及YC 分別獨立表示可經取代之次甲基,
LA 、LB 及LC 分別獨立表示可經取代之亞甲基或鍵結鍵,
RA1 、RA2 、RB1 、RB2 、RC1 及RC2 分別獨立表示可經取代之C4-10 烷基]
所表示之化合物或其鹽。
更佳為列舉下述結構式所表示之陽離子性脂質。
[化12]
[化13]
[化14]
[化15]
[化16]
[化17]
[化18]
[化19]
[化20]
[化21]
以及該等之鹽。
作為上述陽離子性脂質中之更佳者,可列舉下述結構式所表示之陽離子性脂質。
[化22]
[化23]
[化24]
以及該等之鹽。
於另一較佳實施態樣中,可列舉下述式(II)所表示之陽離子性脂質(以下亦稱為「化合物(II)」)。
[化25]
[式中,
n表示2~5之整數,
R表示直鏈狀C1-5 烷基、直鏈狀C7-11 烯基或直鏈狀C11 二烯基,
波浪線分別獨立為順式或反式之鍵]
所表示之化合物或其鹽。
n表示2~5之整數,
R表示直鏈狀C1-5 烷基、直鏈狀C7-11 烯基或直鏈狀C11 二烯基,
波浪線分別獨立為順式或反式之鍵]
所表示之化合物或其鹽。
更佳為列舉下述結構式所表示之陽離子性脂質。
[化26]
[化27]
[化28]
[化29]
[化30]
[化31]
[化32]
[化33]
[化34]
[化35]
[化36]
以及該等之鹽。
作為上述陽離子性脂質中之更佳者,可列舉下述結構式所表示之陽離子性脂質。
[化37]
[化38]
[化39]
以及該等之鹽。
化合物(II)例如可藉由以下之製法製造。化合物(II)中之波浪線兩者為順式鍵之化合物、及波浪線之一者或兩者為反式鍵之化合物均可藉由與以下所示之製法相同之製法製造。尤其於酯化時,藉由使用對應於目標化合物(II)之結構之適宜原料,能夠獲得所需結構之化合物(II)。又,化合物(II)之鹽可藉由適當地與無機鹽基、有機鹽基、有機酸、鹼性或酸性胺基酸進行混合而獲得。
[化40]
上述製造方法中之各步驟所使用之原料或試劑、以及所獲得之化合物分別可形成鹽。
於各步驟中獲得之化合物為游離化合物之情形時,可藉由公知方法轉化為目標鹽。相反地,於各步驟中獲得之化合物為鹽之情形時,可藉由公知方法轉化為游離體或其他種類之目標鹽。
各步驟中獲得之化合物可直接以反應液用於下一反應,或獲得粗產物後再用於下一反應,或依據常規方法採用濃縮、晶化、再結晶、蒸餾、溶劑萃取、分餾、層析等分離手段自反應混合物單離及/或純化各步驟中獲得之化合物。
於各步驟之原料或試劑之化合物有市售之情形時,可直接使用市售品。
各步驟之反應之反應時間根據所使用之試劑或溶劑而不同,於無特別記載之情況下,通常為1分鐘~48小時、較佳為10分鐘~8小時。
各步驟之反應之反應溫度根據所使用之試劑或溶劑而不同,於無特別記載之情況下,通常為-78℃~300℃、較佳為-78℃~150℃。
各步驟之反應之壓力根據所使用之試劑或溶劑而不同,於無特別記載之情況下,通常為1氣壓~20氣壓、較佳為1氣壓~3氣壓。
於各步驟之反應時,可使用例如Biotage公司製造之Initiator等微波(Microwave)合成裝置。反應溫度根據所使用之試劑或溶劑而不同,於無特別記載之情況下,通常為室溫~300℃,較佳為室溫~250℃,更佳為50℃~250℃。反應時間根據所使用之試劑或溶劑而不同,於無特別記載之情況下,通常為1分鐘~48小時,較佳為1分鐘~8小時。
於各步驟之反應中,於無特別記載之情況下,使用相對於基質為0.5當量~20當量、較佳為0.8當量~5當量之試劑。於使用試劑作為觸媒之情形時,使用相對於基質為0.001當量~1當量、較佳為0.01當量~0.2當量之試劑。於將試劑兼用作反應溶劑之情形時,試劑係使用溶劑量。
於各步驟之反應時,於無特別記載之情況下,該等反應係於無溶劑、或者溶解或懸浮於適宜溶劑中之條件下進行。作為溶劑之具體例,可列舉以下者。
醇類:甲醇、乙醇、異丙醇、異丁醇、第三丁醇、2-甲氧基乙醇等;
醚類:二乙醚、二異丙醚、二苯醚、四氫呋喃、1,2-二甲氧基乙烷等;
芳香族烴類:氯苯、甲苯、二甲苯等;
飽和烴類:環己烷、己烷、庚烷等;
醯胺類:N,N-二甲基甲醯胺、N-甲基吡咯啶酮等;
鹵化烴類:二氯甲烷、四氯化碳等;
腈類:乙腈等;
亞碸類:二甲基亞碸等;
芳香族有機鹼類:吡啶等;
酸酐類:乙酸酐等;
有機酸類:甲酸、乙酸、三氟乙酸等;
無機酸類:鹽酸、硫酸等;
酯類:乙酸乙酯、乙酸異丙酯等;
酮類:丙酮、甲基乙基酮等;
水。
可將兩種以上之上述溶劑以適宜比例混合使用。
醚類:二乙醚、二異丙醚、二苯醚、四氫呋喃、1,2-二甲氧基乙烷等;
芳香族烴類:氯苯、甲苯、二甲苯等;
飽和烴類:環己烷、己烷、庚烷等;
醯胺類:N,N-二甲基甲醯胺、N-甲基吡咯啶酮等;
鹵化烴類:二氯甲烷、四氯化碳等;
腈類:乙腈等;
亞碸類:二甲基亞碸等;
芳香族有機鹼類:吡啶等;
酸酐類:乙酸酐等;
有機酸類:甲酸、乙酸、三氟乙酸等;
無機酸類:鹽酸、硫酸等;
酯類:乙酸乙酯、乙酸異丙酯等;
酮類:丙酮、甲基乙基酮等;
水。
可將兩種以上之上述溶劑以適宜比例混合使用。
於各步驟之反應中使用鹼之情形時,可使用例如以下所示之鹼。
無機鹼類:氫氧化鈉、氫氧化鉀、氫氧化鎂等;
鹼性鹽類:碳酸鈉、碳酸鈣、碳酸氫鈉等;
有機鹼類:三乙基胺、二乙基胺、吡啶、4-二甲基胺基吡啶、N,N-二甲基苯胺、1,4-二氮雜雙環[2.2.2]辛烷、1,8-二氮雜雙環[5.4.0]-7-十一烯、咪唑、哌啶等;
金屬烷氧化物類:乙醇鈉、第三丁醇鉀、第三丁醇鈉等;
鹼金屬氫化物類:氫化鈉等;
金屬醯胺類:醯胺鈉、二異丙基醯胺鋰、雙(三甲基矽烷)胺基鋰等;
有機鋰類:正丁基鋰、第二丁基鋰等。
鹼性鹽類:碳酸鈉、碳酸鈣、碳酸氫鈉等;
有機鹼類:三乙基胺、二乙基胺、吡啶、4-二甲基胺基吡啶、N,N-二甲基苯胺、1,4-二氮雜雙環[2.2.2]辛烷、1,8-二氮雜雙環[5.4.0]-7-十一烯、咪唑、哌啶等;
金屬烷氧化物類:乙醇鈉、第三丁醇鉀、第三丁醇鈉等;
鹼金屬氫化物類:氫化鈉等;
金屬醯胺類:醯胺鈉、二異丙基醯胺鋰、雙(三甲基矽烷)胺基鋰等;
有機鋰類:正丁基鋰、第二丁基鋰等。
於各步驟之反應中使用酸或酸性觸媒之情形時,可使用例如以下所示之酸或酸性觸媒。
無機酸類:鹽酸、硫酸、硝酸、氫溴酸、磷酸等;
有機酸類:乙酸、三氟乙酸、檸檬酸、對甲苯磺酸、10-樟腦磺酸等;
路易斯酸:三氟化硼二乙醚錯合物、碘化鋅、無水氯化鋁、無水氯化鋅、無水氯化鐵等。
有機酸類:乙酸、三氟乙酸、檸檬酸、對甲苯磺酸、10-樟腦磺酸等;
路易斯酸:三氟化硼二乙醚錯合物、碘化鋅、無水氯化鋁、無水氯化鋅、無水氯化鐵等。
只要無特別記載,各步驟之反應係依據公知方法,例如:第5版實驗化學講座,第13卷~19卷(日本化學會(編));新實驗化學講座,第14卷~15卷(日本化學會(編));精密有機化學 修訂第2版(L. F. Tietze, Th. Eicher,南江堂);修訂 有機人名反應機理及其應用(東鄉秀雄(著),講談社);ORGANIC SYNTHESES Collective Volume I~VII(John Wiley & SonsInc);Modern Organic Synthesis in the Laboratory A Collection of Standard Experimental Procedures(Jie Jack Li(著),OXFORD UNIVERSITY出版);Comprehensive Heterocyclic Chemistry III, Vol.1~Vol.14(Elsevier Japan股份有限公司);有機合成中人名反應之戰略性應用(富岡清監(譯),化學同人發行);Comprehensive Organic Transformations(VCH Publishers Inc.)1989年刊等中記載之方法進行。
於各步驟中,官能基之保護或去保護反應係依據公知方法,例如:Wiley-Interscience公司2007年刊「Protective Groups in Organic Synthesis, 4thEd.」(Theodora W. Greene、Peter G. M. Wuts著);Thieme公司2004年刊「Protecting Groups 3rdEd.」(P. J. Kocienski(著))等中記載之方法進行。
作為醇等之羥基或酚性羥基之保護基,例如可列舉:甲氧基甲醚、苄醚、對甲氧基苄醚、第三丁基二甲基矽醚、第三丁基二苯基矽醚、四氫吡喃醚等醚型保護基;乙酸酯等羧酸酯型保護基;甲磺酸酯等磺酸酯型保護基;碳酸第三丁酯等碳酸酯型保護基等。
作為醛之羰基之保護基,例如可列舉:二甲基縮醛等縮醛型保護基;環狀1,3-二㗁烷等環狀縮醛型保護基等。
作為酮之羰基之保護基,例如可列舉:二甲基縮酮等縮酮型保護基;環狀1,3-二㗁烷等環狀縮酮型保護基;O-甲基肟等肟型保護基;N,N-二甲基腙等腙型保護基等。
作為羧基之保護基,例如可列舉:甲酯等酯型保護基;N,N-二甲基醯胺等醯胺型保護基等。
作為硫醇之保護基,例如可列舉:苄基硫醚等醚型保護基;硫代乙酸酯、硫代碳酸酯、硫代胺基甲酸酯等酯型保護基等。
作為胺基、或咪唑、吡咯、吲哚等芳香族雜環之保護基,例如可列舉:胺基甲酸苄酯等胺基甲酸酯型保護基;乙醯胺等醯胺型保護基;N-三苯基甲基胺等烷基胺型保護基、甲磺醯胺等磺醯胺型保護基等。
保護基之去除可藉由公知方法,例如使用酸、鹼、紫外光、肼、苯基肼、N-甲基二硫代胺基甲酸鈉、氟化四丁基銨、乙酸鈀、鹵化三烷基矽烷(例如碘化三甲基矽烷、溴化三甲基矽烷)之方法或還原法等進行。
於各步驟中進行還原反應之情形時,作為使用之還原劑,可列舉:氫化鋁鋰、三乙醯氧基硼氫化鈉、氰基硼氫化鈉、氫化二異丁基鋁(DIBAL-H)、硼氫化鈉、四甲基三乙醯氧基硼氫化銨等金屬氫化物類;硼烷四氫呋喃錯合物等硼烷類;雷氏鎳;雷氏鈷;氫;甲酸等。例如可於存在氫或甲酸之條件下使用雷氏鎳或雷氏鈷。於還原碳-碳雙鍵或三鍵之情形時,有使用鈀-碳或林德拉(Lindlar)觸媒等觸媒之方法。
於各步驟中進行氧化反應之情形時,作為使用之氧化劑,可列舉:間氯過苯甲酸酸(MCPBA)、過氧化氫、第三丁基過氧化氫等過酸類;過氯酸四丁基銨等過氯酸鹽類;氯酸鈉等氯酸鹽類;亞氯酸鈉等亞氯酸鹽類;過碘酸鈉等過碘酸類;氧碘苯等高原子價碘試劑;二氧化錳、過錳酸鉀等含有錳之試劑;四乙酸鉛等鉛類;氯鉻酸吡啶鎓(PCC)、二鉻酸吡啶鎓(PDC)、瓊斯試劑等含有鉻之試劑;N-溴琥珀醯亞胺(NBS)等鹵化合物類;氧;臭氧;三氧化硫-吡啶錯合物;四氧化鋨;二氧化硒;2,3-二氯-5,6-二氰基-1,4-苯醌(DDQ)等。
於各步驟中進行自由基環化反應之情形時,作為使用之自由基起始劑,可列舉:偶氮二異丁腈(AIBN)等偶氮化合物;4-4'-偶氮雙-4-氰基戊酸(ACPA)等水溶性自由基起始劑;存在空氣或氧氣之條件下之三乙基硼;過氧化苯甲醯等。又,作為使用之自由基反應試劑,可列舉:三丁基錫烷、三(三甲基矽烷基)矽烷、1,1,2,2-四苯基乙矽烷、二苯基矽烷、碘化釤等。
於各步驟中進行維蒂希(Wittig)反應之情形時,作為使用之Wittig試劑,可列舉亞烷基磷烷類等。亞烷基磷烷類可藉由公知方法,例如使鏻鹽與強鹼進行反應而製備。
於各步驟中進行霍納爾-
埃蒙斯(Horner-Emmons)反應之情形時,作為使用之試劑,可列舉:二甲基膦醯基乙酸甲酯、二乙基膦醯基乙酸乙酯等膦醯基乙酸酯類;鹼金屬氫化物類、有機鋰類等鹼。
於各步驟中進行傅里德-克拉夫茨(Friedel-Crafts)反應之情形時,作為使用之試劑,可列舉:路易斯酸、與醯氯或烷基化劑(例如鹵化烷基類、醇、烯烴類等)。或者,亦可使用有機酸或無機酸代替路易斯酸,亦可使用乙酸酐等酸酐代替醯氯。
於各步驟中進行芳香族親核取代反應之情形時,作為試劑,可使用親核劑(例如胺類、咪唑等)與鹼(例如鹼性鹽類、有機鹼類等)。
於各步驟中進行利用碳陰離子之親核加成反應、利用碳陰離子之親核1,4-加成反應(麥克爾(Michael)加成反應)、或利用碳陰離子之親核取代反應之情形時,作為用以產生碳陰離子之鹼,可列舉:有機鋰類、金屬烷氧化物類、無機鹼類、有機鹼類等。
於各步驟中進行格林尼亞(Grignard)反應之情形時,作為Grignard試劑,可列舉:溴化苯基鎂等芳基鎂鹵化物類;溴化甲基鎂、溴化異丙基鎂等烷基鎂鹵化物類。Grignard試劑可藉由公知方法,例如以醚或四氫呋喃作為溶劑,使鹵化烷基或鹵化芳基與金屬鎂進行反應而製備。
於各步驟中進行克腦文蓋爾(Knoevenagel)縮合反應之情形時,作為試劑,可使用被兩個拉電子基夾持之活性亞甲基化合物(例如丙二酸、丙二酸二乙酯、丙二腈等)及鹼(例如有機鹼類、金屬烷氧化物類、無機鹼類)。
於各步驟中進行維爾斯邁爾-哈克(Vilsmeier-Haack)反應之情形時,作為試劑,可使用磷醯氯與醯胺衍生物(例如N,N-二甲基甲醯胺等)。
於各步驟中進行醇類、烷基鹵化物類、磺酸酯類之疊氮化反應之情形時,作為使用之疊氮化劑,可列舉:疊氮磷酸二苯酯(DPPA)、疊氮三甲基矽烷、疊氮化鈉等。例如於將醇類進行疊氮化之情形時,有使用疊氮磷酸二苯酯與1,8-二氮雜雙環[5,4,0]十一碳-7-烯(DBU)之方法或使用疊氮三甲基矽烷與路易斯酸之方法等。
於各步驟中進行還原胺化反應之情形時,作為使用之還原劑,可列舉:三乙醯氧基硼氫化鈉、氰基硼氫化鈉、氫、甲酸等。於基質為胺化合物之情形時,作為使用之羰基化合物,可列舉:多聚甲醛、以及乙醛等醛類、環己酮等酮類。於基質為羰基化合物之情形時,作為使用之胺類,可列舉:氨、甲基胺等一級胺;二甲基胺等二級胺等。
於各步驟中進行光延反應之情形時,作為試劑,可使用偶氮二羧酸酯類(例如偶氮二羧酸二乙酯(DEAD)、偶氮二羧酸二異丙酯(DIAD)等)及三苯膦。
於各步驟中進行酯化反應、醯胺化反應或脲化反應之情形時,作為使用之試劑,可列舉:醯氯、醯溴等醯鹵體;酸酐、活性酯體、硫酸酯體等經活化之羧酸類。作為羧酸之活化劑,可列舉:1-乙基-3-(3-二甲基胺基丙基)碳二醯亞胺鹽酸鹽(WSCD)等碳二醯亞胺系縮合劑;4-(4,6-二甲氧基-1,3,5-三𠯤-2-基)-4-甲基嗎啉鎓氯化物-n水合物(DMT-MM)等三𠯤系縮合劑;1,1-羰基二咪唑(CDI)等碳酸酯系縮合劑;疊氮磷酸二苯酯(DPPA);苯并三唑-1-基氧基-三(二甲基胺基)鏻鹽(BOP試劑);碘化2-氯-1-甲基吡啶鎓(向山試劑);亞硫醯氯;氯甲酸乙酯等鹵代甲酸低級烷基酯;O-(7-氮雜苯并三唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸鹽(HATU);硫酸;或者該等之組合等。於使用碳二醯亞胺系縮合劑之情形時,可進而於反應中添加1-羥基苯并三唑(HOBt)、N-羥基琥珀醯亞胺(HOSu)、二甲基胺基吡啶(DMAP)等添加劑。
於各步驟中進行偶合反應之情形時,作為使用之金屬觸媒,可列舉:乙酸鈀(II)、四(三苯基膦)鈀(0)、二氯雙(三苯基膦)鈀(II)、二氯雙(三乙基膦)鈀(II)、三(二亞苄基丙酮)二鈀(0)、氯化[1,1'-雙(二苯基膦基)二茂鐵]鈀(II)、乙酸鈀(II)等鈀化合物;四(三苯基膦)鎳(0)等鎳化合物;氯化三(三苯基膦)銠(III)等銠化合物;鈷化合物;氧化銅、碘化銅(I)等銅化合物;鉑化合物等。可進而於反應中添加鹼,作為此種鹼,可列舉:無機鹼類、鹼性鹽類等。
於各步驟中進行硫羰基化反應之情形時,作為硫羰基化劑,代表性地使用五硫化二磷,除五硫化二磷以外,亦可使用2,4-雙(4-甲氧基苯基)-1,3,2,4-二硫二磷雜環丁烷-2,4-二硫化物(勞森(Lowesson)試劑)等具有1,3,2,4-二硫二磷雜環丁烷-2,4-二硫化物結構之試劑。
於各步驟中進行沃爾-齊格勒(Wohl-Ziegler)反應之情形時,作為使用之鹵化劑,可列舉:N-碘琥珀醯亞胺、N-溴琥珀醯亞胺(NBS)、N-氯琥珀醯亞胺(NCS)、溴、磺醯氯等。藉由進而於反應中添加熱、光、過氧化苯甲醯、偶氮二異丁腈等自由基起始劑,可使反應加速。
於各步驟中進行羥基之鹵化反應之情形時,作為使用之鹵化劑,可列舉氫鹵酸與無機酸之醯鹵化物,具體而言,若為氯化,可列舉:鹽酸、亞硫醯氯、氧氯化磷等,若為溴化,可列舉:48%氫溴酸等。又,亦可採用藉由三苯膦與四氯化碳或四溴化碳等之作用而由醇獲得鹵化烷基體之方法。或可採用如將醇轉化為磺酸酯後使之與溴化鋰、氯化鋰或碘化鈉反應之經過兩階段反應而合成鹵化烷基體之方法。
於各步驟中進行阿爾布佐夫(Arbuzov)反應之情形時,作為使用之試劑,可列舉:溴乙酸乙酯等鹵化烷基類;亞磷酸三乙酯或亞磷酸三(異丙基)酯等亞磷酸酯類。
於各步驟中進行磺酯化反應之情形時,作為使用之磺化劑,可列舉:甲磺醯氯、對甲苯磺醯氯、甲磺酸酐、對甲苯磺酸酐、三氟甲磺酸酐等。
於各步驟中進行水解反應之情形時,作為試劑,可使用酸或鹼。又,於進行第三丁酯之酸水解反應之情形時,有添加甲酸或三乙基矽烷等以還原捕捉副生成之第三丁基陽離子之情況。
於各步驟中進行脫水反應之情形時,作為使用之脫水劑,可列舉:硫酸、五氧化二磷、氧氯化磷、N,N'-二環己基碳二醯亞胺、氧化鋁、多磷酸等。
於另一較佳實施態樣中,可列舉下述式(III)所表示之陽離子性脂質(以下亦稱為「化合物(III)」)。
[化41]
[式中,
n1表示2~6之整數,
n2表示0~2之整數,
n3表示0~2之整數,
L表示-C(O)O-或-NHC(O)O-,
Ra表示直鏈狀C5-13 烷基、直鏈狀C13-17 烯基或直鏈狀C17 二烯基,
Rb表示直鏈狀C2-9 烷基,
Rc表示氫原子或直鏈狀C2-9 烷基,
Rd表示氫原子或直鏈狀C2-9 烷基,
Re表示直鏈狀C2-9 烷基,
Rf表示直鏈狀C2-9 烷基]
所表示之化合物或其鹽。
n1表示2~6之整數,
n2表示0~2之整數,
n3表示0~2之整數,
L表示-C(O)O-或-NHC(O)O-,
Ra表示直鏈狀C5-13 烷基、直鏈狀C13-17 烯基或直鏈狀C17 二烯基,
Rb表示直鏈狀C2-9 烷基,
Rc表示氫原子或直鏈狀C2-9 烷基,
Rd表示氫原子或直鏈狀C2-9 烷基,
Re表示直鏈狀C2-9 烷基,
Rf表示直鏈狀C2-9 烷基]
所表示之化合物或其鹽。
更佳為列舉下述結構式所表示之陽離子性脂質。
[化42]
[化43]
[化44]
[化45]
[化46]
[化47]
[化48]
[化49]
[化50]
[化51]
[化52]
[化53]
[化54]
[化55]
[化56]
[化57]
[化58]
[化59]
以及該等之鹽。
作為上述陽離子性脂質中之更佳者,可列舉下述結構式所表示之陽離子性脂質。
[化60]
[化61]
以及該等之鹽。
化合物(III)例如可藉由以下之製法製造。尤其於酯化時,藉由使用對應於目標化合物(III)之結構之適宜原料,能夠合成所需結構之化合物(I)。又,化合物(III)之鹽可藉由適當地與無機鹼、有機鹼、有機酸、鹼性或酸性胺基酸進行混合而獲得。
[化62]
[化63]
[化64]
[化65]
以上之式中,P1 、P2 、P3 、P4 、P5 及P6 分別獨立表示保護基;
化合物(A)表示式:;
化合物(B)表示式:,R1表示;
化合物(C)表示式:,R2表示;
其它各記號表示與上述相同之含義。
以上之式中,P1 、P2 、P3 、P4 、P5 及P6 分別獨立表示保護基;
化合物(A)表示式:;
化合物(B)表示式:,R1表示;
化合物(C)表示式:,R2表示;
其它各記號表示與上述相同之含義。
上述製造方法中之各步驟之反應所使用之原料或試劑、以及反應條件可與化合物(II)之製造方法中之上述各者相同。
於另一實施態樣中,可列舉WO2011/153493中記載之下述結構式所表示之陽離子性脂質。
[化66]
[化67]
[化68]
[化69]
[化70]
[化71]
[化72]
[化73]
[化74]
[化75]
[化76]
[化77]
[化78]
[化79]
[化80]
[化81]
[化82]
[化83]
[化84]
[化85]
[化86]
[化87]
[化88]
[化89]
[化90]
[化91]
[化92]
[化93]
[化94]
[化95]
[化96]
[化97]
[化98]
[化99]
[化100]
[化101]
[化102]
[化103]
[化104]
[化105]
[化106]
[化107]
[化108]
[化109]
[化110]
[化111]
[化112]
[化113]
[化114]
[化115]
[化116]
[化117]
[化118]
及該等之鹽。
作為上述陽離子性脂質中之更佳者,可列舉下述結構式所表示之陽離子性脂質。
[化119]
以及該等之鹽。
於另一實施態樣中,可列舉WO2013/126803中記載之下述結構式所表示之陽離子性脂質。
[化120]
[化121]
[化122]
[化123]
[化124]
以及該等之鹽。
作為上述陽離子性脂質中之更佳者,可列舉下述結構式所表示之陽離子性脂質。
[化125]
及其鹽。
於另一實施態樣中,可列舉Dong等人按照(Proc Natl Acad Sci U S A. 2014 Apr 15; 111(15): 5753)中記載之下述反應圖解(scheme)所合成之陽離子性脂質K-E12、H-A12、Y-E12、G-O12、K-A12、R-A12、cKK-E12、cPK-E12、PK1K-E12、PK500-E12、cQK-E12、cKK-A12、KK-A12、PK-4K-E12、cWK-E12、PK500-O12、PK1K-O12、cYK-E12、cDK-E12、cSK-E12、cEK-E12、cMK-E12、cKK-O12、cIK-E12、cKK-E10、cKK-E14及cKK-E16。
[化126]
作為上述陽離子性脂質中之更佳者,可列舉:cKK-E12、cKK-E14。
於另一實施態樣中,可列舉Love KT等人按照(Proc Natl Acad Sci U S A. 2010 May 25;107(21):9915)中記載之下述反應圖解所合成之陽離子性脂質C14-98、C18-96、C14-113、C14-120、C14-120、C14-110、C16-96、C12-200。
[化127]
作為上述陽離子性脂質中之更佳者,可列舉:C14-110、C16-96及C12-200。
於尤佳實施態樣中,可列舉下述式(I)所表示之陽離子性脂質(以下亦稱為「化合物(I)」)。
[化128]
[式中,
L1 為C1-22 伸烷基、C2-22 伸烯基或C3-22 伸二烯基,
n為0或1之整數,
R1 為
(1)氫原子、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(4)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R2 為-CH2 -O-CO-R5 、-CH2 -CO-O-R5 或-R5 ,
R3 為-CH2 -O-CO-R6 、-CH2 -CO-O-R6 或-R6 ,
R4 為氫原子、-CH2 -O-CO-R7 、-CH2 -CO-O-R7 或-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R8 及R9 分別獨立表示C1-6 烷基]
或其鹽。
L1 為C1-22 伸烷基、C2-22 伸烯基或C3-22 伸二烯基,
n為0或1之整數,
R1 為
(1)氫原子、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(4)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R2 為-CH2 -O-CO-R5 、-CH2 -CO-O-R5 或-R5 ,
R3 為-CH2 -O-CO-R6 、-CH2 -CO-O-R6 或-R6 ,
R4 為氫原子、-CH2 -O-CO-R7 、-CH2 -CO-O-R7 或-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基,
R8 及R9 分別獨立表示C1-6 烷基]
或其鹽。
L1
為C1-22
伸烷基、C2-22
伸烯基或C3-22
伸二烯基。
L1 較佳為C1-22 伸烷基。 L1 更佳為C1-12 伸烷基。 L1 進而較佳為C1-6 伸烷基。
L1 較佳為C1-22 伸烷基。 L1 更佳為C1-12 伸烷基。 L1 進而較佳為C1-6 伸烷基。
n為0或1之整數。
n較佳為1之整數。
n較佳為1之整數。
R1
為
(1)氫原子、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(4)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基。
R1 較佳為
(1)氫原子、
(2)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)、或者、
(3)可經1個或2個直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基)取代之直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基)。
R1 尤佳為氫原子。
(1)氫原子、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(4)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基。
R1 較佳為
(1)氫原子、
(2)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)、或者、
(3)可經1個或2個直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基)取代之直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基)。
R1 尤佳為氫原子。
R2
為-CH2
-O-CO-R5
、-CH2
-CO-O-R5
或-R5
。
R2 較佳為-CH2 -O-CO-R5 或-R5 。
R2 更佳為-CH2 -O-CO-R5 。
R2 較佳為-CH2 -O-CO-R5 或-R5 。
R2 更佳為-CH2 -O-CO-R5 。
R3
為-CH2
-O-CO-R6
、-CH2
-CO-O-R6
或-R6
。
R3 較佳為-CH2 -O-CO-R6 或-R6 。
R3 更佳為-CH2 -O-CO-R6 。
R3 較佳為-CH2 -O-CO-R6 或-R6 。
R3 更佳為-CH2 -O-CO-R6 。
R4
為氫原子、-CH2
-O-CO-R7
、-CH2
-CO-O-R7
或-R7
。
R4 較佳為氫原子或-CH2 -O-CO-R7 。
R4 更佳為-CH2 -O-CO-R7 。
R4 較佳為氫原子或-CH2 -O-CO-R7 。
R4 更佳為-CH2 -O-CO-R7 。
R5
、R6
及R7
分別獨立為
(1)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基。
R5 、R6 及R7 較佳為分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)、或者
(3)直鏈狀之C3-22 二烯基(較佳為直鏈狀之C4-18 二烯基)。
R5 、R6 及R7 更佳為分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、或者
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)。
(1)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、
(2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者
(3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基。
R5 、R6 及R7 較佳為分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)、或者
(3)直鏈狀之C3-22 二烯基(較佳為直鏈狀之C4-18 二烯基)。
R5 、R6 及R7 更佳為分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、或者
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)。
R8
及R9
分別獨立為C1-6
烷基。
R8 及R9 分別獨立為C1-3 烷基(較佳為甲基)。
R8 及R9 分別獨立為C1-3 烷基(較佳為甲基)。
化合物(I)較佳為如下化合物:於上述式(I)中,
L1 為C1-22 伸烷基(較佳為C1-12 伸烷基,更佳為C1-6 伸烷基),
n為1之整數,
R1 為
(1)氫原子、
(2)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)、或者
(3)可經1個或2個直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基)取代之直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基),
R2 為-CH2 -O-CO-R5 或-R5 ,
R3 為-CH2 -O-CO-R6 或-R6 ,
R4 為氫原子或-CH2 -O-CO-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)、或者
(3)直鏈狀之C3-22 二烯基(較佳為直鏈狀之C4-18 二烯基),且
R8 及R9 分別獨立為C1-6 烷基(較佳為C1-3 烷基、尤佳為甲基)。
L1 為C1-22 伸烷基(較佳為C1-12 伸烷基,更佳為C1-6 伸烷基),
n為1之整數,
R1 為
(1)氫原子、
(2)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C6-12 烷基)、或者
(3)可經1個或2個直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基)取代之直鏈狀之C2-22 烯基(較佳為直鏈狀之C6-12 烯基),
R2 為-CH2 -O-CO-R5 或-R5 ,
R3 為-CH2 -O-CO-R6 或-R6 ,
R4 為氫原子或-CH2 -O-CO-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)、或者
(3)直鏈狀之C3-22 二烯基(較佳為直鏈狀之C4-18 二烯基),且
R8 及R9 分別獨立為C1-6 烷基(較佳為C1-3 烷基、尤佳為甲基)。
化合物(I)更佳為如下化合物:於上述式(I)中,
L1 為C1-12 伸烷基(較佳為C1-6 伸烷基),
n為1之整數,
R1 為氫原子,
R2 為-CH2 -O-CO-R5 ,
R3 為-CH2 -O-CO-R6 ,
R4 為-CH2 -O-CO-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)、或者
(3)直鏈狀之C3-22 二烯基(較佳為直鏈狀之C4-18 二烯基),且
R8 及R9 分別獨立為C1-6 烷基(較佳為C1-3 烷基、尤佳為甲基)。
L1 為C1-12 伸烷基(較佳為C1-6 伸烷基),
n為1之整數,
R1 為氫原子,
R2 為-CH2 -O-CO-R5 ,
R3 為-CH2 -O-CO-R6 ,
R4 為-CH2 -O-CO-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基)、或者
(3)直鏈狀之C3-22 二烯基(較佳為直鏈狀之C4-18 二烯基),且
R8 及R9 分別獨立為C1-6 烷基(較佳為C1-3 烷基、尤佳為甲基)。
化合物(I)更佳為如下化合物:於上述式(I)中,
L1 為C1-6 伸烷基,
n為1之整數,
R1 為氫原子,
R2 為-CH2 -O-CO-R5 ,
R3 為-CH2 -O-CO-R6 ,
R4 為-CH2 -O-CO-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、或者
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基),且
R8 及R9 分別獨立為C1-3 烷基(較佳為甲基)。
L1 為C1-6 伸烷基,
n為1之整數,
R1 為氫原子,
R2 為-CH2 -O-CO-R5 ,
R3 為-CH2 -O-CO-R6 ,
R4 為-CH2 -O-CO-R7 ,
R5 、R6 及R7 分別獨立為
(1)可經1個或2個直鏈狀之C1-22 烷基(較佳為直鏈狀之C1-10 烷基)取代之直鏈狀之C1-22 烷基(較佳為直鏈狀之C4-18 烷基)、或者
(2)直鏈狀之C2-22 烯基(較佳為直鏈狀之C4-18 烯基),且
R8 及R9 分別獨立為C1-3 烷基(較佳為甲基)。
作為上述各結構式所表示之化合物之鹽,較佳為藥理學上容許之鹽,例如可列舉:與無機鹼之鹽(例如鈉鹽、鉀鹽等鹼金屬鹽;鈣鹽、鎂鹽等鹼土金屬鹽;鋁鹽、銨鹽)、與有機鹼之鹽(例如與三甲基胺、三乙基胺、吡啶、甲基吡啶、乙醇胺、二乙醇胺、三乙醇胺、胺丁三醇[三(羥基甲基)甲基胺]、第三丁基胺、環己基胺、苄基胺、二環己基胺、N,N-二苄基乙二胺之鹽)、與無機酸之鹽(例如與氫氟酸、鹽酸、氫溴酸、氫碘酸、硝酸、硫酸、磷酸之鹽)、與有機酸之鹽(與甲酸、乙酸、三氟乙酸、鄰苯二甲酸、反丁烯二酸、草酸、酒石酸、順丁烯二酸、檸檬酸、琥珀酸、蘋果酸、甲磺酸、苯磺酸、對甲苯磺酸之鹽)、與鹼性胺基酸之鹽(與精胺酸、離胺酸、鳥胺酸之鹽)或與酸性胺基酸之鹽(與天冬胺酸、麩胺酸之鹽)。
陽離子性脂質於本發明之脂質奈米粒子中存在之全部脂質中所占之比率(莫耳%)例如為約10%~約80%,較佳為約20%~約70%,更佳為約40%~約60%,但並不限定於該等。
上述陽離子性脂質可僅使用1種,亦可將2種以上組合使用。於使用複數個陽離子性脂質之情形時,較佳為以陽離子性脂質整體計成為上述比率。
上述陽離子性脂質可僅使用1種,亦可將2種以上組合使用。於使用複數個陽離子性脂質之情形時,較佳為以陽離子性脂質整體計成為上述比率。
(c)非陽離子性脂質 於本說明書中,所謂「非陽離子性脂質」意指陽離子性脂質以外之脂質,於生理學pH等所選擇之pH下不帶有純淨正電荷之脂質。作為本發明之脂質奈米粒子中使用之非陽離子性脂質,例如可列舉:磷脂質、類固醇類、PEG脂質等。
作為磷脂質,例如只要為穩定地保持核酸、且不會抑制與細胞膜(原生質膜及細胞器膜)之融合以提高編碼CAR或外源性TCR之核酸向標靶免疫細胞內之傳遞性者,並無特別限定,例如可列舉:磷脂醯膽鹼、磷脂醯乙醇胺、磷脂醯絲胺酸、磷脂醯肌醇、磷脂酸、棕櫚醯油醯磷脂醯膽鹼、溶血磷脂醯膽鹼、溶血磷脂醯乙醇胺、二棕櫚醯磷脂醯膽鹼、二油醯磷脂醯膽鹼、二硬脂醯磷脂醯膽鹼或二亞油醯磷脂醯膽鹼等。
作為較佳之磷脂質,可列舉:二硬脂醯磷脂醯膽鹼(DSPC)、二油醯磷脂醯膽鹼(DOPC)、二棕櫚醯磷脂醯膽鹼(DPPC)、二油醯磷脂醯甘油(DOPG)、棕櫚醯油醯磷脂醯甘油(POPG)、二棕櫚醯磷脂醯甘油(DPPG)、二油醯磷脂醯乙醇胺(DOPE)、棕櫚醯油醯磷脂醯膽鹼(POPC)、棕櫚醯油醯磷脂醯乙醇胺(POPE)、及二油醯磷脂醯乙醇胺4-(N-順丁烯二醯亞胺甲基)-環己烷-1-羧酸酯(DOPE-mal),更佳為DOPC、DPPC、POPC及DOPE。
磷脂質於本發明之脂質奈米粒子中存在之全部脂質中所占之比率(莫耳%)例如可為約0%~約90%,較佳為約5%~約30%,更佳為約8%~約15%。
上述磷脂質可僅使用1種,亦可將2種以上組合使用。於使用複數個磷脂質之情形時,較佳為以磷脂質整體計成為上述比率。
上述磷脂質可僅使用1種,亦可將2種以上組合使用。於使用複數個磷脂質之情形時,較佳為以磷脂質整體計成為上述比率。
作為類固醇類,可列舉:膽固醇、5α-膽固烷醇、5β-腎固醇(coprostanol)、膽固醇基-(2'-羥基)乙醚、膽固醇基-(4'-羥基)丁醚、6-酮膽甾烷醇、5α-膽甾烷、膽甾烯酮(cholestenone)、5α-膽固烷酮(cholestanone)、5β-膽固烷酮、癸酸膽固醇酯,較佳為膽固醇。
於存在類固醇類之情形時,其於本發明之脂質奈米粒子中存在之全部脂質中所占之比率(莫耳%)例如可為約10%~約60%,較佳為約12%~約58%,更佳為約20%~約55%。
上述類固醇類可僅使用1種,亦可將2種以上組合使用。於使用複數個類固醇類之情形時,較佳為以類固醇類整體計成為上述比率。
上述類固醇類可僅使用1種,亦可將2種以上組合使用。於使用複數個類固醇類之情形時,較佳為以類固醇類整體計成為上述比率。
於本說明書中,所謂「PEG脂質」意指聚乙二醇(PEG)與脂質之任意之複合體。作為PEG脂質,只要為例如具有能夠抑制本發明之脂質奈米粒子之凝集之效果者,則並無特別限定,例如可列舉:與二烷基氧基丙基鍵結之PEG(PEG-DAA)、與二醯基甘油鍵結之PEG(PEG-DAG)(例如SUNBRIGHT GM-020(日油))、與磷脂醯乙醇胺等磷脂質鍵結之PEG(PEG-PE)、與腦醯胺複合之PEG(PEG-Cer)、與膽固醇複合之PEG(PEG-cholesterol)或該等之衍生物、或該等之混合物、mPEG2000-1,2-二-O-烷基-sn3-胺甲醯基甘油酯(arbomoylglyceride)PEG-C-DOMG)、1-[8'-(1,2-二肉豆蔻醯基-3-丙氧基)-甲醯胺-3',6-二氧雜辛基]胺甲醯基-ω-甲基-聚(乙二醇)(2KPEG-DMG)等。作為較佳之PEG脂質,可列舉:PEG-DGA、PEG-DAA、PEG-PE、PEG-Cer、及該等之混合物,更佳為選自由PEG-二癸氧基丙基複合體、PEG-二月桂氧基丙基複合體、PEG-二肉豆蔻氧基丙基複合體、PEG-二棕櫚氧基丙基複合體、PEG-二硬脂氧基丙基複合體所組成之群中之PEG-DAA複合體、及該等之混合物。
作為PEG之自由末端,除甲氧基以外,亦可使用用以與下述T細胞靶向配體結合之順丁烯二醯亞胺基或N-羥基丁二醯亞胺基等。作為具有用以與T細胞靶向配體結合之官能基之PEG脂質(本說明書中有時稱為「末端反應性PEG脂質」),例如可使用SUNBRIGHT DSPE-020MA或SUNBRIGHT DSPE-020MA(日油)。
作為PEG之自由末端,除甲氧基以外,亦可使用用以與下述T細胞靶向配體結合之順丁烯二醯亞胺基或N-羥基丁二醯亞胺基等。作為具有用以與T細胞靶向配體結合之官能基之PEG脂質(本說明書中有時稱為「末端反應性PEG脂質」),例如可使用SUNBRIGHT DSPE-020MA或SUNBRIGHT DSPE-020MA(日油)。
PEG脂質於本發明之脂質奈米粒子中存在之全部脂質中所占之比率(莫耳%)例如可為約0%~約20%,較佳為約0.1%~約5%,更佳為約0.7%~約2%。
末端反應性PEG脂質於上述全部PEG脂質中所占之比率(莫耳%)例如可為約10%~約100%,較佳為約20%~約100%,更佳為約30%~約100%。 上述PEG脂質可僅使用1種,亦可將2種以上組合使用。於使用複數個PEG脂質之情形時,較佳為以PEG脂質整體計成為上述比率。
末端反應性PEG脂質於上述全部PEG脂質中所占之比率(莫耳%)例如可為約10%~約100%,較佳為約20%~約100%,更佳為約30%~約100%。 上述PEG脂質可僅使用1種,亦可將2種以上組合使用。於使用複數個PEG脂質之情形時,較佳為以PEG脂質整體計成為上述比率。
本發明之脂質奈米粒子係用於對免疫細胞、尤其是負責獲得性免疫中之細胞免疫之T細胞、或者負責自然免疫之NK細胞、單核球、巨噬細胞、樹狀細胞等、以及作為具有NK細胞性質之T細胞之NKT細胞中,基因導入CAR或外源性TCR並使之表現。因此,尤其為了於活體內效率良好地向標靶免疫細胞進行傳遞,本發明之脂質奈米粒子可進而含有能夠將脂質奈米粒子靶向免疫細胞、尤其是T細胞之配體。
(d)能夠將脂質奈米粒子靶向T細胞之配體 作為能夠將本發明之脂質奈米粒子靶向T細胞之配體,只要為能夠特異性識別於T細胞上特異性表現或高表現之表面分子者,則並無特別限制,較佳為包含針對CD3、CD4、CD8或CD28之抗體之1個以上之抗原結合區者,作為更佳例,可列舉包含抗CD3抗體及/或抗CD28抗體之抗原結合區者。又,作為尤其於活體內向T細胞進行傳遞之情況下之較佳例,可列舉僅包含抗CD3抗體之抗原結合區者。此處,所謂「抗原結合區」係與構成上述CAR之抗原結合區含義相同,然而,CAR由於需以編碼其之核酸之形式製備故存在制約,通常多使用單鏈抗體,但T細胞靶向配體之抗原結合區係以蛋白質之狀態含有於本發明之脂質奈米粒子,因此不僅可使用單鏈抗體,亦可較佳地使用例如完全抗體分子、Fab、F(ab')2
、Fab'、Fv、還原抗體(rIgG)、dsFv、sFv、二聚體、三聚體等其他任意之抗體片段。尤其於活體內向標靶免疫細胞進行傳遞之情況下,可較佳地使用不具有Fc部分之Fab'。該等抗體片段可藉由利用還原劑(例如2-巰基乙醇、二硫蘇糖醇)或肽酶(例如木瓜酶、胃蛋白酶、無花果酶)對完全抗體(例如IgG)進行處理、或利用基因重組操作而製備。
若T細胞靶向配體為完全抗體分子,則可使用市售之抗CD3、CD4、CD8、CD28抗體等,或亦可自產生該抗體之細胞之培養液進行單離。另一方面,於該配體為上述任一種抗原結合區(抗體片段)之情形時,可藉由與取得編碼構成上述CAR之抗原結合區之核酸之方法相同之方法,單離出編碼抗CD3、CD4、CD8、CD28抗體等之抗原結合區之核酸,使用該核酸重組生產該抗原結合區。
於本發明之脂質奈米粒子中,T細胞靶向配體只要存在於該脂質奈米粒子之表面上,則可以任意樣式與外殼結合,例如於包含末端反應性PEG脂質作為非陽離子性脂質之情形時,可附加於PEG之末端。例如藉由使於末端導入有順丁烯二醯亞胺基之PEG脂質(例如SUNBRIGHT DSPE-020MA)與上述還原抗體之硫醇基進行反應,可製備經配體(抗體)標記之脂質奈米粒子(有時稱為「抗體-LNP」)。
於將本發明之脂質奈米粒子用於向T細胞以外之免疫細胞、例如NK細胞或樹狀細胞等中進行基因導入之情形時,即便脂質奈米粒子表面不具有用以靶向該等免疫細胞之配體,脂質奈米粒子亦可效率良好地進行傳遞,但亦可使脂質奈米粒子表面具有針對在各免疫細胞表面表現之分子之適宜之靶向配體。例如於NK細胞之情形時,可列舉包含針對CD16、CD56之抗體之抗原結合區者等,但並不限定於該等。
2. 本發明之脂質奈米粒子之製造
本發明之脂質奈米粒子可採用例如US9, 404, 127中記載之方法製造。於脂質奈米粒子進而含有T細胞靶向配體之情形時,可藉由在製作脂質奈米粒子後與T細胞靶向配體進行化學結合而製造。或可如WO2016/021683之記載,藉由例如以下方式製造:製備溶解有上述成分(b)與(c)之有機溶劑溶液,與溶解有(a)之水或緩衝液進行混合,藉此製成脂質奈米粒子後,與T細胞靶向配體進行化學結合。作為陽離子性脂質、磷脂質、膽固醇及PEG脂質之混合比(莫耳比),例如可列舉40~60:0~20:0~50:0~5,但並不限定於該等。又,於調配PEG脂質作為非陽離子性脂質、並將T細胞靶向配體附加於PEG之末端之情形時,PEG脂質與該配體之調配比(莫耳比)例如可為20:1~1:20。上述PEG脂質中可以約10%~約100%之比率(莫耳%)包含末端反應性PEG。上述混合可採用移液管或微小流體混合系統(例如Asia microfluidic system(Syrris)或Nanoassemblr(Precision Nanosystems))進行。可對所獲得之脂質粒子進行凝膠過濾純化、或透析及殺菌過濾。 有機溶劑溶液中之全部脂質成分之濃度較佳為0.5~100 mg/mL。
作為有機溶劑,例如可列舉:甲醇、乙醇、1-丙醇、2-丙醇、1-丁醇、第三丁醇、丙酮、乙腈、N,N-二甲基甲醯胺、二甲基亞碸、或該等之混合物。該有機溶劑亦可含有0~20%之水或緩衝液。作為緩衝液,可列舉:酸性緩衝液(例如乙酸緩衝液、檸檬酸緩衝液)、或中性緩衝液(例如4-(2-羥基乙基)-1-哌𠯤乙磺酸(HEPES)緩衝液、三(羥基甲基)胺基甲烷(Tris)緩衝液、磷酸緩衝液、磷酸緩衝生理鹽水(PBS))。
於採用微小流體混合系統進行混合之情形時,較佳為混合相對於有機溶劑溶液1體積份為1~5體積份之水或緩衝液。又,於該系統中,混合液(有機溶劑溶液與水或緩衝液之混合液)流速較佳為0.1~10 mL/min,溫度較佳為4~45℃。
於以如上方式製造脂質粒子分散液時,藉由預先於水或緩衝液中添加編碼CAR或外源性TCR之核酸,可製造包含成分(a)~(d)之分散液。此處,該核酸較佳為以於水或緩衝液中之濃度成為0.05~2.0 mg/mL之方式添加。
又,本發明之脂質奈米粒子亦可藉由將脂質粒子分散液與該核酸利用自身公知方法進行混合而製造。 本發明之脂質奈米粒子中之該核酸之含量較佳為1~20重量%。該含量可採用Quant-iTTM Ribogreen(註冊商標)(Invitrogen)進行測定。又,本發明之脂質奈米粒子中之該核酸之包封率可根據因有無添加界面活性劑(例如Triton-X100)而產生之螢光強度之差來算出。
又,本發明之脂質奈米粒子亦可藉由將脂質粒子分散液與該核酸利用自身公知方法進行混合而製造。 本發明之脂質奈米粒子中之該核酸之含量較佳為1~20重量%。該含量可採用Quant-iTTM Ribogreen(註冊商標)(Invitrogen)進行測定。又,本發明之脂質奈米粒子中之該核酸之包封率可根據因有無添加界面活性劑(例如Triton-X100)而產生之螢光強度之差來算出。
分散介質可藉由透析而置換至水或緩衝液中。透析係使用區分分子量10~20K之超過濾膜,於4℃~室溫下實施。可反覆進行透析。透析亦可利用切向流過濾(Tangential Flow Filtration)。
藉由如上方式獲得之本發明之脂質奈米粒子中之核酸與脂質之比率(重量比)為約0.01~約0.2。
本發明之脂質奈米粒子之平均粒徑較佳為10~200 nm。該脂質粒子之平均粒徑例如可藉由使用Zetasizer Nano ZS(Malvern Instruments)進行自相關函數之累積量解析而算出。
3. 導入有本發明之脂質奈米粒子之活體外免疫細胞
本發明提供一種藉由使本發明之脂質奈米粒子與自生物體採集之免疫細胞(於本說明書中亦稱為「活體外免疫細胞」)接觸,而向該T細胞內導入編碼CAR或外源性TCR之核酸,從而製造表現該CAR或外源性TCR之活體外免疫細胞之方法,以及藉由該方法獲得之活體外免疫細胞。此處,作為「免疫細胞」,只要為具有能夠藉由某種作用機制抑制癌細胞等靶細胞(病原細胞)之能力之細胞(所謂免疫效應細胞),則並無特別限制,例如可列舉:負責獲得性免疫中之細胞免疫之T細胞、或者負責自然免疫之NK細胞、單核球、巨噬細胞、樹狀細胞等、以及作為具有NK細胞性質之T細胞之NKT細胞等。於一較佳實施態樣中,免疫細胞可為T細胞。於本說明書中,自生物體採集之T細胞亦稱為「活體外T細胞」。另一方面,於另一較佳實施態樣中,免疫細胞可為NK細胞、巨噬細胞、樹狀細胞等負責自然免疫之細胞。認為,T細胞即便於HLA型一致之情況下,亦在相當程度上有因同種異系(異體)移植而引起GVHD(Graft-versus-host disease,移植物抗宿主疾病)之風險,相對於此,異體NK細胞等則不會引起GVHD。因此,若預先準備各種HLA型之異體活體外免疫細胞,則能夠現成(off-the-shelf)地使用。關於CAR-NK細胞,例如於US2016/0096892、Mol Ther. 25(8): 1769-1781 (2017)等中有記載,關於CAR-樹狀細胞、CAR-巨噬細胞等,例如於WO2017/019848、eLIFE. 2018 e36688等中有記載。 又,於另一形態下,本發明提供一種含有本發明之脂質奈米粒子而成之CAR或外源性TCR之表現誘導用組合物。
作為導入本發明之脂質奈米粒子之免疫細胞(例如T細胞),若是包含免疫細胞(例如T細胞)或其前驅細胞之細胞群,則可為經單離之特定之免疫細胞(例如T細胞),亦可為例如淋巴細胞或包含多能性細胞之淋巴細胞之前驅細胞之類的非均質細胞群。於本發明中,所謂「淋巴細胞」意指脊椎動物之免疫系統中之白血球之亞型之一,作為淋巴細胞,可列舉:T細胞、B細胞、自然殺手細胞(NK細胞)。較佳為經單離純化之T細胞。於本發明中,所謂「T細胞」意指於淋巴器官或末梢血液中等可見之白血球之一種,具有主要於胸腺分化成熟而表現TCR之特徵的一類淋巴細胞。作為本發明可使用之T細胞,例如可列舉:作為CD8陽性細胞之細胞毒殺性T細胞(CTL)、作為CD4陽性細胞之輔助性T細胞、抑制性T細胞、效應T細胞等,較佳為細胞毒殺性T細胞。
上述淋巴細胞可自人或非人哺乳動物之例如末梢血液、骨髓及臍帶血進行採集。於將導入有本發明之脂質奈米粒子之活體外免疫細胞(例如活體外T細胞)用於癌等疾病之治療之情形時,該細胞群較佳為自治療對象本人、或與治療對象HLA型一致之供體採集。
作為包含多能性細胞之淋巴細胞之前驅細胞,例如可列舉:胚胎幹細胞(embryonic stem cell:ES細胞)、人工多能性幹細胞(induced pluripotent stem cell:iPS細胞)、胚胎腫瘤細胞(EC細胞)、胚胎生殖幹細胞(EG細胞)、造血幹細胞、喪失自我複製能力之多能性前驅細胞(multipotent progenitor:MMP)、骨髓共同淋巴樣前驅細胞(common myelo-lymphoid progenitor)(MLP)、骨髓系前驅細胞(MP)、粒細胞單核前驅細胞(GMP)、巨噬細胞-樹狀細胞前驅細胞(MDP)、樹狀細胞前驅細胞(DCP)等。可藉由自身公知方法使多能性細胞等未分化細胞分化為各種免疫細胞、例如T細胞。
使本發明之脂質奈米粒子與活體外免疫細胞接觸之方法並無特別限定,例如於通常之免疫細胞之培養基中添加本發明之脂質奈米粒子即可。或為了提高導入效率,可併用例如磷酸鈣共沈澱法、PEG法、電穿透法、顯微注射法、脂轉染法等。
尤其於本發明之脂質奈米粒子包含編碼外源性TCR之核酸作為活性成分之情形時,就外源性TCR之表現上升、錯配TCR之出現之抑制、或自反應性之抑制之觀點而言,可利用siRNA抑制該T細胞原本表現之內源性之TCRα鏈及TCRβ鏈之表現。於將上述核酸應用於該方法之情形時,為了避免siRNA對外源性TCR產生效果,較佳為將編碼該TCR之核酸之鹼基元列設為與抑制內源性之TCRα鏈及TCRβ鏈之表現之siRNA所作用之RNA對應之鹼基元列不同的序列(密碼子改變型序列)。該等方法例如記載於國際公開第2008/153029號。上述鹼基元列可藉由對天然取得之編碼TCR之核酸導入沉默突變、或化學合成人工設計之核酸而製作。或為了避免與內源性之TCR鏈發生錯配,可將編碼外源性TCR之核酸之恆定區之部分或整體置換為源自人以外之動物、例如小鼠之恆定區。
4. 含有本發明之脂質奈米粒子、或導入有該脂質奈米粒子之活體外免疫細胞而成之醫藥
本發明提供一種含有本發明之脂質奈米粒子、或導入有該脂質奈米粒子之活體外免疫細胞(例如活體外T細胞)而成之醫藥(以下簡記為「本發明之醫藥」)。 (4-1.含有導入有本發明之脂質奈米粒子之活體外免疫細胞而成之醫藥) 導入有本發明之脂質奈米粒子之免疫細胞(例如T細胞)藉由表現CAR或外源性TCR,可特異性識別表現該CAR或外源性TCR所特異性識別之表面抗原之細胞而將其殺傷(例如誘導細胞凋亡)。因此,藉由含有編碼識別作為表面抗原之於癌細胞等疾病細胞上特異性表現或表現亢進之表面分子之CAR或外源性TCR之核酸作為活性成分,導入有本發明之脂質奈米粒子之活體外免疫細胞可用於癌等疾病之預防或治療,可安全地對哺乳動物(人或其他哺乳動物(例如:小鼠、大鼠、倉鼠、兔、貓、狗、牛、羊、猴)。較佳為人)進行投予。
(4-2.含有本發明之脂質奈米粒子而成之醫藥) 含有本發明之脂質奈米粒子而成之本發明之醫藥較佳為將該脂質奈米粒子與公知之藥學上容許之載體(包括:賦形劑、稀釋劑、增量劑、結合劑、潤滑劑、流動助劑、崩解劑、界面活性劑等)或常用之添加劑等進行混合而以醫藥組合物之形式製備。賦形劑可列舉業者熟知之例如磷酸緩衝生理鹽水(例如0.01M磷酸鹽、0.138M NaCl、0.0027M KCl,pH值7.4),含有鹽酸鹽、氫溴酸鹽、磷酸鹽、硫酸鹽等無機酸鹽之水溶液,生理鹽液,二醇或乙醇等溶液,及乙酸鹽、丙酸鹽、丙二酸鹽、苯甲酸鹽等有機酸之鹽等。又,亦可使用濕潤劑或乳化劑等輔助劑、及pH緩衝劑。進而,亦可使用懸浮劑、保存劑、穩定化劑及分散劑等製劑輔助劑等。又,上述醫藥組合物亦可為用於在使用前利用適宜之無菌液體進行再構成之乾燥形態。該醫藥組合物可根據製備之形態(錠劑、丸劑、膠囊劑、散劑、顆粒劑、糖漿劑、乳劑、懸浮液等經口投予劑;注射劑、點滴劑、外用劑、栓劑等非經口投予劑)等而實施全身或局部、經口投予或非經口投予。於非經口投予之情形時,可實施靜脈投予、皮內投予、皮下投予、直腸投予、經皮投予等。又,於以注射劑型使用之情形時,亦可添加容許之緩衝劑、溶解輔助劑、等張劑等。
作為含有本發明之脂質奈米粒子而成之本發明之醫藥之投予量,例如每次平均每1 kg體重,於以編碼CAR或外源性TCR之核酸量計0.001 mg~10 mg之範圍內投予。例如於對人患者投予之情形時,對體重60 kg之患者於0.001~50 mg之範圍內投予。上述投予量為例示,可根據所使用之核酸之種類或投予路徑、投予對象或患者之年齡、體重、症狀等適當選擇投予量。
藉由對哺乳動物(人或其他哺乳動物(例如:小鼠、大鼠、倉鼠、兔、貓、狗、牛、羊、猴)。較佳為人)投予含有本發明之脂質奈米粒子而成之本發明之醫藥,可於該動物體內之免疫細胞、例如T細胞(於本說明書中亦稱為「活體內免疫細胞」、「活體內T細胞」)中誘導CAR或外源性TCR之表現,該活體內免疫細胞特異性識別表現CAR或外源性TCR所靶向之表面抗原之癌細胞等而將該疾病細胞殺傷,藉此顯示針對該疾病之預防或治療效果。
於以導入有本發明之脂質奈米粒子之活體外免疫細胞作為活性成分之醫藥之情形時,在將該免疫細胞投予至對象前可使用適宜之培養基及/或刺激分子進行培養及/或刺激。作為刺激分子,可列舉:細胞激素類、適宜之蛋白質、其他成分等,但並不限定於該等。例如於T細胞之情形時,作為細胞激素類,例如例示IL-2、IL-7、IL-12、IL-15、IFN-γ等,可較佳地使用IL-2。IL-2於培養基中之濃度並無特別限定,例如宜為0.01~1×105
U/mL,更宜為1~1×104
U/mL。又,作為適宜之蛋白質,例如例示CD3配體、CD28配體、抗IL-4抗體。又,此外,亦可添加凝集素等淋巴細胞刺激因子。進而,亦可於培養基中添加血清或血漿。該等於培養基中之添加量並無特別限定,例示0體積%~20體積%,又,可根據培養階段相應變更血清或血漿之使用量。亦可例如階段性減小血清或血漿濃度地使用。作為血清或血漿之來源,自體或非自體均可,但就安全性之觀點而言,較佳為源於自體者。
以導入有本發明之脂質奈米粒子之活體外免疫細胞作為活性成分之醫藥較佳為非經口地投予至對象而使用。作為非經口之投予方法,可列舉:靜脈內、動脈內、肌肉內、腹腔內、及皮下投予等方法。投予量根據對象之狀態、體重、年齡等適當選擇,通常以細胞數計,對於體重60 kg之對象,每次以通常1×106
~1×1010
個、較佳為1×107
~1×109
個、更佳為5×107
~5×108
個之方式投予。又,可一次性投予,亦可分複數次投予。可將以導入有本發明之脂質奈米粒子之活體外免疫細胞作為活性成分之本發明之醫藥製成適於非經口投予之公知形態,例如注射或注入劑。該醫藥可適當包含藥理學上容許之賦形劑。作為藥理學上容許之賦形劑,可列舉上述所記載者。為了穩定地維持細胞,該醫藥亦可包含生理鹽水、磷酸緩衝生理鹽水(PBS)、培養基等。作為培養基,並無特別限定,可列舉:RPMI、AIM-V、X-VIVO10等培養基,但並不限定於該等。又,亦可基於穩定化之目的而對該醫藥添加醫藥學上容許之載體(例如:人血清白蛋白)、保存劑等。
本發明之醫藥可作為癌之預防或治療藥。本發明之醫藥之適用對象之癌並無特別限制,例如可列舉:急性淋巴細胞性癌、腺泡狀橫紋肌肉瘤、膀胱癌、骨癌、腦癌(例如神經管胚細胞瘤)、乳癌、肛門、肛門廔管或肛門直腸之癌、眼癌、肝內膽管之癌、關節癌、頸部、膽囊或胸膜之癌、鼻、鼻腔或中耳之癌、口腔癌、外陰癌、慢性骨髄性癌、結腸癌、食道癌、子宮頸癌、纖維肉瘤、消化道類癌腫瘤、頭頸部癌(例如頭頸部扁平上皮癌)、下咽頭癌、腎癌、喉頭癌、白血病(例如急性淋巴母細胞性白血病、急性淋巴細胞性白血病、慢性淋巴細胞性白血病、急性骨髄性白血病)、液性腫瘤、肝癌、肺癌(例如非小細胞肺癌)、淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤、彌漫性大細胞型B細胞淋巴瘤、濾泡性淋巴瘤)、惡性間皮瘤、肥胖細胞瘤、黑色素瘤、多發性骨髓瘤、上咽頭癌、卵巢癌、胰腺癌;腹膜、網膜及腸間膜之癌;咽頭癌、前列腺癌、直腸癌、腎癌、皮膚癌、小腸癌、軟組織癌、實體腫瘤、胃癌、睾丸癌、甲狀腺癌、尿管癌等,但並不限定於該等。
以下,列舉實施例而更具體地說明本發明,但該等僅為例示,本發明並不限定於該等。
[實施例]
[實施例]
實施例1 (抗體之還原處理) 於9.21 mg/ml之抗CD3抗體(Bio X Cell公司)111 μl中混合10 mM之DTT(Dithiothreitol,二硫蘇糖醇)水溶液12.3 μl。相同地,於6.73 mg/ml之IgG2a抗體(Bio X Cell公司)149 μl中混合10 mM之DTT水溶液16.6 μl。各抗體與DTT之混合液係利用vortex進行混合,於室溫下進行30分鐘反應。利用HPLC(管柱:TSKgel G2000SWXL 7.8 mm×30 cm,TOSOH公司,流動相:PBS)對反應液進行分餾,而獲得包含還原抗體之分取液。使用Amicon 0.5 ml-10K對分取液進行超濃縮。濃縮液中之抗體蛋白質濃度及硫醇基濃度分別藉由230 nm之吸光度及使用N-(7-二甲基胺基-4-甲基香豆素-3-基)順丁烯二醯亞胺(DACM)之螢光呈色反應而測定。再者,還原抗CD3抗體之產量為176 μl,蛋白質濃度1.75 mg/ml,硫醇基濃度5.14 μM;還原IgG2a抗體之產量為86 μl,蛋白質濃度5.19 mg/ml,硫醇基濃度45.1 μM。
實施例2 (順丁烯二醯亞胺(Maleimide)-LNP之製備) 將脂質混合物(陽離子性脂質:DPPC:膽固醇:SUNBRIGHT GM-020:SUNBRIGHT DSPE-020MA=60:10.6:28:1.4:1(莫耳比))溶解於90% EtOH、10% 水中,而獲得7.0 mg/ml之脂質溶液。作為陽離子性脂質,以59.1:0.9(莫耳比)混合使用WO2016/021683中記載之3-戊基辛酸3-((5-(二甲基胺基)戊醯基)氧基)-2,2-雙(((3-戊基辛醯基)氧基)甲基)丙基酯(化合物7)與N,N,N-三甲基-5-側氧基-5-(3-((3-戊基辛醯基)氧基)-2,2-雙(((3-戊基辛醯基)氧基)甲基)丙氧基)戊烷-1-碘化鋁(化合物8)。使編碼具有4-1BB與CD3ζ作為細胞內訊號傳遞區之靶向CD19之CAR的mRNA溶解於10 mM 2-𠰌啉基乙磺酸(MES)緩衝液(pH值5.0)中而獲得0.2 mg/ml之核酸溶液。於室溫下利用Nanoassemblr裝置(Precision Nanosystems公司)將所獲得之脂質溶液及核酸溶液以流速比3 ml/min:6 ml/min進行混合,而獲得包含組合物之分散液。對於所獲得之分散液,使用Slyde-Α-Lyzer(20k之區分分子量,Thermo Scientific公司),於水中在室溫下進行1小時透析,於PBS中在4℃下進行48小時透析。繼而,使用0.2 μm之針筒過濾器(syringe filter)(Iwaki)進行過濾,以4℃保存。
實施例3 (還原抗體與順丁烯二醯亞胺-LNP之結合反應) 於順丁烯二醯亞胺-LNP分散液中以還原抗體相對於順丁烯二醯亞胺之莫耳濃度成為1/20之方式混合還原抗體溶液,於室溫下靜置4小時。其後,以4℃保管直至純化步驟。
實施例4 (抗體-LNP之凝膠過濾純化) 將還原抗體與順丁烯二醯亞胺-LNP之反應液載入凝膠過濾管柱Sepharose CL-4B(Cat N0.17-0150-01/GE Healthcare),以D-PBS(-)作為流動相,進行分餾。繼而,藉由測定各餾份(fraction)之蛋白質濃度而鑑定包含目標抗體-LNP之組份。利用0.2 μm之針筒過濾器對抗體-LNP進行過濾,以4℃保存。
實施例5 (向CD8+ T細胞之活體外轉染) 採集C57BL/6J小鼠之脾臟,分散於ACK裂解緩衝液(lysing buffer)(Biosource)中,藉此獲得小鼠脾細胞。將所獲得之小鼠脾細胞於含有1 ng/ml介白素(interleukin)7及2 μg/ml刀豆素(concavalin)A之RPMI-1640完全培養基(complete RPMI 1640 Medium)中培養2天,藉由利用Ficoll密度梯度離心去除死細胞及使用CD8陰性分選套組(Negative Isolation Kit)(Stemcell Technologies)進行處理,而分離小鼠CD8+ T細胞。使所獲得之小鼠CD8+ T細胞分散於含有10 ng/ml介白素2及抗體-LNP之RPMI-1640完全培養基中進行培養,藉此使小鼠CD8+ T細胞轉染CAR或外源性TCR。 藉由相同方式自購入之人初代培養T細胞分離CD8+ T細胞,使人CD8+ T細胞轉染CAR或外源性TCR。
實施例6
(CAR-T細胞之試管內(in vitro)細胞抑制性評價) 將作為抑制性評價細胞之強制表現CD19之人慢性骨髄性白血病細胞株K562細胞利用膜染料(membrane dye)PKH-26(Sigma-Aldrich)進行標記,利用包含10%胎牛血清(fetal calf serum)之RPMI培養基清洗後,以1×10^5 cells/ml分散於相同之培養基中進行培養。將經標記之抑制性評價細胞分注於96孔盤中進行培養,混合CAR-T細胞後於37℃下培養3小時,使用磷脂結合蛋白V-亮紫421(Annexin V-Brilliant Violet 421)(BioLegend)染色後進行流式細胞儀分析,藉此定量發生細胞凋亡之細胞。
(CAR-T細胞之試管內(in vitro)細胞抑制性評價) 將作為抑制性評價細胞之強制表現CD19之人慢性骨髄性白血病細胞株K562細胞利用膜染料(membrane dye)PKH-26(Sigma-Aldrich)進行標記,利用包含10%胎牛血清(fetal calf serum)之RPMI培養基清洗後,以1×10^5 cells/ml分散於相同之培養基中進行培養。將經標記之抑制性評價細胞分注於96孔盤中進行培養,混合CAR-T細胞後於37℃下培養3小時,使用磷脂結合蛋白V-亮紫421(Annexin V-Brilliant Violet 421)(BioLegend)染色後進行流式細胞儀分析,藉此定量發生細胞凋亡之細胞。
實施例7
(活體內抗癌活性評價試驗) 對6週齡之NOD-SCID小鼠於尾靜脈投予穩定表現螢光素酶之K562-CD19細胞,經過1週飼育而製作小鼠血液癌模型。繼而,每週一次、連續三週於尾靜脈投予藉由使用抗體-LNP於活體外轉染編碼CAR之核酸而獲得之人CAR-T細胞1×10^6 cells,利用活體內發光成像系統IVIS(PerkinElmer)進行測定,評價因CAR-T細胞而產生之癌細胞減少情況。
(活體內抗癌活性評價試驗) 對6週齡之NOD-SCID小鼠於尾靜脈投予穩定表現螢光素酶之K562-CD19細胞,經過1週飼育而製作小鼠血液癌模型。繼而,每週一次、連續三週於尾靜脈投予藉由使用抗體-LNP於活體外轉染編碼CAR之核酸而獲得之人CAR-T細胞1×10^6 cells,利用活體內發光成像系統IVIS(PerkinElmer)進行測定,評價因CAR-T細胞而產生之癌細胞減少情況。
實施例8
(使用陽離子性脂質之順丁烯二醯亞胺-LNP之製備)
將脂質混合物(陽離子性脂質:DPPC:膽固醇:SUNBRIGHT GM-020:SUNBRIGHT DSPE-020MA=60:10.6:28:1.4:1(莫耳比))溶解於90% EtOH、10% 25 mM乙酸緩衝液(pH值4.0)中,而獲得10 mg/ml之脂質溶液。作為陽離子性脂質,使用(9Z)-十四-9-烯酸3-((5-(二甲基胺基)戊醯基)氧基)-2,2-雙(((9Z)-十四-9-烯醯氧基)甲基)丙基酯(化合物12)、(9Z,9'Z)雙-十四-9-烯酸2-(((4-(二甲基胺基)丁醯基)氧基)甲基)-2-((十二醯氧基)甲基)丙烷-1,3-二基酯(化合物21)、及二癸酸2-(((4,5-二丁基壬氧基)氧基)甲基)-2-(((5-(二甲基胺基)戊醯基)氧基)甲基)丙烷-1,3-二基酯(化合物35)。使編碼靶向CD19之CAR之pcDNA3.1-hCD19CAR溶解於10 mM 2-𠰌啉基乙磺酸(MES)緩衝液(pH值5.5)中而獲得0.2 mg/ml之核酸溶液。再者,pcDNA3.1-hCD19CAR係將自WO2013/126712引用之CD19 IgG4 28z序列組入至pcDNA3.1(Thermo Fisher Scientific)之多選殖位點(multi cloning site)而製作。於室溫下利用Nanoassemblr裝置(Precision Nanosystems公司)將所獲得之脂質溶液及核酸溶液以流速比3 ml/min:6 ml/min進行混合,而獲得包含組合物之分散液。對於所獲得之分散液,使用Slyde-Α-Lyzer(20k之區分分子量,Thermo Scientific公司),於水中在室溫下進行1小時透析,於PBS中在4℃下進行48小時透析。繼而,使用0.2 μm之針筒過濾器(Iwaki)進行過濾,以4℃保存。
(使用陽離子性脂質之順丁烯二醯亞胺-LNP之製備)
將脂質混合物(陽離子性脂質:DPPC:膽固醇:SUNBRIGHT GM-020:SUNBRIGHT DSPE-020MA=60:10.6:28:1.4:1(莫耳比))溶解於90% EtOH、10% 25 mM乙酸緩衝液(pH值4.0)中,而獲得10 mg/ml之脂質溶液。作為陽離子性脂質,使用(9Z)-十四-9-烯酸3-((5-(二甲基胺基)戊醯基)氧基)-2,2-雙(((9Z)-十四-9-烯醯氧基)甲基)丙基酯(化合物12)、(9Z,9'Z)雙-十四-9-烯酸2-(((4-(二甲基胺基)丁醯基)氧基)甲基)-2-((十二醯氧基)甲基)丙烷-1,3-二基酯(化合物21)、及二癸酸2-(((4,5-二丁基壬氧基)氧基)甲基)-2-(((5-(二甲基胺基)戊醯基)氧基)甲基)丙烷-1,3-二基酯(化合物35)。使編碼靶向CD19之CAR之pcDNA3.1-hCD19CAR溶解於10 mM 2-𠰌啉基乙磺酸(MES)緩衝液(pH值5.5)中而獲得0.2 mg/ml之核酸溶液。再者,pcDNA3.1-hCD19CAR係將自WO2013/126712引用之CD19 IgG4 28z序列組入至pcDNA3.1(Thermo Fisher Scientific)之多選殖位點(multi cloning site)而製作。於室溫下利用Nanoassemblr裝置(Precision Nanosystems公司)將所獲得之脂質溶液及核酸溶液以流速比3 ml/min:6 ml/min進行混合,而獲得包含組合物之分散液。對於所獲得之分散液,使用Slyde-Α-Lyzer(20k之區分分子量,Thermo Scientific公司),於水中在室溫下進行1小時透析,於PBS中在4℃下進行48小時透析。繼而,使用0.2 μm之針筒過濾器(Iwaki)進行過濾,以4℃保存。
(順丁烯二醯亞胺-LNP之核酸濃度測定、與順丁烯二醯亞胺推測濃度之算出)
利用0.5%曲拉通(Triton)X-100溶解順丁烯二醯亞胺-LNP,使用Quant-iT™ PicoGreen™雙鏈DNA檢測套組(dsDNA Assay Kit)(Thermo Fisher Scientific)測定pDNA濃度。又,將未添加曲拉通X-100而測得之pDNA濃度設為未經LNP包封之pDNA之濃度,算出pDNA向LNP之包封率。順丁烯二醯亞胺推測濃度係藉由所測得之pDNA濃度乘以順丁烯二醯亞胺-PEG-脂質(DSPE-020MA)之添加比率而算出。所獲得之各值示於表1。
利用0.5%曲拉通(Triton)X-100溶解順丁烯二醯亞胺-LNP,使用Quant-iT™ PicoGreen™雙鏈DNA檢測套組(dsDNA Assay Kit)(Thermo Fisher Scientific)測定pDNA濃度。又,將未添加曲拉通X-100而測得之pDNA濃度設為未經LNP包封之pDNA之濃度,算出pDNA向LNP之包封率。順丁烯二醯亞胺推測濃度係藉由所測得之pDNA濃度乘以順丁烯二醯亞胺-PEG-脂質(DSPE-020MA)之添加比率而算出。所獲得之各值示於表1。
[表1]
(2種混合還原抗體與順丁烯二醯亞胺-LNP之結合反應)
將使用DTT還原之抗人CD3抗體(anti-human CD3 antibody)(BE0001-2,BioXCell)及抗人/猴CD28抗體(anti-human/monkey CD28 antibody)(BE0248,BioXCell)等量地混合,以相對於順丁烯二醯亞胺-LNP之順丁烯二醯亞胺成為1/20莫耳量之方式進行混合。順丁烯二醯亞胺-LNP與還原抗體之濃度與體積示於表2。將混合液於室溫下靜置4小時後,以4℃保管直至純化步驟。
將使用DTT還原之抗人CD3抗體(anti-human CD3 antibody)(BE0001-2,BioXCell)及抗人/猴CD28抗體(anti-human/monkey CD28 antibody)(BE0248,BioXCell)等量地混合,以相對於順丁烯二醯亞胺-LNP之順丁烯二醯亞胺成為1/20莫耳量之方式進行混合。順丁烯二醯亞胺-LNP與還原抗體之濃度與體積示於表2。將混合液於室溫下靜置4小時後,以4℃保管直至純化步驟。
[表2]
(抗體-LNP之凝膠過濾純化)
將還原抗體與順丁烯二醯亞胺-LNP之反應液載入凝膠過濾管柱Sepharose CL-4B(Cat N0.17-0150-01/GE Healthcare),以D-PBS(-)作為流動相,進行分餾。繼而,藉由測定各餾份之蛋白質濃度而鑑定包含目標抗體-LNP之組份。利用0.2 μm之針筒過濾器對抗體-LNP進行過濾,以4℃保存。所獲得之抗體-LNP之粒徑係利用Zetasizer Nano ZS(Malvern Panalytical)進行測定。核酸濃度與抗體蛋白質濃度係分別使用Quant-iT™ PicoGreen™雙鏈DNA檢測套組(Thermo Fisher Scientific)及ATTO-TAG™ FQ胺-衍生化套組(Amine-Derivatization Kit)(Thermo Fisher Scientific)進行測定。各分析結果之值示於表3。
將還原抗體與順丁烯二醯亞胺-LNP之反應液載入凝膠過濾管柱Sepharose CL-4B(Cat N0.17-0150-01/GE Healthcare),以D-PBS(-)作為流動相,進行分餾。繼而,藉由測定各餾份之蛋白質濃度而鑑定包含目標抗體-LNP之組份。利用0.2 μm之針筒過濾器對抗體-LNP進行過濾,以4℃保存。所獲得之抗體-LNP之粒徑係利用Zetasizer Nano ZS(Malvern Panalytical)進行測定。核酸濃度與抗體蛋白質濃度係分別使用Quant-iT™ PicoGreen™雙鏈DNA檢測套組(Thermo Fisher Scientific)及ATTO-TAG™ FQ胺-衍生化套組(Amine-Derivatization Kit)(Thermo Fisher Scientific)進行測定。各分析結果之值示於表3。
[表3]
實施例9
(使用抗體-LNP之向人初代培養T細胞之CD19 CAR轉染試驗)
利用培養基將人泛性T細胞(Human Pan-T Cells)(AccuCell人外周血泛性T細胞(human peripheral blood pan-T cells),負向選擇(Negative selection))製備成1.1×106 cells/ml,以90 μl/well接種至96孔盤。培養基係使用以30 ng/ml之濃度添加有重組(recombinant)IL-2(Thermo Fisher Scientific)之X-VIVO10(Lonza)。繼而,於培養基中添加已利用PBS將pcDNA3.1-hCD19CAR濃度稀釋成30 μg/ml之抗體-LNP 10 μl,於37℃、5%CO2 培養箱中進行3天及6天之培養。
(使用抗體-LNP之向人初代培養T細胞之CD19 CAR轉染試驗)
利用培養基將人泛性T細胞(Human Pan-T Cells)(AccuCell人外周血泛性T細胞(human peripheral blood pan-T cells),負向選擇(Negative selection))製備成1.1×106 cells/ml,以90 μl/well接種至96孔盤。培養基係使用以30 ng/ml之濃度添加有重組(recombinant)IL-2(Thermo Fisher Scientific)之X-VIVO10(Lonza)。繼而,於培養基中添加已利用PBS將pcDNA3.1-hCD19CAR濃度稀釋成30 μg/ml之抗體-LNP 10 μl,於37℃、5%CO2 培養箱中進行3天及6天之培養。
(利用流式細胞儀之CD19CAR表現評價)
將於96孔盤中培養之人初代培養T細胞回收至1.5 ml管內,添加2 μl之重組人CD19蛋白(Recombinant human CD19 protein)(Fc Chimera Active)(生物素(Biotin))(Abcam),於冰上靜置30分鐘。繼而,藉由添加200 μl之含1%FBS之細胞清洗緩衝液(Cell Wash)(BD)及300×g、5分鐘離心而進行清洗,清洗2次後,去除上清液,使細胞分散於100 μl之1%FBS細胞清洗緩衝液。於細胞之分散液中添加0.2 μl之亮紫421鏈親和素(Brilliant Violet 421 Streptavidin),以移液(pipetting)方式進行混合後,於冰上靜置30分鐘。對經染色之細胞實施3次利用200 μl之1%FBS細胞清洗緩衝液及離心之清洗,進行過濾器過濾後,分散於200 μl之1%FBS細胞清洗緩衝液中,利用LSRFortessa(BD)進行流式細胞儀解析。
將已利用hCD3/hCD28-化合物12-pcDNA3.1-hCD19CAR進行轉染之人初代培養T細胞藉由流式細胞儀解析而獲得之CD19 CAR表現解析結果示於圖1。抗體-LNP添加後3天及6天之CD19 CAR陽性率分別為51.9%及41.7%,以與利用病毒載體之基因導入相比亦為充分之效率獲得CAR陽性細胞。
將已利用hCD3/hCD28-化合物21-pcDNA3.1-hCD19CAR及hCD3/hCD28-化合物35-pcDNA3.1-hCD19CAR進行轉染之人初代培養T細胞藉由流式細胞儀解析而獲得之CD19 CAR表現解析結果示於圖2。抗體-LNP添加後3天之CD19 CAR陽性率分別為5.12%及47.0%。
將於96孔盤中培養之人初代培養T細胞回收至1.5 ml管內,添加2 μl之重組人CD19蛋白(Recombinant human CD19 protein)(Fc Chimera Active)(生物素(Biotin))(Abcam),於冰上靜置30分鐘。繼而,藉由添加200 μl之含1%FBS之細胞清洗緩衝液(Cell Wash)(BD)及300×g、5分鐘離心而進行清洗,清洗2次後,去除上清液,使細胞分散於100 μl之1%FBS細胞清洗緩衝液。於細胞之分散液中添加0.2 μl之亮紫421鏈親和素(Brilliant Violet 421 Streptavidin),以移液(pipetting)方式進行混合後,於冰上靜置30分鐘。對經染色之細胞實施3次利用200 μl之1%FBS細胞清洗緩衝液及離心之清洗,進行過濾器過濾後,分散於200 μl之1%FBS細胞清洗緩衝液中,利用LSRFortessa(BD)進行流式細胞儀解析。
將已利用hCD3/hCD28-化合物12-pcDNA3.1-hCD19CAR進行轉染之人初代培養T細胞藉由流式細胞儀解析而獲得之CD19 CAR表現解析結果示於圖1。抗體-LNP添加後3天及6天之CD19 CAR陽性率分別為51.9%及41.7%,以與利用病毒載體之基因導入相比亦為充分之效率獲得CAR陽性細胞。
將已利用hCD3/hCD28-化合物21-pcDNA3.1-hCD19CAR及hCD3/hCD28-化合物35-pcDNA3.1-hCD19CAR進行轉染之人初代培養T細胞藉由流式細胞儀解析而獲得之CD19 CAR表現解析結果示於圖2。抗體-LNP添加後3天之CD19 CAR陽性率分別為5.12%及47.0%。
實施例10
(利用抗體-LNP轉染CD19 CAR之人初代培養T細胞之癌細胞抑制性評價)
將使用DELFIA細胞抑制性分析套組(Perkin Elmer)標記之人前B細胞(pre-B cell)株NALM-6、及人伯基特淋巴瘤細胞株Daudi作為靶細胞(target cell),以1×104 cell/100 μl/well之細胞密度接種至U形底96孔板(96-well U bottom plate)。培養基係使用含10%FBS之RPMI(無酚紅(phenol red free))。繼而,將藉由其他項目中記載之方法利用hCD3/hCD28-化合物12-pcDNA3.1-hCD19CAR轉染CD19CAR之人初代培養T細胞(抗體-LNP添加後3天之CD19 CAR陽性率為19%)作為效應細胞(effector cell),以與靶細胞之細胞數比成為0~16之方式分散添加於100 μl之培養基中。靶細胞與效應細胞混合後經過3小時後,回收20 μl之培養上清液。於所回收之培養上清液中添加20 μl之銪(Eu)溶液,根據自受到抑制之靶細胞釋出之螯合劑TDA與Eu之複合體所發出之螢光強度,計算細胞損傷率。
將藉由添加轉染CD19 CAR之人初代培養T細胞而產生之Nalm-6及Daudi之細胞損傷率示於圖3。
(利用抗體-LNP轉染CD19 CAR之人初代培養T細胞之癌細胞抑制性評價)
將使用DELFIA細胞抑制性分析套組(Perkin Elmer)標記之人前B細胞(pre-B cell)株NALM-6、及人伯基特淋巴瘤細胞株Daudi作為靶細胞(target cell),以1×104 cell/100 μl/well之細胞密度接種至U形底96孔板(96-well U bottom plate)。培養基係使用含10%FBS之RPMI(無酚紅(phenol red free))。繼而,將藉由其他項目中記載之方法利用hCD3/hCD28-化合物12-pcDNA3.1-hCD19CAR轉染CD19CAR之人初代培養T細胞(抗體-LNP添加後3天之CD19 CAR陽性率為19%)作為效應細胞(effector cell),以與靶細胞之細胞數比成為0~16之方式分散添加於100 μl之培養基中。靶細胞與效應細胞混合後經過3小時後,回收20 μl之培養上清液。於所回收之培養上清液中添加20 μl之銪(Eu)溶液,根據自受到抑制之靶細胞釋出之螯合劑TDA與Eu之複合體所發出之螢光強度,計算細胞損傷率。
將藉由添加轉染CD19 CAR之人初代培養T細胞而產生之Nalm-6及Daudi之細胞損傷率示於圖3。
實施例11 (還原Fab'之製備) 與Mleimide-LNP結合之還原Fab'係使用Pierce F(ab')2製備套組(Preparation Kit)(Thermo Fisher Scientific),由抗小鼠CD3ε抗體(anti-mouse CD3ε antibody)(BE0001-1,BioXCell)及抗小鼠CD28抗體(anti-mouse CD28 antibody)(BE0015-1,BioXCell)所製備。由7.86 mg/ml之抗小鼠CD3ε抗體0.5 ml獲得1 ml之1.75 mg/ml之F(ab')2。由3.86 mg/ml之抗小鼠CD28抗體0.5 ml獲得0.62 ml之0.97 mg/ml之F(ab')2。對各F(ab')2以40 mM之濃度混合作為還原劑之2-胺基乙硫醇對甲苯磺酸鹽,於37℃下靜置1小時。利用Zeba脫鹽離心柱(Spin Desalting Columns), 7K MWCO-0.5 ml(Thermo Fischer Scientific)純化所獲得之Fab',以4℃保存直至與順丁烯二醯亞胺-LNP進行反應。再者,Fab'之蛋白質濃度及硫醇基濃度分別藉由230 nm之吸光度及使用N-(7-二甲基胺基-4-甲基香豆素-3-基)順丁烯二醯亞胺(DACM)之螢光呈色反應而測定。
實施例12 (還原Fab'與順丁烯二醯亞胺-LNP之結合反應) 經還原之抗小鼠CD3ε Fab'及抗小鼠CD28 Fab'係以相對於藉由上述方法製備之順丁烯二醯亞胺-LNP之順丁烯二醯亞胺成為1/20莫耳量之方式進行混合。將混合液於室溫下靜置4小時後,以4℃保管直至純化步驟。
實施例13 (還原Fab'-LNP之凝膠過濾純化) 將還原Fab'與順丁烯二醯亞胺-LNP之反應液載入凝膠過濾管柱Sepharose CL-4B(Cat No.17-0150-01/GE Healthcare),以D-PBS(-)作為流動相,進行分餾。繼而,藉由測定各餾份之蛋白質濃度而鑑定包含目標Fab'-LNP之組份。利用0.2 μm之針筒過濾器對Fab'-LNP進行過濾,以4℃保存。所獲得之抗體-LNP之粒徑係利用Zetasizer Nano ZS(Malvern Panalytical)進行測定。核酸濃度與抗體蛋白質濃度係分別使用Quant-iT™ PicoGreen™雙鏈DNA檢測套組(Thermo Fisher Scientific)及ATTO-TAG™ FQ胺-衍生化套組(Thermo Fisher Scientific)進行測定。 [產業上之可利用性]
本發明之脂質奈米粒子不僅於活體外、且於活體內亦能夠效率良好地選擇性地向T細胞導入CAR或外源性TCR,因此,可提供製造成本較低之CAR-T或TCR-T細胞療法。又,由於不使用病毒載體,故可避免因病毒蛋白引起之抗原性問題,為一種極其有用之癌免疫療法之新穎平台(platform)。
本申請案係基於2017年12月27日於日本提出申請之日本專利申請2017-252616,至此將其全部內容歸入本說明書中。
圖1表示已利用hCD3/hCD28-化合物12-pcDNA3.1-hCD19CAR進行轉染之人初代培養T細胞藉由流式細胞儀解析而獲得之CD19 CAR表現解析結果。
圖2表示已利用hCD3/hCD28-化合物21-pcDNA3.1-hCD19CAR及hCD3/hCD28-化合物35-pcDNA3.1-hCD19CAR進行轉染之人初代培養T細胞藉由流式細胞儀解析而獲得之CD19 CAR表現解析結果。
圖3表示藉由添加轉染有CD19 CAR之人初代培養T細胞而產生之Nalm-6及Daudi之細胞損傷率。
Claims (34)
- 一種脂質奈米粒子,其包含以下之(a)~(c): (a)編碼嵌合抗原受體或外源性T細胞受體之核酸; (b)陽離子性脂質;及 (c)非陽離子性脂質。
- 如請求項1之脂質奈米粒子,其中上述陽離子性脂質為 式(I)所表示之化合物或其鹽: [式中, L1 為C1-22 伸烷基、C2-22 伸烯基或C3-22 伸二烯基, n為0或1之整數, R1 為 氫原子、 可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、 可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者 可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基, R2 為-CH2 -O-CO-R5 、-CH2 -CO-O-R5 或-R5 , R3 為-CH2 -O-CO-R6 、-CH2 -CO-O-R6 或-R6 , R4 為氫原子、-CH2 -O-CO-R7 、-CH2 -CO-O-R7 或-R7 , R5 、R6 及R7 分別獨立為 (1)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C1-22 烷基、 (2)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C2-22 烯基、或者 (3)可經選自直鏈狀之C1-22 烷基及直鏈狀之C2-22 烯基中之1個或2個取代基取代之直鏈狀之C3-22 二烯基, R8 及R9 分別獨立表示C1-6 烷基]。
- 如請求項1之脂質奈米粒子,其中上述核酸為mRNA或DNA。
- 如請求項1之脂質奈米粒子,其中上述非陽離子性脂質為磷脂質、膽固醇及/或PEG脂質。
- 如請求項1之脂質奈米粒子,其中上述脂質奈米粒子於表面具有能夠靶向T細胞之配體。
- 如請求項5之脂質奈米粒子,其中上述配體為包含選自由針對CD3之抗體、針對CD4之抗體、針對CD8之抗體及針對CD28之抗體所組成之群中之1種以上之抗體之抗原結合區的配體。
- 如請求項5之脂質奈米粒子,其中上述配體為包含針對CD3之抗體及/或針對CD28之抗體之抗原結合區的配體。
- 如請求項5之脂質奈米粒子,其中上述配體為包含針對CD3之抗體及針對CD28之抗體之抗原結合區的配體。
- 一種醫藥,其含有如請求項1之脂質奈米粒子而成。
- 如請求項9之醫藥,其係癌之預防或治療藥。
- 如請求項9之醫藥,其向活體內免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並誘導其之表現。
- 如請求項9之醫藥,其向活體內T細胞中基因導入嵌合抗原受體或外源性T細胞受體並誘導其之表現。
- 一種向哺乳動物之活體內免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:對該哺乳動物投予如請求項1之脂質奈米粒子。
- 一種向哺乳動物之活體內T細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:對該哺乳動物投予如請求項1之脂質奈米粒子。
- 一種針對哺乳動物之癌之預防或治療方法,其特徵在於:對該哺乳動物投予如請求項1之脂質奈米粒子。
- 如請求項1之脂質奈米粒子,其用於癌之預防、治療。
- 一種如請求項1之脂質奈米粒子之用途,其用於製造癌之預防、治療劑。
- 一種嵌合抗原受體或外源性T細胞受體表現誘導用組合物,其含有如請求項1之脂質奈米粒子而成。
- 一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,其係於包含活體外免疫細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,其係於包含活體外T細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種醫藥,其含有表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞而成,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種醫藥,其含有表現嵌合抗原受體或外源性T細胞受體之活體外T細胞而成,該活體外T細胞係於包含活體外T細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 如請求項21之醫藥,其係癌之預防或治療藥。
- 如請求項21之醫藥,其係細胞凋亡誘導藥。
- 一種向活體外免疫細胞中基因導入嵌合抗原受體或外源性T細胞受體並使之表現之方法,其特徵在於:於包含活體外免疫細胞之培養液中添加如請求項1之脂質奈米粒子。
- 一種使活體外T細胞表現嵌合抗原受體或外源性T細胞受體之方法,其特徵在於:於包含活體外T細胞之培養液中添加如請求項1之脂質奈米粒子。
- 一種癌之預防或治療方法,其特徵在於:對哺乳動物投予表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種癌之預防或治療方法,其特徵在於:對哺乳動物投予表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,該活體外T細胞係於包含活體外T細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞,其用於癌之預防、治療,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞,其用於癌之預防、治療,該活體外T細胞係於包含活體外T細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞之用途,其用於製造癌之預防、治療劑,該活體外免疫細胞係於包含活體外免疫細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種表現嵌合抗原受體或外源性T細胞受體之活體外T細胞之用途,其用於製造癌之預防、治療劑,該活體外免疫細胞係於包含活體外T細胞之培養液中添加如請求項1之脂質奈米粒子而獲得。
- 一種含有表現嵌合抗原受體或外源性T細胞受體之活體外免疫細胞而成之醫藥之製造方法,其包括如下步驟:於包含活體外免疫細胞之培養液中添加如請求項1之脂質奈米粒子。
- 一種含有表現嵌合抗原受體或外源性T細胞受體之活體外T細胞而成之醫藥之製造方法,其包括如下步驟:於包含活體外T細胞之培養液中添加如請求項1之脂質奈米粒子。
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