TW202110875A - 病毒載體及其在授受性細胞療法之應用 - Google Patents
病毒載體及其在授受性細胞療法之應用 Download PDFInfo
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Abstract
一種載體,含有:編碼蛋白質Z1之第一核苷酸序列S1;編碼蛋白質Z2之第二核苷酸序列S2;編碼蛋白質Y1之第三核苷酸序列S3;及編碼蛋白質Y2之第四核苷酸序列S4,其中Z1及Z2形成第一二聚體且Y1及Y2形成第二二聚體,其中該第一二聚體Z1Z2不同於該第二二聚體Y1Y2。
Description
本發明係關於T細胞製造。在一態樣中,本發明係關於使用多順反子盒在單一載體中表現複數種蛋白之T細胞製造。更確切地,本發明係關於共表現TCRαβ及CD8αβ之T細胞之T細胞製造及該等T細胞在接受性細胞療法之應用。
作為遺傳性、獲得性或傳染性疾病之潛在療法的人類淋巴球之基因工程需要轉基因之有效轉移及表現。在針對癌症之接受性免疫療法之情況下,自然存在及/或重組之抗瘤T細胞受體(T-cell receptor;TCR)已被用於賦予正常T細胞或腫瘤浸潤之淋巴球抗瘤反應性。
Morgan等人(J Immunol
。2003年9月15日;171(6): 3287–32百分比)揭示由雙順反子RNA表現之抗gp100 TCR,其中編碼TCRβ鏈之第一基因之表現由末端長重複(long terminal repeat;LTR)控制且編碼TCRα鏈之第二基因由內部核糖體進入位點(internal ribosome entry site;IRES)操縱。用此抗gp100 TCR基因工程化之CD4+ T細胞係抗原反應性的。
Cohen等人(J Immunol
。2005年11月1日;175(9): 5799–5808)揭示用於共表現連結p53表位之TCRα鏈及TCRβ鏈兩者之雙順反子逆轉錄病毒載體。編碼TCRα鏈之第一基因之表現由LTR控制且編碼TCRβ鏈之第二基因由IRES操縱。p53 TCR轉導之淋巴球能夠利用高結合性(avidity)特別地辨識肽脈衝APC以及用野生型或突變型p53蛋白轉染之HLA-A2.1+細胞。
Hughes等人(Hum Gene Ther
。2005年4月;16(4): 457–472)揭示用於共表現抗MART-1 TCR之各種雙順反子逆轉錄病毒載體。編碼TCRα鏈之第一基因之表現由LTR控制且編碼TCRβ鏈之第二基因由IRES操縱,或反之亦然。另外,編碼TCRα鏈之第一基因之表現由LTR控制且編碼TCRβ鏈之第二基因由PGK啟動子操縱,或反之亦然。用此等載體轉導之T細胞展示出高活性T細胞效應物功能。
Zhao等人(J Immunol
。2005年4月1日;174(7): 4415–4423)揭示用於共表現NY-ESO-1 TCR之雙順反子逆轉錄病毒載體。編碼TCRα鏈之第一基因之表現由LTR控制且編碼TCRβ鏈之第二基因由IRES操縱,或編碼TCRα鏈之第一基因之表現由LTR控制且編碼TCRβ鏈之第二基因PGK啟動子操縱。轉導淋巴球可有效地辨識且殺死HLA-A2-及NY-ESO-1-陽性黑色素瘤細胞株。
Morgan等人(Gene Therapy
(2008) 15,1411–1423)揭示雙順反子慢病毒載體,該等雙順反子慢病毒載體組合弗林裂解位點與跟隨有2A核糖體跳躍肽之胺基酸間隔基(GSG或SGSG (SEQ ID NO: 8))以表現抗gp100 TCR或抗MART-1 TCR。當間隔基序列藉由添加合成V5肽而增大時,標籤順序蛋白處理加速,此產生能夠調解轉導淋巴球中之高水平TCR表現之慢病毒載體。
仍需要基因傳遞系統以在接受性細胞療法中實現安全且高效之轉基因表現。
在一態樣中,本發明提供一種包括載體之基因傳遞系統,該載體包含:編碼蛋白質Z1之第一核苷酸序列S1、編碼蛋白質Z2之第二核苷酸序列S2、編碼蛋白質Y1之第三核苷酸序列S3及編碼蛋白質Y2之第四核苷酸序列S4,其中Z1及Z2形成第一二聚體且Y1及Y2形成第二二聚體,其中該第一二聚體Z1Z2不同於該第二二聚體Y1Y2,且其中該基因傳遞系統在適應性細胞療法中使用。
在另一態樣中,該S1、S2、S3及S4可以選自以下各者之5’至3’定向串聯排列:S1-S2-S3-S4、S1-S2-S4-S3、S1-S3-S2-S4、S1-S3-S4-S2、S1-S4-S3-S2、S1-S4-S2-S3、S2-S1-S3-S4、S2-S1-S4-S3、S2-S3-S1-S4、S2-S3-S4-S1、S2-S4-S3-S1、S2-S4-S1-S3、S3-S1-S2-S4、S3-S1-S4-S2、S3-S2-S1-S4、S3-S2-S4-S1、S3-S4-S1-S2、S3-S4-S2-S1、S4-S1-S2-S3、S4-S1-S3-S2、S4-S2-S1-S3、S4-S2-S3-S1、S4-S3-S1-S2或S4-S3-S2-S1。
在另一態樣中,該載體可進一步包括編碼2A肽之第五核苷酸序列S5及編碼連接肽之第六核苷酸序列S6,其中S5及S6定位在S1與S2之間、S1與S3之間、S1與S4之間、S2與S3之間、S2與S4之間及/或S3與S4之間。
在另一態樣中,該2A肽可選自P2A (SEQ ID NO: 3)、T2A (SEQ ID NO: 4)、E2A (SEQ ID NO: 5)或F2A (SEQ ID NO: 6)。
在另一態樣中,該連接肽可為GSG或SGSG (SEQ ID NO: 8)。
在另一態樣中,該載體可包括編碼弗林肽(SEQ ID NO: 2)之第七核苷酸序列S7,其中S7定位在S1與S2之間、S1與S3之間、S1與S4之間、S2與S3之間、S2與S4之間及/或S3與S4之間。
在另一態樣中,該載體可進一步包括選自土撥鼠PRE (Woodchuck PRE;WPRE)或B型肝炎病毒(hepatitis B virus;HBV) PRE (HPRE)之轉錄後調節元件(post-transcriptional regulatory element;PRE)序列。
在另一態樣中,該載體可進一步包括控制S1、S2、S3、S4、S5、S6及/或S7之轉錄之啟動子序列,其中該啟動子序列係選自以下各者:巨細胞病毒(cytomegalovirus;CMV)啟動子、磷酸甘油酯激酶(phosphoglycerate kinase;PGK)啟動子、髓磷脂鹼性蛋白(myelin basic protein;MBP)啟動子、膠質原纖維酸性蛋白(glial fibrillary acidic protein;GFAP)啟動子、含骨髓增生肉瘤病毒增強物之改造MoMuLV LTR (MNDU3)、泛素C啟動子、EF-1 α啟動子或鼠類幹細胞病毒(Murine Stem Cell Virus;MSCV)啟動子。
在另一態樣中,該第一二聚體Z1Z2可選自SEQ ID NO: 13及14、15及16、17及18、19及20、21及22、23及24、25及26、27及28、29及30、31及32、33及34、35及36、37及38、39及40、41及42、43及44、45及46、47及48、49及50、51及52、53及54、55及56、57及58、59及60、61及62、63及64、65及66、67及68、69及70、71及72、73及74、75及76、77及78、79及80、81及82、83及84、85及86、87及88或89及90。
在另一態樣中,該第二二聚體Y1及Y2係在SEQ ID NO: 11及12中闡述。
在另一態樣中,該定向為S2-S1-S4-S3。
在另一態樣中,該載體具有選自以下各者之序列:PTE WPRE (SEQ ID NO: 91)、TPE WPRE (SEQ ID NO: 92)或PTE fn WPRE (SEQ ID NO: 93)。
在另一態樣中,該定向為S4-S3-S2-S1。
在另一態樣中,該載體具有序列PTE CD8 TCR WPRE (SEQ ID NO: 94)。
在另一態樣中,該病毒載體係選自腺病毒、痘病毒、α病毒、沙粒狀病毒、黃病毒、棒狀病毒、逆轉錄病毒、慢病毒、皰疹病毒、副黏液病毒或小核糖核酸病毒。
在另一態樣中,該載體經選自以下各者之病毒之包膜蛋白假型化:本地貓內源病毒(RD114)、RD114之嵌合型式(RD114TR)、長臂猿白血病病毒(gibbon ape leukemia virus;GALV)、GALV之嵌合型式(GALV-TR)、雙嗜性鼠類白血病病毒(murine leukemia virus;MLV 4070A)、桿狀病毒(GP64)、水泡性口炎病毒(vesicular stomatitis virus;VSV-G)、雞瘟病毒(fowl plague virus;FPV)、伊波拉病毒(Ebola virus;EboV)、狒狒逆轉錄病毒包膜醣蛋白(baboon retroviral envelope glycoprotein;BaEV)或淋巴細胞性脈絡叢腦膜炎病毒(lymphocytic choriomeningitis virus;LCMV)。
在另一態樣中,該載體經水泡性口炎病毒(VSV-G)之包膜蛋白假型化。
在一個態樣中,本發明係關於一種製備用於免疫療法之T細胞之方法,該方法包括以下步驟:自人類受試者之血液樣本分離T細胞;在胺基雙膦酸酯存在之情況下活化該等分離之T細胞;用本文中所描述之載體轉導該等活化之T細胞;及擴增該等轉導之T細胞。
在另一態樣中,可自白血球分離術人類樣本分離該等T細胞。
在另一態樣中,該胺基雙膦酸酯可選自帕米膦酸(pamidronic acid)、阿侖膦酸(alendronic acid)、唑來膦酸(zoledronic acid)、利塞膦酸(risedronic acid)、伊班膦酸(ibandronic acid)、英卡膦酸(incadronic acid)、前述膦酸之鹽及/或前述膦酸之水合物。
在另一態樣中,該活化可進一步在人重組白細胞介素2 (interleukin 2;IL-2)及人重組白細胞介素15 (interleukin 15;IL-15)存在之情況下進行。
在另一態樣中,該擴增可在IL-2及IL-15存在之情況下進行。
在另一態樣中,該等T細胞可為γδ T細胞。
在另一態樣中,該第一二聚體Z1Z2及該第二二聚體Y1Y2共表現在該等擴增之T細胞之表面上。
在另一態樣中,本發明係關於一種藉由以上態樣之方法製備的擴增之T細胞群體。
在一個態樣中,本發明係關於一種治療患癌症之病人之方法,該方法包含以下步驟:向該病人投藥包含本文中所描述的擴增之T細胞之該群體之組合物,其中該等T細胞殺死在表面上呈現與MHC分子複合之肽之癌細胞,其中該肽係選自SEQ ID NO: 98至255中之任一者,其中該癌症係選自由以下各者組成之群組:非小細胞肺癌、小細胞肺癌、黑色素瘤、肝癌、乳癌、子宮癌、Merkel氏細胞癌、胰腺癌、膽囊癌、膽管癌、結腸直腸癌、膀胱癌、腎癌、白血病、卵巢癌、食道癌、腦癌、胃癌及前列腺癌。
在另一態樣中,該組合物進一步包括佐劑。
在另一態樣中,該佐劑係選自以下各者中之一或多者:抗CD40抗體、咪喹莫特(imiquimod)、雷西莫特(resiquimod)、GM-CSF、環磷醯胺、舒尼替尼(sunitinib)、貝伐單抗(bevacizumab)、阿特珠單抗(atezolizumab)、干擾素α、干擾素β、CpG寡核苷酸及衍生物、poly-(I:C)及衍生物、RNA、西地那非(sildenafil)、具有聚(乳酸交酯共-乙交酯) (poly(lactide co-glycolide);PLG)之顆粒調配物、病毒體、白細胞介素(IL)-1、IL-2、IL-4、IL-7、IL-12、IL-13、IL-15、IL-21及IL-23。
在一個態樣中,本發明係關於一種在患癌症之病人中引出免疫反應之方法,該方法包含以下步驟:向該病人投藥包含本文中所描述的擴增之T細胞之該群體之組合物,其中該等T細胞殺死在表面上呈現與MHC分子複合之肽之癌細胞,其中該肽係選自SEQ ID NO: 98至255中之任一者,且其中該癌症係選自由以下各者組成之群組:非小細胞肺癌、小細胞肺癌、黑色素瘤、肝癌、乳癌、子宮癌、Merkel氏細胞癌、胰腺癌、膽囊癌、膽管癌、結腸直腸癌、膀胱癌、腎癌、白血病、卵巢癌、食道癌、腦癌、胃癌及前列腺癌。
在另一態樣中,該免疫反應包含細胞毒性T細胞反應。
在一態樣中,本發明提供藉由在本文中所描述之方法中利用斯他汀(statin)來製備T細胞之方法。在另一態樣中,本發明提供藉由在斯他汀存在之情況下活化T細胞來製備T細胞之方法。
在又一態樣中,本發明係關於一種製備用於免疫療法之T細胞之方法,該方法包括以下步驟:在斯他汀存在之情況下活化該等T細胞;用本發明之載體轉導該等活化之T細胞,其中該載體可經水泡性口炎病毒(VSV-G)之包膜蛋白假型化;及擴增該等轉導之T細胞。
在另一態樣中,該等T細胞可包括αβ T細胞、γδ T細胞及/或自然殺手T細胞。
在另一態樣中,斯他汀可選自阿托伐他汀(atorvastatin)、西伐他汀(cerivastatin)、達伐他汀(dalvastatin)、氟多他汀(fluindostatin)、氟伐他汀(fluvastatin)、美伐他汀(mevastatin)、普伐他汀(pravastatin)、辛伐他汀(simvastatin)、維洛他汀(velostatin)及瑞舒伐他汀(rosuvastatin)。
在一態樣中,本發明係關於一種製備用於免疫療法之T細胞之方法,該方法包括以下步驟:活化該等T細胞;用本發明之載體轉導該等活化之T細胞;及擴增該等轉導之T細胞。
在另一態樣中,該活化可在抗CD3抗體及抗CD28抗體存在之情況下進行。
在另一態樣中,該擴增可在IL-7及IL-15存在之情況下進行。
如本文中所使用,術語「自剪切2A肽」係指藉由防止普通肽鍵在甘胺酸與最後一個脯胺酸之間形成而共平移地作用的相對短之肽(長度為約20個胺基酸,視病毒起源而定),從而導致核糖體跳至下一個密碼子,及Gly與Pro之間的初生肽剪切。在裂解之後,短2A肽保持融合至「上游」蛋白質之C端,而脯胺酸添加至「下游」蛋白質之N端。自剪切2A肽可選自豬捷申病毒-1 (P2A)、馬鼻炎A病毒(E2A)、一點褐翅蛾病毒(T2A)、口蹄疫病毒(F2A)或其任何組合(參見例如Kim等人之PLOS One 6:e18556,2011,包括2A核酸及胺基酸序列之參考文獻之內容係以全文引用之方式併入本文中)。藉由在對2A序列進行自剪切之前添加連接序列(GSG或SGSG (SEQ ID NO: 8)),此可實現生物活性蛋白(例如,TCR)之有效合成。
如本文中所使用,術語「啟動子」係指通常位於基因上游(朝向有意義鏈(sense strand)之5′區域)的DNA之調節區域,該調節區域允許基因之轉錄。啟動子含有藉由被稱為轉錄因子之蛋白辨識之特定DNA序列及回應元。此等因子結合至啟動子序列,從而甄選RNA聚合酶,自基因之編碼區域合成RNA。舉例而言,本文中所使用之啟動子序列可選自巨細胞病毒(cytomegalovirus;CMV)啟動子、磷酸甘油酯激酶(phosphoglycerate kinase;PGK)啟動子、髓磷脂鹼性蛋白(myelin basic protein;MBP)啟動子、膠質原纖維酸性蛋白(glial fibrillary acidic protein;GFAP)啟動子、含骨髓增生肉瘤病毒增強物之改造MoMuLV LTR (MNDU3)、泛素C啟動子、EF-1 α啟動子或鼠類幹細胞病毒(Murine Stem Cell Virus;MSCV)啟動子。
如本文中所使用之術語「組成型啟動子」可包括在大部分時間引導大部分細胞或組織中之基因之轉錄的調節序列。在一些非限制性實施例中,組成型啟動子可選自由以下各者組成之群組:MSCV啟動子、泛素C (Ubiqitin C;Ubc)啟動子、CMV啟動子、EF-1 α啟動子、PGK啟動子、β-肌動蛋白啟動子及ROSA26啟動子。
在一些實施例中,啟動子可為可誘導型啟動子。可誘導型啟動子之活性可回應於信號而增大或減小。舉例而言,可誘導型啟動子可回應於信號(諸如T細胞活化或異丙基β-D-1-硫代半乳哌喃糖苷(isopropyl β-D-1-thiogalactopyranoside;IPTG))之出現而促進轉錄。可誘導型啟動子可回應於信號(諸如硫酸鹽)之缺少而促進轉錄。在此等情形中之一者中,轉錄量可以或不可與信號之量或信號之缺乏成比例。適合原核宿主細胞之可誘導型啟動子之實例可包括(但不限於) NFAT、CD69、lac、tac、trc、trp、pho、recA、tetA、nar、phage PL、cspA、T7及PBAD啟動子(參見Terpe K. 2006 Appl. Microbiol. Biotechnol. 72:211;參考文獻之內容係以全文引用之方式併入)。
在一些實施例中,可誘導型啟動子可包括活化T細胞之核因子(nuclear factor of activated T cell;NFAT)/AP1轉錄回應元(transcriptional response element;TRE)。在辨識同源肽/MHC1複合後,NFAT可經歷至核之Ca2+依賴易位,其中NFAT促進庇護NFAT TRE之基因之轉錄。合適之NFAT TRE係此項技術中熟知的且包括人IL2啟動子NFAT TRE (Macian等人(2001) Oncogene, 2001年4月30日;20(19):2476-89)。Zhang等人(「用於轉移性黑色素瘤之免疫療法的用編碼白細胞介素12之可誘導型基因基因工程化之腫瘤浸潤淋巴球(Tumor-Infiltrating Lymphocytes Genetically Engineered with an Inducible Gene Encoding Interleukin-12 for the Immunotherapy of Metastatic Melanoma)」,Clin. Cancer Res. 21:2278-2288, 2015)描述表現係由可誘導型NFAT啟動子驅動的經基因工程化以隱匿單鏈IL12之人腫瘤浸潤淋巴球(tumor-infiltrating lymphocyte;TIL)在臨床試驗中之應用。此等列舉之參考文獻之內容係以全文引用之方式併入。
在一些實施例中,可誘導型啟動子可包括例如如美國5,759,805中所揭示之CD69啟動子,美國5,759,805之內容係以全文引用之方式併入。CD69可處於早期的在活化T細胞上誘導之此等新合成之細胞-表面活化分子中。CD69表現可在60分鐘之T細胞刺激內觀察到,但在休眠細胞中可能不出現。CD69表現亦可在胸腺細胞、B細胞、自然殺手(natural killer;NK)細胞及嗜中性球上可誘導。位於小鼠CD69啟動子上游50 kb內的被稱為CNS1至CNS4之四個非編碼區域可對T細胞及B細胞中之CD69活化之發育及時間控制有用。CNS2區域可充當強度增強物。Kulemzin等人(「對人T細胞及嵌合抗原受體(chimeric antigen receptor;CAR) NK細胞株中之可誘導型轉基因表現之穩定整合報導的設計及分析(Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and chimeric antigen receptor (CAR) NK cell lines)」,BMC Medical Genomics 2019, 12(Suppl 2):44, 88-95;參考文獻之內容係以全文引用之方式併入)描述,在原生T細胞之情況下,活化可誘導之CD69啟動子變體提供最高倍數誘導。此啟動子因此可用於表現經活化但未休眠之人T細胞或CAR T細胞中之蛋白。
在一些實施例中,可誘導型啟動子可為IPTG可誘導啟動子。IPTG可誘導啟動子可指以回應於IPTG或可促進自乳糖操縱子之轉錄之任何其他乳糖衍生物(例如,別乳糖)的方式促進轉錄之任何多核苷酸序列。IPTG可誘導啟動子之許多實例係此項技術中已知,包括(但不限於) tac (例如,tacI、tacII等)啟動子、lac啟動子及其衍生物(例如,lacUV5、taclac,諸如此類)。
在一態樣中,含編碼CD8 α鏈、CD8 β鏈、TCR α鏈及TCR β鏈之序列的4合1病毒載體(例如,慢病毒載體)之表現可由組成型或可誘導型啟動子驅動。舉例而言,第5A圖展示含PTE CD8 TCR WPRE (SEQ ID NO: 94)之4合1病毒載體,PTE CD8 TCR WPRE具有編碼CD8 α (SEQ ID NO: 12)及CD8 β (SEQ ID NO: 13)之密碼子最佳化序列,該等密碼子最佳化序列位於編碼TCR (例如,TCR R11KE α鏈(SEQ ID NO: 13)及R11KE β鏈(SEQ ID NO: 14))之序列上游且由組成型MSCV啟動子(SEQ ID NO: 1)驅動。上文描述之相同編碼序列亦可由可誘導型啟動子(例如,NFAT、CD69或IPTG啟動子)驅動。
在另一態樣中,含編碼融合蛋白、TCR α鏈及TCR β鏈之序列之3合1病毒載體之表現可由組成型或可誘導型啟動子驅動。舉例而言,第5B圖展示含CD8aCD4Fusion.TCR WPRE (SEQ ID NO: 256)之病毒載體,CD8aCD4Fusion.TCR WPRE具有:編碼融合蛋白之密碼子最佳化序列,其中CD8α胞外域與CD4跨膜域及CD4胞內域融合;及編碼TCR R11KE α鏈(SEQ ID NO: 13)及R11KE β鏈(SEQ ID NO: 14)之序列,由MSCV啟動子(SEQ ID NO: 1)驅動。第5C圖展示含CD8bCD4Fusion.TCR WPRE (SEQ ID NO: 257)之病毒載體,CD8bCD4Fusion.TCR WPRE具有:編碼融合蛋白之密碼子最佳化序列,其中CD8β胞外域與CD4跨膜域及CD4胞內域融合;及編碼TCR R11KE α鏈(SEQ ID NO: 13)及R11KE β鏈(SEQ ID NO: 14)之序列,由MSCV啟動子(SEQ ID NO: 1)驅動。第5D圖展示含CD8bCD8aFusion.TCR WPRE (SEQ ID NO: 258)之病毒載體,CD8bCD8aFusion.TCR WPRE具有:編碼融合蛋白之序列,其中CD8β胞外域與CD8α跨膜域及CD8α胞內域融合;及編碼TCR R11KE α鏈(SEQ ID NO: 13)及R11KE β鏈(SEQ ID NO: 14)之序列,由MSCV啟動子(SEQ ID NO: 1)驅動。上文所描述之相同編碼序列亦可由可誘導型啟動子(例如,NFAT、CD69或IPTG啟動子)驅動。
在一態樣中,本發明之4合1病毒載體之表現可由雙向組成型及/或可誘導型啟動子。舉例而言,第6A圖展示含PGK.CD8.EF1a.TCR (SEQ ID NO: 259)之4合1病毒載體,PGK.CD8.EF1a.TCR具有:位於編碼TCR R11KE α鏈及R11KE β鏈之序列上游的編碼CD8 α鏈及CD8 β鏈之密碼子最佳化序列,其中編碼CD8 α鏈及CD8 β鏈之序列及編碼TCR R11KE α鏈及R11KE β鏈之序列可由雙向啟動子(例如,PGK啟動子及EF-1 α啟動子)隔開。PGK啟動子可定位在編碼CD8 α鏈及CD8 β鏈之密碼子最佳化序列的3’末端以驅動CD8 α鏈及CD8β鏈之表現。EF-1 α啟動子可定位在編碼TCR R11KE α鏈及R11KE β鏈之序列的5’末端以驅動TCR R11KE α鏈及R11KE β鏈之表現。
第6B圖展示含PGK.TCR.EF1a.CD8 (SEQ ID NO: 260)之另一4合1病毒載體,PGK.TCR.EF1a.CD8具有位於編碼CD8 α鏈及CD8 β鏈之密碼子最佳化序列上游的編碼TCR R11KE α鏈及R11KE β鏈之序列,其中編碼TCR R11KE α鏈及R11KE β鏈之序列及編碼CD8 α鏈及CD8 β鏈之序列可由雙向啟動子(例如,PGK啟動子及EF-1 α啟動子)隔開。PGK啟動子可定位在編碼TCR R11KE α鏈及R11KE β鏈之序列的3’末端以驅動TCR R11KE α鏈及R11KE β鏈之表現。EF-1 α啟動子可定位在編碼CD8 α鏈及CD8 β鏈之密碼子最佳化序列的5’末端以驅動CD8 α鏈及CD8β鏈之表現。
本發明之一些實施例可包括含編碼TCR α鏈及TCR β鏈之序列及編碼其他蛋白質之序列之病毒載體,其他蛋白質諸如細胞介素(包括但不限於IL-1、IL-2、IL-6、IL-7、IL-10、IL-12、IL-15、IL-18及IL-21)、IL-15/IL-15受體(IL-15 receptor;IL-15R)融合蛋白、顯性陰性TGF β受體(DN TGFbRII)及/或轉化生長因子-β受體之胞外域。在一些實施例中,此等編碼序列可由啟動子或雙向啟動子驅動。
第7圖展示含位於編碼細胞介素之序列上游的編碼TCR α鏈及TCR β鏈之序列之病毒載體,其中編碼TCR α鏈及TCR β鏈之序列及編碼細胞介素之序列可由雙向啟動子隔開。雙向啟動子可自5’至3’方向以組成型-組成型、組成型-可誘導型、可誘導型-組成型或可誘導型-可誘導型定向排列。舉例而言,組成型啟動子(例如,MSCV、PGK或EF1 α啟動子)可定位在編碼TCR α鏈及TCR β鏈之序列的3’末端以驅動TCR α鏈及TCRβ鏈之表現。可誘導型啟動子(例如,NFAT、CD69或IPTG啟動子)可定位在編碼細胞介素之序列的5’末端以驅動細胞介素之表現。第8A圖展示可誘導型NFAT啟動子,其中最小IL-2啟動子定位在編碼IL-12 (例如,IL-12 α (p35)/IL-12β (p40)融合蛋白(SEQ ID NO: 261))之序列的5’末端以驅動第7圖所示之病毒載體中的12 α (p35)/IL-12β (p40)融合蛋白之表現。第8B圖展示可誘導型CD69啟動子,其中CNS1及CNS2增強物元件定位在編碼IL-12 (例如,IL-12 α (p35)/IL-12β (p40)融合蛋白 (SEQ ID NO: 262))之序列的5’末端以驅動第7圖所示之病毒載體中的12 α (p35)/IL-12β (p40)融合蛋白之表現。第8C圖展示可誘導型NFAT啟動子,其中最小IL-2啟動子定位在編碼IL-18 (例如,IL-18 變體 1 (SEQ ID NO: 263))之序列的5’末端以驅動第7圖所示之病毒載體中的IL-18變體1之表現。第8D圖展示可誘導型CD69啟動子,其中CNS1及CNS2增強物元件定位在編碼IL-18 (例如,IL-18變體1 (SEQ ID NO: 264))之序列的5’末端以驅動第7圖所示之病毒載體中的IL-18變體1之表現。
在一態樣中,本發明提供具有CD8β-CD8α-TCRβ-TCRα之5’末端至3’末端方向定向之4合1構築體。在另一態樣中,本發明提供具有CD8β-CD8α-TCRα-TCRβ之5’末端至3’末端方向定向之4合1構築體。在另一態樣中,本發明提供具有CD8α-CD8β-TCRβ-TCRα之5’末端至3’末端方向定向之4合1構築體。在另一態樣中,本發明提供具有CD8α-CD8β-TCRα-TCRβ之5’末端至3’末端方向定向之4合1構築體。
在一態樣中,本發明提供具有不包括TCRβ-TCRα-CD8α-CD8β之5’末端至3’末端方向定向之4合1構築體。在另一態樣中,本發明提供具有不包括TCRβ-TCRα-CD8β-CD8α之5’末端至3’末端方向定向之4合1構築體。在另一態樣中,本發明提供具有不包括TCRα-TCRβ-CD8α-CD8β之5’末端至3’末端方向定向之4合1構築體。在另一態樣中,本發明提供具有不包括TCRα-TCRβ-CD8β-CD8α之5’末端至3’末端方向定向之4合1構築體。
在一態樣中,本發明提供具有CD8β-CD8α-TCRβ-TCRα之5’末端至3’末端方向定向之4合1構築體。在一非限制性態樣中,本發明提供具有不包括TCRβ-TCRα-CD8α-CD8β之5’末端至3’末端方向定向之4合1構築體。
在一些實施例中,本發明之病毒載體可含有編碼TCR α鏈及TCR β鏈之序列及編碼TGF-β抑制因子(例如,顯性陰性TGF β受體(dominant-negative TGF beta receptor;DN TGFbRII)及/或轉化生長因子-β受體之胞外域)之序列。第9A圖展示含位於編碼DN TGFbRII之序列上游的編碼TCR α鏈及TCR β鏈之序列之病毒載體,其中編碼TCR α鏈及TCR β鏈之序列及編碼DN TGFbRII之序列可由雙向啟動子隔開。舉例而言,第9A圖展示:組成型啟動子(例如,MSCV、Ubc、CMV、EF-1 α及PGK啟動子)可定位在編碼TCR α鏈及TCR β鏈之序列的3’末端以驅動TCR α鏈及TCRβ鏈之表現;且另一組成型啟動子可定位在編碼DN TGFbRII之序列的5’末端以驅動DN TGFbRII之表現。
替代地,第9B圖展示含組成型啟動子(例如,MSCV、Ubc、CMV、EF-1 α及PGK啟動子)之病毒載體,該組成型啟動子定位在位於編碼TCR α鏈及TCR β鏈之序列上游的編碼DN TGFbRII之序列之5’末端以驅動DN TGFbRII、TCR α鏈及TCR β鏈之表現。上文描述之相同編碼序列亦可由可誘導型啟動子(例如,NFAT、CD69或IPTG啟動子)驅動。
如本文中所使用,術語「順反子」係指DNA分子的規定一個多肽鏈之形成(即用於一個多肽鏈之編碼)之切片。舉例而言,「雙順反子」係指DNA分子的規定兩個多肽鏈之形成(即用於兩個多肽鏈之編碼)之兩個切片;「三順反子」係指DNA分子的規定三個多肽鏈之形成(即用於三個多肽鏈之編碼)之三個切片;等。
如本文中所使用,術語「多順反子RNA」或「多順反子RNA」係指含有用於轉譯至若干蛋白質之基因資訊之RNA。相比之下,單順反子RNA含有用於僅轉譯單個蛋白質之基因資訊。在本發明之上下文中,自實例2至實例4中之慢病毒轉錄之多順反子RNA可轉譯成四種蛋白質(4合1):TCRα鏈、TCRβ鏈、CD8α鏈及CD8β鏈;或轉譯成兩種蛋白(2合1):TCRα鏈及TCRβ鏈或CD8α鏈及CD8β鏈。
如本文中所使用,術語「串聯排列」係指核酸序列上的基因單行地一個接另一個或在另一個後面連續地排列。基因在核酸序列連續地連接在一起,而每一基因之編碼股(有意義股)在核酸序列上連接在一起。
如本文中所使用,術語「有意義股」係指基因的轉譯或可轉譯至蛋白質中之DNA股。當基因在核酸序列中相對於啟動子定向在「有意義方向」上時,「有意義股」位於在啟動子下游之5’末端,其中編碼蛋白質之核酸之第一密碼子接近啟動子且最後一個密碼子遠離啟動子。
如本文中所使用,術語「病毒載體」係指包括病毒來源之至少一種元素且具有封裝至病毒載體顆粒中之能力的核酸載體構築體,且編碼至少一外源性核酸。載體及/或顆粒可用於將任何核酸轉移至體外或體內之細胞中之目的。病毒載體之眾多形式係此項技術中已知的。術語「病毒體」用於指代單一之傳染性病毒顆粒。「病毒載體」、「病毒載體顆粒」及「病毒顆粒」亦指DNA或RNA核及蛋白殼如同存在於細胞外的完全病毒顆粒。舉例而言,病毒載體可選自腺病毒、痘病毒、α病毒、沙粒狀病毒、黃病毒, 棒狀病毒、逆轉錄病毒、慢病毒、皰疹病毒、副黏液病毒或小核糖核酸病毒。
術語「T細胞」或「T淋巴球」係技術認可的且意欲包括胸腺細胞、初始T淋巴球、未成熟T淋巴球、成熟T淋巴球、休眠T淋巴球或活化T淋巴球。適合在特定實施例中使用之T細胞之說明性群體包括(但不限於)輔助T細胞(HTL;CD4+ T細胞)、細胞毒性T細胞(CTL;CD8+ T細胞)、CD4+CD8+ T細胞、CD4-CD8- T細胞、自然殺手T細胞、表現αβ TCR之T細胞(αβ T細胞)、表現γδ TCR之T細胞(γδ T細胞)或T細胞之任何其他子集。適合在特定實施例中使用之T細胞之其他說明性群體包括(但不限於)表現以下標記中之一或多者之T細胞:CD3、CD4、CD8、CD27、CD28、CD45RA、CD45RO、CD62L、CD127、CD197及HLA-DR,且在必要時可藉由陽性或陰性選擇技術進一步分離。
術語「斯他汀」、「伐他汀」或如本文中可互換地使用之「3-羥基-3-甲基戊二醯基輔酶A (3-hydroxy-3-methylglutaryl coenzyme A;HMG-CoA)還原酶抑制劑」係指抑制酵素3-羥基-3-甲基戊二醯基輔酶A (HMG- CoA)還原酶之藥劑。此酵素係在將HMG-CoA轉換至甲羥戊酸時涉及,該轉換係膽固醇生物合成中之步驟之一。此抑制容易根據熟習此項技術者熟知之標準檢定來判定。
根據本發明可使用之較佳斯他汀包括:阿托伐他汀,揭示於美國專利第4,681,893號中;阿托伐他汀鈣,揭示於美國專利第5,273,995中號;西伐他汀,揭示於美國專利第5,502,199號中;達伐他汀,揭示於美國專利第5,316,765號中;氟多他汀,揭示於美國專利第4,915,954號中;氟伐他汀,揭示於美國專利第4,739,073號中;洛伐他汀(lovastatin),揭示於美國專利第4,231,938號中;美伐他汀,揭示於美國專利第3,983,140號中;普伐他汀,揭示於美國專利第4,346,227號中;辛伐他汀,揭示於美國專利第4,444,784號中;維洛他汀,揭示於美國專利第4,448,784號及美國專利第4,450,171號中;及瑞舒伐他汀,揭示於美國6,858,618及美國7,511,140中,此等參考文獻中之每一者之內容係以全文引用之方式併入本文中。代表性3-羥基-3-甲基戊二醯基輔酶A還原酶抑制劑可包括阿托伐他汀、阿托伐他汀鈣(亦稱為Liptor®)、洛伐他汀(稱為Mevacor®)、普伐他汀(亦稱為Pravachol®)、辛伐他汀(亦稱為Zocor®)及瑞舒伐他汀。
在一個態樣中,本發明係關於可用於轉基因表現之T細胞(例如,腫瘤浸潤淋巴球、CD8+ T細胞、CD4+ T細胞及γδ T細胞)的活化、轉導及/或擴增。在另一態樣中,本發明係關於當消耗α-TCR及/或β-TCR陽性細胞時的γδ T細胞之活化、轉導及擴增。
在一態樣中,可自在體外培養之複雜樣本分離γδ T細胞。在另一態樣中,可活化且擴增整個PBMC群體,諸如單核細胞、αβ T細胞、B細胞及NK細胞之特定細胞群體先前未耗盡。在另一態樣中,富集γδ T細胞群體可在其特定活化及擴增之前產生。在另一態樣中,γδ T細胞之活化及擴增可在原生或工程化APC不存在之情況下執行。在另外態樣中,來自腫瘤標本之γδ T細胞之分離及擴增可使用以下各者執行:固定化γδ T細胞分裂素,包括特定於γδ TCR之抗體;及其他γδ TCR活化劑,包括凝集素。在另一態樣中,來自腫瘤標本之γδ T細胞之分離及擴增可在以下各者不存在之情況下執行:γδ T細胞分裂素,包括特定於γδ TCR之抗體;及其他γδ TCR活化劑,包括凝集素。
在一態樣中,自受試者(例如,人類受試者)之白血球分離物分離γδ T細胞。在另一態樣中,γδ T細胞未自周邊血液單核細胞(peripheral blood mononuclear cell;PBMC)分離。
在一態樣中,分離之γδ T細胞可回應於與一或多個抗原接觸而快速地擴增。一些γδ T細胞(諸如Vγ9Vδ2+ T細胞)可回應於與一些抗原(如萜焦磷酸酯、烷基胺及組織培養期間之代謝物或微生物萃取物)接觸而在體外快速地擴增。受刺激γδ T細胞可展現可促進γδ T細胞自複雜樣本分離之眾多抗原呈現、共刺激及黏著分子。可在體外用至少一種抗原刺激複雜樣本內之γδ T細胞1天、2天、3天、4天、5天、6天、7天或另一合適時間段。利用合適抗原刺激γδ T細胞可使γδ T細胞群體在體外擴增。
可用於在體外刺激來自複雜樣本之γδ T細胞之擴增的抗原之非限制性實例可包括以下各者:萜焦磷酸酯(諸如異戊烯基焦磷酸酯(isopentenyl pyrophosphate;IPP))、烷基胺、人微生物病原體之代謝物、共生細菌之代謝物、甲基-3-丁烯基-1-焦磷酸酯(2M3B1PP), (E)-4-羥基-3-甲基-丁-2-烯基焦磷酸酯(HMB-PP)、焦磷酸乙酯(ethyl pyrophosphate;EPP)、焦磷酸法尼酯(farnesyl pyrophosphate;FPP)、磷酸二甲烯丙酯(dimethylallyl phosphate;DMAP)、焦磷酸二甲烯丙酯(dimethylallyl pyrophosphate;DMAPP)、乙基-腺苷三磷酸(ethyl-adenosine triphosphate;EPPPA)、焦磷酸香葉酯(geranyl pyrophosphate;GPP)、香葉基焦磷酸香葉酯(geranylgeranyl pyrophosphate;GGPP)、異戊烯基-腺苷三磷酸(isopentenyl-adenosine triphosphate;IPPPA), monoethyl phosphate (MEP)、焦磷酸單乙酯(monoethyl pyrophosphate;MEPP)、3-甲醯基-1-丁基-焦磷酸酯(TUBAg 1)、X-焦磷酸酯 (TUBAg 2), 3-甲醯基-1-丁基-尿核苷三磷酸(TUBAg 3), 3-甲醯基-1-丁基-去氧胸苷三磷酸(TUBAg 4)、單乙基烷基胺、焦磷酸烯丙酯、焦磷酸巴豆酯、二甲烯丙基-γ-尿核苷三磷酸、巴豆基-γ-尿核苷三磷酸、烯丙基-γ-尿核苷三磷酸、乙胺、異丁胺、二級丁胺、異-戊胺及含氮雙磷酸酯。
γδ T細胞之活化及擴增可使用本文中所描述之活化及共刺激試劑執行以觸發特定γδ T細胞增生及持續群體。在一態樣中,來自不同培養物之γδ T細胞之活化及擴增可達成不同的單株或混合多株群體子集。在另一態樣中,不同促效劑可用於識別提供特定γδ活化信號之試劑。在另一態樣中,提供特定γδ活化信號之試劑可為針對γδ TCR之不同單株抗體(monoclonal antibody;MAb)。在另一態樣中,可使用在不誘發細胞能量及細胞凋亡之情況下幫助觸發特定γδ T細胞增生之伴生共刺激試劑。此等共刺激試劑可包括結合至表現在γδ細胞上之受體之配位子,諸如NKG2D、CD161、CD70、JAML、DNAX附屬分子-1 (DNAX accessory molecule-1;DNAM-1)、ICOS、CD27、CD137、CD30、HVEM、SLAM、CD122、DAP及CD28。在另一態樣中,共刺激試劑可為特定於CD2分子及CD3分子上之獨特表位之抗體。CD2及CD3在表現於αβ或γδ T細胞上時可具有不同的構形結構。在另一態樣中,CD3及CD2之特定抗體可引起γδ T細胞之不同活化。
γδ T細胞群體可在γδ T細胞之工程化之前在體外擴增。可用於促進γδ T細胞群體在體外之擴增之試劑的非限制性實例可包括抗CD3或抗CD2、抗CD27、抗CD30、抗CD70、抗OX40抗體、IL-2、IL-15、IL-12、IL-9、IL-33、IL-18或IL-21、CD70 (CD27配位子)、植物凝集素(phytohaemagglutinin;PHA)、刀豆蛋白(concavalin A;ConA)、商陸(pokeweed;PWM)、蛋白花生凝集素(protein peanut agglutinin;PNA)、大豆凝集素(soybean agglutinin;SBA)、兵豆凝集素(Les Culinaris Agglutinin;LCA)、豌豆凝集素(Pisum Sativum Agglutinin;PSA)、大蝸牛凝集素(Helix pomatia agglutinin;HPA)、禾豆凝集素(Vicia graminea Lectin;VGA)或能夠刺激T細胞增生之另一合適分裂素。
可藉由γδ T細胞之基因工程化來增強γδ T細胞辨識廣效抗原之能力。在一態樣中,γδ T細胞可經工程化以提供辨識在體內選擇之抗原之通用同種異體療法(universal allogeneic therapy)。γδ T細胞之基因工程化可包括將表現將抗原結合及T細胞活化功能兩者組合至單一受體中之腫瘤辨識部分(諸如αβ TCR、γδ TCR、嵌合抗原受體(chimeric antigen receptor;CAR))之構築體、其抗原結合片段或淋巴球活化域穩定地整合至分離之γδ T細胞之基因組(細胞介素(例如,IL-15、IL-12、IL-2、IL-7、IL-21、IL-18、IL-19、IL-33、IL-4、IL-9、IL-23或IL1β))中以離體及在體內增強T細胞增生、生存及功能。分離之γδ T細胞之基因工程化亦可包括自分離之γδ T細胞之基因組(諸如MHC基因座(loci))中之一或多個內在基因刪除或擾亂基因表現。
工程化γδ T細胞可用各種方法產生。舉例而言,編碼包含腫瘤辨識或另一類型之辨識部分之表現盒的多核苷酸可藉由以下各者穩定地引入至γδ T細胞中:轉位子/轉位酶系統或基於病毒之基因轉移系統,諸如慢病毒或逆轉錄病毒系統;或另一合適方法,諸如轉染、電穿孔、轉導、脂質轉染、磷酸鈣(CaPO4
)、諸如有機改性矽酸鹽(Ormosil)之奈米工程化物質、病毒傳遞方法(包括腺病毒、逆轉錄病毒、慢病毒、腺相關病毒或另一合適方法)。許多病毒方法已被用於人類基因療法,諸如WO 1993020221中描述之方法,WO 1993020221之內容係以全文引用之方式併入本文中。可用於工程化γδ T細胞之病毒方法之非限制性實例可包括γ-逆轉錄病毒、腺病毒、慢病毒、單純性皰疹病毒、牛痘病毒、養痘病毒或腺病毒相關聯之病毒方法。
在一態樣中,本文中所描述之構築體及載體將與在2018年11月26日申請之美國16/200,308中所描述之方法一起使用,美國16/200,308之內容係以全文引用之方式併入。
在一態樣中,病毒係指自然存在之病毒以及人工病毒。根據本發明之一些實施例之病毒可為包膜或無包膜病毒。小病毒(諸如AAV)係無包膜病毒之實例。在一較佳實施例中,病毒可為包膜病毒。在較佳實施例中,病毒可為逆轉錄病毒且特別為慢病毒。可促進真核細胞之病毒感染之病毒包膜蛋白可包括HIV-1衍生之慢病毒載體(lentiviral vector;LV),該等慢病毒載體用來自水泡性口炎病毒(vesicular stomatitis virus;VSV-G)、改造貓內在 逆轉錄病毒(RD114TR) (SEQ ID NO: 97)及改造長臂猿白血病病毒(gibbon ape leukemia virus;GALVTR) 之包膜醣蛋白(GP)假型化。此等包膜蛋白可高效地促進包括腺相關病毒(adeno-associated virus;AAV)的諸如小病毒之其他病毒之進入,由此表明該等病毒之廣義效率。舉例而言,可使用其他病毒包膜蛋白,包括Moloney鼠類白血病病毒(murine leukemia virus;MLV) 4070 env (諸如在Merten等人之J. Virol. 79:834-840, 2005中描述;參考文獻之內容係以引用方式併入本文中)、RD114 env、嵌合包膜蛋白 RD114pro或RDpro (其為藉由用HIV-1基質/蛋白殼(MA/CA)裂解序列置換RD114之R肽裂解序列建構之RD114-HIV 嵌合體,諸如在Bell等人之Experimental Biology and Medicine(2010; 235: 1269–1276)中描述;參考文獻之內容係以引用方式併入本文中)、桿狀病毒GP64 env (諸如在Wang等人之J. Virol. (81:10869-10878, 2007)中描述;參考文獻之內容係以引用方式併入本文中)或GALV env (諸如在Merten等人之J. Virol.(79:834-840, 2005)中描述;參考文獻之內容係以引用方式併入本文中)或其衍生物。
本發明之實施例係基於如下發現:單一慢病毒盒可用於自單一多順反子mRNA產生表現兩個不同二聚體之至少四種個別單體蛋白之單一慢病毒載體,以便在細胞表面上共表現該等二聚體。舉例而言,慢病毒載體之單一拷貝之整合足以轉變γδ T細胞以共表現TCRαβ及CD8αβ。
在一個態樣中,本發明係關於含多順反子盒之載體,該多順反子盒在能夠表現多於一種、多於兩種、多於三種、多於四種基因、多於五種基因或多於六種基因之單一載體內,其中由此等基因編碼之多肽可彼此相互作用,或可形成二聚體。二聚體可為同二聚體(即,兩個相同蛋白質形成二聚體)或異二聚體(即,兩個結構不同之蛋白質形成二聚體)。
在一個態樣中,慢病毒載體可含有編碼蛋白質Z1之第一核苷酸序列S1、編碼蛋白質Z2之第二核苷酸序列S2、編碼蛋白質Y1之第三核苷酸序列S3及編碼蛋白質Y2之第四核苷酸序列S4,其中Z1及Z2形成第一二聚體且Y1及Y2形成第二二聚體,其中第一二聚體Z1Z2不同於第二二聚體Y1Y2。
在一個態樣中,第一慢病毒載體可含有編碼二聚體Z1Z2之雙順反子盒(2合1),且第二慢病毒載體可含有編碼二聚體Y1Y2之雙順反子盒(2合1)。在2合1載體中,S1及S2可以S1-S2或S2-S1之5’至3’定向串聯排列。同樣地,在2合1載體中,S3及S4可以S3-S4或S4-S3之5’至3’定向串聯排列。Z1與Z2或Y1與Y2可由一或多個自剪切2A肽分開。
在另一態樣中,單個慢病毒載體(4合1)可編碼不同的二聚體Z1Z2及Y1Y2兩者,其中Z1、Z2、Y1及Y2可由一或多個自剪切2A肽隔開。舉例而言,S1、S2、S3及S4可以選自以下各者之5’至3’定向串聯排列:S1-S2-S3-S4、S1-S2-S4-S3、S1-S3-S2-S4、S1-S3-S4-S2、S1-S4-S3-S2、S1-S4-S2-S3、S2-S1-S3-S4、S2-S1-S4-S3、S2-S3-S1-S4、S2-S3-S4-S1、S2-S4-S3-S1、S2-S4-S1-S3、S3-S1-S2-S4、S3-S1-S4-S2、S3-S2-S1-S4、S3-S2-S4-S1、S3-S4-S1-S2、S3-S4-S2-S1、S4-S1-S2-S3、S4-S1-S3-S2、S4-S2-S1-S3、S4-S2-S3-S1、S4-S3-S1-S2或S4-S3-S2-S1。
在一態樣中,二聚體Z1Z2可為具有TCRα鏈及TCRβ鏈之TCR。
在一態樣中,能夠與本文中所描述之構築體、方法及實施例一起使用之TCR及抗原結合蛋白包括例如表2中列出之彼等TCR及抗原結合蛋白(SEQ ID NO: 13至90)及在以下各者中描述之彼等TCR及抗原結合蛋白:美國公開案20170267738、美國公開案20170312350、美國公開案20180051080、美國公開案20180164315、美國公開案20180161396、美國公開案20180162922、美國公開案20180273602、美國公開案20190016801、美國公開案20190002556、美國公開案20190135914、美國專利10,538,573、美國專利10,626,160、美國公開案20190321478、美國公開案20190256572、美國專利10,550,182、美國專利10,526,407、美國公開案20190284276、美國公開案20190016802及美國專利10,583,573,此等公開案及其中所描述之序列列表中之每一者的內容係以全文引用之方式併入本文中。
在另一態樣中,二聚體Z1Z2可為選自以下各者之TCRα鏈及TCRβ鏈:R11KEA (SEQ ID NO: 13及14)、R20P1H7 (SEQ ID NO: 15及16)、R7P1D5 (SEQ ID NO: 17及18)、R10P2G12 (SEQ ID NO: 19及20)、R10P1A7 (SEQ ID NO: 21及22)、R4P1D10 (SEQ ID NO: 23及24)、R4P3F9 (SEQ ID NO: 25及26)、R4P3H3 (SEQ ID NO: 27及28)、R36P3F9 (SEQ ID NO: 29及30)、R52P2G11 (SEQ ID NO: 31及32)、R53P2A9 (SEQ ID NO: 33及34)、R26P1A9 (SEQ ID NO: 35及36)、R26P2A6 (SEQ ID NO: 37及38)、R26P3H1 (SEQ ID NO: 39及40)、R35P3A4 (SEQ ID NO: 41及42)、R37P1C9 (SEQ ID NO: 43及44)、R37P1H1 (SEQ ID NO: 45及46)、R42P3A9 (SEQ ID NO: 47及48)、R43P3F2 (SEQ ID NO: 49及50)、R43P3G5 (SEQ ID NO: 51及52)、R59P2E7 (SEQ ID NO: 53及54)、R11P3D3 (SEQ ID NO: 55及56)、R16P1C10 (SEQ ID NO: 57及58)、R16P1E8 (SEQ ID NO: 59及60)、R17P1A9 (SEQ ID NO: 61及62)、R17P1D7 (SEQ ID NO: 63及64)、R17P1G3 (SEQ ID NO: 65及66)、R17P2B6 (SEQ ID NO: 67及68)、R11P3D3KE (SEQ ID NO: 69及70)、R39P1C12 (SEQ ID NO: 71及72)、R39P1F5 (SEQ ID NO: 73及74)、R40P1C2 (SEQ ID NO: 75及76)、R41P3E6 (SEQ ID NO: 77及78)、R43P3G4 (SEQ ID NO: 79及80)、R44P3B3 (SEQ ID NO: 81及82)、R44P3E7 (SEQ ID NO: 83及84)、R49P2B7 (SEQ ID NO: 85及86)、R55P1G7 (SEQ ID NO: 87及88)或R59P2A7 (SEQ ID NO: 89及90)。在一態樣中,該等序列展現SEQ ID NO: 13至90中之任一者之至少約90%、至少約95%或至少約98%。
表1展示當肽與MHC分子複合時TCR結合至之肽的實例。
表1
TCR名稱 | 肽(名稱/序列/SEQ ID NO:) |
R20P1H7、R7P1D5、R10P2G12 | MAG-003 (KVLEHVVRV) (SEQ ID NO: 215) |
R10P1A7 | IGF2BP3-001 (KIQEILTQV) (SEQ ID NO: 123) |
R4P1D10、R4P3F9、R4P3H3 | COL6A3-002 (FLLDGSANV) (SEQ ID NO: 238) |
R36P3F9、R52P2G11、R53P2A9 | DCAF4L2-001 (ILQDGQFLV) (SEQ ID NO: 193) |
R26P1A9、R26P2A6、R26P3H1、R35P3A4、R37P1C9、R37P1H1、R42P3A9、R43P3F2、R43P3G5、R59P2E7 | MAGEA1-003 (KVLEYVIKV) (SEQ ID NO: 202) |
R11KEA、R11P3D3、R16P1C10、R16P1E8、R17P1A9、R17P1D7、R17P1G3、R17P2B6、R11P3D3KE | PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) |
R39P1C12、R39P1F5、R40P1C2、R41P3E6、R43P3G4、R44P3B3、R44P3E7、R49P2B7、R55P1G7、R59P2A7 | SPINK2-001 (ALSVLRLAL) (SEQ ID NO: 248) |
在一態樣中,能夠與本文中所描述之方法及實施例一起使用之腫瘤相關聯抗原(tumor associated antigen;TAA)肽包括例如表3中列出之TAA肽及在以下各者中描述之彼等TAA肽:美國公開案20160187351、美國公開案20170165335、美國公開案20170035807、美國公開案20160280759、美國公開案20160287687、美國公開案20160346371、美國公開案20160368965、美國公開案20170022251、美國公開案20170002055、美國公開案20170029486、美國公開案20170037089、美國公開案20170136108、美國公開案20170101473、美國公開案20170096461、美國公開案20170165337、美國公開案20170189505、美國公開案20170173132、美國公開案20170296640、美國公開案20170253633、美國公開案20170260249、美國公開案20180051080、 美國公開案第20180164315號、美國公開案20180291082、美國公開案20180291083、美國公開案20190255110、美國專利9,717,774、美國專利9,895,415、美國公開案20190247433、美國公開案20190292520、 美國公開案20200085930、美國專利10,336,809、美國專利10,131,703、美國專利10,081,664、美國專利10,081,664、美國專利10,093,715、10,583,573及US20200085930,此等公開案、序列及其中描述之序列列表中之每一者的內容係以全文引用之方式併入本文中。
在另一態樣中,二聚體Z1Z2可為T細胞二聚傳信模組,諸如CD3δ/ε、CD3γ/ε及CD247 ζ/ζ或ζ/η、TCRα可變區域(Vα)與TCRβ可變區域(Vβ)之二聚體、免疫球蛋白重鏈可變區域(VH)與免疫球蛋白輕鏈可變區域(VL)之二聚體、Vα與VH之二聚體、Vα與VL之二聚體、Vβ與VH之二聚體或Vβ與VL之二聚體。
在另一態樣中,Y1Y2可為CD8α鏈及CD8β鏈或任何其他合適之二聚膜受體,較佳為在CD8+ T細胞中及/或在CD4+ T細胞中表現之彼等二聚體。
弗林係普遍存在之似枯草桿菌蛋白酶前蛋白轉化酶,該轉化酶之天然基質包括特定血清蛋白及生長因子受體,諸如似胰島素生長因子受體。用於弗林裂解之一致序列為RXXR (SEQ ID NO: 7),但實際裂解之可能性取決於基質三級結構及直接圍繞辨識位點之胺基酸。添加弗林裂解位點加上連接序列(GSG或SGSG (SEQ ID NO: 8))可實現高效率之基因表現。
在一個態樣中,串聯排列的弗林連接子-2A肽之核苷酸序列可定位在Z1與Z2之間、Z1與Y1之間、Z1與Y2之間、Z2與Y1之間、Z2與Y2之間及/或Y1與Y2之間。弗林可具有RXXR之一致序列(SEQ ID NO: 7),例如,RAKR (SEQ ID NO: 2)。該連接序列可為GSG或SGSG (SEQ ID NO: 8)。該2A肽可選自 P2A (SEQ ID NO: 3)、T2A (SEQ ID NO: 4)、E2A (SEQ ID NO: 5)、F2A (SEQ ID NO: 6)或其任何組合。
在另一態樣中,串聯排列的連接子-2A肽之核苷酸序列可定位在Z1與Z2之間、Z1與Y1之間、Z1與Y2之間、Z2與Y1之間、Z2與Y2之間及/或Y1與Y2之間。該連接序列可為GSG或SGSG (SEQ ID NO: 8)。該2A肽可選自P2A (SEQ ID NO: 3)、T2A (SEQ ID NO: 4)、E2A (SEQ ID NO: 5)、F2A (SEQ ID NO: 6)或其任何組合。
在一態樣中,工程化(或轉導)之γδ T細胞可在不受抗原呈現細胞或胺基雙膦酸酯刺激之情況下離體擴增。本發明之抗原反應性工程化T細胞可離體及在體內擴增。在另一態樣中,本發明之工程化γδ T細胞之活躍群體可在不受抗原呈現細胞、抗原肽、非肽分子或小分子化合物(諸如胺基雙膦酸酯)抗原刺激之情況下離體擴增,但使用特定抗體、細胞介素、分裂素或融合蛋白(諸如IL-17 Fc融合蛋白、MICA Fc融合蛋白及CD70 Fc融合蛋白)。可在γδ T細胞群體之擴增中使用的抗體之實例包括抗CD3、抗CD27、抗CD30、抗CD70、抗OX40、抗NKG2D或抗CD2抗體,細胞介素之實例可包括IL-2、IL-15、IL-12、IL-21、IL-18、IL-9、IL-7及/或IL-33,且分裂素之實例可包括CD70、人CD27之配位子、植物凝集素 (phytohaemagglutinin;PHA)、刀豆蛋白A (concavalin A;ConA)、商陸分裂素(pokeweed mitogen;PWM)、蛋白花生凝集素(protein peanut agglutinin;PNA)、大豆凝集素(soybean agglutinin;SBA)、兵豆凝集素(les culinaris agglutinin;LCA)、豌豆 凝集素(pisum sativum agglutinin;PSA)、大蝸牛凝集素(Helix pomatia agglutinin;HPA)、禾豆凝集素(Vicia graminea Lectin;VGA)或能夠刺激T細胞增生之另一合適分裂素。在另一態樣中,工程化γδ T細胞群體可在少於60天、少於48天、少於36天、少於24天、少於12天或少於6天內擴增。在另一態樣中,可將工程化γδ T細胞群體可擴增約7天至約49天、約7天至約42天、約7天至約35天、約7天至約28天、約7天至約21天或約7天至約14天。
在另一態樣中,本發明提供將用於接受性轉移療法的工程化γδ T細胞之群組進行離體擴增之方法。本發明之工程化γδ T細胞可離體擴增。本發明之工程化γδ T細胞可在不用APC活化或無與APC共培養及胺基磷酸酯之情況下在體外擴增。
治療方法
可投藥含本文中所描述之工程化γδ T細胞之組合物以用於預防及/或治療性治療。在治療性應用中,可以足以治癒或至少部分地中止疾病或狀況之症狀的量向已遭受疾病或狀況之受試者投藥醫藥組合物。亦可投藥工程化γδ T細胞以減小使狀況發展、縮小或惡化之可能性。用於治療用途的工程化γδ T細胞群體之有效量可基於疾病或狀況之嚴重性及過程、先前療法、受試者之健康狀態、體重及/或對藥物之反應及/或治療醫師之判斷改變。
本發明之組合物亦可包括一或多種佐劑。佐劑係非特定地增強或賦予免疫反應(例如,由CD8-陽性T細胞及輔助T (helper-T;TH)細胞引起的對抗原之免疫反應)的物質,且因此可認為在本發明之藥物中有用。合適之佐劑包括(但不限於) 1018 ISS、鋁鹽、AMPLIVAX®、AS15、BCG、CP-870,893、CpG7909、CyaA、dSLIM、鞭毛蛋白或自鞭毛蛋白衍生之TLR5配位子、FLT3配位子、GM-CSF、IC30、IC31、咪喹莫特(ALDARA®)、雷西莫特、ImuFact IMP321、如IL-2、IL-13、IL-21之白細胞介素、干擾素α或干擾素β或其聚乙二醇化衍生物、IS Patch、ISS、ISCOMATRIX、ISCOMs、JuvImmune®、LipoVac、MALP2、MF59、單磷醯脂質A、Montanide IMS 1312、Montanide ISA 206、Montanide ISA 50V、Montanide ISA-51、油包水及水包油乳液、OK-432、OM-174、OM-197-MP-EC、ONTAK、OspA、PepTel®載體系統、聚(乳酸交酯共-乙交酯) [poly(lactide co-glycolide);PLG]為主及聚葡萄糖微粒、乳鐵蛋白SRL172、病毒體及其他似病毒顆粒、YF-17D、VEGF捕捉劑、R848、β-葡聚醣、Pam3Cys、阿奎拉QS21刺激子(其衍生自皂素、紛歧桿菌萃取物及合成細菌細胞壁模擬),及其他專屬佐劑(諸如Ribi's Detox、Quil或Superfos)。諸如Freund或GM-CSF之佐劑係較佳的。特定用於樹突細胞之幾種免疫佐劑(例如,MF59)及其製備先前已描述(Allison and Krummel, 1995)。亦可使用細胞介素(cytokine)。多種細胞介素已直接連接,從而影響至淋巴組織(例如,TNF-)之樹突細胞遷移,使樹突細胞變成針對T淋巴球之有效抗原呈現細胞(例如,GM-CSF、IL-1及IL-4)之成熟加速(美國專利第5,849,589號,該美國專利特別地以全文引用之方式併入本文中)且充當免疫佐劑(例如,IL-12、IL-15、IL-23、IL-7、IFN-α、IFN-β) (Gabrilovich等人,1996)。
亦已報道CpG免疫刺激寡核苷酸以增強佐劑在疫苗調定中之效應。不受理論限制,CpG寡核苷酸藉由經由類鐸受體(Toll-like receptor;TLR)、主要為TLR9活化先天(非適應)免疫系統而起作用。CpG觸發之TLR9 活化增強對廣泛多種抗原之抗原特異性體液及細胞反應,該等抗原包括預防及治療性疫苗兩者中之肽或蛋白質抗原、活或死病毒、樹突細胞疫苗、自體細胞疫苗及多醣共軛物。更重要地,該活化增強樹突細胞成熟及分化,從而導致TH1細胞之增強活化及強細胞毒性T淋巴球(cytotoxic T-lymphocyte;CTL)產生,即使不存在CD4 T細胞幫助。由TLR9刺激誘發之TH1偏倚即使在正常促進TH2偏倚之疫苗佐劑存在之情況下亦得以維持,疫苗佐劑諸如明礬或不完全弗氏佐劑(incomplete Freund’s adjuvant;IFA)。CpG寡核苷酸在經調配或與其他佐劑共投藥或為諸如微粒、奈米顆粒、脂質乳液或類似調配物之調配物時展示更高之佐劑活性,CpG寡核苷酸係在抗原相對弱時誘發強反應尤其必需的。CpG寡核苷酸亦加速免疫反應且使抗原劑量能夠減小近似兩個數量級,具有對一些實驗中的無CpG之全劑量疫苗之相當大抗體反應(Krieg, 2006)。US 6,406,705 B1描述CpG寡核苷酸、非核酸佐劑及用於誘發抗原特異性免疫反應之抗原之組合使用。CpG TLR9拮抗劑係Mologen (Berlin, Germany)之雙主幹環圈免疫調節劑(double Stem Loop Immunomodulator;dSLIM),此係本發明之醫藥組合物之較佳成分。亦可使用其他TLR結合分子,諸如RNA結合TLR 7、TLR 8及/或TLR 9。
有用佐劑之其他實例包括(但不限於)化學改性CpG (例如CpR、Idera)、諸如poly(I:C)及其衍生物之dsRNA類似物(例如AmpliGen®、Hiltonol®、聚(ICLC)、聚(IC-R)、聚(I:C12U)、非CpG細菌DNA或RNA以及免疫活性小分子及抗體,諸如環磷醯胺、舒尼替尼、免疫查核點抑制劑,包括易普利單抗(ipilimumab)、納武單抗(nivolumab)、帕博利珠單抗(pembrolizumab)、阿特珠單抗、阿維魯單抗(avelumab)、德瓦魯單抗(durvalumab)及西米普利單抗(cemiplimab)、Bevacizumab®、西樂葆(celebrex)、NCX-4016、西地那非、他達拉非(tadalafil)、伐地那非(vardenafil)、索拉非尼(sorafenib)、替莫唑胺(temozolomide)、替西羅莫司(temsirolimus)、XL-999、CP-547632、pazopanib、VEGF捕捉劑、ZD2171、AZD2171、抗CTLA4、以免疫系統之關鍵結構為目標之其他抗體(例如抗CD40、抗TGFβ、抗TNFα受體)及SC58175,該等抗體可起治療作用及/或充當佐劑。在本發明之上下文中有用之佐劑及添加劑之量及濃度可容易由熟習此項技術者在不過度實驗之情況下判定。
較佳佐劑為抗CD40、咪喹莫特、雷西莫特、GM-CSF、環磷醯胺、舒尼替尼、貝伐單抗、阿特珠單抗、干擾素α、干擾素β、CpG寡核苷酸及衍生物、poly-(I:C)及衍生物、RNA、西地那非及具有聚(乳酸交酯共-乙交酯) (poly(lactide co-glycolide);PLG)之顆粒調配物、病毒體及/或白細胞介素(IL)-1、IL-2、IL-4、IL-7、IL-12、IL-13、IL-15、IL-21及IL-23。
在一較佳實施例中,根據本發明之醫藥組合物,該佐劑係選自由以下各者組成之群組:諸如顆粒性白血球巨噬細胞群落刺激因子(Granulocyte Macrophage Colony Stimulating Factor;GM-CSF, sargramostim)之群落刺激因子、環磷醯胺、咪喹莫特、雷西莫特及干擾素α。
在一較佳實施例中,根據本發明之醫藥組合物,該佐劑係選自由以下各者組成之群組:諸如顆粒性白血球巨噬細胞群落刺激因子(Granulocyte Macrophage Colony Stimulating Factor;GM-CSF, sargramostim)之群落刺激因子、環磷醯胺、咪喹莫特及雷西莫特。在根據本發明之醫藥組合物之一較佳實施例中,該佐劑為環磷醯胺、咪喹莫特或雷西莫特。更佳佐劑為Montanide IMS 1312、Montanide ISA 206、Montanide ISA 50V、Montanide ISA-51、聚ICLC (Hiltonol®)及抗CD40 mAB或其組合。
本發明之工程化γδ T細胞可用於治療需要治療狀況(例如,本文中所描述之癌症)之受試者。
用γδ T細胞治療受試者之狀況(例如,病痛)之方法可包括向受試者投藥治療有效量之工程化γδ T細胞。本發明之γδ T細胞可以各種方案(例如,時序、濃度、劑量、治療的間隔及/或調配物)。在接收本發明之工程化γδ T細胞之前,受試者亦可經過例如化療、輻射或兩者之組合預先處理。工程化γδ T細胞群體亦可在向受試者投藥之前冷凍或低溫保存。工程化γδ T細胞群體可包括表現相同、不同腫瘤辨識部分或相同與不同腫瘤辨識部分之組合的兩個或更多細胞。舉例而言,工程化γδ T細胞群體可包括經設計以辨識不同抗原或同一抗原中之不同表位的若干不同工程化γδ T細胞。
本發明之γδ T細胞可用於治療各種狀況。在一態樣中,本發明之工程化γδ T細胞可用於治療癌症,包括實性瘤及血液系統惡性腫瘤。癌症之非限制性實例包括:急性淋巴母細胞白血病、急性骨髓性白血病、腎上腺皮質癌、AIDS相關癌、AIDS相關淋巴瘤、肛門癌、闌尾癌、星狀細胞瘤、神經胚細胞瘤、基底細胞瘤、膽管癌、膀胱癌、骨癌、腦腫瘤(諸如小腦星狀細胞瘤、大腦星狀細胞瘤/惡性神經膠瘤、室管膜瘤、髓母細胞瘤、小腦幕上原始神經外胚層腫瘤、視覺路徑及下視丘神經膠質瘤、乳癌、支氣管腺瘤、Burkitt氏淋巴瘤、原發灶不明瘤、中樞神經系統淋巴瘤、小腦星狀細胞瘤)、子宮頸癌、兒童期癌、慢性淋巴球性白血病、慢性骨髓性白血病、慢性骨髓增生疾病、大腸癌、皮膚T細胞淋巴瘤、結締組織增生性小圓細胞瘤、子宮內膜癌、室管膜瘤、食道癌、Ewing氏肉瘤、生殖細胞腫瘤、膽囊癌、胃癌、胃腸道類癌腫瘤、胃腸道間質瘤、神經膠質瘤、毛細胞白血病、頭頸癌、心臟癌、肝細胞(肝)癌、Hodgkin氏淋巴瘤、咽下癌、眼內黑色素瘤、胰島細胞癌、Kaposi氏肉瘤、腎癌、喉頭癌、唇及口腔癌、脂肪肉瘤、肝癌、肺癌(諸如非小細胞及小細胞肺癌)、淋巴瘤、白血病、巨球蛋白血症、骨肉瘤之惡性纖維組織細胞瘤、髓母細胞瘤、黑色素瘤、間皮瘤、原發灶隱匿之轉移性鱗狀頸癌、口癌、多發性內分泌瘤症候群、骨髓發育不良症候群、骨髓性白血病、鼻腔及副鼻腔癌、鼻咽癌、神經胚細胞瘤、非Hodgkin氏淋巴瘤、非小細胞肺癌、口腔癌、口咽癌、骨肉瘤/骨惡性纖維組織細胞瘤、卵巢癌、卵巢上皮癌、卵巢生殖細胞腫瘤、胰腺癌、胰腺癌胰島細胞、副鼻腔及鼻腔癌、副甲狀腺癌、陰莖癌、咽癌、嗜鉻細胞瘤、松果體星狀細胞瘤、松果體胚細胞瘤、垂體腺瘤、胸膜肺胚細胞瘤、漿細胞贅瘤、原發性中樞神經系統淋巴瘤、前列腺癌、直腸癌、腎細胞瘤、腎盂及輸尿管移行細胞癌、視網膜母細胞瘤、橫紋肌肉瘤、唾液腺癌、肉瘤、皮膚癌、merkel氏細胞皮膚癌、小腸癌、軟組織肉瘤、鱗狀細胞癌、胃癌、T細胞淋巴瘤、喉癌、胸腺瘤、胸腺癌、甲狀腺癌、滋養細胞腫瘤(妊娠性)、原發部位不明癌、尿道癌、子宮肉瘤、陰道癌、陰門癌、Waldenstrm巨球蛋白血症及Wilms 腫瘤。
在一態樣中,本發明之工程化γδ T細胞可用於治療傳染性疾病。在另一態樣中,本發明之工程化γδ T細胞可用於治療傳染性疾病,傳染性疾病可由病毒導致。在又一態樣中,本發明之工程化γδ T細胞可用於治療免疫疾病,諸如自體免疫病。
可在狀況臨床發作之前、期間及之後為受試者提供利用本發明之γδ T細胞之治療。可在疾病臨床發作之後1天、1週、6個月、12個月或2年之後為受試者提供治療。可在疾病臨床發作之後為受試者提供治療持續多於1天、1週、1個月、6個月、12個月、2年、3年、4年、5年、6年、7年、8年、9年、10年或更久。可在疾病臨床發作之後為受試者提供治療持續少於1天、1週、1個月、6個月、12個月或2年。治療亦可包括在臨床實驗中治療人類。治療可包括向受試者投藥包含本發明之工程化γδ T細胞之醫藥組合物。
在另一態樣中,本發明之工程化γδ T細胞至受試者之投藥可調節內在淋巴球在受試者身體中之活性。在另一態樣中,向受試者投藥工程化γδ T細胞可將抗原提供至內原T細胞且可提高免疫反應。在另一態樣中,記憶T細胞可為CD4+ T細胞。在另一態樣中,記憶T細胞可為CD8+ T細胞。在另一態樣中,本發明之工程化γδ T細胞至受試者之投藥可活化另一免疫細胞之細胞毒性。在另一態樣中,另一免疫細胞可為CD8+ T細胞。在另一態樣中,另一免疫細胞可為自然殺手T細胞。在另一態樣中,本發明之工程化γδ T細胞至受試者之投藥可抑制調節T細胞。在另一態樣中,調節T細胞可為FOX3+ Treg細胞。在另一態樣中,調節T細胞可為FOX3− Treg細胞。活性可由本發明之工程化γδ T細胞調節之細胞的非限制性實例可包括:造血幹細胞;B細胞;CD4;CD8;紅血球;白血細胞;樹突細胞,包括樹突狀抗原呈現細胞;白血球;巨噬細胞;記憶B細胞;記憶T細胞;單核細胞;自然殺手細胞;嗜中性球顆粒性白血球;T輔助細胞;及T殺手細胞。
在大部分骨髓移植期間,可習知地使用環磷醯胺與全身照射之組合以防止受試者之免疫系統對移植物中的造血幹細胞(hematopietic stem cell;HSC)之排斥。在一態樣中,可執行利用白細胞介素-2 (interleukin-2;IL-2)離體培養供體骨髓,以增強供體骨髓中之殺手淋巴球之產生。白細胞介素-2 (interleukin-2;IL-2)係野生型淋巴球之生長、增生及分化可能必需的細胞介素。對γδ T細胞至人中之接受性轉移的當前研究可需要將γδ T細胞與白細胞介素-2共投藥。然而,低劑量及高劑量之IL-2可具有高毒性之副作用。IL-2毒性在多個器官/系統中顯現,在心、肺、腎及中樞神經系統中最明顯。在另一態樣中,本發明提供在不共投藥初始細胞介素或其改造型式(諸如IL-2、IL-15、IL-12、IL-21)之情況下向受試者投藥工程化γδ T細胞之方法。在另一態樣中,可在不共投藥IL-2之情況下向受試者投藥工程化γδ T細胞。在另一態樣中,可在程序(諸如不共投藥IL-2之骨髓移植)期間向受試者投藥工程化γδ T細胞。.
投藥方法
可以任何次序或同時地向受試者投藥一個或多個工程化γδ T細胞群體。若同時投藥,則多個工程化γδ T細胞可以單一的統一形式(諸如靜脈注射)或以多種形式(例如,多次靜脈輸液,皮下注射(s.c)、注射物或藥片)提供。工程化γδ T細胞可一起或分開地包裝在單一封裝或複數個封裝中。工程化γδ T細胞中之一者或全部可以多個劑量給出。若不同時進行,則多個劑量之間的時序可改變至多達約一週、一個月、兩個月、三個月、四個月、五個月、六個月或約一年。在另一態樣中,在向受試者投藥之後,工程化γδ T細胞可在受試者之身體內、在體外擴增。可冷凍工程化γδ T細胞以利用同一細胞製備提供用於多種治療之細胞。本發明之工程化γδ T細胞及包含其之醫藥組合物可封裝為套件。套件可包括關於工程化γδ T細胞及包含其之組合物的使用之說明(例如,書面說明)。
在另一態樣中,一種治療癌症之方法包含向受試者投藥治療有效量之工程化γδ T細胞,其中該投藥治療癌症。在另外實施例中,可投藥治療有效量之工程化γδ T細胞持續至少約10秒、30秒、1分鐘、10分鐘、30分鐘、1小時、2小時、3小時、4小時、5小時、6小時、12小時、24小時、2天、3天、4天、5天、6天、1週、2週、3週、1個月、2個月、3個月、4個月、5個月、6個月或1年。在另一態樣中,可投藥治療有效量之工程化γδ T細胞持續至少一週。在另一態樣中,可投藥治療有效量之工程化γδ T細胞持續至少兩週。
本文中所描述之工程化γδ T細胞可在疾病或狀況出現之前、期間或之後投藥,且投藥含工程化γδ T細胞之醫藥組合物之時序可改變。舉例而言,工程化γδ T細胞可用作預防且可視狀況或疾病之趨勢而連續地向受試者投藥,以便減小疾病或狀況發生之可能性。可在症狀發作期間或在症狀發作之後儘可能快地向受試者投藥工程化γδ T細胞。工程化γδ T細胞之投藥可在症狀發作時、在症狀發作的前3小時內、在症狀發作的前6小時內、在症狀發作的前24小時內、在症狀發作的前48小時內或症狀發作之任何時間段內立即開始。初始投藥可經由任何實用路徑,諸如使用本文中所描述之任何調配物藉由本文中所描述之任何路徑。在另一態樣中,本發明之工程化γδ T細胞之投藥可為靜脈投藥。只要在癌症、傳染性疾病、免疫疾病、敗血症發作之後或在骨髓移植時可行,即可投藥一或多個劑量之工程化γδ T細胞,且持續治療免疫疾病所需之時間長度,例如約24小時至約48小時、約48小時至約1週、約1週至約2週、約2週至約1個月、約1月至約3個月。對於癌症治療,可在癌症發作之後多年且在其他治療之前或之後才投藥一或多個劑量之工程化γδ T細胞。在另一態樣中,可將工程化γδ T細胞投藥持續至少約10分鐘、30分鐘、1小時、2小時、3小時、4小時、5小時、6小時、12小時、24小時、至少48小時、至少72小時、至少96小時、至少1週、至少2週、至少3週、至少4週、至少1個月、至少2個月、至少3個月、至少4個月、至少5個月、至少6個月、至少7個月、至少8個月、至少9個月、至少10個月、至少11個月、至少12個月、至少1年、至少2年、至少3年、至少4年或至少5年。治療之長度可針對每一受試者改變。
保存
在一態樣中,γδ T細胞可在冷凍介質中進行調配且置放在諸如液氮冷凍庫(-196℃)或超低溫度冷凍庫(-65℃、-80℃、-120℃或-150℃)之低溫儲存單元中,以長期儲存至少約1個月、2個月、3個月、4個月、5個月、6個月、1年、2年、3年或至少5年。冷凍介質可含有二甲基亞碸(dimethyl sulfoxide;DMSO)及/或氯化鈉(NaCl)及/或葡萄糖及/或硫酸聚葡萄糖及/或羥乙澱粉(hydroyethyl starch;HES),具有生理性pH緩衝劑以將pH維持在約6.0至約6.5之間、約6.5至約7.0之間、約7.0至約7.5之間、約7.5至約8.0之間或約6.5至約7.5之間。可將低溫保存之γδ T細胞解凍且藉由利用如本文中所描述之抗體、蛋白質、肽及/或細胞介素之刺激進行進一步處理。可將低溫保存之γδ T細胞解凍且用如本文中所描述之病毒載體(包括逆轉錄病毒、腺相關病毒(adeno-associated virus;AAV)及慢病毒載體)或非病毒手段(包括RNA、例如轉位子之DNA及蛋白質)進行基因改造。可進一步低溫保存經改造γδ T細胞以在冷凍介質中以約每ml至少101、102、103、104、105、106、107、108、109或至少約1010個細胞形成數量為至少約1、5、10、100、150、200、500小瓶之細胞庫。低溫保存之細胞庫可保持其官能性且可解凍且進一步受刺激且擴增。在另一態樣中,解凍之細胞可在合適之密封容器(諸如細胞培養袋及/或生物反應器)中受刺激且擴增,以產生大量細胞作為同種細胞產品。低溫保存之 γδ T細胞可將其生物功能在低溫儲存條件下維持至少約6個月、7個月、8個月、9個月、10個月、11個月、12個月、13個月、15個月、18個月、20個月、24個月、30個月、36個月、40個月、50個月或至少約60個月。在另一態樣中,在調配物中不可使用防腐劑。可將低溫保存之γδ T細胞解凍且作為同種現貨細胞產品輸注至多個病人中。
在一態樣中,本文中所描述之工程化γδ T細胞可以如下之量存在於組合物中:至少1×103
個細胞/ml、至少2×103
個細胞/ml、至少3×103
個細胞/ml、至少4×103
個細胞/ml、至少5×103
個細胞/ml、至少6×103
個細胞/ml、至少7×103
個細胞/ml、至少8×103
個細胞/ml、至少9×103
個細胞/ml、至少1×104
個細胞/ml、至少2×104
個細胞/ml、至少3×104
個細胞/ml、至少4×104
個細胞/ml、至少5×104
個細胞/ml、至少6×104
個細胞/ml、至少7×104
個細胞/ml、至少8×104
個細胞/ml、至少9×104
個細胞/ml、至少1×105
個細胞/ml、至少2×105
個細胞/ml、至少3×105
個細胞/ml、至少4×105
個細胞/ml、至少5×105
個細胞/ml、至少6×105
個細胞/ml、至少7×105
個細胞/ml、至少8×105
個細胞/ml、至少9×105
個細胞/ml、至少1×106
個細胞/ml、至少2×106
個細胞/ml、至少3×106
個細胞/ml、至少4×106
個細胞/ml、至少5×106
個細胞/ml、至少6×106
個細胞/ml、至少7×106
個細胞/ml、至少8×106
個細胞/ml、至少9×106
個細胞/ml、至少1×107
個細胞/ml、至少2×107
個細胞/ml、至少3×107
個細胞/ml、至少4×107
個細胞/ml、至少5×107
個細胞/ml、至少6×107
個細胞/ml、至少7×107
個細胞/ml、至少8×107
個細胞/ml、至少9×107
個細胞/ml、至少1×108
個細胞/ml、至少2×108
個細胞/ml、至少3×108
個細胞/ml、至少4×108
個細胞/ml、至少5×108
個細胞/ml、至少6×108
個細胞/ml、至少7×108
個細胞/ml、至少8×108
個細胞/ml、至少9×108
個細胞/ml、至少1×109
個細胞/ml,或更多,約1×103
個細胞/ml至約至少1×108
個細胞/ml、約1×105
個細胞/ml至約至少1×108
個細胞/ml或約1×106
個細胞/ml至約至少1×108
個細胞/ml。
在一態樣中,本文中描述之方法可用於生產根據本發明之一態樣之自體或外源產品。
在一態樣中,本文中所描述之載體、構築體或序列可包含SEQ ID NO: 1至97及265至266中之任一者的約80%、約85%、約90%、約85%、約96%、約97%、約98%或約99%。「與參考序列具有至少85%一致性」之序列係在整個長度上與參考序列之整個長度具有85%或更高、特別90%、91%、92%、93%、94%、95%、96%、97%、98%或99%之序列一致性的序列。
在本申請案之上下文中,使用全域配對對準(即在整個長度上對兩個序列進行比較)來計算「一致性百分比」。用於比較兩個或更多序列之一致性之方法係此項技術中熟知的。可例如使用«針»程序,該程序使用Needleman-Wunsch全域對準演算法(Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453)以找出當考慮整體長度時的兩個序列之最優對準(包括間隙)。該針程序例如可在ebi.ac.uk World Wide Web網站獲得,且在隨後公開案(EMBOSS: The European Molecular Biology Open Software Suite
(2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276—277)中描述。根據本發明,使用具有等於10.0之「Gap Open」參數、等於0.5之「間隙延長」參數及Blosum62基質的EMBOSS:針(全域)程序來計算兩種多肽之間的一致性百分比。
由「至少80%、85%、90%、95%、96%、97%、98%或99%等同」於參考序列之胺基酸序列組成的蛋白質可包含突變,諸如與參考序列相比之刪除、插入及/或取代。在取代之情況下,由與參考序列具有至少80%、85%、90%、95%、96%、97%、98%或99%一致性之胺基酸序列組成的蛋白質可對應於自不同於參考序列之種衍生的同源序列。
「胺基酸取代」可為保守或非保守的。較佳地,取代為保守取代,其中一種胺基酸被具有類似結構及/或化學性質之另一種胺基酸取代。
在一實施例中,保守取代可包括由Dayhoff在「The Atlas of Protein Sequence and Structure. Vol. 5」(Natl. Biomedical Research),中描述之彼等保守取代,參考文獻之內容係以全文引用之方式併入。舉例而言,在一態樣中,屬以下群組中之一者之胺基酸可彼此交換,因此構成保守交換:群組1:丙胺酸(A)、脯胺酸(P)、甘胺酸(G)、天冬醯胺酸(N)、絲胺酸(S)、蘇胺酸(T);群組2:半胱胺酸(C)、絲胺酸(S)、酪胺酸(Y)、蘇胺酸(T);群組3:纈胺酸(V)、異白胺酸(I)、白胺酸(L)、甲硫胺酸(M)、丙胺酸(A)、苯丙胺酸(F);群組4:離胺酸(K)、精胺酸(R)、組胺酸(H);群組5:苯丙胺酸(F)、酪胺酸(Y)、色胺酸(W)、組胺酸(H);及群組6:天冬胺酸(D)、麩胺酸(E)。在一態樣中,保守胺基酸取代可選自以下各者:T→A、G→A、A→I、T→V、A→M、T→I、A→V、T→G及/或T→S。
在一另外實施例中,保守胺基酸取代可包括胺基酸被同類別之另一胺基酸取代,例如,(1)非極性:Ala、Val、Leu、Ile、Pro、Met、Phe、Trp;(2)不帶電荷極性:Gly、Ser、Thr、Cys、Tyr、Asn、Gln;(3)酸性:Asp、Glu;及(4)鹼性:Lys、Arg、His。其他保守胺基酸取代亦可如下所述地進行:(1)芳族:Phe、Tyr、His;(2)質子供體:Asn、Gln、Lys、Arg、His、Trp;及(3)質子受體:Glu、Asp、Thr、Ser、Tyr、Asn、Gln (參見例如美國專利第10,106,805號,該美國專利之內容係以全文引用之方式併入)。
在另一實施例中,保守取代可根據表1進行。用於預測對蛋白改性之容忍度的方法可在例如Guo等人之Proc. Natl. Acad. Sci.,USA,101(25):9205-9210 (2004)中發現,參考文獻之內容係以全文引用之方式併入。
表 A
:保守胺基酸取代
保守胺基酸取代 | |
胺基酸 | 取代(其他取代係此項技術中已知的) |
Ala | Ser,Gly,Cys |
Arg | Lys,Gln,His |
Asn | Gln,His,Glu,Asp |
Asp | Glu,Asn,Gln |
Cys | Ser,Met,Thr |
Gln | Asn,Lys,Glu,Asp,Arg |
Glu | Asp,Asn,Gln |
Gly | Pro,Ala,Ser |
His | Asn,Gln,Lys |
Ile | Leu,Val,Met,Ala |
Leu | Ile,Val,Met,Ala |
Lys | Arg,Gln,His |
Met | Leu,Ile,Val,Ala,Phe |
Phe | Met,Leu,Tyr,Trp,His |
Ser | Thr,Cys,Ala |
Thr | Ser,Val,Ala |
Trp | Tyr,Phe |
Tyr | Trp,Phe,His |
Val | Ile,Leu,Met,Ala,Thr |
在一態樣中,本文中所描述之序列可包括1、2、3、4、5、10、15、20、25或30次胺基酸或核苷酸突變、取代、刪除。在一態樣中,SEQ ID NO: 1至97及265至266中之任一者可包括1、2、3、4、5、10、15、20、25或30次突變、取代或刪除。在又一態樣中,突變或取代係保守胺基酸取代。
在另一實施例中,保守取代可為表B中示出的在「保守取代」標題下之彼等取代。若此等取代導致生物活性之變化,則可引入更多實質性變化(在表B中表示為「例示性取代」且在必要時篩選產品。
表B:胺基酸取代
胺基酸取代 | ||
原始殘基(自然存在之胺基酸) | 保守取代 | 例示性取代 |
Ala (A) | Val | Val;Leu;Ile |
Arg (R) | Lys | Lys;Gln;Asn |
Asn (N) | Gln | Gln;His;Asp;Lys;Arg |
Asp (D) | Glu | Glu;Asn |
Cys (C) | Ser | Ser;Ala |
Gln (Q) | Asn | Asn;Glu |
Glu (E) | Asp | Asp;Gln |
Gly (G) | Ala | Ala |
His (H) | Arg | Asn;Gln;Lys;Arg |
Ile (I) | Leu | Leu;Val;Met;Ala;Phe;正白胺酸 |
Leu (L) | Ile | 正白胺酸;Ile;Val;Met;Ala;Phe |
Lys (K) | Arg | Arg;Gln;Asn |
Met (M) | Leu | Leu;Phe;Ile |
Phe (F) | Tyr | Leu;Val;Ile;Ala;Tyr |
Pro (P) | Ala | Ala |
Ser (S) | Thr | Thr |
Thr (T) | Ser | Ser |
Trp (W) | Tyr | Tyr;Phe |
Tyr(Y) | Phe | Trp;Phe;Thr;Ser |
Val(V) | Leu | Ile;Leu;Met;Phe;Ala;正白胺酸 |
實例1
表2. DNA及蛋白序列
表3. TAA肽序列
SEQ ID NO: | 描述 | 序列 |
1 | MSCV啟動子 | Tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcact |
2 | 弗林 | RAKR |
3 | P2A | ATNFSLLKQAGDVEENPGP |
4 | T2A | EGRGSLLTCGDVEENPGP |
5 | E2A | QCTNYALLKLAGDVESNPGP |
6 | F2A | VKQTLNFDLLKLAGDVESNPGP |
7 | 弗林一致序列 | RXXR |
8 | 連接子 | SGSG |
9 | WPRE | cagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
10 | X蛋白啟動子 | Ggggaagctgacgtcctttcc |
11 | CD8 α鏈 | MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV |
12 | CD8 β鏈 | MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQPQGEGISGTFVPQCLHGYYSNTTTSQKLLNPWILKT |
13 | R11KEA α鏈 | MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
14 | R11KE β鏈 | MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRETMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
15 | R20P1H7 α鏈 | MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQGENSGYSTLTFGKGTMLLVSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
16 | R20P1H7 β鏈 | MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLGPGLAAYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
17 | R7P1D5 α鏈 | MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEYSSASKIIFGSGTRLSIRPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
18 | R7P1D5 β鏈 | MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRANTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
19 | R10P2G12 α鏈 | MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEGNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
20 | R10P2G12 β鏈 | MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSSGSHQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
21 | R10P1A7 α鏈 | MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESKETRLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
22 | R10P1A7 β鏈 | MLLLLLLLGPGISLLLPGSLAGSGLGAWSQHPSVWICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARAGGHEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWVWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
23 | R4P1D10 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFHDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
24 | R4P1D10 β鏈 | MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVASAYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
25 | R4P3F9 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
26 | R4P3F9 β鏈 | MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
27 | R4P3H3 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKAGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
28 | R4P3H3 β鏈 | MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLLTSGGDNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
29 | R36P3F9 α鏈 | METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATVSNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
30 | R36P3F9 β鏈 | MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSSTSGGLSGETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
31 | R52P2G11 α鏈 | MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSAYGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
32 | R52P2G11 β鏈 | MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLGSPDGNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
33 | R53P2A9 α鏈 | MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYNSYAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
34 | R53P2A9 β鏈 | MGPGLLCWVLLCLLGAGPVDAGVTQSPTHLIKTRGQQVTLRCSPISGHKSVSWYQQVLGQGPQFIFQYYEKEERGRGNFPDRFSARQFPNYSSELNVNALLLGDSALYLCASSLDGTSEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
35 | R26P1A9 α鏈 | METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCLIGASGSRLTFGEGTQLTVNPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
36 | R26P1A9 β鏈 | MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSYFGWNEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
37 | R26P2A6 α鏈 | MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDVSGGYNKLIFGAGTRLAVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
38 | R26P2A6 β鏈 | MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASTTPDGTDEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
39 | R26P3H1 α鏈 | MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDMNRDDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
40 | R26P3H1 β鏈 | MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSRAEGGEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
41 | R35P3A4 α鏈 | MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASPTGGYNKLIFGAGTRLAVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
42 | R35P3A4 β鏈 | MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSLGGASQEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
43 | R37P1C9 α鏈 | MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILFNFNKFYFGSGTKLNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
44 | R37P1C9 β鏈 | MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSSGETNEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
45 | R37P1H1 α鏈 | MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFGYSGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
46 | R37P1H1 β鏈 | MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSNEGQGWEAEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
47 | R42P3A9 α鏈 | MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVHNFNKFYFGSGTKLNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
48 | R42P3A9 β鏈 | MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLLGQGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
49 | R43P3F2 α鏈 | MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSNNNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
50 | R43P3F2 β鏈 | MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSPTGTSGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
51 | R43P3G5 α鏈 | MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALNRDDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
52 | R43P3G5 β鏈 | MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRLPSRTYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
53 | R59P2E7 α鏈 | METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVNSDYKLSFGAGTTVTVRANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
54 | R59P2E7 β鏈 | MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGLGTGDYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
55 | R11P3D3 α鏈 | MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
56 | R11P3D3 β鏈 | MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATlLYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
57 | R16P1C10 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVISNFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
58 | R16P1C10 β鏈 | MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPWDSPNEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
59 | R16P1E8 α鏈 | MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAAGNKLTFGGGTRVLVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
60 | R16P1E8 β鏈 | MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSYTNQGEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
61 | R17P1A9 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVLNQAGTALIFGKGTTLSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
62 | R17P1A9 β鏈 | MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAETGPWLGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
63 | R17P1D7 α鏈 | MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRWAQGGSEKLVFGKGTKLTVNPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
64 | R17P1D7 β鏈 | MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATELWSSGGTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
65 | R17P1G3 α鏈 | IMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVGPSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
66 | R17P1G3 β鏈 | MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSPGGSGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
67 | R17P2B6 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVVSGGGADGLTFGKGTHLIIQPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
68 | R17P2B6 β鏈 | MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGRGGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
69 | R11P3D3KE α鏈 | MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
70 | R11P3D3KE β鏈 | NNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATlLYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
71 | R39P1C12 α鏈 | TYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIDNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
72 | R39P1C12 β鏈 | MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSQLNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF |
73 | R39P1F5 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNNARLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
74 | R39P1F5 β鏈 | MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGANEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
75 | R40P1C2 α鏈 | MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYLNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
76 | R40P1C2 β鏈 | MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEMTAVGQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
77 | R41P3E6 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFT AQLNKASQYVSLLIRDSQPSDSATYLCAAFSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
78 | R41P3E6 β鏈 | MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSQYTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
79 | R43P3G4 α鏈 | MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNGGDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
80 | R43P3G4 β鏈 | MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGALEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
81 | R44P3B3 α鏈 | MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGLYNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
82 | R44P3B3 β鏈 | MGCRLLCCVVFCLLQAGPLDTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKFLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSLGDRGYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
83 | R44P3E7 α鏈 | MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEINNNARLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
84 | R44P3E7 β鏈 | MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSPPDQNTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
85 | R49P2B7 α鏈 | MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSEGALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTTGATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVRIFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
86 | R49P2B7 β鏈 | MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLMGELTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
87 | R55P1G7 α鏈 | MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMMGDTGTASKLTFGTGTRLQVTLDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
88 | R55P1G7 β鏈 | MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFGGYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
89 | R59P2A7 α鏈 | VKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS |
90 | R59P2A7 β鏈 | MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSGLVAEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG |
91 | PTE WPRE | tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcagcggccgccccgggtcgacgctaccaccatggactcttggaccttctgctgcgtgagcctgtgcatcctggtggccaagcacacagacgccggcgtgatccagtcccctaggcacgaggtgaccgagatgggccaggaggtgacactgcgctgtaagccaatctctggccacaacagcctgttttggtatagggagaccatgatgcgcggcctggagctgctgatctacttcaataacaatgtgcccatcgacgattccggcatgcctgaggatcggttttctgccaagatgcccaatgccagcttctccacactgaagatccagcctagcgagccaagagactccgccgtgtatttttgcgcctctagcccaggcagcaccgatacacagtacttcggaccaggaaccaggctgacagtgctggaggacctgaagaacgtgttcccccctgaggtggccgtgtttgagccctctgaggccgagatcagccacacccagaaggccaccctggtgtgcctggcaaccggcttctatcctgatcacgtggagctgtcctggtgggtgaacggcaaggaggtgcacagcggcgtgtccacagacccacagcccctgaaggagcagccagccctgaatgatagccggtattgcctgtcctctcggctgagagtgtccgccaccttttggcagaacccccggaatcacttcagatgtcaggtgcagttttacggcctgtccgagaacgatgagtggacccaggaccgggccaagcctgtgacacagatcgtgtctgccgaggcatggggaagagcagactgtggcttcacctctgagagctaccagcagggcgtgctgagcgccaccatcctgtatgagatcctgctgggcaaggccacactgtacgccgtcctggtctccgctctggtgctgatggcaatggtcaaaagaaaagatagtcggggacgggccaagagatctggcagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccccatggagaagaatcccctggctgcccccctgctgatcctgtggtttcacctggactgcgtgtcctctatcctgaatgtggaacagagcccacagagcctgcacgtgcaggagggcgactccaccaacttcacatgctcttttcctagctccaacttctacgccctgcactggtacagaaaggagaccgcaaagtccccagaggccctgttcgtgatgacactgaacggcgatgagaagaagaagggccgcatcagcgccaccctgaatacaaaggagggctactcctatctgtacatcaagggctcccagcctgaggactctgccacctatctgtgcgccctgtacaacaataacgatatgcggtttggcgccggcaccagactgacagtgaagccaaacatccagaatccagaccccgccgtgtatcagctgcgggacagcaagtctagcgataagagcgtgtgcctgttcaccgactttgattctcagacaaacgtgagccagtccaaggacagcgacgtgtacatcaccgacaagacagtgctggatatgagaagcatggacttcaagtctaacagcgccgtggcctggtccaataagtctgatttcgcctgcgccaatgcctttaataactccatcatccccgaggataccttctttccttctccagagtcctcttgtgacgtgaagctggtggagaagtctttcgagaccgatacaaacctgaattttcagaacctgagcgtgatcggcttcaggatcctgctgctgaaggtggccggctttaatctgctgatgaccctgaggctgtggagctcccgggccaagagatctggcagcggcgagggcagaggcagcctgctgacctgcggcgacgtggaggagaaccccggccccatgcgcccgagactgtggcttctgctcgccgcgcaactgactgtcctgcacggaaacagcgtgctgcagcagacaccggcctacatcaaagtgcagaccaacaagatggtcatgctgtcctgcgaggccaagatttccctctccaacatgcggatctattggttgcggcagagacaggcgccttcctcggactcccaccatgagttcttggccctgtgggactccgccaagggaactattcacggcgaagaagtggaacaggagaagatcgccgtgtttcgcgatgcctcccgctttatactgaatctgacctccgtgaagcccgaagatagcgggatctacttttgcatgattgtgggctcacccgaactgaccttcgggaagggcactcagctgagcgtggtggacttcctccccactaccgcccaacccactaagaagtcaaccctgaagaagcgggtttgcagactcccacggccggaaacgcagaagggtccgctgtgttccccgatcaccctggggctccttgtggctggagtgctggtccttctggtgtcccttggcgtcgccattcacctctgctgccggagaaggagggccagactgaggttcatgaagcagcctcagggagaggggatcagtggcactttcgtgccacaatgcctccatggctactattccaacaccaccacctcgcaaaagctgctgaacccctggatcctgaaaacccgggccaagagatctggcagcggccagtgcaccaactacgccctgctgaagctggccggcgacgtggagagcaaccccggccccatggcgcttcccgtgaccgcactcctgttgccccttgccctgctgttgcacgccgcacgaccttcccaattccgggtgtcccctctggatcgcacctggaacctcggggaaacggtggagctcaagtgtcaagtcctcctgtcgaacccgaccagcggatgcagctggctgttccagccgagaggagctgccgcctcacccaccttcctcctgtacttgagccagaacaagccgaaggccgctgagggtctggacacccagcgcttctcgggcaaacggctgggagacacttttgtgctgactctctccgacttccggcgggagaacgagggctactacttctgctctgcgctctccaattcaatcatgtacttctcacacttcgtgccggtgttcctgcctgccaagcccaccactactccggcacccagacctccaactcccgctcccaccatcgcgtcccaacccctttcgctgcgccctgaagcgtgtcggcctgctgctggaggagccgtgcatacccgcggtctggacttcgcgtgcgacatctacatttgggcccctttggctggcacctgtggagtgctgctcctgtcccttgtgatcaccctgtactgcaaccaccggaataggcggagagtctgcaagtgtccgcggcctgtcgtgaagtcaggagataagccgagcctgtccgcacgctacgtgtgaaccggtccgcagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
92 | TPE WPRE | tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcagcggccgccccgggtcgacgctaccaccatggactcttggaccttctgctgcgtgagcctgtgcatcctggtggccaagcacacagacgccggcgtgatccagtcccctaggcacgaggtgaccgagatgggccaggaggtgacactgcgctgtaagccaatctctggccacaacagcctgttttggtatagggagaccatgatgcgcggcctggagctgctgatctacttcaataacaatgtgcccatcgacgattccggcatgcctgaggatcggttttctgccaagatgcccaatgccagcttctccacactgaagatccagcctagcgagccaagagactccgccgtgtatttttgcgcctctagcccaggcagcaccgatacacagtacttcggaccaggaaccaggctgacagtgctggaggacctgaagaacgtgttcccccctgaggtggccgtgtttgagccctctgaggccgagatcagccacacccagaaggccaccctggtgtgcctggcaaccggcttctatcctgatcacgtggagctgtcctggtgggtgaacggcaaggaggtgcacagcggcgtgtccacagacccacagcccctgaaggagcagccagccctgaatgatagccggtattgcctgtcctctcggctgagagtgtccgccaccttttggcagaacccccggaatcacttcagatgtcaggtgcagttttacggcctgtccgagaacgatgagtggacccaggaccgggccaagcctgtgacacagatcgtgtctgccgaggcatggggaagagcagactgtggcttcacctctgagagctaccagcagggcgtgctgagcgccaccatcctgtatgagatcctgctgggcaaggccacactgtacgccgtcctggtctccgctctggtgctgatggcaatggtcaaaagaaaagatagtcggggacgggccaagagatctggcagcggcgagggcagaggcagcctgctgacctgcggcgacgtggaggagaaccccggccccatggagaagaatcccctggctgcccccctgctgatcctgtggtttcacctggactgcgtgtcctctatcctgaatgtggaacagagcccacagagcctgcacgtgcaggagggcgactccaccaacttcacatgctcttttcctagctccaacttctacgccctgcactggtacagaaaggagaccgcaaagtccccagaggccctgttcgtgatgacactgaacggcgatgagaagaagaagggccgcatcagcgccaccctgaatacaaaggagggctactcctatctgtacatcaagggctcccagcctgaggactctgccacctatctgtgcgccctgtacaacaataacgatatgcggtttggcgccggcaccagactgacagtgaagccaaacatccagaatccagaccccgccgtgtatcagctgcgggacagcaagtctagcgataagagcgtgtgcctgttcaccgactttgattctcagacaaacgtgagccagtccaaggacagcgacgtgtacatcaccgacaagacagtgctggatatgagaagcatggacttcaagtctaacagcgccgtggcctggtccaataagtctgatttcgcctgcgccaatgcctttaataactccatcatccccgaggataccttctttccttctccagagtcctcttgtgacgtgaagctggtggagaagtctttcgagaccgatacaaacctgaattttcagaacctgagcgtgatcggcttcaggatcctgctgctgaaggtggccggctttaatctgctgatgaccctgaggctgtggagctcccgggccaagagatctggcagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccccatgcgcccgagactgtggcttctgctcgccgcgcaactgactgtcctgcacggaaacagcgtgctgcagcagacaccggcctacatcaaagtgcagaccaacaagatggtcatgctgtcctgcgaggccaagatttccctctccaacatgcggatctattggttgcggcagagacaggcgccttcctcggactcccaccatgagttcttggccctgtgggactccgccaagggaactattcacggcgaagaagtggaacaggagaagatcgccgtgtttcgcgatgcctcccgctttatactgaatctgacctccgtgaagcccgaagatagcgggatctacttttgcatgattgtgggctcacccgaactgaccttcgggaagggcactcagctgagcgtggtggacttcctccccactaccgcccaacccactaagaagtcaaccctgaagaagcgggtttgcagactcccacggccggaaacgcagaagggtccgctgtgttccccgatcaccctggggctccttgtggctggagtgctggtccttctggtgtcccttggcgtcgccattcacctctgctgccggagaaggagggccagactgaggttcatgaagcagcctcagggagaggggatcagtggcactttcgtgccacaatgcctccatggctactattccaacaccaccacctcgcaaaagctgctgaacccctggatcctgaaaacccgggccaagagatctggcagcggccagtgcaccaactacgccctgctgaagctggccggcgacgtggagagcaaccccggccccatggcgcttcccgtgaccgcactcctgttgccccttgccctgctgttgcacgccgcacgaccttcccaattccgggtgtcccctctggatcgcacctggaacctcggggaaacggtggagctcaagtgtcaagtcctcctgtcgaacccgaccagcggatgcagctggctgttccagccgagaggagctgccgcctcacccaccttcctcctgtacttgagccagaacaagccgaaggccgctgagggtctggacacccagcgcttctcgggcaaacggctgggagacacttttgtgctgactctctccgacttccggcgggagaacgagggctactacttctgctctgcgctctccaattcaatcatgtacttctcacacttcgtgccggtgttcctgcctgccaagcccaccactactccggcacccagacctccaactcccgctcccaccatcgcgtcccaacccctttcgctgcgccctgaagcgtgtcggcctgctgctggaggagccgtgcatacccgcggtctggacttcgcgtgcgacatctacatttgggcccctttggctggcacctgtggagtgctgctcctgtcccttgtgatcaccctgtactgcaaccaccggaataggcggagagtctgcaagtgtccgcggcctgtcgtgaagtcaggagataagccgagcctgtccgcacgctacgtgtgaaccggtccgcagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
93 | PTE fn WPRE | tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcagcggccgccccgggtcgacgctaccaccatggactcttggaccttctgctgcgtgagcctgtgcatcctggtggccaagcacacagacgccggcgtgatccagtcccctaggcacgaggtgaccgagatgggccaggaggtgacactgcgctgtaagccaatctctggccacaacagcctgttttggtatagggagaccatgatgcgcggcctggagctgctgatctacttcaataacaatgtgcccatcgacgattccggcatgcctgaggatcggttttctgccaagatgcccaatgccagcttctccacactgaagatccagcctagcgagccaagagactccgccgtgtatttttgcgcctctagcccaggcagcaccgatacacagtacttcggaccaggaaccaggctgacagtgctggaggacctgaagaacgtgttcccccctgaggtggccgtgtttgagccctctgaggccgagatcagccacacccagaaggccaccctggtgtgcctggcaaccggcttctatcctgatcacgtggagctgtcctggtgggtgaacggcaaggaggtgcacagcggcgtgtccacagacccacagcccctgaaggagcagccagccctgaatgatagccggtattgcctgtcctctcggctgagagtgtccgccaccttttggcagaacccccggaatcacttcagatgtcaggtgcagttttacggcctgtccgagaacgatgagtggacccaggaccgggccaagcctgtgacacagatcgtgtctgccgaggcatggggaagagcagactgtggcttcacctctgagagctaccagcagggcgtgctgagcgccaccatcctgtatgagatcctgctgggcaaggccacactgtacgccgtcctggtctccgctctggtgctgatggcaatggtcaaaagaaaagatagtcggggatctggcagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccccatggagaagaatcccctggctgcccccctgctgatcctgtggtttcacctggactgcgtgtcctctatcctgaatgtggaacagagcccacagagcctgcacgtgcaggagggcgactccaccaacttcacatgctcttttcctagctccaacttctacgccctgcactggtacagaaaggagaccgcaaagtccccagaggccctgttcgtgatgacactgaacggcgatgagaagaagaagggccgcatcagcgccaccctgaatacaaaggagggctactcctatctgtacatcaagggctcccagcctgaggactctgccacctatctgtgcgccctgtacaacaataacgatatgcggtttggcgccggcaccagactgacagtgaagccaaacatccagaatccagaccccgccgtgtatcagctgcgggacagcaagtctagcgataagagcgtgtgcctgttcaccgactttgattctcagacaaacgtgagccagtccaaggacagcgacgtgtacatcaccgacaagacagtgctggatatgagaagcatggacttcaagtctaacagcgccgtggcctggtccaataagtctgatttcgcctgcgccaatgcctttaataactccatcatccccgaggataccttctttccttctccagagtcctcttgtgacgtgaagctggtggagaagtctttcgagaccgatacaaacctgaattttcagaacctgagcgtgatcggcttcaggatcctgctgctgaaggtggccggctttaatctgctgatgaccctgaggctgtggagctcccgggccaagagaggcagcggcgagggcagaggcagcctgctgacctgcggcgacgtggaggagaaccccggccccatgcgcccgagactgtggcttctgctcgccgcgcaactgactgtcctgcacggaaacagcgtgctgcagcagacaccggcctacatcaaagtgcagaccaacaagatggtcatgctgtcctgcgaggccaagatttccctctccaacatgcggatctattggttgcggcagagacaggcgccttcctcggactcccaccatgagttcttggccctgtgggactccgccaagggaactattcacggcgaagaagtggaacaggagaagatcgccgtgtttcgcgatgcctcccgctttatactgaatctgacctccgtgaagcccgaagatagcgggatctacttttgcatgattgtgggctcacccgaactgaccttcgggaagggcactcagctgagcgtggtggacttcctccccactaccgcccaacccactaagaagtcaaccctgaagaagcgggtttgcagactcccacggccggaaacgcagaagggtccgctgtgttccccgatcaccctggggctccttgtggctggagtgctggtccttctggtgtcccttggcgtcgccattcacctctgctgccggagaaggagggccagactgaggttcatgaagcagcctcagggagaggggatcagtggcactttcgtgccacaatgcctccatggctactattccaacaccaccacctcgcaaaagctgctgaacccctggatcctgaaaacccgggccaagagatctggcagcggccagtgcaccaactacgccctgctgaagctggccggcgacgtggagagcaaccccggccccatggcgcttcccgtgaccgcactcctgttgccccttgccctgctgttgcacgccgcacgaccttcccaattccgggtgtcccctctggatcgcacctggaacctcggggaaacggtggagctcaagtgtcaagtcctcctgtcgaacccgaccagcggatgcagctggctgttccagccgagaggagctgccgcctcacccaccttcctcctgtacttgagccagaacaagccgaaggccgctgagggtctggacacccagcgcttctcgggcaaacggctgggagacacttttgtgctgactctctccgacttccggcgggagaacgagggctactacttctgctctgcgctctccaattcaatcatgtacttctcacacttcgtgccggtgttcctgcctgccaagcccaccactactccggcacccagacctccaactcccgctcccaccatcgcgtcccaacccctttcgctgcgccctgaagcgtgtcggcctgctgctggaggagccgtgcatacccgcggtctggacttcgcgtgcgacatctacatttgggcccctttggctggcacctgtggagtgctgctcctgtcccttgtgatcaccctgtactgcaaccaccggaataggcggagagtctgcaagtgtccgcggcctgtcgtgaagtcaggagataagccgagcctgtccgcacgctacgtgtgaaccggtccgcagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
94 | PTE CD8 TCR WPRE | tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcagcggccgccccgggtcgacgctaccaccatgcgcccgagactgtggcttctgctcgccgcgcaactgactgtcctgcacggaaacagcgtgctgcagcagacaccggcctacatcaaagtgcagaccaacaagatggtcatgctgtcctgcgaggccaagatttccctctccaacatgcggatctattggttgcggcagagacaggcgccttcctcggactcccaccatgagttcttggccctgtgggactccgccaagggaactattcacggcgaagaagtggaacaggagaagatcgccgtgtttcgcgatgcctcccgctttatactgaatctgacctccgtgaagcccgaagatagcgggatctacttttgcatgattgtgggctcacccgaactgaccttcgggaagggcactcagctgagcgtggtggacttcctccccactaccgcccaacccactaagaagtcaaccctgaagaagcgggtttgcagactcccacggccggaaacgcagaagggtccgctgtgttccccgatcaccctggggctccttgtggctggagtgctggtccttctggtgtcccttggcgtcgccattcacctctgctgccggagaaggagggccagactgaggttcatgaagcagcctcagggagaggggatcagtggcactttcgtgccacaatgcctccatggctactattccaacaccaccacctcgcaaaagctgctgaacccctggatcctgaaaacccgggccaagagatctggcagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccccatggcgcttcccgtgaccgcactcctgttgccccttgccctgctgttgcacgccgcacgaccttcccaattccgggtgtcccctctggatcgcacctggaacctcggggaaacggtggagctcaagtgtcaagtcctcctgtcgaacccgaccagcggatgcagctggctgttccagccgagaggagctgccgcctcacccaccttcctcctgtacttgagccagaacaagccgaaggccgctgagggtctggacacccagcgcttctcgggcaaacggctgggagacacttttgtgctgactctctccgacttccggcgggagaacgagggctactacttctgctctgcgctctccaattcaatcatgtacttctcacacttcgtgccggtgttcctgcctgccaagcccaccactactccggcacccagacctccaactcccgctcccaccatcgcgtcccaacccctttcgctgcgccctgaagcgtgtcggcctgctgctggaggagccgtgcatacccgcggtctggacttcgcgtgcgacatctacatttgggcccctttggctggcacctgtggagtgctgctcctgtcccttgtgatcaccctgtactgcaaccaccggaataggcggagagtctgcaagtgtccgcggcctgtcgtgaagtcaggagataagccgagcctgtccgcacgctacgtgcgggccaagagatctggcagcggcgagggcagaggcagcctgctgacctgcggcgacgtggaggagaaccccggccccatggactcttggaccttctgctgcgtgagcctgtgcatcctggtggccaagcacacagacgccggcgtgatccagtcccctaggcacgaggtgaccgagatgggccaggaggtgacactgcgctgtaagccaatctctggccacaacagcctgttttggtatagggagaccatgatgcgcggcctggagctgctgatctacttcaataacaatgtgcccatcgacgattccggcatgcctgaggatcggttttctgccaagatgcccaatgccagcttctccacactgaagatccagcctagcgagccaagagactccgccgtgtatttttgcgcctctagcccaggcagcaccgatacacagtacttcggaccaggaaccaggctgacagtgctggaggacctgaagaacgtgttcccccctgaggtggccgtgtttgagccctctgaggccgagatcagccacacccagaaggccaccctggtgtgcctggcaaccggcttctatcctgatcacgtggagctgtcctggtgggtgaacggcaaggaggtgcacagcggcgtgtccacagacccacagcccctgaaggagcagccagccctgaatgatagccggtattgcctgtcctctcggctgagagtgtccgccaccttttggcagaacccccggaatcacttcagatgtcaggtgcagttttacggcctgtccgagaacgatgagtggacccaggaccgggccaagcctgtgacacagatcgtgtctgccgaggcatggggaagagcagactgtggcttcacctctgagagctaccagcagggcgtgctgagcgccaccatcctgtatgagatcctgctgggcaaggccacactgtacgccgtcctggtctccgctctggtgctgatggcaatggtcaaaagaaaagatagtcggggacgggccaagagatctggcagcggccagtgcaccaactacgccctgctgaagctggccggcgacgtggagagcaaccccggccccatggagaagaatcccctggctgcccccctgctgatcctgtggtttcacctggactgcgtgtcctctatcctgaatgtggaacagagcccacagagcctgcacgtgcaggagggcgactccaccaacttcacatgctcttttcctagctccaacttctacgccctgcactggtacagaaaggagaccgcaaagtccccagaggccctgttcgtgatgacactgaacggcgatgagaagaagaagggccgcatcagcgccaccctgaatacaaaggagggctactcctatctgtacatcaagggctcccagcctgaggactctgccacctatctgtgcgccctgtacaacaataacgatatgcggtttggcgccggcaccagactgacagtgaagccaaacatccagaatccagaccccgccgtgtatcagctgcgggacagcaagtctagcgataagagcgtgtgcctgttcaccgactttgattctcagacaaacgtgagccagtccaaggacagcgacgtgtacatcaccgacaagacagtgctggatatgagaagcatggacttcaagtctaacagcgccgtggcctggtccaataagtctgatttcgcctgcgccaatgcctttaataactccatcatccccgaggataccttctttccttctccagagtcctcttgtgacgtgaagctggtggagaagtctttcgagaccgatacaaacctgaattttcagaacctgagcgtgatcggcttcaggatcctgctgctgaaggtggccggctttaatctgctgatgaccctgaggctgtggagctcctgaaccggtccgcagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
95 | R11KE WPRE | tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcagcggccgccccgggtcgacgctaccaccatggactcttggaccttctgctgcgtgagcctgtgcatcctggtggccaagcacacagacgccggcgtgatccagtcccctaggcacgaggtgaccgagatgggccaggaggtgacactgcgctgtaagccaatctctggccacaacagcctgttttggtatagggagaccatgatgcgcggcctggagctgctgatctacttcaataacaatgtgcccatcgacgattccggcatgcctgaggatcggttttctgccaagatgcccaatgccagcttctccacactgaagatccagcctagcgagccaagagactccgccgtgtatttttgcgcctctagcccaggcagcaccgatacacagtacttcggaccaggaaccaggctgacagtgctggaggacctgaagaacgtgttcccccctgaggtggccgtgtttgagccctctgaggccgagatcagccacacccagaaggccaccctggtgtgcctggcaaccggcttctatcctgatcacgtggagctgtcctggtgggtgaacggcaaggaggtgcacagcggcgtgtccacagacccacagcccctgaaggagcagccagccctgaatgatagccggtattgcctgtcctctcggctgagagtgtccgccaccttttggcagaacccccggaatcacttcagatgtcaggtgcagttttacggcctgtccgagaacgatgagtggacccaggaccgggccaagcctgtgacacagatcgtgtctgccgaggcatggggaagagcagactgtggcttcacctctgagagctaccagcagggcgtgctgagcgccaccatcctgtatgagatcctgctgggcaaggccacactgtacgccgtcctggtctccgctctggtgctgatggcaatggtcaaaagaaaagatagtcggggacgggccaagagatctggcagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccccatggagaagaatcccctggctgcccccctgctgatcctgtggtttcacctggactgcgtgtcctctatcctgaatgtggaacagagcccacagagcctgcacgtgcaggagggcgactccaccaacttcacatgctcttttcctagctccaacttctacgccctgcactggtacagaaaggagaccgcaaagtccccagaggccctgttcgtgatgacactgaacggcgatgagaagaagaagggccgcatcagcgccaccctgaatacaaaggagggctactcctatctgtacatcaagggctcccagcctgaggactctgccacctatctgtgcgccctgtacaacaataacgatatgcggtttggcgccggcaccagactgacagtgaagccaaacatccagaatccagaccccgccgtgtatcagctgcgggacagcaagtctagcgataagagcgtgtgcctgttcaccgactttgattctcagacaaacgtgagccagtccaaggacagcgacgtgtacatcaccgacaagacagtgctggatatgagaagcatggacttcaagtctaacagcgccgtggcctggtccaataagtctgatttcgcctgcgccaatgcctttaataactccatcatccccgaggataccttctttccttctccagagtcctcttgtgacgtgaagctggtggagaagtctttcgagaccgatacaaacctgaattttcagaacctgagcgtgatcggcttcaggatcctgctgctgaaggtggccggctttaatctgctgatgaccctgaggctgtggagctcctgaaccggtccgcagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
96 | CD8 WPRE | tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcagcggccgccccgggtcgacgctaccaccatgcgcccgagactgtggcttctgctcgccgcgcaactgactgtcctgcacggaaacagcgtgctgcagcagacaccggcctacatcaaagtgcagaccaacaagatggtcatgctgtcctgcgaggccaagatttccctctccaacatgcggatctattggttgcggcagagacaggcgccttcctcggactcccaccatgagttcttggccctgtgggactccgccaagggaactattcacggcgaagaagtggaacaggagaagatcgccgtgtttcgcgatgcctcccgctttatactgaatctgacctccgtgaagcccgaagatagcgggatctacttttgcatgattgtgggctcacccgaactgaccttcgggaagggcactcagctgagcgtggtggacttcctccccactaccgcccaacccactaagaagtcaaccctgaagaagcgggtttgcagactcccacggccggaaacgcagaagggtccgctgtgttccccgatcaccctggggctccttgtggctggagtgctggtccttctggtgtcccttggcgtcgccattcacctctgctgccggagaaggagggccagactgaggttcatgaagcagcctcagggagaggggatcagtggcactttcgtgccacaatgcctccatggctactattccaacaccaccacctcgcaaaagctgctgaacccctggatcctgaaaacccgggccaagagatctggcagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccccatggcgcttcccgtgaccgcactcctgttgccccttgccctgctgttgcacgccgcacgaccttcccaattccgggtgtcccctctggatcgcacctggaacctcggggaaacggtggagctcaagtgtcaagtcctcctgtcgaacccgaccagcggatgcagctggctgttccagccgagaggagctgccgcctcacccaccttcctcctgtacttgagccagaacaagccgaaggccgctgagggtctggacacccagcgcttctcgggcaaacggctgggagacacttttgtgctgactctctccgacttccggcgggagaacgagggctactacttctgctctgcgctctccaattcaatcatgtacttctcacacttcgtgccggtgttcctgcctgccaagcccaccactactccggcacccagacctccaactcccgctcccaccatcgcgtcccaacccctttcgctgcgccctgaagcgtgtcggcctgctgctggaggagccgtgcatacccgcggtctggacttcgcgtgcgacatctacatttgggcccctttggctggcacctgtggagtgctgctcctgtcccttgtgatcaccctgtactgcaaccaccggaataggcggagagtctgcaagtgtccgcggcctgtcgtgaagtcaggagataagccgagcctgtccgcacgctacgtgtgaaccggtccgcagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
97 | RD114TR | MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMTNQKWKCRVTPKISPSGGELQNCPCNTFQDSMHSSCYTEYRQCRRINKTYYTATLLKIRSGSLNEVQILQNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQIIPPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGNNMAYTYLPQNWTRLCVQASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYANKSGIVRNKIRTLQEELQKRRESLASNPLWTGLQGFLPYLLPLLGPLLTLLLILTIGPCVFNRLVQFVKDRISVVQALVLTQQYHQLKPL |
265 | WPREmut1 | cagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc |
266 | WPREmut2 | gagcatcttaccgccatttatacccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatggtggggcaatcatttacattttttgggatatgtaattactagttcaggtgtattgccacaagacaaacttgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaactttgttgctccttttacgctgtgtggatttgctgctttattgcctctgtatcttgctattgcttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgtctctttttgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtc |
SEQ ID NO: | 胺基酸序列 | SEQ ID NO: | 胺基酸序列 | SEQ ID NO: | 胺基酸序列 |
98 | YLYDSETKNA | 151 | LLWGHPRVALA | 204 | SLLNQPKAV |
99 | HLMDQPLSV | 152 | VLDGKVAVV | 205 | KMSELQTYV |
100 | GLLKKINSV | 153 | GLLGKVTSV | 206 | ALLEQTGDMSL |
101 | FLVDGSSAL | 154 | KMISAIPTL | 207 | VIIKGLEEITV |
102 | FLFDGSANLV | 155 | GLLETTGLLAT | 208 | KQFEGTVEI |
103 | FLYKIIDEL | 156 | TLNTLDINL | 209 | KLQEEIPVL |
104 | FILDSAETTTL | 157 | VIIKGLEEI | 210 | GLAEFQENV |
105 | SVDVSPPKV | 158 | YLEDGFAYV | 211 | NVAEIVIHI |
106 | VADKIHSV | 159 | KIWEELSVLEV | 212 | ALAGIVTNV |
107 | IVDDLTINL | 160 | LLIPFTIFM | 213 | NLLIDDKGTIKL |
108 | GLLEELVTV | 161 | ISLDEVAVSL | 214 | VLMQDSRLYL |
109 | TLDGAAVNQV | 162 | KISDFGLATV | 215 | KVLEHVVRV |
110 | SVLEKEIYSI | 163 | KLIGNIHGNEV | 216 | LLWGNLPEI |
111 | LLDPKTIFL | 164 | ILLSVLHQL | 217 | SLMEKNQSL |
112 | YTFSGDVQL | 165 | LDSEALLTL | 218 | KLLAVIHEL |
113 | YLMDDFSSL | 166 | VLQENSSDYQSNL | 219 | ALGDKFLLRV |
114 | KVWSDVTPL | 167 | HLLGEGAFAQV | 220 | FLMKNSDLYGA |
115 | LLWGHPRVALA | 168 | SLVENIHVL | 221 | KLIDHQGLYL |
116 | KIWEELSVLEV | 169 | YTFSGDVQL | 222 | GPGIFPPPPPQP |
117 | LLIPFTIFM | 170 | SLSEKSPEV | 223 | ALNESLVEC |
118 | FLIENLLAA | 171 | AMFPDTIPRV | 224 | GLAALAVHL |
119 | LLWGHPRVALA | 172 | FLIENLLAA | 225 | LLLEAVWHL |
120 | FLLEREQLL | 173 | FTAEFLEKV | 226 | SIIEYLPTL |
121 | SLAETIFIV | 174 | ALYGNVQQV | 227 | TLHDQVHLL |
122 | TLLEGISRA | 175 | LFQSRIAGV | 228 | SLLMWITQC |
123 | KIQEILTQV | 176 | ILAEEPIYIRV | 229 | FLLDKPQDLSI |
124 | VIFEGEPMYL | 177 | FLLEREQLL | 230 | YLLDMPLWYL |
125 | SLFESLEYL | 178 | LLLPLELSLA | 231 | GLLDCPIFL |
126 | SLLNQPKAV | 179 | SLAETIFIV | 232 | VLIEYNFSI |
127 | GLAEFQENV | 180 | AILNVDEKNQV | 233 | TLYNPERTITV |
128 | KLLAVIHEL | 181 | RLFEEVLGV | 234 | AVPPPPSSV |
129 | TLHDQVHLL | 182 | YLDEVAFML | 235 | KLQEELNKV |
130 | TLYNPERTITV | 183 | KLIDEDEPLFL | 236 | KLMDPGSLPPL |
131 | KLQEKIQEL | 184 | KLFEKSTGL | 237 | ALIVSLPYL |
132 | SVLEKEIYSI | 185 | SLLEVNEASSV | 238 | FLLDGSANV |
133 | RVIDDSLVVGV | 186 | GVYDGREHTV | 239 | ALDPSGNQLI |
134 | VLFGELPAL | 187 | GLYPVTLVGV | 240 | ILIKHLVKV |
135 | GLVDIMVHL | 188 | ALLSSVAEA | 241 | VLLDTILQL |
136 | FLNAIETAL | 189 | TLLEGISRA | 242 | HLIAEIHTA |
137 | ALLQALMEL | 190 | SLIEESEEL | 243 | SMNGGVFAV |
138 | ALSSSQAEV | 191 | ALYVQAPTV | 244 | MLAEKLLQA |
139 | SLITGQDLLSV | 192 | KLIYKDLVSV | 245 | YMLDIFHEV |
140 | QLIEKNWLL | 193 | ILQDGQFLV | 246 | ALWLPTDSATV |
141 | LLDPKTIFL | 194 | SLLDYEVSI | 247 | GLASRILDA |
142 | RLHDENILL | 195 | LLGDSSFFL | 248 | ALSVLRLAL |
143 | YTFSGDVQL | 196 | VIFEGEPMYL | 249 | SYVKVLHHL |
144 | GLPSATTTV | 197 | ALSYILPYL | 250 | VYLPKIPSW |
145 | GLLPSAESIKL | 198 | FLFVDPELV | 251 | NYEDHFPLL |
146 | KTASINQNV | 199 | SEWGSPHAAVP | 252 | VYIAELEKI |
147 | SLLQHLIGL | 200 | ALSELERVL | 253 | VHFEDTGKTLLF |
148 | YLMDDFSSL | 201 | SLFESLEYL | 254 | VLSPFILTL |
149 | LMYPYIYHV | 202 | KVLEYVIKV | 255 | HLLEGSVGV |
150 | KVWSDVTPL | 203 | VLLNEILEQV |
實例2
γδ T細胞製造
為了分離γδ T細胞,在一態樣中,可自受試者或自受試者之複雜樣本分離γδ T細胞。在一態樣中,複雜樣本可為周邊血液樣本、臍帶血樣本、腫瘤、幹細胞前體、腫瘤生物檢體、組織、淋巴液或來自直接接觸外部環境或自幹前體細胞衍生的受試者之上皮位點。可自受試者之複雜樣本直接分離γδ T細胞,例如,藉由利用流動式細胞測量術技術將表現一或多個細胞表面標記之γδ T細胞分類。野生型γδ T細胞可展現可與γδ T細胞相關聯之眾多抗原辨識、抗原呈現、共刺激及黏著分子。一或多個細胞表面標記(諸如特異性γδ TCR、抗原辨識、抗原呈現、配位子、黏著分子或共刺激分子)可用於自複雜樣本分離野生型γδ T細胞。與γδ T細胞相關聯或由γδ T細胞表現之各種分子可用於自複雜樣本分離γδ T細胞,例如,分離Vδ1+、Vδ2+、Vδ3+細胞或其任何組合混合群體。
舉例而言,可例如利用血球分離機(包括Ficoll-Paque™ PLUS (GE Healthcare)系統或另一合適之裝置/系統)自受試者收集周邊血液單核細胞。γδ T細胞或γδ T細胞之所要亞群可利用(例如,利用流動式細胞測量術技術)自收集之樣本純化。臍帶血細胞亦可在受試者誕生期間自臍帶血獲得。
表現在收集之γδ T細胞上的細胞表面標記之陽性及/或陰性選擇可用於自周邊血液樣本、臍帶血樣本、腫瘤、腫瘤生物檢體、組織、淋巴液或自受試者之上皮樣本直接分離γδ T細胞或表現類似細胞表面標記之γδ T細胞群體。舉例而言,可基於以下各者之陽性或陰性表現而自複雜樣本分離γδ T細胞:CD2、CD3、CD4、CD8、CD24、CD25、CD44、Kit、TCR α、TCR β、TCR α、TCR δ、NKG2D、CD70、CD27、CD30、CD16、CD337 (NKp30)、CD336 (NKp46)、OX40、CD46、CCR7及其他合適之細胞表面標記。
第1圖展示根據本發明之一實施例之γδ T細胞製造。此程序可包括自白血球分離術產品收集或獲得白血球或PBMC。白血球分離術可包括自供體收集全血且使用血球分離機來分離成分。血球分離機分離出所要之血液成分且將剩餘部分返還至供體之循環。舉例而言,可使用血球分離設備來收集白血球、血漿及血小板,且將紅血球及嗜中性球返還至供體之循環。在此程序中可使用可購得之白血球分離術產品。獲得白血球之另一方法為自膚色血球層(buffy coat)獲得白血球。為了分離膚色血球層,自供體獲得抗凝全血且進行離心分離。在離心分離之後,血液分成血漿、紅血球及膚色血球層。膚色血球層係位於血漿與紅血球層之間的層。血球分離收集可導致與藉由膚色血球層收集達成之結果相比純度較高且單核細胞含量顯著增加。利用白血球分離術可能達成之單核細胞含量通常可為自膚色血球層獲得之單核細胞含量之20倍。為了豐富單核細胞,進一步分離可能需要使用Ficoll梯度。
為了耗盡來自PBMC之αβ T細胞,可藉由磁性分離(例如,使用經抗αβ TCR抗體塗佈之CliniMACS®磁珠)自PBMC分離αβ TCR表現細胞,接著低溫保存αβ TCR-T細胞耗盡之PBMC。為了製造「現貨」T細胞產品,可將低溫保存的αβ TCR-T細胞耗盡之PBMC解凍,且在胺基雙膦酸酯(例如,唑來膦酸)及/或異戊烯基焦磷酸鹽 (isopentenyl pyrophosphate;IPP)及/或細胞介素(例如,白細胞介素2 (interleukin 2;IL-2)、白細胞介素15 (interleukin 15;IL-15)及/或白細胞介素18 (interleukin 18;IL-18))及/或其他活化劑(例如,類鐸受體2 (Toll-like receptor;TLR2) 配位子)存在之情況下以小/中等規模(例如,24至4至6孔盤或T75/T175燒瓶)或以大規模(例如,50 ml至100公升袋)活化1天至10天(例如,2天至7天)。
第1圖展示:經活化T細胞可藉由用病毒載體(諸如慢病毒載體)進行轉導來工程化,該病毒載體將感興趣之外源性基因(諸如針對特定癌抗原及CD8之αβ TCR)表現至分離之γδ T細胞中。轉導可實行一次或多次以達成小規模(例如,24至4至6孔盤)或中等/大規模之穩定轉基因表現持續½天至5天(例如,1天)。
第1圖進一步展示:轉導或工程化γδ T細胞之擴增可在細胞介素(例如,IL-2、IL-15、IL-18及其他細胞介素)存在之情況下以小/中等規模(例如,燒瓶/G-Rex)或以大規模(例如,50 ml至100公升袋)實行持續7天至35天(例如7天至28天)。接著可低溫保存擴增之轉導T細胞產品以作為用於輸注至病人中之「現貨」T細胞產品。
實例3
慢病毒病毒載體
本文中所使用之慢病毒載體含有先前示出以增強載體功能之若干元件,該等元件包括用於改良之複製及核輸入的中央多嘌呤區(central polypurine tract;cPPT)、來自鼠類幹細胞病毒(Murine Stem Cell Virus;MSCV) (SEQ ID NO: 1)之啟動子(該啟動子已展示出減少某些細胞型中之載體緘默)、用於改良之轉錄終止的土撥鼠肝炎病毒轉錄後回應元件(woodchuck hepatitis virus posttranscriptional responsive element;WPRE) (SEQ ID NO: 9),且主鏈係可具有改良之安全性、持續基因表現及反緘默性質之刪除3’-LTR 自失活(self-inactivating;SIN)載體設計(Yang等人,Gene Therapy
(2008) 15, 1411–1423,參考文獻之內容係以全文引用之方式併入)。
在一態樣中,本文中所描述之載體、構築體或序列包含WPRE之突變形式。在另一態樣中,本文中所描述之序列或載體包含WPRE型式1之突變(例如,WPREmut1 (SEQ ID NO: 265))或WPRE型式2之突變(例如,WPREmut2 (SEQ ID NO: 266))。在一態樣中,WPRE突變體包含至多一種突變、至多兩種突變、至多三種突變、至少四種突變或至多五種突變。在一態樣中,本文中所描述之載體、構築體或序列不包含WPRE。在另一態樣中,本發明提供SEQ ID NO: 91至96中之一者中的一種、兩種、三種、四種、五種、十種或20種取代。
在另一態樣中,本文中所描述之載體、構築體或序列不包括X蛋白啟動子。
為了在轉導γδ T細胞中獲得TCRαβ及CD8αβ之最佳共表現水平,產生具有各種設計之慢病毒載體。第2圖展示T細胞可用表現TCRαβ或CD8αβ之兩種單獨慢病毒載體(2合1)及共表現TCRαβ及CD8αβ之單一慢病毒載體(4合1)轉導。在4合1載體中,編碼TCRα鏈、TCRβ鏈、CD8α鏈及CD8β鏈之核苷酸可以各種次序混洗。如此產生之各種4合1載體可用於轉導γδ T細胞,接著使用此項技術中已知之技術(例如,流動式細胞測量術)量測該等轉導細胞之TCR/CD8共表現水平。
為了產生共表現TCRαβ及CD8αβ之慢病毒載體,編碼弗林連接子(GSG或SGSG (SEQ ID NO: 8))-2A肽之核苷酸可定位在TCRα鏈與TCRβ鏈之間、CD8α鏈與CD8β鏈之間及TCR鏈與CD8鏈之間以實現高效率之基因表現。2A肽可選自 P2A (SEQ ID NO: 3)、T2A (SEQ ID NO: 4)、E2A (SEQ ID NO: 5)或F2A (SEQ ID NO: 6)。
慢病毒病毒載體亦可含有轉錄後調節元件(post-transcriptional regulatory element;PRE),諸如土撥鼠PRE (Woodchuck post-transcriptional regulatory element;WPRE) (SEQ ID NO: 9),以藉由提高核及細胞質mRNA水平兩者來增強轉基因之表現。包括以下各者之一或多個調節元件亦可使用及/或與WPRE組合以提高轉基因表現:小鼠RNA運輸元件(RNA transport element;RTE)、1型猿逆轉錄病毒(simian retrovirus type 1;SRV-1)之組成型運輸元件(constitutive transport element;CTE)及人熱休克蛋白70之5′未轉譯區域(Hsp70 5′UTR)。
慢病毒載體可用RD114TR (SEQ ID NO: 97)假型化,RD114TR係含融合至鼠類白血病病毒之細胞質尾(cytoplasmic tail;TR)的貓內源病毒(RD114)之胞外及跨膜域之嵌合醣蛋白。亦可使用其他病毒包膜蛋白(諸如VSV-G env、MLV 4070A env、RD114 env、嵌合包膜蛋白RD114pro、桿狀病毒GP64 env或GALV env)或其衍生物。
第3圖展示共表現TCRαβ (R11KEA)及CD8αβ之四種不同4合1載體(即,PTE WPRE (SEQ ID NO: 91)、TPE WPRE (SEQ ID NO: 92)、PTE fn WPRE (SEQ ID NO: 93)及PTE CD8 TCR WPRE (SEQ ID NO: 94)),且兩種2合1載體(即,表現TCRαβ (R11KEA)之R11KE WPRE (SEQ ID NO: 95)及表現CD8αβ. TCRαβ (R11KEA)之CD8 WPRE (SEQ ID NO: 96))結合至與MHC分子複合之PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147)。
實例4
TCR及CD8之共表現
自供體1及供體2獲得之γδ T細胞係藉由第1圖所示之程序製造。在用唑來膦酸、IL2及IL15活化後之第3天或第6天,用經RD114TR假型化之慢病毒(例如,PTE WPRE (SEQ ID NO: 91)、TPE WPRE (SEQ ID NO: 92)、PTE fn WPRE (SEQ ID NO: 93)及PTE CD8 TCR WPRE (SEQ ID NO: 94)轉導γδ T細胞,接著使用流動式細胞測量術量測R11KEA及CD8之共表現水平。經由流動式細胞測量術使用特定於TCR (Vβ8)及CD8 (CD8α)之抗體來評估轉導效率。
第4圖展示:在來自供體1之γδ T細胞中,由利用PTE CD8 TCR WPRE轉導產生的R11KEA及CD8之共表現水平(即,40.5% (第3天)及18.5% (第6天))比來自利用PTE WPRE (29.6% (第3天),16.2% (第6天))、TPE WPRE (30.8% (第3天),11.0% (第6天))及PTE fn WPRE (33.0% (第3天),15.0% (第6天))轉導的共表現水平高。在來自供體2之γδ T細胞中,由利用PTE WPRE轉導產生的在活化後第6天的R11KEA及CD8之共表現水平(即,18.8%)比來自利用TPE WPRE (14.2%)、PTE fn WPRE (14.7%)及PTE CD8 TCR WPRE (17.2%)轉導的共表現水平高。作為對照,在分開地用2合1載體(即,TCRαβ (R11KEA)或CD8 WPRE)轉導之γδ T細胞中偵測R11KEA及CD8之背景水平。
實例5
對4合1病毒載體(例如,慢病毒載體)之轉基因表現及官能性之影響,該等4合1病毒載體含位於載體中之不同位置處的編碼CD8αβ鏈之序列及編碼TCRαβ鏈之序列。
WO 2019/204662描述表現外源性CD8αβ共受體及一或多個外源性工程化抗原受體(例如,TCR)之CD4+細胞。表4展示WO 2019/204662中所描述之4合1構築體與根據本發明之態樣之4合1構築體之間的比較。
表4
WO 2019/204662 | 本文中描述之態樣 | |
轉基因之定向(自5’末端至3’末端方向) | TCRβ-TCRα-CD8α-CD8β | CD8β-CD8α-TCRβ-TCRα |
CD8αβ序列之來源 | GenBank | 密碼子最佳化(用於增強表現) |
2A連接子 | 2A | 2A + 弗林連接子(用於促進感興趣基因之殘餘2A序列之有效裂解) |
細胞型 | CD4+細胞及CD8+細胞 | γδ T細胞(低(0-20%) CD8表現且無CD4 表現) |
病毒及假型 | 逆轉錄病毒及RD114 | 慢病毒及RD114TR及VSV-G |
本發明之核酸分子之開讀框(open reading frame;ORF)可至少部分地經密碼子最佳化。密碼子最佳化係基於如下發現:轉譯效率可由轉移RNA (transfer RNA;tRNAs)在細胞中出現之不同頻率判定。因此,本發明之核酸分子之開讀框與對應野生型編碼區域相比可經過改造,使得野生型序列的針對在細胞中相對稀有之tRNA編碼之至少一個密碼子可用來交換針對tRNA編碼的密碼子,該密碼子在細胞中相對頻繁且與相對稀有之tRNA攜帶相同胺基酸。藉由此改造,本發明之核酸分子之開讀框可經改造,使得密碼子(可獲得該等密碼子之頻繁出現之tRNA)可置換對應於稀有tRNA之密碼子。換言之,根據本發明,藉由此改造,野生型開讀框的針對稀有tRNA編碼之所有密碼子可用來交換針對tRNA編碼之密碼子,該密碼子在細胞中更頻繁且與稀有tRNA攜帶相同胺基酸。哪些tRNA相對頻繁地出現在細胞中且相比之下哪些tRNA相對很少出現係熟習此項技術者已知的;例如,Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666,參考文獻之內容係以全文引用之方式併入本文中。在一些實施例中,本發明之核酸分子之開讀框可較佳相對於本發明之核酸分子待表現所在之系統、較佳相對於本發明之核酸分子待轉譯所在之系統經密碼子最佳化。較佳地,本發明之核酸分子之開讀框的密碼子使用可根據哺乳動物密碼子用途、更佳根據人密碼子用途加以密碼子最佳化。較佳地,開讀框可經密碼子最佳化且經G/C內含物改造。
為了判定哪個轉基因定向提供更好的轉基因表現及官能性,各自含有位於編碼CD8αβ鏈之序列上游的編碼TCRαβ鏈之序列之三種4合1病毒載體(例如,PTE.WPRE (SEQ ID NO: 91)、TPE.WPRE (SEQ ID NO: 92)及PTE.fn.WPRE (SEQ ID NO: 93))及含有位於編碼TCRαβ鏈之序列上游的編碼CD8αβ鏈之序列之一種4合1病毒載體(例如,PTE.CD8.TCR.WPRE (SEQ ID NO: 94))經轉導至γδ T細胞中,接著使用經螢光標記之抗CD8抗體及經螢光標記之抗TCR Vβ8 (Vb8)抗體進行螢光活化之細胞分類(fluorescence-activated cell sorting;FACS)分析以偵測CD8及TCR各自在細胞表面上之表現。
第10圖及第11圖展示:基於CD8+Vb8+雙重陽性細胞之% (第10圖)及CD8之MFI或Vb8之MFI (第11圖),與用含PTE.WPRE、TPE.WPRE或PTE.fn.WPRE之4合1病毒載體轉導的供體相比,自用含PTE.CD8.TCR.WPRE之4合1病毒載體轉導的供體3及供體4獲得之γδ T細胞在製造第14天產生CD8及TCR兩者在細胞表面上之最高表現。
CD8及TCR兩者在用含PTE.CD8.TCR.WPRE之4合1病毒載體轉導的γδ T細胞之細胞表面上之高表現與該等細胞之體外殺傷活性很好地相關。舉例而言,第12圖展示:與用含PTE.WPRE、TPE.WPRE或PTE.fn.WPRE之4合1病毒載體轉導的供體相比,自用含PTE.CD8.TCR.WPRE之4合1病毒載體轉導的供體3獲得之γδ T細胞展現對高靶肽呈現細胞株UACC257 (頂部檢測組合)及低靶肽呈現細胞株U2OS (底部檢測組合)兩者之最佳殺傷活性,第13A圖至第13C圖展示在靶細胞(例如,UACC257 (第13A圖)、U2OS (第13B圖)及靶陰性細胞株MCF-7 (第13C圖))存在之情況下的用含PTE.CD8.TCR.WPRE、PTE.WPRE、TPE.WPRE或PTE.fn.WPRE之4合1病毒載體轉導的對應γδ T細胞之IFN-γ分泌量。
第14圖展示:與用含PTE.WPRE、TPE.WPRE或PTE.fn.WPRE之4合1病毒載體轉導的供體相比,自用含PTE.CD8.TCR.WPRE之4合1病毒載體轉導的供體4獲得之γδ T細胞亦展現對UACC257 (頂部檢測組合)及U2OS (底部檢測組合)兩者之最佳殺傷活性,第15A圖至第15C圖展示在靶細胞(例如,UACC257 (第15A圖)、U2OS (第15B圖)及MCF-7 (第15C圖))存在之情況下的用含PTE.CD8.TCR.WPRE、PTE.WPRE、TPE.WPRE或PTE.fn.WPRE之4合1病毒載體轉導的對應γδ T細胞之IFN-γ分泌量。
第16圖展示:與用PTE.WPRE、TPE.WPRE及PTE.fn.WPRE轉導的供體類似,自用含PTE.CD8.TCR.WPRE之4合1病毒載體轉導之供體3及供體4獲得之γδ T細胞產生小於0.6的每細胞整合載體之拷貝。每細胞整合載體之此低拷貝數在安全需求之極限內,即,小於每細胞整合載體之5拷貝數。
第17A圖及第17B圖展示:自用含PTE.CD8.TCR.WPRE之4合1病毒載體轉導的供體3及供體4分別獲得之γδ T細胞達成可比得上用含PTE.WPRE、TPE.WPRE或PTE.fn.WPRE之4合1病毒載體轉導的供體的在製造之第14天之細胞擴增之水平。
為了判定轉導γδ T細胞之記憶細胞表現型,用異藻藍蛋白(allophycocyanin;APC)-Cy7-標記之抗CD45RA抗體及BV421標記之抗CCR7抗體將細胞染色,接著進行FACS分析以判定存在於轉導γδ T細胞中之Tcm、初始T細胞、TemRA及Teff之%。第18A圖展示此分析之一實例。
第18B圖展示:自用含PTE.CD8.TCR.WPRE之4合1病毒載體轉導的供體3及供體4獲得之γδ T細胞達成可比得上用含PTE.WPRE、TPE.WPRE或PTE.fn.WPRE之4合1病毒載體轉導的供體的在製造之第14天之記憶T細胞表現型之水平。
實例6
對用一種4合1病毒載體轉導的細胞對比用兩種2合1病毒載體轉導的細胞中之轉基因表現之影響
第19圖展示由利用含PTE.CD8.TCR.WPRE (120 µl)之4合1慢病毒載體之轉導產生的CD8+TCR+ γδ T細胞(檢測組合B,20.9%)比由利用含CD8.WPRE (120 µl)之2合1慢病毒載體與含R11KE.WPRE (120 µl)之2合1慢病毒載體之混合物之轉導產生的CD8+TCR+ γδ T細胞(檢測組合D,15.5%)多。另一方面,由用含CD8.WPRE (120 µl)之2合1慢病毒載體與含R11KE.WPRE之2合1慢病毒載體(120 µl)之混合物轉導產生的CD8+TCR-γδ T細胞(檢測組合D,28.3%)比用含PTE.CD8.TCR.WPRE之4合1慢病毒病毒載體轉導產生的CD8+TCR-γδ T細胞(檢測組合B, 21.2%)多。類似地,由用含PTE.CD8.TCR.WPRE (240 µl)之4合1慢病毒載體轉導產生的CD8+TCR+ γδ T細胞(檢測組合C,27.7%)比用含CD8.WPRE (240 µl)之2合1慢病毒載體與含R11KE.WPRE之2合1慢病毒載體(240 µl)之混合物轉導產生的CD8+TCR-γδ T細胞(檢測組合E,21.1%)多。另一方面,用含CD8.WPRE (240 µl)之2合1慢病毒載體與含R11KE.WPRE之2合1慢病毒載體(240 µl)之混合物轉導產生的CD8+TCR-γδ T細胞(檢測組合E, 40.2%)比用含PTE.CD8.TCR.WPRE之4合1慢病毒載體轉導產生的CD8+TCR-γδ T細胞(檢測組合C, 24.4%)多。未轉導(Non-transduced;NT) γδ T細胞充當對照物(檢測組合A)。2色染色(2-color staining)係使用APC標記之抗CD8β抗體及藻紅素(phycoerythrin;PE)標記之靶肽/MHC複合四聚物執行。此等結果暗示:利用含編碼CD8αβ及TCRαβ之序列之4合1慢病毒載體(例如,PTE.CD8.TCR.WPRE)之轉導可導致CD8+TCR+ T細胞之數目大於用含編碼CD8αβ之序列之2合1慢病毒載體(例如,CD8.WPRE)與含編碼TCRαβ之序列之2合1慢病毒載體(例如,R11KE.WPRE)的混合物轉導的CD8+TCR+ T細胞。另一方面,利用含編碼CD8αβ之序列之2合1慢病毒載體(例如,CD8.WPRE)與含編碼TCRαβ之序列之2合1慢病毒載體(例如,R11KE.WPRE)的混合物之轉導可導致CD8+TCR- T細胞之數目大於用含編碼CD8αβ及TCRαβ之序列之4合1慢病毒載體(例如,PTE.CD8.TCR.WPRE)轉導的CD8+TCR- T細胞。
第20圖展示:用於轉導的含PTE.CD8.TCR.WPRE之4合1病毒載體之增加量(例如,30 µl、120 µl及240 µl)增強轉導效率,例如,CD8+TCR+ γδ T細胞之%增大,例如,30 µl下為9.6%、120 µl下為20.9%且240 µl下為27.7%。未轉導γδ T細胞充當對照物。2色染色係使用APC標記之抗CD8β抗體及PE標記之靶肽/MHC複合四聚物執行。
實例7
4合1構築體在αβ T細胞中之表現
包括表現重組蛋白(例如,CD8αβ及/或TCRαβ)之工程化αβ T細胞的工程化淋巴球可根據US 2019/0247433中所揭示之方法來製造,US 2019/0247433之內容在此以全文引用之方式併入。舉例而言,第37圖展示T細胞製造程序370
,該T細胞製造程序可包括以下步驟:分離PBMC (371
),其中PBMC可新鮮使用或冷凍儲存直至準備使用,或可為白血球分離術產品(例如,leukopak),或可用作T細胞製造之起始材料及淋巴球群體(例如,αβ TCR+ T細胞、CD8+、CD4+或兩者)之選擇;解凍淋巴球且將淋巴球靜置隔夜(例如,約16小時或約4至6小時) (372
),此可允許凋亡細胞死去且恢復T細胞官能性(若使用新鮮材料,則此步驟可係非必需的);活化淋巴球(373
),該活化可使用抗CD3及抗CD28抗體(可溶解或表面束縛,例如,磁性或可生物分解之珠、固定在培養皿上之抗體);用含編碼重組蛋白(例如,CD8αβ及/或TCRαβ多肽)之序列之病毒載體進行轉導(374
),其中病毒載體可為慢病毒載體或逆轉錄病毒載體,或轉染可藉由非病毒方法執行;及擴增淋巴球,收穫且低溫保存(375
),該步驟可在細胞介素(例如,IL-7及IL-15)、血清(ABS或FBS)及/或低溫保存介質存在之情況下實行。
外源性CD8表現
為了判定用含具有編碼CD8及TCR之序列之4合1構築體之病毒載體轉導的αβ T細胞中之外源性CD8表現,用增加量之LV- PTE.CD8.TCR.WPRE轉導自供體5及供體6獲得之T細胞,接著進行關於淋巴球<單態<活細胞<CD3+群體閘控之FACS以偵測CD4+細胞中之% CD8α+細胞。第21圖展示以下各者:來自供體5之% CD8α+CD4+細胞之增加,自2.87% (未轉導)至14.7% (2.5 µl/1x106
個細胞)、19.5% (5 µl/1x106
個細胞)、21.7% (7.5 µl/1x106
個細胞)及24.1% (10 µl/1x106
個細胞);及來自供體6之% CD8α+CD4+細胞之增加,自1.93% (未轉導)至12.5% (2.5 µl/1x106
個細胞)、17.2% (5 µl/1x106
個細胞)、19.6% (7.5 µl/1x106
個細胞)及20.8% (10 µl/1x106
個細胞)。
外源性TCR表現
為了判定用含具有編碼CD8及TCR之序列之4合1構築體之病毒載體轉導的αβ T細胞中之外源性TCR表現,用增加量之LV- PTE.CD8.TCR.WPRE轉導自供體5及供體6獲得之T細胞,接著進行關於淋巴球<單態<活細胞<CD3+<CD4+CD8+群體閘控之FACS以偵測CD4+CD8+細胞群體中之%靶肽/MHC複合Dextramer203+ (即,TCR+)細胞。第22圖展示:來自供體5之% Dextramer203+細胞增加,自0.32% (未轉導)至41.9% (2.5 µl/1x106
個細胞)、48.3% (5 µl/1x106
個細胞)、54.5% (7.5 µl/1x106
個細胞)及49.5% (10 µl/1x106
個細胞);及來自供體6之% Dextramer203+細胞增加,自0.19% (未轉導)增加至35.5% (2.5 µl/1x106
個細胞)、41.2% (5 µl/1x106
個細胞)、44.6% (7.5 µl/1x106
個細胞)及44.0% (10 µl/1x106
個細胞)。
為了偵測各種αβ T細胞群體中之TCR表現,藉由進行關於淋巴球<單態<活細胞<CD3+<CD4+/-CD8+/-閘控之FACS來分析用LV- PTE.CD8.TCR.WPRE轉導之αβ T細胞。第23圖展示自供體5 (頂部檢測組合)及供體6 (底部檢測組合)獲得之% Dextramer203 (Dex203)+ (即,TCR+)細胞通常在CD4+CD8α+細胞群體中比在CD4-CD8α+細胞群體中高。相比之下,% Dex203+ (即,TCR+)細胞在CD4+CD8α-細胞群體中最少。類似地,第24圖展示自供體5 (頂部檢測組合)及供體6 (底部檢測組合)獲得之% Dextramer203 (Dex203) MFI通常在CD4+CD8α+細胞群體中比在CD4-CD8α+細胞群體中高。相比之下,% Dex203 MFI在CD4+CD8α-細胞群體中最小。此等結果暗示:藉由LV- PTE.CD8.TCR.WPRE編碼之外源性TCR及CD8可共表現在CD4+ T細胞及CD4- T細胞兩者中。
實例8
表現4合1構築體或僅TCR構築體之αβ T細胞之功能分析
第25圖展示用於測試用含4合1構築體(例如,PTE.CD8.TCR.WPRE (LV-CD8.TCR))或僅TCR構築體(例如,R11KE.WPRE (LV-TCR))之慢病毒載體(lentiviral vector;LV)轉導的αβ T細胞之官能性在實驗設計。簡要地說,在第-1天,在96孔盤中接種靶細胞(例如,高抗原表現UACC257+RFP細胞株(陽性對照物)及抗原陰性MCF7+GFP細胞株(陰性對照物))。將供體細胞產品(例如,用LV-CD8.TCR (5 µl/1x106
個細胞)或LV-TCR (2.5 µl/1x106
個細胞)轉導之PBMC (自供體5、供體6、供體7及供體8獲得))解凍且在24孔G-Rex®透氣性快速擴增裝置中靜置隔夜。在第0天,以2:1之效應物細胞對靶細胞(E/T)比率(例如,200,000個效應物細胞:100,000個靶細胞)將供體細胞產品(效應物細胞)與靶細胞(例如,UACC257+RFP及MCF7+GFP)共培養。於在37℃下培養5小時後,以0.5 µl/孔向每一孔添加蛋白轉運抑制劑(例如,GolgiStopTM
(BD Biosciences)),接著在37℃下培養4小時。接著對細胞進行離心分離以收集用於ELISA之上清液以偵測IFN-γ表現且使用胞內細胞介素染色(intracellular cytokine staining;ICS)板收穫用於染色之T細胞,例如,CD3、CD4、CD8、IFN-γ、顆粒酶B及活/死。
CD4-CD8+ T細胞群體
第26圖展示:將自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之分組供體獲得之CD4-CD8α+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)情況下高的% IFN-γ-陽性細胞(頂部檢測組合)及IFN-γ MFI (底部檢測組合)。相比之下,當將用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之CD4-CD8α+ T細胞與抗原陰性MCF7共培養時,觀察到就% IFN-γ-陽性細胞及IFN-γ MFI而言在轉導細胞與未轉導細胞之間無明顯差異。進行關於CD4-CD8α+IFN-γ+ T細胞閘控之FACS。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=4)。此等結果暗示:用LV-TCR或LV-CD8.TCR轉導之CD4-CD8α+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由表現IFN-γ而功能活躍,且轉導細胞對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
第27圖展示:將自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之分組供體獲得之CD4-CD8α+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)情況下高的%顆粒酶B-陽性細胞(頂部檢測組合)及顆粒酶B MFI (底部檢測組合)。相比之下,當將用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之CD4-CD8α+ T細胞與抗原陰性MCF7共培養時,觀察到就%顆粒酶B-陽性細胞及顆粒酶B MFI而言在轉導細胞與未轉導細胞之間無明顯差異。進行關於CD4-CD8α+顆粒酶B+ T細胞閘控之FACS。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=3)。此等結果暗示:用LV-TCR或LV-CD8.TCR轉導之CD4-CD8α+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由表現顆粒酶B而功能活躍,且轉導細胞可對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
CD4+CD8+ T細胞群體
第28圖展示:將自用LV-CD8.TCR (TCR+CD8)轉導之分組供體獲得之CD4+CD8α+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)情況下高的% IFN-γ-陽性細胞(頂部檢測組合)及IFN-γ MFI (底部檢測組合)。相比之下,當將用LV-CD8.TCR (TCR+CD8)轉導之CD4+CD8α+ T細胞與抗原陰性MCF7共培養時,觀察到就% IFN-γ-陽性細胞及IFN-γ MFI而言在轉導細胞與未轉導細胞之間無明顯差異。進行關於針對NT細胞之CD4+CD8α-IFN-γ+及針對LV-CD8.TCR轉導之細胞的CD4+CD8α+IFN-γ+閘控之FACS。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=4)。此等結果暗示:用LV-CD8.TCR轉導之CD4+CD8α+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由表現IFN-γ而功能活躍,且轉導細胞可對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
第29圖展示:將自用LV-CD8.TCR (TCR+CD8)轉導之分組供體獲得之CD4+CD8α+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)情況下高的%顆粒酶B-陽性細胞(頂部檢測組合)及顆粒酶B MFI (底部檢測組合)。相比之下,當將用LV-CD8.TCR (TCR+CD8)轉導之CD4+CD8α+ T細胞與抗原陰性MCF7共培養時,觀察到就%顆粒酶B-陽性細胞及顆粒酶B MFI而言在轉導細胞與未轉導細胞之間無明顯差異。進行關於針對NT細胞之CD4+CD8α-顆粒酶B+及針對LV-CD8.TCR轉導之T細胞的CD4+CD8α+顆粒酶B+閘控之FACS。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=3)。此等結果暗示:用LV-CD8.TCR轉導之CD4+CD8α+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由表現顆粒酶B而功能活躍,且轉導細胞可對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
CD3+ T細胞
第30圖展示:將自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之分組供體獲得之CD3+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)情況下高的% IFN-γ-陽性細胞(頂部檢測組合)及IFN-γ MFI (底部檢測組合)。相比之下,當將用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之CD4-CD8α+ T細胞與抗原陰性MCF7共培養時,觀察到就% IFN-γ-陽性細胞及IFN-γ MFI而言在轉導細胞與未轉導細胞之間無明顯差異。進行關於CD3+ T細胞閘控之FACS。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=4)。此等結果暗示:用LV-TCR或LV-CD8.TCR轉導之CD3+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由表現IFN-γ而功能活躍,且轉導細胞可對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
第31圖展示:將自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之分組供體獲得之CD3+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)情況下高的%顆粒酶B-陽性細胞(頂部檢測組合)及顆粒酶B MFI (底部檢測組合)。相比之下,當將用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之CD3+ T細胞與抗原陰性MCF7共培養時,觀察到就%顆粒酶B-陽性細胞及顆粒酶B MFI而言在轉導細胞與未轉導細胞之間無明顯差異。進行關於CD3+ T細胞閘控之FACS。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=3)。此等結果暗示:用LV-TCR或LV-CD8.TCR轉導之CD3+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由表現顆粒酶B而功能活躍,且轉導細胞可對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
第32圖展示:將自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之分組供體獲得之CD3+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)、僅MCF7細胞及僅UACC257細胞情況下高的IFN-γ隱匿之水平。相比之下,當將用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之CD4-CD8α+ T細胞與抗原陰性MCF7共培養時,觀察到IFN-γ隱匿之水平在轉導細胞與未轉導細胞之間無明顯差異。(效應物與靶細胞比率= 2:1且分組之供體N=4)。此等結果暗示:用LV-TCR或LV-CD8.TCR轉導之CD3+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由隱匿IFN-γ而功能活躍,且轉導細胞可對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
第33圖展示:將自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之個別供體5、供體6、供體7及供體8獲得之CD3+ T細胞與高目標表現UACC257細胞共培養導致比無轉導(NT)、僅MCF7細胞及僅UACC257細胞情況下高的IFN-γ隱匿之水平。相比之下,當將用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導之CD4-CD8α+ T細胞與抗原陰性MCF7共培養時,觀察到IFN-γ隱匿之水平在轉導細胞與未轉導細胞之間無明顯差異。(效應物與靶細胞比率= 2:1)。此等結果暗示:自用LV-TCR或LV-CD8.TCR轉導之個別供體獲得之CD3+ T細胞在接觸高抗原表現靶細胞(例如,UACC257細胞)時例如藉由隱匿IFN-γ而功能活躍,且轉導細胞可對抗原陰性細胞(例如,MCF7細胞)幾乎沒有影響。
實例9
斯他汀對T細胞活化標識之表現之影響
為了判定斯他汀對T細胞活化標識之表現之影響,用斯他汀(例如,阿托伐他汀、普伐他汀或瑞舒伐他汀)處理T細胞,接著進行FACS分析以量測T細胞活化標識(例如,CD25、CD69及hLDLR)之表現。
CD4+ T細胞群體
第34圖展示經阿托伐他汀、普伐他汀或瑞舒伐他汀處理之CD3+CD4+ T細胞中的% CD25+細胞(頂部檢測組合)、% CD69+細胞(中間檢測組合)及% hLDLR+細胞(底部檢測組合)。預活化之細胞、在無斯他汀或DMSO情況下活化之細胞(對照物)及DMSO充當對照物。此等結果展示:儘管阿托伐他汀、普伐他汀及瑞舒伐他汀對% CD4+CD25+細胞及% CD4+CD69+細胞幾乎沒有影響,但斯他汀(例如,阿托伐他汀)可使% CD4+hLDLR+細胞增大。進行關於淋巴球>單態>活/死>CD3+>CD4+閘控之FACS。
CD8+ T細胞群體
第35圖展示經阿托伐他汀、普伐他汀或瑞舒伐他汀處理之CD3+CD8+ T細胞中的% CD25+細胞(頂部檢測組合)、% CD69+細胞(中間檢測組合)及% hLDLR+細胞(底部檢測組合)。預活化之細胞、在無斯他汀或DMSO情況下活化之細胞(對照物)及DMSO充當對照物。此等結果展示:儘管阿托伐他汀、普伐他汀及瑞舒伐他汀對% CD8+CD25+細胞及% CD8+CD69+細胞幾乎沒有影響,但斯他汀(例如,阿托伐他汀)可使% CD8+hLDLR+細胞增大。進行關於淋巴球>單態>活/死>CD3+>CD8+閘控之FACS。
實例10
WPRE對慢病毒滴定量之影響
為了判定WPRE對慢病毒滴定量之影響,產生含野生型(wild type;wt) WPRE (SEQ ID NO: 9) (LV-A)、無WPRE (LV-B)、WPREmut1 (SEQ ID NO: 265) (LV-C)或WPREmut2 (SEQ ID NO: 266) (LV-D)之慢病毒載體(LV)。用LV-A、LV-B、LV-C或LV-D轉染HEK293T細胞,接著使用此項技術中已知之方法進行滴定量判定。第36圖展示此等慢病毒載體之滴定量順序如下:LV-C > LV-D ≥ LV-A > LV-B。此等結果暗示:WPREmut1及WPREmut2可對改良慢病毒載體生產有用。
本發明之優點可包括產生在單一載體中共表現多個轉基因(例如,4種多肽)之病毒載體,及產生共表現TCRαβ及CD8αβ之γδ T細胞以作為用於接受性細胞療法的安全且靶特定之「現貨」T細胞產品。
說明書中列舉之所有參考文獻係以引用方式併入本文中,好像每一參考文獻係特定且個別地指示為以引用方式併入。任何參考文獻之引用係用於在申請日之前揭示且不應解釋為如下許可:本發明無權憑藉在先發明而比此參考文獻提前。
370:T細胞製造程序
371,372,373,374,375:步驟
第1圖展示根據本發明之一個實施例之γδ T細胞製造程序。γδ T細胞製造可包括:收集或獲得白血球或PBMC (例如,白血球分離術產品);自PBMC或白血球分離術產品消耗αβ T細胞,接著進行γδ T細胞之活化、轉導及擴增。
第2圖展示根據本發明之一些實施例之利用開讀框(open reading frame;ORF)混洗之轉導策略。
第3圖展示根據本發明之一些實施例之慢病毒構築體。
第4圖展示在用唑來膦酸、IL-2及IL-15活化3天或6天后的經用於轉導γδ T細胞之RD114TR假型化之慢病毒。經由流動式細胞測量術使用特定於TCR (Vβ8)及CD8 (CD8α)之抗體來評估轉導效率。
第5A圖展示根據本發明之一實施例之構築體。
第5B圖展示根據本發明之另一實施例之構築體。
第5C圖展示根據本發明之另一實施例之構築體。
第5D圖展示根據本發明之另一實施例之構築體。
第6A圖展示根據本發明之另一實施例之構築體。
第6B圖展示根據本發明之另一實施例之構築體。
第7圖展示根據本發明之一些實施例之構築體之示意圖。
第8A圖展示根據本發明之一實施例之構築體。
第8B圖展示根據本發明之另一實施例之構築體。
第8C圖展示根據本發明之另一實施例之構築體。
第8D圖展示根據本發明之另一實施例之構築體。
第9A圖展示根據本發明之一些實施例之構築體之示意圖。
第9B圖展示根據本發明之一些實施例之構築體之示意圖。
第10圖展示用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的% CD8+TCR+ γδ T細胞。未轉導(Non-transduced;NT)細胞充當對照物。
第11圖展示用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的γδ T細胞中之CD8及TCR之中位數螢光強度(median fluorescence intensity;MFI)。未轉導(Non-transduced;NT)細胞充當對照物。
第12圖展示自用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的供體3獲得之γδ T細胞在高抗原表現腫瘤細胞株(例如,UACC257 (頂部檢測組合))中或在低抗原表現腫瘤細胞株(例如,U2OS (底部檢測組合))中之腫瘤殺傷活性,如藉由Incucyte細胞毒性分析所測定。僅靶(Target only)及未轉導(non-transduced)細胞充當對照物。
第13A圖至第13C圖展示自用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的供體3獲得之γδ T細胞在高抗原表現腫瘤細胞株(例如,UACC257 (第13A圖))中、在低抗原表現腫瘤細胞株(例如,U2OS (第13B圖))中或在抗原陰性腫瘤細胞株(例如,MCF-7 (第13C圖))中的干擾素(interferon;IFN)-γ隱匿之量。未轉導細胞充當對照物。
第14圖展示自用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的供體4獲得之γδ T細胞在高抗原表現腫瘤細胞株(例如,UACC257 (頂部檢測組合))中或在低抗原表現腫瘤細胞株(例如,U2OS (底部檢測組合))中之腫瘤殺傷活性,如藉由Incucyte細胞毒性分析所測定。僅靶及未轉導細胞充當對照物。
第15A圖至第15C圖展示自用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的供體4獲得之γδ T細胞在高抗原表現腫瘤細胞株(例如,UACC257 (第15A圖))中、在低抗原表現腫瘤細胞株(例如,U2OS (第15B圖))中或在抗原陰性腫瘤細胞株(例如,MCF-7 (第15C圖))中之IFN-γ隱匿之量。未轉導細胞充當對照物。
第16圖展示用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的γδ T細胞中之病毒載體之拷貝數。未轉導細胞充當對照物。
第17A圖及第17B圖展示自用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的供體3 (第17A圖)或供體4 (第17B圖)獲得之γδ T細胞之倍數擴增(fold expansion)。未轉導(Non-transduced;NT)細胞充當對照物。
第18A圖展示根據本發明之一些實施例的藉由流動式細胞測量術測定之γδ T細胞之記憶表現型。
第18B圖展示用含PTE.CD8.TCR.WPRE、PTE.WPRE、PTE.Fn.WPRE或TPE.WPRE之病毒載體轉導的γδ T細胞之記憶表現型。未轉導(Non-transduced;NT)細胞充當對照物。
第19圖展示用含PTE.CD8.TCR.WPRE (檢測組合B (120 µl)及檢測組合C (240 µl))之單一慢病毒載體(lentiviral vector;LV)轉導或用一種含R11KE.WPRE且另一種含CD8,WPRE (檢測組合D及E)之兩種單獨慢病毒載體(病毒載體之量增大,例如,R11KE.WPRE及CD8,WPRE各自120 µl (檢測組合D)及R11KE.WPRE及CD8,WPRE各自240 µl (檢測組合E))轉導的γδ T細胞之間的轉導效率之比較。未轉導(Non-transduced;NT)細胞充當對照物。
第20圖展示用增大量之含PTE.CD8.TCR.WPRE之病毒載體(例如,30 µl、120 µl及240 µl)轉導之γδ T細胞中的增強之轉導效率。未轉導細胞充當對照物。
第21圖展示使用表現本發明之4合1構築體(例如,LV- PTE.CD8.TCR.WPRE)之慢病毒載體(lentiviral vector;LV)之各種稀釋物的自供體5及供體6獲得之CD4+ T細胞中之強制CD8表現。
第22圖展示使用表現本發明之4合1構築體(例如,LV- PTE.CD8.TCR.WPRE)之LV之各種稀釋物的對CD4+ T細胞中之TCR表現之偵測。
第23圖展示在自用本發明之4合1構築體(例如,LV- PTE.CD8.TCR.WPRE)轉導的供體5 (頂部檢測組合)及供體6 (底部檢測組合)獲得之CD4+及/或CD8+ T細胞中的%靶肽/MHC複合Dextramer203 (Dex203)+。
第24圖展示在自用本發明之4合1構築體(例如,LV- PTE.CD8.TCR.WPRE)轉導的供體5 (頂部檢測組合)及供體6 (底部檢測組合)獲得之CD4+及/或CD8+T細胞中的Dex203 MFI。
第25圖展示根據本發明之一個實施例的用於測試用4合1構築體或僅TCR構築體轉導的T細胞之官能性之實驗設計。
第26圖展示自用含R11KE.WPRE之慢病毒載體(LV-TCR) (TCR)或含PTE.CD8.TCR.WPRE之慢病毒載體(LV-CD8.TCR) (TCR+CD8)轉導的分組供體獲得之CD4-CD8α+ T細胞中的增加之% IFN-γ-陽性細胞(頂部檢測組合)及增加之IFN-γ MFI (底部檢測組合),接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=4)。
第27圖展示自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導的分組供體獲得之CD4-CD8α+ T細胞中的增加之%顆粒酶B-陽性細胞(頂部檢測組合)及增加之顆粒酶B MFI (底部檢測組合),接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=3)。
第28圖展示自用LV-CD8.TCR (TCR+CD8)轉導或未轉導(without transduction;NT)的分組供體獲得之CD4+CD8α+ T細胞中的增加之% IFN-γ-陽性細胞(頂部檢測組合)及增加之IFN-γ MFI (底部檢測組合),接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。(效應物與靶細胞比率= 2:1且分組之供體N=4)。
第29圖展示自用LV-CD8.TCR (TCR+CD8)轉導或未轉導(NT)的分組供體獲得之CD4+CD8α+ T細胞中的增加之%顆粒酶B-陽性細胞(頂部檢測組合)及增加之顆粒酶B MFI (底部檢測組合),接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。(效應物與靶細胞比率= 2:1且分組之供體N=4)。
第30圖展示自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導的分組供體獲得之CD3+ T細胞中的增加之% IFN-γ-陽性細胞(頂部檢測組合)及增加之IFN-γ MFI (底部檢測組合),接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=4)。
第31圖展示自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導的分組供體獲得之CD3+ T細胞中的增加之%顆粒酶B-陽性細胞(頂部檢測組合)及增加之顆粒酶B MFI (底部檢測組合),接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。未轉導(Non-transduced;NT)細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=3)。
第32圖展示自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導的分組供體獲得之CD3+ T細胞中的增加之IFN-γ隱匿,接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。未轉導(Non-transduced;NT)細胞、UACC257細胞及MCF7細胞充當對照物。(效應物與靶細胞比率= 2:1且分組之供體N=4)。
第33圖展示自用LV-TCR (TCR)或LV-CD8.TCR (TCR+CD8)轉導的個別供體5、6、7及8獲得之CD3+ T細胞中的增加之IFN-γ隱匿,接著將該等T細胞與高目標表現UACC257細胞共培養,以將該等T細胞和與非目標表現MCF7共培養之T細胞進行比較。未轉導(Non-transduced;NT)細胞、僅UACC257細胞及僅MCF7細胞充當對照物。(效應物與靶細胞比率= 2:1)。
第34圖展示經阿托伐他汀、普伐他汀或瑞舒伐他汀治療之CD3+CD4+ T細胞中的% CD25+細胞(頂部檢測組合)、% CD69+細胞(中間檢測組合)及%人類低密度脂蛋白受體(human low-density lipoprotein receptor;hLDLR)+細胞(底部檢測組合)。預活化之細胞、在無斯他汀或DMSO情況下活化之細胞(對照物)及DMSO充當對照物。
第35圖展示經阿托伐他汀、普伐他汀或瑞舒伐他汀治療之CD3+CD8+ T細胞中的% CD25+細胞(頂部檢測組合)、% CD69+細胞(中間檢測組合)及% hLDLR+細胞(底部檢測組合)。預活化之細胞、在無斯他汀或DMSO情況下活化之細胞(對照物)及DMSO充當對照物。
第36圖展示根據本發明之一個實施例的慢病毒載體之滴定量。
第37圖展示根據本發明之一個實施例的T細胞製造程序。
國內寄存資訊(請依寄存機構、日期、號碼順序註記)
無
國外寄存資訊(請依寄存國家、機構、日期、號碼順序註記)
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Claims (51)
- 一種載體,包含編碼CD8α多肽之核苷酸序列S1、編碼CD8β多肽之核苷酸序列S2、編碼T細胞受體(TCR)α多肽之核苷酸序列S3及編碼TCRβ多肽之核苷酸序列S4,其中該等核苷酸序列係以選自以下各者之5’至3’定向串聯排列:S1-S2-S3-S4、S1-S2-S4-S3、S2-S1-S3-S4、S2-S1-S4-S3、S3-S4-S1-S2、S3-S4-S2-S1、S4-S3-S1-S2及S4-S3-S2-S1。
- 如請求項1所述之載體,其中該等核苷酸序列係以選自以下各者之5’至3’定向串聯排列:S1-S2-S3-S4、S1-S2-S4-S3、S2-S1-S3-S4及S2-S1-S4-S3。
- 如請求項2所述之載體,其中該等核苷酸序列係以S2-S1-S4-S3之5’至3’定向串聯排列。
- 如請求項1至3中任一項所述之載體,其中該等CD8αβ多肽包含與SEQ ID NO: 11具有至少90%一致性且與SEQ ID NO: 12具有至少90%一致性之胺基酸序列。
- 如請求項1至3中任一項所述之載體,其中該等TCRαβ多肽包含與選自以下各者之胺基酸序列具有至少90%一致性之胺基酸序列:SEQ ID NO: 13及14、15及16、17及18、19及20、21及22、23及24、25及26、27及28、29及30、31及32、33及34、35及36、37及38、39及40、41及42、43及44、45及46、47及48、49及50、51及52、53及54、55及56、57及58、59及60、61及62、63及64、65及66、67及68、69及70、71及72、73及74、75及76、77及78、79及80、81及82、83及84、85及86、87及88或89及90。
- 如請求項1至3中任一項所述之載體,其中該等TCRαβ多肽包含與SEQ ID NO: 13具有至少90%一致性且與SEQ ID NO:14具有至少90%一致性之胺基酸序列。
- 如請求項1至3中任一項所述之載體,進一步包含編碼2A肽之核苷酸序列S5及編碼連接肽之核苷酸序列S6,其中S5及S6定位在S1與S2之間、S1與S3之間、S1與S4之間、S2與S3之間、S2與S4之間及/或S3與S4之間。
- 如請求項7所述之載體,其中該2A肽係選自P2A (SEQ ID NO: 3)、T2A (SEQ ID NO: 4)、E2A (SEQ ID NO: 5)或F2A (SEQ ID NO: 6)。
- 如請求項7所述之載體,其中該連接肽為GSG或SGSG (SEQ ID NO: 8)。
- 如請求項1至3中任一項所述之載體,進一步包含編碼弗林肽(SEQ ID NO: 2)之核苷酸序列S7,其中S7定位在S1與S2之間、S1與S3之間、S1與S4之間、S2與S3之間、S2與S4之間及/或S3與S4之間。
- 一種載體,包含編碼蛋白質Z1之第一核苷酸序列S1、編碼蛋白質Z2之第二核苷酸序列S2、編碼蛋白質Y1之第三核苷酸序列S3及編碼蛋白質Y2之第四核苷酸序列S4,其中Z1及Z2形成第一二聚體且Y1及Y2形成第二二聚體,其中該第一二聚體Z1Z2在結構上不同於該第二二聚體Y1Y2,其中該載體在用於適應性細胞療法之基因傳遞系統中。
- 如請求項11所述之載體,其中該S1、S2、S3及S4係以選自以下各者之5’至3’定向串聯排列:S1-S2-S3-S4、S1-S2-S4-S3、S1-S3-S2-S4、S1-S3-S4-S2、S1-S4-S3-S2、S1-S4-S2-S3、S2-S1-S3-S4、S2-S1-S4-S3、S2-S3-S1-S4、S2-S3-S4-S1、S2-S4-S3-S1、S2-S4-S1-S3、S3-S1-S2-S4、S3-S1-S4-S2、S3-S2-S1-S4、S3-S2-S4-S1、S3-S4-S1-S2、S3-S4-S2-S1、S4-S1-S2-S3、S4-S1-S3-S2、S4-S2-S1-S3、S4-S2-S3-S1、S4-S3-S1-S2或S4-S3-S2-S1。
- 如請求項11或12所述之載體,進一步包含編碼2A肽之第五核苷酸序列S5及編碼連接肽之第六核苷酸序列S6,其中S5及S6定位在S1與S2之間、S1與S3之間、S1與S4之間、S2與S3之間、S2與S4之間及/或S3與S4之間。
- 如請求項13所述之載體,其中該2A肽係選自P2A (SEQ ID NO: 3)、T2A (SEQ ID NO: 4)、E2A (SEQ ID NO: 5)或F2A (SEQ ID NO: 6)。
- 如請求項13所述之載體,其中該連接肽為GSG或SGSG (SEQ ID NO: 8)。
- 如請求項11或12中任一項所述之載體,進一步包含編碼弗林肽(SEQ ID NO: 2)之第七核苷酸序列S7,其中S7定位在S1與S2之間、S1與S3之間、S1與S4之間、S2與S3之間、S2與S4之間及/或S3與S4之間。
- 如請求項11或12中任一項所述之載體,進一步包含選自土撥鼠PRE (WPRE)或B型肝炎病毒(HBV) PRE (HPRE)之轉錄後調節元件(PRE)序列。
- 如請求項11或12中任一項所述之載體,進一步包含控制該S2至該S7之轉錄之啟動子序列,其中該啟動子序列係選自巨細胞病毒(CMV)啟動子、磷酸甘油酯激酶(PGK)啟動子、髓磷脂鹼性蛋白(MBP)啟動子、膠質原纖維酸性蛋白(GFAP)啟動子、含骨髓增生肉瘤病毒增強物之改造MoMuLV LTR (MNDU3)、泛素C啟動子、EF-1 α啟動子或鼠類幹細胞病毒(MSCV)啟動子。
- 如請求項11或12中任一項所述之載體,其中該第一二聚體Z1Z2係選自SEQ ID NO: 13及14、15及16、17及18、19及20、21及22、23及24、25及26、27及28、29及30、31及32、33及34、35及36、37及38、39及40、41及42、43及44、45及46、47及48、49及50、51及52、53及54、55及56、57及58、59及60、61及62、 63及64、65及66、67及68、69及70、71及72、73及74、75及76、77及78、79及80、81及82、83及84、85及86、87及88或89及90。
- 如請求項11或12中任一項所述之載體,其中該第二二聚體Y1及Y2為SEQ ID NO: 11及12。
- 如請求項11或12中任一項所述之載體,其中該定向為S2-S1-S4-S3。
- 如請求項21所述之載體,包含選自以下各者之序列:PTE WPRE (SEQ ID NO: 91)、TPE WPRE (SEQ ID NO: 92)或PTE fn WPRE (SEQ ID NO: 93)。
- 如請求項11或12中任一項所述之載體,其中該定向為S4-S3-S2-S1。
- 如請求項23所述之載體,包含序列PTE CD8 TCR WPRE (SEQ ID NO: 94)。
- 如請求項11或12中任一項所述之載體,該載體為選自以下各者之病毒載體:腺病毒、痘病毒、α病毒、沙粒狀病毒、黃病毒、棒狀病毒、逆轉錄病毒、慢病毒、皰疹病毒、副黏液病毒或小核糖核酸病毒。
- 如請求項25所述之載體,其中該載體經選自以下各者之病毒之包膜蛋白假型化:本地貓內源病毒(RD114)、RD114之嵌合型式(RD114TR)、長臂猿白血病病毒(GALV)、GALV之嵌合型式(GALV-TR)、雙嗜性鼠類白血病病毒(MLV 4070A)、桿狀病毒(GP64)、水泡性口炎病毒(VSV-G)、雞瘟病毒(FPV)、伊波拉病毒(EboV)或狒狒逆轉錄病毒包膜醣蛋白(BaEV)、淋巴細胞性脈絡叢腦膜炎病毒(LCMV)。
- 一種製備用於免疫療法之T細胞之方法,包含以下步驟: 自人類受試者之血液樣本分離T細胞; 在胺基雙膦酸酯存在之情況下活化該等分離之T細胞; 用如請求項11至26中任一項所述之載體轉導該等活化之T細胞;及 擴增該等轉導之T細胞。
- 如請求項27所述之方法,其中自白血球分離術人類樣本分離該等T細胞。
- 如請求項27或28所述之方法,其中該胺基雙膦酸酯係選自帕米膦酸、阿侖膦酸、唑來膦酸、利塞膦酸、伊班膦酸、英卡膦酸、前述膦酸中任一者之鹽及/或其水合物。
- 如請求項27或28所述之方法,其中該活化在人重組白細胞介素2 (IL-2)及人重組白細胞介素15 (IL-15)存在之情況下進一步進行。
- 如請求項27或28所述之方法,其中該擴增在IL-2及IL-15存在之情況下進行。
- 如請求項27或28所述之方法,其中該等T細胞為γδ T細胞。
- 如請求項27或28所述之方法,其中該第一二聚體Z1Z2及該第二二聚體Y1Y2共表現在該等擴增之T細胞之表面上。
- 一種藉由如請求項27至32中任一項所述之方法製備之擴增之T細胞群體。
- 一種包含如請求項34所述之擴增之T細胞群體的組合物在製造用於治療患癌症之病人之藥物之應用, 其中該等T細胞殺死在表面上呈現與MHC分子複合之肽之癌細胞,其中該肽係選自SEQ ID NO: 98至255, 其中該癌症係選自由以下各者組成之群組:非小細胞肺癌、小細胞肺癌、黑色素瘤、肝癌、乳癌、子宮癌、Merkel氏細胞癌、胰腺癌、膽囊癌、膽管癌、結腸直腸癌、膀胱癌、腎癌、白血病、卵巢癌、食道癌、腦癌、胃癌及前列腺癌。
- 如請求項35所述之應用,其中該組合物進一步包含佐劑。
- 如請求項36所述之應用,其中該佐劑係選自抗CD40抗體、咪喹莫特、雷西莫特、GM-CSF、環磷醯胺、舒尼替尼、貝伐單抗、阿特珠單抗、干擾素α、干擾素β、CpG寡核苷酸及衍生物、poly-(I:C)及衍生物、RNA、西地那非、具有聚(乳酸交酯共-乙交酯) (PLG)之顆粒調配物、病毒體、白細胞介素(IL)-1、IL-2、IL-4、IL-7、IL-12、IL-13、IL-15、IL-21及IL-23。
- 一種包含如請求項34所述之擴增之T細胞群體之組合物在製造用於在患癌症之病人中引出免疫反應之藥物之應用, 其中該等T細胞殺死在表面上呈現與MHC分子複合之肽之癌細胞,其中該肽係選自SEQ ID NO: 98至255, 其中該癌症係選自由以下各者組成之群組:非小細胞肺癌、小細胞肺癌、黑色素瘤、肝癌、乳癌、子宮癌、Merkel氏細胞癌、胰腺癌、膽囊癌、膽管癌、結腸直腸癌、膀胱癌、腎癌、白血病、卵巢癌、食道癌、腦癌、胃癌及前列腺癌。
- 如請求項38所述之應用,其中該組合物進一步包含佐劑。
- 如請求項39所述之應用,其中該佐劑係選自抗CD40抗體、咪喹莫特、雷西莫特、GM-CSF、環磷醯胺、舒尼替尼、貝伐單抗、阿特珠單抗、干擾素α、干擾素β、CpG寡核苷酸及衍生物、poly-(I:C)及衍生物、RNA、西地那非、具有聚(乳酸交酯共-乙交酯) (PLG)之顆粒調配物、病毒體、白細胞介素(IL)-1、IL-2、IL-4、IL-7、IL-12、IL-13、IL-15、IL-21及IL-23。
- 如請求項38至40中任一項所述之應用,其中該免疫反應包含細胞毒性T細胞反應。
- 一種製備用於免疫療法之T細胞之方法,包含以下步驟: 在斯他汀存在之情況下活化該等T細胞, 用如請求項11至26中任一項所述之載體轉導該等活化之T細胞, 其中該載體經VSV-G之包膜蛋白假型化,及 擴增該等轉導之T細胞。
- 如請求項42所述之方法,其中該等T細胞包含αβ T細胞、γδ T細胞及/或自然殺手T細胞。
- 如請求項42或43所述之方法,其中該斯他汀係選自阿托伐他汀、西伐他汀、達伐他汀、氟多他汀、氟伐他汀、美伐他汀、普伐他汀、辛伐他汀、維洛他汀及瑞舒伐他汀。
- 一種製備用於免疫療法之T細胞之方法,包含以下步驟: 活化該等T細胞, 用如請求項11至26中任一項所述之載體轉導該等活化之T細胞,及 擴增該等轉導之T細胞。
- 如請求項45所述之方法,其中該等T細胞包含αβ T細胞、γδ T細胞及/或自然殺手T細胞。
- 如請求項45或46所述之方法,其中該等T細胞包含αβ T細胞。
- 如請求項47所述之方法,其中該活化在抗CD3抗體及抗CD28抗體存在之情況下進行。
- 如請求項47或48所述之方法,其中該擴增在IL-7及IL-15存在之情況下進行。
- 一種藉由如請求項42至49中任一項所述之方法製備之擴增之T細胞群體。
- 一種包含如請求項50所述之擴增之T細胞群體之組合物在製造用於治療患癌症之病人之藥物之應用, 其中該等T細胞殺死在表面上呈現與MHC分子複合之肽之癌細胞,其中該肽係選自SEQ ID NO: 98至255, 其中該癌症係選自由以下各者組成之群組:非小細胞肺癌、小細胞肺癌、黑色素瘤、肝癌、乳癌、子宮癌、Merkel氏細胞癌、胰腺癌、膽囊癌、膽管癌、結腸直腸癌、膀胱癌、腎癌、白血病、卵巢癌、食道癌、腦癌、胃癌及前列腺癌。
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