TW202340457A - 同種異體治療細胞 - Google Patents
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Abstract
本揭露提供同種異體細胞,例如當用於治療患者之疾病時,會導致移植物抗宿主疾病(GVHD)之風險降低、最小化或不存在,及CD4、CD8、及NK細胞排斥之風險降低、最小化或不存在。可對同種異體細胞進行基因工程改造以降低MHC I類及/或MHC II類分子之表現或活性,諸如剔除RFX5基因。亦提供製備及使用此類同種異體細胞之方法。
Description
本揭露關於細胞療法之領域,且更具體而言為經基因工程改造之免疫細胞。
T細胞療法仰賴經富集或經修飾人類T細胞以靶向及殺滅患者之癌細胞。為了增加T細胞靶向及殺滅特定癌細胞之能力,已開發出方法來工程改造T細胞以表現將T細胞對準特定目標癌細胞之建構體。包含能夠與特定腫瘤抗原交互作用之結合域的嵌合抗原受體(CAR)及經工程改造T細胞受體(TCR)讓T細胞能夠靶向及殺滅表現該特定腫瘤抗原之癌細胞。
CD19定向之嵌合抗原受體T細胞(CAR T細胞)已證明在治療B細胞惡性病之範圍中的強效抗腫瘤功效。然而,自體CAR T療法存在技術、製造、及商業限制,這可能會限制其臨床應用的全部潛力。同種異體CAR-T療法係克服自體療法之固有限制並提供用於臨床用途之「現成(off-the-shelf)」方法的替代策略。
原則上,同種異體CAR T療法採用來自健康人類供體之T細胞,該等T細胞隨後經歷基因修飾以賦予抗腫瘤抗原之特異性。另外,亦引入基因編輯以預防移植物抗宿主疾病(GVHD)及患者之免疫系統對同種異體CAR T細胞之排斥。GVHD主要歸因於供體T細胞上之T細胞受體(TCR)αβ蛋白與接受者患者細胞上錯配之人類白血球抗原(HLA)分子之間的相互作用。反之,宿主之內源性CD8
+T細胞可相互作用,並消除攜帶錯配之主要組織相容性複合體(MHC) I類分子的供體T細胞移植物。
已努力發展適合現成使用之低免疫原性細胞,尤其是免疫細胞,諸如T細胞及NK細胞。在一個實例中,β-2-微球蛋白(B2M)係MHC I類分子之主要組分,在同種異體T細胞中去活化。B2M去活化可消除MHC I類,咸信會減少或預防宿主中錯配之CD8 T細胞的排斥反應。然而,觀察到的是,B2M剔除CAR T細胞易受宿主之NK細胞殺滅,因為NK細胞經刺激且殺滅缺乏MHC I類活性之同種異體T細胞。
在各種實施例中,本揭露提供優於B2M剔除之細胞工程方法。該方法可實現最小或不具有GVHD之風險,及最小或不具有CD4、CD8、及NK細胞排斥之風險,同時保持相當或甚至改良之治療活性。
根據本揭露之一個實施例,提供一種同種異體細胞,其經基因工程改造以減少MHC I類或MHC II類之表現或活性。在一些實施例中,MHC I類表現或活性減少但未消除。在一些實施例中,MHC II類表現及/或活性可減少或甚至消除。亦提供用於製備此類細胞之方法。
本揭露之一個實施例提供一種經分離之人類免疫細胞,其經工程改造以具有相較於對應之非經工程改造免疫細胞低10%至80%的MHC I類活性或表現。在一些實施例中,相較於對應之非經工程改造免疫細胞,該細胞具有至少低20%或至少40%之MHC II類活性或表現。在一些實施例中,相較於對應之非經工程改造免疫細胞或參考細胞,該細胞具有至少低75%之MHC II類活性或表現。
在一些實施例中,對應或參考免疫細胞係尚未經工程改造以具有降低之MHC I類及/或MHC II類分子表現的免疫細胞。在一些實施例中,對應或參考免疫細胞係經工程改造以具有降低之MHC I類表現但不降低MHC II類表現的免疫細胞。
在一些實施例中,細胞係T細胞或NK細胞,或任何其他免疫細胞,諸如單核球或巨噬細胞,或衍生自諸如iPSC之幹細胞之細胞。在一些實施例中,細胞包含編碼嵌合抗原受體(CAR)或T細胞受體(TCR)之外源性多核苷酸。在一些實施例中,CAR識別CD19及/或CD20。在一些實施例中,CAR包含SEQ ID NO:26或27之胺基酸序列或與SEQ ID NO: 26或27具有至少約90%序列同一性之序列。
在一些實施例中,CAR識別CLL-1。在一些實施例中,CAR包含SEQ ID NO:65至76或78至83中任一項之胺基酸序列或與SEQ ID NO:65至76或78至83中任一項具有至少約90%序列同一性之序列。
在一些實施例中,RFX5(調節因子X5)、TAP1(抗原加工相關轉運蛋白1)、TAP2(抗原肽轉運蛋白2)、或CIITA(II類、主要組織相容性複合體、反式活化子)中之至少一者之內源性基因係去活化或減少。在一些實施例中,內源性基因之兩個等位基因皆去活化。在一些實施例中,RFX5在細胞中去活化。在一些實施例中,TAP1、TAP2、及CIITA之內源性基因未經工程改造。
在一些實施例中,TRAC(T細胞受體α恆定)之內源性基因進一步去活化或減少。在一些實施例中,B2M(β-2-微球蛋白)之內源性基因未經工程改造,或其中細胞具有正常的B2M活性。
在一些實施例中,去活化使用(a)內源性基因之編輯,(b)抑制性RNA之表現,或(c)抑制劑,較佳為抗體來達成。在一些實施例中,該編輯係藉由CRISPR/Cas9、鋅指核酸酶(ZFN)、TALEN、MegaTAL、大範圍核酸酶、Cpf1、同源重組、單股去氧寡核苷酸(ssODN)、或鹼基編輯。
亦提供用於細胞之方法,但不限於此。在一些實施例中,在編輯基因(諸如RFX5)之前將CAR或TCR引入細胞。亦提供使用細胞治療疾病之方法。
序列表
本申請案含有以XML檔案格式電子提交之序列表,且其全文特此以引用方式併入本文中。該XML副本(建立於2023年2月22日)的檔名為K-1138-TW-NP_SL.xml且檔案大小為147,558位元組。
相關申請案之交互參照
本申請案主張2022年2月28日申請之美國臨時專利申請案第63/314,848號之優先權權益,其特此以引用方式全文併入本文中。
定義
為了能更輕易理解本揭露,以下先定義某些用語。下列用語及其他用語之額外定義係在本說明書中各處闡述。
除非有具體陳述或自上下文中明顯可知,如本文中所使用,用語「或(or)」係理解為涵括性的且同時涵蓋「或(or)」與「及(and)」。
在本文中,將使用用語「及/或(and/or)」之處認為是具體揭露兩個指定特徵或組分之各者(包含或不包含另一者)。因此,如用於諸如「A及/或B」之詞組中的用語「及/或」在本文中係意欲包括A及B;A或B;A(單獨);及B(單獨)。同樣地,如用於諸如「A、B、及/或C (A, B, and/or C)」之詞組中的用語「及/或(and/or)」係意欲涵蓋下列態樣之各者:A、B、及C;A、B、或C;A或C;A或B;B或C;A及C;A及B;B及C;A(單獨);B(單獨);及C(單獨)。
除非特定陳述或從上下文顯而易見,否則用語「約(about)」係指藉由所屬技術領域中具有通常知識者所判定之特定值或組成之可接受誤差範圍內之一值或組成,其將部分取決於該值或組成如何測量或判定,即,測量系統之限制。舉例而言,「約」或「基本上包含(comprising essentially of)」可意指根據所屬技術領域中之實踐之一或多個標準差內。「約」或「基本上包含」可意指至多10%之範圍(即±10%)。因此,「約」可理解為在10%、9%、8%、7%、6%、5%、4%、3%、2%、1%、0.5%、0.1%、0.05%、0.01%、或0.001%內大於或小於所述值。例如,約5 mg可包括在4.5 mg與5.5 mg之間的任何量。此外,特別是關於生物系統或程序,用語可意指至多一個數量級或至多5倍之值。除非另外說明,否則在本揭露中提供特定值或組成時,應假設「約」或「基本上包含」之意義係在該特定值或組成之可接受誤差範圍內。
「投予(Administering)」係指使用所屬技術領域中具有通常知識者已知的任何各種方法及遞送系統將藥劑實體引入至對象,諸如本文所揭示之經修飾之T細胞。用於本文所揭示之配方的例示性投予途徑包括靜脈內、肌內、皮下、腹膜內、脊椎、或其他腸胃外投予途徑,例如藉由注射或輸注。片語「腸胃外投予(parenteral administration)」意指除腸內及局部(topical)投予以外的投予模式,通常藉由注射,且包括但不限於靜脈內、肌內、動脈內、鞘內、淋巴內(intralymphatic)、病灶內、囊內、眶內、心內、皮內、腹膜內、經氣管、皮下、表皮下(subcuticular)、關節內、囊下、蜘蛛膜下、脊椎內、硬膜外、及胸骨內注射及輸注、以及體內電穿孔。在一些實施例中,配方經由非腸胃外途徑投予,例如經口。其他非腸胃外途徑包括局部、上皮或黏膜投予途徑,例如,鼻內、陰道內、直腸、舌下、或局部。亦可執行投予,例如一次、多次、及/或經過一或多個延伸週期。
用語「經活化(activated)」及「活化(activation)」係指已經足夠刺激以誘導可偵測細胞增生之T細胞的狀態。在一個實施例中,活化亦可與誘導之細胞介素產生及可偵測效應功能相關。用語「活化之T細胞(activated T cell)」尤其係指增殖之T細胞。透過單獨TCR產生的信號可能不足以完全活化T細胞,且亦可能需要一或多個二級或共刺激信號。因此,T細胞活化包含透過TCR/CD3複合物之初級刺激信號及一或多個二級共刺激信號。共刺激可藉由已接受初級活化信號之T細胞(諸如透過TCR/CD3複合物之刺激)的增生及/或細胞介素產生來證明。
用語「同種異體(allogeneic)」係指衍生自一個個體之任何材料,其接著引入相同物種之另一個體,例如同種異體T細胞移植。
用語「抗體(antibody)」(Ab)包括但不限於特異性結合至抗原之醣蛋白免疫球蛋白。一般而言,且抗體可包含至少兩個重(H)鏈及兩個輕(L)鏈,其等藉由二硫鍵或其抗原結合分子互相連接。各H鏈包含重鏈可變區(在本文中縮寫為VH)及重鏈恆定區。重鏈恆定區包含三個恆定域,CH1、CH2、及CH3。各輕鏈包含輕鏈可變區(在本文中縮寫為VL)及輕鏈恆定區。輕鏈恆定區包含一個恆定域,CL。VH及VL區可進一步細分成高度變異區,稱為互補決定區(CDR),其中散布稱為架構區(FR)之更具保守性的區。各VH及VL包含三個CDR及四個FR,以下列順序從胺基端排列到羧基端:FR1、CDR1、FR2、CDR2、FR3、CDR3、及FR4。重鏈及輕鏈之可變區含有與抗原交互作用之結合域。Ab之恆定區可介導免疫球蛋白與宿主組織或因子之結合,包括免疫系統之各種細胞(例如效應細胞)及經典補體系統之第一組分(C1q)。一般而言,人類抗體係大約150 kD之四聚體藥劑,由兩個同一之重(H)鏈多肽(各自約50 kD)及兩個同一之輕(L)鏈多肽(各自約25 kD)組成,其等彼此締合成一般稱為「Y形狀」結構。重鏈及輕鏈藉由單一二硫鍵彼此相連或連接;其他兩個二硫鍵將重鏈鉸鏈區彼此連接,使得二聚體彼此連接並形成四聚體。天然產生之抗體亦經醣化,例如在CH2域上。
「抗原結合分子(antigen binding molecule)」、「抗原結合部分(antigen binding portion)」、「抗原結合片段(antigen binding fragment)」、或「抗體片段(antibody fragment)」係指包含衍生分子之抗體之抗原結合部分(例如CDR)之任何分子。抗原結合分子可包括抗原互補決定區(CDR)。抗體片段之實例包括但不限於形成自抗原結合分子之Fab、Fab'、F(ab')2、及Fv片段、dAb、線性抗體、scFv抗體、及多特異性抗體。肽體(peptibody)(亦即,包含肽結合域之Fc融合分子)係合適抗原結合分子之另一個實例。在一些實施例中,抗原結合分子結合至腫瘤細胞上之抗原。在一些實施例中,抗原結合分子結合至涉及過度增生性疾病之細胞上的抗原或結合至病毒或細菌抗原。在某些實施例中,抗原結合分子係嵌合抗原受體(CAR)或經工程改造之T細胞受體(TCR)。在某些實施例中,抗原結合分子結合至:2B4 (CD244)、4-1BB、5T4、A33抗原、腺癌抗原、腎上腺素受體β3 (ADRB3)、A激酶錨定蛋白4 (AKAP-4)、α胎兒蛋白(AFP)、間變性淋巴瘤激酶(ALK)、雄性激素受體、B7H3 (CD276)、β2整合素、BAFF、B淋巴瘤細胞、B細胞突變抗原(BCMA)、bcr-abl(由斷點簇集區(BCR)及Abelson鼠白血病病毒致癌基因同源物1 (Abl)所組成之致癌基因融合蛋白)、BhCG、骨髓基質細胞抗原2 (BST2)、CCCTC結合因子(鋅指蛋白質)樣(BORIS或印記位點調節物兄弟)、BST2、C242抗原、9-0-乙醯基-CA19-9標記、CA-125、CAEX、鈣網蛋白、碳酸酐酶9 (CAIX)、C-MET、CCR4、CCR5、CCR8、CD2、CD3、CD4、CD5、CD8、CD7、CD10、CD16、CD19、CD20、CD22、CD23(IgE受體)、CD24、CD25、CD27、CD28、CD30 (TNFRSF8)、CD33、CD34、CD38、CD40、CD40L、CD41、CD44、CD44V6、CD49f、CD51、CD52、CD56、CD63、CD70、CD72、CD74、CD79a、CD79b、CD80、CD84、CD96、CD97、CD100、CD123、CD125、CD133、CD137、CD138、CD150、CD152 (CTLA-4)、CD160、CD171、CD179a、CD200、CD221、CD229、CD244、CD272 (BTLA)、CD274 (PDL-1、B7H1)、CD279 (PD-1)、CD352、CD358、CD300分子樣家族成員f (CD300LF)、癌胚抗原(CEA)、密連蛋白6 (CLDN6)、C型凝集素樣分子(CLL-1或CLECL1)、C型凝集素域家族12成員A (CLEC12A)、巨細胞病毒(CMV)感染之細胞抗原、CNT0888、CRTAM (CD355)、CS-1(亦稱為CD2子集1、CRACC、CD319、及19A24)、CTLA-4、週期蛋白B l、染色體X開讀框61 (CXORF61)、細胞色素P450 1B 1 (CYP1B1)、DNAM-1 (CD226)、橋粒芯蛋白4、DR3、DR5、E-鈣黏素新表位、上皮生長因子受體(EGFR)、EGF1R、上皮生長因子受體變體III (EGFRvIII)、上皮醣蛋白2 (EGP-2)、上皮醣蛋白40 (EGP-40)、含EGF樣模組之黏液素樣激素受體樣2 (EMR2)、突變型延伸因子2 (ELF2M)、內皮唾液酸蛋白、上皮細胞黏附分子(EPCAM)、A型蝶素受體2 (EphA2)、蝶素B2、受體酪胺酸蛋白質激酶erb-B2,3,4 (erb-B2,3,4)、ERBB、ERBB2 (Her2/neu)、ERG(跨膜絲胺酸蛋白酶2 (TMPRSS2) ETS融合基因)、ETA、位於染色體12p上之ETS易位變體基因6 (ETV6-AML)、IgA受體之Fc片段(FCAR或CD89)、纖維母細胞活化蛋白α (FAP)、FBP、Fc受體樣5 (FCRL5)、胎兒乙醯膽鹼受體(AChR)、纖連蛋白外域B、Fms樣酪胺酸激酶3 (FLT3)、葉酸結合蛋白(FBP)、葉酸受體1、葉酸受體α、葉酸受體β、Fos相關抗原1、岩藻醣基、岩藻醣基GM1;GM2、神經節苷脂G2 (Gd2)、神經節苷脂GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l -4)bDGlcp(l- l)Cer)、o-乙醯基-GD2神經節苷脂(OAcGD2)、GITR (TNFRSF 18)、GM1、神經節苷脂GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer)、GP 100、globoH糖基神經醯胺之六醣部分(GloboH)、醣蛋白75、磷脂醯肌醇蛋白聚醣3 (GPC3)、醣蛋白100 (gplOO)、GPNMB、G蛋白偶聯受體20 (GPR20)、G蛋白偶聯受體C類5組成員D (GPRC5D)、A型肝炎病毒細胞受體1 (HAVCR1)、人類表皮生長因子受體2 (HER-2)、HER2/neu、HER3、HER4、HGF、高分子量黑色素瘤相關抗原(HMWMAA)、人類乳突病毒E6 (HPV E6)、人類乳突病毒E7 (HPV E7)、突變型熱休克蛋白70-2 (mut hsp70-2)、人類分散因子受體激酶、人類端粒酶反轉錄酶(hTERT)、HVEM、ICOS、類胰島素生長因子受體1(IGF-1受體)、IGF-I、IgGl、免疫球蛋白λ樣多肽1 (IGLL1)、IL-6、介白素11受體α (IL- l lRa)、IL-13、介白素13受體次單元α2(IL- 13Ra2或CD213A2)、類胰島素生長因子I受體(IGF1-R)、整合素α5β1、整合素ανβ3、腸羧基酯酶、κ輕鏈、KCS1、激酶插入域受體(KDR)、KIR、KIR2DL1、KIR2DL2、KIR2DL3、KIR3DL2、KIR-L、KG2D配體、KIT (CD117)、KLRGI、LAGE-la、LAG3、淋巴球特異性蛋白酪胺酸激酶(LCK)、白血球免疫球蛋白樣受體亞家族A成員2 (LILRA2)、豆莢蛋白、白血球相關免疫球蛋白樣受體1 (LAIR1)、路易士(Y)抗原、LeY、LG、LI細胞黏附分子(LI-CAM)、LIGHT、LMP2、淋巴球抗原6複合物、LTBR、基因座K 9 (LY6K)、Ly-6、淋巴球抗原75 (LY75)、黑色素瘤癌症睪丸抗原1 (MAD-CT-1);黑色素瘤癌症睪丸抗原2 (MAD-CT-2)、MAGE、黑色素瘤相關抗原1 (MAGE-A1)、由T細胞識別之MAGE-A3黑色素瘤抗原1(MelanA或MARTI)、MelanA/MARTl、間皮素、MAGE A3、黑色素瘤細胞凋亡抑制因子(ML-IAP)、黑色素瘤特異性硫酸軟骨素蛋白聚醣(MCSCP)、MORAb-009、MS4A1、黏液素1 (MUCl)、MUC2、MUC3、MUC4、MUC5AC、MUC5b、MUC7、MUC16、黏液素CanAg、II型密拉氏管抑制物質(MIS)受體、v-myc禽骨髓瘤細胞病毒致癌基因神經母細胞瘤衍生性同源物(MYCN)、N-羥乙醯神經胺酸、N-乙醯基葡萄糖胺基轉移酶V (NA17)、神經細胞黏附分子(NCAM)、NKG2A、NKG2C、NKG2D、NKG2E配體、NKR-P IA、NPC-1C、NTB-A、乳腺分化抗原(NY-BR-1)、NY-ESO-1、致癌胎兒抗原(h5T4)、嗅覺受體51E2 (OR51E2)、OX40、漿細胞抗原、poly SA、前頂體素結合蛋白sp32 (OY-TES l)、p53、p53突變體、泛連接蛋白(PANX3)、前列腺酸性磷酸酶(PAP)、成對盒蛋白Pax-3 (PAX3)、成對盒蛋白Pax-5 (PAX5)、前列腺癌腫瘤抗原1(PCTA-1或半乳糖凝集素8)、PD-1H、血小板衍生生長因子受體α (PDGFR-α)、PDGFR-β、PDL192、PEN-5、磷脂絲胺酸、胎盤特異性蛋白1 (PLAC1)、聚唾液酸、前列腺酶、前列腺癌細胞、前列腺蛋白、蛋白酶絲胺酸21(睪素或PRSS21)、蛋白酶3 (PR1)、前列腺幹細胞抗原(PSCA)、前列腺特異性膜抗原(PSMA)、蛋白酶體(前體、巨蛋白因子)次單元β型、晚期糖化終產物受體(RAGE-1)、RANKL、Ras突變體、Ras同源物家族成員C (RhoC)、RON、受體酪胺酸激酶樣孤兒受體1 (ROR1)、腎遍在蛋白1 (RU1)、腎遍在蛋白2 (RU2)、肉瘤易位斷點、由T細胞識別之鱗狀細胞癌抗原3 (SART3)、SAS、SDC1、SLAMF7、唾液酸基路易士黏附分子(sLe)、Siglec-3、Siglec-7、Siglec-9、音蝟因子(SHH)、精子蛋白17 (SPA17)、階段特異性胚胎抗原4 (SSEA-4)、STEAP、sTn抗原、滑膜肉瘤X斷點2 (SSX2)、存活素、腫瘤相關醣蛋白72 (TAG72)、TCR5y、TCRa、TCRB、TCRγ交替讀框蛋白(TARP)、端粒酶、TIGIT、TNF-α前驅物、腫瘤內皮標記1 (TEM1/CD248)、腫瘤內皮標記7相關蛋白(TEM7R)、肌腱蛋白C (tenascin C)、TGF-β1、TGF-β2、轉麩醯胺酸酶5 (TGS5)、促血管生成素結合細胞表面受體2 (Tie 2)、TIM1、TIM2、TIM3、Tn Ag、TRAIL-R1、TRAIL-R2、酪胺酸酶相關蛋白2 (TRP-2)、促甲狀腺激素受體(TSHR)、腫瘤抗原CTAA16.88、酪胺酸酶、ROR1、TAG- 72、尿溶蛋白2 (UPK2)、VEGF-A、VEGFR-1、血管內皮生長因子受體2 (VEGFR2)、及波形蛋白、威爾姆氏腫瘤蛋白(WT1)、或X抗原家族成員1A (XAGE1)。特異性結合至所述抗原之胺基酸序列係所屬技術領域中已知的,或可使用所屬技術領域中已知之方法製備;實例包括免疫球蛋白、免疫球蛋白之可變區(例如,可變片段(「Fv」)或二價可變片段(「Fab」))、單鏈抗體等。在某些實施例中,抗原結合分子係特異性結合抗原之抗體片段,包括其一或多個互補決定區(CDR)。在進一步實施例中,抗原結合分子係單鏈可變片段(scFv)。在一些實施例中,抗原結合分子包含親合性多聚體(avimer)或由其所組成。
用語「可變區(variable region)」或「可變域(variable domain)」可互換使用。可變區一般係指抗體之一部分,通常係輕鏈或重鏈之一部分,一般係成熟重鏈中胺基端的約110至120個胺基及成熟輕鏈中約90至115個胺基酸,其在抗體之間的序列差異很大,且係用於特定抗體對其特定抗原之結合及特異性。序列之變異性集中在稱為互補決定區(CDR)之區中,而可變域中更高度保守的區稱為架構區(FR)。不希望受任何特定機制或理論束縛,咸信輕鏈及重鏈之CDR主要負責抗體與抗原之交互作用及特異性。在某些實施例中,可變區係人類可變區。在某些實施例中,可變區包含嚙齒動物或鼠類CDR及人類架構區(FR)。在特定實施例中,可變區係靈長類(例如非人類靈長類)可變區。在某些實施例中,可變區包含嚙齒動物或鼠類CDR及靈長類(例如非人類靈長類)架構區(FR)。
用語「VL」及「VL域(VL domain)」可互換使用以指抗體或其抗原結合分子之輕鏈可變區。
用語「VH」及「VH域(VH domain)」可互換使用以指抗體或其抗原結合分子之重鏈可變區。
CDR之一些定義係通常使用:Kabat編號、Chothia編號、AbM編號、或contact編號。AbM定義係Oxford Molecular之AbM抗體模型化軟體所使用之兩者之間之妥協。Contact定義係基於可用複雜晶體結構之分析。
「抗原(antigen)」係指可刺激人類或動物產生抗體或T細胞反應之化合物、組成物或物質,包括組成物(諸如包括腫瘤特異性蛋白質之一者),該等組成物被注射或吸收至人類或動物中。抗原與特定體液或細胞免疫之產物反應,包括藉由異源抗原(諸如所揭示之抗原)所誘導者。「目標抗原(target antigen)」或「所關注之目標抗原(target antigen of interest)」係在其他正常(所欲)細胞之表面上未實質上發現之抗原,且本文所涵蓋之TCR或CAR之結合域經設計以結合至其。所屬技術領域中具有通常知識者將容易地理解,任何大分子包括幾乎所有蛋白質或肽可作為抗原。抗原可內源性表現,即藉由基因體DNA表現,或可重組表現。抗原可對特定組織(諸如癌細胞)具有特異性,或其可廣泛表現。此外,較大分子之片段可充當抗原。在一個實施例中,抗原係腫瘤抗原。在一個特定實施例中,抗原係下列者的全部或片段:2B4 (CD244)、4-1BB、5T4、A33抗原、腺癌抗原、腎上腺素受體β3 (ADRB3)、A激酶錨定蛋白4 (AKAP-4)、α胎兒蛋白(AFP)、間變性淋巴瘤激酶(ALK)、雄性激素受體、B7H3 (CD276)、β2整合素、BAFF、B淋巴瘤細胞、B細胞突變抗原(BCMA)、bcr-abl(由斷點簇集區(BCR)及Abelson鼠白血病病毒致癌基因同源物1 (Abl)所組成之致癌基因融合蛋白)、BhCG、骨髓基質細胞抗原2 (BST2)、CCCTC結合因子(鋅指蛋白質)樣(BORIS或印記位點調節物兄弟)、BST2、C242抗原、9-0-乙醯基-CA19-9標記、CA-125、CAEX、鈣網蛋白、碳酸酐酶9 (CAIX)、C-MET、CCR4、CCR5、CCR8、CD2、CD3、CD4、CD5、CD8、CD7、CD10、CD16、CD19、CD20、CD22、CD23(IgE受體)、CD24、CD25、CD27、CD28、CD30 (TNFRSF8)、CD33、CD34、CD38、CD40、CD40L、CD41、CD44、CD44V6、CD49f、CD51、CD52、CD56、CD63、CD70、CD72、CD74、CD79a、CD79b、CD80、CD84、CD96、CD97、CD100、CD123、CD125、CD133、CD137、CD138、CD150、CD152 (CTLA-4)、CD160、CD171、CD179a、CD200、CD221、CD229、CD244、CD272 (BTLA)、CD274 (PDL-1、B7H1)、CD279 (PD-1)、CD352、CD358、CD300分子樣家族成員f (CD300LF)、癌胚抗原(CEA)、密連蛋白6 (CLDN6)、C型凝集素樣分子(CLL-1或CLECL1)、C型凝集素域家族12成員A (CLEC12A)、巨細胞病毒(CMV)感染之細胞抗原、CNT0888、CRTAM (CD355)、CS-1(亦稱為CD2子集1、CRACC、CD319、及19A24)、CTLA-4、週期蛋白B l、染色體X開讀框61 (CXORF61)、細胞色素P450 1B 1 (CYP1B1)、DNAM-1 (CD226)、橋粒芯蛋白4、DR3、DR5、E-鈣黏素新表位、上皮生長因子受體(EGFR)、EGF1R、上皮生長因子受體變體III (EGFRvIII)、上皮醣蛋白2 (EGP-2)、上皮醣蛋白40 (EGP-40)、含EGF樣模組之黏液素樣激素受體樣2 (EMR2)、突變型延伸因子2 (ELF2M)、內皮唾液酸蛋白、上皮細胞黏附分子(EPCAM)、A型蝶素受體2 (EphA2)、蝶素B2、受體酪胺酸蛋白質激酶erb-B2,3,4 (erb-B2,3,4)、ERBB、ERBB2 (Her2/neu)、ERG(跨膜絲胺酸蛋白酶2 (TMPRSS2) ETS融合基因)、ETA、位於染色體12p上之ETS易位變體基因6 (ETV6-AML)、IgA受體之Fc片段(FCAR或CD89)、纖維母細胞活化蛋白α (FAP)、FBP、Fc受體樣5 (FCRL5)、胎兒乙醯膽鹼受體(AChR)、纖連蛋白外域B、Fms樣酪胺酸激酶3 (FLT3)、葉酸結合蛋白(FBP)、葉酸受體1、葉酸受體α、葉酸受體β、Fos相關抗原1、岩藻醣基、岩藻醣基GM1;GM2、神經節苷脂G2 (Gd2)、神經節苷脂GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l -4)bDGlcp(l- l)Cer)、o-乙醯基-GD2神經節苷脂(OAcGD2)、GITR (TNFRSF 18)、GM1、神經節苷脂GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer)、GP 100、globoH糖基神經醯胺之六醣部分(GloboH)、醣蛋白75、磷脂醯肌醇蛋白聚醣3 (GPC3)、醣蛋白100 (gplOO)、GPNMB、G蛋白偶聯受體20 (GPR20)、G蛋白偶聯受體C類5組成員D (GPRC5D)、A型肝炎病毒細胞受體1 (HAVCR1)、人類表皮生長因子受體2 (HER-2)、HER2/neu、HER3、HER4、HGF、高分子量黑色素瘤相關抗原(HMWMAA)、人類乳突病毒E6 (HPV E6)、人類乳突病毒E7 (HPV E7)、突變型熱休克蛋白70-2 (mut hsp70-2)、人類分散因子受體激酶、人類端粒酶反轉錄酶(hTERT)、HVEM、ICOS、類胰島素生長因子受體1(IGF-1受體)、IGF-I、IgGl、免疫球蛋白λ樣多肽1 (IGLL1)、IL-6、介白素11受體α (IL- l lRa)、IL-13、介白素13受體次單元α2(IL- 13Ra2或CD213A2)、類胰島素生長因子I受體(IGF1-R)、整合素α5β1、整合素ανβ3、腸羧基酯酶、κ輕鏈、KCS1、激酶插入域受體(KDR)、KIR、KIR2DL1、KIR2DL2、KIR2DL3、KIR3DL2、KIR-L、KG2D配體、KIT (CD117)、KLRGI、LAGE-la、LAG3、淋巴球特異性蛋白酪胺酸激酶(LCK)、白血球免疫球蛋白樣受體亞家族A成員2 (LILRA2)、豆莢蛋白、白血球相關免疫球蛋白樣受體1 (LAIR1)、路易士(Y)抗原、LeY、LG、LI細胞黏附分子(LI-CAM)、LIGHT、LMP2、淋巴球抗原6複合物、LTBR、基因座K 9 (LY6K)、Ly-6、淋巴球抗原75 (LY75)、黑色素瘤癌症睪丸抗原1 (MAD-CT-1);黑色素瘤癌症睪丸抗原2 (MAD-CT-2)、MAGE、黑色素瘤相關抗原1 (MAGE-A1)、由T細胞識別之MAGE-A3黑色素瘤抗原1(MelanA或MARTI)、MelanA/MARTl、間皮素、MAGE A3、黑色素瘤細胞凋亡抑制因子(ML-IAP)、黑色素瘤特異性硫酸軟骨素蛋白聚醣(MCSCP)、MORAb-009、MS4A1、黏液素1 (MUCl)、MUC2、MUC3、MUC4、MUC5AC、MUC5b、MUC7、MUC16、黏液素CanAg、II型密拉氏管抑制物質(MIS)受體、v-myc禽骨髓瘤細胞病毒致癌基因神經母細胞瘤衍生性同源物(MYCN)、N-羥乙醯神經胺酸、N-乙醯基葡萄糖胺基轉移酶V (NA17)、神經細胞黏附分子(NCAM)、NKG2A、NKG2C、NKG2D、NKG2E配體、NKR-P IA、NPC-1C、NTB-A、乳腺分化抗原(NY-BR-1)、NY-ESO-1、致癌胎兒抗原(h5T4)、嗅覺受體51E2 (OR51E2)、OX40、漿細胞抗原、poly SA、前頂體素結合蛋白sp32 (OY-TES l)、p53、p53突變體、泛連接蛋白(PANX3)、前列腺酸性磷酸酶(PAP)、成對盒蛋白Pax-3 (PAX3)、成對盒蛋白Pax-5 (PAX5)、前列腺癌腫瘤抗原1(PCTA-1或半乳糖凝集素8)、PD-1H、血小板衍生生長因子受體α (PDGFR-α)、PDGFR-β、PDL192、PEN-5、磷脂絲胺酸、胎盤特異性蛋白1 (PLAC1)、聚唾液酸、前列腺酶、前列腺癌細胞、前列腺蛋白、蛋白酶絲胺酸21(睪素或PRSS21)、蛋白酶3 (PR1)、前列腺幹細胞抗原(PSCA)、前列腺特異性膜抗原(PSMA)、蛋白酶體(前體、巨蛋白因子)次單元β型、晚期糖化終產物受體(RAGE-1)、RANKL、Ras突變體、Ras同源物家族成員C (RhoC)、RON、受體酪胺酸激酶樣孤兒受體1 (ROR1)、腎遍在蛋白1 (RU1)、腎遍在蛋白2 (RU2)、肉瘤易位斷點、由T細胞識別之鱗狀細胞癌抗原3 (SART3)、SAS、SDC1、SLAMF7、唾液酸基路易士黏附分子(sLe)、Siglec-3、Siglec-7、Siglec-9、音蝟因子(SHH)、精子蛋白17 (SPA17)、階段特異性胚胎抗原4 (SSEA-4)、STEAP、sTn抗原、滑膜肉瘤X斷點2 (SSX2)、存活素、腫瘤相關醣蛋白72 (TAG72)、TCR5y、TCRa、TCRB、TCRγ交替讀框蛋白(TARP)、端粒酶、TIGIT、TNF-α前驅物、腫瘤內皮標記1 (TEM1/CD248)、腫瘤內皮標記7相關蛋白(TEM7R)、肌腱蛋白C (tenascin C)、TGF-β1、TGF-β2、轉麩醯胺酸酶5 (TGS5)、促血管生成素結合細胞表面受體2 (Tie 2)、TIM1、TIM2、TIM3、Tn Ag、TRAIL-R1、TRAIL-R2、酪胺酸酶相關蛋白2 (TRP-2)、促甲狀腺激素受體(TSHR)、腫瘤抗原CTAA16.88、酪胺酸酶、ROR1、TAG- 72、尿溶蛋白2 (UPK2)、VEGF-A、VEGFR-1、血管內皮生長因子受體2 (VEGFR2)、及波形蛋白、威爾姆氏腫瘤蛋白(WT1)、或X抗原家族成員1A (XAGE1)。「目標(target)」係藉由結合模體、抗原結合系統、CAR、或抗原結合劑(例如抗體)結合之任何分子。
用語「自體(autologous)」係指任何衍生自相同個體之材料,該材料之後會再重新引入至該個體。例如,本文所述之經工程改造之自體細胞療法(eACT
™)方法涉及自患者收集淋巴球,接著將其工程改造以表現例如CAR構築體,接著投予回同一位患者。
「嵌合抗原受體(chimeric antigen receptor)」或「CAR」係指經工程改造以包含結合模體之分子及活化免疫細胞(例如T細胞,諸如初始T細胞、中央記憶T細胞、效應記憶T細胞、或其組合)在抗原結合時之手段。CAR亦稱為人工T細胞受體、嵌合T細胞受體或嵌合免疫受體。在一些實施例中,CAR包含結合模體、胞外域、跨膜域、一或多個共刺激域、及胞內信號傳導域。已經基因工程改造以表現嵌合抗原受體之T細胞可稱為CAR T細胞。「胞外域(extracellular domain)」(或「ECD」)係指多肽之部分,其當多肽存在於細胞膜中時,應理解為位於細胞膜外之胞外空間中。
如本文所用,用語「胞外配位體結合域(extracellular ligand-binding domain)」係指能夠結合配位體(例如細胞表面分子)之寡肽或多肽。例如,可選擇胞外配體結合域以識別作用為與特定疾病狀態(例如癌症)相關之目標細胞上之細胞表面標記的配體。可作用為配體之細胞表面標記之實例包括與病毒、細菌、及寄生蟲感染、自體免疫疾病、及癌細胞相關者。
CAR之結合域可接著為「間隔子(spacer)」或「鉸鏈(hinge)」,其係指移動遠離效應細胞表面之抗原結合域以實現適當細胞/細胞接觸、抗原結合及活化(Patel et al., Gene Therapy, 1999; 6: 412-419)。CAR中之鉸鏈區通常在跨膜(TM)與結合域之間。在某些實施例中,鉸鏈區係免疫球蛋白鉸鏈區,且可為野生型免疫球蛋白鉸鏈區或改變之野生型免疫球蛋白鉸鏈區。用於本文所描述之CAR中之其他例示性鉸鏈區包括衍生自類型1膜蛋白(諸如CD8α、CD4、CD28、及CD7)之胞外區的鉸鏈區,其可係來自此等分子之野生型鉸鏈區,或可經改變。
「跨膜(transmembrane)」區或域係CAR之一部分,其將胞外結合部分錨定至免疫效應細胞之質膜,並促進結合域與目標抗原之結合。跨膜域可係CD3ζ跨膜域,然而可採用之其他跨膜域包括由CD8α、CD4、CD28、CD45、CD9、CD16、CD22、CD33、CD64、CD80、CD86、CD134、CD137、及CD154獲得者。在一個實施例中,跨膜域係CD137之跨膜域。在某些實施例中,跨膜域係合成,其中其將主要包括疏水性殘基,諸如白胺酸及纈胺酸。
「胞內信號傳導域(intracellular signaling domain)」或「信號傳導域(signaling domain)」係指嵌合抗原受體蛋白之部分,其參與將有效CAR結合至目標抗原之訊息轉導至免疫效應細胞內部以引發效應細胞功能,例如活化、細胞介素產生、增生、及細胞毒性活性,包括細胞毒性因子至結合CAR之目標細胞的釋放、或與胞外CAR域結合之抗原引發的其他細胞反應。用語「效應功能(effector function)」係指細胞之特定功能。T細胞之效應功能,例如可係細胞溶解活性或包括細胞介素之分泌之幫助或活性。因此,用語「胞內信號傳導域(intracellular signaling domain)」或「信號傳導域(signaling domain)」在本文中可互換使用,指蛋白質之部分,其轉導效應功能信號且引導細胞以執行特定功能。雖然通常可採用整個胞內信號傳導域,但在許多情況下,不必使用整個域。在使用胞內信號傳導域之截短部分的程度,只要其轉導效應功能信號,則可使用此類截短部分代替整個域。用語胞內信號傳導域意欲包括足以轉導效應功能信號之胞內信號傳導域之任何截短部分。胞內信號傳導域亦稱為「信號轉導域」,且通常源自人類CD3或FcRy鏈之部分。
已知透過單獨T細胞受體產生之信號不足於完全活化T細胞且亦需要二級或共刺激信號。因此,T細胞活化可藉由兩種不同類別之細胞質信號傳導序列介導:透過T細胞受體起始抗原依賴性初級活化者(初級細胞質信號傳導序列)及以抗原非依賴性方式作用者,以提供二級或共刺激信號(二級細胞質信號傳導序列)。共刺激方式作用之細胞質信號傳導序列可含有稱為基於免疫受體酪胺酸之活化模體或ITAM之信號傳導模體。
含有特別用於本揭露之初級細胞質信號傳導序列之ITAM的實例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、及CD66d者。
如本文所用,用語「共刺激信號傳導域(costimulatory signaling domain)」或「共刺激域(costimulatory domain)」係指包含共刺激分子之胞內域之CAR之部分。共刺激分子係除抗原受體或Fc受體以外之細胞表面分子,其提供T淋巴球在結合至抗原時為有效活化及功能所需之第二信號。此類共刺激分子之實例包括CD27、CD28、4-1 BB (CD137)、0X40 (CD134)、CD30、CD40、PD-1、ICOS (CD278)、LFA-1、CD2、CD7、LIGHT、NKD2C、B7-H2、及特異性結合CD83之配體。因此,雖然本揭露提供衍生自CD3zeta、及4-1 BB之例示性共刺激域,其他共刺激域係考慮搭配本文所述之CAR使用。納入一或多種共刺激信號傳導域可增強T細胞表現CAR受體之功效及擴增。胞內信號傳導及共刺激信號傳導域可以任何順序串聯連接至跨膜域之羧基端。
儘管經工程改造以含有來自CD3或FcRγ之信號傳導域的基於scFv之CAR已經顯示遞送用於T細胞活化及效應功能之強效信號,在不存在伴隨共刺激信號之情況下,其不足以引起促使T細胞存活及擴增的信號。含有衍生自CD3ζ或FcRγ之結合域、鉸鏈、跨膜、及信號傳導域之其他CAR與一或多個共刺激信號傳導域(例如衍生自CD28、CD137、CD134、及CD278之胞內共刺激域)一起可更有效地引導抗腫瘤活性以及在體外、及動物模型及癌症患者中增加CAR表現性T細胞中之細胞介素分泌、裂解活性、存活、及增生(Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106:3360-3365)。
「共刺激信號(costimulatory signal)」係指與初級信號(諸如TCR/CD3連接)組合之信號引起T細胞反應,諸如但不限於關鍵分子之增生及/或上調或下調。
「共刺激配體(costimulatory ligand)」包括特異性結合T細胞上之同源共刺激分子的抗原呈現細胞上之分子。共刺激配體之結合提供介導T細胞反應之信號,包括但不限於增生、活化、分化、及類似者。除了刺激分子所提供之初級信號外,共刺激配體亦藉由T細胞受體(TCR)/CD3複合物與裝載肽之主要組織相容性複合體(MHC)分子的結合誘導信號。共刺激配體可包括但不限於3/TR6、4-1BB配體、結合鐸(Toll)配體受體之促效劑或抗體、B7-1 (CD80)、B7-2 (CD86)、CD30配體、CD40、CD7、CD70、CD83、疱疹病毒進入介導物(herpes virus entry mediator, HVEM)、人類白血球抗原G (HLA-G)、ILT4、免疫球蛋白樣轉錄本(ILT) 3、可誘導型共刺激配體(ICOS-L)、細胞間黏附分子(ICAM)、與B7-H3特異性結合之配體、淋巴毒素β受體、MHC I類鏈相關蛋白A (MICA)、MHC I類鏈相關蛋白B (MICB)、OX40配體、PD-L2、或程式性死亡(PD) L1。共刺激配體包括但不限於與存在於T細胞上之共刺激分子特異性結合之抗體,諸如但不限於4-1BB、B7-H3、CD2、CD27、CD28、CD30、CD40、CD7、ICOS、與CD83特異性結合之配體、淋巴球功能相關抗原1 (LFA-1)、自然殺手細胞受體C (NKG2C)、OX40、PD-1、或腫瘤壞死因子超家族成員14(TNFSF14或LIGHT)。
「共刺激分子(costimulatory molecule)」係與共刺激配體特異性結合之T細胞上之同源結合夥伴(partner),從而介導T細胞之共刺激反應,諸如但不限於增生。共刺激分子包括但不限於「共刺激分子」係與共刺激配體特異性結合之T細胞上之同源結合夥伴,從而介導T細胞之共刺激反應,諸如但不限於增生。共刺激分子包括但不限於4-1BB/CD137、B7-H3、BAFFR、BLAME (SLAMF8)、BTLA、CD33、CD45、CD100 (SEMA4D)、CD103、CD134、CD137、CD154、CD16、CD160 (BY55)、CD18、CD19、CD19a、CD2、CD22、CD247、CD27、CD276 (B7-H3)、CD28、CD29、CD3 (α; β;δ;ε;γ;ζ)、CD30、CD37、CD4、CD4、CD40、CD49a、CD49D、CD49f、CD5、CD64、CD69、CD7、CD80、CD83配體、CD84、CD86、CD8α、CD8β、CD9、CD96 (Tactile)、CD11a、CD11b、CD11c、CD11d、CDS、CEACAM1、CRT AM、DAP-10、DNAM1 (CD226)、Fcγ受體、GADS、GITR、HVEM (LIGHTR)、IA4、ICAM-1、ICAM-1、ICOS、Ig α (CD79a)、IL2R β、IL2R γ、IL7R α、整合素、ITGA4、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB2、ITGB7、ITGBl、KIRDS2、LAT、LFA-1、LFA-1、LIGHT、LIGHT(腫瘤壞死因子超家族成員14;TNFSF14)、LTBR、Ly9 (CD229)、淋巴球功能相關抗原-1 (LFA-1 (CDl la/CD18)、MHC I類分子、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80 (KLRF1)、OX40、PAG/Cbp、PD-1、PSGL1、SELPLG (CD162)、信號傳導淋巴球性活化分子、SLAM (SLAMF1; CD150; IPO-3)、SLAMF4 (CD244; 2B4)、SLAMF6 (NTB-A; Lyl08)、SLAMF7、SLP-76、TNF、TNFr、TNFR2、鐸配體受體、TRANCE/RANKL、VLA1、或VLA-6、或其片段、截短、或組合。
「保守性胺基酸取代(conservative amino acid substitution)」係其中胺基酸殘基被具有類似側鏈之胺基酸殘基置換者。所屬技術領域中已定義具有側鏈之胺基酸殘基之家族。此等家族包括具有鹼性側鏈之胺基酸(例如離胺酸、精胺酸、組胺酸)、酸性側鏈(例如天冬胺酸、麩胺酸)、未帶電極性側鏈(例如甘胺酸、天冬醯胺酸、麩醯胺酸、絲胺酸、蘇胺酸、酪胺酸、半胱胺酸、色胺酸)、非極性側鏈(例如丙胺酸、纈胺酸、白胺酸、異白胺酸、脯胺酸、苯丙胺酸、甲硫胺酸)、β-支鏈側鏈(例如蘇胺酸、纈胺酸、異白胺酸)、及芳族側鏈(例如酪胺酸、苯丙胺酸、色胺酸、組胺酸)。在某些實施例中,抗體或其抗原結合分子之(多個)CDR內或(多個)架構區內的一或多個胺基酸殘基可用具有類似側鏈之胺基酸殘基置換。通常而言,若兩個序列在對應位置中含有保守性胺基酸取代,則通常將該兩個序列視為「實質上類似的(substantially similar)」。舉例而言,某些胺基酸通常分類為「疏水性(hydrophobic)」或「親水性(hydrophilic)」胺基酸,及/或具有「極性(polar)」或「非極性(non-polar)」側鏈。用一個胺基酸取代相同類型的另一胺基酸可視為保守性取代。例示性胺基酸分類概述於以下
表 1中:
表
1. 胺基酸分類
胺基酸 | 3 字母 | 性質 | 性質 | 疏水性指數 |
丙胺酸 | Ala | 非極性 | 中性 | 1.8 |
精胺酸 | Arg | 極性 | 陽性 | -4.5 |
天冬醯胺酸 | Asn | 極性 | 中性 | -3.5 |
天冬胺酸 | Asp | 極性 | 陰性 | -3.5 |
半胱胺酸 | Cys | 非極性 | 中性 | 2.5 |
麩胺酸 | Glu | 極性 | 陰性 | -3.5 |
麩醯胺酸 | Gln | 極性 | 中性 | -3.5 |
甘胺酸 | Gly | 非極性 | 中性 | -0.4 |
組胺酸 | His | 極性 | 陽性 | -3.2 |
異白胺酸 | Ile | 非極性 | 中性 | 4.5 |
白胺酸 | Leu | 非極性 | 中性 | 3.8 |
離胺酸 | Lys | 極性 | 陽性 | -3.9 |
甲硫胺酸 | Met | 非極性 | 中性 | 1.9 |
苯丙胺酸 | Phe | 非極性 | 中性 | 2.8 |
脯胺酸 | Pro | 非極性 | 中性 | -1.6 |
絲胺酸 | Ser | 極性 | 中性 | -0.8 |
蘇胺酸 | Thr | 極性 | 中性 | -0.7 |
色胺酸 | Trp | 非極性 | 中性 | -0.9 |
酪胺酸 | Tyr | 極性 | 中性 | -1.3 |
纈胺酸 | Val | 非極性 | 中性 | 4.2 |
「T細胞受體」或「TCR」係指存在於T細胞表面上之抗原識別分子。在正常T細胞發展期間,四個TCR基因α、β、γ、及δ之各者可重新排列導致高度不同之TCR蛋白質。
用語「異源(heterologous)」意指來自非天然存在之序列的任何來源。例如,作為共刺激蛋白質之一部分包括的異源序列係非天然存在之胺基酸,亦即與野生型人類共刺激蛋白質不一致。例如,異源核苷酸序列係指野生型人類共刺激蛋白質編碼序列以外的核苷酸序列。
用語「同一性(identity)」係指聚合分子之間的整體相關性,例如在核酸分子之間(例如DNA分子及/或RNA分子)及/或多肽分子之間。與兩個所提供之多肽序列之間的同一性百分比計算的方法係已知的。例如可藉由比對二個序列以用於最佳比較目的來執行兩個核酸或多肽序列之同一性百分比之計算(例如可在第一及第二序列中之一或兩者中引入間隙以用於最佳比對,且可出於比較目的忽略非同一性序列)。接著比較對應位置處之核苷酸或胺基酸。當第一序列中之位置被與第二序列中之對應位置同一之殘基(例如核苷酸或胺基酸)佔據時,則分子在彼位置處係同一的。兩個序列之間的同一性百分比係序列共有的相同位置數目之函數,其可選地考慮間隙數目及各間隙之長度,可能需要引入間隙以達到兩個序列之最佳比對。可使用數學演算法(諸如BLAST(基本局部比對搜尋工具))實現序列之比較或比對及兩個序列之間的同一性百分比之判定。在一些實施例中,若其序列係至少25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、或99%同一(例如85%至90%、85%至95%、85%至100%、90%至95%、90%至100%、或95%至100%),則聚合分子視為彼此「同源」。
免疫療法之T細胞可來自所屬技術領域中已知之任何來源。例如,T細胞可體外分化自造血幹細胞群,或T細胞可獲自對象。T細胞可得自例如周邊血液單核細胞(PBMC)、骨髓、淋巴結組織、臍帶血、胸腺組織、來自感染部位之組織、腹水、胸膜積水、脾臟組織、及腫瘤。此外,T細胞可衍生自所屬技術領域中可用之一或多種T細胞系。T細胞亦可使用所屬技術領域中具有通常知識者已知之任何數量之技術,諸如FICOLL
™分離及/或血球分離來自對象收集之血液之單位獲得。T細胞療法單離T細胞之額外方法揭示於美國專利公開案第2013/0287748號,其以全文以引用方式併入本文中。
「患者(patient)」包括任何罹患癌症(例如,淋巴瘤或白血病)之人類。用語「對象(subject)」及「患者」在本文中可互換使用。
用語「對象」及「患者」包括人類及非人類動物對象以及具有正式診斷病症者、未經正式辨識病症者、接受醫療注意者、及在發展病症之風險下者。
用語「醫藥上可接受(pharmaceutically acceptable)」係指分子或組成物當向接受者投予時,對其接受者無害,或對其接受者的益處超過任何有害效應。關於用於調配如本文所揭示之組成物的載劑、稀釋劑、或賦形劑,醫藥上可接受之載劑、稀釋劑、或賦形劑必須與組成物之其他成分相容且對其接受者無害,或對接受者的益處必須超過任何有害效應。用語「醫藥上可接受之載劑(pharmaceutically acceptable carrier)」意指醫藥上可接受之材料、組成物、或媒劑,諸如液體或固體填充劑、稀釋劑、賦形劑、或溶劑包封材料,其涉及將藥劑自身體之一個部分攜帶或運輸至另一部分(例如自一個器官至另一個)。在醫藥組成物中存在之各載體在與配方之其他成分相容之情況下必須係「可接受(acceptable)」,且對患者無害,或必須對接受者的益處超過任何有害效應。可用作醫藥上可接受之載劑之材料的一些實例包含:糖,諸如乳糖、葡萄糖、及蔗糖;澱粉,諸如玉米澱粉及馬鈴薯澱粉;纖維素及其衍生物,諸如羧甲基纖維素鈉、乙基纖維素、及乙酸纖維素;粉末狀黃蓍膠;麥芽;明膠;滑石;賦形劑,諸如可可脂及栓劑蠟;油,諸如花生油、棉籽油、紅花子油、芝麻油、橄欖油、玉米油、及黃豆油;二醇,諸如丙二醇;多元醇,諸如甘油、山梨醇、甘露醇、及聚乙二醇;酯,諸如油酸乙酯及月桂酸乙酯;洋菜;緩衝劑,諸如氫氧化鎂及氫氧化鋁;藻酸;無致熱原的水;等張鹽水;林格氏液;乙醇;pH緩衝溶液;聚酯、聚碳酸酯及/或聚酐;及醫藥配方中採用的其他非毒性相容物質。
用語「醫藥組成物(pharmaceutical composition)」係指其中活性劑與一或多種醫藥上可接受之載劑調配在一起之組成物。在一些實施例中,活性劑以適於治療方案中投予之單位劑量存在,該治療方案展示當向相關對象或群體投予時達成預定治療效果之統計顯著機率。在一些實施例中,醫藥組成物可經調配以固體或液體形式投予,包含但不限於適用於以下形式:口服投予,例如灌劑(水性或非水性溶液或懸浮液)、錠劑,例如用於經頰、舌下、及全身性吸收者、丸劑、散劑、顆粒劑、施用於舌頭之糊劑;腸胃外投予,例如藉由皮下、肌肉內、靜脈內或硬膜外注射作為例如無菌溶液或懸浮液或持續釋放配方;局部施用,例如作為乳膏、軟膏或控制釋放貼片、或施用至皮膚、肺或口腔之噴霧;陰道內或直腸內,例如作為栓劑、乳膏或泡沫;舌下;眼;經皮膚;或經鼻、肺及其他黏膜表面。
用語「降低(reducing)」及「減少(decreasing)」在本文中可互換使用,並且指示任何小於原始者之改變。「降低」及「減少」係相對用語,需要測量前後之間的比較。「降低」及「減少」包括完全耗盡。
用語「參考(reference)」描述與其進行比較之標準或控制。舉例而言,在一些實施例中,將所關注之藥劑、動物、個體、群體、樣本、序列、或值與作為藥劑、動物、個體、群體、樣本、序列、或值之參考或對照相比較。在一些實施例中,將所關注之測試、測量或判定與參考或對照實質上同時測試、測量及/或判定。在一些實施例中,參考或對照係歷史參考或對照,該歷史參考或對照可選地實施在有形介質中。通常,參考或對照係在與評估對象相當的條件或情況下判定或表徵的。當存在足夠的相似性以證明對所選擇之參考或對照的依賴及/或比較是合理的。
「調節T細胞(regulatory T cell)」(「Treg」、「Treg細胞」、或「Tregs」)係指參與控制某些免疫活性(例如自體免疫、過敏、及對感染之反應)之CD4+ T淋巴球譜系。調節T細胞可調節T細胞群之活性,且亦可影響某些先天性免疫系統細胞類型。Treg可藉由生物標記CD4、CD25、及Foxp3之表現及CD127之低表現識別。天然存在之Treg細胞通常構成約5%至10%周邊CD4+ T淋巴球。然而,腫瘤微環境內之Treg細胞(亦即腫瘤浸潤Treg細胞),可能佔總CD4+ T淋巴球群之多達20至30%。
治療劑(例如經工程改造之CAR T細胞)之「治療有效量(therapeutically effective amount)」、「有效劑量(effective dose)」、「有效量(effective amount)」、或「治療有效劑量(therapeutically effective dosage)」係任何量,當單獨或與另一治療劑組合使用時,保護對象免受疾病之發作或促進疾病回歸,表現為疾病症狀之嚴重程度降低、疾病無症狀期之頻率及持續時間增加,或預防由於疾病折磨造成的損害或殘疾。促進疾病回歸之治療劑之能力可使用所屬技術領域中具有通常知識者已知之各種方法評估,諸如在臨床試驗期間在人類對象中、在預測人類功效的動物模型系統中、或藉由在體外檢定中檢定藥劑之活性。
用語「轉導(transduction)」及「轉導(transduced)」係指藉由病毒載體將外來DNA引入細胞中之程序(參見Jones et al., 「Genetics: principles and analysis,」 Boston: Jones & Bartlett Publ. (1998))。在一些實施例中,載體係反轉錄病毒載體、DNA載體、RNA載體、腺病毒載體、桿狀病毒載體、艾司坦-巴爾(Epstein Barr)病毒載體、乳多泡病毒載體、牛痘病毒載體、單純疱疹病毒載體、腺病毒相關載體、慢病毒載體、或其任何組合。
對象之「治療(treatment)」或「治療(treating)」係指向對象進行之任何類型之干預或過程,或向對象投予活性劑,目的係反轉、緩解、改善、抑制、減緩或預防症狀、併發症或病況之發作、進展、發展、嚴重程度或再發、或與疾病相關聯之生物化學指示。在一個實施例中,「治療(treatment)」或「治療(treating)」包括部分緩解。在另一實施例中,「治療」包括完全緩解。在一些實施例中,治療可係未展現出相關疾病、病症及/或病況之徵象之對象,及/或僅展現出疾病、病症及/或病況之早期徵象之對象。在一些實施例中,此類治療可係展現出相關疾病、病症及/或病況之一或多種建立徵象之對象。在一些實施例中,治療可係已診斷患有相關疾病、病症及/或病況之對象。在一些實施例中,治療可係已知具有一或多個敏感性因子之對象,該一或多個敏感性因子與相關疾病、病症及/或病況之發展風險增加的統計學上相關。
用語「載體(vector)」係指經修飾以包含或合併所提供核酸序列之接受者核酸分子。一種類型之載體係「質體(plasmid)」,其係指額外DNA可連接之環狀雙股DNA分子。另一種類型之載體係病毒載體,其中額外DNA區段可連接至病毒基因體中。某些載體能夠在其等被引入的宿主細胞中自主複製(例如具有細菌複製起源之細菌載體及游離基因(episomal)哺乳動物載體)。其他載體(例如非游離基因哺乳動物載體)在引入宿主細胞中後可整合至宿主細胞之基因體中,且藉此與宿主基因體一起複製。此外,某些載體包含直接表現可操作性地連接至其之插入基因之序列。此類載體在本文中可稱為「表現載體(expression vector)」。標準技術可用於載體之工程改造,例如,見於Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)),其以引用方式併入本文中。
「鋅指DNA結合蛋白」(或結合域)係一種蛋白質,或較大蛋白質中之域,其透過一或多個鋅指以序列特異性方式結合DNA,鋅指係結合域內的胺基酸序列區域,其結構透過鋅離子之配位而穩定。因此,多指ZFP之各鋅指皆包括識別螺旋區域,用於結合主鏈內的DNA。用語鋅指DNA結合蛋白通常縮寫為鋅指蛋白或ZFP。用語「鋅指核酸酶(zinc finger nuclease)」包括一個ZFN以及一對ZFN(該對之成員稱為「左及右」或「第一及第二」或「對」),其二聚化以裂解目標基因。
「TALE DNA結合域」或「TALE」係包含一或多個TALE重複域/單元之多肽。各包含重複可變雙殘基(RVD)之重複域參與TALE與其同源目標DNA序列之結合。單個「重複單元」(亦稱為「重複」)之長度通常為33至35個胺基酸,且與天然存在之TALE蛋白內的其他TALE重複序列至少展現出一些序列同源性。TALE蛋白可經設計以使用重複單元內之典型或非典型RVD結合至目標位點。參見例如美國專利第8,586,526號及第9,458,205號。鋅指及TALE DNA結合域可係「經工程改造」以結合至預定核苷酸序列,例如經由工程改造(改變一或多個胺基酸)天然存在的鋅指蛋白的識別螺旋區域,或藉由工程改造參與DNA結合之胺基酸(重複可變雙殘基或RVD區域)。因此,經工程改造之鋅指蛋白或TALE蛋白係非天然存在之蛋白質。工程改造鋅指蛋白及TALE之方法之非限制性實例係設計及選擇。經設計之蛋白質係不存在於自然界中之蛋白質,其設計/組成主要來自理性標準。設計之理性標準包括應用取代規則及電腦化演算法來處理儲存現有ZFP或TALE設計(典型和非典型RVD)及綁定資料之資訊的資料庫中的資訊。參見例如美國專利第9,458,205號;第8,586,526號;第6,140,081號;第6,453,242號;及第6,534,261號;亦參見國際專利公開案WO 98/53058;WO 98/53059;WO 98/53060;WO 02/016536;及WO 03/016496。用語「TALEN」包括一個TALEN以及一對TALEN(該對之成員稱為「左及右」或「第一及第二」或「對」),其二聚化以裂解目標基因。
CRISPR/Cas(簇集之規律間隔的短回文重複序列/CRISPR相關蛋白)系統,因為其無與倫比的編輯效率、方便性、及在生物體中之潛在應用,自其構想以來一直是最強大的基因體編輯工具。在嚮導RNA (gRNA)之引導下,Cas核酸酶可在各種細胞(細胞系及自活生物體之細胞)之目標基因體位點產生DNA雙股斷裂(DSB)。此等DSB接著藉由內源性DNA修復系統修復,可用以執行所欲的基因體編輯。
最近開發之鹼基編輯器(BE)將CRISPR/Cas系統與APOBEC(載脂蛋白B mRNA編輯酶、催化多肽樣)胞嘧啶去胺酶家族整合在一起,大幅增強CRISPR/Cas9介導之基因校正的效率。透過與Cas9內切酶(nCas9)或催化死亡Cas9 (dCas9)融合,大鼠APOBEC1 (rA1)之胞嘧啶(C)去胺活性可係有目的地定向到基因體中之目標鹼基,並在此等鹼基上催化C至胸腺嘧啶(T)取代。
先導編輯(PE)係基因體編輯技術,藉由該技術可修飾活生物體之基因體。先導編輯直接將新的遺傳資訊寫入目標DNA位點。其使用融合蛋白,由融合至經工程改造之反轉錄酶的催化損害的核酸內切酶(例如Cas9)及先導編輯嚮導RNA (pegRNA)組成,能夠識別目標位點並提供新的基因資訊以替換目標DNA核苷酸。先導編輯介導了目標插入、缺失、及鹼基至鹼基之轉化,而不需要雙股斷裂(DSB)或供體DNA模板。
RFX5 剔除細胞
來自健康供體之同種異體供體細胞有可能提供可依需求應用之現成細胞產品,與自體細胞相比,成本更低。隨著基因編輯技術之進步,已嘗試剔除某些基因,以開發適合現成使用(off-shelf use)之低免疫原性細胞。
β-2-微球蛋白(β2M或B2M)係MHC I類分子之重要組分。B2M之缺失可消除MHC I類,這已經證實可降低或預防宿主中錯配之CD8 T細胞的排斥反應。
本發明人已開發一種同種異體抗CD19 CAR T細胞產品,其中TCR α恆定(TRAC)基因座及B2M兩者之部分由鋅指核酸酶(ZFN)刪除,導致細胞表面之蛋白質表現降低。然而,觀察到的是,此等經編輯之CAR T細胞對宿主之NK細胞敏感,因為NK細胞受到刺激且殺滅缺乏MHC I類表現之同種異體T細胞。
因此,本發明人尋找替代的基因編輯方法,該些方法優於B2M剔除且可實現最小化或不具有GVHD風險,及最小化或不具有CD4、CD8、及NK細胞排斥之風險,同時保持相當或甚至改良之治療活性。
根據本揭露之一個實施例,提供一種同種異體細胞,其經基因工程改造以減少MHC I類或MHC II類之表現或活性。在一些實施例中,MHC I類表現或活性減少但未消除。在一些實施例中,MHC II類表現及/或活性可減少或甚至消除。亦提供用於製備此類細胞之方法。
在一個實施例中,相較於參考同種異體細胞(例如,未經如此工程改造之細胞,諸如僅具有TRAC剔除之T細胞),MHC I類表現或活性減少至少10%、或至少20%、30%、40%、50%、60%、70%、80%、或90%。在一個實施例中,相較於參考同種異體細胞,MHC I類表現或活性保持在至少5%、或至少10%、15%、20%、30%、40%、50%、60%、70%、80%、或90%。在一個實施例中,相較於參考同種異體細胞,MHC I類表現或活性係約5%至90%、10%至80%、20%至80%、20%至70%、30%至70%、30%至60%、40%至60%、10%至60%、10%至50%、20%至60%、20%至50%、20%至40%、10%至40%、或10%至30%。
在一個實施例中,相較於參考同種異體細胞,MHC II類表現或活性減少至少10%、或至少20%、30%、40%、50%、60%、70%、80%、或90%。在一個實施例中,相較於參考同種異體細胞,MHC II類表現或活性保持在至少5%、或至少10%、15%、20%、30%、40%、50%、60%、70%、80%、或90%。在一個實施例中,相較於參考同種異體細胞,MHC II類表現或活性係約5%至90%、10%至80%、20%至80%、20%至70%、30%至70%、30%至60%、40%至60%、10%至60%、10%至50%、20%至60%、20%至50%、20%至40%、10%至40%、或10%至30%。
在一個實施例中,相較於參考同種異體細胞,MHC I類及II類兩者之表現或活性減少至少10%、或至少20%、30%、40%、50%、60%、70%、80%、或90%。在一個實施例中,相較於參考同種異體細胞,MHC I類及II類兩者之表現或活性保持在至少5%、或至少10%、15%、20%、30%、40%、50%、60%、70%、80%、或90%。
在一些實施例中,參考免疫細胞尚未經工程改造為具有降低之MHC I類及/或MHC II類分子之表現。在一些實施例中,參考免疫細胞已經工程改造為具有降低之MHC I類表現,但不降低MHC II類表現(例如,TRAC及B2M剔除)。在一些實施例中,參考免疫細胞已藉由引入表現CAR或TCR之外源構築體而經工程改造,但尚未經工程改造為具有降低之MHC I類或MHC II類分子之表現。在一些實施例中,參考免疫細胞係自健康供體之非轉導(NTD)細胞,其尚未經工程改造為具有降低之MHC I類或MHC II類分子之表現。
在一些實施例中,MHC I類之表現或活性降低或減弱,而MHC II類之活性表現被剔除或消除。
用語MHC I類亦指人類白血球抗原(HLA) I類。MHC I類分子之實例包括但不限於B2M、個別HLA分子(例如,HLA-A、-B、-C、-E、-G)、TAP1、TAP2、及/或與裸淋巴球症候群I (Bare Lymphocyte Syndrome I) (BLSI)相關之基因。
用語MHC II類亦指人類白血球抗原(HLA) II類。MHC II類分子之實例包括但不限於轉錄因子(例如,RFXANK、RFXS、RFXAP、或RFX5)或反式活化因子(CHTA)、與BLS II相關之基因、及/或個別HLA分子(例如,HLA-DP、-DQ、-DR、-DiVI、-DO -α及β鏈)。
研究了多種候選基因,包括RFX5(調節因子X5)、TAP1(與抗原加工相關之轉運蛋白1)、TAP2(抗原肽轉運蛋白2)、及CIITA(II類,主要組織相容性複合體、反式活化因子)之單個剔除。發現RFX5單個剔除達成了經工程改造之細胞中MHC I類之最佳下調及MHC II類表現之消除。MHC I類下調足以降低錯配之CD8 T細胞之排斥,但不促進NK細胞之殺滅。MHC II類表現之消除亦降低錯配之CD4 T細胞之反應。同樣重要的是,如實驗實例中所展示,剔除RFX5之CAR細胞具有極佳的可製造性及CAR官能性。
因此,本揭露之一個實施例提供一種用於製備在誘導移植物抗宿主疾病(GVHD)或宿主排斥中活性降低之同種異體細胞的方法。在一些實施例中,該方法需要降低(或完全消除)參與MHC I類及/或II類表現之基因的表現/活性。
在一些實施例中,基因選自RFX5(調節因子X5)、TAP1(與抗原加工相關之轉運蛋白1)、TAP2(抗原肽轉運蛋白2)、或CIITA(II類,主要組織相容性複合體,反式活化因子)。在一些實施例中,基因係RFX5,且TAP1、TAP2、及CIITA中之至少一者亦經完全或部分地去活化或抑制。在一些實施例中,僅RFX5之表現或活性在細胞中降低,而TAP1、TAP2、及CIITA無基因變化。
在一些實施例中,細胞中B2M(β-2-微球蛋白)之內源性基因未經工程改造。亦即,未對B2M基因座進行基因編輯,且未將抑制劑引入細胞。
在一些實施例中,細胞進一步經工程改造以降低(或完全消除)TRAC(T細胞受體α恆定)之表現/活性。
在一些實施例中,細胞衍生自健康供體。在一些實施例中,細胞衍生自/分化自幹細胞,諸如誘導性多能性幹細胞(iPSC)。
在一些實施例中,細胞係衍生自健康供體之T細胞。
用於減少細胞中基因之表現
/ 活性之技術
降低或消除基因之表現或活性之方法係所屬技術領域中已知的。在一些實施例中,此類降低或消除包括基因(例如RFX5)產生之任何可偵測的減少。在某些實例中,相較於對照組(在未抑制RFX5之對應細胞中偵測到的RFX5的量),細胞中可偵測的RFX5減少至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少75%、至少80%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%(諸如減少40%至90%、40%至80%、或50%至95%)。
在某些實施例中,相較於對照組(在未抑制TCR之對應細胞中偵測到的TCR的量),細胞中可偵測的TCR減少至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少75%、至少80%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%(諸如減少40%至90%、40%至80%、或50%至95%)。
在某些實施例中,相較於對照組(在未抑制B2M之對應細胞中偵測到的B2M的量),細胞中可偵測的B2M減少至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少75%、至少80%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%(諸如減少40%至90%、40%至80%、或50%至95%)。
在某些實施例中,基因表現之降低或消除藉由直接抑制基因而發生(例如,減弱或剔除RFX5基因可減少或消除RFX5之表現或活性)。在其他實施例中,基因表現之降低或消除藉由間接抑制基因而發生(例如,減弱或剔除RFX5基因可減少MHC I類分子之表現或活性)。
減少百分比及增加百分比可藉由所屬技術領域中已知之方法計算。作為非限制性實例,相對於參考或對應細胞(例如,不包含RFX5 KO之細胞),在經編輯之細胞(例如,包含RFX5 KO之細胞)中分子之表現或活性減少或降低的百分比可藉由減去參考/對應細胞值減去經編輯之細胞值,將該量除以參考值,接著乘以100得到降低百分比來計算出。若百分比為負,則可意謂增加而不是減少。
在一些實施例中,基因之表現或活性可用適合的抑制劑,諸如小分子抑制劑、抑制性RNA(例如,siRNA、shRNA)或抗體降低。在一些實施例中,此可藉由在目標基因之一或兩個等位基因上對目標基因進行基因編輯而達成。
在某些實施例中,使用DNA結合域降低RFX5中之一或多者之表現,例如耦合至核酸酶域,特異性結合至RFX5基因中之目標位點並介導在目標位點之突變,從而減少官能性RFX5之表現。任何DNA結合域皆可用於本文中所揭示之組成物及方法,包括但不限於鋅指DNA結合域、TALE DNA結合域、CRISPR/Cas核酸酶之DNA結合部分(sgRNA)、或自大範圍核酸酶之DNA結合域。
在某些實施例中,DNA結合域包含鋅指蛋白。較佳地,鋅指蛋白質係非天然存在的,因為其經工程改造以結合至所選之目標位點。相較於天然存在之鋅指蛋白,經工程改造之鋅指結合域可具有新穎的結合特異性。工程方法包括但不限於合理設計及各種類型之選擇。合理設計包括例如,使用包含三聯體(或四聯體)核苷酸序列及個別鋅指胺基酸序列之資料庫,其中,各三聯體或四聯體核苷酸序列與一或多個結合特定三聯體或四聯體序列之鋅指胺基酸序列相關。
通常,ZFP包括至少三個指狀物。某些ZFP包括四個、五個、或六個指狀物。包括三個指狀物之ZFP通常識別包括9或10個核苷酸之目標位點;包括四個指狀物之ZFP通常識別包括12至14個核苷酸之目標位點;而具有六個指狀物之ZFP可識別包括18至21個核苷酸之目標位點。ZFP亦可係包括一或多個調節域之融合蛋白,該些域可係轉錄活化或抑制域。
在一些實施例中,DNA結合域可衍生自核酸酶。舉例而言,歸巢核酸內切酶及大範圍核酸酶,諸如I-Scel、I-CeuI、PI-PspI、PI-Sce、I-SceIV、I-CsmI、I-PanI、I-SceII、I-PpoI、I-SceIII、I-TevI、I-TevII、及I-TevIII之識別序列係已知的。另外,歸巢核酸內切酶及大範圍核酸酶之DNA結合特異性可經工程改造以結合非天然目標位點。
在一些實施例中,TALEN包含核酸內切酶(例如FokI)裂解結構域或裂解半結構域。在其他實施例中,TALE核酸酶係mega TAL。此等mega TAL核酸酶係包含TALE DNA結合域及大範圍核酸酶裂解域之融合蛋白。大範圍核酸酶裂解域作為單體具有活性,且不需要二聚化即可發揮活性。
在某些實施例中,DNA結合域係CRISPR/Cas核酸酶系統之部分,包括結合至DNA之單個嚮導RNA (sgRNA)。編碼系統之RNA組分的CRISPR(簇集之規律間隔的短回文重複序列)基因座及編碼蛋白質之cas(CRISPR相關)基因座構成CRISPR/Cas核酸酶系統之基因序列。微生物宿主中之CRISPR基因座含有CRISPR相關(Cas)基因以及能夠對CRISPR介導之核酸裂解之特異性進行程式化的非編碼RNA元素的組合。
可適用於本揭露之細胞及方法的sgRNA可使用CRISPR設計工具來識別。例示性sgRNA序列係顯示於
表 2中。
表2 :例示性嚮導RNA
嚮導RNA | 序列(5'到3') |
TRAC gRNA – 1 | CTTCAAGAGCAACAGTGCTG (SEQ ID NO:84) |
TRAC gRNA – 2 | ctctcagctggtacacggca (SEQ ID NO:85) |
TRAC gRNA – 3 | TCTCTCAGCTGGTACACGGC (SEQ ID NO:86) |
TRAC gRNA – 4 | GCTGGTACACGGCAGGGTCA (SEQ ID NO:87) |
B2M gRNA – 1 | GGCCGAGATGTCTCGCTCCG (SEQ ID NO:88) |
B2M gRNA – 2 | CGCGAGCACAGCTAAGGCCA (SEQ ID NO:89) |
B2M gRNA – 3 | Gagtagcgcgagcacagcta (SEQ ID NO:90) |
B2M gRNA – 4 | AAGTCAACTTCAATGTCGGA (SEQ ID NO:91) |
RFX5 gRNA – 1 | TCGGAGCCTCTGAAGAAGGG (SEQ ID NO:92) |
RFX5 gRNA – 2 | GCCAAGTACACTGAGCAATG (SEQ ID NO:93) |
RFX5 gRNA – 3 | CAGGGGTGGCATAGACACCA (SEQ ID NO:94) |
RFX5 gRNA – 4 | Gccaagtacactgagcaatg (SEQ ID NO:95) |
II型CRISPR係表徵最充分的系統中之一者,且在四個連續步驟中進行靶向DNA雙股斷裂。首先,自CRISPR基因座轉錄兩種非編碼RNA,pre-crRNA陣列及tracrRNA。其次,tracrRNA與pre-crRNA之重複區域雜交,並介導pre-crRNA加工成含有個別間隔序列之成熟crRNA。第三,成熟crRNA:tracrRNA複合物經由crRNA上之間隔物與目標DNA上與原間隔物相鄰基序(protospacer adjacent motif, PAM)相鄰的原間隔物之間的瓦生-克立克鹼基配對(Watson-Crick base-pairing),將功能域(例如核酸酶(諸如Cas))引導至目標DNA,這是目標識別之額外需求。最後,Cas9介導目標DNA之裂解以在原間隔物內產生雙股斷裂。CRISPR/Cas系統之活性包含以下三個步驟:(i)在稱為「適應」之程序中,將外來DNA序列插入CRISPR陣列以預防未來攻擊,(ii)相關蛋白質之表現,以及陣列之表現及加工,接著(iii) RNA介導之外來核酸干擾。因此,在細菌細胞中,數種所謂的「Cas」蛋白質參與CRISPR/Cas系統之自然功能,且在諸如插入外來DNA等功能中充分作用。
核酸酶之非限制性實例包括大範圍核酸酶、TALEN、及鋅指核酸酶。核酸酶可包含異源DNA結合及裂解域(例如鋅指核酸酶;具有異源裂解域之大範圍核酸酶DNA結合域)或替代地,可改變天然存在之核酸酶之DNA結合域以結合所選取之目標位點(例如,已經工程改造以結合至不同於同源結合位點之大範圍核酸酶)。
嵌合抗原受體或
T 細胞受體之表現
經工程改造之細胞,尤其是免疫細胞(諸如T細胞、NK細胞、及其他免疫細胞類型)亦可用經設計以表現CAR之載體進行基因工程改造,該些CAR將細胞毒性重導至腫瘤細胞。CAR係組合了基於抗體之對於目標抗原(例如腫瘤抗原)的特異性與T細胞受體活化胞內域的分子,以產生展現特定抗腫瘤細胞免疫活性之嵌合蛋白。
本文所涵蓋之CAR包含結合至特定目標抗原之胞外域(亦稱為結合域或抗原特異性結合域)、跨膜域、及胞內信號傳導域。CAR之特徵為其重導免疫效應細胞特異性之能力,藉以利用單株抗體、可溶性配體、或細胞特異性共受體來觸發增生、細胞介素產生、吞噬作用或分子產生(其可以主要組織相容性(MHC)非依賴性方式介導表現目標抗原之細胞的細胞死亡)。
在一些實施例中,CAR包括胞外結合域,其包括但不限於特異性結合目標抗原之抗體或其抗原結合片段、連繫配體、共受體之胞外域。
作為非限制性實例,目標抗原可包括:HPV–16 E6與HPV–16 E7、α葉酸受體、5T4、α
vβ
6整合素、BCMA、TACI、B7–H3、B7–H6、CAIX、CD19、CD20、CD22、CD28、CD30、CD33、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD137 (4–1BB)、CD138、CD171、CEA、CSPG4、CLL-1、EGFR、EGFR家族包括ErbB2 (HERII)、EGFRvIII、EGP2、EGP40、EPCAM、EphA2、EpCAM、FAP、胎兒AchR、FRa、GD2、GD3、磷脂醯肌醇蛋白聚糖3 (GPC3)、HLA–Al+MAGEI、HLA–A2 + MAGE1、HLAA3 + MAGE1、HLA–Al + NY–ES0–1、HLA–A2 + NY–ES0–1、HLA–A3 + NY–ES0–1、IL–llRα、IL–13Rα2、λ、路易士–Y、κ、間皮素、Mucl、Muc16、NCAM、NKG2D配體、NYE-S0–1、PRAME、PSCA、PSMA、RORI、SSX、生存素、TAG72、TEM、及VEGFRII;一或多個鉸鏈域或間隔域;跨膜域,包括但不限於自CD8α、CD4、CD45、PD-1、及CD152之跨膜域;一或多個細胞內共刺激信號傳導域,包括但不限於自CD28、CD54 (ICAM)、CD134 (OX40)、CD137 (41BB)、CD152 (CTLA4)、CD273 (PD–L2)、CD274 (PD–L1)、及CD278 (ICOS)之細胞內共刺激信號傳導域;及自CD3ζ或FcRγ之主要信號傳導域。在一個實施例中,CAR結合至腫瘤抗原,其包含CLL-1、CD19、CD20、CD28、CD137 (4–1BB)、磷脂醯肌醇蛋白聚糖3 (GPC3)、PSCA、或PSMA。在某些實施例中,CAR結合CD19。在某些實施例中,CAR結合CD20。在某些實施例中,CAR包括結合CD19之第一scFv及結合CD20之第二scFv。實例CD19或CD20結合序列提供於
表 3。
表
3. 抗原結合序列實例
名稱 | 序列 |
抗CD20 v01 VH/VL | SEQ ID NO:1 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGGSWYSNWFDPWGQGTMVTVSS SEQ ID NO:2 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDRSLPPTFGGGTKVEIK |
抗CD20 v02 VH/VL | SEQ ID NO:3 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGIHWNWIRQPPGKGLEWIGDIDTSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGQESATYLGMDVWGQGTTVTVSS SEQ ID NO:4 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQLYTYPFTFGGGTKVEIK |
抗CD20 v03 VH/VL | SEQ ID NO:5 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSS SEQ ID NO:6 DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK |
抗CD20 v04 VH/VL | SEQ ID NO:7 QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSS SEQ ID NO:8 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIK |
抗CD20 v05 VH/VL | SEQ ID NO:9 QVQLQESGPGLVKPSETLSLTCTVSGGSISSPDHYWGWIRQPPGKGLEWIGSIYASGSTFYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSS SEQ ID NO:10 DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK |
抗CD20 v06 VH/VL | SEQ ID NO:11 QITLKESGPTLVKPTQTLTLTCTFSGFSLDTEGVGVGWIRQPPGKALEWLALIYFNDQKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTAVYYCARDTGYSRWYYGMDVWGQGTTVTVSS SEQ ID NO:12 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYAYPITFGGGTKVEIK |
抗CD20 v07 VH/VL | SEQ ID NO:13 QVQLQQWGAGLLKPSETLSLTCAVYGGSFEKYYWSWIRQPPGKGLEWIGEIYHSGLTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS SEQ ID NO:14 DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSFPWTFGGGTKVEIK |
抗CD20 v08 VH/VL | SEQ ID NO:15 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSRYVWSWIRQPPGKGLEWIGEIDSSGKTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS SEQ ID NO:16 DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSFPWTFGGGTKVEIK |
抗CD20 v09 VH/VL | SEQ ID NO:17 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYAWSWIRQPPGKGLEWIGEIDHRGFTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS SEQ ID NO:18 DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSFPWTFGGGTKVEIK |
抗CD20 v10 VH/VL | SEQ ID NO:19 QVQLQQWGAGLLKPSETLSLTCAVYGGSFQKYYWSWIRQPPGKGLEWIGEIDTSGFTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVGRYSYGYYITAFDIWGQGTTVTVSS SEQ ID NO:20 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSFPFTFGGGTKVEIK |
抗CD19 VH/VL v01 | SEQ ID NO:21 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS SEQ ID NO:22 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT |
抗CD19 VH/VL v02 | SEQ ID NO:23 EVQLVESGGGLVQPGRSLRLSCTASGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:24 DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPDQAPKLLIKHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGQGTKLEIK |
抗CD19 scFv | SEQ ID NO:25 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS |
抗CD20/抗CD19雙順反子CAR v01 | SEQ ID NO:26 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIKAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR |
抗CD20/抗CD19雙特異性CAR v02 | SEQ ID NO:27 MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIKGGGGSGKPGSGEGGSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSSGGGGSGKPGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPDQAPKLLIKHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR |
抗CD20/抗CD19雙順反子CAR v01 | SEQ ID NO:28 ATGCTGCTGCTGGTGACATCTCTGCTGCTTTGCGAGCTGCCCCACCCTGCCTTCCTGCTTATCCCCGACATTCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCTTAGGAGATAGAGTTACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAATACCTGAACTGGTATCAGCAGAAGCCCGACGGCACTGTGAAACTGCTTATTTACCACACCTCCAGACTGCACAGCGGCGTTCCCAGCAGATTCTCTGGCAGCGGATCTGGAACCGACTACAGCCTCACCATCTCCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACACTGCCCTACACCTTCGGAGGAGGAACCAAGCTGGAGATCACCGGGGGAGGAGGCTCTGGAGGCGGCGGATCAGGAGGAGGGGGATCTGAGGTTAAGCTGCAGGAGAGCGGCCCTGGCCTGGTGGCTCCTAGCCAATCTTTATCTGTGACCTGCACTGTGTCCGGCGTTAGCCTGCCCGATTATGGCGTTTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTTATCTGGGGCAGCGAGACCACATACTACAACAGCGCCCTGAAGAGCAGACTTACGATTATCAAGGACAACAGCAAGAGCCAGGTTTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCTAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGAACAAGCGTTACCGTTAGCAGCGCTGCTGCACTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTTAAGGGCAAGCACCTGTGCCCCAGCCCTCTGTTCCCTGGACCTTCTAAGCCTTTCTGGGTTCTGGTGGTGGTCGGCGGCGTTTTAGCCTGTTACAGCCTTCTGGTGACTGTGGCCTTCATCATCTTTTGGGTTAGAAGCAAGAGAAGCAGACTGCTCCACAGCGACTACATGAACATGACCCCCAGACGGCCTGGCCCCACCAGAAAGCATTACCAGCCCTACGCTCCTCCCAGAGACTTCGCCGCCTACAGGAGCAGAGTTAAATTCAGCAGATCCGCCGATGCCCCCGCTTACCAACAGGGACAAAACCAGCTGTACAATGAGCTCAACCTGGGGAGAAGAGAAGAATACGACGTTCTGGATAAGAGAAGGGGCAGAGATCCCGAAATGGGGGGCAAGCCCAGACGCAAGAACCCTCAGGAGGGGCTTTACAACGAACTGCAGAAGGATAAGATGGCTGAGGCTTACTCGGAGATTGGGATGAAGGGGGAGAGAAGGCGGGGCAAGGGACACGATGGCTTATACCAGGGGCTGAGCACCGCCACCAAGGACACATACGACGCTCTTCATATGCAGGCTCTGCCCCCAAGAAGGGCTAAGAGATCTGGCTCTGGCGAGGGCAGAGGCAGCTTGCTTACATGTGGCGATGTGGAGGAGAACCCCGGGCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGCTTCAGCTCCAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCAGCTCCAGCTATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTAGAATGGATCGGCTCGATATATTACTCCGGCAGCACCTACTATAACCCCAGCTTGAAGAGCCGGGTTACCATTTCTGTGGACACATCAAAGAACCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGCATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGTTCCACCAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGATATACAGATGACACAGAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATCGTGTAACGATCACCTGCCGGGCCTCTCAGAGCATCAACTCCTACCTCAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAATTACTCATCTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACCCTCACTATCTCCAGCTTGCAGCCCGAGGATTTCGCCACTTATTACTGTCAGCAGAGCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGA |
抗CD20/抗CD19雙特異性CAR v02 | SEQ ID NO:29 ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAACAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGCCAGCAAAGCCTCGCCGACCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAAGGGGGGGGTGGAAGTGGGAAGCCTGGCAGCGGCGAGGGCGGCAGTCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGAGACTGACTACAGCAGCGGAATGGGATACGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCTCCTCAGGCGGTGGCGGCAGTGGGAAGCCTGGCAGCGATATTCAAATGACCCAGTCCCCGTCCTCCCTGAGTGCCTCCGTCGGTGACCGTGTTACGATTACCTGCCGTGCGAGCCAAGACATCTCTAAATACCTGAACTGGTATCAGCAAAAACCGGATCAGGCACCGAAACTGCTGATCAAACATACCTCACGTCTGCACTCGGGTGTGCCGAGCCGCTTTAGTGGTTCCGGCTCAGGTACCGATTACACCCTGACGATCAGCTCTCTGCAGCCGGAAGACTTTGCCACGTATTACTGCCAGCAAGGTAATACCCTGCCGTATACGTTCGGCCAAGGTACCAAACTGGAAATCAAAGGGGGGGGTGGAAGTGGGGGCGGTGGCAGCGGCGGTGGCGGCAGTGAAGTGCAGCTGGTTGAAAGCGGTGGTGGTCTGGTTCAACCGGGTCGTTCCCTGCGTCTGTCATGTACGGCGAGTGGTGTCTCCCTGCCGGACTATGGCGTGTCCTGGATTCGTCAGCCGCCGGGTAAAGGCCTGGAATGGATTGGTGTCATCTGGGGCAGTGAAACCACGTATTACAACTCGGCCCTGAAAAGCCGTTTCACCATCTCTCGCGATAACAGTAAAAATACGCTGTACCTGCAGATGAATAGCCTGCGCGCGGAAGACACCGCCGTTTACTACTGCGCAAAACATTACTACTACGGTGGCAGCTATGCTATGGATTACTGGGGTCAAGGCACGCTGGTCACCGTTTCGTCAGCCGCTGCCCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGATGA |
抗CD19 CAR | SEQ ID NO:30 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR |
在各種實施例中,結合模體可包含本揭露之重鏈可變域(例如與
表 3中所示之重鏈可變域具有至少75%序列同一性,例如至少80%、85%、90%、95%、或100%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)、本揭露之輕鏈可變域(例如與
表 3中所示之輕鏈可變域具有至少75%序列同一性,例如至少80%、85%、90%、95%、或100%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)、及連接子(參見例如Whitlow et al. Protein Eng. 1993 Nov; 6(8):989-95.)。在各種實施例中,結合模體可包含前導序列或信號序列、本揭露之重鏈可變域(例如與
表 3中所示之重鏈可變域具有至少75%序列同一性,例如至少80%、85%、90%、95%、或100%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)、本揭露之輕鏈可變域(例如與
表 3中所示之輕鏈可變域具有至少75%序列同一性,例如至少80%、85%、90%、95%、或100%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)、及連接子。
若提供一種結合模體之胺基酸或核苷酸序列,其包含本揭露之重鏈可變域及本揭露之輕鏈可變域,則連接兩個可變域之連接子鑑於本揭露自序列將係顯而易見的且已知於所屬技術領域中。若提供一種結合模體之胺基酸或核苷酸序列,其包含本揭露之重鏈可變域及本揭露之輕鏈可變域,則前導序列鑑於本揭露將係顯而易見的且已知於所屬技術領域中。為避免疑問,本揭露之重鏈可變域及輕鏈可變域可以任何定向存在,例如其中重鏈可變域係輕鏈可變域之C端之定向,或其中重鏈可變域係輕鏈可變域之N端之定向。例示性抗CD20結合模體顯示於
表 4 中。 表
4. 例示性抗
CD20 結合模體序列
名稱 | 序列 |
(Ab1) | SEQ ID NO: 31 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCTATTACTGGAGCTGGATCCGGCAGCCTCCTGGAAAAGGATTAGAATGGATCGGCGAGATAGACCACAGCGGGAGCACAAACTACAACCCCAGCCTGAAATCGCGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGGCGGAGGCTCCTGGTACAGCAACTGGTTCGATCCTTGGGGCCAAGGCACCATGGTGACCGTTTCCAGCGGCTCTACAAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCAGCACAAAGGGCGACATCCAGATGACACAGAGCCCCAGCACCCTTAGCGCCTCTGTGGGAGATAGGGTTACCATTACCTGCAGGGCTTCCCAGAGCATCAGCAGCTGGCTGGCATGGTATCAACAGAAGCCTGGCAAGGCTCCCAAGCTGCTCATCTATGACGCCTCCAGCCTGGAAAGCGGGGTTCCCTCCAGATTTAGCGGCTCAGGCTCCGGAACAGAGTTCACCCTTACCATCTCTAGCCTGCAACCCGACGACTTCGCTACTTATTACTGTCAACAAGACAGAAGCTTGCCCCCCACATTCGGCGGAGGGACCAAGGTTGAGATCAAG |
(Ab2) | SEQ ID NO:32 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCATCCACTGGAACTGGATCCGGCAGCCTCCTGGCAAAGGCCTTGAATGGATCGGCGATATCGACACCAGCGGCTCCACCAACTACAACCCCAGCCTGAAATCGAGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGACTGGGCCAGGAAAGCGCTACCTACCTTGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACTGTTAGCTCTGGCTCAACAAGCGGCAGCGGCAAGCCTGGCTCAGGAGAAGGAAGCACAAAGGGCGACATTGTAATGACTCAGAGCCCCGACAGCCTGGCCGTTAGCTTAGGCGAAAGGGCTACAATCAATTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTCGCATGGTATCAACAGAAGCCAGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCTTCCACCAGAGAGAGCGGGGTTCCCGATAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACGCTCACAATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGTTGTACACCTACCCCTTCACATTCGGCGGAGGCACCAAGGTTGAGATCAAG |
(Ab3) | SEQ ID NO:33 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGCTTCAGCTCCAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCAGCTCCAGCTATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTAGAATGGATCGGCTCGATATATTACTCCGGCAGCACCTACTATAACCCCAGCTTGAAGAGCCGGGTTACCATTTCTGTGGACACATCAAAGAACCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGCATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGTTCCACCAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGATATACAGATGACACAGAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATCGTGTAACGATCACCTGCCGGGCCTCTCAGAGCATCAACTCCTACCTCAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAATTACTCATCTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACCCTCACTATCTCCAGCTTGCAGCCCGAGGATTTCGCCACTTATTACTGTCAGCAGAGCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAG |
(Ab4) | SEQ ID NO:34 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGCTTGTGCAGAGCGGAGCTGAAGTTAAGAAGCCTGGCGCCTCTGTGAAGGTTAGCTGCAAGGCCAGCGGCTACACATTCAAGGAATATGGCATCTCCTGGGTTAGGCAGGCTCCCGGCCAAGGCTTAGAATGGATGGGCTGGATCTCCGCCTACTCCGGCCACACCTACTACGCCCAGAAGCTTCAGGGCAGGGTTACCATGACCACCGACACCAGCACCTCTACCGCCTATATGGAGCTGAGGAGCCTGAGATCGGACGACACAGCTGTGTATTACTGCGCCAGAGGCCCCCACTACGACGACTGGTCTGGATTTATCATCTGGTTCGACCCCTGGGGGCAGGGCACCCTGGTCACAGTTTCTTCTGGCTCCACCAGCGGAAGCGGCAAGCCAGGCTCAGGCGAAGGATCTACAAAAGGCGACATCCAAATGACACAGAGCCCCAGCAGCTTGAGCGCCTCCGTTGGCGACAGAGTTACAATCACCTGCAGGGCCTCTCAGAGCATCAGCAGCTATTTGAATTGGTATCAACAGAAGCCAGGAAAGGCCCCTAAGCTGCTCATCTACGCTGCCAGCTCGCTCCAATCTGGCGTTCCTAGCAGATTTAGCGGCTCCGGCAGCGGCACAGACTTTACTCTTACCATTAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTATTGCCAGCAAAGCTACAGATTCCCTCCCACCTTTGGCCAGGGCACAAAGGTTGAGATCAAG |
(Ab5) | SEQ ID NO:35 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCTCTCCCGACCATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTGGAATGGATCGGCAGCATCTACGCCAGCGGCAGCACATTCTACAACCCCTCGCTCAAAAGCAGGGTTACTATTTCTGTGGACACAAGCAAAAATCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGGATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGCTCCACAAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGACATTCAAATGACCCAAAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATAGGGTTACCATTACCTGCAGGGCCAGCCAAAGCATCAACTCCTACCTAAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAACTACTCATTTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCAGCGGTTCTGGAACAGATTTCACTCTCACAATATCTTCGCTGCAGCCCGAGGATTTCGCTACCTACTATTGCCAGCAATCCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAG |
(Ab6) | SEQ ID NO:36 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGATCACATTAAAAGAGAGCGGACCTACACTGGTGAAGCCCACCCAAACGCTTACCCTCACCTGCACCTTTAGCGGGTTCAGCCTGGACACAGAGGGCGTTGGCGTTGGATGGATCAGGCAGCCTCCTGGCAAAGCCCTCGAATGGCTTGCCCTCATCTACTTCAACGACCAGAAGAGATACAGCCCCTCCTTAAAATCTCGGCTCACAATCACCAAAGACACAAGCAAAAATCAGGTTGTGCTCACCATGACCAACATGGACCCTGTGGACACCGCTGTGTACTACTGTGCCAGAGACACCGGCTACAGCAGATGGTACTACGGGATGGACGTTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGCTCTACAAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCAGCACAAAGGGCGACATCCAGATGACGCAATCCCCCAGCTCTGTGAGCGCCTCTGTGGGAGACAGAGTTACAATCACATGCCGGGCCTCCCAGGGCATCAGCTCTTGGCTGGCATGGTATCAACAGAAGCCTGGCAAGGCTCCCAAGCTGCTCATCTATGCCGCCTCCTCCTTACAATCTGGAGTTCCCTCCAGGTTCAGCGGGAGCGGCTCAGGAACAGACTTCACCCTTACCATCTCTAGCCTGCAACCCGAGGACTTCGCTACTTATTACTGTCAGCAGGCCTACGCCTACCCCATCACATTCGGCGGAGGAACAAAGGTTGAGATCAAG |
(Ab7) | SEQ ID NO:37 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCGAGAAATACTACTGGAGCTGGATCCGGCAGCCTCCCGGCAAAGGCTTAGAATGGATCGGCGAGATTTATCACAGCGGGCTCACCAACTACAACCCCAGCCTGAAATCTCGAGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTAGATACGACAGCAGCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACTGTCTCCTCTGGATCTACCAGCGGCAGCGGCAAGCCTGGATCTGGCGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCTGTGTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCTTGGTATCAACAGAAGCCTGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATCGGTTTAGCGGCTCCGGCTCAGGAACCGATTTCACCCTCACTATCTCCAGCCTCCAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAG |
(Ab8) | SEQ ID NO:38 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCCGCTATGTGTGGAGCTGGATCCGGCAGCCTCCTGGCAAAGGCCTTGAATGGATCGGAGAGATAGACAGCAGCGGCAAGACCAACTACAACCCCAGCCTGAAATCACGCGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTAGATACGACAGCTCCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACAGTTAGCTCTGGAAGCACCAGCGGCTCCGGCAAGCCTGGATCTGGTGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCTGTGTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCATGGTATCAACAGAAGCCTGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATCGCTTTAGCGGCAGCGGTTCTGGCACCGATTTCACTCTTACAATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAG |
(Ab9) | SEQ ID NO:39 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCTACGCTTGGAGCTGGATTAGACAGCCTCCTGGCAAAGGACTAGAATGGATCGGAGAGATCGACCACAGAGGCTTCACCAACTACAACCCCAGCCTGAAATCCAGAGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGGGTTAGATACGACAGCAGCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACGGTTAGCTCTGGATCTACCAGCGGCAGCGGCAAGCCTGGCTCAGGAGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCCGTTTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCATGGTATCAACAGAAGCCAGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATAGATTTTCGGGATCAGGCTCCGGCACCGATTTCACTCTTACGATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAG |
(Ab10) | SEQ ID NO:40 ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCCAGAAATACTACTGGAGCTGGATCCGGCAGCCTCCCGGCAAAGGCTTAGAATGGATCGGAGAGATAGACACCAGCGGCTTCACCAACTACAACCCCAGCCTGAAATCTAGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTGGCAGATACAGCTACGGCTACTACATCACCGCCTTCGACATTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGAAGCACTAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCTCAACCAAGGGCGACATCGTGATGACACAGAGCCCCGACTCTCTGGCTGTGTCCCTGGGAGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTGGCATGGTATCAACAGAAGCCTGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCTTCCACCAGAGAATCAGGCGTTCCAGACAGGTTCTCCGGCTCGGGTTCAGGCACAGACTTCACCCTTACCATCTCTTCCCTGCAGGCCGAAGATGTGGCCGTTTACTACTGTCAGCAGCACTACAGCTTCCCTTTCACATTCGGCGGAGGCACCAAGGTTGAGATCAAG |
編碼結合模體、鉸鏈、及共刺激域之例示性核苷酸序列提供於
表 5 中。
表
5 :編碼抗
CD20 結合模體、鉸鏈、及共刺激域之核苷酸序列
SEQ ID NO | 序列 |
41 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGCTTCAGCTCCAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCAGCTCCAGCTATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTAGAATGGATCGGCTCGATATATTACTCCGGCAGCACCTACTATAACCCCAGCTTGAAGAGCCGGGTTACCATTTCTGTGGACACATCAAAGAACCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGCATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGTTCCACCAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGATATACAGATGACACAGAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATCGTGTAACGATCACCTGCCGGGCCTCTCAGAGCATCAACTCCTACCTCAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAATTACTCATCTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACCCTCACTATCTCCAGCTTGCAGCCCGAGGATTTCGCCACTTATTACTGTCAGCAGAGCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
42 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCTCTCCCGACCATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTGGAATGGATCGGCAGCATCTACGCCAGCGGCAGCACATTCTACAACCCCTCGCTCAAAAGCAGGGTTACTATTTCTGTGGACACAAGCAAAAATCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGGATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGCTCCACAAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGACATTCAAATGACCCAAAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATAGGGTTACCATTACCTGCAGGGCCAGCCAAAGCATCAACTCCTACCTAAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAACTACTCATTTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCAGCGGTTCTGGAACAGATTTCACTCTCACAATATCTTCGCTGCAGCCCGAGGATTTCGCTACCTACTATTGCCAGCAATCCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
43 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGATCACATTAAAAGAGAGCGGACCTACACTGGTGAAGCCCACCCAAACGCTTACCCTCACCTGCACCTTTAGCGGGTTCAGCCTGGACACAGAGGGCGTTGGCGTTGGATGGATCAGGCAGCCTCCTGGCAAAGCCCTCGAATGGCTTGCCCTCATCTACTTCAACGACCAGAAGAGATACAGCCCCTCCTTAAAATCTCGGCTCACAATCACCAAAGACACAAGCAAAAATCAGGTTGTGCTCACCATGACCAACATGGACCCTGTGGACACCGCTGTGTACTACTGTGCCAGAGACACCGGCTACAGCAGATGGTACTACGGGATGGACGTTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGCTCTACAAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCAGCACAAAGGGCGACATCCAGATGACGCAATCCCCCAGCTCTGTGAGCGCCTCTGTGGGAGACAGAGTTACAATCACATGCCGGGCCTCCCAGGGCATCAGCTCTTGGCTGGCATGGTATCAACAGAAGCCTGGCAAGGCTCCCAAGCTGCTCATCTATGCCGCCTCCTCCTTACAATCTGGAGTTCCCTCCAGGTTCAGCGGGAGCGGCTCAGGAACAGACTTCACCCTTACCATCTCTAGCCTGCAACCCGAGGACTTCGCTACTTATTACTGTCAGCAGGCCTACGCCTACCCCATCACATTCGGCGGAGGAACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
44 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCCAGAAATACTACTGGAGCTGGATCCGGCAGCCTCCCGGCAAAGGCTTAGAATGGATCGGAGAGATAGACACCAGCGGCTTCACCAACTACAACCCCAGCCTGAAATCTAGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTGGCAGATACAGCTACGGCTACTACATCACCGCCTTCGACATTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGAAGCACTAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCTCAACCAAGGGCGACATCGTGATGACACAGAGCCCCGACTCTCTGGCTGTGTCCCTGGGAGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTGGCATGGTATCAACAGAAGCCTGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCTTCCACCAGAGAATCAGGCGTTCCAGACAGGTTCTCCGGCTCGGGTTCAGGCACAGACTTCACCCTTACCATCTCTTCCCTGCAGGCCGAAGATGTGGCCGTTTACTACTGTCAGCAGCACTACAGCTTCCCTTTCACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
45 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCGAGAAATACTACTGGAGCTGGATCCGGCAGCCTCCCGGCAAAGGCTTAGAATGGATCGGCGAGATTTATCACAGCGGGCTCACCAACTACAACCCCAGCCTGAAATCTCGAGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTAGATACGACAGCAGCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACTGTCTCCTCTGGATCTACCAGCGGCAGCGGCAAGCCTGGATCTGGCGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCTGTGTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCTTGGTATCAACAGAAGCCTGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATCGGTTTAGCGGCTCCGGCTCAGGAACCGATTTCACCCTCACTATCTCCAGCCTCCAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
46 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCCGCTATGTGTGGAGCTGGATCCGGCAGCCTCCTGGCAAAGGCCTTGAATGGATCGGAGAGATAGACAGCAGCGGCAAGACCAACTACAACCCCAGCCTGAAATCACGCGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTAGATACGACAGCTCCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACAGTTAGCTCTGGAAGCACCAGCGGCTCCGGCAAGCCTGGATCTGGTGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCTGTGTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCATGGTATCAACAGAAGCCTGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATCGCTTTAGCGGCAGCGGTTCTGGCACCGATTTCACTCTTACAATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
47 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCTACGCTTGGAGCTGGATTAGACAGCCTCCTGGCAAAGGACTAGAATGGATCGGAGAGATCGACCACAGAGGCTTCACCAACTACAACCCCAGCCTGAAATCCAGAGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGGGTTAGATACGACAGCAGCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACGGTTAGCTCTGGATCTACCAGCGGCAGCGGCAAGCCTGGCTCAGGAGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCCGTTTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCATGGTATCAACAGAAGCCAGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATAGATTTTCGGGATCAGGCTCCGGCACCGATTTCACTCTTACGATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
48 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCTATTACTGGAGCTGGATCCGGCAGCCTCCTGGAAAAGGATTAGAATGGATCGGCGAGATAGACCACAGCGGGAGCACAAACTACAACCCCAGCCTGAAATCGCGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGGCGGAGGCTCCTGGTACAGCAACTGGTTCGATCCTTGGGGCCAAGGCACCATGGTGACCGTTTCCAGCGGCTCTACAAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCAGCACAAAGGGCGACATCCAGATGACACAGAGCCCCAGCACCCTTAGCGCCTCTGTGGGAGATAGGGTTACCATTACCTGCAGGGCTTCCCAGAGCATCAGCAGCTGGCTGGCATGGTATCAACAGAAGCCTGGCAAGGCTCCCAAGCTGCTCATCTATGACGCCTCCAGCCTGGAAAGCGGGGTTCCCTCCAGATTTAGCGGCTCAGGCTCCGGAACAGAGTTCACCCTTACCATCTCTAGCCTGCAACCCGACGACTTCGCTACTTATTACTGTCAACAAGACAGAAGCTTGCCCCCCACATTCGGCGGAGGGACCAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
49 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGCTTGTGCAGAGCGGAGCTGAAGTTAAGAAGCCTGGCGCCTCTGTGAAGGTTAGCTGCAAGGCCAGCGGCTACACATTCAAGGAATATGGCATCTCCTGGGTTAGGCAGGCTCCCGGCCAAGGCTTAGAATGGATGGGCTGGATCTCCGCCTACTCCGGCCACACCTACTACGCCCAGAAGCTTCAGGGCAGGGTTACCATGACCACCGACACCAGCACCTCTACCGCCTATATGGAGCTGAGGAGCCTGAGATCGGACGACACAGCTGTGTATTACTGCGCCAGAGGCCCCCACTACGACGACTGGTCTGGATTTATCATCTGGTTCGACCCCTGGGGGCAGGGCACCCTGGTCACAGTTTCTTCTGGCTCCACCAGCGGAAGCGGCAAGCCAGGCTCAGGCGAAGGATCTACAAAAGGCGACATCCAAATGACACAGAGCCCCAGCAGCTTGAGCGCCTCCGTTGGCGACAGAGTTACAATCACCTGCAGGGCCTCTCAGAGCATCAGCAGCTATTTGAATTGGTATCAACAGAAGCCAGGAAAGGCCCCTAAGCTGCTCATCTACGCTGCCAGCTCGCTCCAATCTGGCGTTCCTAGCAGATTTAGCGGCTCCGGCAGCGGCACAGACTTTACTCTTACCATTAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTATTGCCAGCAAAGCTACAGATTCCCTCCCACCTTTGGCCAGGGCACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
50 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCATCCACTGGAACTGGATCCGGCAGCCTCCTGGCAAAGGCCTTGAATGGATCGGCGATATCGACACCAGCGGCTCCACCAACTACAACCCCAGCCTGAAATCGAGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGACTGGGCCAGGAAAGCGCTACCTACCTTGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACTGTTAGCTCTGGCTCAACAAGCGGCAGCGGCAAGCCTGGCTCAGGAGAAGGAAGCACAAAGGGCGACATTGTAATGACTCAGAGCCCCGACAGCCTGGCCGTTAGCTTAGGCGAAAGGGCTACAATCAATTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTCGCATGGTATCAACAGAAGCCAGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCTTCCACCAGAGAGAGCGGGGTTCCCGATAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACGCTCACAATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGTTGTACACCTACCCCTTCACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
51 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGCTTCAGCTCCAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCAGCTCCAGCTATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTAGAATGGATCGGCTCGATATATTACTCCGGCAGCACCTACTATAACCCCAGCTTGAAGAGCCGGGTTACCATTTCTGTGGACACATCAAAGAACCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGCATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGTTCCACCAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGATATACAGATGACACAGAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATCGTGTAACGATCACCTGCCGGGCCTCTCAGAGCATCAACTCCTACCTCAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAATTACTCATCTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACCCTCACTATCTCCAGCTTGCAGCCCGAGGATTTCGCCACTTATTACTGTCAGCAGAGCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
52 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCTCTCCCGACCATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTGGAATGGATCGGCAGCATCTACGCCAGCGGCAGCACATTCTACAACCCCTCGCTCAAAAGCAGGGTTACTATTTCTGTGGACACAAGCAAAAATCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGGATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGCTCCACAAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGACATTCAAATGACCCAAAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATAGGGTTACCATTACCTGCAGGGCCAGCCAAAGCATCAACTCCTACCTAAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAACTACTCATTTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCAGCGGTTCTGGAACAGATTTCACTCTCACAATATCTTCGCTGCAGCCCGAGGATTTCGCTACCTACTATTGCCAGCAATCCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
53 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGATCACATTAAAAGAGAGCGGACCTACACTGGTGAAGCCCACCCAAACGCTTACCCTCACCTGCACCTTTAGCGGGTTCAGCCTGGACACAGAGGGCGTTGGCGTTGGATGGATCAGGCAGCCTCCTGGCAAAGCCCTCGAATGGCTTGCCCTCATCTACTTCAACGACCAGAAGAGATACAGCCCCTCCTTAAAATCTCGGCTCACAATCACCAAAGACACAAGCAAAAATCAGGTTGTGCTCACCATGACCAACATGGACCCTGTGGACACCGCTGTGTACTACTGTGCCAGAGACACCGGCTACAGCAGATGGTACTACGGGATGGACGTTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGCTCTACAAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCAGCACAAAGGGCGACATCCAGATGACGCAATCCCCCAGCTCTGTGAGCGCCTCTGTGGGAGACAGAGTTACAATCACATGCCGGGCCTCCCAGGGCATCAGCTCTTGGCTGGCATGGTATCAACAGAAGCCTGGCAAGGCTCCCAAGCTGCTCATCTATGCCGCCTCCTCCTTACAATCTGGAGTTCCCTCCAGGTTCAGCGGGAGCGGCTCAGGAACAGACTTCACCCTTACCATCTCTAGCCTGCAACCCGAGGACTTCGCTACTTATTACTGTCAGCAGGCCTACGCCTACCCCATCACATTCGGCGGAGGAACAAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
54 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCCAGAAATACTACTGGAGCTGGATCCGGCAGCCTCCCGGCAAAGGCTTAGAATGGATCGGAGAGATAGACACCAGCGGCTTCACCAACTACAACCCCAGCCTGAAATCTAGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTGGCAGATACAGCTACGGCTACTACATCACCGCCTTCGACATTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGAAGCACTAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCTCAACCAAGGGCGACATCGTGATGACACAGAGCCCCGACTCTCTGGCTGTGTCCCTGGGAGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTGGCATGGTATCAACAGAAGCCTGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCTTCCACCAGAGAATCAGGCGTTCCAGACAGGTTCTCCGGCTCGGGTTCAGGCACAGACTTCACCCTTACCATCTCTTCCCTGCAGGCCGAAGATGTGGCCGTTTACTACTGTCAGCAGCACTACAGCTTCCCTTTCACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
55 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCGAGAAATACTACTGGAGCTGGATCCGGCAGCCTCCCGGCAAAGGCTTAGAATGGATCGGCGAGATTTATCACAGCGGGCTCACCAACTACAACCCCAGCCTGAAATCTCGAGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTAGATACGACAGCAGCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACTGTCTCCTCTGGATCTACCAGCGGCAGCGGCAAGCCTGGATCTGGCGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCTGTGTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCTTGGTATCAACAGAAGCCTGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATCGGTTTAGCGGCTCCGGCTCAGGAACCGATTTCACCCTCACTATCTCCAGCCTCCAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
56 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCCGCTATGTGTGGAGCTGGATCCGGCAGCCTCCTGGCAAAGGCCTTGAATGGATCGGAGAGATAGACAGCAGCGGCAAGACCAACTACAACCCCAGCCTGAAATCACGCGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTAGATACGACAGCTCCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACAGTTAGCTCTGGAAGCACCAGCGGCTCCGGCAAGCCTGGATCTGGTGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCTGTGTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCATGGTATCAACAGAAGCCTGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATCGCTTTAGCGGCAGCGGTTCTGGCACCGATTTCACTCTTACAATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
57 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCTACGCTTGGAGCTGGATTAGACAGCCTCCTGGCAAAGGACTAGAATGGATCGGAGAGATCGACCACAGAGGCTTCACCAACTACAACCCCAGCCTGAAATCCAGAGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGGGTTAGATACGACAGCAGCGACAGCTATTACTACAGCTATGACTACGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACGGTTAGCTCTGGATCTACCAGCGGCAGCGGCAAGCCTGGCTCAGGAGAAGGAAGCACAAAGGGCGACATTGTGCTCACCCAGAGCCCCGACAGCCTGGCCGTTTCTTTAGGCGAAAGGGCTACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTTGCATGGTATCAACAGAAGCCAGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCCTCTAGCAGAGAGAGCGGGGTTCCCGATAGATTTTCGGGATCAGGCTCCGGCACCGATTTCACTCTTACGATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGAGCTATAGCTTCCCCTGGACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
58 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCTATTACTGGAGCTGGATCCGGCAGCCTCCTGGAAAAGGATTAGAATGGATCGGCGAGATAGACCACAGCGGGAGCACAAACTACAACCCCAGCCTGAAATCGCGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGGCGGAGGCTCCTGGTACAGCAACTGGTTCGATCCTTGGGGCCAAGGCACCATGGTGACCGTTTCCAGCGGCTCTACAAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCAGCACAAAGGGCGACATCCAGATGACACAGAGCCCCAGCACCCTTAGCGCCTCTGTGGGAGATAGGGTTACCATTACCTGCAGGGCTTCCCAGAGCATCAGCAGCTGGCTGGCATGGTATCAACAGAAGCCTGGCAAGGCTCCCAAGCTGCTCATCTATGACGCCTCCAGCCTGGAAAGCGGGGTTCCCTCCAGATTTAGCGGCTCAGGCTCCGGAACAGAGTTCACCCTTACCATCTCTAGCCTGCAACCCGACGACTTCGCTACTTATTACTGTCAACAAGACAGAAGCTTGCCCCCCACATTCGGCGGAGGGACCAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
59 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGCTTGTGCAGAGCGGAGCTGAAGTTAAGAAGCCTGGCGCCTCTGTGAAGGTTAGCTGCAAGGCCAGCGGCTACACATTCAAGGAATATGGCATCTCCTGGGTTAGGCAGGCTCCCGGCCAAGGCTTAGAATGGATGGGCTGGATCTCCGCCTACTCCGGCCACACCTACTACGCCCAGAAGCTTCAGGGCAGGGTTACCATGACCACCGACACCAGCACCTCTACCGCCTATATGGAGCTGAGGAGCCTGAGATCGGACGACACAGCTGTGTATTACTGCGCCAGAGGCCCCCACTACGACGACTGGTCTGGATTTATCATCTGGTTCGACCCCTGGGGGCAGGGCACCCTGGTCACAGTTTCTTCTGGCTCCACCAGCGGAAGCGGCAAGCCAGGCTCAGGCGAAGGATCTACAAAAGGCGACATCCAAATGACACAGAGCCCCAGCAGCTTGAGCGCCTCCGTTGGCGACAGAGTTACAATCACCTGCAGGGCCTCTCAGAGCATCAGCAGCTATTTGAATTGGTATCAACAGAAGCCAGGAAAGGCCCCTAAGCTGCTCATCTACGCTGCCAGCTCGCTCCAATCTGGCGTTCCTAGCAGATTTAGCGGCTCCGGCAGCGGCACAGACTTTACTCTTACCATTAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTATTGCCAGCAAAGCTACAGATTCCCTCCCACCTTTGGCCAGGGCACAAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
60 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTGCAGCAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCAGCGGCATCCACTGGAACTGGATCCGGCAGCCTCCTGGCAAAGGCCTTGAATGGATCGGCGATATCGACACCAGCGGCTCCACCAACTACAACCCCAGCCTGAAATCGAGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGACTGGGCCAGGAAAGCGCTACCTACCTTGGCATGGATGTGTGGGGGCAGGGCACCACCGTTACTGTTAGCTCTGGCTCAACAAGCGGCAGCGGCAAGCCTGGCTCAGGAGAAGGAAGCACAAAGGGCGACATTGTAATGACTCAGAGCCCCGACAGCCTGGCCGTTAGCTTAGGCGAAAGGGCTACAATCAATTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTCGCATGGTATCAACAGAAGCCAGGCCAGCCTCCCAAGCTGCTCATCTACTGGGCTTCCACCAGAGAGAGCGGGGTTCCCGATAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACGCTCACAATCAGCAGCTTACAGGCCGAGGATGTGGCTGTCTACTATTGTCAGCAGTTGTACACCTACCCCTTCACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCTGCTGCATTGGATAATGAAAAATCGAACGGCACAATCATTCATGTGAAGGGCAAACACCTGTGTCCCAGCCCCTTGTTCCCAGGACCTAGCAAGCCTTTTTGGGTTCTCGTGGTGGTGGGCGGCGTTCTGGCTTGCTACTCTCTACTTGTAACTGTCGCATTTATTATATTCTGGGTTAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
61 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGCTTCAGCTCCAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCAGCTCCAGCTATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTAGAATGGATCGGCTCGATATATTACTCCGGCAGCACCTACTATAACCCCAGCTTGAAGAGCCGGGTTACCATTTCTGTGGACACATCAAAGAACCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGCATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGTTCCACCAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGATATACAGATGACACAGAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATCGTGTAACGATCACCTGCCGGGCCTCTCAGAGCATCAACTCCTACCTCAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAATTACTCATCTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCTCCGGTTCTGGAACAGATTTCACCCTCACTATCTCCAGCTTGCAGCCCGAGGATTTCGCCACTTATTACTGTCAGCAGAGCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
62 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAAGAGAGCGGACCTGGCTTAGTGAAGCCCAGCGAAACCCTGTCCCTCACCTGCACCGTTTCTGGCGGAAGCATCAGCTCTCCCGACCATTACTGGGGATGGATCAGGCAGCCCCCTGGCAAGGGTTTGGAATGGATCGGCAGCATCTACGCCAGCGGCAGCACATTCTACAACCCCTCGCTCAAAAGCAGGGTTACTATTTCTGTGGACACAAGCAAAAATCAGTTCAGCCTGAAGCTGAGCTCTGTGACTGCCGCCGACACAGCTGTGTACTACTGTGCCAGAGAGACAGACTACTCCAGCGGGATGGGCTACGGCATGGATGTGTGGGGACAAGGAACCACCGTTACTGTGAGCAGCGGCTCCACAAGCGGCTCAGGCAAGCCTGGCTCAGGAGAAGGAAGCACCAAGGGGGACATTCAAATGACCCAAAGCCCCTCCAGCCTGTCCGCCAGCGTTGGCGATAGGGTTACCATTACCTGCAGGGCCAGCCAAAGCATCAACTCCTACCTAAATTGGTATCAACAGAAGCCAGGCAAGGCCCCCAAACTACTCATTTACGCCGCCAGCAGCTTACAGAGCGGGGTTCCCTCTAGATTCTCCGGCAGCGGTTCTGGAACAGATTTCACTCTCACAATATCTTCGCTGCAGCCCGAGGATTTCGCTACCTACTATTGCCAGCAATCCCTGGCCGACCCCTTCACATTCGGCGGAGGCACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
63 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGATCACATTAAAAGAGAGCGGACCTACACTGGTGAAGCCCACCCAAACGCTTACCCTCACCTGCACCTTTAGCGGGTTCAGCCTGGACACAGAGGGCGTTGGCGTTGGATGGATCAGGCAGCCTCCTGGCAAAGCCCTCGAATGGCTTGCCCTCATCTACTTCAACGACCAGAAGAGATACAGCCCCTCCTTAAAATCTCGGCTCACAATCACCAAAGACACAAGCAAAAATCAGGTTGTGCTCACCATGACCAACATGGACCCTGTGGACACCGCTGTGTACTACTGTGCCAGAGACACCGGCTACAGCAGATGGTACTACGGGATGGACGTTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGCTCTACAAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCAGCACAAAGGGCGACATCCAGATGACGCAATCCCCCAGCTCTGTGAGCGCCTCTGTGGGAGACAGAGTTACAATCACATGCCGGGCCTCCCAGGGCATCAGCTCTTGGCTGGCATGGTATCAACAGAAGCCTGGCAAGGCTCCCAAGCTGCTCATCTATGCCGCCTCCTCCTTACAATCTGGAGTTCCCTCCAGGTTCAGCGGGAGCGGCTCAGGAACAGACTTCACCCTTACCATCTCTAGCCTGCAACCCGAGGACTTCGCTACTTATTACTGTCAGCAGGCCTACGCCTACCCCATCACATTCGGCGGAGGAACAAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
64 | ggtaccCCCGGgCCCATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCTCAGGTTCAGTTACAACAATGGGGAGCTGGCCTGTTAAAGCCCAGCGAAACCCTGTCCCTCACCTGCGCTGTGTATGGCGGAAGCTTCCAGAAATACTACTGGAGCTGGATCCGGCAGCCTCCCGGCAAAGGCTTAGAATGGATCGGAGAGATAGACACCAGCGGCTTCACCAACTACAACCCCAGCCTGAAATCTAGGGTTACAATCTCTGTGGACACAAGCAAGAATCAGTTCTCCCTGAAGCTGAGCAGCGTTACTGCCGCCGACACAGCTGTGTACTATTGCGCCAGAGTTGGCAGATACAGCTACGGCTACTACATCACCGCCTTCGACATTTGGGGCCAAGGCACCACTGTGACCGTTTCCAGCGGAAGCACTAGCGGCAGCGGGAAACCTGGTTCTGGAGAGGGCTCAACCAAGGGCGACATCGTGATGACACAGAGCCCCGACTCTCTGGCTGTGTCCCTGGGAGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCGTTCTGTACAGCAGCAACAACAAGAACTACCTGGCATGGTATCAACAGAAGCCTGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCTTCCACCAGAGAATCAGGCGTTCCAGACAGGTTCTCCGGCTCGGGTTCAGGCACAGACTTCACCCTTACCATCTCTTCCCTGCAGGCCGAAGATGTGGCCGTTTACTACTGTCAGCAGCACTACAGCTTCCCTTTCACATTCGGCGGAGGCACCAAGGTTGAGATCAAGGCAGCTGCTTTCGTGCCTGTGTTCCTGCCTGCTAAGCCCACCACCACTCCTGCTCCAAGACCTCCTACCCCCGCTCCTACAATCGCCAGCCAACCTCTGAGCCTGAGACCGGAGGCATGCAGACCTGCGGCAGGGGGAGCAGTTCACACAAGAGGCTTGGACTTCGCTTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGCGGAGTTCTTCTTCTTAGCCTGGTGATCACCCTGTACTGCAACCACAGAAACAGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat |
鉸鏈可衍生自天然來源或合成來源。在一些實施例中,本揭露之抗原結合系統可包含係來自、或衍生自(例如包含所有或片段)CD2、CD3δ、CD3ε、CD3γ、CD4、CD7、CD8.alpha.、CD8.beta.、CD11a (ITGAL)、CD11b (ITGAM)、CD11c (ITGAX)、CD11d (ITGAD)、CD18 (ITGB2)、CD19 (B4)、CD27 (TNFRSF7)、CD28、CD28T、CD29 (ITGB1)、CD30 (TNFRSF8)、CD40 (TNFRSF5)、CD48 (SLAMF2)、CD49a (ITGA1)、CD49d (ITGA4)、CD49f (ITGA6)、CD66a (CEACAM1)、CD66b (CEACAM8)、CD66c (CEACAM6)、CD66d (CEACAM3)、CD66e (CEACAM5)、CD69 (CLEC2)、CD79A(B細胞抗原受體複合物相關α鏈)、CD79B(B細胞抗原受體複合物相關β鏈)、CD84 (SLAMF5)、CD96 (Tactile)、CD100 (SEMA4D)、CD103 (ITGAE)、CD134 (OX40)、CD137 (4–1BB)、CD150 (SLAMF1)、CD158A (KIR2DL1)、CD158B1 (KIR2DL2)、CD158B2 (KIR2DL3)、CD158C (KIR3DP1)、CD158D (KIRDL4)、CD158F1 (KIR2DL5A)、CD158F2 (KIR2DL5B)、CD158K (KIR3DL2)、CD160 (BY55)、CD162 (SELPLG)、CD226 (DNAM1)、CD229 (SLAMF3)、CD244 (SLAMF4)、CD247 (CD3–ζ)、CD258 (LIGHT)、CD268 (BAFFR)、CD270 (TNFSF14)、CD272 (BTLA)、CD276 (B7–H3)、CD279 (PD–1)、CD314 (NKG2D)、CD319 (SLAMF7)、CD335 (NK–p46)、CD336 (NK–p44)、CD337 (NK–p30)、CD352 (SLAMF6)、CD353 (SLAMF8)、CD355 (CRTAM)、CD357 (TNFRSF18)、可誘導型T細胞共刺激劑(ICOS)、LFA–1 (CD11a/CD18)、NKG2C、DAP–10、ICAM–1、NKp80 (KLRF1)、IL–2Rβ、IL–2Rγ、IL–7Rα、LFA1–1、SLAMF9、LAT、GADS (GrpL)、SLP-76 (LCP2)、PAG1/CBP、a CD83配位體、Fcγ受體、MHC 1類分子、MHC 2類分子、TNF受體蛋白、免疫球蛋白蛋白質、細胞介素受體、整合素、活化NK細胞受體、或Toll配位體受體、或其片段或組合。在某些實施例中,CAR不包含CD28鉸鏈。
跨膜域可衍生自天然或合成來源。在來源係天然之情況下,域可衍生自任何膜結合或跨膜蛋白。例示性跨膜域可衍生自(例如可包含至少一跨膜域)T細胞受體之α、β、或ζ鏈、CD28、CD3ε、CD3δ、CD3γ、CD45、CD4、CD5、CD7、CD8、CD8α、CD8β、CD9、CD11a、CD11b、CD11c、CD11d、CD16、CD22、CD27、CD33、CD37、CD64、CD80、CD86、CD134、CD137、TNFSFR25、CD154、4-1BB/CD137、活化NK細胞受體、免疫球蛋白、B7-H3、BAFFR、BLAME (SLAMF8)、BTLA、CD100 (SEMA4D)、CD103、CD160 (BY55)、CD18、CD19、CD19a、CD2、CD247、CD276 (B7-H3)、CD29、CD30、CD40、CD49a、CD49D、CD49f、CD69、CD84、CD96 (Tactile)、CDS、CEACAM1、CRT AM、細胞介素受體、DAP-10、DNAM1 (CD226)、Fcγ受體、GADS、GITR、HVEM(LIGHTR)、IA4、ICAM-1、ICAM-1、Igα (CD79a)、IL-2Rβ、IL-2Rγ、IL-7Rα、可誘導型T細胞共刺激劑(ICOS)、整合素、ITGA4、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB2、ITGB7、ITGB1、KIRDS2、LAT、LFA-1、LFA-1、與CD83、LIGHT、LIGHT、LTBR、Ly9 (CD229)結合之配位體、淋巴球功能相關抗原1 (LFA-1; CD1-1a/CD18)、MHC第1型分子、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80 (KLRF1)、OX-40、PAG/Cbp、程式性死亡1 (PD-1)、PSGL1、SELPLG (CD162)、信號傳導淋巴球性活化分子(SLAM蛋白)、SLAM (SLAMF1; CD150; IPO-3)、SLAMF4 (CD244; 2B4)、SLAMF6 (NTB-A; Ly108)、SLAMF7、SLP-76、TNF受體蛋白、TNFR2、TNFSF14、鐸配體受體、TRANCE/RANKL、VLA1、或VLA-6、或其片段、截短、或組合。在一些實施例中,跨膜域可為合成(且可例如包含主要疏水性殘基,諸如白胺酸及纈胺酸)。在一些實施例中,苯丙胺酸之三聯體、色胺酸、及纈胺酸包含在合成跨膜域之各端處。在一些實施例中,跨膜域直接鏈接或連接至細胞質域。在一些實施例中,短寡肽或多肽連接子(例如在長度之2與10個胺基酸之間)可形成跨膜域與胞內域之間的連接。在一些實施例中,連接子係甘胺酸-絲胺酸雙聯體。
在一些實施例中,信號傳導域及/或活化域包含基於免疫受體酪胺酸之活化模體(ITAM)。含ITAM之細胞質信號傳導序列之實例包含衍生自TCRζ、FcRγ、FcRβ、CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、及CD66d者(參見例如Love et al., Cold Spring Harb.Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol.27:591-619 (2009))。
CAR可包含共刺激信號傳導域,例如以增加信號傳導效力。參見美國專利第7,741,465、及6,319,494號,以及Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012);Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol.Toxicol.56:59-83 (2016)。透過單獨TCR產生的信號可能不足以完全活化T細胞,且二級或共刺激信號可增加活化。因此,在一些實施例中,信號傳導域進一步包含活化一或多種免疫細胞效應功能(例如本文所述之天然免疫細胞效應功能)的一或多個額外信號傳導域(例如共刺激信號傳導域)。在一些實施例中,可使用此類共刺激信號傳導域之一部分,只要該部分轉導效應功能信號即可。在一些實施例中,本文所述之細胞質域包含T細胞輔助受體(或其片段)之一或多個細胞質序列。此類T細胞共受體之非限制性實例包含CD27、CD28、4-1BB (CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴球功能相關抗原1 (LFA-1)、MYD88、CD2、CD7、LIGHT、NKG2C、B7-H3、及與CD83結合之配體。
在某些實施例中,本文所涵蓋之CAR可包含各種域之間的連接子殘基,例如在VH及VL域之間,添加用於分子之適當間隔構形。本文所涵蓋之CAR可包含一、兩個、三個、四個、或五個或更多個連接子。在一些實施例中,連接子之長度係約1至約25個胺基酸、約5至約20個胺基酸、或約10至約20個胺基酸、或任何中介長度之胺基酸。在一些實施例中,連接子係1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或更多個胺基酸長。
在一些實施例中,本文所涵蓋之CAR包含胞內信號傳導域。「胞內信號傳導域」係指CAR之部分,其參與將有效CAR結合至目標抗原之訊息轉導至免疫效應細胞內部以引發效應細胞功能,例如活化、細胞介素產生、增生、及細胞毒性活性,包括細胞毒性因子至結合CAR之目標細胞的釋放、或與胞外CAR域結合之抗原引發的其他細胞反應。在一些實施例中,信號傳導域及/或活化域包含基於免疫受體酪胺酸之活化模體(ITAM)。含ITAM之細胞質信號傳導序列之實例包含衍生自TCRζ、FcRγ、FcRβ、CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、及CD66d者(參見例如Love et al., Cold Spring Harb.Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol.27:591-619 (2009))。在某些實施例中,合適的信號傳導域包含但不限於4-1BB/CD137、活化NK細胞受體、免疫球蛋白蛋白質、B7-H3、BAFFR、BLAME (SLAMF8)、BTLA、CD100 (SEMA4D)、CD103、CD160 (BY55)、CD18、CD19、CD19a、CD2、CD247、CD27、CD276 (B7-H3)、CD28、CD29、CD3δ、CD3ε、CD3γ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8α、CD8β、CD96 (Tactile)、CD11a、CD11b、CD11c、CD11d、CDS、CEACAM1、CRT AM、細胞介素受體、DAP-10、DNAM1 (CD226)、Fcγ受體、GADS、GITR、HVEM (LIGHTR)、IA4、ICAM-1、ICAM-1、Igα (CD79a)、IL-2Rβ、IL-2Rγ、IL-7Rα、可誘導型T細胞共刺激劑(ICOS)、整合素、ITGA4、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB2、ITGB7、ITGB1、KIRDS2、LAT、LFA-1、LFA-1、與CD83結合之配體、LIGHT、LIGHT、LTBR、Ly9 (CD229)、Ly108)、淋巴球功能相關抗原1 (LFA-1; CD1-1a/CD18)、MHC第1型分子、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80 (KLRF1)、OX-40、PAG/Cbp、程式性死亡1 (PD-1)、PSGL1、SELPLG (CD162)、信號傳導淋巴球性活化分子(SLAM蛋白)、SLAM (SLAMF1; CD150; IPO-3)、SLAMF4 (CD244; 2B4)、SLAMF6 (NTB-A)、SLAMF7、SLP-76、TNF受體蛋白、TNFR2、TNFSF14、鐸配體受體、TRANCE/RANKL、VLA1、或VLA-6、或其片段、截短、或組合。
CAR之組分可使用生物技術用於等效組分之常規技術交換或「對換(swapped)」。在各種實施例中,本揭露提供根據SEQ ID NO: 31至40中任一項之結合模體或與SEQ ID NO: 31至40中任一項具有至少75%同一性之序列(例如至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)組合(例如,相鄰地融合至)鉸鏈,可選地進一步組合(例如,相鄰地融合至)共刺激域。其若干非限制性實例提供於SEQ ID NO: 42至64或與SEQ ID NO: 42至64中任一項具有至少75%同一性之序列(例如至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)。
雙順反子CAR可包含第一CAR序列及第二CAR序列,其表現為單一多肽,包含第一及第二CAR之間的可裂解連接子。可裂解連接子之實例包括但不限於弗林蛋白酶-GSG-T2A(參見例如Chng等人. MAbs. 2015 Mar-Apr; 7(2): 403–412,其關於可裂解連接子以引用方式併入本文中;亦參見Guedan等人. Mol Ther Methods Clin Dev. 2019 Mar 15; 12: 145–156,其關於雙順反子CAR設計子以引用方式併入本文中)、2A連接子(例如T2A)、類2A連接子或其功能等效物、及其組合。在一些實施例中,連接子包括甲吡啶(picornaviral)類2A連接子、豬鐵士古病毒(teschovirus) (P2A)、病毒(T2A)之CHYSEL序列或其組合、變異體、及功能等效物。
例示性抗CD20/抗CD19雙順反子CAR可具有或包含各別在SEQ ID NO: 28及26中所闡述之核苷酸及胺基酸序列,或與SEQ ID NO: 28及26具有至少75%同一性之序列(例如至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)。
雙特異性CAR可係包含第一結合模體及第二結合模體之單一多肽。例示性抗CD20/抗CD19雙特異性CAR可具有或包含各別在SEQ ID NO: 29及27中所闡述之核苷酸及胺基酸序列,或與SEQ ID NO: 29及27具有至少75%同一性之序列(例如至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、或至少99%同一性;例如85至90%、85至95%、85至100%、90至95%、90至100%、或95至100%)。
本揭露尤其提供結合CD20及第二目標抗原(例如CD19)之雙特異性抗體。雙特異性抗體包含具有結合第一目標抗原之第一結合模體及結合第二目標抗原之第二結合模體的抗體。在一些實施例中,雙特異性抗體包含本揭露之抗CD20結合模體及本揭露之抗CD19結合模體。在一些實施例中,雙特異性抗體包含抗CD20結合模體,其包含本揭露之抗CD20重鏈可變域及本揭露之抗CD20輕鏈可變域;以及抗CD19結合模體,其包含抗CD19重鏈可變域及抗CD19輕鏈可變域。
本揭露包含編碼本文所提供之抗CD20結合模體及/或抗CD19結合模體之核酸。本揭露包含編碼抗體之核酸,其包含但不限於編碼結合模體(例如,抗CD20結合模體及抗CD19結合模體)之核酸。本揭露包含編碼本文所提供之抗原結合系統之核酸,其包含但不限於編碼雙順反子與雙特異性嵌合抗原受體(例如,結合CD20及CD19之雙順反子及雙特異性嵌合抗原受體)之核酸。
本揭露包含載體,其包含本揭露之核酸及/或編碼本揭露之多肽。在各種實施例中,本揭露包含載體,其包含編碼本文所提供之抗CD20結合模體及/或抗CD19結合模體之核酸。在各種實施例中,本揭露包含載體,其包含編碼本文所提供之抗體之核酸,包含但不限於編碼結合模體分子(例如,抗CD20結合模體或抗CD19結合模體)之核酸。在各種實施例中,本揭露包含載體,其包含編碼本文所提供之一或多種抗原結合系統之核酸,包含但不限於編碼雙順反子或雙特異性嵌合抗原受體(例如,結合CD20及CD19之雙順反子及雙特異性嵌合抗原受體)之核酸。
在某些實施例中,本揭露之CAR結合CLL-1。CLL-1 CAR胺基酸序列實例提供於
表 6中。
表
6 :例示性
CLL-1 CAR 胺基酸序列
CLL-1 CAR | 序列 |
CLL-1 CAR 1 | QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 65) |
CLL-1 CAR 2 | QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (SEQ ID NO. 66) |
CLL-1 CAR 3 | QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (SEQ ID NO:67) |
CLL-1 CAR 4 | DIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:68) |
CLL-1 CAR 5 | DIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 69) |
CLL-1 CAR 6 | DIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTADTAVYYCVSLVYCGGDCYSGFDYWGQGTLVTVSSAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 70) |
CLL-1 CAR 7 | QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGFYWSWIRQHPGKGLEWIGYIHHSGSTHYNPSLKSRVTISIDTSKNLFSLRLSSVTAADTAVYYCASLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSR FSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 71) |
CLL-1 CAR 8 | QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGFYWSWIRQHPGKGLEWIGYIHHSGSTHYNPSLKSRVTISIDTSKNLFSLRLSSVTAADTAVYYCASLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 72) |
CLL-1 CAR 9 | QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGFYWSWIRQHPGKGLEWIGYIHHSGSTHYNPSLKSRVTISIDTSKNLFSLRLSSVTAADTAVYYCASLVYCGGDCYSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSFTCQASQDINNFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPFTFGGGTKVEIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:73) |
CLL-1 CAR 10 | QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTVTEDTSTDTAYMELSSLRSEDTAVYYCATESRGIGWPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLISGASSLKSGVPSRFSGSGSGTDFTLTISSLPPEDFATYYCQQSYSTPITFGQGTRLEIKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:74) |
CLL-1 CAR 11 | QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTVTEDTSTDTAYMELSSLRSEDTAVYYCATESRGIGWPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLISGASSLKSGVPSRFSGSGSGTDFTLTISSLPPEDFATYYCQQSYSTPITFGQGTRLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:75) |
CLL-1 CAR 12 | QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTVTEDTSTDTAYMELSSLRSEDTAVYYCATESRGIGWPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLISGASSLKSGVPSRFSGSGSGTDFTLTISSLPPEDFATYYCQQSYSTPITFGQGTRLEIKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:76) |
CLL-1 CAR 13 | QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:78) |
CLL-1 CAR 14 | QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:79) |
CLL-1 CAR 15 | QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:80) |
CLL-1 CAR 16 | EIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSAAALDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:81) |
CLL-1 CAR 17 | EIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:82) |
CLL-1 CAR 18 | EIVMTQSPATLSVSPGERATLSCRASQSVSSLLTWYQQKPGQAPRLLIFGASTRATGIPARFSGSGSGTGFTLTISSLQSEDFAVYYCQQYDTWPFTFGPGTKVDFKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYVDSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCARERYSGRDYWGQGTLVTVSSAAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:83) |
在一些實施例中,本揭露之CAR包含與SEQ ID NO: 65至76或78至83中任一項之胺基酸序列具有至少75%同一性、至少80%同一性、至少85%同一性、至少90%同一性、至少95%同一性、至少96%同一性、至少97%同一性、至少98%同一性、至少99%同一性、或100%同一性的胺基酸序列。
在一些實施例中,本揭露之CAR或TCR可進一步包含前導序列或肽(在本文中亦稱為「信號肽」)。在某些實施例中,前導肽包含與胺基酸序列MALPVTALLLPLALLLHAARP (SEQ ID NO: 96)至少約75%、至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%、或100%同一性的胺基酸序列。在一些實施例中,前導肽包含SEQ ID NO: 96之胺基酸序列。
用語「效應功能(effector function)」係指細胞之特定功能。T細胞之效應功能,例如可係細胞溶解活性或包括細胞介素之分泌之幫助或活性。因此,用語「胞內信號傳導域」係指蛋白質之部分,其轉導效應功能信號且引導細胞以執行特定功能。雖然通常可採用整個胞內信號傳導域,但在許多情況下,不必使用整個域。在使用胞內信號傳導域之截短部分的程度,只要其轉導效應功能信號,則可使用此類截短部分代替整個域。用語胞內信號傳導域意欲包括足以轉導效應功能信號之胞內信號傳導域之任何截短部分。
在一些實施例中,細胞可經工程改造以表現外源性T細胞受體(TCR)。可針對TCR庫對目標抗原之選擇性來篩選TCR庫。以此方式,可選擇、選殖對目標抗原具有高結合性及反應性之天然TCR,隨後將其引入用於過繼免疫療法之T細胞群中。
在本文所述之一個實施例中,藉由引入編碼TCR次單元之多核苷酸來修飾T細胞,該多核苷酸可形成賦予T細胞對表現目標抗原之腫瘤細胞具有特異性的TCR。在一些實施例中,相較於天然存在之次單元,次單元具有一或多個胺基酸取代、缺失、插入、或修飾,只要次單元保留形成TCR之能力,TCR賦予轉染之T細胞歸巢至目標細胞的能力,並參與免疫學相關細胞介素信號傳導。TCR亦可以高結合性結合顯示相關腫瘤相關肽之目標細胞,且可選地在體內介導對呈現相關肽之目標細胞的有效殺滅。
編碼TCR之核酸可自其等在T細胞之(天然存在)染色體中之天然環境中分離出來,且可併入如本文其他地方所述之適合載體中。核酸及包含其等之載體皆可轉移至細胞中,該細胞可係T細胞。接著經修飾之T細胞能夠表現由經轉導之核酸或核酸編碼之一或多條TCR鏈(且在一些態樣中為兩條鏈)。在一些實施例中,TCR係外源性TCR,因為其被引入到通常不表現所引入TCR之T細胞中。TCR之一態樣是其對於由主要組織相容性基因複合物(MHC)或類似免疫組分所呈現之腫瘤抗原具有高結合性。與TCR相比,CAR係經工程改造而以MHC非依賴性之方式結合目標抗原。
由本文所述之核酸編碼之蛋白質可表現為具有附接至TCR之α鏈或β鏈之胺基末端或羧基末端部分的額外多肽,只要所附接之額外多肽不會干擾α鏈或β鏈形成功能性T細胞受體及MHC依賴性抗原識別之能力。
由本文所涵蓋之TCR所識別的抗原包括但不限於癌症抗原,包括在血液科癌症與實體腫瘤兩者及病毒誘導性癌症上之抗原。其他說明性抗原包括但不限於HPV致癌蛋白,包括HPV–16 E6與HPV–16 E7、α葉酸受體、5T4、α
vβ
6整合素、BCMA、TACI、B7–H3、B7–H6、CAIX、CD19、CD20、CD22、CD28、CD30、CD33、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD137 (4–1BB)、CD138、CD171、CEA、CSPG4、CLL-1、EGFR、EGFR家族包括ErbB2 (HERII)、EGFRvIII、EGP2、EGP40、EPCAM、EphA2、EpCAM、FAP、胎兒AchR、FRa、GD2、GD3、磷脂醯肌醇蛋白聚糖3 (GPC3)、HLA–Al+MAGEI、HLA–A2 + MAGE1、HLAA3 + MAGE1、HLA–Al + NY–ES0–1、HLA–A2 + NY–ES0–1、HLA–A3 + NY–ES0–1、IL–11Rα、IL–13Rα2、λ、路易士–Y、κ、間皮素、Mucl、Muc16、NCAM、NKG2D配體、NY-ES0–1、PRAME、PSCA、PSMA、RORI、SSX、生存素、TAG72、TEM、及VEGFRII。
在一些實施例中,在細胞經工程改造以降低MHC I類及/或II類表現或活性之後,將編碼CAR或TCR之多核苷酸引入細胞。在一些實施例中,在細胞經工程改造以降低MHC I類及/或II類表現或活性之前,將編碼CAR或TCR之多核苷酸引入細胞。在一些實施例中,兩輪工程改造至少間隔一天進行(不在同一天或24小時內)。
治療
本揭露之細胞,例如同種異體細胞,可用於治療各種疾病及病況,特別是癌症。在一個實施例中,癌症可包含威爾姆氏腫瘤、尤文氏肉瘤、神經內分泌腫瘤、膠質母細胞瘤、神經母細胞瘤、黑色素瘤、皮膚癌、乳癌、結腸癌、直腸癌、前列腺癌、肝癌、腎癌、胰臟癌、肺癌、膽道癌、子宮頸癌、子宮內膜癌、食道癌、胃癌、頭頸癌、甲狀腺髓樣癌、卵巢癌、神經膠質瘤、淋巴瘤、白血病、骨髓瘤、急性淋巴胚細胞白血病、急性骨髓性白血病、慢性淋巴球性白血病、慢性骨髓性白血病、霍奇金氏淋巴瘤、非霍奇金氏淋巴瘤、及膀胱癌。
在一些實施例中,本揭露之細胞可用於治療骨髓性疾病,包括但不限於急性骨髓性白血病(AML)、慢性骨髓性白血病(CML)、慢性粒單核細胞白血病(CMML)、幼年型粒單核細胞白血病、非典型慢性骨髓性白血病、急性前骨髓細胞白血病(APL)、急性單核細胞白血病、急性紅細胞白血病、急性巨核細胞白血病、骨髓增生不良症候群(MDS)、骨髓增生性病症、髓樣腫瘤、髓樣肉瘤)、母細胞性漿細胞樣樹突細胞腫瘤(blastic plasmacytoid dendritic cell neoplasm) (BPDCN)、或其組合。額外疾病包括發炎及/或自體免疫疾病,諸如類風濕性關節炎、牛皮癬、過敏、哮喘、克隆氏症、IBD、IBS、纖維肌增生症、肥大細胞增多症、狼瘡、及腹腔疾病。
在一些實施例中,本揭露之細胞可用於治療由B細胞引起的癌症,例如B細胞淋巴瘤。在一些實施例中,本揭露之細胞可用於治療非特指型之瀰漫性大B細胞淋巴瘤(DLBCL)、原發性縱膈腔大B細胞淋巴瘤、高級別B細胞淋巴瘤、或由濾泡淋巴瘤引起之DLBCL。
本文所述之另一實施例係一種治療有需要之對象之癌症的方法,其包含投予有效量,例如治療有效量之包含本揭露之細胞的組成物。投予之量及頻率將藉由患者之病況及患者之疾病之類型及嚴重程度等因素來判定,但適當的劑量可藉由臨床試驗判定。在一些實施例中,癌症之特徵在於表現由CAR或TCR分子靶向之抗原,諸如CD19及/或CD20。
在一些實施例中,癌症之特徵在於表現由CAR或TCR分子靶向之抗原,諸如CLL-1。
在其他實施例中,提供一種方法,其包含向有需要之患者投予治療有效量的本文所涵蓋之經修飾之T細胞或包含其之組成物,單獨或與一或多種治療劑組合。在某些實施例中,本揭露之細胞用於治療有患癌風險之患者。因此,本揭露提供治療或預防癌症之方法,其包含向有需要之對象投予治療有效量之本揭露之經修飾之T細胞。
將理解的是,CAR
+/CAR-T
+/TCR
+細胞之目標劑量可在約1×10
6至約2×10
10細胞/kg之範圍內(例如,約1×10
6細胞/kg、約2×10
6細胞/kg、約3×10
6細胞/kg、約4×10
6細胞/kg、約5×10
6細胞/kg、約6×10
6細胞/kg、約7×10
6細胞/kg、約8×10
6細胞/kg、約9×10
6細胞/kg、約1×10
7細胞/kg、約2×10
7細胞/kg、約3×10
7細胞/kg、約4×10
7細胞/kg、約5×10
7細胞/kg、約6×10
7細胞/kg、約7×10
7細胞/kg、約8×10
7細胞/kg、約9×10
7細胞/kg、約1×10
8細胞/kg、約2×10
8細胞/kg、約3×10
8細胞/kg、約4×10
8細胞/kg、約5×10
8細胞/kg、約6×10
8細胞/kg、約7×10
8細胞/kg、約8×10
8細胞/kg、約9×10
8細胞/kg、約1×10
9細胞/kg、約2×10
9細胞/kg、約3×10
9細胞/kg、約4×10
9細胞/kg、約5×10
9細胞/kg、約6×10
9細胞/kg、約7×10
9細胞/kg、約8×10
9細胞/kg、約9×10
9細胞/kg、1×10
10細胞/kg、或約2×10
10細胞/kg)。將理解的是,高於及低於此範圍之劑量可能適於某些對象。
所屬技術領域中具有通常知識者將認識到,可能需要多次投予本揭露之組成物,以影響所欲療法。舉例而言,組成物可在1週、2週、3週、1個月、2個月、3個月、4個月、5個月、6個月、1年、2年、5年、10年、或更久內投予1次、2次、3次、4次、5次、6次、7次、8次、9次、或10次、或更多次。
在一個實施例中,向有需要之對象投予有效量之組成物以增加對象對癌症之細胞免疫反應。免疫反應可包括藉由能夠殺死感染細胞、調控T細胞、及輔助T細胞反應之細胞毒性T細胞介導之細胞免疫反應。主要由能夠活化B細胞從而導致抗體產生之輔助T細胞介導之體液免疫反應亦可被誘導。各種技術可用於分析由本揭露之組成物所誘導之免疫反應的類型,該等技術在所屬技術領域中熟知;例如,
Current Protocols in Immunology,由下列者編輯:John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.
用於投予本文所述之細胞組成物之方法包括任何方法,其可有效導致再引入經離體基因修飾之免疫效應細胞,其在對象中直接表現TCR或CAR,或再引入免疫效應細胞之經基因修飾之先驅細胞,其在引入對象中時分化成表現TCR或CAR之成熟免疫效應細胞。一種方法包含將周邊血液T細胞用根據本揭露之核酸構築體離體轉導、及將經轉導之細胞返回對象中。
本文所述之另一實施例係使癌細胞與本揭露之經工程改造免疫細胞接觸之方法,其中癌細胞生長被抑制或降低。
儘管前述揭露已藉由說明及實例之方式進行詳細說明以用於清晰理解目的,然而根據本揭露之教示對所屬技術領域中具有通常知識者將顯而易見可在不背離隨附申請專利範圍之精神或範圍內進行某些變化及修改。僅以說明性的方式而非限制性的方式提供以下實例。所屬技術領域中具有通常知識者將容易地識別許多非臨界參數,其可改變或修改以產生類似的結果。
本說明書中所提及之所有出版物、專利、及專利申請案係以引用方式併入本文中,有如特定及個別指示以引用方式併入各個別出版物、專利、及專利申請案。然而,在本文中引用參考文獻不應解讀為承認此類參考文獻對本揭露而言係先前技術。在以引用方式併入之參考文獻中所提供之任何定義及用語不同於本文中所提供之用語及論述之情況下,則以本文之用語及定義為準。本說明書中各處所引用之所有參考文獻之內容係以引用方式明示併入本文中。
實例實例
1.RFX5剔除
CAR T細胞之測試
此實例評估研究級鋅指核酸酶(ZFN)之體外功效,其靶向RFX5(調節因子X5)之缺失及其提供保護免受T細胞及NK細胞介導之排斥反應之能力,用於同種異體(MHC錯配)CAR-T細胞產物。
方法:將健康供體T細胞用CD19/CD20雙順反子CAR(具有慢病毒載體(LLV))轉導,接著對靶向TRAC(T細胞受體α恆定)及RFX5之ZFN進行電穿孔。經編輯及未經編輯之CAR T細胞擴增14天,並在第7天及第10天檢查CAR、TCRαβ、MHC I類、及MHC II類表現之表現。大部分細胞在第10天被冷凍,用於所有功能性及等位反應性檢定,而小部分細胞進行至第14天以確認細胞活力,且在整個製造中在類似於未經編輯之細胞之經編輯細胞條件下維持擴增。CAR官能性藉由CAR-T細胞與CD19/CD20陽性腫瘤目標細胞系(Raji WT),在各種E:T比率下之細胞毒性(24及48小時)、增殖(120小時)、及細胞介素分泌(24小時)之共培養來評估。CAR-T持久性及細胞毒性功能藉由每3至4天以1:1 E:T比率用CD19/CD20陽性Raji MHC KO腫瘤細胞連續刺激CAR-T細胞來評估。另外,亦測量MHC錯配之CD8 T細胞及NK細胞對經編輯及未經編輯之CAR T細胞之殺滅。
結果:RFX5 KO CAR-T細胞具有與β2M KO CAR-T細胞類似的可製造性及CAR官能性。如
表 7A 至7C所示,RFX5剔除(KO)同種異體(Allo) CAR-T細胞具有良好的活力及與B2M KO Allo CAR-T細胞類似之擴增。
表
7A. 原始細胞計數
(x10
6)
表
7B. 倍數擴增
表
7C. 活力
天數 | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO |
3 | 9.9 | 9.9 | 9.9 | 9.9 |
5 | 44.4 | 41.6 | 9.8 | 10.2 |
7 | 172.15 | 163.6 | 5.5 | 34.01 |
10 | 544.4 | 651 | 244.5 | 244.5 |
12 | 1.7 | 2.4 | 4.4 | 3.6 |
14 | 1.9 | 11.2 | 19.5 | 15.8 |
天數 | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO |
3 | 1 | 1 | 1 | 1 |
5 | 4.48 | 4.20 | 0.99 | 1.03 |
7 | 17.39 | 16.53 | 0.56 | 3.44 |
10 | 54.99 | 65.76 | 24.70 | 24.70 |
天數 | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO |
0 | 96.1 | 96.1 | 96.1 | 96.1 |
3 | 89.7 | 88 | 88 | 88 |
5 | 88.1 | 89 | 86 | 89 |
7 | 93.3 | 93.2 | 90.2 | 91.5 |
10 | 98 | 92.1 | 95.8 | 95.6 |
12 | 96.3 | 94.3 | 89.2 | 91.2 |
14 | 97 | 91.2 | 95.6 | 94.3 |
此外,如
表 8所示
,RFX5或B2M KO不影響外源性CAR分子之表現。
表
8.CAR 表現
CD19 CAR % | CD4/CD19 CAR+ | CD8/CD19 CAR+ |
NTD | 0.022 | 0.031 |
LVV | 88.6 | 82.2 |
LVV TRAC + B2M KO | 83.9 | 68.8 |
LVV TRAC + RFX5 KO | 85.7 | 64.8 |
CD19 CAR MFI | CD4/CD19 CAR+ MFI | CD8/CD19 CAR+ MFI |
LVV | 707 | 557 |
LVV TRAC + B2M KO | 580 | 455 |
LVV TRAC + RFX5 KO | 880 | 555 |
CD20 CAR % | CD4/CD20 CAR+ | CD8/CD20 CAR+ |
NTD | 0 | 0 |
LVV | 91.8 | 88.9 |
LVV TRAC + B2M KO | 88.7 | 78.8 |
LVV TRAC + RFX5 KO | 88.1 | 72.1 |
CD20 CAR MFI | CD4/CD20 CAR+ MFI | CD8/CD20 CAR+ MFI |
LVV | 7461 | 6583 |
LVV TRAC + B2M KO | 6497 | 4780 |
LVV TRAC + RFX5 KO | 7649 | 4387 |
表9A 至9C顯示CD19/CD20 B2M KO與RFX5 KO CAR-T具有類似活性。
表
9A. 連續殺滅
- Raji MHC I/II KO 系
表
9B. 細胞介素產量
(pg/mL) - 24 小時,
Raji WT 系
表
9C. 細胞毒性
- 48 小時,
Raji WT 細胞系
輪 | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO | |||||||
1 | 65.69 | 64.76 | 61.46 | 72.92 | 76.29 | 74.80 | 67.96 | 65.05 | 71.57 | |
2 | 27.72 | 27.72 | 28.40 | 73.39 | 70.41 | 73.64 | 81.88 | 76.21 | 69.51 | |
3 | 0 | 0 | 0 | 11.18 | 12.75 | 4.73 | -1.38 | 25.03 | 25.79 | |
4 | 0 | 0 | 0 | 3.86 | 4.69 | -2.87 | 8.68 | 14.17 | 10.16 | |
NTD | LVV | LVV TRAC + B2M KO | |||||||
IFNγ | 551.1 | 753.8 | 485.1 | 214065 | 176671 | 177473 | 142278 | 144393 | 150414 |
IL-2 | 45.9 | 61.6 | 51.8 | 3998 | 3699.6 | 3496.5 | 11345.7 | 11630.8 | 12364.3 |
TNFα | 9.4 | 9.8 | 9.5 | 1035.2 | 852.7 | 842.9 | 1431.3 | 1420.3 | 1367.7 |
LVV TRAC + RFX5 KO | |||
IFNγ | 129964.4 | 89862.3 | 93004.4 |
IL-2 | 12061.9 | 11343.9 | 12480.2 |
TNFα | 1471.4 | 1327.9 | 1483.1 |
E:T 比: | NTD | LVV | LVV TRAC + B2M KO | ||||||
1:1 | -29.97 | -26.23 | -20.40 | 98.39 | 97.01 | 97.92 | 94.47 | 90.29 | 89.78 |
1:3 | -11.48 | -9.09 | 3.94 | 77.91 | 70.96 | 72.92 | 51.53 | 51.60 | 51.89 |
1:9 | -19.16 | -5.43 | -10.04 | 42.99 | 35.88 | 40.60 | 27.87 | 30.67 | 23.84 |
E:T 比: | LVV TRAC + RFX5 KO | ||
1:1 | 93.47 | 88.24 | 95.19 |
1:3 | 52.22 | 48.35 | 53.96 |
1:9 | 24.45 | 24.42 | 24.03 |
RFX5 KO CAR T細胞減少HLA I類表現,具有與β2M KO CAR T細胞類似之MFI,這降低了宿主同種異體反應性反應(
表 10)。該表顯示RFX5 KO導致MHC I類敲落(下調),而B2M KO導致MHC I類剔除(消除)。同時,在TRAC + B2M KO與TRAC + RFX5 KO細胞兩者中觀察到類似之TCR KO。MHC I類MFI與I類剔除及敲落細胞類似。然而,僅在TRAC + RFX5 KO細胞中觀察到有效之MHC II類KO。
表
10.TCR 及
MHC 分子表現
TCRab | CD4 | CD8 |
NTD | 98.7 | 97.6 |
LVV | 93.2 | 90.6 |
LVV TRAC + B2M KO | 1.71 | 1.96 |
LVV TRAC + RFX5 KO | 1.6 | 2.36 |
I 類 | ||
NTD | 99.7 | 100 |
LVV | 99.9 | 100 |
LVV TRAC + B2M KO | 10.3 | 10.6 |
LVV TRAC + RFX5 KO | 56.9 | 63.5 |
II 類 | ||
NTD | 72.2 | 89.1 |
LVV | 95.5 | 99.2 |
LVV TRAC + B2M KO | 93.5 | 97.6 |
LVV TRAC + RFX5 KO | 14 | 22 |
I 類MFI+ | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO |
第10天 | 3845 | 6372 | 2455 | 1546 |
相較於剔除β2M,剔除RFX5提供了免受宿主NK殺滅之增強保護,以及相較於未經編輯之CAR-T細胞,降低了自錯配之宿主CD8 T細胞之排斥(
表 11A 至11B)。
表 11A 至11B顯示RFX5 KO提供了免受宿主NK之保護且降低CD8排斥。
表 11A:相較於B2M KO,RFX5 KO在3個供體中3個皆改良NK保護;
表 11B:RFX5 KO在1/3錯配供體中將CD8降低至與B2M KO類似之水平,且相較於2/3錯配供體中未經編輯之細胞,CD8排斥降低。RFX5 KO細胞展現出缺乏MHC II類表現(
表 10),預計這會最大限度地減少宿主CD4 T細胞之反應。
表
11A. NK 殺滅
表
11B. CD8 殺滅
NK 檢定 | NK 供體1 | NK 供體2 | NK 供體3 | ||||||
NTD | 26.3 | 29 | 31.1 | 33.1 | 28.7 | 29.1 | 30.5 | 30.8 | |
LVV | 32.3 | 31.6 | 33.1 | 37.6 | 34.4 | 36.7 | 40.2 | 34.9 | 37.5 |
LVV TRAC + B2M KO | 67.1 | 61.9 | 71.2 | 70.7 | 72.5 | 72.8 | 84.3 | 85.5 | 86.5 |
LVV TRAC + RFX5 KO | 43.8 | 36.7 | 40.3 | 53.8 | 52.8 | 50 | 48 | 51 | 51.3 |
CD8 檢定 | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO | ||||||||
CD8供體1 | 90.5 | 97 | 98.1 | 94.2 | 87.5 | 91.3 | 28.7 | 26.4 | 28.3 | 56.6 | 51.6 | 69.2 |
CD8供體2 | 93.6 | 93.5 | 93.5 | 80.8 | 80.7 | 88.4 | 18.1 | 24.5 | 21.5 | 53.1 | 45.6 | 64.3 |
CD8供體3 | 78.2 | 73.3 | 76.6 | 43.2 | 41.4 | 50.9 | 23.4 | 26.5 | 23.4 | 28.9 | 30.1 | 33.7 |
此實例表明,轉錄因子RFX5之缺失允許產生具有低水平之MHC I類及不存在MHC II類表現之CAR-T細胞,導致MHC錯配之CD8 T細胞及NK細胞之殺滅降低。
實例
2. 同種異體
TRAC-RFX5 抗
CD19/CD20 CAR 之臨床測試
將開發針對CD19及CD20之健康供體衍生之同種異體(HD Allo)產品,其用於治療癌症,無論患者之HLA類型如何,利用鋅指核酸酶(ZFN)剔除基因。該產品將包含針對CD19及CD20之健康供體衍生之T細胞。使用ZFN,TRAC基因將被破壞以解決移植物抗宿主疾病(GVHD)之風險,並且RFX5基因將被破壞以降低MHC I類及II類之表現,以最小化宿主排斥。
TRAC及RFX5基因可替代地藉由本文所述或所屬技術領域中已知之基因編輯方法中之任一者破壞(例如,經由sgRNA及CRISPR/cas9)。
所有修改旨在達成以下基本產品屬性:(i)相對於具有TRAC及B2M基因座破壞之同種異體抗CD19/CD20 CAR(同種異體TRAC-B2M CD19/CD20)及習知的非基因編輯之抗CD19 CAR T細胞,體內活性曲線相當或有所改良,(ii) GVHD風險最小化到零,以及(iii)如使用體外檢定所證實,患者之內源性CD4及CD8 T細胞以及NK細胞排斥之風險最小化。預計將達成以下目標。
− 如藉由腫瘤細胞清除所證明,相對於同種異體TRAC/B2M KO CD19/CD20 CAR-T細胞,體內活性相當或有所改良;
− 相較於同種異體TRAC-B2M KO CD19/CD20 CAR-T細胞,CAR表現及細胞擴增動力學(體內及在製造期間)相當或有所改良;
− 如藉由降低NK細胞對同種異體CAR T細胞之殺滅所證明,相對於同種異體TRAC-B2M KO CD19/CD20 CAR-T細胞,降低了體外錯配之NK細胞排斥;及
− 如藉由降低殺滅及/或細胞介素分泌所證明,相對於未經編輯之抗CD19或抗CD19/20 CAR-T細胞,降低了體外錯配之CD4 T細胞識別及CD8 T細胞排斥。
實例
3.RFX5 剔除
CAR T 細胞之測試
此實例評估研究級規律間隔重複短迴文序列簇(CRISPR)及靶向RFX5基因缺失之CRISPR相關蛋白9 (Cas9) (CRISPR/Cas9)核糖核蛋白之體外功效,及其提供保護免受T細胞及NK細胞介導之排斥的能力,用於同種異體(MHC錯配之)CAR-T細胞產品。
方法:用CLL-1 CAR(具有慢病毒載體(LLV))轉導健康供體T細胞,接著用靶向TRAC(T細胞受體α恆定)及B2M(β-2-微球蛋白)、或靶向TRAC及RFX5之單個嚮導RNA (sgRNA)對CRISPR/Cas9核糖核蛋白進行電穿孔。將β2M剔除(KO)同種異體(Allo) CAR-T細胞(即TRAC + B2M KO,其中TRAC及B2M皆被剔除)、RFX5 KO Allo CAR-T細胞(即TRAC + RFX5 KO,其中TRAC及RFX5皆被剔除)、未經編輯之CAR-T細胞(即LVV)、及未經轉導之對照細胞(即NTD)擴增10天,並在第10天檢查CAR、TCRαβ、MHCI類、及MHC II類表現。在第10天將細胞冷凍用於所有功能性及等位反應性檢定。測量MHC錯配之NK細胞對經編輯及未經編輯之CAR T細胞之殺滅。
結果:RFX5 KO CAR-T細胞具有與β2M KO CAR-T細胞類似的可製造性及CAR官能性。如
表 12A 至12C所示,RFX5剔除(KO)同種異體(Allo) CAR-T細胞具有良好的活力及與B2M KO Allo CAR-T細胞類似之擴增。
表
12A. 原始細胞計數
(x10
6)
表
12B. 倍數擴增
表
12C. 活力
天數 | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO |
3 | 9 | 8 | 8 | 8 |
5 | 54 | 47 | 37 | 40 |
7 | 258 | 239 | 205 | 227 |
10 | 876 | 712 | 720 | 828 |
天數 | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO |
3 | 1 | 1 | 1 | 1 |
5 | 5.95 | 6.15 | 4.82 | 5.15 |
7 | 28.68 | 31.22 | 26.61 | 29.48 |
10 | 97.33 | 93.14 | 93.49 | 107.53 |
天數 | NTD | LVV | LVV TRAC + B2M KO | LVV TRAC + RFX5 KO |
0 | 93.0 | |||
3 | 93.2 | 93.5 | 90.0 | 90.5 |
5 | 94.5 | 93.6 | 95.0 | 95.9 |
7 | 96.8 | 96.1 | 96.4 | 96.7 |
10 | 96.2 | 89.9 | 90.7 | 91.9 |
此外,如
表 13所示
,RFX5或B2M KO不影響外源性CAR分子之表現。
表
13. 第
10 天之
CAR 表現
CLL1 CAR % | CD4/CLL1 CAR+ | CD8/CLL1 CAR+ |
NTD | 0.032 | 0.033 |
LVV | 90.8 | 92.5 |
LVV TRAC + B2M KO | 91.1 | 92.8 |
LVV TRAC + RFX5 KO | 91.9 | 93.7 |
表14顯示RFX5 KO導致MHC I類敲落(下調)。同時,在TRAC + B2M KO與TRAC + RFX5 KO細胞兩者中觀察到類似之TCR KO。然而,僅在TRAC + RFX5 KO細胞中觀察到有效之MHC II類KO。
表
14. 第
10 天之
TCR 及
MHC 分子表現
TCRab | CD4 | CD8 |
NTD | 90.9 | 86.9 |
LVV | 95.3 | 92.7 |
LVV TRAC + B2M KO | 1.45 | 2.03 |
LVV TRAC + RFX5 KO | 1.51 | 2.24 |
I 類 | ||
NTD | 98.81 | 99.31 |
LVV | 98.67 | 99 |
LVV TRAC + B2M KO | 16.19 | 16.2 |
LVV TRAC + RFX5 KO | 66.44 | 53.5 |
II 類 | ||
NTD | 67.2 | 79.2 |
LVV | 67.3 | 93.4 |
LVV TRAC + B2M KO | 63.6 | 90.5 |
LVV TRAC + RFX5 KO | 12.4 | 18.8 |
相較於剔除β2M,剔除RFX5提供了免受宿主NK殺滅之增強保護(
表 15)。表15:相較於B2M KO,RFX5 KO在3個供體中3個皆改良NK保護。
表
15. NK 殺滅(死亡
% )
NK 檢定 | NK 供體1 | NK 供體2 | NK 供體3 | ||||||
NTD | 17.5 | 17 | 15.9 | 22.6 | 20.6 | 21.2 | 14.9 | 14.8 | 15 |
LVV | 35.3 | 31.7 | 34.7 | 41.4 | 40.7 | 38.4 | 30.9 | 33.1 | 32 |
LVV TRAC + B2M KO | 70.9 | 71.4 | 68.2 | 74.3 | 70.8 | 73.4 | 61.8 | 62.8 | 65.6 |
LVV TRAC + RFX5 KO | 34.9 | 35.8 | 37.1 | 44.1 | 41.1 | 42.4 | 36.5 | 33.7 | 33 |
此實例表明,轉錄因子RFX5之缺失允許產生具有低水平之MHC I類及不存在MHC II類表現之CAR-T細胞,相較於B2M KO細胞,這導致NK細胞之殺滅降低。
實例
4.RFX5 剔除
CAR T 細胞之測試
此實例將評估RFX5 KO CAR T細胞在B細胞淋巴瘤及急性骨髓性白血病之小鼠模型中之體內功效。
方法:藉由實例1及3中所述之方法產生的RFX5 KO CAR-T細胞及對應的對照將用於體內研究。將評估6至7週齡的雌性NSG小鼠,5隻小鼠/組。將測試兩種劑量的CAR T細胞:1x10
6或2x10
6CAR+T細胞/小鼠及5x10
6或10x10
6CAR+ T細胞/小鼠。將表現相關抗原之腫瘤細胞經由側尾靜脈靜脈內植入6至7週齡雌性NSG小鼠體內。
在腫瘤植入之後,將對以下B細胞淋巴瘤或AML荷瘤小鼠之組進行評估:(1)無治療;(2)用非轉導(NTD) T細胞治療;(3)用未經編輯之CAR-T細胞治療;(4)用同種異體RFX5及TRAC KO CAR-T細胞治療;及(5)用同種異體B2M及TRAC KO CAR-T細胞治療。
將藉由生物發光(生物發光與腫瘤負荷正相關)及動物存活率評估T細胞產品對腫瘤負荷之效應來評估腫瘤殺滅。RFX5 KO CAR-T細胞擴增動力學及持久性將藉由在CAR-T細胞輸注之後24小時採集之血液樣本的離體分析來評估,接著每週取樣以追蹤CAR表現、TCR、MHC I類、MHC II類、及其他相關T細胞表型標記。
* * *
雖然已描述數個實施例,但顯然本揭露及實例可提供利用本文所述之組成物及方法或由本文所述之組成物及方法涵蓋之其他實施例。因此,將理解的是,其範圍由可自本揭露及隨附申請專利範圍中理解的內容所界定,而非由以實例之方式表示之實施例所界定。
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TW202340457A_112107109_SEQL.xml
Claims (35)
- 一種經分離之人類免疫細胞,其經工程改造以具有(i)相較於參考細胞,MHC I類活性或表現低10%至80%;(ii)相較於參考細胞,MHC II類活性或表現至少低75%;及(iii)編碼嵌合抗原受體(CAR)或T細胞受體(TCR)之外源性多核苷酸。
- 如請求項1之免疫細胞,其係T細胞或NK細胞。
- 如請求項1或2之免疫細胞,其中RFX5(調節因子X5)、TAP1(抗原加工相關轉運蛋白1)、TAP2(抗原肽轉運蛋白2)、或CIITA(II類、主要組織相容性複合體、反式活化子)之內源性基因係去活化或缺乏。
- 如請求項3之免疫細胞,其中該內源性基因之兩個等位基因皆去活化或缺乏。
- 如請求項3之免疫細胞,其中RFX5之該內源性基因去活化或缺乏。
- 如請求項3之免疫細胞,其中TAP1、TAP2、及CIITA之該內源性基因未經工程改造。
- 如請求項1或2之免疫細胞,其中TRAC(T細胞受體α恆定)之該內源性基因被進一步去活化。
- 如請求項1或2之免疫細胞,其中B2M(β-2-微球蛋白)之該內源性基因未經工程改造,或其中該細胞具有B2M之正常活性。
- 如請求項1之免疫細胞,其中該CAR識別CD19及/或CD20。
- 如請求項1之免疫細胞,其中該CAR識別CLL-1。
- 如請求項9之免疫細胞,其中該CAR包含SEQ ID NO:26或27之胺基酸序列,或與SEQ ID NO:26或27具有90%序列同一性之序列。
- 如請求項10之免疫細胞,其中該CAR包含SEQ ID NO:65至76或78至83之胺基酸序列,或與SEQ ID NO:65至76或78至83具有90%序列同一性之序列。
- 如請求項4之免疫細胞,其中該去活化使用(a)該內源性基因之編輯,(b)抑制性RNA之表現,或(c)抑制劑,較佳為抗體來達成。
- 如請求項13之免疫細胞,其中該編輯係藉由CRISPR/Cas9、鋅指核酸酶(ZFN)、TALEN、MegaTAL、大範圍核酸酶、Cpf1、同源重組、單股去氧寡核苷酸(ssODN)、或鹼基編輯。
- 如請求項1之免疫細胞,其中該參考免疫細胞未經編輯為具有降低之MHC I類表現或降低之MHC II類表現。
- 一種用於製備在誘導移植物抗宿主疾病(GVHD)或宿主排斥中活性降低之同種異體細胞的方法,包含對細胞進行工程改造,使該細胞相較於參考細胞具有降低10%至80%的MHC I類表現或活性,及相較於參考細胞具有降低至少75%的MHC II類表現或活性。
- 如請求項16之方法,該方法在該同種異體細胞中降低RFX5(調節因子X5)、TAP1(抗原加工相關轉運蛋白1)、TAP2(抗原肽轉運蛋白2)、或CIITA(II類、主要組織相容性複合體、反式活化子)之該表現或活性。
- 如請求項17之方法,其中該內源性基因之兩個等位基因皆去活化或缺乏。
- 如請求項17或18之方法,其降低該細胞中RFX5之該表現或活性。
- 如請求項16之方法,其中TAP1、TAP2、及CIITA之該內源性基因未經工程改造。
- 如請求項16至18中任一項之方法,該方法進一步包含降低該細胞中之TRAC(T細胞受體α恆定)之該表現或活性。
- 如請求項16至18中任一項之方法,其中該細胞中之B2M(β-2-微球蛋白)之內源性基因未經工程改造。
- 如請求項16至18中任一項之方法,其中該降低使用(a)基因之編輯,(b)抑制性RNA之表現,或(c)抑制劑,較佳為抗體來達成。
- 如請求項23之方法,其中該編輯係藉由CRISPR/Cas9、鋅指核酸酶(ZFN)、TALEN、MegaTAL、大範圍核酸酶、Cpf1、同源重組、單股去氧寡核苷酸(ssODN)、或鹼基編輯。
- 如請求項16至18中任一項之方法,其中該同種異體細胞係T細胞或NK細胞。
- 如請求項16之方法,該方法進一步包含向該同種異體細胞引入編碼嵌合抗原受體(CAR)或T細胞受體(TCR)之外源性多核苷酸。
- 如請求項26之方法,其中該CAR識別CD19及/或CD20。
- 如請求項27之方法,其中該CAR包含SEQ ID NO:26或27之胺基酸序列,或與SEQ ID NO:26或27具有90%同一性之胺基酸序列。
- 如請求項26之方法,其中該CAR識別CLL-1。
- 如請求項29之方法,其中該CAR包含SEQ ID NO:65至76或78至83之胺基酸序列,或與SEQ ID NO:65至76或78至83具有90%序列同一性之序列。
- 如請求項26至30中任一項之方法,其中在編輯該基因之前將該CAR或TCR引入該細胞。
- 一種如請求項1至15中任一項之免疫細胞用於製備藥物之用途,其中該藥物係用於治療有需要之患者之癌症。
- 如請求項32之用途,其中該免疫細胞最初並非源自該患者。
- 如請求項32之用途,其中該癌症係淋巴瘤、白血病、骨髓瘤、急性淋巴球性白血病、急性骨髓性白血病、慢性淋巴球性白血病、慢性骨髓性白血病、霍奇金氏淋巴瘤、或非霍奇金氏淋巴瘤。
- 一種使癌細胞與如請求項1至15中任一項之免疫細胞接觸之活體外方法,其中該癌細胞生長被抑制或降低。
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GB9710807D0 (en) | 1997-05-23 | 1997-07-23 | Medical Res Council | Nucleic acid binding proteins |
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SG10201510092QA (en) | 2010-12-09 | 2016-01-28 | Univ Pennsylvania | Use of chimeric antigen receptor-modified t cells to treat cancer |
CA2854819C (en) | 2011-11-16 | 2022-07-19 | Sangamo Biosciences, Inc. | Modified dna-binding proteins and uses thereof |
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CN116063550A (zh) * | 2020-09-02 | 2023-05-05 | 南京北恒生物科技有限公司 | 靶向nk激活性受体的嵌合抗原受体 |
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