TW202317991A - 目標胜肽與mhc分子間結合特性之特性描述方法 - Google Patents
目標胜肽與mhc分子間結合特性之特性描述方法 Download PDFInfo
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Abstract
本發明關於一種目標胜肽與已知細胞類型MHC分子間結合特性之特性描述方法,該方法包含以下步驟:(i) 提供二或多個細胞,所述細胞之特性為在其表面展現 MHC分子,(ii) 將所述二或多個細胞分別放入二或多個容器,因此每一容器包含一或多個細胞,(iii) 在不同容器中加入目標胜肽之不同變體,其中所述胜肽之變體經標記且具有相同胺基酸序列,但在標記類型及其濃度方面互不相同,並使細胞與之接觸,藉此於不同容器中在細胞表面形成胜肽MHC複合體,(iv)分離出如此形成之胜肽MHC複合體,以及(v) 判定所形成不同胜肽MHC複合體之濃度(圖1)。
Description
本發明係關於目標胜肽(peptide of interest)與MHC分子間結合特性之特性描述方法。
主要組織相容複合體(MHC)是多數脊椎動物所共有六號染色體上之基因簇,用以編碼不同基因,對組織相容性及後天免疫系統具有重要影響。在人體中,此基因簇通常亦稱為人類白血球抗原(HLA)。MHC第一型分子表達於哺乳動物體內除紅血球以外之所有細胞。其主要功能為將源自細胞內或內吞蛋白質之短肽呈遞至胞毒性T淋巴細胞(CTL) (參見「Boniface and Davis, 1995」、「Goldberg and Rizzo, 2015b」、「Gruen and Weissman, 1997」、「Rock and Shen, 2005」)。除T細胞受體 (TCR)之外,CTL亦表達CD8輔助受體。當一CTL之CD8受體對接至目標細胞上之MHC第一型分子時,若CTL之TCR符合MHC第一型分子與所呈遞胜肽複合體所代表之表位,CTL會藉由釋出細胞溶解酵素或使細胞經歷由細胞凋亡造成之計畫性細胞死亡而觸發目標細胞裂解參見「Boniface and Davis, 1995」、「Delves and Roitt, 2000」、「Lustgarten et al., 1991」)。因此,MHC第一型分子可幫助調節細胞免疫力,做為人體對抗細胞內病原體之主要手段,例如病毒及包括L型細菌或志賀氏屬(Shigella)及立克次體屬(Rickettsia)等細菌種類在內之某些細菌(參見「Goldberg and Rizzo, 2015b」、「Madden et al., 1993」、「Ray et al., 2009」)。此程序對於免疫反應及對例如癌症等腫瘤疾病之防禦亦極為重要 (參見「Coley, 1991」、「Coulie et al., 2014」、「Urban and Schreiber, 1992」)。
異源二聚結構之MHC第一型分子係由編碼在MHC 基因簇中之多態重α亞基與在MHC基因座之外位於染色體15號之小型恆定β2微球蛋白(β2m)亞基所組成。多態α鏈具有由三個結構域α1、α2及α3所構成之N端胞外區域、使MHC分子得以連接細胞表面之穿膜螺旋及短胞質尾。其中兩個結構域α1與α2在兩個長α螺旋之間形成胜肽結合槽,槽底是由八個β股構成。類免疫球蛋白域α3涉及與CD8輔助受體之相互作用。恆定β2m使複合體具有穩定性,且參與 CD8輔助受體對胜肽MHC第一型分子複合體之辨識。β2m與α亞基為非共價鍵結,且為位於胜肽結合槽底部之若干口袋所固定。在不同人類HLA對偶基因中可能大相逕庭之胺基酸(AA)側鏈填補結合槽之中央最寬部分,而保守側鏈則於結合槽之狹窄兩端成簇。多態胺基酸殘基完全左右個別HLA分子所綁定胜肽之生物化學特性(參見「Boniface and Davis, 1995」、「Falk et al., 1991」、「Goldberg and Rizzo, 2015a」、「Rammensee et al., 1995」)。
在人體中,MHC第一型基因簇之特徵為其多態性(polymorphism)及多基因性(polygenicity)。每一染色體將HLA-A、-B及-C對偶基因共同編碼以構成HLA第一型單倍型。因此,最多六個標準HLA第一型分子可表達在同一細胞表面; HLA-A、-B與 -C同種異型(allotype)之實施例組合示於下表。IPD-IMGT/HLA 資料庫(3.41.1版,2021-6-11) 於2021年6月共包含6,766種HLA-A對偶基因 (4,064種蛋白質)、7,697種 HLA-B對偶基因 (4,962種蛋白質)及6,621種HLA-C對偶基因 (3,831種蛋白質) (參見「Robinson et al., 2015」)。
HLA-A | HLA-B | HLA-C |
A*02:01 | B*40:02 | C*03:04 |
A*24:02 | B*52:01 | C*12:02 |
MHC分子是允許免疫系統結合以辨識並耐受其本身(自動辨識)之組織抗原。MHC分子 並對於與MHC異二聚體複合並作為潛在外來抗原型態呈遞予T細胞之胞內胜肽具有分子伴侶之功能(參見「Felix and Allen, 2007」、「Stern and Wiley, 1994」)。
MHC分子與TCR及不同輔助受體相互作用,以於抗原結合親和力及特異性以及訊號轉導效力等方面優化TCR與抗原相互作用之結合條件(參見「Boniface and Davis, 1995」、「Gao et al., 2000」、「Lustgarten et al., 1991」)。
MHC胜肽複合體本質上為自體抗原/同種抗原之複合體。結合時,T細胞原則上容許自體抗原,但接觸同種抗原時則會發生作用。當此原則崩壞時即會出現疾病狀態(特別是自體免疫) (參見「Basu et al., 2001」、「Felix and Allen, 2007」、「Whitelegg et al., 2005」)。
在MHC第一型分子上,細胞呈遞之胞質溶膠胜肽主要是來自蛋白質轉換及缺陷核醣體產物之自體胜肽(參見「Goldberg and Rizzo, 2015b」、「Schwanhausser et al., 2011, 2013」、「Yewdell, 2003」、「Yewdell et al., 1996」)。此等胜肽多半具有延長構造,通常為8至12個胺基酸殘基長,但亦可能稍長於此(參見「Guo et al., 1992」、「Madden et al., 1993」、「Rammensee, 1995」)。在受包括病毒及微生物等細胞內病原體感染時及癌變過程中,外源蛋白或與惡性轉化有關之蛋白亦會在MHC第一型分子所承載之蛋白酶體中降解,並進一步展現於細胞表面(參見「Goldberg and Rizzo, 2015b」、「Madden et al., 1993」、「Urban and Schreiber, 1992」)。此外,一種名為交叉呈遞之現象可將胞外抗原裝載於MHC第一型分子,藉此可使樹突細胞(dendritic cell,DC) 啟動初始CTL (參見「Rock and Shen, 2005」)。T細胞可偵測在MHC分子中展現0.1%-1%之胜肽,且能以此喚起免疫反應 (參見「Davenport et al., 2018」、「Sharma and Kranz, 2016」、「Siller-Farfan and Dushek, 2018」、「van der Merwe and Dushek, 2011」)。
由MHC第一型分子展現之胜肽依其起源而可分為「腫瘤相關胜肽」 (tumor-associated peptide,TUMAP)、「病毒衍生胜肽」或更常見之「病原體衍生胜肽」(參見「Coulie et al., 2014」、「Freudenmann et al., 2018」、「Kirner et al., 2014」、「Urban and Schreiber, 1992」)。
目前技術已將MHC第一型分子、藉其所呈遞之胜肽與T細胞受體間之相互影響用於治療介入,包括 (i) 疫苗接種、(ii) TCR療法及(iii)過繼性T細胞療法 (參見「Dahan and Reiter, 2012」、「He et al., 2019」、「Hilf et al., 2019」、「Kuhn et al., 2019」、「Rosenberg et al., 2011」、「Velcheti and Schalper, 2016」)。
TUMAP之疫苗接種已用於預備並啟動免疫系統以對抗癌症。其活化反應之過程包含疫苗接種、引發、擴散與排除。於疫苗接種步驟,將TUMAP連同佐藥/免疫調節劑經皮膚內施用,以創造發炎反應而召集免疫細胞(樹突細胞)並促其成熟。於引發步驟,再次施用TUMAP使其結合真皮內樹突細胞DC,並於此將之載入MHC第一型分子。而後DC遷移至淋巴結,在此活化 (「啟動」)初始T細胞,使T細胞經由其TCR特別辨識疫苗中所用之TUMAP。T細胞備妥後,其數量會迅速增加(株落增殖),並離開淋巴結,而開始搜尋在其MHC上出現與引發時促使活化者完全相同TUMAP之腫瘤細胞。一旦覓得目標細胞,T細胞即對腫瘤細胞展開溶解/凋亡攻擊(參見「Hilf et al., 2019」、「Kirner et al., 2014」、「Molenkamp et al., 2005」) 。
於過繼性T細胞療法中,先分離出病患本身之T細胞,任選地富集具有所需抗原特異性的克隆,於體外擴增,而後再次注入病患體內。分離出之自體取得T細胞可再經修改以表達經再造後可辨識特定病源體產生胜肽或腫瘤相關胜肽之TCR。以此方式,可教導此等T細胞結合疾病部位之細胞,並對目標細胞發起溶解/凋亡攻擊。亦可對配備有嵌合抗原受體(chimeric antigen receptor,CAR)之T細胞加入共激分子(例如CD40配體),以進一步強化觸發之抗腫瘤免疫反應(參見「Kuhn et al., 2019」、「Rosenberg et al., 2011」)。
另一類替代治療方式採用經改造之可溶TCR,使其呈遞於MHC上時辨識特定病源體產生胜肽或腫瘤相關胜肽(參見「Dahan and Reiter, 2012」、「 He et al., 2019」)。此種TCR帶有能夠結合T細胞之免疫調節部分,此係對T細胞上大量存在之CD3分子具有親和力之抗體片段。藉由此一機制,可將T細胞導向疾病所在之處並對目標細胞展開溶解/凋亡攻擊(參見「Chang et al., 2016」、「Dao et al., 2015」、「He et al., 2019」)。可溶TCR勝於抗體類(免疫)療法之最大優勢在於將潛在目標範圍擴大至胞內蛋白質,而非僅限於一般抗體形式所可觸及之細胞表面抗原(參見「Dahan and Reiter, 2012」、「He et al., 2019」)。
為開發治療能夠辨識胜肽MHC複合體之治療體,必須利用適當檢測方法確認實體對胜肽MHC複合體或對所述複合體呈現細胞所展現之結合性質有何特質。亦需要對此種實體進行例如細胞殺傷活性等能力之判定。且需要能夠建立劑量反應曲線以利判定例如半抑制濃度(IC50)等劑量依賴效應。並需要能夠使用標準化細胞株以利達成特定待調查胜肽以及不同胜肽間之最大可再現性。由於可做為潛在目標之胜肽數量幾乎無限,而多數此類胜肽尚未為人所發現,因此亦需要適用於所有可經MHC 展現之胜肽之檢測系統。
參照納入
本案所引用一切出版品、專利、專利申請及其他文件無論出於任何目的而予以參照者,其全部內容均視同納入本文,如同個別所述出版品、專利、專利申請及其他文件各自出於任何目的而在引用下所納入之程度。若在此併入之一或多個參考文獻與本案之教示有所衝突,應以本說明書之教示為準。
由於在此所述裝置及方法可能有所變化,本發明並不限於所述裝置特定零組件或所述方法特定程序步驟,合先敘明。應知在此所用術語僅為描述特定實施例之用,不具現制性質。除非上下文另有明確指定,否則在說明書及請求項中所用「一」及「該」等單數形態應包含單數及/或複數指稱。亦應知,由數值所界定之參數範圍視同包含其用以界定之數值。
亦須說明者,應知在此所揭露之實施例不應理解為互不相關之單獨實施例。一實施例中所述特徵亦等同於揭露在本案之其他實施例。若一實施例提及之特定功能未於另一實施例中提及,則熟悉此技藝人士應知,所述功能未必不能出現於上述之另一實施例。熟悉此技藝人士應理解,本案精神在於使另一實施例亦具有所述功能,唯出於維持說明書簡明之理由及篇幅限制之考量,未予重複說明。
此外,在此所引用先前技術文件,尤其是揭露標準或慣例方法之先前技術文件,其內容係經引用而納入本文。在此情況下,引用納入之主要目的在於提供可據以實施之充分揭露,並避免冗長贅述。
根據本發明之第一態樣,一種目標胜肽與已知細胞類型MHC分子間結合特性之特性描述方法包含以下步驟:
a) 提供二或多個特徵為在其表面展現MHC分子之細胞;
b) 將所述二或多個細胞分別放入二或多個容器中,使得每一容器包含一或多個細胞;
c) 於上述不同容器中加入(=「裝載」)一目標胜肽之不同變體,其中所述胜肽之該等變體係經標記且具有相同之胺基酸序列,但具有不同
(i) 標記類型;及
(ii) 濃度;
並使所述細胞與之接觸,藉此於所述不同容器中在該等細胞之表面形成胜肽MHC複合體;
d) 分離出如此形成之胜肽MHC複合體;及
e) 判定步驟c)中所形成不同胜肽MHC複合體之濃度。
是以藉此方法能夠判定已知細胞類型或細胞株上所載(亦即與膜結合MHC複合)胜肽之未知濃度,因而同時取得多種濃度之胜肽MHC複合體 (「pMHC」)。
Stopfer等人於自然通訊 | (2020) 11: 2760所揭露之方法雖看似相同,但並無法提供目標胜肽與MHC分子間結合特性之特性描述,更遑論判定不同胜肽MHC複合體之濃度。並且,其是將胜肽載入以重組方式生產並摺疊之非膜結合MHC。
使用例如Stopfer等人(2020)之ELISA較容易評估可溶MHC或可溶pMHC單體之濃度。但ELISA無法用於本案發明人所欲進行之膜結合pMHC複合體 (亦即「loaded」細胞或細胞株)量化。並且,Stopfer等人(2020)亦提及ELISA在此用途上之其他缺點,包括紫外線介導MHC單體及ELISA對照試劑目前不易購得,僅限於少數常見人類第一型等位基因。此外,作者一再強調其利用胜肽特異性多點校正曲線校正每一細胞之拷貝平均數(第2頁右欄倒數第二段)。
反之,本案發明人制訂如請求項1所述之方法以快速準確評估膜結合pMHC之絕對豐度,從而可透過實驗達成胜肽性質及數量之控制。換言之,胜肽於本發明中之作用並非建立內部校正曲線,而是在單次檢測中評估多種裝載濃度之豐度。
應知本發明方法之範圍包含用於調查二或甚至更多種類目標胜肽與MHC間結合特性之實施例。
「裝載」程序包含將一或多種能夠結合MHC之目標胜肽加入環繞細胞之培養基中。
在一實施例中,所添加之胜肽會與業已與MHC 結合之胜肽在結合上進行競爭。若存在過量,基於解離平衡,所添加之胜肽將實質上取代業已與MHC結合之胜肽。
於另一實施例中,所用細胞為包含功能上「缺乏」之MHC者,如本文他處所述。此等功能上「缺乏」之MHC能夠直接結合添加(「載入」)於周圍培養基中之胜肽。
在此,「目標胜肽之變體」與「胜肽變體」為同義用語。
如在此所用,「MHC分子」關於展現於脊椎動物細胞之蛋白類型,可在基於細胞之免疫系統中發揮作用。一般而言,免疫系統根據MHC在其表面所呈現之胜肽判定MHC為自己或非己。
例如,人體具有三類MHC,亦即MHC第一型(第Ia型包括HLA-A、HLA-B、HLA-C等等單倍型;第Ib型包括HLA-E、HLA-F、HLA-G等等單倍型)、MHC第二型(包括HLA-DM、-DO、-DP、-DQ、-DR等等單倍型)。
MHC 第一型 | MHC 第二型 | |
分子結構 | α1、α2、α3 + ß2 微球蛋白 | ß1、ß2 + α1、α2 |
細胞種類 | 全身細胞 | 抗原呈現細胞 (Antigen-presenting cells ,APC) |
作用對象 | CD8 +胞毒T細胞 | CD4 +輔助T細胞 |
典型胜肽長度 | 8 – 10 AA | 13 – 25 AA |
典型胜肽起源 | 經過抗原加工之細胞內胜肽 | 外源性胜肽 |
例如,小鼠體內至少有兩種MHC,亦即MHC第一型(第Ia型包括H-2K、H-2D、H-2L等等單倍型,第 1b 型包括 Qa-2、Qa-1等等單倍型)及MHC第二型 (包括 I-A、I-E等等單倍型)。
根據一種實施例,所述MHC分子為MHC第一型。
異二聚性MHC第一型分子是由一個在MHC基因簇內編碼之多型性重α亞基及一個基因位於染色體15上MHC座以外之小型不變體β2-微球蛋白(β2m)亞基所構成。多型性α鏈包含:具有α1、α2及α3等三域之N端胞外區;使MHC分子能夠附連於細胞表面之穿膜螺旋;以及短細胞質尾。其中,α1與α2兩域在二長α螺旋之間形成胜肽結合槽,其槽底係由八個β股所形成。免疫球蛋白樣域α3涉及與CD8輔受體之相互作用。不變體β2m提供複合體穩定性並參與CD8輔受體對胜肽MHC第一型複合體之辨識。β2m與α亞基為非共價鍵結,且為位於胜肽結合槽底部之若干口袋所固定。在不同人類 HLA 等位基因間中可能大相逕庭之胺基酸(AA)側鏈填滿結合槽之中央最寬部位,而保守側鏈則於結合槽之狹窄兩端成簇。多型性胺基酸殘基完全左右個別HLA分子中所存在胜肽之生物化學性質(參見「Boniface and Davis, 1995」、「Falk et al., 1991」、「Goldberg and Rizzo, 2015a」、「Rammensee et al., 1995」)。
在人體中,MHC第一型基因簇之特徵為其多態性(polymorphism)及多基因性(polygenicity)。每一染色體將HLA-A、-B及-C對偶基因共同編碼以構成HLA第一型單倍型。因此,最多六個標準HLA第一型分子可表達在同一細胞表面; HLA-A、-B與 -C同種異型(allotype)之實施例組合示於下表。IPD-IMGT/HLA 資料庫(3.41.1版,2021-6-11) 於2021年6月共包含6,766種HLA-A對偶基因 (4,064種蛋白質)、7,697種 HLA-B對偶基因 (4,962種蛋白質)及6,621種HLA-C對偶基因 (3,831種蛋白質) (參見「Robinson et al., 2015」)。
HLA-A | HLA-B | HLA-C |
A*02:01 | B*40:02 | C*03:04 |
A*24:02 | B*52:01 | C*12:02 |
在多因子疾病發展中,遺傳易感性(genetic predisposition)代表一包括個體HLA對偶基因之組成之共同因素。例如僵直性脊椎炎(HLA-B*27)、乳糜瀉(HLA-DQA1*05:01–DQB1*02:01或HLA-DQA1*03:01–DQB1*03:02)、猝睡症 (HLA-DQB1*06:02)或第一型糖尿病(HLA-DRB1*04:01–DQB1*03:02)等自體免疫失調皆與HLA素有淵源(參見「Caillat-Zucman, 2009」)。再者,特定HLA同種異型顯然於對人類免疫缺乏病毒或瘧疾寄生物之接觸傳染風險及感染途徑方面有所影響(參見「Hill et al., 1991」、「The International HIV Controllers Study et al., 2010」、「Trachtenberg et al., 2003」)。此外,癌症免疫療法之反應取決於個別HLA基因型: 雖然HLA-A、-B及 -C 對偶基因之最大雜合性能夠助長對於檢查點抑制劑之反應,但HLA-B*15:01據報會減弱新抗原導向的CTL反應(參見「Chowell et al. , 2018」)。
MHC分子為組織抗原,使免疫系統能夠與其結合、對其辨識並加以容受(自動辨識)。對於受MHC異二聚體複合並以潛在外來抗原之型態呈遞至T細胞之細胞內胜肽,MHC分子亦具有分子伴侶之功能 ((參見「Felix and Allen, 2007」、「Stern and Wiley, 1994」)。
MHC分子與TCR及各種輔受體相互作用下可在抗原結合親和力及特異性方面優化TCR與抗原相互作用時之結合條件,以及訊號轉導效果(參見「Boniface and Davis, 1995」、「Gao et al., 2000」、「Lustgarten et al., 1991」)。
MHC胜肽複合體本質上為自體抗原/同種抗原之複合體。於結合時,T細胞原則上應容許自體抗原,但接觸同種抗原時則會展現活性。當此原則遭到打破時,疾病狀態(特別是自體免疫疾病)即隨之產生(參見「Basu et al., 2001」、「Felix and Allen, 2007」、「Whitelegg et al., 2005」)。
在MHC第一型分子上,細胞呈遞之胞質溶膠胜肽主要是來自蛋白質轉換即缺陷核糖體產物之自體胜肽(參見「Goldberg and Rizzo, 2015b」、「Schwanhausser et al., 2011, 2013」、「Yewdell, 2003」、「Yewdell et al., 1996」)。此等胜肽通常具有延長構造,長度通常為8至12個胺基酸殘基,但亦可調整為較此略長之形態(參見「Guo et al., 1992」、「Madden et al., 1993」、「Rammensee, 1995」)。在受包括病毒及微生物等細胞內病原體感染時及癌變過程中,外部來源蛋白質或與惡性轉化相關之蛋白質亦會在MHC第一型分子所承載之蛋白酶體中降解,並進而顯現於細胞表面 (參見「Goldberg and Rizzo, 2015b」、「Madden et al., 1993」、「Urban and Schreiber, 1992」)。此外,透過名為交叉呈遞之現象,胞外抗原可載於MHC第一型,經由樹突細胞(DC) 啟動初始CTL(Rock and Shen, 2005)。T細胞能夠偵測在MHC分子上以0.1%-1% 顯現之胜肽,即便少量仍可喚起免疫反應(參見「Davenport et al., 2018」、「Sharma and Kranz, 2016」、「Siller-Farfan and Dushek, 2018」、「van der Merwe and Dushek, 2011」)。
根據多種實施例,所述目標胜肽之長度介於8至15個胺基酸殘基之間。
上述胜肽通常受例如HLA-A或HLA-B 同種異型等MHC第一型分子結合。
此等MHC第一型分子具有位於其α1及α2域之胜肽結合槽(見圖11A),其中,所述待展現胜肽係經由所謂錨著殘基固定而不移動。視HLA 同種異型而異,個別胜肽可能經由二、三或四個錨著殘基固定。不同HLA同種異型於主錨與側錨間有所差異。
下表中顯示針對所選HLA同種異型,在個別錨著位置(主錨為粗體,側錨為斜體)結合9-mer胜肽之胺基酸偏好。亦參見圖11B,其中顯示非特異性HLA 同種異型之所謂序列標誌,顯示於包括 P2及 P9在內不同位置之偏好。
HLA 同種異型 | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | 主 錨著殘基 | 側 錨著 殘 基 |
A*01:01 | T | D | Y | P2、P3、P9 | |||||||
S | E | ||||||||||
L | |||||||||||
A*02:01 | L | E | L | V | P2、P9 | P4、P6 | |||||
D | V | L | |||||||||
P | I | ||||||||||
A*03:01 | R | V | L | K | P2、P9 | P1、P7 | |||||
K | L | I | |||||||||
A | I | V | |||||||||
T | |||||||||||
A*24:02 | Y | F | P2、P9 | ||||||||
L | |||||||||||
I | |||||||||||
B*07:02 | R | P | R | L | P2、P9 | P1、P3 | |||||
S | A | ||||||||||
A | |||||||||||
B*08:01 | L | K | K | L | P5、P9 | P2、P3 | |||||
P | L | R | |||||||||
I | R | ||||||||||
A | |||||||||||
B*44:02 | E | E | W | P2、P9 | P1 | ||||||
A | F | ||||||||||
S | Y | ||||||||||
B*44:03 | E | E | Y | P2、P9 | P1 | ||||||
A | F | ||||||||||
S | W | ||||||||||
L |
對於除9-mers以外之結合胜肽,胺基酸在P5被插入或移除以表示相應的基序。下表就HLA-A*02:01及長度為8 至13個AA之胜肽例示其錨著及側錨位置之胺基酸偏好。
8-mer | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | |||||
L | E | L | V | ||||||||||
D | V | L | |||||||||||
P | I | ||||||||||||
9-mer | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | ||||
L | E | L | V | ||||||||||
D | V | L | |||||||||||
P | I | ||||||||||||
10-mer | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | |||
L | E | L | V | ||||||||||
D | V | L | |||||||||||
P | I | ||||||||||||
11-mer | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | P11 | ||
L | E | L | V | ||||||||||
D | V | L | |||||||||||
P | I | ||||||||||||
12-mer | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | P11 | P12 | |
L | E | L | V | ||||||||||
D | V | L | |||||||||||
P | I | ||||||||||||
13-mer | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | P11 | P12 | P13 |
L | E | L | V | ||||||||||
D | V | L | |||||||||||
P | I |
基於上述,根據若干實施例,所述目標胜肽具有以下序列基序: XmA1XnA2Xo,其中,
X為一y蛋白胺基酸;
A
1為一選自以下項目所構成群組之胺基酸:T、A、E、I、L、P、S、V、Y;
A
2為一選自以下項目所構成群組之胺基酸:Y、F、I、K、L、V、W;
m為一介於1至10間之整數;
n是6;
o為一介於≥ 1至≤ 10間之整數;且
m + o ≤ 7。
根據其他實施例,所述目標胜肽為腫瘤相關胜肽(tumor-associated peptide, TUMAP)或疾病相關胜肽。
顧名思義,腫瘤相關胜肽(tumor-associated peptide, TUMAP)或疾病相關胜肽為存在於罹癌或患其他疾病細胞表面而非健康細胞表面之胜肽,或指存在於罹癌或患其他疾病細胞之豐度遠高於在健康細胞表面上之豐度者。
根據其他實施例,所述目標胜肽之變體為同位素標記(「類同位素分子」)。
根據其他實施例,所述同位素標記包含至少一同位素標記胺基酸。
一般而言,每種胺基酸皆有多種同位素標記變體存在並可供購買。但在一實施例中,胺基酸A及G從未經同位素標記。
根據其他實施例,目標胜肽之不同變體於同位素標記之類型方面互不相同。
所述不同特別是包括同位素標記胺基酸殘基之類型及/或個別胜肽中同位素標記胺基酸殘基之總量。
根據本發明一種實施例,所述胜肽MHC複合體係經免疫親和強化(immunoaffinity enrichment)而分離。
免疫親和強化亦稱為「免疫沉澱(immunoprecipitation)」,其方法可見於例如(Caron et al., 2015)與(Freudenmann et al., 2018)、(Kowalewski and Stevanović, 2013)及(Kasuga, 2013),其內容為使本發明可據以實施之目的而併入本文。
HLA第一型及第二型胜肽通常係自細胞裂解物分離而出。透過機械方式使細胞懸浮液均質化後裂解,以利用例如NP-40、Triton X-100、CHAPS、脫氧膽酸鈉(sodium deoxycholate)或IGEPAL CA-630等非變性清潔劑為佳。裂解緩衝液可包含蛋白酶抑制劑,以免HLA-胜肽複合體降解。
澄清後之裂解物可再接受例如使用下述MHC結合多肽之免疫親和層析。所述MHC結合多肽可共價接合於例如瓊脂糖(sepharose)或瓊脂糖純化(agarose)樹脂等基質,或非共價附連於蛋白質A或蛋白質G。可使用多種市售交聯解決方案,例如CNBr活化瓊脂糖或具有經乙醛活化4%瓊脂糖珠之AminoLinkTM連接樹脂。裂解物有時是在以蛋白質A或蛋白質G 進行免疫親和層析前自天然抗體預先洗出(precleared)。
另一種MHC分子之免疫親和強化方法為利用弱酸洗提使(載入之)胜肽自MHC分子釋出(參見「Freudenmann et al., 2018; Storkus et al., 1993」)。
根據一種實施例,所述免疫親和強化係利用MHC結合多肽執行。
此處之重點在於區分「MHC結合多肽」與「pMHC結合蛋白」。MHC結合多肽對MHC 具有特異性,不論是否已有胜肽與MHC結合在先,且不論先行結合胜肽之序列或結構,一律與MHC結合。pMHC結合蛋白則特別結合於固定之胜肽MHC (pMHC)複合體,取決於包含胜肽序列或結構在內之因素。因此,MHC結合多肽可用於例如MHC或pMHC之免疫親和強化,不論胜肽具有何種序列或結構,而pMHC結合蛋白可用為專與特定胜肽MHC (pMHC)複合體結合以喚起生理反應之治療體。
根據一種實施例,所述免疫親和強化係利用MHC特異性抗體執行。
為分離並分析由MHC所呈遞之胜肽,通常使用對於目標MHC分子具有特異性之單株抗體(參見「Freudenmann et al., 2018」)。所需說明者,此種抗體與MHC之結合不受已與MHC結合之個別胜肽所影響。不同HLA同種異型可用不同抗體,雖然抗體所結合之HLA 同種異型各不相同。下表列出對人類或鼠科 MHC具有特定性之此等抗體:
無性繁殖系 | 特異性 | 出處 |
W6/32 | HLA‑A、‑B、‑C | (Barnstable et al., 1978) |
B1.23.2 | HLA‑B、‑C | (Rebai et al., 1983) |
BB7.2 | HLA‑A*02 | (Parham and Brodsky, 1981) |
GAP-A3 | HLA‑A*03 | (Berger et al., 1982) |
ME1 | HLA‑B*07、‑B*27、‑Bw22 | (Ellis et al., 1982) |
Tü39 | HLA‑DR、‑DP、‑DQ | (Maeda and Hirata, 1984) |
B7/21 | HLA‑DP | (Robbins et al., 1987; Royston et al., 1981) |
L243 | HLA‑DR | (Lampson and Levy, 1980) |
LB3.1 | HLA‑DR | (Gorga et al., 1986) |
Spv‑L3 | HLA‑DQ | (Spits et al., 1983) |
IVD‑12 | HLA‑DQ | (Kolstad et al., 1987) |
Y‑3 | H‑2K b | (Hämmerling et al., 1982; Jones and Janeway, 1981) |
B8‑24‑3 | H‑2K b | (Köhler et al., 1981) |
20‑8‑4S | H‑2K b | (Ozato and Sachs, 1981) |
28‑8‑6S | H‑2K b | (Ozato and Sachs, 1981) |
5F1 | H‑2K b | (Hämmerling et al., 1982; Sherman and Randolph, 1981) |
B22‑249.R1 | H2‑D b | (Lemke et al., 1979) |
28‑14‑8S | H‑2D b | (Ozato and Sachs, 1981) |
27‑11‑13S | H‑2D b | (Ozato and Sachs, 1981) |
M5/114 | Ia | (Bhattacharya et al., 1981) |
其他適用之抗體可如(Sidney et al., 2013)所揭露,其內容為使本發明可據以實施之目的經引用而併入本文。
根據一種實施例,分離出胜肽MHC複合體後,自MHC沖提所述胜肽。
如在此所用,「沖提」係指胜肽自胜肽MHC複合體釋出之程序,「沖提液」係指包含有沖提胜肽之培養基。
HLA複合體之沖提可經由以例如0.1–0.2% TFA (三氟乙酸)、10%乙酸等強酸處理,或以0.1–0.2 N乙酸處理後進行熱變性處理而達成。上述兩種方案皆會導致MHC分子變性,將所結合之胜肽釋出。
除卻上述免疫親和強化繼以胜肽沖提之方案外,亦可利用弱酸沖提(MAE),使非共價結合β2-微球蛋白及胜肽自細胞表面之MHC複合體分離而出,如此即可自完整細胞分離出MHC所結合之胜肽。通常可使用例如檸檬酸鹽磷酸鹽緩衝液等較低酸鹼值(如 pH 3.3)緩衝液處理約1分鐘。MAEr能夠以較少純化步驟分離出與MHC結合之胜肽,無需使用清潔劑,且不會出現使低親和力胜肽優先喪失之偏誤。然而,與細胞膜經由靜液力交互作用之汙染胜肽亦可能在弱酸處理下沖提出來。亦可藉由分析例如使用β2微球蛋白缺失細胞株之等效陰性對照組而對此種胜肽與MHC結合胜肽加以區分。
根據一種實施例,係於沖提液中判定所述不同胜肽變體之濃度,藉此判定步驟c)中所形成不同胜肽MHC複合體之濃度。
由於胜肽與MHC間之化學計量為1:1,胜肽濃度等於步驟c)所形成胜肽MHC複合體濃度(但應注意複合體可能在純化過程中減少)。
不同胜肽變體在沖提液中之濃度可利用例如WO2016107740A1中所描述之 LC-MS/MS進行判定,該案之內容僅為使本發明可據以實施之目的而併入本文。
在一實施例中,所述方法包含:
(1) 判定各製備物之細胞數,其中,細胞係接觸或已接觸目標胜肽之不同變體及濃度;
(2) 添加已知數量之目標胜肽,隨選結合於步驟a)製備物之MHC,以直接在組織均質化(「突峰I」)後進行為佳;
(3) 分離或純化形成之胜肽MHC複合體;
(4) 將胜肽自MHC中沖提;
(5) 添加已知數量之待定量目標胜肽至所述胜肽沖提液(「突峰II」)為內部校正物;
(6)對所述目標胜肽執行質譜分析以產生 ;
(i) 一步驟(3)分離效率訊號;
(ii) 一步驟(5)內部校正物訊號;及
(iii) 一目標胜肽總量訊號;及
(7) 基於步驟(6)取得訊號與;
(i) 取得細胞數;
(ii) 步驟(2)加入之已知數量之待定量所述目標胜肽及/或胜肽MHC複合體;及
(iii) 步驟(5)加入之已知數量之待定量MHC胜肽配體,其中,所述定量包含計算步驟(5)內部校正物訊號與目標胜肽訊號間之比率並將此比率與校正曲線比較;
以定量所述MHC 配體。
可選地,所述定量進一步包含基於與相同用量內部校正物之比率產生胜肽特異性校正曲線,並判定所述待定量之目標胜肽之定量下限(LLOQ),藉此,若量化數量高於設定之LLOQ,即可達成細胞上目標胜肽之絕對定量。
根據一種實施例,本發明之方法進一步包含判定步驟a)之製備物中至少一種MHC分子之數量。判定至少一種MHC分子數量之方法揭露於例如 DE1020211051428及請求其優先權之PCT申請。
根據一種實施例,本發明方法包含判定
(i)細胞在步驟b)中所接觸目標胜肽之濃度與
(ii) 步驟c)所形成不同胜肽MHC複合體濃度
間之比率。
根據本發明一種實施例,就每一胜肽變體,判定與之接觸之細胞數。
此一目的可經由多種方法達成,例如利用顯微鏡及/或光學影像處理進行細胞或細胞核計數、光度計DNA判定、螢光DNA定量PCR、利用LC-MS之組織蛋白定量或自動細胞計數(利用例如CASY儀器)。
根據本發明一種實施例,計算而得之比率為該等細胞於步驟b)中所接觸之胜肽濃度 (µg mL-1或nM)對每一細胞中pMHC複合體內胜肽拷貝數。
此等比率可基於以下判定
(i) 細胞在步驟b)中所接觸之目標胜肽已知濃度,
(ii) 步驟c)所形成不同胜肽MHC複合體之濃度,如步驟d)中所判定者,及隨選參酌
(iii) 與個別胜肽變體接觸之細胞數。
根據不同比率,可制訂出校正曲線或公式,因而能夠預測細胞是否接觸一定濃度之目標胜肽,以及整體或每一細胞將會形成多少胜肽MHC複合體。
如此同樣十分有助於為確認例如以下特性而使載有目標胜肽之細胞接觸一或多個 pMHC結合體之實驗:
所述pMHC結合體在結合檢測或生物檢測中之能力,及
所述pMHC結合體對個別胜肽MHC複合體之親和力或特異性。
根據本發明一種實施例,不同胜肽變體之濃度係於所述一或多個細胞上經由至少一選自以下項目所構成群組之方法判定:
質譜法 (MS)
串聯式質譜法 (MS/MS)
液相層析串聯式質譜法 (LC-MS,LC-MS/MS)。
利用質譜法(MS)確認先前與MHC結合胜肽之濃度可參考例如其內容經引用併入本文之WOWO2016107740,及其內容經引用併入本文之「Freudenmann et al., 2018」。其中,以液相層析串聯式質譜法(LC-MS/MS)尤為適合。
利用液相層析串聯式質譜法(LC-MS/MS)為胜肽定序之具體過程為預分餾複合體胜肽溶液後進行MS。取得MS1光譜後選出足數胜肽進行分餾,產出MS2光譜。預分餾通常經由層析步驟執行,例如反相或SCX (強陽離子交換)層析分離。
MS定序通常是利用碰撞誘發解離(collision induced dissociation, CID)或光束型高能碰撞解離 (higher-energy CID, HCD)達成。上述方法產生之胜肽片段離子可用於自動化資料庫搜尋策略或從頭測序(de novo)分析以釐清胜肽序列。但上述方法係針對胰蛋白酶肽優化,其所產生之光譜通常缺乏足以肯定辨識對象之片段資訊,因此並不適用於HLA配體。故而有例如電子轉移/高能誘發解離(electron-transfer/higher-energy-induced dissociation, EThCD)等混合分餾法之提出。MS取得策略之總體探討可參見「Caron et al., 2015」或「Schumacher et al., 2017」,前開文件之內容為使本發明可據以實施之目的而併入本文。
根據本發明一種實施例,構成所述胜肽MHC複合體之該胜肽為並非由一已建立之細胞系所呈現之胜肽。
取自KLK3之胜肽即為一例。至今未見任何論述關於在其表面表達KLK3及/或展現衍生自KLK3之MHC受限胜肽。一種上述KLK3衍生MHC受限胜肽示於SEQ ID NO 1。
對於可能在例如癌症治療(例如經由適當治療體,例如過繼性 T細胞、可溶T細胞受體(TCR)或TCR模擬抗體)等方面代表重要目標之胜肽,可能難以開發評估其能否做為有效治療選項之適當體外系統或細胞式檢測。但利用本發明之方法,便可人工制訂呈現此等胜肽之細胞,研究其與不同治療體接觸後之反應,據以取得劑量反應曲線。
惟本發明之方法亦可用於實際上為既有細胞株所呈現之胜肽,例如可於PRAME、胜肽
為針對可在體外與胜肽MHC複合體結合之治療體調查其活性及能力,必須利用適當模型系統將目標胜肽呈現於其MHC分子。理想上,呈現之多寡,亦即每一目標細胞之胜肽拷貝數,應與在代表調查藥物產品最終為安全有效之病患原生組織中所觀察到之數量相當。
同樣,亦可能有研究是針對不含目標胜肽但(以特定數量)呈現 (可能)脫靶胜肽之細胞探討其脫靶效應(off-target effects) (參見「Liu et al., 2020」)。但有時建立模型系統並不可行,例如細胞株完全未呈現目標胜肽或在所需拷貝數範圍內未呈現目標胜肽之情形。
根據本發明一種實施例,二或多個以在其表面展現MHC分子為其特性之細胞係缺乏胜肽抗原加工及/或胜肽抗原呈現。
此類細胞株(幾乎)皆缺乏胜肽之內生MHC呈現。
根據本發明一種實施例,所述細胞缺乏胜肽抗原加工及/或呈現之原因是缺乏抗原加工相關轉運體(transporter associated with antigen processing, TAP)。
與抗原加工相關轉運體 (TAP)之蛋白複合體屬於ATP結合盒轉運體家族。其作用是將胞質溶膠胜肽遞送至內質網(endoplasmic reticulum, ER)中,使胜肽可在此與初生MHC第一型分子結合。
TAP結構由兩種蛋白質構成:TAP-1 (NCBI基因:6890)及 TAP-2 (NCBI基因:6891),各有一疏水區及一ATP結合區。兩者組成之異二聚體產生四域轉運體。
此類細胞是自外部載入目標MHC結合胜肽之主要對象。外部添加合成胜肽促進MHC第一型組合並/或結合於且穩定化空缺MHC第一型I-β2m異二聚體(參見「Lewis et al., 1996」、「 Liu et al., 2020」、「Ljunggren et al., 1991」、「Salter and Cresswell,1986」、「Townsend et al., 1989」)。其可為自然表達(空缺) MHC分子,亦可經由轉染導入至MHC缺失細胞 (參見「DeMars et al., 1984」、「Lewis et al., 1996」、「Riberdy and Cresswell, 1992」)。
根據本發明一種實施例,所述細胞缺乏胜肽抗原加工及/或呈現導致其細胞表面產生功能上「空缺」之第一型MHC之表達。
如在此所用,「空缺」MHC意指細胞於其表面呈現之MHC並未帶有結合之T細胞表位胜肽。此種機能性「空缺」MHC能夠與加入(「載入」)周圍培養基之個別胜肽結合。
根據本發明實施例,所述細胞係選自下列項目所構成之群組:
T2 (174xCEM.T2)
RMA-S
B-LCL 721.174或B-LCL 721.180
C1R-T134K
T2為取自淋巴瘤之細胞株,因缺乏TAP而在細胞表面表達少量HLA-A2,且僅能夠呈現外源胜肽。外源胜肽結合至HLA-A2可提升HLA-A2-胜肽複合體之穩定性,且可利用抗體螢光染色方式測得。
RMA-S突變細胞株在第一型組合上有所缺陷,因而其在細胞表面表達第一型分子之程度大幅降低。
可用於本發明之細胞株及其特性列於下表:
細胞株 | 抗原加工 機轉失常 | 出處 |
B-LCL 721.174 B-LCL 721.180 (人類 B淋巴母細胞株) | 功能性第二型基因同源性缺失(包括 TAP) HLA第一型及第二型表達大幅減少 | (DeMars et al., 1984; Erlich et al., 1986) |
.174xCEM.T2 (‘T2’;人類 B-LCL 721.174與人類 T-LCL CEMR.3之混合) | 功能性第二型基因同源性缺失(包括 TAP) 正常HLA第一型重鏈及β2m, HLA組合缺陷/空缺HLA分子 | (Erlich et al., 1986; Riberdy and Cresswell,1992; Salter and Cresswell, 1986; Salter et al., 1985) |
C1R-T134K (以HLA-A2.1 T134K轉染之人類 B淋巴母細胞株) | C1R: HLA第一型分子低度表達(HLA-A*03、-Cw3、-Bw62缺失 /HLA-A*02轉錄缺陷/ HLA-B*35轉譯缺陷/HLA-Cw4組合缺陷 HLA-A2.1 T134K:α2域突變導致無法與TAP作用。HLA組合缺陷/缺乏HLA分子 | (Lewis et al., 1996; Storkus et al., 1987; Zemmour, 1996; Zemmour et al., 1992) |
RMA-S (鼠科動物T細胞淋巴瘤) | TAP缺陷 MHC第一型表達降低、MHC第一型組合缺陷/空缺第一型分子 | (Ljunggren and Karre, 1985; Ljunggren et al., 1991; Townsend et al., 1989) |
在利用抗原加工機轉失常細胞株之外,亦可自外部裝載其他具有目標胜肽且天生或經轉染後而就目標HLA 同種異型為陽性之細胞株(例如NCIH1755、T98G、Hs695T)。
在此情況下,裝載胜肽會與已結合於細胞MHC之胜肽競爭地位。
根據本發明一種實施例,所述方法進一步包含對至少一部分已接觸目標胜肽之細胞進行檢測,藉此釐清pMHC結合蛋白或pMHC結合細胞與如此形成胜肽MHC複合體之相互作用特性。
此處之重點在於區分「MHC結合多肽」與「pMHC結合蛋白」。MHC結合多肽對MHC 具有特異性,不論是否已有胜肽與MHC結合在先,且不論先行結合胜肽之序列或結構,一律與MHC結合。pMHC結合蛋白則特別結合於固定之胜肽MHC (pMHC)複合體,取決於包含胜肽序列或結構在內之因素。因此,MHC結合多肽可用於例如MHC或pMHC之免疫親和強化,不論胜肽具有何種序列或結構,而pMHC結合蛋白可用為專與特定胜肽MHC (pMHC)複合體結合以喚起生理反應之治療體。
根據本發明一種實施例,所述方法進一步包含判定關聯於pMHC結合蛋白或pMHC結合細胞與pMHC間相互作用之劑量反應關係。
根據本發明一種實施例,所述檢測為生物檢測。
上述生物檢測例如為功能檢測,例如細胞激素釋出檢測。
所用檢測方式可為ELISPOT。於此類檢測中,是將T細胞與依據本發明所產出之載有胜肽之抗原呈遞細胞共同培養。若T細胞包含能夠與載有胜肽之抗原呈遞細胞中胜肽MHC複合體結合之匹配 T細胞受體,T細胞將例如釋出干擾素γ。後者可經由例如於反應孔中塗佈抗干擾素抗體之方式量化。上述方案有時稱為ELISPOT (酵素結合免疫吸附斑點分析法(enzyme liked immunospot))。以此方式可在目標胜肽量與細胞激素釋出量間建立劑量反應曲線。ELISPOT檢測亦可用於偵測腫瘤壞死因子α、白血球介素-(IL-)4 IL-5、IL-6、IL-10、IL-12、顆粒球巨噬細胞株刺激因子,甚至是顆粒酶B分泌淋巴細胞。參閱「Bercovici et al., 2000」,其內容為使本發明可據以實施之目的而經引用併入本文以供參考。
或可利用其中融合有抗CD3抗體之可溶性雙功能T細胞受體。將T細胞受體與細胞共同培養,洗去未鍵結之T細胞受體,將T細胞加入並與結合之雙功能T細胞受體相接,使其釋出細胞激素,再加以定量。
另一種檢測方式為胞內細胞激素之流式細胞術分析(flow cytometric analyses)。此種檢測是測量培養上清液中之胞內細胞激素含量。當受到例如孟寧素(monensin)或布雷菲德菌素A(brefeldin A)等分泌抑制劑作用時,T細胞 於抗原活化之際會在其細胞質中累積細胞激素。淋巴細胞經固定透化後,以細胞計數法對胞內細胞激素進行定量。此項技術可判定產生之細胞激素、產生此等細胞激素之細胞種類以及每一細胞產生之細胞激素量。同樣參閱「Bercovici et al., 2000」,其內容為使本發明可據以實施之目的而經引用併入本文。
另一生物檢測方法為細胞毒性檢測,其係測量由細胞毒性T細胞 (Cytotoxic T lymphocyte, CTL)所引起之目標細胞裂解。CTL裂解之黃金標準素為51Cr釋出檢測(51Cr-release assay),其係關於將51Cr加入至目標細胞,並測量裂解細胞所釋出之51Cr量。小鼠或人類CTL活性 之偵測通常仰賴細胞毒性檢測,其係關於以同源配體(通常為展現於已知類型細胞表面之MHC第一型受限胜肽)刺激外周血單個核細胞 (peripheral blood mononuclear cells, PBMC)或脾臟細胞,並在一週時間中加入IL-2使其擴張,而後測試其對載有51Cr細胞之裂解能力。
另一檢測方式為細胞毒性檢測, 其係對CTL所致目標細胞裂解進行測量。CTL裂解效率可經由測量瀕死或凋亡細胞所釋出之上清液中之乳酸去氫酶(lactate dehydrogenase, LDH)多寡而加以量化。
另一種可用檢測為細胞毒性檢測,其係對由CLT引起之目標細胞裂解進行測量。因此,目標細胞之基因發生改變,而於構成上表達冷光素酶。於目標細胞裂解時,可藉由在上清液中添加特定基質並測量化學發光訊號而得知冷光素酶活性。
根據本發明一種實施例,所述生物檢測為細胞激素釋出檢測。
根據本發明一種實施例,所述檢測為體外檢測。
根據本發明一種實施例,所述體外檢測為表面電漿共振檢測。
表面電漿共振 (surface plasmon resonance, SPR)是正負介電材料之間介面上由入射光引起之傳導電子共振振盪。許多用於測量材料對平面金屬(通常為金或銀)表面或對金屬奈米粒子表面吸附能力之標準工具均是以SPR為基礎。SPR生物感測器可用於分子在親和力及化學動力方面相互作用之有效濃度判定及特性描述。Biacore 即為SPR生物感測器之一例。
根據本發明其他實施例,所述體外檢測為下列之一:
LDH細胞毒性檢測
冷光素酶細胞毒性檢測
51CR釋出檢測。
LDH細胞毒性檢測為簡單可靠之細胞毒性量化方法。乳酸去氫酶(LDH)為一存在於多種細胞類型中的胞質酶。質膜受損後會將LDH釋出至細胞培養基中。利用耦合酵素反應可對培養基中之胞外LDH進行定量,在所述耦合酵素反應中,乳酸在LDH催化下經NAD+還原為NADH而轉換為丙酮酸鹽。而後黃遞酶(diaphorase)利用NADH將四唑鹽(tetrazolium salt, INT)還原為可於490 nm測量之紅色甲䐶產物。甲䐶形成之多寡與釋出至培養基中且代表細胞毒性之LDH量成正比。
冷光素酶細胞毒素檢測之作用為測量係包種群中死亡細胞之相對數量。此類檢測是測量膜損傷細胞(membrane-compromised cells)釋出蛋白酶時內部細胞蛋白酶(死亡細胞蛋白酶)之胞外活性。利用細胞不可滲透冷光胜肽基質(例如AAF胺基螢光素(AAF-aminoluciferin))測量死亡細胞蛋白酶活性。釋出之胺基螢光素產物經檢測試劑中所含冷光素酶產生冷光。AAF胺基螢光素基質無法通過活細胞之完好細胞膜,因此不會產生任何來自活體細胞種群之可察知訊號。發光量與承受胞毒壓力細胞之比例成正比。
鉻51 (
51Cr)釋出檢測普遍用於細胞毒性之精準量化,尤其常用於腫瘤及病毒細胞溶解研究中。檢測目的為判定回應感染或藥物治療所產生之淋巴細胞數量。
目標細胞上標記之
51Cr會於細胞溶解時自目標細胞釋出。對樣本進行離心處理並收集上清液後可分離出標記。經離心程序取得之上清液可直接於γ計數器中計數,或於微量盤中混以閃爍液(scintillation cocktail)(或於LumaPlate™乾燥)後在液相閃爍計數中計數。
根據本發明實施例,所述pMHC結合蛋白係選自下列項目所構成之群組:
T細胞受體,或其目標結合片段,或
TCR模擬抗體,或其目標結合片段。
適用於上述目的之T細胞受體例如見於WO2019012141A1者,該案之內容經引用併入本文。此類TCR尤其是具有TCR α及β鏈,且無跨膜域。對個別pMHC複合體之特異性是由TCR α 及β鏈之可變域決定,特別是取決於其中所含之互補決定區(CDR)。再者,此類T細胞受體可包含作用性部分,如促炎性細胞激素,或CD3結合部分,如抗CD3 抗體或抗體片段。此一機轉將T細胞轉向疾病部位,並對目標細胞展開細胞溶解/凋亡攻擊(參見「Chang et al., 2016」、「Dao et al., 2015」、「He et al., 2019」)。此外,所述T細胞受體可包含能夠延長血中半衰期之部分,例如Fc域。其他適用於上述目的之T細胞受體可例如為EP3112376A1中所揭露者,該案之內容經引用併入本文。
TCR模擬抗體(亦稱為TCR模擬物)是針對pMHC複合體特異性結合之抗體。適用於上述目的之TCR模擬抗體可參考例如「Chames et al., 2000」、「Denkberg et al., 2003」、「Neethling et al., 2008」、「Willemsen et al., 2005」。
根據本發明一種實施例,所述pMHC結合細胞為一T細胞。
過繼性T細胞療法係分離出病患本身之T細胞,隨選豐富化而使其無性繁殖系具有針對目標胜肽之特異性,並在體外擴大後再次輸入病患體內。
根據本發明實施例,所述T細胞為經改造或非經改造而包含同源或異源 T細胞受體之T細胞。
如在此所用,「同源T細胞受體」意指個別T細胞或T細胞無性繁殖系天然產生之T細胞受體。
如在此所用,「異源T細胞受體」意指經過改造而可辨識或天生即可辨識目標胜肽之T細胞受體。所述 TCR係經重組方式導入T細胞中。如此一來,此等T細胞係受「重新編程」而可結合於細胞之疾病部位並對目標細胞進行溶解/凋亡攻擊。
於某一實施例中,可將配備有嵌合抗原受體 (CAR) 之T細胞加入例如CD40 配體等輔助刺激分子中,以進一步提升觸發之抗腫瘤免疫反應(參見「Kuhn et al., 2019」、「Rosenberg et al., 2011」)。
雖然本發明已於圖式及以上敘述中詳細說明,但此等圖說及敘述僅屬說明或範例性質,不具限制性;本發明並不限於上述實施例。熟悉此技藝人士於實施所請發明時可經由對圖面、揭露內容及請求項之研究而另會並得出對於所述實施例之其他變化。於請求項中,「包含」一詞並不排除其他元件或步驟,且不定冠詞「一」並不排除複數。不同依附項中雖引述特定測量值,但不表示不可利用此等測量值之組合發揮優點。請求項中之所有參考符號不應對本發明之範圍構成限制。
在此揭露之所有胺基酸序列皆是自N端至C端顯示。
實例1:KLK3胜肽
實驗詳情
前列腺特異抗原(Prostate-specific antigen, PSA),亦稱為γ精原蛋白(gamma-seminoprotein)或激肽釋放酶-3 (kallikrein-3, KLK3)、P-30抗原,是人體中由KLK3基因所編碼之糖蛋白酶。PSA屬於激肽釋放酶相關肽酶家族(kallikrein-related peptidase family),由前列腺上皮細胞分泌。
PSA於射精時產生,用於液化精液凝塊中之精液,使精子能夠自由游動。據信此物質亦有助於溶解子宮頸黏液,以利精子進入子宮。
PSA少量存在於具有健康前列腺之男性血清中,但會隨前列腺癌或其他前列腺疾病之發生而增加。PSA不僅可做為前列腺癌指標,亦可用於探測前列腺炎或良性前列腺增生(benign prostatic hyperplasia)。
取自KLK3之MHC受限胜肽已揭露於例如「US10449238B2」,其內容經引用併入本文。
1. 類同位素分子之選擇與合成
共選出 7種類同位素分子,其中六種用於 T2裝載實驗(突峰 I),一種做為下游LC/MS分析(表1)之內標準品(突峰 II)。個別胜肽序列中標記位置之選擇基於兩項標準,必須滿足其中至少一者方能達成清晰之偵測及定量:
1. 於所選組合中之獨特前體質量
2. 相較於其他等壓胜肽/類同位素分子,具有共同前體質量,但因受標記胺基酸位置之故,為獨特之片段離子/轉化
為避免非等壓前體離子之共分離,非等壓類同位素分子之最小質量差應為至少3 Da,於主要雙電荷前體離子轉譯為1.5 Th。
[表1]
胜肽名稱 | 序列 | 註記 |
KLK3胜肽_P5 | SLFHP*EDTGQV | T2裝載 |
KLK3胜肽_V11 | SLFHPEDTGQV* | T2裝載 |
KLK3胜肽_F3 | SLF*HPEDTGQV | T2裝載 |
KLK3胜肽_L2V11 | SL*FHPEDTGQV* | T2裝載 |
KLK3胜肽_L2P5 | SL*FHP*EDTGQV | 內標準品 |
KLK3胜肽_L2P5V11 | SL*FHP*EDTGQV* | T2裝載 |
KLK3胜肽_L2F3V11 | SL*F*HPEDTGQV* | T2裝載 |
同位素標記胜肽合成後再以C18-HPLC純化至至少85 %純度。凍乾後之胜肽以1至2 mg mL
-1之濃度回溶於10% DMSO中。回溶後之胜肽儲備液儲存於-80 °C以供後續分析使用。
2. 類同位素分子特異性校正曲線之取得
為將測得之相對離子強度轉換為絕對度量,於兩次技術重複中取得類同位素分子特異性外部校正曲線。因此,總計六種類同位素分子(表1 ;依上述方式回溶)混合至最終濃度 20 pmol µL
-1。隨後再將儲備液滴定加入5%甲酸,繼而進行LC/MS分析。以每次LC/MS注射5 fmol之定量加入內標準品 KLK3胜肽_L2P5。樣本使用ACQUITY UPLC BEH C18 管柱(美國麻州米爾福德Waters 公司之75 μm × 250 mm管柱),以從1至34.5% ACN之梯度在35分鐘時間內於反相層析儀(美國麻州米爾福德Waters 公司之nanoAcquity UPLC)進行分離。在選定前體離子之碰撞誘發解離(CID)後,採用線上耦接 Orbitrap Fusion Tribrid 質譜儀 (美國麻州沃爾瑟姆Thermo Fisher Scientific公司)以排程平行反應監測(sPRM)模式進行質譜分析。並利用Skyline (參見「MacLean et al., 2010」)進行後續峰界整合。
3. T2細胞培養與初始胜肽裝載
自DSMZ (ACC 598,批號 #2)取得T2細胞株。將細胞置於加有10%熱滅活FBS (Gibco,#10270106)且不含抗生素之RPMI-1640培養基(Gibco,#A1049101) 中,在含有5%二氧化碳之加濕大氣環境中培養。細胞以每2-3天1:4或1:6之比率繼代培養。胜肽裝載實驗前兩天(48小時)將細胞培養基改為包含10%熱滅活人類血清(C.C. Pro, #S-41-M)而非FBS之RPMI-1640培養基(Gibco, #A1049101)。而後將含有人類血清之RPMI-1640培養基用於胜肽裝載實驗。
收穫細胞時確認用於實驗之細胞共為100或120百萬個。將總量分配於五或六個50-ml容量 Falcon管,每管最後包含20百萬個細胞。離心步驟(1300 rpm,7分鐘)後,將每組之20百萬個沉澱細胞重新懸浮於16 ml之類同位素分子稀釋液(每百萬細胞800 µl胜肽稀釋液)並移至T75培養燒瓶,再於含5%二氧化碳之加濕大氣中以37 °C 培養兩小時。培養過程中,每20-30分鐘輕搖六支T75燒瓶。以多種同位素培養兩小時後,自不同T75培養燒瓶收集對應細胞懸浮液,移至乾淨50-ml容量Falcon管進行後續清洗步驟。以磷酸鹽緩衝液(phosphate buffered saline, PBS)將細胞清洗兩次(所有離心步驟均是以1300 rpm持續7分鐘)。二次清洗離心後,去除上清液,將各組沉澱細胞重新懸浮於5 ml之PBS中。隨後將來自六支離心管之細胞懸浮液匯總為一個樣本 (置於乾淨50-ml Falcon管)。使用額外之 15 ml PBS沖洗六支離心管以收集所有殘餘細胞。而後將細胞懸浮液以1300 rpm離心7分鐘,去除上清液,將最終細胞沉澱物直接放置於乾冰上。
4. 細胞毒性檢驗與胜肽裝載
利用共培養設置對校正後T2細胞之TCR進行功能親和力評估。以OKT3及CD28預刺激CD8
+T細胞,三天後再以TCR mRNA對其進行電穿孔。於共培養當天,對冷光素酶轉導T2細胞裝載不同濃度之KLK3衍生胜肽類同位素分子。簡言之,將2x10
7百萬T2細胞在加入不同濃度胜肽之40 ml RPMI + 10% HS中以37 °C及5%二氧化碳之條件培養2小時。培養後,清洗並收穫細胞。一部分細胞用於共培養設置,其餘細胞用於以AbsQuant® (參見「WO2016107740A1」所述方法,該案內容為使本發明可據以實施之目的經引用併入本文)判定絕對拷貝數。以1:1之比率播種T細胞及載有胜肽之T2細胞,培養24小時候收穫上清液。分析上清液是否存在有由受胜肽特異性T細胞所殺傷之凋亡/壞死T2細胞所釋出之冷光素酶。添加特定基質後,在微量盤分析儀中測量化學發光訊號,藉此判定上清液中存在之冷光素酶量。並計算受測TCR之半殺滅效率以評估功能親和力。
5. HLA/胜肽親和力純化與 LC/MS
將樣本置於液態氮中急凍後存放於-80 °C以待後續分離。在CHAPS 清潔劑緩衝液中完成細胞裂解後,使用耦接CNBr活化 Sepharose樹脂(德國弗萊堡GE Healthcare Europe)之抗體 BB7.2 (德國蒂賓根大學免疫學系) (參見「Falk et al., 1991」)對胜肽-HLA複合體進行免疫沉澱。利用酸處理將胜肽自抗體樹脂中洗出,並透過超濾加以純化。在 LC/MS分析前,加入每次注射5 fmol用量之內標準品 KLK3胜肽_L2P5。按上述方式進行LC/MS資料取得及資料分析。
6. 結果
結果示於圖1至6。
對七種KLK3胜肽類同位素分子進行同步LC/MS分析,每種使用總計5 fmol之注射量,分析結果一如預期,亦即所有胜肽異構型均可明確辨識,即等壓之類同位素分子可辨識於MS1等級(圖2),類同位素分子特異性片段離子系列造成之碰撞誘發解離(CID)後形成之獨特b離子系列可辨識於MS2等級(圖2,下方)。於個別類同位素分子之後續滴定中,KLK3胜肽_L2P5保持在5 fmol,因此可取得類同位素分子特異性校正曲線(圖3)。利用此等校正曲線可進一步將MS訊號轉換為絕對胜肽量(參見「WO2016107740A1」所述方法,該案內容為使本發明可據以實施之目的經引用併入本文)。具有5種類同位素分子細胞之T2之最終濃度0.1 µg / mL (亦即~ 81 nM)胜肽裝載顯示CV 為11%之高再現性 (「生物複製」)且分別轉譯為每一細胞~10,000 拷貝之絕對KLK3胜肽豐度(圖4)。為進一步評估動態範圍,於0.0001至1 µg / mL (亦即~ 0.081 nM至813 nM) 之範圍滴定測量類同位素分子,並將類同位素分子裝載於空載T2細胞。結果顯示R
2為0.9988之極高線性,並顯示T2細胞按比例載有KLK3胜肽,亦即在測試之設定範圍內,提高十倍之胜肽濃度轉譯為提高十倍之絕對豐度。KLK3胜肽L2F3V11 (濃度0.1 µg mL
-1)確認在此濃度下每一細胞最高可達 10,000 拷貝之胜肽豐度之先前發現。
根據胜肽裝載T2細胞如上述之絕對胜肽豐度評估,配合同時進行之細胞毒性檢測,可進一步評比受測TCR 之相對功能親和力高低,並可將此範圍轉譯為呈現KLK3胜肽之各自拷貝數估計值(圖6)。
實例2:PRAME胜肽
實驗詳情
優先表達於腫瘤中之黑色素瘤抗原為人體中受 PRAME 基因編碼之蛋白質。針對此基因,發明人取五種編碼相同蛋白質之拼接轉錄變體加以觀察。
此基因編碼之抗原主要表達於人類黑色素瘤,且可由溶胞性T淋巴細胞辨識。除睪丸以外,其他正常細胞並不表達此抗原。此一表達模式類似於例如MAGE、BAGE及GAGE等其他腫瘤睪丸(cancer-testis, CT)抗原。但不同於其他 CT 抗原,此抗原亦表達於急性白血病(acute leukemias)。PRAME在腫瘤組織內過度表達,但在正常身體組織中數量較少,此一對比使其成為癌症治療研究中備受關注之目標。近年來,免疫療法已為癌症治療開啟嶄新時代,各種新穎抗原特定性免疫療法方案應運而生。針對PRAME特定性免疫療法之研究主要涉及疫苗及細胞免疫療法。
PRAME能夠抑制維生素A酸傳訊及由維生素A酸所介導之分化與凋亡。PRAME在三陰性乳癌中之過度表達則會經由導入上皮細胞間質轉化而促進癌細胞移動。
取自PRAME 之MHC受限胜肽已揭露於例如「US10934338B2」,其內容經引用併入本文。
1. 類同位素分子之選擇與合成
共選出八種類同位素分子,其中七種用於 T2、Hs695T及 T98G裝載實驗,一種做為下游LC/MS分析(表2)之內標準品。個別胜肽序列中標記位置之選擇基於兩項標準,必須滿足其中至少一者方能達成清晰之偵測及定量:
1. 於所選組合中之獨特前體質量
2. 相較於其他等壓胜肽/類同位素分子,具有共同前體質量,但因受標記胺基酸位置之故,為獨特之片段離子/轉化
為避免非等壓前體離子之共分離,非等壓類同位素分子之最小質量差應為至少3 Da,於主要雙電荷前體離子轉譯為1.5 Th。
[表2]
胜肽名稱 | 序列 | 註記 |
PRAME胜肽_L3 | SLL*QHLIGL | T2、Hs695T、T98G裝載 |
PRAME胜肽_L9 | SLLQHLIGL* | T2、T98G裝載 |
PRAME胜肽_L3L6 | SLL*QHL*IGL | 內標準品 |
PRAME胜肽_L3L9 | SLL*QHLIGL* | T2裝載 |
PRAME胜肽_L2L3L9 | SL*L*QHLIGL* | T2、Hs695T、T98G裝載 |
PRAME胜肽_L3L6I7 | SLL*QHL*I*GL | T2、Hs695T、T98G裝載 |
PRAME胜肽_L2L3L6I7 | SL*L*QHL*I*GL | T2、Hs695T、T98G裝載 |
PRAME胜肽_L3L6I7L9 | SLL*QHL*I*GL* | T2、Hs695T、T98G裝載 |
同位素標記胜肽合成後再以C18-HPLC純化至至少85 %純度。凍乾後之胜肽以1至2 mg mL
-1之濃度回溶於 10% DMSO中。回溶後之胜肽儲備液儲存於-80 °C以供後續分析使用。
2. 類同位素分子特異性校正曲線之取得
為將測得之相對離子強度轉換為絕對度量,於兩次技術重複中取得類同位素分子特異性外部校正曲線。因此,總計七種類同位素分子(表2 ;依上述方式回溶)混合至最終濃度 20 pmol µL
-1。隨後再將儲備液滴定加入5%甲酸,繼而進行LC/MS分析。以每次LC/MS注射100 fmol之定量加入內標準品 PRAME胜肽_L3L6。樣本使用ACQUITY UPLC BEH C18 管柱(美國麻州米爾福德Waters 公司之75 μm × 250 mm管柱),以從1至34.5% ACN之梯度在35分鐘時間內於反相層析儀(美國麻州米爾福德Waters 公司之nanoAcquity UPLC)進行分離。在選定前體離子之碰撞誘發解離(CID)後,採用線上耦接 Orbitrap Fusion Tribrid 質譜儀 (美國麻州沃爾瑟姆Thermo Fisher Scientific公司)以排程平行反應監測(sPRM)模式進行質譜分析。並利用Skyline (參見「MacLean et al., 2010」)進行後續峰界整合。
3. T2細胞培養與初始胜肽裝載
自DSMZ (ACC 598,批號 #2)取得T2細胞株。將細胞置於加有10%熱滅活FBS (Gibco,#10270106)且不含抗生素之RPMI-1640培養基(Gibco,#A1049101) 中,在含有5%二氧化碳之加濕大氣環境中培養。細胞以每2-3天1:4或1:6之比率繼代培養。胜肽裝載實驗前兩天(48小時)將細胞培養基改為包含10%熱滅活人類血清(C.C. Pro, #S-41-M)而非FBS之RPMI-1640培養基(Gibco, #A1049101)。而後將含有人類血清之RPMI-1640培養基用於胜肽裝載實驗。
收穫細胞時確認用於實驗之細胞共為 100或120百萬個。將總量分配於五或六個50-ml容量 Falcon管,每管最後包含20百萬個細胞。離心步驟(1300 rpm,7分鐘)後,將每組之20百萬個沉澱細胞重新懸浮於16 ml之類同位素分子稀釋液(每百萬細胞800 µl胜肽稀釋液)並移至T75培養燒瓶,再於含5%二氧化碳之加濕大氣中以37 °C 培養兩小時。培養過程中,每20-30分鐘輕搖六支T75燒瓶。以多種同位素培養兩小時後,自不同T75培養燒瓶收集對應細胞懸浮液,移至乾淨50-ml容量Falcon管進行後續清洗步驟。以PBS將細胞清洗兩次(所有離心步驟均是以1300 rpm持續7分鐘)。二次清洗離心後,去除上清液,將各組沉澱細胞重新懸浮於5 ml之PBS中。隨後將來自六支離心管之細胞懸浮液匯總為一個樣本 (置於乾淨50-ml Falcon管)。使用額外之 15 ml PBS沖洗六支離心管以收集所有殘餘細胞。而後將細胞懸浮液以1300 rpm離心7分鐘,去除上清液,將最終細胞沉澱物直接放置於乾冰上。
4. T98G之細胞毒性檢驗與 T98G及Hs695T之胜肽裝載
利用共培養設置對對載有PRAME 之T98G細胞進行功能分析。於共培養當日,對T98G (或Hs695T)細胞裝載不同濃度之PRAME胜肽類同位素分子。參閱「PCT/EP2020/050936」,其關於共培養詳情之內容為使本發明可據以實施之目的經引用併入本文。簡言之,將2.5x10
7T98G (或2x10
7Hs695T)細胞在加有不同濃度胜肽之 40 ml DMEM + 10% FCS 中以 37 °C及5% 二氧化碳之條件培養2小時。培養後,清洗並收穫細胞。一部分 細胞(0.5x107) 用於共培養設置 (T98G) ,其餘細胞(Hs695T為所有細胞) 以AbsQuant® (參見「WO2016107740A1」所述方法,該案內容為使本發明可據以實施之目的經引用併入本文)判定絕對拷貝數。以10:1之比率播種人類外周血單個核細胞(PBMC)及載有胜肽之T98G細胞,在TCER之存在下培養48小時,直到收穫上清液。分析上清液是否存在有由受胜肽特異性T細胞所殺傷之凋亡/壞死T98G細胞所釋出之乳酸去氫酶(LDH)。添加特定基質後,在微量盤分析儀中測量比色訊號,藉此判定上清液中存在之LDH量。
5. HLA/胜肽親和力純化與LC/MS
將樣本置於液態氮中急凍後存放於-80 °C以待後續分離。在CHAPS 清潔劑緩衝液中完成細胞裂解後,使用耦接CNBr活化 Sepharose樹脂(德國弗萊堡GE Healthcare Europe)之抗體 BB7.2 (德國蒂賓根大學免疫學系) (參見「Falk et al., 1991」)對胜肽-HLA複合體進行免疫沉澱。利用酸處理將胜肽自抗體樹脂中洗出,並透過超濾加以純化。在 LC/MS分析前,加入每次注射100 fmol用量之內標準 PRAME胜肽_L3L6。按上述方式進行LC/MS資料取得及資料分析。
6. 結果
結果示於圖7至圖10。
對八種PRAME胜肽類同位素分子進行同步LC/MS分析,每種使用總計100 fmol之注射量,分析結果一如預期,亦即所有胜肽異構型均可明確辨識,即等壓之類同位素分子可辨識於MS1等級(圖7,上方),類同位素分子特異性片段離子系列造成之碰撞誘發解離(CID)後形成之獨特b離子系列可辨識於MS2等級(圖7,下方)。於個別類同位素分子之後續滴定中,PRAME胜肽_L3L6保持在100 fmol,因此可取得類同位素分子特異性校正曲線(圖8A)。利用此等校正曲線可進一步將MS訊號轉換為絕對胜肽量(參見「WO2016107740A1」所述方法,該案內容為使本發明可據以實施之目的經引用併入本文)。為進一步評估動態範圍,於0.0001至1 µg / mL (亦即~ 0.1 nM至1000 nM) 之範圍滴定測量六種類同位素分子,並將類同位素分子裝載於空載T2細胞。結果顯示R2 為0.9935之極高線性,並顯示T2細胞按比例載有PRAME胜肽,亦即在測試之設定範圍內,提高十倍之胜肽濃度轉譯為提高十倍之絕對豐度(圖8B)。相同作法套用於載有胜肽之Hs695T (R
2為0.9890,圖8C)及 T98G (圖9)細胞,呈現裝載濃度1 nM (~0.001 µg / mL) PRAME胜肽之可察知拷貝數。根據胜肽裝載T98G細胞如上述之絕對胜肽豐度評估,配合同時進行之細胞毒性檢測,可進一步評比受測TCER之相對功能親和力高低(圖10),並可將此範圍轉譯為呈現PRAME胜肽之各自拷貝數估計值(圖9)。
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序列
以下序列構成本申請揭露內容之一部分。隨同申請亦提交有符合WIPO ST 25規定之電子序列表。為免疑義,若下表序列與電子序列表之序列有所出入,應以此表序列為準。
SEQ ID No | 序列 | 檢索限定詞 |
1 | SLFHPEDTGQV | KLK3衍生胜肽 |
2 | SLLQHLIGL | PRAME衍生胜肽 |
[圖1]顯示本發明方法部分元素之總體原理。
[圖2]顯示以同位素方式選擇之KLK3胜肽變體(「類同位素分子」)間在前體離子 (全MS)或結果片段離子(MS/MS;例如2+ 前體離子配合618.30 m/z) 之多寡差異。
[圖3]顯示針對已知胜肽(在此範例中,取自KLK3 (SLFHPEDTGQV),n = 6)之所選同位素標記變體(「類同位素分子」)之校正曲線產生方式。於質譜儀讀數中可識別出不同變體。
基於上述校正曲線,可將已知實驗中測得之MS訊號轉換為個別同位素標記變體之濃度。所根據之方法可見於,例如,WO2016107740A1,其內容僅為使本發明可據以實施之目的而併入本文。
[圖4]中池化樣本 (n=5)顯示使 T2細胞載有相同濃度之不同類型同位素分子時可產生之高度可再現性CV為 ~ 11% (「生物」重複)。
[圖5]顯示以下兩者間之滴定曲線: (a)目標胜肽(不同濃度且以不同方式標記之胜肽變體)與(b)就目標胜肽取得之結果MS訊號,以T2細胞標準化至細胞數。
[圖6]細胞毒性檢測。功能親和力(EC50)是經評估TCR轉染T細胞對載有KLK3胜肽之T2細胞殺滅效率而得知。冷光素酶組成性表達T2細胞裝載滴定量之KLK3胜肽類同位素分子後與經特定TCR轉染之CD8+ T細胞共同培養。測量瀕死T2細胞所釋出上清液中之冷光素酶活性,據以分析殺傷效果。以載入無關NYESO胜肽之T2細胞為陰性對照並以針對NYESO胜肽之TCR為陽性對照,如圖所示。X符號代表經免疫沉澱後由LC/MS測量而得的個別T2胜肽裝載濃度每一細胞之絕對拷貝數。
[圖7]顯示經同位素方式選定PRAME胜肽變體 (「類同位素分子」)間之前體離子(全MS)或結果片段離子 (MS/MS; 例如2+ 前體離子配合507.83 m/z)含量差異。
[圖8]顯示針對已知胜肽(在此範例中,取自PRAME (SLLQHLIGL),n = 7)之所選同位素標記變體(「類同位素分子」)之校正曲線產生方式。於質譜儀讀數中可是別出不同變體。
基於上述校正曲線,可將已知實驗中測得之MS訊號轉換為個別同位素標記變體之濃度。所根據之方法可見於,例如,WO2016107740A1,其內容僅為使本發明可據以實施之目的而併入本文。
[圖9]顯示以下兩者間之滴定曲線:(a) 目標胜肽(不同濃度且以不同方式標記之胜肽變體)與(b) 就目標胜肽取得之結果MS訊號,分別以T2細胞(圖9A)、Hs695T細胞 (圖9B)或 T98G細胞 (圖9C) 標準化至細胞數。
[圖10]顯示PRAME胜肽裝載之功能分析,及測試載有PRAME胜肽之T98G細胞配合外周血單個核細胞(PBMC)在滴定PRAME特異可溶T細胞受體 (「TCER」)存在下產生之殺滅效率;所述TCER係如「PCT/EP2020/050936」中所揭露者,為使本發明可據以實施之目的,該案內容經引用併入本文。T98G細胞載以滴定量之PRAME胜肽類同位素分子,而後與具有兩種不同施體之PBMC及在進行PRAME特異性TCER之滴定下共培養。測量瀕死T98G細胞所釋出上清液中之乳酸去氫酶(LDH)含量,據以分析細胞毒性。
[圖11A]以範例方式說明由α1及α2域所形成之MHC第一型分子胜肽結合槽,其具有結合胜肽之所謂錨著殘基(anchor residues)。
[圖11B]提供非特異性HLA第一型同種異型之序列標誌,顯示在包括錨著殘基(anchor residues)P2及P9等不同位置之胺基酸偏好。
[序列表] <![CDATA[<110> 德商英麥提克生物技術股份有限公司(Immatics Biotechnologies GmbH)]]> <![CDATA[<120> 目標胜肽與MHC分子間結合特性之特性描述方法]]> <![CDATA[<130> ID 47564]]> <![CDATA[<160> 2 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 1]]> Ser Leu Phe His Pro Glu Asp Thr Gly Gln Val 1 5 10 <![CDATA[<210> 2]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 2]]> Ser Leu Leu Gln His Leu Ile Gly Leu 1 5
Claims (30)
- 一種目標胜肽與已知細胞類型MHC分子間結合特性之特性描述方法,該方法包含以下步驟: a) 提供二或多個特徵為在其表面展現MHC分子之細胞; b) 將所述二或多個細胞分別放入二或多個容器中,使得每一容器包含一或多個細胞; c) 於上述不同容器中加入(=「裝載」)一目標胜肽之不同變體,其中所述胜肽之該等變體係經標記且具有相同之胺基酸序列,但具有不同之 (i) 標記類型;及 (ii) 濃度, 並使所述細胞與之接觸,藉此於所述不同容器中在該等細胞之表面形成胜肽MHC複合體; d) 分離出如此形成之胜肽MHC複合體;及 e) 判定步驟c)中所形成不同胜肽MHC複合體之濃度。
- 如請求項1所述之方法,其中,該MHC分子為MHC第一型。
- 如請求項1或2所述之方法,其中,該目標胜肽之長度介於8至15個胺基酸殘基之間。
- 如以上任一請求項所述之方法,其中,該目標胜肽具有以下序列基序 : X mA 1X nA 2X o,其中, X為一蛋白胺基酸; A 1為一選自以下項目所構成群組之胺基酸:T、A、E、I、L、P、S、V及Y; A 2為一選自以下項目所構成群組之胺基酸:Y、F、I、K、L、V及W; m為一介於1與10間之整數; n是6; o為一介於≥ 1與≤ 10間之整數;且 m + o ≤ 7。
- 如以上任一請求項所述之方法,其中,該目標胜肽為一腫瘤相關胜肽(TUMAP)。
- 如以上任一請求項所述之方法,其中,所述目標胜肽之該等變體係經同位素標記(「類同位素分子」)。
- 如請求項6所述之方法,其中,所述同位素標記包含至少一同位素標記胺基酸。
- 如請求項6或7所述之方法,其中,所述目標胜肽之該等變體在同位素標記之類型上互不相同。
- 如以上任一請求項所述之方法,其中,該胜肽MHC複合體係經由免疫親和強化而分離。
- 如請求項9所述之方法,其中,所述免疫親和強化係使用一MHC結合多肽執行。
- 如請求項10所述之方法,其中,該免疫親和強化係使用一MHC特異性抗體執行。
- 如以上任一請求項所述之方法,其中,待分離所述胜肽MHC複合體後,自該等MHC沖提出該等胜肽。
- 如請求項12所述之方法,其中,係於沖提液中判定所述不同胜肽變體之濃度,藉此判定步驟c)中所形成不同胜肽MHC複合體之濃度。
- 如以上任一請求項所述之方法,其中,包括判定 (i) 該等細胞於步驟b)中所接觸之至少一目標胜肽之濃度與 (ii) 步驟c)中所形成不同胜肽MHC複合體之濃度 兩者之比率。
- 如以上任一請求項所述之方法,其中,還包括,就每一所述胜肽變體,判定與其接觸之細胞數。
- 如請求項14或15所述之方法,其中,該計算而得之比率為該等細胞於步驟b)中所接觸之胜肽濃度 (µg mL-1或nM)對每一細胞中pMHC複合體內胜肽拷貝數之比率。
- 如以上任一請求項所述之方法,其中,所述不同胜肽變體之濃度係於所述一或多個細胞上經由至少一選自以下項目所構成群組之方法判定: 質譜法 (MS); 串聯式質譜法 (MS/MS);及 液相層析串聯式質譜法 (LC-MS,LC-MS/MS)。
- 如以上任一請求項所述之方法,其中,構成所述胜肽MHC複合體之該胜肽為並非由一已建立之細胞系所呈現之胜肽。
- 如以上任一請求項所述之方法,其中,該二或多個以在其表面展現MHC分子為其特性之細胞係缺乏胜肽抗原加工及/或胜肽抗原呈現。
- 如請求項19所述之方法,其中,該細胞缺乏胜肽抗原加工及/或呈現之原因是缺乏抗原加工相關性傳遞蛋白(TAP)。
- 如請求項19或20所述之方法,其中,該細胞缺乏胜肽抗原加工及/或呈現導致其細胞表面產生功能上「空缺」之第一型MHC之表達。
- 如請求項19至21中任一項所述之方法,其中,該細胞係選自下列項目所構成之群組: T2 (174xCEM.T2); RMA-S; B-LCL 721.174或B-LCL 721.180;及 C1R-T134K。
- 如以上任一請求項所述之方法,還包含對至少一部分與已所述目標胜肽接觸之該等細胞進行一檢測,該檢測可顯示一pMHC結合蛋白或一pMHC結合細胞對上述形成胜肽MHC複合體之相互作用之特性。
- 如以上任一請求項所述之方法,包含判定關聯於該pMHC結合蛋白或該pMHC結合細胞與該pMHC間相互作用之劑量反應關係。
- 如請求項23或24所述之方法,其中,該檢測為一生物檢測。
- 如請求項23至25中任一項所述之方法,其中,該生物檢測為一細胞激素釋出檢測。
- 如請求項23至26中任一項所述之方法,其中,該檢測為一體外檢測。
- 如請求項23至24及27中任一項所述之方法,其中,該體外檢測為一表面電漿共振檢測。
- 如請求項23至28中任一項所述之方法,其中,該pMHC結合蛋白係選自下列項目所構成之群組: 一T細胞受體,或其目標結合片段;及 一TCR模擬抗體,或其目標結合片段。
- 如請求項23至28中任一項所述之方法,其中,該pMHC結合細胞為一T細胞。
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