TWI394834B - 作為編碼抗原和蛋白質毒素之核苷酸序列的載體之微生物、製造彼之方法及彼之用途 - Google Patents
作為編碼抗原和蛋白質毒素之核苷酸序列的載體之微生物、製造彼之方法及彼之用途 Download PDFInfo
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Description
本發明關於作為編碼抗原和蛋白質毒素之異源核苷酸序列的載體之微生物,製造彼之方法以及對應之質體或表現載體。這些微生物可作為藥物,尤其是作為用於治療不同腫瘤之腫瘤疫苗。
癌症之免疫療法代表地種腫瘤治療法的可能選項。利用不同方法之多樣臨床試驗係集中於其在患者體內之效力。原則上,被動及主動免疫療法間係有所區別。
主動免疫療法係針對誘導與疫苗相關之腫瘤特異性免疫反應。後者目前在臨床上係利用數種不同之方法探查。例如:有類稱為全細胞疫苗者,其原料為直接自患者取得(自體)或自適當之細胞株衍生(異源)之腫瘤細胞。然後,通常係將這些細胞去活化,差異處理後再重新施予於患者身上。
相反地,抗原特異性疫苗含有一(或更多)腫瘤特異性抗原、部分該抗原或編碼該特異性抗原之DNA以及抗獨特型疫苗。通常,這些疫苗並非單獨隔離而係與適當之載體一起注射。因此,一方面係使用不同之典型佐劑,但亦可與生物免疫刺激劑(諸如細胞活素)組合。
為了進行免疫刺激可應用含有與免疫刺激劑(諸如破傷風毒素)聯結之抗原的方法。再者,有些嘗試係將抗原
與樹突細胞一起組合施予。最後,有些嘗試係使用具有病毒或細菌載體之重組活疫苗。
很長一段時間係使用由作為佐劑之細菌毒素(諸如破傷風毒素、志賀毒素、致死毒素或霍亂毒素)與抗原所組成之融合蛋白質來作為疫苗,尤其是可用來對抗感染者(Freytag及Clements,1999)。另外,亦可使用天然毒素(其通常與標靶細胞特異性分子(諸如腫瘤細胞之細胞表面分子)融合)以破壞標靶細胞。
在此方面,具天然毒素之融合蛋白質(其通常包含酶性單位及蛋白質-結合結構區)在作為佐劑時可發展其理想之作用(Freytag及Clements,1999)。藉由這些疫苗可取得令人滿意之免疫反應,即使是在黏膜(尤其是口腔)免疫後亦是。使用這些融合蛋白質之麻煩為天然毒素具高度毒性,因此無法在人體中設立(Holmgren,等人,2005)。
因此,一連串之研究全部係關於將毒素去毒性並同時保存佐劑之效果。然而,由於在大部分情況中,佐劑之效果與造成毒性效果之酶促活性相一致(Lycke,等人,1992),去毒性無法直接執行,即使某些毒素顯示出未損失其酶促、佐劑活性(Hormozi,等人,1999;Lycke,等人,1992)。
在霍亂毒素(CT)之案例中有數種去毒性之嘗試(Agren et al., 1999;Byun et al., 2001;Eriksson et al., 2004;Kweon et al., 2002;Sanchez et al., 2002),然而
,霍亂毒素作為黏膜佐劑很普遍(Freytag及Clements)。因此,總結上述,可有效誘導抗體反應(主要為黏膜IgA),使抗體反應中與毒素相關之受第Ⅱ類MHC限制的T細胞支援增加為該用於疫苗(其由蛋白質-抗原及經融合或共同投服之毒素所組成)之黏膜佐劑的主要先決條件(Freytag及Clements)。
在霍亂毒素(尤其是其B-次單位(CtxB))方面係以作為佐劑之形式進行測試,因其負責結合GM-1受體且當單獨分離時並不顯示毒性效果(Holmgren,等人)。具CtxB之蛋白質融合物的特徵為主要誘導Th2免疫反應。這些為T細胞反應,其主要由細胞活素(諸如IL-4或IL-6)決定其特徵,且其主要誘出抗體,但其幾乎不或最多有限地起始一細胞免疫反應,尤其是細胞毒性T細胞反應(CTL)(Holmgren,等人)。
另外,CtxB作為黏膜佐劑可引起蛋白質-抗原系統性耐受性。系統性耐受性一詞係描述抗原特異性淋巴球(尤其是T細胞或B細胞)之竭盡或去活化。因此,此種方法不能用來誘導系統性免疫反應(Holmgren,等人)。
相對於黏膜施予,腹膜內或皮下施予毒素-抗原融合蛋白質可引起系統性以及低細胞毒性反應。確實,此已用於模型系統中之腫瘤疫苗接種(參考,如(Becerra,等人,2003))。然而,這類反應亦可以純化之抗原本身取得且主要係取決於所使用之佐劑。除了在模型中所測得之CTL反應非常弱外,甚至並無證據顯示該保護作用係倚賴
這些效果。再者,該抗原並不經由口服施予而僅能經由直接注射(s.c.、i.d.、i.m.、i.p.)抗原來施予。
融合至去毒性之毒素的抗原蛋白質若以口服腫瘤疫苗之形式施予時通常為無效。主要的原因為(若有)其僅能誘導出低水準之系統性免疫反應或者,在CtxB之情況中,甚至誘導出系統性耐受性及誘導出黏膜限制之抗體及Th2型免疫反應。
例如:McSorley,等人證明以CtxB-抗原融合蛋白質(對腫瘤疫苗而言,其代表引起系統性反應之較佳方式)進行鼻部免疫化時宜容耐Th1細胞並將其去活化,然而,Th2細胞則不受影響(McSorley,等人1998)。Th2反應之特徵在於主要為製造IL-4或IL-6之T-輔助者細胞。尤其是,這些細胞活素負責啟動B細胞製造抗體,這些抗體可在大部分之習知疫苗中提供保護作用。相反的,Th1 T細胞大部分分泌IL-2及IFN-γ這類細胞活素而藉此參與細胞性免疫反應。根據免疫策略之目的來啟動以抗體為主之Th2免疫反應(稱為Th2偏移)或以細胞為主之Th1反應(稱為Th1偏移)非常地重要。
相對地,系統性誘導具有製造IFN-γ之T輔助細胞的Th1為主之細胞性免疫反應及誘導細胞毒性T細胞(CTL)對腫瘤免疫療法而言是必要的。
習知技藝顯示出毒素可作為佐劑。尤其是,霍亂毒素(CT)顯示強烈之佐劑效果。然而,此作用在毒素被去毒性時很快地明顯減弱。在其次單位CtxB方面,口服施予
甚至可誘導系統性耐受性。此問題可經由鼻部施予來部分避免。然而,若應用鼻部投服則出現其他問題,尤其是,諸如:關於GM-1受體在腦中之表現的與CtxB次單位相關的問題(van Ginkel,等人,2000),這些問題反應在特別嚴重之副作用的風險將增加(Mutsch,等人,2004)。
為了克服這些問題,可製造共同表現毒素及(異源)抗原的重組活疫苗。此選擇在感染疫苗(即,針對特殊病原,如:結核病病原,且意圖誘導對抗該病原之免疫力的疫苗)方面已在努力。
在發展這類感染疫苗之過程中曾利用數種重組細菌株來產生為存活或去活化疫苗形式之經口投服的與適當(異源)抗原融合之重組毒素。在大部分情況中,產生之菌株僅表現重組毒素。因此,這些方法主要僅針對毒素本身(誘導抗體反應)及,必然地,表現毒素之菌株(病原)產生免疫性。這類疫苗並不適合作為腫瘤療法中可能使用之腫瘤疫苗,因而,不令人意外地,相關試驗中並未提及任何這類可能之應用(Reveneau et al., 2002)(Vertiev et al., 2001)(Freytag and Clements, 1999;Jackson et al., 1996)。
關於這類感染疫苗,在誘導抗體反應之過程中,毒素並不作為佐劑而是誘導較佔上風之黏膜抗體免疫反應。這些感染疫苗試驗不包括或僅有非常微弱之系統性免疫反應。相對於腫瘤疫苗,這類疫苗並不尋求誘導細胞性免疫反
應,尤其是細胞毒性T細胞。正常情況下,表現毒素之細菌病原顯示胞外存活形態,因此,並不會造成CTL活化,即:由這類感染疫苗提供之保護作用為CTL獨立性。
下列細菌菌株已用於上述之感染疫苗試驗中:重組之乳桿菌(Reveneau et al., 2002)、李斯特氏菌(Vertiev et al., 2001)、炭疽桿菌(Mendelson et al., 2005;Mesnage et al., 1999)、志賀氏菌(Anderson et al., 2000;Tzschaschel et al., 1996b)、大腸桿菌(Walker et al., 1992)、弧菌(Butterton et al., 1995;Chen et al., 1998;Thungapathra et al., 1999)及沙門氏菌(Jackson et al., 1996)。
另外,亦嘗試藉由表面遞呈來增強黏膜免疫反應(Konieczny et al., 2000)或以異源抗原(沙門氏菌、志賀氏菌:(Garmory et al.,2003;Orr et al., 1999;Su et al., 1991;Tzschaschel et al., 1996a;Tzschaschel et al., 1996b)、耶爾森氏菌:(Sory and Cornelis, 1990)、弧菌(Ryan et al., 1997a)、大腸桿菌:(Zhu et al., 2006))之形式分泌毒素。然而,在這些額外案例中,毒素本身代表該免疫反應所針對之抗原。
因此,需注意:毒素並非作為佐劑,所描述之研究亦不企圖將毒素與另外之分別(異源)抗原一起以融合蛋白質之形式表現。再者,具CT或CtxB之融合蛋白質並非以這些系統產生且亦不建議如此。
因此,上述文件中所提及之融合蛋白質為肽分泌訊號
(諸如HlyA)與毒素蛋白質之融合蛋白質,而非由毒素與(異源)抗原所組成之融合蛋白質。
這些研究之主要目的僅為取得理想之黏膜免疫反應(Tzschaschel et al., 1996a)。誘導系統性免疫反應並非尋求之目標。若最後進行測量,則由於這類模型中之保護作用僅由抗體傳介(比較,如:[31]),因此,系統性免疫反應之分析僅限於抗體(尤其是系統性IgA)。這些研究對於系統性細胞性免疫反應(尤其是細胞毒性T細胞反應)之誘導作用並無描述。與分泌訊號融合原係分別用來增加毒素之溶解度及增強其穩定性,因為強烈、單純之胞質性表現通常會產生不溶性聚集物(Gentschev et al., 2002a)。
於此觀點,某些作者觀察到具分泌訊號之融合蛋白質可造成快速之胞質性降解(Tzschaschel et al., 1996a),然而,其他人則觀察到安定作用(Orr et al., 1999)。因此,習知技藝在此處是有些矛盾的。目前為止,先前以分泌之毒素(毒素+分泌訊號)進行的實驗係針對增加穩定性,而此點顯然並未在所有案例中達到。
當然,其中一種理由在於表現強度及質體穩定性不同。Tzschaschel,等人描述所使用之質體系統非常不穩定,且無選擇的,僅可在一些細菌中找列。該作者經由迷你轉位子(transposon)利用染色體整合來作為可能之解決方法(Tzschaschel et al., 1996a;Tzschaschel et al., 1996b)。
然而,這類系統擁有數種不利之處。一方面,整合點並未限定,此可導致宿主株有令人不滿意的表型轉變(如:增加/減少鄰接基因之表現)。另一方面,使用單一基因組複本的預期表現水準低,此對致免疫力具負面效果。畢竟,染色體轉位子整合相當不穩定,因其造成重複成分處之經常性的自發性切除。
如上述,習知技藝在分泌之異源毒素的穩定性方面有些矛盾。
Garmory,等人甚至假設分泌異源抗原在致免疫性方面並無任何特殊益處(Garmony,等人,2002;Roland,等人,2005)。在其他案例中,靜脈內投服後確實觀察到對分泌之毒素的系統性抗體反應增加,但口服施予後則不。完全相反地,口服施予甚至間接地產生問題(Roland,等人,2005)。
最終,以製造破傷風毒素之革蘭氏陽性菌株(植物乳桿菌)進行之研究顯示出與分泌毒素之菌株(毒素之呈現方式為結合在細菌之細胞膜或包含在細胞質內)相較下,其在誘導系統性抗體反應上並無顯著差異(Reveneau,等人,2002)。
先前研究中並未研究亦未描述系統性細胞性免疫反應(尤其是細胞毒性T細胞)及所產生之保護作用。
因此,若分泌作為佐劑之毒素代表一種優點,則上述以毒素為基礎之感染疫苗的習知技藝便不能表示對誘導系統性(細胞性)免疫反應的顧慮。完全相反地,更確切地
說,上述研究偏重於指出穩定性之問題並提出分泌之異源毒素之遺漏的優點。習知技藝中完全未描述或提出由分泌訊號、毒素及異源抗原所組成之融合蛋白質。
在上述章節中提出描述細菌載體之習知技藝,該細菌載體異源地表現毒素且可作為感染疫苗。在其列舉之實例中主要係進行關於毒素之表現或穩定性以及其溶解度之改良,例如:插入強表現啟動因子或將毒素與分泌訊號融合。
其他作者亦研究活疫苗中之毒素與異源抗原的融合物。在這些案例中,毒素大部分係作為佐劑。在某些案例中(如(Brossier,等人,2000)),異源抗原係作為佐劑而毒素係作為適當之抗原。
然而,需注意:在所提及之案例中,毒素-抗原基因融合構造物僅表現於細胞質或周質中。毒素-抗原構造物既不融合至額外之分泌訊號(此可使其完全分泌)亦不直接分泌。
在這些構成毒素-抗原基因融合構造物之過程中係使用重組之大腸桿菌(Clemens et al., 2004)、炭疽桿菌(Brossier al., 2000)、志賀氏菌(Koprowski et al., 2000;Ranallo et al., 2005;Zheng et al., 2005)及弧菌(Silva et al., 2003)。在沙門氏菌(摘要於(Garmory et al., 2002))方面,亦有CT變種與抗原(Hajishengallis et al., 1996;Huang et al., 2000)或其他毒素與抗原(Barry et al., 1996;Cardenas and Clements, 1993;
Chabalgoity et al., 1997;Chabalgoity et al., 1996;Chabalgoity et al., 2000;Chabalgoity et al., 1995;Chacon et al., 1996;Jagusztyn-Krynicka et al., 1993;Khan et al., 1994a;Khan et al., 1994b;Lee et al., 2000;POgonka et al., 2003;Schodel et al., 1990;Smerdou et al.,1996;Ward et al., 1999;Wu et al., 2000)之融合物的描述。
在大部分之這些案例中,主要之焦點係在誘導黏膜(抗體)免疫反應而在誘導系統性免疫反應方面僅選擇皮下施予而不選擇口服施予[36]。
一些以沙門氏菌作為援助菌株之工作僅限於用來決定菌株之特徵(Gomez-Duarte et al., 1995;Jagusztyn-Krynicka et al., 1993),其他僅分析黏膜及/或系統性抗體反應及/或保護作用(Barry et al., 1996;Cardenas and Clements,1993;Dunstan et al., 2003;Hajishengallis et al., 1996;Harokopakis et al., 1997;Khan et al., 1994a;Khan et al., 1994b;Pogonka et al., 2003;Smerdou et al., 1996;Somner et al., 1999)。在所有這些其中僅偶然使用抗原與破傷風毒素之融合物且只作為感染疫苗之案例中並無系統性細胞性免疫反應,尤其是細胞毒性T細胞反應之研究。
因此,從免疫學觀點來看,從那些研究中無法取得關於作為腫瘤疫苗之可能用途的結論,因其焦點係設定在由抗體傳介之作為感染疫苗的效果。
包含對研究過之免疫反應的同型(isotype)分析的研究調查顯示出更具相關性。事實上,在這些案例中並不直接測量細胞性免疫反應,僅有抗體反應之同型變化形廓提供免疫反應之Th1/Th2偏移的結論。如IgG1之抗體同型與Th2反應有關而如IgG2a之抗體同型則與Th1反應有關。如已提及者,Th1反應為以細胞為主之免疫反應,而Th2反應主要係代表由體液抗體驅動之反應。再者,那些研究並未描述腫瘤疫苗亦未提出任何作為抗腫瘤劑之用途。
一種以作為載體之沙門氏菌(其表現破傷風毒素-抗原融合蛋白質)為基礎之感染疫苗研究已在狗體內進行。在狗體內誘導之低抗體反應在抗體變化形廓方面顯示出Th1偏移,因此,其為與細胞型免疫反應較有關之反應(Chabalgoity et al., 2000)。無可否認地,狗之免疫學的研究很少,因此,狗之抗體的變化形廓可以提供之關於Th1偏移的資訊有多少並不清楚。
然而,由相同團體利用可相比擬之構造物在小鼠中進行之研究顯示出IgG1及IgG2a具相同水準之抗體變化形廓,此暗示一種混合之Th1/Th2反應。
有趣的是,現存之抗破傷風毒素的免疫力(由於先前進行之免疫處理,其可在大部分人類中找到)可另外強烈地誘導出IgG1,而IgG2a則很難誘導出。此清楚地指出明確的th2偏移(Chabalgoity et al., 1995)。因此,人類使用以破傷風毒素為基礎之活疫苗作為腫瘤疫苗時頗具傷
害性。因可預期的是,在大部分這些顯示破傷風毒素特異性反應的患者中可誘導出強烈之以Th2抗體為主的反應。
僅很少數之研究亦分析細胞性免疫反應且對具有及不具有毒素融合物之基因構造物進行比較。例如:在一種案例中係在沙門氏菌中比較具有及不具有破傷風毒素之抗原融合物(Lee,等人)。其中,破傷風毒素抗原融合構造物主要係增加總體抗體水準,然而,Th1/Th2變化形廓幾乎沒有改變。即使是該抗原特異性CD4+
T細胞在典型Th1細胞活素(諸如:分別為IFN-γ及IL-2)的分泌情形亦僅顯示些微之分岐性。由相同團體稍早進行之研究亦測量細胞性IFN-γ之水準。然而,並未與不具破傷風毒素之構造物(Chabalgoity,等人)進行比較。其他以不同之革蘭氏陰性細菌載體(如,志賀氏菌)(Koprowski et al., ;Ranallo et al.,; Zheng et al.)或弧菌(Campos et al., ;Ryan et al.)進行之研究亦未分別分析同型及細胞性免疫反應。
總之,可確定的是,所提出之研究清楚聚焦在誘導由抗體驅動之體液免疫反應。確實,其使用基因毒素抗原構造物,但這些構造物並不帶有分泌訊號且亦不被直接分泌。然而,絕無系統性細胞免疫反應之分析,尤其是細胞毒性T細胞反應。再者,這類細胞性細胞毒性T細胞反應無法自體液抗體反應推定且若抗體反應係由Th1/Th2組成,則無法偵測。
不過,這些細胞性細胞毒性T細胞免疫反應對腫瘤疫
苗接種療法確實非常重要。
因此,就僅限於黏膜感染疫苗之毒素-抗原融合物而言,發展中之技藝的現況並不允許任何關於任何這類構造物於作為腫瘤疫苗上的可能用途的聲明。
如上述,基因融合構造物表現時可不需分泌系統之協助。毒素及毒素抗原構造物通常分別位於細胞質及周質(即,介於二種膜之間)。為了誘導有效之細胞性免疫反應,毒素必須可讓抗原-遞呈細胞(APC)自由接近。通常,天然毒素係在革蘭氏陰性細菌之周質中製造。此對單純之黏膜免疫反應而言已是足夠,因周質毒素可自結腸中之周質流出,因此亦為可接近者(Hunt and Hardy, 1991)。然而,若載體瞄準結腸外之抗原-遞呈細胞(諸如集合淋巴結(Peyer's Patch)或淋巴器官,如:淋巴結或脾臟),則此並無意義。
原則上,二種因子對腫瘤疫苗之效力具關鍵性:Th1-型細胞性免疫反應之誘導及固有免疫系統之組成分(諸如NK細胞、NKT-細胞及γ-δ T細胞,其對腫瘤療法之效力而言很重要)的參與(Dunn et al., 2004)。
這些固有免疫系統之組成分的重要性落在多種層級。經適當活化之NR-及γ-δ T細胞可局部製造大量IFN-γ。此干擾素(其亦由特異性Th1偏極之T細胞製造)在腫瘤療法中有多種功能。其中心功能之一為抑制血管生成,其中斷腫瘤之氧及營養供應,使腫瘤飢餓。再者,NX細胞擁有辨識第Ⅰ類MHC分子的受體。若這些分子呈現在
細胞上則NK細胞將受到抑制。
在誘導特異性細胞毒性T細胞之疫苗方面,這些CTLs可殺死腫瘤細胞。若腫瘤細胞失去其表現第Ⅰ類MHC分子的能力(此在腫瘤中經常發生)則特異性細胞毒性T細胞失效。因此,在此案例中,對NK細胞之抑制作用終止,NK細胞即可直接排除腫瘤細胞。
因此,若腫瘤疫苗能有效地誘導出二種組成分即為理想情況。關於融合之毒素佐劑的Th1-Th2偏移方面有些矛盾之數據。如稍早所討論者,以分離方式施予之毒素抗原融合構造物可明顯誘導出強烈之Th2偏極的免疫反應。某些作者對活疫苗載體之描述仍為低度之Th2偏移;其他作者則見到輕微之Th1偏移。
然而,這些數據亦完全以非分泌之構造物為基礎。藉由這類感染疫苗誘導固有之免疫力從未曾經過比較或考慮。
如同已提出者,主要理由為現存之疫苗為黏膜感染疫苗,而非腫瘤疫苗。因此,對Th1免疫反應、CTL免疫反應及固有之免疫系統反應之誘導並非焦點。相反的,考慮腫瘤疫苗時,誘導這些免疫反應是不可少的。
有趣的是,在細胞生物學中通常係使用天然毒素作為傳信通路之抑制劑。因此,在各毒素中,天然百日咳毒素顯示出可抑制NK細胞之特殊細胞凋亡樣式,但霍亂毒素則不。一種不同研究顯示出霍亂毒素(但其B-次單位則不)阻斷特殊之NK細胞功能(Poggi,等人,1996)。天然
百日咳毒素係用來抑制淋巴球之趨化作用(Spangrude,等人,1985)。即便這些研究與腫瘤疫苗接種無關,本技藝之技術熟習人士亦可歸結出使用毒素作為腫瘤疫苗有害之結論,因對腫瘤療法具關鍵性之固有免疫系統之反應係受到抑制而非受誘導。
其他研究證實天然百日咳毒素(但非去活化之百日咳毒素)之類的毒素能有效地誘導固有免疫系統之組成分。若真考慮對抗腫瘤之免疫療法時,此表示需使用天然毒素。然而,由於毒性原因,這類投藥是不可行的。再者,這類誘導將不可避免地引致由Th2指導之次級免疫反應,此將對腫瘤療法有害(Boyd,等人,2005)。因此,在誘導固有免疫反應方面,習知技藝既未描述亦不曾考慮接種腫瘤疫苗。相反地,文獻中之評論分析甚至不利於在腫瘤療法中使用毒素。
然而,有趣的為一種毒素或其次單位與其他刺激劑之協同作用的分析(諸如以低甲基化之CpG再現單位(CpGODN)(Holmgren,等人,2005)或脂多醣(LPS)免疫刺激DNA寡核苷酸)。在LPS之案例中顯示出原先主要係透過毒素之B-次單位而增加對單核細胞之誘導,然而,整體來說,其係被整個毒素(全毒素(holotoxin))抑制(Hajishengallis et al., 2004)。然而,這些研究完全倚賴使用純化之毒素-抗原-融合構造物(其中係加入LPS或CpG之類的物質作為佐劑)。再者,僅對可誘導適合之免疫反應但不直接攻擊腫瘤之巨噬細胞進行分析。因此,這
些研究在誘導固有免疫系統之組成分(尤其是可直接攻擊腫瘤之NK細胞)方面並無重要性。
然而,其他研究顯示NK細胞可被活化並被毒素(像銅綠假單胞菌外毒素A)以趨化方式攻擊(Muhlen,等人,2004)。根據該實驗系統,Th1反應亦可被誘導,雖然,NK細胞及Th1反應很可能因此受到抑制(Michalkiewicz et al., 1999)。然而,這些分析主要係瞄準腸毒素A之肝毒性的分析且與接種腫瘤疫苗無關。有趣的是,該效果為高度之劑量倚賴性,只要稍微改變劑量即可使效果逆轉。再者,作者無法揭露何種反應可在活體內有效發生。因此,從該數據無法預測若使用毒素或甚至是去毒性之毒素作為佐劑時可能發生何種效果。
總之,免疫反應強烈倚賴所應用之特殊系統。在大部分案例中,黏膜抗感染疫苗係瞄準黏膜處之免疫系統局部運作,以誘導有效之黏膜免疫反應(Lycke,2005)。然而,這些研究並不包括研發腫瘤疫苗。另外,這些研究缺乏關於誘導系統性細胞性免疫反應(尤其是腫瘤疫苗接種所必要之細胞毒性T細胞反應)之資訊。
分泌異源抗原已經證明可提供有利於系統性免疫反應之益處(Hess,等人,1996)。然而,所描述之經分泌的抗原並非經分泌之毒素-抗原構造物;其中並未使用毒素或其次單位。在基因轉殖腫瘤模型中藉此取得之免疫反應亦高度受限(Gentschev,等人,2005)。確實,此案例中可誘導微弱之抗體及細胞毒性T細胞反應,以藉此部分
防止腫瘤惡化。然而,不僅是免疫反應本身受限,還有保護作用本身亦受限。同樣地,這些腫瘤疫苗接種研究缺乏與非分泌形之構造物的比較。再者,腫瘤疫苗接種並未進行比較性研究(Hess,等人,1996)且相對於進一步之研究,其未見關於分泌之任何益處(Garmory,等人,2002;Roland,等人,2005)。
總之,如前述,習知技藝對分泌之看法相當矛盾且對腫瘤療法中分泌形毒素-抗原構造物並無任何關於潛在利益的建議。完全相反的,現有文獻之評論分析較不贊同這類用途。
細菌毒素(Todar,2002):在一種化學水準下有二種類型之細菌毒素,脂多醣類(其與革蘭氏陰性細菌之細胞壁結合)及蛋白質類(其自細菌細胞釋出且可在遠離細菌生長部位之組織部位作用)。與細胞結合之脂多醣(LPS)毒素係稱為內毒素而胞外擴散性毒素則稱為外毒素。
外毒素為存活細菌在指數生長期間所分泌之典型的可溶性蛋白質,但在某些案例中其係經由細菌細胞溶解而釋出。毒素通常係由引起該與毒素相關之疾病的特殊菌株所特別製造(如:僅破傷風桿菌製造破傷風毒素;僅白喉桿菌製造白喉毒素)。革蘭氏陽性細菌及革蘭氏陰性細菌均製造可溶性蛋白質毒素。
一般而言,現存之蛋白質(外-)毒素有三種類別:(i)第Ⅰ型毒素(超抗原),其結合至宿主細胞表面並調
節免疫反應,但不轉位入細胞內,(ii)第Ⅱ型毒素(打洞毒素(pore-forming toxins)),其作用在宿主細胞膜並使宿主細胞滲漏及死亡,(iii)第Ⅲ型毒素(A-B毒素),其經由一特異性受體結合至宿主細胞並轉位入細胞內,在其中變成活性並修飾宿主細胞之蛋白質或其他組成分。
如上述指出,在宿主細胞內於胞內作用之第Ⅲ型毒素係由二種組成分組成:一種組成分(次單位A)造成毒素之酶活性;另一種組成分(次單位B)與結合至宿主細胞膜上之特殊受體有關並可將酶轉移通過細胞膜。酶組成分需待其自天然(A+B)毒素釋出後才具有活性。分離之A次單位具有酶活性但缺乏結合及進入細胞之能力。分離之B次單位可結合靶的細胞(甚至阻斷天然毒素之結合),但其不具毒性。
毒素次單位可有多種不同之合成及排列方式:A+B表示該毒素係以在靶的細胞表面交互作用之二種分別的蛋白質次單位形式合成及分泌;A-B或A-5B或AB5表示A及B次單位係分別合成,但在分泌期間藉由非共價鍵聯結並結合至其靶的;5B或B5表示該蛋白質之結合結構區係由5個相同之次單位所組成。AB或A/B代表毒素以單肽形式合成,分成可藉由蛋白質水解裂解而分開之A及B結構區的毒素。AB或A/B毒素之實例為白喉毒素、外毒素A、肉毒桿菌毒素及破傷風毒素。A-5B或AB5毒素之實例為霍亂毒素及志賀毒素,而炭疽毒素LF及炭疽毒素EF為
A-B毒素之實例。
習知技藝之其他相關文件包含下列:Michl,等人描述細菌及細菌毒素於作為實體腫瘤治療劑之用途。其中揭示毒素-抗原融合構造物及這類構造物之細菌靶向,研究白喉毒素(ET)、假單胞菌外毒素A(PE)及產氣莢膜桿菌腸毒素(CPE)之用途。然而,作者並未提及霍亂毒素,亦未證明或明示由細菌遞送之毒素-抗原融合構造物分泌(Michl及Gress,2004)。
Lahiri提出關於不同細菌毒素之概述,並討論其種種用途。雖然作者提及毒素-抗原融合構造物,但其並未談及霍亂毒素及分泌之毒素-抗原融合構造物的細菌靶向(Lahiri,2000)。
Lavelle,等人揭示作為免疫調節藥物之感染劑分子。該作者亦提及霍亂毒素-抗原融合蛋白質,但這類構造物僅直接以蛋白質之形式施予,並不藉由經遺傳修改之活疫苗施予。(Lavelle,等人,2004)。
WO 01/74383係針對嵌合形抗原-腸毒素黏膜免疫原亦提及霍亂毒素次單位A2及B之用途。然而,這類嵌合形免疫原總是同時包含A2及B次單位,且企圖用於黏膜免疫,而非用於腫瘤治療中。
WO 02/077249描述用於將異源蛋白質遞送至特異突變之靶的細胞的非致命性小腸結腸炎耶爾森氏菌突變株。其中亦提及霍亂毒素次單位A1之用途,但該專利文件並未提及分泌且僅提及感染病及感染狀態之治療。
WO 2004/018630揭示編碼雙股真核細胞表現盒之重組雙股RNA噬菌體。其中雖然提及霍亂毒素次單位A,但該文件並無進一步之相關內容。
Holmgren,等人概述黏膜免疫及佐劑之領域。尤其是,其討論霍亂毒素作為黏膜佐劑之效果,但未揭示關於遺傳表現系統或活疫苗之資訊,亦未提及腫瘤療法(Holmgren,等人,2003)。
Holmgren及Czerkinsky亦概述黏膜免疫性及疫苗。然而,此文章亦僅限於抗感染,並未討論腫瘤療法或明示於此領域之可能用途(Holmgren及Czerkinsky,2005)。
Freytag及Clements之另一評論討論黏膜佐劑在抗感染免疫療法中之應用。雖然提及霍亂毒素作為黏膜佐劑,但作者並未提及分泌之毒素-抗原構造物及腫瘤療法為可能之投藥領域(Freytag及Clements,2005)。
Shaw及Starnbach描述使用改良之細菌毒素遞送疫苗抗原。然而,該文章並未提及霍亂毒素,且為了疫苗接種之原因而受限於直接施予毒素-抗原融合蛋白質(Shaw及Starnbach,2003)。
WO 03/072789針對作為編碼用於治療腫瘤之細胞抗原之核苷酸序列的載體之微生物。雖然該專利文件提及分泌及於腫瘤治療領域中之用途,但其完全未提及細菌毒素及融合蛋白質。
Gentschev、Dietrich和Goebel以及Gentschev,等人描述細菌靶向及其於腫瘤疫苗研發中之用途。然而,此二
篇文章並未提及細菌毒素及融合蛋白質於腫瘤療法中之用途(Gentschev,等人,2002a;Gentschev,等人,2002b)。
WO 98/23763揭示表現大腸桿菌溶血素B及D次單位以及包含融合至hlyA之異源抗原的融合多肽之霍亂弧菌細胞。其進一步描述表現霍亂毒素次單位B及分泌訊號序列之融合多肽、異源抗原和霍亂毒素A2次單位之霍亂弧菌疫苗株。最後,其揭示一種包含融合至困難梭菌毒素A或毒素B次單位之抗原部分的霍亂毒素B次單位。然而,該專利申請案並未提及蛋白質毒素加上異源性非-蛋白質毒素抗原之融合蛋白質於腫瘤療法中之用途。
Dietrich與同事們討論二種疫苗遞送工具-溶血素A及李斯特菌素-其可用於細胞介導之免疫性。然而,其並未提及蛋白質毒素-異源抗原融合蛋白質或共同-表現(Dietrich,等人,2003)。
Gentschev,等人描述大腸桿菌之α-溶血素分泌系統於傷寒沙門氏菌Ty21a疫苗株中遞送抗原之用途。然而,該作者並未提及細菌毒素及融合蛋白質於腫瘤療法中之用途(Gentschev,等人,2004)。
WO 02/47727係針對包含蛋白質毒素之B次單位的治療劑。該文件僅揭示CtxB及EtxB與病毒抗原之融合蛋白質。其中未提及細菌疫苗或細菌疫苗遞送。
Cheng-hua S與同事們描述霍亂毒素B次單位與HBV PreS2抗原決定部位之基因融合物以及該融合蛋白質在直
接免疫研究中之抗原性。然而,其中未提及細菌疫苗或細菌疫苗遞送(Cheng-hua,等人,1995)。
Sanchez,等人揭示當與DNA疫苗經由皮下共同投服時,霍亂毒素B次單位基因可增強黏膜免疫球蛋白A、Th1型及CD8+
細胞毒性反應(Sanchez,等人,2004)。然而,此方法中,作者使用DNA作為載體,其本身可作為Th1促進佐劑。另外,蛋白質係由宿主細胞製造且非直接或經由細菌載體遞送,因此,可被真核細胞直接利用。
WO 01/29233係針對嵌合型致免疫組成物及編碼彼之核酸。然而,其中未提及細菌疫苗或細菌疫苗遞送。。
WO 2007/044406係關於利用以SopE攜帶型Ⅲ分泌訊號為根據之細菌抗原遞送系統來刺激免疫反應的方法。然而,該專利申請案中未提及細菌毒素及融合蛋白質於腫瘤療法中之用途。
總之,從習知技藝中可歸結出:天然毒素因為其毒性強因此不能用於人類。再者,將其應用於腫瘤療法中是有害的,因固有之免疫系統(尤其是NK細胞)的反應將受到抑制。然而,本免疫反應為成功之腫瘤療法的關鍵組成分,因腫瘤細胞經常失去其表現第Ⅰ類MHC分子的能力,因此,對於CTL之辨識及攻擊具抗性。
另一方面,使用單獨之去毒性毒素次單位或融合至(異源)抗原蛋白質之去毒性毒素次單位可造成被強烈減弱之佐劑效果及/或甚至引起免疫系統的系統性耐受性以及黏膜限制性抗體和Th2型免疫反應。
再者,分泌(異源)抗原-毒素融合蛋白質(其僅在抗感染疫苗(即,瞄準抗原或甚至是毒素本身)之過程中才被提及)不僅顯示出對細胞質表現無助益且一般而言並不適合。
總之,未曾提出腫瘤治療方法,亦未描述或達成以製造IFN-γ之T-輔助細胞來系統性誘導以Th1為主之細胞免疫反應(誘導CTL)或活化固有免疫反應,而這些均為腫瘤療法所必需。
本發明之目的係提供新穎之腫瘤疫苗,藉由此疫苗可誘導強烈之系統性細胞性免疫系統反應並可取得有效之腫瘤療法。
令人驚訝地,於一觀點中,本發明之目的可藉由提供包含下列組成分之作為編碼抗原和蛋白質毒素之核苷酸序列的載體之微生物來解決:(Ⅰ)至少一種編碼至少一種野生型或突變之蛋白質的至少一種完全或部分抗原之核苷酸序列;及(Ⅱ)至少一種編碼至少一種蛋白質毒素及/或至少一種蛋白質毒素次單位之核苷酸序列;及(Ⅲ)a)至少一種編碼至少一種轉運系統(其可使組成分(Ⅰ)及組成分(Ⅱ)之表現產物表現在該微生物之外表面上及/或可使組成分(Ⅰ)及組成分(Ⅱ)之表現產物分泌);及/或至少一種訊號序列(其可使組成分(Ⅰ
)及組成分(Ⅱ)之表現產物分泌)之核苷酸序列;及/或b)選擇性地,至少一種編碼至少一種用於於哺乳動物細胞之細胞溶質中溶解微生物及用於胞內釋出質體或表現載體(其包含在該溶解之微生物內)之蛋白質的核苷酸序列;及(Ⅳ)至少一種編碼至少一種用於表現一或多種組成分(Ⅰ)至(Ⅲ)之活化序列的核苷酸序列,其中該活化序列可在該微生物中被活化及/或為組織細胞特異性、腫瘤細胞特異性、巨噬細胞特異性、樹突細胞特異性、淋巴細胞特異性、功能特異性或非細胞特異性;其中該組成分(Ⅰ)至(Ⅳ)中之任一可出現一次或數次且若組成分(Ⅰ)至(Ⅳ)中之一種組成分出現數次,則其可彼此各自獨立地為相同或相異。
於一較佳體系中係提供包含上述組成分(Ⅰ)至(Ⅳ)之微生物,其中組成分(Ⅰ)及組成分(Ⅱ)並不相同,亦即組成分(Ⅰ)並不編碼至少一種編碼至少一種蛋白質毒素及/或至少一種蛋白質毒素次單位之核苷酸序列。
在本發明之過程中,與組成分(Ⅳ)相關之‘‘組織細胞-特異性”一詞係指在靶的組織細胞中被特異活化之活化序列,諸如激素倚賴性啟動子(如:在前列腺組織中者)。
在本發明之過程中,與組成分(Ⅳ)相關之“腫瘤細胞-特異性”一詞係指在腫瘤細胞中被特異活化之活化序
列,諸如被腫瘤特異性致癌基因之作用活化的啟動因子成分。
在本發明之過程中,與組成分(Ⅳ)相關之“巨噬細胞-特異性”一詞係指在巨噬細胞中被特異活化之活化序列,諸如編碼巨噬細胞-特異性基因之啟動子成分(如:編碼F4/80之基因)。
在本發明之過程中,與組成分(Ⅳ)相關之“樹突細胞-特異性”一詞係指在樹突細胞中被特異活化之活化序列,諸如控制B7.1之表現的啟動子成分。“樹突細胞-特異性”一詞等同“樹突型-細胞-特異性”一詞,即,其具相同意義且均指樹突細胞。
在本發明之過程中,與組成分(Ⅳ)相關之“淋巴細胞-特異性”一詞係指在淋巴系之細胞中被特異活化之成分,如:調節T細胞中CD3分子之表現的啟動子成分或調節成熟B細胞中CD20之表現的啟動子成分。
在本發明之過程中,與組成分(Ⅳ)相關之“功能-特異性”一詞係指在細胞(如:已喪失p53表現之腫瘤細胞)中被特異活化之活化序列,或在細菌中根據,如:細胞定位或氧之壓力而被活化之活化序列。
在本發明之過程中,與組成分(Ⅳ)相關之“非細胞-特異性”一詞係指到處活躍之活化序列,諸如組成上活性之細菌啟動子。
在本發明之過程中,“核苷酸序列”一詞係指dsDNA、ssDNA、dsRNA、ssRNA或dsDNA/RNA雜交體。宜為
dsDNA。
在本發明之過程中,“抗原”一詞係指與抗體反應之分子,即,可產生抗體者。一些抗原本身不會誘出抗體之製造;僅那些可誘導抗體製造者稱為免疫原。在本發明之目的中係欲包含所有種類之已知抗原。藉由資料庫及/或在不造成過度負擔下進行實驗審查來取得關於可能之抗原的必要資訊為本技藝之技術熟習人士所知之學問。抗原之實例為:(尤其是)細胞抗原、組織細胞特異性抗原(如:腫瘤衍生來源之組織細胞)、細胞蛋白質抗原、病毒抗原、病毒蛋白質抗原,等。宜為蛋白質抗原。更宜為異源抗原或外來抗原,即,非本發明之各別微生物的內生性抗原,或非本發明之各別微生物所天然表現,但藉由標準分子生物法引入其中的抗原。
在本發明之過程中,“完全抗原”一詞係指與根據上述定義之抗體反應之完全分子。完全抗原之實例為,如:全長蛋白質,其亦為較佳者。
在本發明之過程中,“部分抗原”一詞係指與根據上述定義之抗體反應之分子的特殊部分。部分抗原可為,如:蛋白質再現單位(motives),諸如在蛋白質內之胺基酸環、蛋白質激酶結構區、抗原決定部位,等。宜為蛋白質激酶結構區及抗原決定部位,後者為抗體所能辨識之抗原的特殊位置(亦稱為抗原決定簇)。
在本發明之過程中,與蛋白質相關之“野生型”及“經突變”等詞係分別指由其“天然”支配性胺基酸序列(
由各別之核苷酸序列編碼)所組成之蛋白質及與該野生型序列相較下,在其胺基酸序列(由各別之核苷酸序列編碼)中具有一或多種突變之蛋白質。較佳地,野生型及/或突變之蛋白質係衍生自腫瘤細胞。在部分抗原方面,更佳者為包含突變之序列,即,宜選擇包含一或多種突變之抗原決定部位(如:B-Raf V600E抗原決定部位)。
在本發明之意義中,微生物係指細菌、革蘭氏陽性細菌、革蘭氏陰性細菌及真核細胞,後者包含單細胞寄生蟲、酵母菌、腫瘤細胞及細胞株細胞,諸如啤酒酵母、利什曼屬、自患者自體衍生之腫瘤細胞及腫瘤細胞株。這類微生物通常係作為載體,以轉移對微生物而言為外來(異源或異質)之核苷酸序列。較佳地,使用毒性減弱之細菌,例如:攜帶被刪除或去活化之aroA、aro、asd、gal、pur、cya、crp、phoP/Q、omp基因之細菌或溫度敏感突變體或抗生素倚賴性突變體(Cardenas及Clements,1992)。更適合作為包含上述組成分(Ⅰ)至(Ⅳ)之微生物為那些一可克服小腸黏膜,作為載體之革蘭氏陰性、減毒、兼性細胞內細菌(如:沙門氏菌屬或志賀氏菌屬)。
於一較佳體系中係提供包含上述組成分(Ⅰ)至(Ⅳ)之微生物,其中該微生物係選自“細菌、革蘭氏陽性細菌、革蘭氏陰性細菌及真核細胞”且宜選自“埃希氏菌屬、大腸桿菌、沙門氏菌屬、傷寒沙門氏菌、鼠傷寒沙門氏菌、耶爾森氏菌屬、小腸結腸炎耶爾森氏菌、弧菌屬、霍亂弧菌、李斯特菌屬、單核球增多性李斯特菌、志賀氏菌
屬、福氏志賀氏菌(Shigella flexneri)“,較佳地,其中該微生物之毒性被減弱。更佳地,上述定義之微生物中係排除霍亂弧菌。
在本發明之目的中,與微生物相關之“屬”一詞係欲包含指定菌屬中之所有成員(包括種、亞種,等)。例如:“沙門氏菌屬“一詞係欲包含沙門氏菌屬中之所有成員,諸如:傷寒沙門氏菌及鼠傷寒沙門氏菌。
於另一較佳體系中係提供根據上述定義之微生物,其中該組成分(Ⅰ)之至少一種野生型或突變之蛋白質的至少一種完全或部分抗原係選自下列野生型蛋白質及彼之已知突變體:“受體”受體之胞外、跨膜或胞內部分;黏著分子;黏著分子之胞外、跨膜或胞內部分;訊號轉導蛋白質;細胞週期蛋白質;轉錄因子;分化蛋白質;胚胎蛋白質;病毒蛋白質;過敏原;微生物致病原之蛋白質;真核生物致病原之蛋白質;睾丸癌抗原蛋白質;腫瘤抗原蛋白質;及/或組織-細胞特異性蛋白質”,其中該組織細胞係選自:甲狀腺、乳腺、唾液腺、淋巴結、乳腺、胃黏膜、腎臟、卵巢、前列腺、子宮頸、膀胱黏膜及神經”。
在突變之蛋白質方面,突變可為致癌性且可能造成其原始細胞功能之損失或取得。
這類抗原在細胞中控制細胞生長及細胞分裂,並出現在正常細胞之細胞膜上(例如:第Ⅰ類MHC分子)。在腫瘤細胞中,這些抗原常過度表現或特異突變。這類突變可具有致癌基因遏制子之功能限制或因此將原癌基因活化
成致癌基因並可單獨涉入腫瘤生長或,通常在腫瘤生長中過度表現。這類細胞抗原出現在腫瘤細胞膜上,因此代表在腫瘤細胞上之抗原,然而,其不會引起影響患者之腫瘤疾病的免疫反應。Rapp (US 5,156,841)已描述致癌蛋白質(即,致癌基因之表現產物)於作為腫瘤疫苗之免疫原的用途。
根據本發明之抗原及其(致癌性)突變的實例為:1)受體,諸如“Her-2/neu、雄激素受體、雌激素受體、乳鐵蛋白受體、中期因子(midkine)受體、EGF受體、ERBB2、ERBB4、TRAIL受體、FAS、TNF α受體、TGF-β受體:ii)訊號轉導蛋白質,諸如c-Raf (Raf-1)、A-Raf、B-Raf、B-Raf V599E、B-Raf V600E、B-Raf KD、B-Raf V600E激酶結構區、B-Raf V600E KD、B-Raf V600E激酶結構區KD、B-Raf激酶結構區、B-Raf激酶結構區KD、Ras、Bcl-2、Bcl-X、Bcl-W、Bfl-1、Brag-1、Mcl-1、A1、Bax、BAD、Bak、Bcl-Xs、Bid、Bik、Hrk、Bcr/abl、Myb、C-Met、IAP1、IAO2、XIAP、ML-IAP LIVIN、存活素、APAF-1;iii)細胞週期控制之蛋白質,諸如細胞週期素D (1-3)、細胞週期素E、細胞週期素A、細胞週期素B、細胞週期素H、Cdk-1、Cdk-2、Cdk-4、Cdk-6、Cdk-7、Cdc25C、p16、p15、p21、p27、p18、pRb、p107、p130、E2F (1-5)、GAAD45、MDM2、PCNA、ARF、PTEN、APC、BRCA、p53及同源物;iv)轉錄因子,諸如C-Myc、NFkB、c-Jun、ATF-2、Sp1;v)
胚胎蛋白質,諸如癌胚抗原、α-胎兒蛋白、MAGE、MAGE-1、MAGE-3、NY-ESO-1、PSCA;vi)分化抗原,諸如MART、Gp100、酪胺酸酶、GRP、TCF-4、髓鞘鹼性蛋白(basic myelin)、α-乳白蛋白、GFAP、前列腺特異性抗原(PSA)、纖維酸性蛋白質、酪胺酸酶、EGR-1、MUC1;vii)病毒性抗原,諸如下列病毒之抗原:HIV、HPV、HCV、HPV、EBV、CMV、HSV、流感病毒、A型流感病毒、A型流感病毒(H5N1)及(H3N2)、B型流感病毒、C型流感病毒;紅血球凝集素(hemagglutinins)、紅血球凝集素H1、紅血球凝集素H5、紅血球凝集素H7、紅血球凝集素HA1(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)、紅血球凝集素HA12(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)、紅血球凝集素HA12C(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)、神經胺基酸酶、微生物抗原:p60、LLO、尿素酶,等。真核細胞病原之抗原:CSP(瘧疾)、calflagin(錐蟲屬)、CPB(碩大利什曼蟲),等。
於另一較佳體系中係提供根據上述定義之微生物,其中該組成分(Ⅰ)之至少一種野生型或突變之蛋白質的至少一種完全或部分抗原係選自下列野生型蛋白質及彼之已知突變體:“Her-2/neu、雄激素受體、雌激素受體、中期因子(midkine)受體、EGF受體、ERBB2、ERBB4、TRAIL受體、FAS、TNF α受體、TGF-β受體、乳鐵蛋白受體、髓鞘鹼性蛋白、α-乳白蛋白、GFAP、纖維酸性蛋
白質、酪胺酸酶、EGR-1、MUC1、c-Raf (Raf-1)、A-Raf、B-Raf、B-Raf V599E、B-Raf V600E、B-Raf KD、B-Raf V600E激酶結構區、B-Raf V600E KD、B-Raf V600E激酶結構區KD、B-Raf激酶結構區、B-Raf激酶結構區KD、N-Ras、K-Ras、H-Ras、Bc1-2、Bc1-X、Bc1-W、Bf1-1、Brag-1、Mc1-1、A1、Bax、BAD、Bak、Bc1-Xs、Bid、Bik、Hrk、Bcr/ab1、Myb、C-Met、IAP1、IAO2、XIAP、ML-IAPLIVIN、存活素、APAF-1、細胞週期素D(1-3)、細胞週期素E、細胞週期素A、細胞週期素B、細胞週期素H、Cdk-1、Cdk-2、Cdk-4、Cdk-6、Cdk-7、Cdc25C、p16、p15、p21、p27、p18、pRb[r1]
、p107、p130、E2F (1-5)、GAAD45、MDM2、PCNA、ARF、PTEN、APC、BRCA、Akt、PI3K、mTOR、p53及同源物、C-Myc、NFkB、c-Jun、ATF-2、Sp1、前列腺特異性抗原(PSA)、癌胚抗原、α-胎兒蛋白、PAP;PSMA;STEAP;MAGE、MAGE-1、MAGE-3、NY-ESO-1、PSCA、MART、Gp100、酪胺酸酶、GRP、TCF-4、HIV病毒、HPV病毒、HCV病毒、HPV病毒、EBV病毒、CMV病毒、HSV病毒、流感病毒、A型流感病毒、A型流感病毒(H5N1)及(H3N2)、B型流感病毒、C型流感病毒之病毒抗原;紅血球凝集素、紅血球凝集素H1、紅血球凝集素H5、紅血球凝集素H7、紅血球凝集素HA1(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)、紅血球凝集素HA12(宜來自A型流感病毒(A/泰國/1 (KAN-1
)2004 (H5N1)、紅血球凝集素HA12C(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)、神經胺基酸酶、[r2]
p60、LLO、尿素酶、CSP、calflagin及/或CPB”。
於另一較佳體系中係提供根據上述定義之微生物,其中該組成分(Ⅰ)之至少一種野生型或突變之蛋白質的至少一種完全或部分抗原係選自下列由野生型蛋白質及彼之已知突變體(括號中之編號)所組成之激酶:AAK1 (NM 014911), AATK (NM 004920), ABL1 (NM 005157), ABL2 (NM 005158), ACK1 (NM 005781), ACVR1 (NM 001105), ACVR1B (NM 020328), ACVR2 (NM 001616), ACVR2B (NM 001106), ACVRL1 (NM 000020), ADCK1 (NM 020421), ADCK2 (NM 052853), ADCK4 (NM 024876), ADCK5 (NM 174922), ADRBK1 (NM 001619), ADRBK2 (NM 005160), AKT1 (NM 005163), AKT2 (NM 001626), AKT3 (NM 005465), ALK (NM 004304), ALK7 (NM 145259), ALS2CR2 (NM 018571), ALS2CR7 (NM 139158), AMHR2 (NM 020547), ANKK1 (NM 178510), ANKRD3 (NM 020639), APEG1 (NM 005876), ARAF (NM 001654), ARK5 (NM 014840), ATM (NM 000051), ATR (NM 001184), AURKA (NM 003600), AURKB (NM 004217), AURKC (NM 003160), AXL (NM 001699), BCKDK (NM 005881), BCR (NM 004327), BIKE (NM 017593), BLK (NM 00171 5), BMPR1A (NM 004329), BMPR1B (NM 001203), BMPR2 (NM 001204), BMX (NM 001721), BRAF (NM 004333), BRD2 (NM 005104), BRD3 (NM 007371), BRD4 (NM 014299), BRDT (NM 001726), BRSK1 (NM 032430), BRSK2 (NM 003957), BTK (NM 000061), BUB1 (NM 004336), BUB1B (NM 001211), CABC1 (NM 020247), CAMK1 (NM 003656), CaMK1b (NM 198452), CAMK1D (NM 020397), CAMK1G (NM 020439), CAMK2A (NM 015981), CAMK2B (NM 001220), CAMK2D (NM 001221), CAMK2G (NM 001222), CAMK4 (NM 001744), CAMKK1 (NM 032294), CAMKK2 (NM 006549), CASK (NM 003688), CCRK (NM 012119), CDC2 (NM 001786), CDC2L1 (NM 001787), CDC2L5 (NM 003718), CDC42BPA (NM 014826), CDC42BPB (NM 006035), CDC7L1 (NM 003503), CDK10 (NM 003674), CDK11 (NM 015076), CDK2 (NM 001798), CDK3 (NM 001258), CDK4 (NM 000075), CDK5 (NM 004935), CDK6 (NM 001259), CDK7 (NM 001799), CDK8 (NM 001260), CDK9 (NM 001261), CDKL1 (NM 004196), CDKL2 (NM 003948), CDKL3 (NM 016508), CDKL4 (NM 001009565), CDKL5 (NM 003159). CHEK1 (NM
001274), CHUK (NM 001278), CIT (NM 007174), CLK1 (NM 004071), CLK2 (NM 003993), CLK3 (NM 003992), CLK4 (NM 020666), CRK7 (NM 016507), CSF1R (NM 005211), CSK (NM 004383), CSNK1A1 (NM 001892), CSNK1D (NM 001893), CSNK1E (NM 001894), CSNK1G1 (NM 022048), CSNK1G2 (NM 001319), CSNK1G3 (NM 004384), CSNK2A1 (NM 001895), CSNK2A2 (NM 001896), DAPK1 (NM 004938), DAPK2 (NM 014326), DAPK3 (NM 001348), DCAMKL1 (NM 004734), DCAMKL2 (NM 152619), DCAMKL3 (XM 047355), DDR1 (NM 013993), DDR2 (NM 006182), DMPK (NM 004409), DMPK2 (NM 017525.1), DYRK1A (NM 001396), DYRK1B (NM 006484), DYRK2 (NM 006482), DYRK3 (NM 003582), DYRK4 (NM 003845), EEF2K (NM 013302), EGFR (NM 005228), EIF2AK3 (NM 004836), EIF2AK4 (NM_001013703), EPHA1 (NM 005232), EPHA10 (NM 001004338), EPHA2 (NM 004431), EPHA3 (NM 005233), EPHA4 (NM 004438), EPHA5 (NM 004439), EPHA6 (XM 114973), EPHA7 (NM 004440), EPHA8 (NM 020526), EPHB1 (NM 004441), EPHB2 (NM 017449), EPHB3 (NM 004443), EPHB4 (NM 004444), EPHB6 (NM 004445), ERBB2 (NM 004448), ERBB3 (NM 001982), ERBB4 (NM 005235), ERK8 (NM 139021), ERN1 (NM 001433), ERN2 (NM 033266), FASTK (NM 025096), FER (NM 005246), FES (NM 002005), FGFR1 (NM 000604), FGFR2 (NM 022970), FGFR3 (NM 000142), FGFR4 (NM 022963), FGR (NM 005248), FLJ23074 (NM 025052), FLJ23119 (NM 024652), FLJ23356 (NM 032237), FLT1 (NM 002019), FLT3 (NM 004119), FLT4 (NM 002020), FRAP1 (NM 004958), FRK (NM 002031), FYN (NM 002037), GAK (NM 005255), GPRK5 (NM 005308), GPRK6 (NM 002082), GPRK7 (NM 139209), GRK4 (NM 005307), GSG2 (NM 031965), GSK3A (NM 019884), GSK3B (NM 002093), GUCY2C (NM 004963), GUCY2D (NM 000180), GUCY2F (NM 001522), H11 (NM 014365), HAK (NM 052947), HCK (NM 002110), HIPK1 (NM 152696), HIPK2 (NM 022740), HIPK3 (NM 005734), HIPK4 (NM 144685), HRI (NM 014413), HUNK (NM 014586), ICK (NM 016513), IGF1R (NM 000875), IKBKB (NM 001556), IKBKE (NM 014002), ILK (NM 004517), INSR (NM 000208), INSRR (NM 014215), IRAKI (NM 001569), IRAK2 (NM 001570), IRAK3 (NM 007199), IRAK4 (NM 016123), ITK (NM 005546), JAK1 (NM 002227), JAK2 (NM 004972), JAK3 (NM 000215), KDR (NM 002253), KIS (NM 144624), KIT (NM 000222). KSR (XM 290793), KSR2 (NM 173598), LAK(NM 025144), LATS1 (NM 004690), LATS2 (NM 014572), LCK (NM 005356), LIMK1 (NM 016735), LIMK2 (NM 005569), LMR3 (XM 055866), LMTK2 (NM 014916), LOC149420 (NM 152835), LOC51086 (NM 015978), LRRK2 (XM 058513), LTK (NM 002344), LYN (NM 002350), MAK (NM 005906), MAP2K1 (NM 002755), MAP2K2 (NM 030662), MAP2K3 (NM 002756), MAP2K4 (NM 003010), MAP2K5 (NM 002757), MAP2K6 (NM 002758), MAP2K7 (NM 005043), MAP3K1 (XM 042066), MAP3K10 (NM 002446),
MAP3K11 (NM 002419), MAP3K12 (NM 006301), MAP3K13 (NM 004721), MAP3K14 (NM 003954), MAP3K2 (NM 006609), MAP3K3 (NM 002401), MAP3K4 (NM 005922), MAP3K5 (NM 005923), MAP3K6 (NM 004672), MAP3K7 (NM 003188), MAP3K8 (NM 005204), MAP3K9 (NM 033141), MAP4K1 (NM 007181), MAP4K2 (NM 004579), MAP4K3 (NM 003618), MAP4K4 (NM 145686), MAP4K5 (NM 006575), MAPK1 (NM 002745), MAPK10 (NM 002753), MAPK11 (NM 002751), MAPK12 (NM 002969), MAPK13 (NM 002754), MAPK14 (NM 001315), MAPK3 (NM 002746), MAPK4 (NM 002747), MAPK6 (NM 002748), MAPK7 (NM 002749), MAPK8 (NM 002750), MAPK9 (NM 002752), MAPKAPK2 (NM 032960), MAPKAPK3 (NM 004635), MAPKAPK5 (NM 003668), MARK (NM 018650), MARK2 (NM 017490), MARKS (NM 002376), MARK4 (NM 031417), MAST1 (NM 014975), MAST205 (NM 015112), MASTS (XM 038150), MAST4 (XM 291141), MASTL (NM 032844), MATK (NM 139355), MELK (NM 014791), MERTK (NM 006343), MET (NM 000245), MGC33182 (NM 145203), MGC42105 (NM 153361 ), MGC43306 (C9ofr96), MGC8407 (NM 024046), MIDORI (NM 020778), MINK (NM 015716), MKNK1 (NM 003684), MKNK2 (NM 017572), MLCK (NM 182493), MLK4 (NM 032435), MLKL (NM 152649), MOS (NM 005372), MST1R (NM 002447), MST4 (NM 016542), MUSK (NM 005592), MYLK (NM 053025), MYLK2 (NM 033118), MYO3A (NM 017433), MYO3B (NM 138995), NEK1 (NM 012224), NEK10 (NM 152534), NEK11 (NM 024800), NEK2 (NM 002497), NEK3 (NM 002498), NEK4 (NM 003157), NEK5 (MGC75495), NEK6 (NM 014397), NEK7 (NM 133494), NEK8 (NM 178170), NEK9 (NM 033116), NLK (NM 016231), NPR1 (NM 000906), NPR2 (NM 003995), NRBP (NM 013392), NRBP2 (NM 178564), NRK (NM 198465), NTRK1 (NM 002529), NTRK2 (NM 006180), NTRK3 (NM 002530), OBSCN (NM 052843), OSR1 (NM 005109), PACE-1 (NM 020423), PAK1 (NM 002576), PAK2 (NM 002577). PAK3 (NM 002578), PAK4 (NM 005884), PAK6 (NM 020168), PAK7 (NM 020341), PASK (NM 015148), PCTK1 (NM 006201), PCTK2 (NM 002595), PCTK3 (NM 212503), PDGFRA (NM 006206), PDGFRB (NM 002609), PDK1 (NM 002610), PDK2 (NM 002611), PDK3 (NM 005391), PDK4 (NM 002612), PDPK1 (NM 002613), PFTK1 (NM 012395), PHKG1 (NM 006213), PHKG2 (NM 000294), PIK3R4 (NM 014602), PIM1 (NM 002648), PIM2 (NM 006875), PIM3 (NM 001001852), PINK1 (NM 032409), PKE (NM 173575), PKMYT1 (NM 004203), pknbeta (NM 013355), PLK (NM 005030), PLK3 (NM 004073), PRKAA1 (NM 006251), PRKAA2 (NM 006252), PRKACA (NM 002730), PRKACB (NM 002731), PRKACG (NM 002732), PRKCA (NM 002737), PRKCB1 (NM 002738), PRKCD (NM 006254), PRKCE (NM 005400), PRKCG (NM 002739), PRKCH (NM 006255), PRKCI (NM 002740), PRKCL1 (NM 002741), PRKCL2 (NM 006256), PRKCM (NM 002742), PRKCN (NM 005813), PRKCQ (NM 006257), PRKCZ (NM 002744), PRKD2 (NM 016457), PRKDC (NM 006904), PRKG1 (NM 006258), PRKG2
(NM 006259), PRKR (NM 002759), PRKWNK1 (NM 018979), PRKWNK2 (NM 006648), PRKWNK3 (NM 020922), PRKWNK4 (NM 032387), PRKX (NM 005044), PRKY (NM 002760), PRPF4B (NM 003913), PSKH1 (NM 006742), PSKH2 (NM 033126), PTK2 (NM 005607), PTK2B (NM 004103), PTK6 (NM 005975), PTK7 (NM 002821), PTK9 (NM 002822), PTK9L (NM 007284), PXK (NM 017771). QSK (NM 025164), RAD53 (NM 007194), RAF1 (NM 002880), RAGE (NM 014226), RET (NM 020975), RHOK (NM 002929), RIOK1 (NM 031480), RIOK2 (NM 018343), RIPK1 (NM 003804), RIPK2 (NM 003821), RIPK3 (NM 006871), RIPK5 (NM 015375), RNASEL (NM 021133), ROCK1 (NM 005406), ROCK2 (NM 004850), ROR1 (NM 005012), ROR2 (NM 004560), ROS1 (NM 002944), RPS6KA1 (NM 002953), RPS6KA2 (NM 021135), RPS6KA3 (NM 004586), RPS6KA4 (NM 003942), RPS6KA5 (NM 004755), RPS6KA6 (NM 014496), RPS6KB1 (NM 003161), RPS6KB2 (NM 003952), RPS6KC1 (NM 012424), RPS6KL1 (NM 031464), RYK (NM 002958), SBK (XM 370948), SCYL1 (NM 020680), SCYL2 (NM 017988), SGK (NM 005627), SgK069 (SU SgK069), SgK085 (XM 373109), SgK110 (SU SgK110), SGK2 (NM 016276), SgK223 (XM 291277), SgK269 (XM 370878), SgK424 (CGP SgK424), SgK493 (SU_SgK493), SgK494 (NM 144610), SgK495 (NM 032017), SGKL (NM 013257), SK681 (NM 001001671), SLK (NM 014720), SMG1 (NM 015092), SNARK (NM 030952), SNF1LK (NM 173354), SNF1LK2 (NM 015191), SNK (NM 006622), SNRK (NM 017719), SRC (NM 005417), SRMS (NM 080823), SRPK1 (NM 003137), SRPK2 (NM 003138), SSTK (NM 032037), STK10 (NM 005990), STK11 (NM 000455), STK16 (NM 003691), STK17A (NM 004760), STK17B (NM 004226), STK18 (NM 014264), STK19 (NM 032454), STK22B (NM 053006), STK22C (NM 052841), STK22D (NM 032028). STK23 (NM 014370), STK24 (NM 003576), STK25 (NM 006374), STK3 (NM 006281), STK31 (NM 031414), STK32B (NM 018401), STK33 (NM 030906), STK35 (NM 080836), STK36 (NM 015690), STK38 (NM 007271), STK38L (NM 015000), STK39 (NM 013233), STK4 (NM 006282), STLK5 (NM 001003787), STYK1 (NM 018423), SUDD (NM 003831), SYK (NM 003177), TAF1 (NM 138923), TAF1L (NM 153809), TAO1 (NM 004783), TAOK1 (NM 020791), TAOK3 (NM 016281), TBCK (NM 033115), TBK1 (NM 013254), TEC (NM 003215), TEK (NM 000459). TESK1 (NM 006285), TESK2 (NM 007170), TEX14 (NM 031272), TGFBR1 (NM 004612), TGFBR2 (NM 003242), TIE (NM 005424), TIF1 (NM 003852), TLK1 (NM 012290), TLK2 (NM 006852), TNIK (NM 015028), TNK1 (NM 003985), TOPK (NM 018492), TP53RK (NM 033550), TRAD (NM 007064), TRIB1 (NM 025195), TRIB2 (NM 021643), TRIB3 (NM 021158), TRIM28 (NM 005762), TRIM33 (NM 015906), TRIO (NM 007118), TRPM6 (NM 017662), TRPM7 (NM 017672), TRRAP (NM 003496), TSSK4 (NM 174944), TTBK1 (NM 032538), TTBK2 (NM 173500), TTK (NM 003318), TTN (NM 003319),
TXK (NM 003328), TYK2 (NM 003331), TYRO3 (NM 006293), ULK1 (NM 003565), ULK2 (NM 014683), ULK3 (NM 015518), ULK4 (NM 017886), VRK1 (NM 003384), VRK2 (NM 006296), VRK3 (NM 016440), WEE1 (NM 003390), Wee1B (NM 173677), YANK1 (NM 145001), YES1 (NM 005433), ZAK (NM 016653), and/or ZAP70 (NM 001079)".
本發明之過程中,“過敏原”一詞係指如此文所定義之引出過敏性及/或過敏反應之完全或部分抗原。實例有:Der p 5(蝨)、Bet v 1(花粉)、Ph1 p 1(草粉)、Asp f 1/a(曲黴)、PLA 2(蜜蜂)、Hev b(乳膠)(Schmid-Grendelmeier and Crameri, 2001)。
微生物及真核病原之抗原及睾丸癌抗原列於上表中。
本發明之過程中,根據組成分(Ⅱ)之蛋白質毒素及/或其次單位宜為細菌蛋白質毒素,更宜為外毒素。細菌外毒素之實例為第Ⅰ型毒素(超抗原)、第Ⅱ型毒素(孔洞形成毒素)及第Ⅲ型毒素(A-B毒素)。
於一較佳體系中係提供根據上述定義之微生物,其中組成分(Ⅱ)係選自下列:“細菌毒素、腸毒素、外毒素、第Ⅰ型毒素、第Ⅱ型毒素、第Ⅲ型毒素、第Ⅳ型毒素、第Ⅴ型毒素、RTX毒素、AB毒素、AB毒素、A/B毒素、A+B毒素、A-5B毒素及/或AB5毒素”。
於另一較佳體系中係提供根據上述定義之微生物,其中組成分(Ⅱ)係選自下列:“腺苷酸環化酶毒素、炭疽病毒素、炭疽病毒素(EF)、炭疽病毒素(LF)、肉毒桿菌毒素、霍亂毒素(CT、CtX)、霍亂毒素次單位B(CTB、CtxB)、白喉毒素(DT、Dtx)、大腸桿菌LT毒素
、大腸桿菌熱不穩定性腸毒素(LT)、大腸桿菌熱不穩定性腸毒素次單位B(LTB)、大腸桿菌ST毒素、大腸桿菌熱穩定性腸毒素(ST)、紅斑毒素、剝脫素毒素、外毒素A、產氣萊膜腸毒素、百日咳毒素(PT、Ptx)、志賀毒素(ST、Stx)、志賀毒素次單位B(STB、StxB)、志賀樣毒素、葡萄球菌腸毒素、破傷風毒素(TT)、中毒休克症候群毒素(TSST-1)、Vero毒素(VT)、困難梭菌(Clostridium difficile)之毒素A(TA)及毒素B(TB)、索氏梭菌(Clostridium sordellii)之致死毒素(LT)及出血性毒素(HT)、諾維梭菌(Clostridium novyi)之α毒素(AT)”。
然而,若使用霍亂毒素或其次單位CtxB作為根據本發明之組成分(Ⅱ)的毒素,則宜不使用霍亂弧菌作為載體(微生物)。
於一較佳體系中係提供根據上述定義之微生物,其中組成分(Ⅰ)及組成分(Ⅱ)係連接在一起以使由此二種組成分編碼之融合蛋白質表現及/或分泌。較合適的為,此融合蛋白質係選自下列:“CtxB-PSA、CtxB-3-Raf V600E KD(激酶失效)、CtxB-B-Raf V600E激酶結構區、CtxB-B-Raf V600E激酶結構區KD(激酶失效)、CtxB-B-Raf、CtxB-B-Raf KD(激酶失效)、CtxB-B-Raf激酶結構區KD(激酶失效)、CtxB-HA1(流感病毒之紅血球凝集素的次單位1)、CtxB-UA12C”。
分泌作用為一種分離、合成製作及自細胞或分泌之化
學物質或一些物質中釋出化學物的過程。分泌作用並非真核細胞所獨有者;其亦出現在細菌及古菌中。ATP結合盒(ABC)型轉運子為生物之所有三種結構區所共有。Sec系統亦為另一保留之分泌系統,其與真核細胞內質網中之易位子(translocon)(包含酵母菌之Sec61易位子複合體及細菌中之Sec Y-E-G複合體)同源。革蘭氏陰性菌具有二種膜,這使得其分泌作用在局部解剖學上較複雜。因此,在革蘭氏陰性細菌中至少有五種專門之分泌系統:
(1)第Ⅰ型分泌系統:其與上述之ATP結合盒轉運子相同。
(2)第Ⅱ型分泌系統:蛋白質穿越內膜係倚賴Sec系統而穿越外膜係倚賴另一特別系統。菌毛(bacterial pili)使用修改之Sec系統,但與第Ⅰ型系統不同。
(3)第Ⅲ型分泌系統(T3SS):其與細菌鞭毛基體同源。其類似於一種分子注射器,透過此分子注射器,細菌(如:志賀菌或耶爾森氏菌)可將蛋白質注射入真核細胞中。細胞溶質中之低Ca2+
濃度開啟調節T3SS之閘門。植物病原中之Hrp系統透過類似機制將生物髮夾(hairpin)注射入植物中。
(4)第Ⅳ型分泌系統,其與細菌之結合機制(及古菌鞭毛)同源。其可轉運DNA及蛋白質。其係在農桿菌(Agrobacterium tumefaciens)中發現,該菌使用此系統來將Ti質體及蛋白質引入發展出冠纓病(腫瘤)之宿主中。幽門螺桿菌使用第Ⅳ型分泌系統將CagA注射入胃上
皮細胞中。百日咳桿菌(其為引起百日咳之媒介)係部分透過第Ⅳ型分泌系統來分泌百日咳毒素。
(5)第Ⅴ型分泌系統(亦稱為自動轉運子系統):其使用Sec系統跨越內膜。使用此通路之蛋白質具有在其C端形成β屏障及插入外膜以將剩餘之肽運出之能力。最後,β屏障可能分裂並留在外膜中。某些人相信這些自動轉運子之剩餘部分將產生類似β屏障之膜孔蛋白(porins)。
細菌以及粒腺體和葉綠體亦使用許多其他特殊轉運系統,諸如雙-精胺酸轉位(Tat)通路,相對於Sec-倚賴性輸出,其運輸經完全摺疊之蛋白質通過膜。系統之名稱來自對瞄準此系統所需之訊號序列中二個連續精胺酸之需求。革蘭氏陰性菌中之分泌涉及藉由合適之分泌系統克服內及外膜,如:Hly第Ⅰ型或第Ⅲ型分泌系統或AIDA自動轉運子。革蘭氏陽性菌中,分泌系統必須克服內膜及細胞壁,而此點在大部分菌株中可經由與合適之分泌訊號融合來達成。
組成分(Ⅲ)a)為至少一種編碼至少一種轉運系統(其可使組成分(Ⅰ)及組成分(Ⅱ)之表現產物表現在微生物之外表面上及/或使組成分(Ⅰ)及組成分(Ⅱ)之表現產物被分泌)。各別組成分可選擇被分泌或表現在微生物之膜上(即,與膜結合)。這類轉運系統為,例如:i)第Ⅰ型分泌系統、第Ⅱ型分泌系統、第Ⅲ型分泌系統、第Ⅳ型分泌系統、第Ⅴ型分泌系統,ii)大腸桿菌之溶
血素轉運系統(訊號)(含有受hly-特異性啟動因子控制之H1yA、HlyB及HlyD之核苷酸序列);下列為欲使用之轉運訊號:在分泌方面-於HlyB及HlyD蛋白質之存在下,C-端HlyA轉運訊號;在膜結合型表現方面-於HlyB蛋白質之存在下,C-端HlyA轉運訊號,iii)大腸桿菌之溶血素轉運系統(訊號)(含有受非hly-特異性細菌啟動因子控制之HlyA、HlyB及HlyD的核苷酸序列),iv)新月柄桿菌(Caulobacter crescentus)之S層(Rsa A)蛋白質之轉運訊號;下列為欲使用之轉運訊號:在分泌及膜結合型表現方面-C-端RsaA轉運訊號,v)大腸桿菌之TolC蛋白質為轉運訊號TolC;下列為欲使用之轉運訊號:在膜結合型表現方面-TolC(大腸桿菌之完整膜蛋白質TolC)之N-端轉運訊號為-種大腸桿菌外膜之多功能孔道形成蛋白質,除了諸如接受大腸桿菌素E1(Morona,等人,1983)及分泌大腸桿菌素V(Fath,等人,1991)之功能外,其亦作為U3噬菌體之受體(Austin,等人,1990);此蛋白質不僅可在大腸桿菌中找到;亦可在許多革蘭氏陰性細菌中找到(Wiener,2000);TolC在外膜之定位及廣泛出現使其為呈現異源抗原之理想候選者,以,如:引起免疫反應。
革蘭氏陽性菌不包含外膜。在這些案例中之分泌作用較簡單且通常不需要提供分泌機制以供通過細胞膜及細胞壁運輸。在革蘭氏陽性菌之案例中,與異源蛋白質在N-端融合之分泌訊號對於分泌作用通常為必要且足夠的。蛋
白質(用於這些蛋白質之訊號序列已有描述)主要包含所有分泌之細菌蛋白質。李斯特菌方面之實例為衍生自李斯特菌素之分泌訊號,p60或ActA。一般而言,類似之方法可應用在真核細胞上,在此真核細胞中,當無滯留訊號存在時,將蛋白質瞄準內質網之訊號序列(例如泛分泌訊號Vtgss)對分泌作用而言為必要且足夠的。
選擇性之組成分(Ⅲ)b)為編碼至少一種用於溶解哺乳動物細胞之細胞溶質中的微生物及用於胞內釋出質體或表現載體(其包含在溶解之微生物中)之蛋白質的至少一種核苷酸序列。這類溶菌蛋白質(細胞內溶素)為,如:李斯特菌特異性溶菌蛋白質,諸如PLY551(Loessner,等人,1995)及/或受李斯特菌啟動子控制之李斯特菌特異性holin蛋白。本發明之一較佳體系係不同組成分(Ⅲ)b)之組合,例如:溶菌蛋白質與holin之組合。
組成分(Ⅲ)a)及/或(Ⅲ)b)二者可彼此各自獨立地為組成上活性。
於一較佳體系中係提供根據上述定義之微生物,其中組成分(Ⅲ)a)係選自:“第Ⅰ型分泌系統、第Ⅱ型分泌系統、第Ⅲ型分泌系統、第Ⅳ型分泌系統、第Ⅴ型分泌系統、大腸桿菌之溶血素轉運系統(訊號)(含有受hly-特異性啟動子控制之HlyA、HlyB及HlyD之核苷酸序列)、大腸桿菌之溶血素轉運系統(訊號)(含有受非hly-特異性細菌啟動因子控制之HlyA、HlyB及HlyD之核苷酸序列)、新月柄桿菌之S層(Rsa A)蛋白質之轉運訊
號、大腸桿菌之TolC蛋白質之轉運訊號、分泌訊號Vtgss及/或衍生自李斯特菌素之分泌訊號,p60及/或ActA”且其中組成分(Ⅲ)b)係選自:‘‘細胞內溶素、革蘭氏陽性細菌之溶菌蛋白質、單核細胞增生李斯特菌之溶菌蛋白質、單核細胞增生李斯特菌之PLY551及/或單核細胞增生李斯特菌之holin蛋白”。
於另一較佳體系中,該相同組成分(Ⅲ)a)可使組成分(Ⅰ)及組成分(Ⅱ)之表現產物表現在微生物之外表面上及/或使組成分(Ⅰ)及組成分(Ⅱ)之表現產物分泌。於此較佳體系中,組成分(Ⅲ)a)為僅編碼一種轉運系統之至少一種核苷酸序列,其可使組成分(Ⅰ)及組成分(Ⅱ)之表現產物伴隨表現在微生物之外表面上及/或使組成分(Ⅰ)及組成分(Ⅱ)之表現產物伴隨分泌,其中這類較佳組成分(Ⅲ)a)為至少一種編碼大腸桿菌之溶血素轉運系統(訊號)(含有受hly-特異性啟動因子控制之HlyA、HlyB及HlyD之核苷酸序列)或編碼大腸桿菌之溶血素轉運系統(訊號)的至少一種核苷酸序列(含有受非hly-特異性細菌啟動因子控制之HlyA、HlyB及HlyD之核苷酸序列)。
於另一較佳體系中係提供根據上述定義之微生物,其中根據組成分(Ⅲ)a),組成分(Ⅰ)及組成分(Ⅱ)之表現產物係被分泌。更合適的,組成分(Ⅰ)及組成分(Ⅱ)係連接在一起,一起表現並以由二種組成分編碼之融合蛋白質的形式分泌。最佳地,此融合蛋白質係選自下列
:“CtxB-PSA、CtxB-B-Raf V600E KD(激酶失效)、CtxB-B-Raf V600E激酶結構區、CtxB-B-Raf V600E激酶結構區KD(激酶失效)、CtxB-B-Raf、CtxB-B-Raf KD(激酶失效)、CtxB-B-Raf激酶結構區KD(激酶失效)、CtxB-HA1(流感病毒之紅血球凝集素的次單位1)、CtxB-HA12C”。
在本發明之過程中,“分泌”一詞係指藉由適當之分泌系統(諸如上述說明者)分泌蛋白質抗原、蛋白質毒素及/或毒素-抗原融合蛋白質通過革蘭氏陰性細菌之二種膜或革蘭氏陽性細菌之內膜及細胞壁進入鄰近環境之作用。
如吾人所知:利用分泌系統(諸如來自大腸桿菌之溶血素第Ⅰ型分泌系統),分泌產物通常在所有細胞部分中找到:細胞膜性、膜結合型,位於自體膜囊泡中及完全分泌入鄰近環境中(Balsalobre,等人,2006),在本發明之過程中分泌作用不需完全。然而完全或幾乎完全之分泌作用(即,完全分泌之分泌產物佔絕大部分)為所需或較佳者。
組成分(Ⅳ)代表編碼至少一種用於表現一或多種組成分(Ⅰ)至(Ⅲ)之活化序列之至少一種核苷酸序列,其中該活化序列可在微生物中被活化及/或為組織細胞特異性、腫瘤細胞特異性、巨噬細胞特異性、樹突細胞特異性、淋巴球特異性、功能特異性或非細胞特異性。
若表現係以膜結合方式結合在微生物外表面上,則宜選擇可在微生物中被活化之活化序列。這類活化序列為,
如:i)組成上活性之啟動子區,諸如具大腸桿菌之β-內醯胺酶基因或大腸桿菌hly基因位之內生性啟動子,tetA基因(Busby及Ebright,1994)之“核糖體結合部位”(RBS)的啟動子區,ii)可被誘導之啟動子,宜為被細胞接受後變成活性之啟動子。單核球增多性李斯特菌之actA啟動子(Dietrich,等人,1998)或鼠傷寒沙門氏菌之pcgC啟動子(Bumann,2001)均屬於此類。
若質體係在微生物溶解入哺乳動物之細胞溶質後釋出,則該活化序列並非細胞特異性,而為組織細胞特異性、細胞週期特異性或功能特異性。較宜為,這類活化序列係經過選擇而可特定於巨噬細胞、樹突細胞及淋巴球中活化。
於另一較佳體系中係提供根據上述定義之微生物,其中
組成分(Ⅰ)係選自下列:B-Raf V600E、B-Raf V600E激酶結構區、B-Raf V600E KD(激酶失效)、B-Raf V600E激酶結構區KD(激酶失效)、B-Raf KD(激酶失效)、B-Raf激酶結構區、B-Raf激酶結構區KD(激酶失效)、前列腺特異性抗原(PSA)、紅血球凝集素HA1(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)、紅血球凝集素HA12(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)、紅血球凝集素HA12C(宜來自A型流感病毒(A/泰國/1 (KAN-1)2004 (H5N1)”;
組成分(Ⅱ)係選自下列:“霍亂毒素次單位B(CTB、CtxB)、大腸桿菌熱不穩定性腸毒素次單位B(LTB)、破傷風毒素(TT)'”;組成分(Ⅲ)a)係選自:“大腸桿菌之HlyA溶血素轉運訊號與Hly分泌系統之組成分(含有受hly-特異性啟動因子控制之H1yA、HlyB及H1yD之核苷酸序列)“;[r3]組成分(Ⅳ)係選自“大腸桿菌hly基因位之內生性啟動因子“;其中組成分(Ⅰ)及組成分(Ⅱ)係連接在一起以使由此二種組成分編碼之融合蛋白質表現且其中該融合蛋白質係經分泌。
上述說明之根據組成分(Ⅰ)至(Ⅳ)及較佳體系之微生物在下文中稱為本發明之微生物。
本發明之微生物非常適合作為腫瘤瞄準過程中之活疫苗而用於腫瘤療法中。此係藉由本發明之微生物進行,其作為遺傳資訊之載體將異源抗原與蛋白質毒素遞送入腫瘤部位,以融合蛋白質之形式表現並於原位被分泌。
本發明之微生物可透過該經遞送、編碼和表現之毒素-抗原融合蛋白質之有效且優越的表現和分泌(即,無胞質性及/或週質性聚集出現)而令人驚異且方便地決定其特徵。
再者,與口服之蛋白質疫苗(其相對地係誘導免疫系統之系統性耐受性)相較下,經由投服本發明之微生物可
出人意表地引出強系統性細胞性免疫系統反應。
最值得注意的是,除了誘導Th1型之系統性細胞性免疫系統反應外,本發明之微生物引起第Ⅰ類MHC限制性CD8+
細胞毒性T細胞(CTL)活化並強烈增強此CTL免疫反應。
再者,除了誘導及/或增加強烈之細胞性系統性Th1及CTL免疫系統反應外,固有之免疫系統(如:NK細胞、NKT細胞及/或γ-δT細胞)亦意外地可被本發明之微生物以協同方式活化。
若使用霍亂毒素作為毒素組成分,本發明之微生物的利處在於人體內無預存之對抗該正常存在之毒素的免疫力(相對於破傷風毒素而言,例如:由兒童期之抗破傷風疫苗接種取得之免疫力)。因此,使用霍亂毒素及/或其次單位(尤其是CtxB)較佳。
本發明之微生物特別適合用於以活疫苗為基礎之標靶腫瘤(免疫)療法的口服投藥。藉此可改良患者之適應性。
然而,本發明之微生物並不僅限於用於腫瘤療法中。原則上,本發明之微生物亦適合治療及/或預防所有那些需要在療法中強制誘導系統性細胞性Th1免疫系統反應的疾病。這類感染性疾病之實例包括,但不限於:HIV、流感、HCV及其他病毒性疾病、結核桿菌病、單核球增多性李斯特菌及其他細菌性疾病。
本發明之微生物極適合用於治療像流感之類的疾病,
此類疾病需藉由組合黏膜免疫系統反應及系統性細胞性免疫反應來提供理想保護。另外,其可用於在過敏性疾病(如:過敏性鼻炎)中特異誘導似Th1免疫性。在這類情況中,該抗原為一種融合至蛋白質毒素組成分之過敏原,且各別疫苗之作用原則為將過敏反應(疾病)中對抗過敏原之以Th2為主的免疫反應轉向Th1免疫反應的免疫反應轉移。
較佳之施予模式為口服。於一較佳體系中,將根據本發明之沙門氏菌株在合適之介質中發酵、收成,然後藉由離心清洗,接著,利用合適之物質調配及穩定,再凍乾之。將凍乾之物質填入抗胃酸之膠囊中,其中含有之存活細胞數宜介於109
至1010
之細菌間。膠囊係藉由液體口服攝入。
或者,將如上述之凍乾細菌與含有緩衝劑(其可中和胃酸)之藥囊一起分發(藥學套組)。於一較佳體系中,此緩衝劑為碳酸鹽緩衝劑。在使用前,以水製備緩衝劑並提取後立即與水混合攝入凍乾細菌。
另一替換法係使用冷凍細菌。在此情況中,清洗後,經由安定劑(宜為蔗糖或甘油)來穩定細菌再將其冷凍並貯存在-80℃,每一劑量之濃度宜介於109
至1010
個細菌。此製劑宜用於如上述之加有碳酸鹽緩衝劑的藥學套組中。
於一較佳體系中係提供包含至少一種本發明微生物(宜為至少一種凍乾之本發明微生物)及藥學上可接受之載體的藥學組成物。
根據本發明之組成分(Ⅰ)至(Ⅳ)係藉由本技藝之技術熟習人士所熟知之方法引入本發明之微生物中。若該微生物代表細菌則將組成分插入質體或表現載體中,再將質體或表現載體轉移入細菌中。適合用於製造質體、表現載體及本發明微生物之分子生物選殖及轉形技術為本技藝之技術熟習人士所熟知且代表例行之實驗工作。
本發明之另一主題係投服含有本發明微生物之藥物製劑。投服係經由局部或系統性途徑,例如:口服、經由口、直腸途徑進入表皮、進入皮下組織、進入肌肉組織、進入體腔、進入器官、進入腫瘤或進入血液循環中。
這類藥物製劑為,例如:在藥師所熟悉之適合用於注射的溶液中的本發明微生物之懸浮液。
本發明之特殊主題為經口或直腸投服根據本發明之藥物,以治療及/或預防疾病。投藥可為一次或數次。在各次投藥中係投服約10至1011
之本發明微生物。若投服此數目之本發明微生物不會引起足夠之免疫反應則必須增加注射之數目。
在投服本發明之微生物後,該遞呈組成分(Ⅰ)的細胞(例如:為腫瘤起源之腫瘤細胞或組織細胞)之耐受性瓦解而觸發針對該腫瘤及/或其組織細胞之強烈系統性免疫反應。根據選擇之組成分(Ⅰ),此細胞性免疫反應係完全針對該腫瘤或亦針對腫瘤細胞,包括該腫瘤細胞源起之組織細胞。
於另一觀點中,本發明之目的已藉由提供包含至少一
種本發明微生物的藥物來解決,該本發明之微生物包含上述之遺傳組成分(Ⅰ)至(Ⅳ)或至少一種如此文所定義之藥學組成物。
於一較佳體系中,本發明之微生物可用於製備用於治療及/或預防選自下列之生理學及/或病理生理學之病況的藥物:“不受控制之細胞分裂、惡性腫瘤、良性腫瘤、實體腫瘤、肉瘤、癌、過度增生病、類癌、尤文氏肉瘤、卡波西氏肉瘤、腦瘤、源自腦及/或神經系統及/或腦脊膜之腫瘤、神經膠質瘤、神經母細胞瘤、胃癌、腎臟癌、腎細胞癌、前列腺癌(prostate cancer)、前列腺癌(prostate carcinomas)、結締組織腫瘤、軟組織肉瘤、胰臟腫瘤、肝腫瘤、頭腫瘤、頸瘤、食道癌、甲狀腺癌、骨肉瘤、腎母細胞腫瘤、胸腺瘤、睾丸癌、肺癌、支氣管癌、乳癌(breast cancer)、乳癌(mamma carcinomas)、小腸癌、結腸直腸腫瘤、結腸癌、直腸癌、婦科腫瘤、卵巢腫瘤(ovary tumors)/卵巢腫瘤(ovarian tumors)、子宮癌、子宮頸癌(cervical cancer)、子宮頸癌(cervix carcinomas)、子宮體癌(cancer of body of uterus)、子宮體癌(corpus carcinomas)、子宮內膜癌、尿膀胱癌、膀胱癌、皮膚癌、基底細胞癌、脊椎瘤(spinalioma)、黑色素瘤、眼內黑色素瘤、白血病、慢性白血病、急性白血病、淋巴瘤、感染、病毒或細菌感染、流行感冒、慢性發炎、器官排斥及/或自體免疫疾病”。
細菌感染包含,但不限於:炭疽熱、細菌性腦膜炎、
肉毒桿菌症、布氏桿菌症、彎曲菌病、貓抓病、霍亂、白喉、流行性斑疹傷寒、膿疱瘡、退伍軍人病、麻瘋病(韓森氏病)、鈎端螺旋體病、李斯特氏菌症、萊姆病、類鼻疽、MRSA感染、諾卡菌症、百日咳(whooping cough)、黑死病、肺炎球菌性肺炎、鸚鵡熱、Q熱、洛磯山斑點熱(RMSF)、沙門氏菌症、猩紅熱、志賀氏菌症、梅毒、破傷風、沙眼、結核病、兔熱病、傷寒、斑疹傷寒、尿道感染、細菌引起之心臟病。
病毒感染包含,但不限於:AIDS、與AIDS相關之併發症(ARC)、水痘(varicella)、一般感冒、巨細胞感染、柯羅拉多壁蝨熱、登革熱、埃博拉出血熱、手足口病、肝炎、單純性疱疹、帶狀疱疹、HPV、流行感冒(flu)、拉薩熱、麻疹、馬爾堡出血熱、感染性單核球過多症、腮腺炎、小兒麻痺症、進行性多病竈白質腦病變(progressive multifocal leukencephalopathy)、鼠疫、德國麻疹、SARS、天花(variola)、病毒性腦炎、病毒性腸胃炎、病毒性腦膜炎、病毒性肺炎、西尼羅河病、黃熱病。
慢性發炎或慢性發炎性疾病包含,但不限於:慢性膽囊炎、支氣管擴張症、類風濕性關節炎、橋本甲狀腺炎、發炎性腸病(潰瘍性結腸炎及克隆氏症)、矽肺症及其他肺塵症。
自體免疫病包含,但不限於:系統性症候群(諸如SLE、修格蓮氏(Sjögren's)症候群)、硬皮症、類風濕
性關節炎和多發性肌炎以及局部症候群(諸如IDDM)、橋本甲狀腺炎、艾迪生氏病、尋常性天疱瘡、牛皮癬、異位性皮膚炎、異位性症候群、氣喘、自體免疫溶血型貧血、多發性硬化症。
包含根據所有此文所描述之用於治療及/或預防如此文所描述和定義之生理學及/或病理生理學病況的較佳體系所定義之至少一種微生物及至少一種藥學組成物的對應藥物亦包括在本發明內。
於另一觀點中,本發明之目的係藉由提供包含如此文所說明之組成分(Ⅰ)至(Ⅳ)的質體或表現載體來解決。攜帶至少一種包含如此文所說明之組成分(Ⅰ)至(Ⅳ)的質體或表現載體的本發明微生物為較佳者。
於另一觀點中,本發明之目的係藉由提供用於製造本發明微生物之方法來解決,其中係製造如此文所說明之質體或表現載體,再以此質體或表現載體將微生物轉形。
於另一觀點中,本發明之目的係藉由提供包含至少一種本發明微生物或如上述之藥學組成物或如上述之藥物以及藥學上可接受之緩衝劑(宜為碳酸鹽緩衝劑)的藥學套組來解決。
所有列舉之參考資料及專利的全部內容在此併為參考資料。本發明藉由下列實例(但不限於此)更詳細地解釋。
實例1:在不同細菌載體株中建構、表現及分泌PSA-CtxB融合蛋白質
為了證明大腸桿菌第Ⅰ型溶血素分泌系統分泌腫瘤抗原(此處為前列腺素特異性抗原(PSA))與作為佐劑之蛋白質毒素組成分(此處為CtxB)之融合蛋白質的可行性及證明其效力,依此文之描述建構PSA-CtxB融合蛋白質。在不同革蘭氏陰性菌株(其可能作為腫瘤療法中之活疫苗株)中測試表現及分泌。分子生物學選殖係以質體/表現載體pMOhly1(此先前已描述)為基礎(Gentschev et al., 20O0;Gentschev et al., 1996, WO 03/072789)。
1A抗卡納黴素pMOhly1-表現載體衍生物之建構方法依(Datsenko and Wanner, 2000)中之描述進行pMOhly1之安苄青黴素抗性盒的取代。
簡單地說,使用同義引物(sense primer)P1 (5'-GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTG
GTGTAGGCTGGAGCTGCTTC-3')和反義引物P2 (5'-GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCC
CATATGAATATCCTCCTTA-3')及作為模版之質體pkD4,進行PCR以製造攜帶卡納黴素抗性基因(KanR
)之片段,此片段鄰接與安苄青黴素抗性基因同源之區(下方劃線處)。
將庇有pKD46質體及靶的質體pMOhly之大腸桿菌株
BW25114在補充以0.2% L-(+)-阿拉伯糖之LB介質(狄夫可(Difco))中,在37℃下生長3-4小時,再以PCR片段轉形。
轉形後,將細菌細胞分佈在含有25微克/毫升卡納黴素之LB瓊脂盤中並在37℃培育一整夜。
第二天,撿出KanR
選殖株並在含有50微克/毫升卡納黴素之LB介質中再培育48小時,以去除所有提供安苄青黴素抗性之質體。
最後,選擇具KanR
及安苄青黴素-敏感表型之選殖株。藉由PCR及定序來確認ApR
基因被KanR
盒取代。所產生之質體稱為pMKhly1。
使用同義引物PSA-Nsi1 (5'-GATTGGTGATGCA
TCCCTCAT-3;Nsi1限制位為下方劃線處)和反義引物PSA-Nsi12 (5'-GGTGCTCATGCAT
TGGCCACG-3')藉由PCR將編碼PSA之DNA片段增數。在Thermal Cycler 60(Biometra, Göttingen,德國)中進行30個循環之PCR:在94℃下1分鐘、54℃下1分鐘及72℃下2分鐘。
以Nsi1
限制酶分解後將攜帶psa基因之DNA片段插入輸出載體pMKhly1之單一Nsil
部位中。自大腸桿菌DH5α(Invitrogene,德國)中分離出所產生之質體pMKhly-PSA,藉由限制分析進行分析並定序。
1C pMKhly-PSA/CtxB質體之選殖方法
使用同義引物Ptac-SaⅡ (5'-AAAAAACGTCAC
GG CTGTGCAGGTCGTAAATCACTGC-3')和反義引物Ptac-NotI (5"-AAAAAAGCGGCCGC
GAAATTGTTATCCGCTCA CAATTCC-3')藉由PCR將編碼來自pGEX-6p-1-質體(Amersham Bioscience,德國)之Ptac-啟動子之201 bpDNA片段增數。在T3熱循環儀(Biometra,德國)中進行30個循環之PCR:在95℃下30秒、55℃下30秒及72℃下90秒。使用同義引物Rbs-NotI-前向(5'-AAAAAAGCGGCCGC
TAAGGATGAATTATGATTAAATTAAA ATTTGG-3')和反義引物ctb-SaⅡ-逆向(5'-TTTATAGTC GAC
TTAATTTGCCATACTAATTGCGGCAATCGC-3')藉由PCR將編碼來自霍亂弧菌EI tor之核糖體結合部位及CtxB的全部編碼序列之413 bpDNA片段增數。在T3熱循環儀(Biometra,德國)中進行PCR:步驟1:30個循環,在95℃下30秒、50℃下30秒及72℃下2分鐘。以Qiaquick PCR純化套組(Qiagen,德國)純化及以Not1
限制酶分解此二種片段後,再將此二片段連接,以產生594 bp之Ptac-ctxB-片段。亦將所產生之片段純化後,再以Sa Ⅱ
限制酶分解之,然後,將Ptac-ctxB-片段插入輸出載體pMKhly-PSA之單一Sa Ⅱ
部位中。自大腸桿菌DH5 α(Invitrogene,德國)中分離出質體pMKhly-PSA/CtxB,再加以分析並定序。
使用同義引物5'ctxB Nsi1 (5'-GCATATGCACATGCA T
CACCTCAAAATATTACTGAT-3')和反義引物3'ctxB Srfl Nsi1 (5'-GGCTTTTTTATATCTTATGCATGCCCGGGC
ATT GCGGCAATCGC-3)(Srfl部位為粗體)藉由PCR將之代表來自霍亂弧菌EI Ior之ctxB基因的~300 bp DNA片段增數。以QIAquick PCR純化套組(Qiagen,德國)純化後,以Nsi1限制酶分解之,將攜帶全ctxB基因但無N-端訊號序列之DNA片段插入輸出載體pMKhly1之單一Nsi1部位中。自大腸桿菌DH5 α (Life Technologies)中分離出所產生之質體pMKhly-CtxB,再分析並定序之。以引物5-PSA-鈍端(5'-GTGGGAGGCTGGGAGTGC-3)和3-PSA-鈍端(5'-GGGGTTGGCCACGATGGT-3'),藉由PCR將來自質體pCDNA3PSA之人類psa基因增數。在Thermal Cycler 60(Biometra,Göttingen,德國)中進行30個循環之PCR:在94℃下1分鐘、56℃下1分鐘及72℃下2分鐘。接著,將0.7kb DNA產物選殖入pMKhly-CtxB之Srfl部位中。藉由限制分析及定序來證明所產生之質體pMKhly-CtxB-PSA。
利用標準之電穿孔程序將質體pMKhly-CtxB-PSA轉形入不同之電勝任(electro-competent)菌株中。撿出抗卡納黴素之單一株落並令其生長在BHI介質(Beckton
Dickinson,美國:小牛腦,從200克泡製,6.0克/升;牛心,從250克泡製,9.8克/升;蛋白腖10.0克/升;氯化鈉5.0克/升;右旋糖2.0克/升;磷酸二鈉2.5克/升)中,密度為每毫升1x109
細胞。生長後,將20毫升培養在Heraeus離心機中於4℃,4000rpm (3000g)離心30分鐘。將18毫升上清液轉移入新管中。接著,加入1.8毫升TCA(三氯醋酸,Applichem,德國),將液體混合並在冰上培育至少1小時。培育後,將上清液在Heraeus離心機中於4℃,4000rpm (3000g)離心30分鐘。倒乾上清液並以1毫升丙酮p.a.(Aplichem,德國)清洗沈澱小丸。將沈澱物在Hereaus離心機中於4℃,4000rpm (3000g)離心10分鐘。將沈澱小丸風乾,在150微升之帶有或不帶有β-巰基乙醇之5x蘭里(Laemmli)緩衝劑(70mM Tris-HCl,pH6.8、40%(體積/體積)甘油、3%(體積/體積)十二烷基硫酸鈉、5%(體積/體積)2-巰基乙醇及0.05%(重量/體積)溴酚藍)中提取(Laemmli,1970)。在SDS PAGE之各道中使用20微升溶液。將分開之蛋白質電泳轉移(西方點墨)至Hybond ECL硝基纖維素膜(Amer-sham-Pharmacia, Little Chalfont, UK)上,並以含1%BSA之PBS(氯化鉀0.20克/升、磷酸二氫鉀0.20克/升、氯化鈉8.00克/升、無水磷酸二氫二鈉1.15克/升)阻斷一整夜。在PBS-吐溫(Tween)0.05%中清洗膜,與多株兔子抗PSA抗體(1:750,DAKO,丹麥)、CtxB抗體(1:1000,Zytomed,德國,柏林)或HlyAs抗體(
Gentschev,等人,1996)一起培育,接著,與HRP-偶合之抗兔子IgG(1/2,000;Dianova,德國,漢堡)培育1小時。以增強之化學發光套組(GE健康照護生命科學(GE Healthcare Life Science,德國)發展西方點墨。
第11圖描述PSA-CtxB融合蛋白質在不同細菌株(包括:大腸桿菌屬、都柏林沙門氏菌、檸檬酸桿菌屬、傷寒沙門氏菌Ty21a、鼠傷寒沙門氏菌屬、尤文氏菌屬(Erwinia spp.)及福氏志賀氏菌屬)中的功能表現及分泌。
表現及分泌PSA-CtxB融合蛋白質之傷寒沙門氏菌Ty21a係存放於德國微生物及細胞培養收集處(DSMZ),編號DSM 19244。
在動物腫瘤模型中,藉由下列實驗證明分泌之融合蛋白質(含有腫瘤抗原與蛋白質毒素)的優異保護效果。在此模型中係將表現及分泌CtxB-PSA融合蛋白質之鼠傷寒沙門氏菌aroA (SL7207)pMKhly-CtxB-PSA與其他對照菌株相比較。
在3週之期間內將DBA/2小鼠進行免疫處置三次。以
細菌進行免疫時,經由胃內施予50微升之7%NaHCO3
來預先處置動物,以增加胃內之pH。預先處置後5-10分鐘,經由胃內途徑施予於體積為100微升之PBS中的5x108
存活之卡納黴素不敏感性細菌。在對照組方面,依(Fensterle,等人,2005)中之描述,以編碼PSA(pcDNA-PSA)之裸出的質體DNA經由肌肉內途徑將小鼠免疫化。
最後一次免疫後7天,依先前發表(Fensterle,等人,1999)中之內容,將小鼠之脾臟通過網篩,再溶解紅血球,以製備免疫化小鼠之脾臟細胞。根據(Fensterle,等人,1999)中所發表之實驗計劃進行ELISPOT分析,以偵測PSA特異性CD8+
T細胞。
簡單地說,在PSA特異性CD8+
T細胞之活體外分析方面,以在100微升碳酸鹽緩衝劑(Fensterle,等人,1999),pH9.6中之5微克/毫升抗老鼠IFN-γ單株抗體(mAb)R4 (PharMingen)塗覆96槽硝基纖維素盤(Millititer HA;Millipore,麻省貝德福市)。在4℃培育一整夜並以在PBS中之1%BSA阻斷之,在每槽100微升RP10(Fensterle,等人,1999)中加入1x105
個來自經免疫接種之小鼠的脾臟細胞。
在CD8+
T細胞反應之分析方面,使用表現PSA之P815細胞株PPSA 24;簡單地說,此細胞株經由CMV啟動子(由質體pCDNA3編碼)表現全長PSA(Fensterle,
等人,2005)。於30 U/毫升IL-2之存在下,在37℃,5%CO2
中培育20-22小時後,清洗培養盤,再與100微升生物素化之抗老鼠IFN-γ mAbXMB1.2(0.25微克/毫升,PharMingen)一起培育2小時。清洗培養盤,於100微升之鹼性磷酸酶-偶合的抗生蛋白鏈菌素(PharMingen)1/20,000稀釋液之存在下,在37℃培育1小時。加入50微升溶於水中之即時可用的受質BCIP/NBT(Sigma,密蘇里州聖路易市)來目視檢查斑點。在放大3倍之解剖顯微鏡下計算斑點。
肽-特異性T細胞(CTL)之頻率係以IFN-γ-分泌細胞/105
脾臟細胞之數目表示。為了分析再次刺激後之T細胞反應,於60 U/毫升重組之IL-2的存在下,以在RP10介質(Fensterle,等人,1999)中的2x106
經放射處理之表現PSA之P815細胞(Fensterle,等人,2005)再度刺激2.5x107
個脾臟細胞5天。依上述,利用與4x105
飼養細胞(=來自未經處理過之DBA/2小鼠的新鮮製備的脾臟細胞)和105
PPSA24細胞混合之不同量之經再次刺激的細胞(每槽105
、3x104
、104
或3x103
)進行ELISPOT分析。
誘導細胞性免疫反應(尤其是CD8+
細胞毒性T細胞)在腫瘤療法之效率中扮演重要角色(Boon et al., 2006;Rosenberg, 2001)。因此,首先測試攜帶pMKhly-CtxB-PSA表現載體之重組菌株鼠傷寒沙門氏菌(SL7207)誘導PSA特異性CD8+
T細胞免疫反應之效率。
為達此目的,依此文之描述,以重組之SL7207/pMKhly-CtxB (n=10)、SL7207/pMKhly-CtxB-PSA (n=10)、SL7207/pMKhly-PSA (n=10)、SL7207/pMKhly-PSA/CtxB (n=10)、SL7207/pMKhly (n=7)經p.o.將64隻10-14週大之雌DBA-2小鼠免疫化,並以裸出的pcDNA3-PSA (n=10)作為陽性對照組。
第12圖顯示ELISPOT數據,其揭露經DNA免疫之小鼠顯示強CD8+
T細胞反應。再度刺激後,在以可分泌融合至CtxB之PSA蛋白質的沙門氏菌進行免疫接種的動物中可偵測到顯著之免疫反應,但以分泌單獨之PSA或分別之PSA和CtxB的沙門氏菌進行免疫接種的動物則不。有趣的是,以分泌單獨之CtxB毒素的沙門氏菌進行免疫接種的動物亦在再度刺激後顯示顯著之免疫反應。這些反應最可能係由於那些非特異辨識靶的細胞株之固有免疫系統的NK細胞或其他細胞。因此,從分泌之單獨的CtxB之數據可歸結出在分泌之CtxB-PSA融合蛋白質(分泌IFN-γ之脾臟細胞)方面所觀察到的免疫反應係由CD8+
T細胞反應及深遠之固有免疫系統(最可能為NK細胞)反應所組成。
為了分析免疫作用之保護能力,根據上述計劃將每組6-7隻小鼠進行免疫處置。在小鼠第三次免疫化後二週,藉由二次s.c.注射1x106
細胞入剔毛之腹部皮膚的每一側
,以PPSA 24(見上述)進行挑戰。在14天之期間內監控小鼠之腫瘤外觀並經由測量最大及最小之腫瘤直徑來評估腫瘤直徑。利用下式以旋轉橢球體(rotation ellipsoid)形式計算腫瘤體積:
藉由單向ANOVA及Dunnett氏多向比較後檢測進行結果之顯著性分析(使用Graph Pad Prism軟體)。後檢測僅在ANOVA結果為顯著時才進行。
第13圖顯示之結果係以平均值+/-SD表示。在第6、9、12及14天觀察到顯著之保護效果。如所預期者,所包含之以裸出之DNA進行的疫苗接種可作為完全保護小鼠免於腫瘤生長之對照組。然而,以裸出之DNA進行之疫苗接種在人體中最多僅顯示出中等效力。考慮細菌構造物時,疫苗株SL7207/pMKhly-CtxB-PSA(表現及分泌CtxB-PSA融合蛋白質)證明為最有效者。其在腫瘤挑戰後第9、12及14天顯著減少腫瘤體積。注意,與第14天之SL7207/pMKhly-CtxB-PSA的數值相較下,SL7207/pMKhly-CtxB株亦減少腫瘤生長。雖然並不顯著,此遞延效果與ELISPOT分析中所測得之細胞反應非常配合。再者,SL7207/pMKhly-PSA株亦在第14天取得顯著之保護效果,此表示本疫苗株亦可誘導低於偵測門檻之T細胞反應。相反地,SL7207/pMKhly-PSA/CtxB疫苗株並不誘導相關效果,且係維持在與單獨之SL7207/pMKhly1相同之範圍
內。
依類似於實例1之程序進行,但使用另一種腫瘤抗原,在激酶結構區具有10aa缺失之致癌基因B-Raf V600E(B-Raf V600E激酶結構區激酶失效(KD),或簡單地記為Braf*KD、更簡單地記為BKD)。依此文之描述建構由此致癌基因及作為佐劑之蛋白質毒素組成分(此處為CtxB)所組成之BKD-CtxB融合蛋白質。在人類疫苗株傷寒沙門氏菌Ty21a中證明其表現及分泌。分子選殖係以先前描述之質體/表現載體pMOhly1為基礎(Gentschev et al., 2005;Gentschev et al., 1996)且選殖程序係類似於本申請案之實例1。所產生之載體稱為pMBKDC。該融合蛋白質之序列列於第10圖中。
第17圖描述利用載體pMBKDC電穿孔入傷寒沙門氏菌Ty21a及其他菌株中之融合蛋白質的表現及分泌情形。
表現及分泌BKD-CtxB融合蛋白質之傷寒沙門氏菌Ty21a菌株係存放於德國微生物及細胞培養收集處(DSMZ),編號DSM 19245。
為了比較活疫苗分泌之毒素-抗原融合蛋白質的免疫
效力,建構一包含廣泛用於免疫研究之抗原(雞卵白蛋白(OVA))和CtxB之融合蛋白質。
使用同義引物Nsi1-OVA-前向5'-CAT GTA TGC ATT AGC CAT GGT ATA CCTGG-3'和反義引物Nsi1-OVA-反向5'-TTT TTT ATG CAT AAG GG AAA CAC CAC ATC TGC C-3',藉由PCR將代表ova
基因(NM205152)之1033bp DNA片段增數。以QIAquick PCR純化套組(Qiagen,德國)純化後,以Nsi1
限制酶分解之,將攜帶全ova
基因,無N-端訊號序列之DNA片段插入輸出載體pMKhly1之單一Nsi1
部位中。自大腸桿菌DH5 α(生命科技(Life Technologies))中分離出所產生之質體pMKhly-Ova,再加以分析並定序。
使用同義引物5'ctxB
Nsi1 (5'-GCATATGCACATGCA T
CACCTCAAAATATTACTGAT-3)和反義引物3'ctxB
Srfl Nsi1 (5'-GGCTTTTTTATATCTTATGCATGCCCGGGC
ATTGCGGCAATCGC-3')(Srfl
部位為粗體),藉由PCR將代表來自霍亂弧菌EI tor之ctxB
基因的~300bp DNA片段增數。以QIAquick PCR純化套組(Qiagen,德國)純化後,以Nsi1
限制酶分解之,將攜帶全ctxB
基因,無N-端訊號序列之DNA片段插入輸出載體pMKhly1之單一Nsi1
部位中。自大腸桿菌DH5 α(生命科技)中分離出所產生之質體pMKhly-CtxB,再加以分析並定序。使用5-OVA-Sfr1 GCC ATC ATG TCA GCT CTA和3-OVA-Sfr1 AGG GGA AAC ACA TCT GCC引物,藉由PCR將來自
pCI-OVA質體之ova基因增數。PCR係在Thermal Cycler 60(Biometra, Göttingen,德國)中進行30個循環:在94℃下1分鐘、49℃下1分鐘及72℃下2分鐘30秒。接著,將1.1-kb DNA產物選殖入質體pMKhly-CtxB之Srfl
部位。藉由限制分析檢查所產生之質體pMKhly-CtxB-OVA並定序。
將此融合蛋白質引出Th1免疫反應之能力與由pMKhly載體編碼之單獨分泌的OVA加上編碼干擾素(IFN-γ)、介白素(IL-12)及趨化素(IP-10)的DNA遞送質體(其編碼受真核細胞啟動子控制的蛋白質)之引出反應的能力相比較。
依上述進行免疫程序及ELISPOT分析。簡單地說,以不同菌株經口將C57BL/6小鼠進行免疫處置三次。最後一次免疫處置後7天,移出脾臟,再刺激5天並在ELISPOT分析中分析。為了再刺激脾臟細胞,使用以SIINFEKL肽(字母代表胺基酸)脈衝處理之細胞、卵白蛋白之H-2b
-限制MHC-I抗原決定部位。
第14圖顯示與共同遞送IFN-γ或IP-10之構造物相較下,包含雞卵白蛋白(OVA)與CtxB之融合蛋白質於誘導OVA特異性CD8+
T細胞反應及可能之固有免疫系統反應上的優越效力。當菌株具有經共同遞送之IL-12構造物時,其顯示出類似之效力。
本發明微生物於表現及分泌任何(非腫瘤)抗原之適合性係透過畜禽疫苗株,鼠傷寒沙門氏菌VacT中該融合至CtxB之禽流感病毒H5N1的溶血素H1蛋白質之表現來證明。
在PCR中使用同義引物5'-HA-G:5'-ATC TGT CAA ATG GAG AAA-3'和反義引物3-HA12C:5'-TAC TCC ACT TAT TTC CTC TCT-3',以將編碼H5,不帶有C-端膜結構區(H12C)之DNA片段增數。接著,將PCR產物選殖入pMKbly-CtxB之Srfl部位。藉由限制分析及定序檢查所產生之pMKhly-CtxB-H12C質體。由抗CtxB及HlyA之多株抗體所進行之免疫點墨法顯示出鼠傷寒沙門氏菌VacT/pMKhly-CtxB-H12C菌株可有效地表現及分泌雜交種H5-蛋白質(第2圖)。在實驗條件下經分泌之H5的量為2-3微克蛋白質/毫升上清液。
令細菌生長在BHI介質中使密度達到每毫升1x109
細胞。將20毫升培養在Heraeus離心機中於4℃,4000rpm (3000g)離心30分鐘。將18毫升上清液轉移入新管中。接著,加入1.8毫升TCA(三氯醋酸,Applichem,德國),將液體混合並在冰上培育至少1小時。培育後,將懸浮液在Heraeus離心機中於4℃,4000rpm (3000g)離心30分鐘。倒乾上清液並以1毫升丙酮p.a.(Applichem,德國)清洗沈澱小丸;將沈澱物在Hereaus離心機中於4℃,4000rpm (3000g)離心10分鐘。將沈澱小丸風乾,在150微升之5x蘭里(Laemmli)緩衝劑中
提取。在SDS PAGE之各道中使用20微升溶液。將分開之蛋白質電泳轉移(西方點墨)至Hybond ECL硝基纖維素膜(Amersham-Pharinacia, Little Chalfont, U.K.)上,並以含1%BSA之PBS阻斷一整夜。在PBS-吐溫0.05%中清洗膜,與多株兔子抗CtxB抗體(1:1000, Zytomed,德國柏林)或HlyAs抗體(Gentschev,等人,1996)一起培育,接著,與HRP-偶合之抗兔子IgG(1/2,000;Dianova,德國,漢堡)一起培育1小時。使用增強之化學發光套組(GE Healthcare Life Science,德國)進行西方點墨。
第15圖描述在畜禽疫苗株,鼠傷寒沙門氏菌VacT中,HA12C-CtxB融合蛋白質之有效表現及功能性分泌作用。
數種革蘭氏陰性細菌均攜帶可用於分泌抗原之第Ⅲ型分泌系統。通常,這些系統可用於直接將異源抗原注射入細胞中。在此實例中,作為分泌訊號(YopE)之耶爾森氏菌毒素組成分係遺傳上融合至作為佐劑之熱不穩定性腸毒素次單位B (LT-B)片段與腫瘤抗原,PSA上。此構造物係欲在合適(宜為減毒)之耶爾森氏菌株中表現,以經由YopE訊號序列,藉由內生性第Ⅲ型分泌機制分泌出融合蛋白質。
在此實例中,將基因融合物YopE18-LT-B-PSA選殖入質體paCYC184之EcoRⅠ部位以將構造物與所使用之cat基因同向使用而產生由該質體編碼之cat啟動因子表現的產物(http://www.fermentas com/techinfo/nucleicacids/mappacyc 184.htm)。為了增強表現,可使用任何啟動因子。
下文中示範一種選殖程序。在PCR反應中,利用下列引物將來自大腸桿菌包含熱不穩定性腸毒素次單位B之染色體DNA或包含此序列(GenBank編號AF242418)之載體增數:Eco-yope18-f(包含EcoRⅠ部位+YopE18訊號序列):5'-CTGAATTC
ATGAAAATATCATCATTTATTTCTACATCAC TGCCCCTGCCGGCATCAGTGTCAAATAAA
GTAAAATGTTTTGTTTTAT
3'(5'區黑:EcoRⅠ部位+3鹼基5',紅:編碼YopE蛋白質第Ⅲ型分泌系統之訊號序列(aal-18)之序列,GenBank編號M92066;italic區:LT-B基因之同源區(GB編號AF242418),鹼基4-28)。
磷酸化之反向引物LT-B-r:5'-GTTTTCCATACTGATTGCCGCAATTGAATTGG-3(GB AF242418之逆向股372-341)。
同時,利用下列引物將如本申請案中所描述之來自載體pMK-CtxB-PSA的PSA序列增數:
磷酸化之前向引物psa-f:5'-GTGGGAGGCTGGGAGTGCGAG-3'反向引物psa-eco-r:5' CCTGAATTC
TTAGACGTGATACCTTGAAGCAC(5'黑:EcoRⅠ部位+3鹼基5',藍:終止密碼子,黑:PSA序列之3'區,本申請案)。
接著,在適當條件下,以DNA連接酶將二種片段連接在一起。連接後,在第二次PCR反應中利用上述之Eco-yope18-f及PSA-Eco-R引物將新片段增數。
利用合適之PCR純化套組將所產生之片段純化,以去除緩衝劑和寡核苷酸,接著,在適當反應條件下以EcoRⅠ酶分解之。利用瓊脂糖凝膠電泳法將所產生之1040 bp片段純化,連接入經EcoRⅠ分解之載體paCYC184。將具四環黴素抗性、cm敏感性株落定序後,選出具有與cm基因同向整合之正確序列的株落並利用電穿孔法將其轉形入合適之耶爾森氏菌株內。此疫苗株將經由其內生性第Ⅲ型分泌系統分泌LT-B-PSA融合物。
革蘭氏陽性及陰性細菌具有不同之分泌必要條件。革蘭氏陰性細菌擁有二層膜,因此,除了分泌訊號外之分泌機制對完全分泌而言是必須的。在革蘭氏陽性細菌方面,分泌訊號序列已足夠。
在此實例中係描述利用p60分泌訊號在李斯特菌中分泌破傷風類毒素PSA融合物的系統。
首先,利用Nsi1分解載體pUC18 (PS)actAOVATinlA(Loeffler,等人,2006),接著,在適當條件下以3'-5'核酸外切酶處理之。最後,藉瓊脂糖凝膠電泳法將片段純化。
同時,利用下列磷酸化引物將來自合適來源(如:Clostridium tetanii
菌株之基因組DNA)之破傷風類毒素片段C(GenBank編號M12739)增數Ltetc for:5'-T
AAAAATCTGGATTGTTGGGTTG-3',而下方劃線之額外鹼基係為了確保融合物之正確框構。
tetc rev:5'-ATCATTTGTCCATCCTTCATCTG-3'。
利用下列磷酸化引物將來自本申請案所描述之質體pMO BKDC的BRAF KD片段增數:br for:5'-GATTGGGAGATTCCTGATG-3' br rev:5'-CCCGTGGACAGGAAACGCACC-3'。
利用合適之PCR純化套組將二種片段純化,接著,在合適條件下,以DNA連接酶將其連接在一起。連接後,利用合適之PCR純化套組將片段再度純化並利用tetc for及br rev引物增數。增數後,藉由瓊脂糖凝膠電泳法將所產生之2280 bp片段純化並連接入pUC18 (PS)actAOVATinlA之純化的Nsi1-3'-5'核酸外切酶片段中。連接並轉形入大腸桿菌後,選出帶有在框構內之片段的株落。接著,利用Pst1及Sac1切質體,利用凝膠電泳法將
2837 bp片段純化並連接入經Pst1和Sac1適當分解且純化之載體pSP118中(Loeffler,等人,2006)。將所產生之載體pSP118-act-TetC-Braf*KD轉形入帶有trpS缺失之李斯特菌株(如:單核球增多性李斯特菌δtrpsδaroA/B)中(Loeffler,等人,2006)。在此設定中,該質體係經由以質體為基礎之trpS(“平衡之致命系結”)穩定而該質體係編碼噬菌體賴胺酸(其導致該菌株之胞內溶解)。act-TetC-Braf*KD盒主要在真核宿主細胞中在actA啟動子(其有些胞內滲漏)之控制下表現(Loeffler,等人,2006)。表現盒之actA訊號序列導致TetC-Braf*KD融合蛋白質分泌。
此實例中係建構一種分泌CtxB-PSA融合蛋白質之真核細胞株(如:腫瘤細胞株)。為了達到此目的,使用全分泌訊號(US 6,733,997),原則上,此全分泌訊號可令對應之表現盒在不同來源之細胞(包括哺乳動物細胞、酵母菌及原核細胞)中分泌。
首先,利用下列引物將來自質體pMO BKDC(本申請案)之CtxB-Braf*KD融合物增數:CB-for:5'-atcGGATCC
TCAAAATATTACTGATTTGTGTGC-3'(小寫字母:間隔子,下方劃線:BamHI部位,大寫字母:CtxB5')
CB-rev:5'-tagGGATCC TTA
GTGGACAGGAAACGCACCATATCC-3'(小寫字母:間隔子,下方劃線:BamHI部位,italic:終止密碼子,大寫字母:BRaf*KD3')。
接著,利用合適之PCR純化套組將PCR產物純化,再接著以BamHI部分分解之(片段含有內BamHI部位)。經由瓊脂糖凝膠電泳法將部分分解之1231 bp片段分離出再接著連接入經BamHI分解及凝膠純化之質體pVtgEGFP(美國專利申請案6733997)中。接著,選出帶有框構內與Vtgss同向之株落,命名為pVtgCtxBRAf。此質體可經由電穿孔法轉形入真核細胞株(此可經由kan/neo選擇盒選擇)。該細胞株可為已建立之細胞株(如:作為異源癌症疫苗之癌症細胞株)或自患者衍生之作為自體癌症疫苗的腫瘤細胞。
在任何情況中,由遺傳上融合至CtxB-Braf*KD之載體pVtgEGFP編碼的分泌訊號Vtgss將可使該融合蛋白質分泌出。
利用類似方法亦可在酵母菌中表現融合蛋白質。例如:證明US 6,733,997之第11圖中所描述之一種修改的選殖策略。首先,藉EagⅠ及Eco47Ⅲ切出如上述之來自質體pVtgCtxBRAf之含有Vtgss-CtxB-Braf*KD融合物的盒並插入載體pBSPGK中(US 6,733,997,第11圖)。接著,以Sac1及HindⅢ分解所產生之載體並依類似於US 6,733,997(第11圖)中所描述之方法將包含PGK成分及
Vtgss-CtxB-Braf*KD融合物之片段整合入載體pYEX-S1的對應區中。所產生之質體可藉由電穿孔法轉形入酵母菌株(如:啤酒酵母)中。該酵母菌將表現及分泌該融合蛋白質且其可用於疫苗接種之目的中。
第1圖描述大腸桿菌溶血素A(hlyA)分泌訊號之核苷酸序列(5'→3')。Nsi1部位處下方劃線。
第2圖描述大腸桿菌溶血素第Ⅰ型轉運系統之大腸桿菌溶血素B (hlyB)的核苷酸序列(5'→3')。
第3圖描述大腸桿菌溶血素第Ⅰ型轉運系統之大腸桿菌溶血素D (hlyD)的核苷酸序列(5'→3')。
第4圖描述不帶有訊號肽之人類前列腺特異性抗原(PSA),編號M26663(現代人前列腺-特異性抗原mRNA,完整cds),100-807區(對應肽:AA26-261)的核苷酸序列(5'→3')。
第5圖描述不帶有訊號肽,包含204-494區之霍亂毒素次單位B (CtxB),編號K01170(霍亂腸毒素次單位A2 (γ)之霍亂弧菌toxA及toxB基因)的核苷酸序列(5'→3')。
第6圖描述含有V600E突變之人類B-raf蛋白質(BRAF)(編號M95712(B-raf蛋白質(BRAF)mRNA,完整編號序列))的B-Raf激酶結構區(B-Raf KD)之1403-2359區(不含1448-1477區(對應肽:AA448-766,不含AA463-472))的核苷酸序列(5'→3')。
第7圖描述人類HLA B27限制之B-Raf V600E抗原
決定部位的核苷酸序列(5'→3')。
第8圖描述CtxB-PSA-HlyA之基因融合構造物的核苷酸序列(5'→3')。
第9圖描述CtxB-B-Raf V600E-HlyA之基因融合構造物的核苷酸序列(5'→3')。
第10圖描述CtxB-B-Raf V600E激酶結構區KD-HlyA之基因融合構造物的核苷酸序列(5'→3')。
第11圖描述在pMKhly-PSA-CtxB之不同菌種重組株中之PSA-CtxB融合蛋白質的功能表現及分泌。
第12圖描述在以不同之活疫苗載體免疫的小鼠中,用於測量CD8+
T-細胞及固有免疫系統(NK細胞)反應之分泌IFN-γ的脾臟細胞。
第13圖描述以不同活疫苗載體進行免疫後反應之腫瘤體積減小情形。
第14圖描述在以不同之活疫苗(其攜帶不同之免疫佐劑與CtxB-OVA融合構造物)免疫化的小鼠中,用於測量CD8+
T-細胞及固有免疫系統(NK細胞)反應之分泌IFN-γ的脾臟細胞。
第15圖描述在畜禽疫苗株中,禽流感紅血球凝集素H1-CtxB融合蛋白質之有效表現及功能性分泌作用,該獸醫疫苗株中包含H5N1紅血球凝集素(HA1+HA2,稱為HA12),無跨膜區(HA12c)之完整序列。活減毒鼠傷寒血清型腸沙門氏菌vacT (gyrA
D87G)及活減毒腸炎血清型腸沙門氏菌vacE(菌株Sm24/Rif12/Ssq)二者均來自
LOHMANN ANIMAL HEALTH GMBH & CO KG。
第16圖描述編碼CtxB-HA12c-HlyA融合蛋白質的核苷酸序列。
第17圖描述利用載體pMBKDC電穿孔入傷寒沙門氏菌Ty21a及其他菌株之融合蛋白質的表現及分泌情形:A)第A1-A3道:利用用於偵測之抗B-Raf抗體,根據TP StMoBKDC分析上清液。第A1道:傷寒沙門氏菌Ty21a pMoBKDC,第A2道:傷寒沙門氏菌Ty21a,第A3道:鼠傷寒沙門氏菌aroA。Braf-V600E kD-CtxB融合蛋白質係以上方箭頭標記且具有約48kDa之預期大小。蛋白質標記:Invitrogen BenchMark預染蛋白梯(Pre-Stained protein ladder),#10748-010。
B)如A中之同等分析,利用新製造之抗-HlyA抗體作為偵測抗體。第1、2道:StMoPc構造物,第3道:傷寒沙門氏菌Ty21a MoBKDC,第4道:大腸桿菌MoBKDC,第5道:傷寒沙門氏菌Ty21a,第6道:鼠傷寒沙門氏菌aroA。蛋白質標記:Invitrogen BenchMark預染蛋白梯,#10748-010,目錄編號1315592。
第18圖描述空載體pMKhly1之完整核苷酸序列(5'→3')。
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Claims (18)
- 一種作為編碼抗原和蛋白質毒素之核苷酸序列的載體之微生物,其包含下列組成分:(I)至少一種編碼至少一種野生型或突變之蛋白質的至少一種完全或部分抗原之核苷酸序列;及(Ⅱ)至少一種編碼至少一種蛋白質毒素及/或至少一種蛋白質毒素次單位之核苷酸序列;及(Ⅲ)a)至少一種編碼至少一種轉運系統(其可使組成分(I)及組成分(Ⅱ)之表現產物表現在該微生物之外表面上及/或可使組成分(I)及組成分(Ⅱ)之表現產物分泌);及/或編碼至少一種訊號序列(其可使組成分(I)及組成分(Ⅱ)之表現產物分泌)之核苷酸序列;及/或b)選擇性地,至少一種編碼至少一種用於於哺乳動物細胞之細胞溶質中溶解微生物及用於胞內釋出質體或表現載體(其包含在該溶解之微生物內)之蛋白質的核苷酸序列;及(Ⅳ)至少一種編碼至少一種用於表現一或多種組成分(I)至(Ⅲ)之活化序列的核苷酸序列,其中該活化序列可在該微生物中被活化及/或為組織細胞特異性、腫瘤細胞特異性、巨噬細胞特異性、樹突細胞特異性、淋巴細胞特異性、功能特異性或非細胞特異性; 其中該組成分(I)至(Ⅳ)中之任一可出現一次或數次且若組成分(I)至(Ⅳ)中之一種組成分出現數次,則其可彼此各自獨立地為相同或相異;其中組成分(I)及組成分(Ⅱ)並非相同,亦即組成分(I)並不編碼至少一種編碼至少一種蛋白質毒素及/或至少一種蛋白質毒素次單位的核苷酸序列;其中組成分(I)及組成分(Ⅱ)並非相同;且其中該微生物係選自大腸桿菌、沙門氏菌屬、耶爾森氏菌屬、弧菌屬、李斯特菌屬、志賀氏菌屬及假單胞菌屬。
- 如申請專利範圍第1項之微生物,其中排除霍亂弧菌作為微生物。
- 如申請專利範圍第1或2項之微生物,其中該組成分(I)之至少一種野生型或突變之蛋白質的至少一種完全或部分抗原係選自下列之野生型蛋白質及彼之已知突變體:“受體;受體之胞外、跨膜或胞內部分;黏著分子;黏著分子之胞外、跨膜或胞內部分;訊號轉導蛋白質;細胞週期蛋白質;轉錄因子;分化蛋白質;胚胎蛋白質;病毒蛋白質;過敏原;微生物致病原之蛋白質;真核生物致病原之蛋白質;腫瘤睾丸抗原蛋白質;腫瘤抗原蛋白質;及/或組織-細胞特異性蛋白質”,其中該組織細胞係選自“甲狀腺、乳腺、唾液腺、淋巴結、乳腺、胃黏膜、腎臟、卵巢、前列腺、子宮頸、膀胱黏膜及神經”。
- 如申請專利範圍第3項之微生物,其中該組成分 (I)之至少一種野生型或突變之蛋白質的至少一種完全或部分抗原係選自下列之野生型蛋白質及彼之已知之突變體:“Her-2/neu、雄激素受體、雌激素受體、中期因子(midkine)受體、EGF受體、ERBB2、ERBB4、TRAIL受體、FAS、TNFα受體、TGF-β受體、乳鐵蛋白受體、髓鞘鹼性蛋白(basic myelin)、α-乳白蛋白、GFAP、纖維酸性蛋白質、酪胺酸酶、EGR-1、MUC1、c-Raf(Raf-1)、A-Raf、B-Raf、B-Raf V599E、B-Raf V600E、B-Raf KD、B-Raf V600E激酶結構區、B-Raf V600E KD、B-Raf V600E激酶結構區KD、B-Raf激酶結構區、B-Raf激酶結構區KD、N-Ras、K-Ras、H-Ras、Bcl-2、Bcl-X、Bcl-W、Bfl-1、Brag-1、Mcl-1、A1、Bax、BAD、Bak、Bcl-Xs、Bid、Bik、Hrk、Bcr/abl、Myb、C-Met、IAP1、IAO2、XIAP、ML-IAP LIVIN、存活素、APAF-1、細胞週期素D(1-3)、細胞週期素E、細胞週期素A、細胞週期素B、細胞週期素H、Cdk-1、Cdk-2、Cdk-4、Cdk-6、Cdk-7、Cdc25C、p16、p15、p21、p27、p18、pRb[r1] 、p107、p130、E2F(1-5)、GAAD45、MDM2、PCNA、ARF、PTEN、APC、BRCA、Akt、PI3K、mTOR、p53及同源物、C-Myc、NFkB、c-Jun、ATF-2、Sp1、前列腺特異性抗原(PSA)、癌胚抗原、α-胎兒蛋白、PAP;PSMA;STEAP;MAGE、MAGE-1、MAGE-3、NY-ESO-1、PSCA、MART、Gp100、酪胺酸酶、GRP、TCF-4、HIV病毒、HPV病毒、HCV病毒、HPV病毒、EBV病毒、 CMV病毒、HSV病毒、流感病毒、A型流感病毒、A型流感病毒(H5N1)及(H3N2)、B型流感病毒、C型流感病毒之病毒抗原;紅血球凝集素(hemagglutinins)、紅血球凝集素H1、紅血球凝集素H5、紅血球凝集素H7、紅血球凝集素HA1(宜來自A型流感病毒(A/泰國/1(KAN-1)2004(H5N1)、紅血球凝集素HA12(宜來自A型流感病毒(A/泰國/1(KAN-1)2004(H5N1)、紅血球凝集素HA12C(宜來自A型流感病毒(A/泰國/1(KAN-1)2004(H5N1)、神經胺基酸酶、[r2] p60、LLO、尿素酶、CSP、calflagin及/或CPB”或者,其中該組成分(I)之至少一種野生型或突變之蛋白質的至少一種完全或部分抗原係選自下列由野生型蛋白質及彼之已知之突變體(括號中之編號)所組成之激酶:AATK(NM 004920),ABL1(NM 005157),ABL2(NM 005158),ACK1(NM 005781),ACVR1(NM 001105),ACVR1B(NM 020328),ACVR2(NM 001616),ACVR2B(NM 001106),ACVRL1(NM 000020),ADCK1(NM 020421),ADCK2(NM 052853),ADCK4(NM 024876),ADCK5(NM 174922),ADRBK1(NM 001619),ADRBK2(NM 005160),AKT1(NM 005163),AKT2(NM 001626),AKT3(NM 005465),ALK(NM 004304),ALK7(NM 145259),ALS2CR2(NM 018571),ALS2CR7(NM 139158),AMHR2(NM 020547),ANKK1(NM 178510),ANKRD3(NM 020639), APEG1(NM 005876),ARAF(NM 001654),ARK5(NM 014840),ATM(NM 000051),ATR(NM 001184),AURKA(NM 003600),AURKB(NM 004217),AURKC(NM 003160),AXL(NM 001699),BCKDK(NM 005881),BCR(NM 004327),BIKE(NM 017593),BLK(NM 001715),BMPR1A(NM 004329),BMPR1B(NM 001203),BMPR2(NM 001204),BMX(NM 001721),BRAF(NM 004333),BRD2(NM 005104),BRD3(NM 007371),BRD4(NM 014299),BRDT (NM 001726),BRSK1(NM 032430),BRSK2(NM 003957),BTK(NM 000061),BUB1(NM 004336),BUB1B(NM 001211),CABC1(NM 020247),CAMK1(NM 003656),CaMK1b(NM 198452),CAMK1D(NM 020397),CAMK1G(NM 020439),CAMK2A(NM 015981),CAMK2B(NM 001220),CAMK2D(NM 001221),CAMK2G(NM 001222),CAMK4(NM 001744),CAMKK1(NM 032294),CAMKK2(NM 006549),CASK(NM 003688),CCRK(NM 012119),CDC2(NM 001786),CDC2L1(NM 001787),CDC2L5(NM 003718),CDC42BPA(NM 014826),CDC42BPB(NM 006035),CDC7L1(NM 003503),CDK10(NM 003674),CDK11(NM 015076),CDK2(NM 001798),CDK3(NM 001258),CDK4(NM 000075),CDK5(NM 004935),CDK6(NM 001259),CDK7(NM 001799),CDK8(NM 001260),CDK9(NM 001261),CDKL1(NM 004196),CDKL2(NM 003948),CDKL3(NM 016508),CDKL4(NM 001009565),CDKL5(NM 003159),CHEK1(NM 001274),CHUK(NM 001278),CIT(NM 007174),CLK1(NM 004071),CLK2(NM 003993),CLK3(NM 003992),CLK4(NM 020666),CRK7(NM 016507),CSF1R(NM 005211),CSK(NM 004383),CSNK1A1(NM 001892),CSNK1D(NM 001893),CSNK1E(NM 001894),CSNK1G1(NM 022048),CSNK1G2(NM 001319),CSNK1G3(NM 004384),CSNK2A1(NM 001895),CSNK2A2(NM 001896),DAPK1(NM 004938),DAPK2(NM 014326),DAPK3(NM 001348),DCAMKL1(NM 004734),DCAMKL2(NM 152619),DCAMKL3(XM 047355),DDR1(NM 013993),DDR2(NM 006182),DMPK(NM 004409),DMPK2(NM 017525.1),DYRK1A(NM 001396),DYRK1B(NM 006484),DYRK2(NM 006482),DYRK3(NM 003582),DYRK4(NM 003845),EEF2K(NM 013302),EGFR(NM 005228),EIF2AK3(NM 004836),EIF2AK4(NM_001013703),EPHA1(NM 005232),EPHA10(NM 001004338),EPHA2(NM 004431),EPHA3(NM 005233),EPHA4(NM 004438),EPHA5(NM 004439),EPHA6(XM 114973),EPHA7(NM 004440),EPHA8(NM 020526),EPHB1(NM 004441),EPHB2(NM 017449),EPHB3(NM 004443),EPHB4(NM 004444),EPHB6(NM 004445),ERBB2(NM 004448),ERBB3(NM 001982),ERBB4(NM 005235),ERK8(NM 139021),ERN1(NM 001433),ERN2(NM 033266),FASTK(NM 025096),FER(NM 005246),FES(NM 002005),FGFR1(NM 000604),FGFR2(NM 022970),FGFR3(NM 000142),FGFR4(NM 022963),FGR(NM 005248),FLJ23074(NM 025052),FLJ23119(NM 024652),FLJ23356(NM 032237),FLT1(NM 002019),FLT3(NM 004119),FLT4(NM 002020),FRAP1(NM 004958),FRK(NM 002031),FYN(NM 002037),GAK(NM 005255),GPRK5(NM 005308),GPRK6(NM 002082),GPRK7(NM 139209),GRK4(NM 005307),GSG2(NM 031965),GSK3A(NM 019884),GSK3B(NM 002093),GUCY2C(NM 004963),GUCY2D(NM 000180),GUCY2F(NM 001522),H11(NM 014365),HAK(NM 052947),HCK(NM 002110),HIPK1(NM 152696),HIPK2(NM 022740),HIPK3(NM 005734),HIPK4(NM 144685),HRI(NM 014413),HUNK(NM 014586),ICK(NM 016513),IGF1R(NM 000875),IKBKB(NM 001556),IKBKE(NM 014002),ILK(NM 004517),INSR(NM 000208),INSRR(NM 014215),IRAK1(NM 001569),IRAK2(NM 001570),IRAK3(NM 007199),IRAK4(NM 016123),ITK(NM 005546),JAK1(NM 002227),JAK2(NM 004972),JAK3(NM 000215),KDR(NM 002253),KIS(NM 144624),KIT(NM 000222),KSR(XM 290793),KSR2(NM 173598),LAK(NM 025144),LATS1(NM 004690),LATS2(NM 014572),LCK(NM 005356),LIMK1(NM 016735),LIMK2(NM 005569),LMR3(XM 055866),LMTK2(NM 014916),LOC149420(NM 152835),LOC51086(NM 015978),LRRK2(XM 058513),LTK(NM 002344),LYN(NM 002350),MAK(NM 005906),MAP2K1(NM 002755),MAP2K2(NM 030662),MAP2K3(NM 002756),MAP2K4(NM 003010),MAP2K5(NM 002757),MAP2K6(NM 002758),MAP2K7(NM 005043),MAP3K1(XM 042066),MAP3K10(NM 002446),MAP3K11(NM 002419),MAP3K12(NM 006301),MAP3K13(NM 004721),MAP3K14(NM 003954),MAP3K2(NM 006609),MAP3K3(NM 002401),MAP3K4(NM 005922),MAP3K5(NM 005923),MAP3K6(NM 004672),MAP3K7(NM 003188),MAP3K8(NM 005204),MAP3K9(NM 033141),MAP4K1(NM 007181),MAP4K2(NM 004579),MAP4K3(NM 003618),MAP4K4(NM 145686),MAP4K5(NM 006575),MAPK1(NM 002745),MAPK10(NM 002753),MAPK11(NM 002751),MAPK12(NM 002969),MAPK13(NM 002754),MAPK14(NM 001315),MAPK3(NM 002746),MAPK4(NM 002747),MAPK6(NM 002748),MAPK7(NM 002749),MAPK8(NM 002750),MAPK9(NM 002752),MAPKAPK2(NM 032960),MAPKAPK3(NM 004635),MAPKAPK5(NM 003668),MARK(NM 018650),MARK2(NM 017490),MARK3(NM 002376),MARK4(NM 031417),MAST1(NM 014975),MAST205(NM 015112),MAST3(XM 038150),MAST4(XM 291141),MASTL(NM 032844),MATK(NM 139355),MELK(NM 014791),MERTK(NM 006343),MET(NM 000245),MGC33182(NM 145203),MGC42105(NM 153361),MGC43306(C9orf96),MGC8407(NM 024046),MIDORI(NM 020778),MINK(NM 015716),MKNK1(NM 003684),MKNK2(NM 017572),MLCK(NM 182493),MLK4(NM 032435),MLKL(NM 152649),MOS(NM 005372),MST1R(NM 002447),MST4(NM 016542),MUSK(NM 005592),MYLK(NM 053025),MYLK2(NM 033118),MYO3A(NM 017433),MYO3B(NM 138995),NEK1(NM 012224),NEK10(NM 152534),NEK11(NM 024800),NEK2(NM 002497),NEK3(NM 002498),NEK4(NM 003157),NEK5(MGC75495),NEK6(NM 014397),NEK7(NM 133494),NEK8(NM 178170),NEK9(NM 033116),NLK(NM 016231),NPR1(NM 000906),NPR2(NM 003995),NRBP(NM 013392),NRBP2(NM 178564),NRK(NM 198465),NTRK1(NM 002529),NTRK2(NM 006180),NTRK3(NM 002530),OBSCN(NM 052843),OSR1(NM 005109),PACE-1(NM 020423),PAK1(NM 002576),PAK2(NM 002577),PAK3(NM 002578),PAK4(NM 005884),PAK6(NM 020168),PAK7(NM 020341),PASK(NM 015148),PCTK1(NM 006201),PCTK2(NM 002595),PCTK3(NM 212503),PDGFRA(NM 006206),PDGFRB(NM 002609),PDK1(NM 002610),PDK2(NM 002611),PDK3(NM 005391),PDK4(NM 002612),PDPK1(NM 002613),PFYK1(NM 012395),PHKG1(NM 006213),PHKG2(NM 000294),PIK3R4(NM 014602),PIM 1(NM 002648),PIM2(NM 006875),PIM3(NM 001001852),PINK1(NM 032409),PKE(NM 173575),PKMYT1(NM 004203),pknbeta(NM 013355),PLK(NM 005030),PLK3(NM 004073),PRKAA1(NM 006251),PRKAA2(NM 006252),PRKACA(NM 002730),PRKACB(NM 002731),PRKACG(NM 002732),PRKCA(NM 002737),PRKCB1(NM 002738),PRKCD(NM 006254),PRKCE(NM 005400),PRKCG(NM 002739),PRKCH(NM 006255),PRKCI(NM 002740),PRKCL1(NM 002741),PRKCL2(NM 006256),PRKCM(NM 002742),PRKCN(NM 005813),PRKCQ(NM 006257),PRKCZ(NM 002744),PRKD2(NM 016457),PRKDC(NM 006904),PRKG1(NM 006258),PRKG2(NM 006259),PRKR(NM 002759),PRKNNK1(NM 018979),PRKWNK2(NM 006648),PRKWNK3(NM 020922),PRKWNK4(NM 032387),PRKX(NM 005044),PRKY(NM 002760),PRPF4B(NM 003913),PSKH1(NM 006742),PSKH2(NM 033126),PTK2(NM 005607),PTK2B(NM 004103),PTK6(NM 005975),PTK7(NM 002821),PTK9(NM 002822),PTK9L(NM 007284),PXK(NM 017771),QSK(NM 025164),RAD53(NM 007194),RAF1(NM 002880),RAGE(NM 014226),RET(NM 020975),RHOK(NM 002929),RIOK1(NM 031480),RIOK2(NM 018343),RIPK1(NM 003804),RIPK2(NM 003821),RIPK3(NM 006871),RIPK5(NM 015375),RNASEL(NM 021133),ROCK1(NM 005406),ROCK2(NM 004850),ROR1(NM 005012),ROR2(NM 004560),ROS1(NM 002944),RPS6KA1(NM 002953),RPS6KA2(NM 021135),RPS6KA3(NM 004586),RPS6KA4(NM 003942),RPS6KA5(NM 004755),RPS6KA6(NM 014496),RPS6KB1(NM 003161),RPS6KB2(NM 003952),RPS6KC1(NM 012424),RPS6KL1(NM 031464),RYK(NM 002958),SBK(XM 370948),SCYL1(NM 020680),SCYL2(NM 017988),SGK(NM 005627),SgK069(SU SgK069),SgK085(XM 373109),SgK110(SU SgK110),SGK2(NM 016276), SgK223(XM 291277),SgK269(XM 370878),SgK424(CGP SgK424),SgK493(SU_SgK493),SgK494(NM 144610),SgK495(NM 032017),SGKL(NM 013257),SK681(NM 001001671),SLK(NM 014720),SMG1(NM 015092),SNARK(NM 030952),SNF1LK(NM 173354),SNF1LK2(NM 015191),SNK(NM 006622),SNRK(NM 017719),SRC(NM 005417),SRMS(NM 080823),SRPK1(NM 003137),SRPK2(NM 003138),SSTK(NM 032037),STK10(NM 005990),STK11(NM 000455),STK16(NM 003691),STK17A(NM 004760),STK17B(NM 004226),STK18(NM 014264),STK19(NM 032454),STK22B(NM 053006),STK22C(NM 052841),STK22D(NM 032028),STK23(NM 014370),STK24(NM 003576),STK25(NM 006374),STK3(NM 006281),STK31(NM 031414),STK32B(NM 018401),STK33(NM 030906),STK35(NM 080836),STK36(NM 015690),STK38(NM 007271),STK38L(NM 015000),STK39(NM 013233),STK4(NM 006282),STLK5(NM 001003787),STYK1(NM 018423),SUDD(NM 003831),SYK(NM 003177),TAF1(NM 138923),TAF1L(NM 153809),TAO1(NM 004783),TAOK1(NM 020791),TAOK3(NM 016281),TBCK(NM 033115),TBK1(NM 013254),TEC(NM 003215),TEK(NM 000459),TESK1(NM 006285),TESK2(NM 007170),TEX14(NM 031272),TGFBR1(NM 004612),TGFBR2(NM 003242),TIE(NM 005424),TIF1(NM 003852),TLK1(NM 012290),TLK2(NM 006852),TNIK(NM 015028),TNK1(NM 003985),TOPK(NM 018492),TP53RK(NM 033550),TRAD(NM 007064),TRIB1(NM 025195),TRIB2(NM 021643),TRIB3(NM 021158),TRIM28(NM 005762),TRIM33(NM 015906),TRIO(NM 007118),TRPM6(NM 017662),TRPM7(NM 017672),TRRAP(NM 003496),TSSK4(NM 174944),TTBK1(NM 032538),TTBK2(NM 173500),TYK(NM 003318),TTN(NM 003319),TXK(NM 003328),TYK2(NM 003331),TYRO3(NM 006293),ULK1(NM 003565),ULK2(NM 014683),ULK3(NM 015518),ULK4(NM 017886),VRK1(NM 003384),VRK2(NM 006296),VRK3(NM 016440),WEE1(NM 003390),Wee1B(NM 173677),YANK1(NM 145001),YES1(NM 005433),ZAK(NM 016653),及/或ZAP70(NM 001079)”。
- 如申請專利範圍第1或2項之微生物,其中組成分(Ⅱ)係選自下列:“細菌毒素、腸毒素、外毒素、第I型毒素、第Ⅱ型毒素、第Ⅲ型毒素、第Ⅳ型毒素、第V型毒素、RTX毒素、AB毒素、A-B毒素、A/B毒素、A+B毒素、A-5B毒素及/或AB5毒素”。
- 如申請專利範圍第5項之微生物,其中組成分 (Ⅱ)係選自下列:“腺苷酸環化酶毒素、炭疽病毒素、炭疽病毒素(EF)、炭疽病毒素(LF)、肉毒桿菌毒素、霍亂毒素(CT、Ctx)、霍亂毒素次單位B(CTB、CtxB)、白喉毒素(DT、Dtx)、大腸桿菌LT毒素、大腸桿菌熱不穩定性腸毒素(LT)、大腸桿菌熱不穩定性腸毒素次單位B(LTB)、大腸桿菌ST毒素、大腸桿菌熱穩定性腸毒素(ST)、紅斑毒素、剝脫素毒素、外毒素A、產氣莢膜腸毒素、百日咳毒素(PT、Ptx)、志賀毒素(ST、Stx)、志賀毒素次單位B(STB、StxB)、志賀樣毒素、葡萄球菌腸毒素、破傷風毒素(TT)、中毒休克症候群毒素(TSST-1)、Vero毒素(VT)、困難梭菌(Clostridium difficile)之毒素A(TA)及毒素B(TB)、索氏梭菌(Clostridium sordellii)之致死毒素(LT)及出血性毒素(HT)、諾維梭菌(Clostridium novyi)之α毒素(AT)”。
- 如申請專利範圍第1或2項之微生物,其中組成分(I)及組成分(Ⅱ)係連接在一起以使由此二種組成分編碼之融合蛋白質表現及/或分泌。
- 如申請專利範圍第7項之微生物,其中該融合蛋白質係選自下列:“CtxB-PSA、CtxB-B-Raf V600E KD、CtxB-B-Raf V600E激酶結構區、CtxB-B-Raf V600E激酶結構區KD、CtxB-B-Raf、CtxB-B-Raf KD、CtxB-B-Raf激酶結構區KD、CtxB-HA1及CtxB-HA12C”。
- 如申請專利範圍第1或2項之微生物,其中組成分 (Ⅲ)a)係選自:“第I型分泌系統、第Ⅱ型分泌系統、第Ⅲ型分泌系統、第Ⅳ型分泌系統、第V型分泌系統、大腸桿菌之溶血素轉運系統(訊號)(含有受hly-特異性啟動子控制之HlyA、HlyB及HlyD之核苷酸序列)、大腸桿菌之溶血素轉運系統(訊號)(含有受非hly-特異性細菌啟動子控制之HlyA、HlyB及HlyD之核苷酸序列)、新月柄桿菌(Caulobacter crescentus)之S層(Rsa A)蛋白質之轉運訊號、大腸桿菌之TolC蛋白質之轉運訊號、分泌訊號Vtgss及/或衍生自李斯特菌素之分泌訊號,p60及/或ActA”且其中組成分(Ⅲ)b)係選自:“細胞內溶素、革蘭氏陽性細菌之溶菌蛋白質、單核細胞增生李斯特菌之溶菌蛋白質、單核細胞增生李斯特菌之PLY551及/或單核細胞增生李斯特菌之holin蛋白”。
- 如申請專利範圍第9項之微生物,其中組成分(Ⅲ)a)為至少一種僅編碼一種轉運系統之核苷酸序列,該轉運系統可使組成分(I)及組成分(Ⅱ)之表現產物伴隨表現在該微生物之外表面上及/或可使組成分(I)及組成分(Ⅱ)之表現產物伴隨分泌,其中該組成分(Ⅲ)a)宜為至少一種編碼大腸桿菌之溶血素轉運系統(訊號)的核苷酸序列(含有受hly-特異性啟動子控制之HlyA、HlyB及HlyD之核苷酸序列)或至少一種編碼大腸桿菌之溶血素轉運系統(訊號)的核苷酸序列(含有受非hly-特異性細菌啟動子控制之HlyA、HlyB及HlyD之核苷酸序列)。
- 如申請專利範圍第1或2項之微生物,其中依組成分(Ⅲ)a),組成分(I)及組成分(Ⅱ)之表現產物係被分泌。
- 如申請專利範圍第1或2項之微生物,其中組成分(I)係選自下列:B-Raf V600E、B-Raf V600E激酶結構區、B-Raf V600E KD、B-Raf V600E激酶結構區KD、B-Raf KD、B-Raf激酶結構區、B-Raf激酶結構區KD、前列腺特異性抗原(PSA)、紅血球凝集素HA1(宜來自A型流感病毒(A/泰國/1(KAN-1)2004(H5N1)、紅血球凝集素HA12(宜來自A型流感病毒(A/泰國/1(KAN-1)2004(H5N1)、紅血球凝集素HA12C(宜來自A型流感病毒(A/泰國/1(KAN-1)2004(H5N1);組成分(Ⅱ)係選自下列:“霍亂毒素次單位B(CTB、CtxB)、大腸桿菌熱不穩定性腸毒素次單位B(LTB)、破傷風毒素(TT)”;組成分(Ⅲ)a)係選自:“大腸桿菌之HlyA溶血素轉運訊號與Hly分泌系統之組成分(含有受hly-特異性啟動子控制之HlyA、HlyB及HlyD之核苷酸序列)”;[r3] 組成分(Ⅳ)係選自“大腸桿菌hly基因位之內生性啟動子”;其中組成分(I)及組成分(Ⅱ)係連接在一起以使由此二種組成分編碼之融合蛋白質表現且其中該融合蛋白質係被分泌。
- 一種藥學組成物,其包含至少一種申請專利範圍第1至12項中任一項之微生物(宜為至少一種經凍乾之微生物)及藥學上可接受之載體(宜為膠囊)。
- 一種藥物,其包含至少一種申請專利範圍第1至12項中任一項之微生物或至少一種申請專利範圍第13項之藥學組成物。
- 一種如申請專利範圍第1至12項中任一項之微生物於製造用於治療及/或預防選自下列之生理學及/或病理生理學症狀之藥物上之用途:“不受控制之細胞分裂、惡性腫瘤、良性腫瘤、實體腫瘤、肉瘤、癌、過度增生病、類癌、尤文氏肉瘤、卡波西氏肉瘤、腦瘤、源自腦及/或神經系統及/或腦脊膜之腫瘤、神經膠質瘤、神經母細胞瘤、胃癌、腎臟癌、腎細胞癌、前列腺癌(prostate cancer)、前列腺癌(prostate carcinomas)、結締組織腫瘤、軟組織肉瘤、胰臟腫瘤、肝腫瘤、頭腫瘤、頸瘤、食道癌、甲狀腺癌、骨肉瘤、腎母細胞腫瘤、胸腺瘤、睾丸癌、肺癌、支氣管癌、乳癌(breast cancer)、乳癌(mamma carcinomas)、小腸癌、結腸直腸腫瘤、結腸癌、直腸癌、婦科腫瘤、卵巢腫瘤(ovary tumors)/卵巢腫瘤(ovarian tumors)、子宮癌、子宮頸癌(cervical cancer)、子宮頸癌(cervix carcinomas)、子宮體癌(cancer of body of uterus)、子宮體癌(corpus carcinomas)、子宮內膜癌、尿膀胱癌、膀胱癌、皮膚癌、基底細胞癌、脊椎瘤(spinalioma)、黑色素瘤、眼 內黑色素瘤、白血病、慢性白血病、急性白血病、淋巴瘤、感染、病毒或細菌感染、流行感冒、慢性發炎、器官排斥及/或自體免疫疾病”。
- 一種質體或表現載體,其包含申請專利範圍第1至12項中任一項之組成分(I)至(Ⅳ)。
- 一種用於製造申請專利範圍第1至12項中任一項之微生物的方法,其中係製造申請專利範圍第16項之質體或表現載體且藉此質體或表現載體將微生物轉形。
- 一種藥學套組,其包含至少一種申請專利範圍第1至12項中任一項之微生物或申請專利範圍第13項之藥學組成物或申請專利範圍第14項之藥物及藥學上可接受之緩衝劑(宜為碳酸鹽緩衝劑)。
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KR101104077B1 (ko) * | 2008-10-14 | 2012-01-11 | 건국대학교 산학협력단 | Egr-1의 발현을 증가시키는 활성을 갖는 신규한 엔터로박테리아속에 속하는 신규균주 및 그 균주를 포함하는 조성물의 항암제로서의 용도 |
CN101480489B (zh) * | 2008-12-03 | 2012-07-04 | 中国人民解放军第三军医大学 | 疟原虫环子孢子蛋白抗肿瘤增殖和迁移的新用途 |
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