TWI447125B - 靶向胜肽用於診斷及治療癌症之套組及方法 - Google Patents
靶向胜肽用於診斷及治療癌症之套組及方法 Download PDFInfo
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Description
本發明關於一種將試劑運輸至癌症細胞以診斷或治療癌症之方法及套組。
醣胺聚多醣(Glycosaminoglycans(GAGs))係由重複雙醣單位所組成之非分支多醣，沿著長度和結構有不同轉譯後修飾。GAGs如肝素/硫酸乙醯肝素(heparin/heparan sulfate(HS))及硫酸軟骨素(chondroitin sulfate(CS))合成係透過絲胺酸-甘胺酸共用模體(serine-glycine concensus motif)沿著核心蛋白(如蛋白多糖)鏈結，並分泌於細胞膜上及胞外基質(extracellular matrix(ECM))中。HS在組織中不會是自由GAG鏈，其與六大類HS蛋白多糖(HSPGs)之核心蛋白連接。這些包括會附於細胞表面糖基化磷脂醯肌醇(glycosylphosphatidylinositol(GPI))的磷脂醯肌醇蛋白聚糖家族(glypicans 1-6)及穿膜多配體蛋白聚糖家族(syndecans 1-4)、串珠素(perlecan)、第十八型膠原蛋白(type XVIII collagen)、人集聚蛋白(agrin)以及HS共受體蛋白多糖β聚糖、神經纖毛蛋白-1、神經纖毛蛋白-2及CD-44變異體(epican)。作為最複雜的生物聚合物之一群，HSPGs提供了結構框架，藉由調節生長因子、細胞介素與形態發生素(morphogen)之梯度形成與信號活性以及胞外基質切除酵素如基質金屬蛋白酵素(matrix metalloproteinases(MMPs))的位置和活性來中介細胞與細胞之間的溝通。HSPGs也會結
合生長因子受體來調節許多細胞成長、發育、分化、形態發生、組織恆定、介質重塑及轉移的生物過程。因此，HSPGs在驅動生物現象如發育、發炎、免疫反應及癌症的分子網絡中扮演著關鍵性的角色。
嗜酸性球在遷移到血液之前為骨髓中發育的顆粒球，它們是白血球及免疫系統的組成部分，負責對抗入侵脊椎動物中的多細胞寄生蟲和感染源。嗜酸性球的循環是遷移至組織後在原位進行嗜酸性去顆粒作用及裂解。組織中嗜酸性球伴隨去顆粒作用的活化會產生新合成原位介白素-5(IL-5)並於胞外釋放鹼性的嗜酸性球顆粒蛋白，包括嗜酸性球陽離子蛋白(eosinophil cationic protein(ECP))與嗜酸性球神經毒素(eosinophil derived neurotoxin(EDN))。在來自各種腫瘤組織包括胸、子宮頸、結腸及肺的組織準備物中檢測嗜酸性球的浸潤，嗜酸性球顆粒蛋白與細胞介素的結合證實其抗癌活性，並可能中介腫瘤細胞凋亡。嗜酸性球並與肥大細胞一起控制過敏及氣喘有關的機制。ECP及EDN也分別被稱為人類核醣核酸酶3(RNase 3)及人類核醣核酸酶2(RNase 2)。ECP主要在包括肝，脾及胎盤的組織中表現，並具有多功能特性，包括核糖核酸分解、細胞毒性、抗細菌、抗病毒、抗寄生蟲及肝素結合活性。此外，在支氣管性氣喘或克隆氏病所造成之組織損傷，發炎組織中會發現ECP於胞外堆積。ECP結合細胞表面GAGs，尤其是支氣管表皮細胞上的HS，並藉由依賴脂肪筏的巨胞飲作用(lipid-rafted dependent macropinocytosis)進入細胞。ECP的結構已經被證實並可精細至高達1.75 Å的解析度，其具有三個α螺旋和五個β股的折疊拓撲學。最有趣的特徵是多達19個面向表面的精胺酸殘基，賦
予ECP極高等電點的基本特性(pI=10.8)，其可促進ECP與細胞表面帶負電荷分子的交互作用。
結構上與肝素相關的HS結合範圍廣泛的不同生長因子，參與各種生理及病理過程，包括營養代謝、傷口癒合、細胞信號傳遞、形態發生、細胞間交互作用(cellular crosstalk)。ECP含有一個主要肝素結合模體RWRCK，其位於α2螺旋與β1股之間的環3區(Fan,T.C.,Chang,H.T.,Chen,I.W.,Wang,H.Y.,and Chang,M.D.(2007)A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein,Traffic 8
,1778-1795)。以這個核心肝素結合模體為基礎，一個具有10個胺基酸的GAG結合胜肽(GBP)(橫跨ECP上殘基32-41)已被證實擁有GAG辨識活性。
上皮細胞癌是一種起源於外胚層和內胚層上皮細胞的惡性腫瘤。當上皮細胞從原有特徵轉型並進入稱為上皮-間質轉化(epithelial-to-mesenchymal transition(EMT))的進程，因失去細胞附著而轉移至第二位置時，細胞表面的GAGs開始不正常表現，改變了各種成長因子受體的結合活性。目前，累積的證據指出蛋白多糖及GAGs在許多癌症細胞中會改變表現(表1)。例如，syndecan-1在骨髓癌病人中過度表現並與預後不佳有關。Syndecan-2通常在結腸癌中過度表現。Syndecan-4在肝細胞癌與惡性間皮瘤中被上調並伴隨有腫瘤細胞的增殖。Glypican-1在乳癌與腦癌(神經膠瘤)中過度表現，而glypican-3則是在肝癌、肺部鱗狀細胞癌、轉移性黑色素瘤、默克細胞癌及卵巢癌中過度表現。
本發明中審視了各種正常及癌細胞株，證實GBP偏好與高轉移腺癌結合，推測是由正常及癌細胞上硫酸化GAG表現程度的不同所造成。本發明也藉由組織微陣列證實GBP能偵測肺腫瘤類型，尤其是腺癌及鱗狀細胞癌。此外，GBP顯示體內腫瘤靶向活性，意味著其靶向至上皮細胞癌的可行性。
除非另有定義，本文中所使用的術語具有其一般普遍為該領域中熟習技藝人士所瞭解的意義。如本申請案全文中所使用，下面術語應具有下面意義：術語「GBP」意指一具有10個胺基酸的醣胺聚多醣(GAG)結合
胜肽，其胺基酸序列為NYRWRCKNQN(SEQ ID NO：1)。
因此，本發明提供一種將試劑運輸至癌症細胞的方法，包含：(a)取得一會結合至癌症細胞的胜肽，其中該胜肽包含一個胺基酸序列為SEQ ID NO：1的癌症靶向模體，其中該胜肽與一所欲靶向至癌症細胞之試劑連接或融合；及(b)曝露該胜肽至一群懷疑有癌症細胞的細胞群中。在一較佳實施例中，該細胞群係在一哺乳動物個體中。較佳地，該哺乳動物個體係一人類個體。在一較佳實施例中，該細胞群係選自組織薄切片、組織厚切片、血液及循環腫瘤細胞所組成之群組。在一較佳實施例中，該癌症係上皮細胞癌。較佳地，該上皮細胞癌係腺癌或鱗狀細胞癌。更佳地，該腺癌係高轉移腺癌。更佳地，該腺癌係選自肺腺癌及結腸腺癌。在一較佳實施例中，所述方法進一步包含在該細胞群中檢測癌症細胞。在一較佳實施例中，所述方法進一步包含診斷癌症。在一較佳實施例中，該試劑係治療劑或造影劑。較佳地，該試劑及胜肽係投予至具有或懷疑具有癌症的個體以治療或顯像。在一較佳實施例中，該治療劑係藥物、化療劑、放射性同位素、促凋亡劑、抗血管生成劑、存活因子、抗凋亡劑、酵素、激素、激素拮抗劑、細胞介素、細胞毒性劑、殺細胞劑、細胞生長抑制劑、生長因子、胜肽、蛋白質、抗生素、抗體、抗體的Fc片段、核酸、抗原、病毒、噬菌體、細菌、脂質體、微粒子、磁珠、微元件、酵母細胞、哺乳動物細胞、細胞或表現載體。較佳地，該試劑係一抗血管生成劑，選自由血小板活化素(thrombospondin)、血管抑素5(angiostatin5)、色素上皮衍生因子、血管收縮素、層黏連蛋白胜肽(laminin peptides)、纖連蛋白胜
肽(fibronectin peptides)、血纖維蛋白溶解酶原活化劑抑制劑(plasminogen activator inhibitors)、組織金屬蛋白酶抑制劑、干擾素、介白素12、血小板因子4、IP-10、Gro-B、血栓粘合素(thrombospondin)、2-甲氧基雌二醇(2-methoxyoestradiol)、增殖素相關蛋白(proliferin-related protein)、羧胺三唑(carboxiamidotriazole)、CM 101、Marimastat、戊糖多硫酸鹽(pentosan polysuiphate)、促血管生成素2(angiopoietin 2)(Regeneron)、α-干擾素、除莠霉素A(herbimycin A)、PNU145156E、16K泌乳素片段、Linomide、Thalidomide、Pentoxifylline、金雀異黃素(genistein)、TNP-470、血管內皮抑素(endostatin)、紫杉醇、Docetaxel、多胺、蛋白酶體抑制劑、激酶抑制劑、訊息胜肽、Accutin、西多福韋(cidofovir)、長春新鹼、博來黴素、AGM-1470、血小板因子4及美諾四環素(minocycline)所組成之群組。在另一實施例中，該試劑係一細胞介素，選自由介白素1(IL-1)、IL-2、IL-5、IL-10、IL-11、IL-12、IL-18、干擾素-γ(IF-γ)、IF-α、IF-β、腫瘤壞死因子-α(TNF-γ)或顆粒球巨噬細胞聚落刺激因子(GM-CSF)所組成之群組。在一較佳實施例中，該造影劑係一追蹤物質，選自由螢光標記物、化學發光蛋白、放射性同位素及磁性奈米粒子所組成之群組。較佳地，該造影劑係一放射性同位素，選自由砈-211、碳-14、鉻-51、氯-36、鈷-57、鈷-58、銅-67、Eu-152、鎵-67、氫-3、碘-123、碘-125、碘-131、銦-111、鐵-59、磷-32、錸-186、錸-188、硒-75、硫-35、鍀-99m及釔-90所組成之群組。在一更佳之實施例中，該會結合至癌症細胞的胜肽係SEQ ID NO：1。
本發明也提供一種用於實施上述方法的套組，包含一會結合至癌
症細胞的胜肽，其中該胜肽包含一個胺基酸序列為SEQ ID NO：1的癌症靶向模體。較佳地，該胜肽為SEQ ID NO：1。
本發明可能以不同的形式來實施，並不僅限於下列文中所提及的實例。下列實施例僅作為本發明不同面向及特點中的代表。
依據先前的研究(Calabro,A.,Benavides,M.,Tammi,M.,Hascall,V.C.,and Midura,R.J.(2000)Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis(FACE),Glycobiology 10
,273-281)，將醣類以2-胺基吖啶酮(AMAC)(Invitrogen，貨號A6289)標記。簡言之，將五十微克的GAGs以冷凍乾燥機乾燥，接著以40 μl 1.25 M 2-胺基吖啶酮(AMAC)/85% DMSO/15%醋酸在25℃培養15分鐘。加入四十微升的1.25 M氰基硼氫化鈉(NaBH3
CN)並在37℃培養16小時。之後，於-20℃在15分鐘的時間內加入720 μl 99%冰冷乙醇，接著在4℃以11,000×g離心5分鐘。以離心式真空濃縮機(Spin Vacuum)和冷凍器(coolsafeTM)小心地移除上清液並冷凍乾燥。將乾燥的沈澱物根據標記的探針強度以適當體積(經常為20 μL或50 μL)的無菌去
離子水重新溶解。將準備好的AMAC標記探針儲存於-80℃中並避免光照。
將AMAC標記探針和蛋白質混合並在25℃培養15分鐘。接著將複合物裝載入1%洋菜凝膠並在含有40 mM三乙酸、1 mM EDTA的pH 8.0緩衝液中進行電泳20至30分鐘。本試驗在黑暗或紅光中進行以避免光照。在UV光下觀察AMAC標記探針並以透照器(ONLY Science Co.,ltd)掃描。
表2為一細胞株列表。細胞培養在添加有熱滅活10%(v/v)胎牛血清(FBS)(Gibco,Invitrogen,USA)及1%(v/v)麩胺酸-青黴素-鏈黴素(biosera)的培養基(Gibco,Invitrogen,USA)中。細胞生長在100-mm盤上並於37℃的5% CO2
中培養。
將細胞塗佈在6孔盤中(2.0×105
細胞/孔)並在培養基中培養。24小時後，將細胞與溶解於培養基中之螢光標記胜肽培養1小時。收集細胞，清洗並懸浮於PBS中。在FACScalibur流式細胞儀(FACS,BD Biosciences,Franklin Lakes,NJ)上對經處理的細胞進行螢光分析，激發波長為488 nm，並收集515-545 nm之間的發射波。
將5 μm的載玻片在60℃烤箱中乾燥1小時。簡言之，將切片以二甲苯(J.T.Baker Phillipsburg,N)脫蠟15分鐘，並在梯度乙醇溶液中復水(rehydrate)。將切片培養在3%過氧化氫於蒸餾水中的溶液中10分鐘以阻斷內源性過氧化物酶的活性。接著清洗切片並將其放置於磷酸鹽緩衝液中10分鐘。使用10 mM pH 6.0檸檬酸緩衝溶液熱處理或0.1%胰蛋白酶處理5分鐘(10E4用)來達到抗原恢復(antigen retrieval)。將載玻片於3% BSA溶液中阻斷(block)1小時並以2 μM
eGFP-GBP融合蛋白培養2小時(eGFP用)之後，將這些切片逐一以10E4抗體(1：200稀釋)及eGFP抗體(1：200稀釋)於4℃培養整夜。將這些切片以超級強化緩衝液(super enhancer buffer)培養20分鐘，並在第二抗體寬譜HRP共軛聚合物(polymer HRP conjugate broad spectrum)中於室溫培養30分鐘，接著以磷酸鹽緩衝液清洗三次。以3,3’-二胺基聯苯胺(DAB,0.2 mg/ml,Pierce,Rockford,IL,USA)培養上色3分鐘，接著以梅爾氏蘇木精(Mayer’s hematoxylin)對比染色1分鐘。接著將每片載玻片以95%酒精及100%酒精浸泡來脫水，之後以二甲苯浸泡15分鐘並蓋上蓋玻片。
根據細胞核或細胞質染色強度(無染色=0；弱染色=1；中度染色=2；強染色=3)以及染色細胞範圍(0-10%=0；11-50%=1；51-80%=2；81-100%=3)給予每個腫瘤一個分數。將染色細胞強度及範圍的正分相乘以得到最終免疫反應分數，最低分為0且最高分為9。
成年雌性Balb/c小鼠購買自台灣的國研院動物中心並在同地飼養。小鼠結腸癌CT-26細胞株購買自ATCC(Rockville,Maryland)。細胞培養在含有10%熱滅活胎牛血清的培養基中，環境為37℃含有5% Co2
的加濕大氣。簡言之，將四至六周大的Balb/c小鼠皮下注射5×105
CT-26細胞(體積為100 μl)。當腫瘤體積生長至約(8-9)±1 mm3
，將小
鼠分成兩組並透過尾巴靜脈注射5 nmol增強型綠色螢光蛋白(eGFP)或eGFP-GBP。注射後1小時，將所有動物以CO2
窒息致死。移出肺、氣管、腎、小腸及腫瘤組織並立刻在10%中性緩衝甲醛中固定。以標準方法處理組織樣本來製備石蠟包埋塊狀樣本(Fan,T.C.,Fang,S.L.,Hwang,C.S.,Hsu,C.Y.,Lu,X.A.,Hung,S.C.,Lin,S.C.,and Chang,M.D.(2008)Characterization of molecular interactions between eosinophil cationic protein and heparin,The Journal of biological chemistry 283
,25468-25474)。將樣本塊切片成6 μm的薄片並用超敏感非生物素HRP檢測系統(BioGenex Laboratories,San Ramon,CA)檢驗，如先前所述(Fan,T.C.,Fang,S.L.,Hwang,C.S.,Hsu,C.Y.,Lu,X.A.,Hung,S.C.,Lin,S.C.,and Chang,M.D.(2008)Characterization of molecular interactions between eosinophil cationic protein and heparin,The Journal of biological chemistry 283
,25468-25474)。同時也用光學顯微鏡(Zeiss-Axioplan,Germany)顯像薄片。
當腫瘤體積生長至約(8-9)±1 mm3
，將每隻小鼠靜脈注射(i.v.)150 μl MNP-GBP(0.06 emu/g)，接著以MRI偵察掃描以準確得到小鼠內部腫瘤的位置。磁性奈米粒子為塗布有葡聚醣的Fe3
O4
奈米粒子(GABC Co.)，平均直徑為52 nm。含有磁性奈米粒子的磁流體的飽和磁化強度為0.06 eum/g。將均勻散佈在PBS中的胜肽-磁性奈米粒子從尾巴靜脈注射進小鼠。注射進每隻小鼠的磁流體體積為150 μl。注射後，以核磁共振影像(MRI)偵測MNP的位置。小鼠注射前及注射一天後，在7-Tesla
系統上進行MRI檢驗。將小鼠以0.5 ml氯胺酮及0.5 ml若朋(rompun)麻醉以進行MR掃描。利用涵蓋整個主動脈的脂肪飽和三維梯度回波脈衝序列(fat-saturated 3D gradient echo pulse sequence(Turbo FLASH))取得多張橫向影像，TR=5.17 ms；TE=2.49 ms；翻轉角度=10°；FOV=256 mm；薄片厚度=2 mm；矩陣=256 x 256；像素大小=1 x 1 x 2 mm；平均數=10。體內MR影像為控制組大鼠及注射組小鼠橫跨腹部大動脈的橫斷面。
所有數據顯示為平均±標準差(SD)，n是進行實驗的數量。利用GraphPad Prism v 4.02(GraphPad Software,USA)以獨立學生氏t檢定(unpaired Student’s t test)進行統計分析來比較兩個平均值。單向變異數分析(ANOVA)被用來測試多種處理之間的差異，接著再進行丹內特測驗法(Dunnett’s test)。P
值小於0.05被視為具有統計意義。
GBP會探測一般的醣胺聚多醣，包括低分子量肝素、硫酸軟骨素及硫酸皮膚素(圖1A)。此外，GBP會識別硫酸化的HS，尤其是O
-硫酸化肝素(圖1B)。肝素中最普遍存在的結構係在IdoA的2-O
位置以及GlcNAc的2-N
、6-O
位置的三硫酸化雙醣。單株抗硫酸乙醯肝素(10E4)抗體會偵測HS上N
-硫酸鹽葡萄糖胺(N
-sulfated glucosamine)的表位
(epitope)。因此，藉由流式細胞儀在各種正常及癌症細胞株中偵測10E4及FITC-GBP的信號以定量確定GBP結合與HSPGs表現之間的相關性(表2)。如表3所示，GBP顯示出與癌症細胞株的高結合數量，如人類肺腺癌H460、肺腺癌CL1-3、肺大細胞癌PC9、人類AGS胃腺癌細胞、人類HCT-116大腸直腸癌細胞、人類HepG2肝細胞癌細胞以及小鼠CT-26結腸腺癌細胞。此外，GBP也會結合至CL3、A549、H157、CL1-5、Caco-2及HT-29。與GBP在Beas-2B細胞中的結合量相比，GBP與癌症細胞株的結合多的多(表3)。此外，GBP在肺腺癌及結腸腺癌的高轉移性腫瘤中比起較低轉移性的腫瘤顯示有較高結合程度，表示GBP喜好連接至選擇性的癌細胞表面在腫瘤擴展時不正常增加的GAGs表現。雖然在正常肺Beas-2B細胞及MRC-5細胞中HS上N
-硫酸鹽葡萄糖胺的表現量比肺癌症細胞株高，GBP辨識所有肺癌細胞株比起一般肺細胞株更為顯著。合併上述，這些觀察結果表示GBP顯著選擇性地結合至高轉移性上皮癌。
使用含有9種來自16個不同組織的癌症實例(食道、胃、結腸、直腸、肝、肺、腎、乳房、子宮頸、卵巢、膀胱、淋巴結、皮膚、腦、前列腺、胰臟)的37例多重器官癌微陣列標本(BCN9636,US Biomax,Rockville,USA)進行免疫組織化學(IHC)篩選。應用單株抗HS抗體及eGFP-GBP來偵測多重癌組織陣列中的信號。表4總結了這些病例的臨床病理特徵。
表4 人類多種癌症組織的組織診斷
在腺癌類中，HS分子在肺組織中高度表現(圖2A)並且在結腸(圖2B)、胃(圖2C)、胰臟(圖2D)、前列腺(圖2E)及直腸(圖2F)中表現較少。同樣地，eGFP-GBP信號在肺組織(圖3A)中也可顯著地觀察到，而在結腸(圖3B)、胃(圖3C)、胰臟(圖3D)、前列腺(圖3E)及直腸(圖3F)中則顯得很少。至於負控制組，抗eGFP抗體顯示無信號(圖3G)。此外，HS分子在肺鱗狀細胞癌組織中高度表現(圖4A)，就像在腺癌肺組織中一樣(圖2A)，但是來自食道(圖4B)及皮膚(圖4C)組織的鱗狀細胞癌僅表現少量的HS。同樣地，eGFP-GBP信號在肺細胞中(圖5A)可被顯然偵測，但在食道(圖5B)及皮膚(圖5C)組織的免疫定位則微弱。在其他癌症中，僅有肝細胞
癌顯示微弱的HS表現(圖6A)及eGFP-GBP信號(圖6B)，但星狀細胞瘤、B細胞淋巴瘤、透明細胞癌、侵襲性乳管癌、移行細胞癌及漿液性腺癌則兩種信號均未顯示。在組織或器官中病理上的增殖伴隨細胞數目的增加，可能是一個異常或癌前病變的跡象。令人驚訝的是，HS表現量(圖7A~7C)和eGFP-GBP結合強度(圖7D~7F)均隨著癌症進展顯然增加，如正常組織(圖7A和7D)、有增殖的正常組織(圖7B和7E)及腺癌(圖7C和7F)的IHC影像所示。總結來說，HS表現量及GBP靶向的染色強度在肺組織的腺癌及鱗狀細胞癌類中顯然比在其他癌症中較強，這提供了一個重要的線索，即GBP可被應用作為一種新的偵測工具來鑑定早期至中期的肺癌。
使用組織陣列中61例肺癌的標本分數來定量分析IHC數據。在這61例中，樣本包括正常肺組織、腺癌、鱗狀細胞癌、細支氣管肺泡癌及大細胞癌(表5)。在腫瘤或非腫瘤區，膜相關的醣胺聚多醣如HS及CS有不同程度的表現。HS及CS在正常肺組織的表面上皮不表現，在固有層中的纖維母細胞也是一樣(圖8A及9A)。
表5 61個檢驗的人類肺組織的組織學診斷
腺癌(圖8B&8C及9B&9C)及鱗狀細胞癌的例子(圖8D&8E及9D&9E)顯示在細胞外膜中HS及CS表現的強染色。此外，比起早期癌症，GBP證實對更侵略的腫瘤進展有較高的辨識度，(圖8C&8E及9C&9E)。細支氣管肺泡癌在膜上顯示有HS及CS的輕微染色(圖8F及9F)。大細胞癌在膜上顯示高度HS表現(圖8G)，但CS表現弱(圖9G)。肺癌的染色分數證實HS及CS在不同類型肺癌表現圖樣的IHC結果(表6)：(1)正常肺組織的染色是100%陰性(4/4)；(2)腺癌為27.3%陰性(6/22)，59.1%弱陽性或中度陽性(13/22)以及13.6%強陽性(3/22)；(3)鱗狀細胞癌的染色是40%弱陽性或中度陽性(6/15)以及60%強陽性(9/15)；(4)細支氣管肺泡癌的染色是20%陰性(2/10)，80%弱陽性或中度陽性(8/10)；(5)大細胞癌的染色是90%弱陽性或中度陽性(9/10)以及10%強陽性(1/10)。由這些結果可推知HS及CS在腺癌及鱗狀細胞癌中相對有高表現量。
GBP已經在先前顯示出體外對肺癌的高度結合。eGFP-GBP的分子探針能力也藉由肺癌組織陣列來評估。在正常肺組織中的血管內皮細胞和纖維母細胞可偵測到微弱的eGFP-GBP信號(圖10A)。同樣地，eGFP-GBP在腺癌(圖10B)及鱗狀細胞癌(圖10C)中的靶向信號展示了非常強的染色強度。此外，eGFP-GBP的靶向強度在細支氣管肺泡癌(圖10D)及大細胞癌(圖10E)中也顯示為強。在不同類型肺癌中eGFP-GBP靶向強度的IHC結果總結於表6：(1)正常肺組織的染色是50%陰性(2/4)及50%弱陽性(2/4)；(2)腺癌的染色是13.6%陰性(3/22)，54.5%弱陽性或中度陽性(12/22)以及31.9%強陽性(7/22)；(3)鱗狀細胞癌的染色是20%陰性(3/15)，46.7%弱陽性或中度陽性(7/15)以及33.3%強陽性(5/15)；(4)細支氣管肺泡癌的染色是40%陰性(4/10)，40%弱陽性或中度陽性(4/10)以及20%
強陽性(2/10)；(5)大細胞癌的染色是90%弱陽性或中度陽性(9/10)以及10%強陽性(1/10)。對這些組織微陣列的分析表示，eGFP-GBP的靶向強度再次被證實與HS及CS在所有肺癌病例中的表現圖樣相似，由此可推測eGFP-GBP會優先靶向至腺癌及鱗狀細胞癌。
小鼠結腸腺癌細胞株CT-26顯示高HSPG表現及高GBP結合活性(表3)。因此，選擇CT-26荷瘤小鼠作為體內GBP靶向模式。將重組eGFP-GBP及eGFP蛋白注射進CT-26荷瘤小鼠1小時後，將小鼠犧牲並作組織解剖。藉由抗eGFP抗體作IHC染色組織切片，驗證eGFP-GBP在體內的靶向活性。eGFP-GBP信號在支氣管上皮(圖11A)、小腸絨毛(圖11C)、腎(圖11E)、肝(圖11G)中僅能微弱偵測到，然而在癌組織(圖11I)中卻很強。腎與肝均被視為是哺乳動物的主要排泄器官，其將大部分的廢物和毒素排出體外。因此，大部分的外源蛋白會在腎(圖11E)與肝(圖11G)中被偵測出。令人驚訝的是，明顯高量的eGFP-GBP信號在CT-26癌組織(圖11I)中被偵測出。對於負控制組(注射eGFP)來說，在支氣管上皮(圖11B)、小腸(圖11D)或CT-26腫瘤組織(圖11J)中並未觀察到由eGFP抗體所偵測到的eGFP信號，然而在排泄器官腎(圖11F)及肝(圖11H)中可偵測到明確的eGFP信號，這是因為循環系統的快速運輸所致。這些數據指出GBP在小鼠模式中也能高度選擇性地辨識腺癌細胞。
磁性奈米粒子(MNP)係應用於核磁共振(MR)顯像分析，其在動物模型中提供了一種非侵入性的即時體內監測器。T2加權的對比MR影像(也稱為弛像增強質子密度加權MR影像)顯示質子信號，尤其是在水上的質子信號。幾個器官如腎、肝及腫瘤組織富含水並且容易藉由T2加權的MRI系統顯像。當塗布有葡聚醣的MNP(Fe3
O4
)(直徑為52 nm)結合至上述組織時，會觀察到信號強度降低。
FITC-GBP(表3)及eGFP-GBP融合蛋白(圖11I)會辨識CT-26，表示MNP-GBP也可能可以在體內辨識癌症細胞。為了測試MNP在CT26小鼠模型中的靶向能力，將背部植入有CT-26腫瘤的12周大雌性Balb/c小鼠靜脈注射入150 μl MNP(0.06 emu/g)。將腫瘤小鼠注射MNP後0、0.5及21小時分別在腎及腫瘤位置顯像，ddH2
O則作為正控制，用於將光亮密度標準化(圖12A、12B及12C，用黃色箭頭指出的圓形信號)。排泄器官腎的MRI對比強度在注射MNP後0.5小時增強(圖12B)但是在注射後21小時恢復原狀(圖12C)。然而，腫瘤的MRI對比強度在注射MNP後0.5小時及21小時均沒有增強(圖12E及圖12F)，顯示單獨MNP並不會靶向至腫瘤組織。腎及腫瘤的MRI對比強度也分別在圖12G及圖12H中量化。
使用與MNP共軛的GBP在MRI系統上作為腫瘤顯像偵測的靶向試劑。為了在結腸癌荷瘤小鼠中監測MNP-GBP的靶向，將背部植入有CT-26腫瘤的12周大雌性Balb/c小鼠靜脈注射入150 μl MNP-GBP(0.06 emu/g)，並且在24小時內收集MRI對比數據。先在注射前偵測
排泄器官腎及CT-26固態腫瘤位置的MRI信號強度，並個別設定為100%(圖13A及13D)。在MNP-GBP注射後，腎的MRI對比在0.5小時內掉了55%(圖13B)並在24小時時回復至100%(圖13C)。而MNP-GBP注射後0.5小時的信號強度並未觀察到不同(圖13E)。然而，注射後24小時顯然可觀察到MNP-GBP結合至腫瘤，而且CT-26腫瘤組織的MRI對比強度跟注射前相比掉了約30%(圖13F)。腎及腫瘤的MRI對比強度在圖13G及圖13H中分別被量化。腫瘤組織學切片以普魯士藍染色，以偵測注射後24小時在腫瘤中的鐵奈米粒子。如同預期，MNP-GBP信號並未在正常組織中被偵測到，但注射後24小時普魯士藍信號顯示出MNP-GBP辨識腫瘤組織的邊緣(圖11I)。重要地是，腫瘤組織學切片在高放大倍率下顯示MNP-GBP選擇性地靶向至CT-26腫瘤組織。這些結果指出GBP會引導MNP定位至腫瘤細胞，強烈支持了GBP不僅在體外結合癌症細胞同時也會在體內靶向至腫瘤細胞的假設。
一個熟知此領域技藝者能很快體會到本發明可很容易達成目標，並獲得所提到之結果及優點，以及那些存在於其中的東西。本發明中之套組及其製造程序與方法乃較佳實施例的代表，其為示範性且不僅侷限於本發明領域。熟知此技藝者將會想到其中可修改之處及其他用途。這些修改都蘊含在本發明的精神中，並在申請專利範圍中界定。
本發明的內容敘述與實施例均揭示詳細，得使任何熟習此技藝者能夠製造及使用本發明，即使其中有各種不同的改變、修飾、及進步
之處，仍應視為不脫離本發明之精神及範圍。
說明書中提及之所有專利及出版品，都以和發明有關領域之一般技藝為準。所有專利和出版品都在此被納入相同的參考程度，就如同每一個個別出版品都被具體且個別地指出納入參考。
在此所適當地舉例說明之發明，可能得以在缺乏任何要件，或許多要件、限制條件或並非特定為本文中所揭示的限制情況下實施。所使用的名詞及表達是作為說明書之描述而非限制，同時並無意圖使用這類排除任何等同於所示及說明之特點或其部份之名詞及表達，但需認清的是，在本發明的專利申請範圍內有可能出現各種不同的改變。因此，應了解到雖然已根據較佳實施例及任意的特點來具體揭示本發明，但是熟知此技藝者仍會修改和改變其中所揭示的內容，諸如此類的修改和變化仍在本發明之申請專利範圍內。
圖1顯示指示濃度的GBP結合至各種醣胺聚多醣(GAGs)的電泳圖譜。將(A)AMAC標記的LMWH、硫酸軟骨素(CS)及硫酸皮膚素(DS)及(B)AMAC標記的LMWH、de
-2-O
-硫酸化肝素、de
-6-O
-硫酸化肝素及N
-乙醯基-de
-O
-硫酸化肝素(0.33 nmol)與指示的倍數莫耳過量GBP預混合，之後在25℃的PBS中與胜肽培養(或沒有胜肽)15分鐘，以進行螢光輔助醣電泳(FACE)。將反應產物在1%洋菜凝膠上分離。反應探針及蛋白顯示於凝膠上方。游離探針與未標記競爭物的相對強度(%)顯示於凝膠下方。
圖2顯示腺癌的多種癌組織中硫酸乙醯肝素(HS)的表現。藉由超敏感非生物素HRP檢測系統(Supersensitive non-biotin HRP detection system)以抗HS抗體進行HS的免疫組織化學(IHC)定位。在肺(A)、結腸(B)、胃(C)、胰臟(D)、前列腺(E)及直腸(F)中檢測HS代表性的IHC染色圖譜(褐色)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B、C、D、E及F均為400×)
圖3顯示腺癌的多種癌組織中eGFP-GBP的靶向結果。藉由超敏感非生物素HRP檢測系統以抗eGFP抗體進行eGFP-GBP的IHC定位。在肺(A)、結腸(B)、胃(C)、胰臟(D)、前列腺(E)及直腸(F)中檢測eGFP-GBP代表性的IHC染色圖譜(褐色)。肺癌組織中所檢測出之未經eGFP-GBP培養的抗eGFP抗體係作為陰性反應(G)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B、C、D、E、F及G均為400×)
圖4顯示鱗狀細胞癌的多種癌組織中HS的表現。藉由超敏感非生物素HRP檢測系統以抗HS抗體進行HS的IHC定位。在肺(A)、食道(B)及皮膚(C)中檢測HS代表性的IHC染色圖譜(褐色)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B及C均為400×)
圖5顯示鱗狀細胞癌的多種癌組織中eGFP-GBP的靶向結果。藉由超敏感非生物素HRP檢測系統以抗eGFP抗體進行eGFP-GBP的IHC定
位。在肺(A)、食道(B)及皮膚(C)中檢測eGFP-GBP代表性的IHC染色圖譜(褐色)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B及C均為400×)
圖6顯示肝細胞癌的肝癌組織中HS的表現及eGFP-GBP的靶向結果。藉由超敏感非生物素HRP檢測系統進行HS及eGFP-GBP的IHC定位。在肝中檢測HS(A)及eGFP-GBP(B)代表性的IHC染色圖譜(褐色)。以蘇木精對比染色將細胞核染成藍色。(放大率：A及B均為400×)
圖7顯示癌症進程中HS表現及eGFP-GBP結合的免疫反應。在癌旁正常肺組織(A及D)、癌旁正常肺組織(基質增生)(B及E)、肺腺癌(C及F)中檢測由抗HS抗體及抗eGFP抗體分別產生的HS的IHC染色圖譜(深褐色)(A至C)及GBP的IHC染色圖譜(深褐色)(D至F)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B、C、D、E及F均為400×)
圖8顯示腫瘤肺組織中HS的表現。藉由超敏感非生物素HRP檢測系統以抗HS抗體進行HS的IHC定位。在正常肺組織(A)、低惡性度(low-graded)腺癌(B)、高惡性度(high-graded)腺癌(C)、低惡性度鱗狀細胞癌(D)、高惡性度鱗狀細胞癌(E)、細支氣管肺泡癌(F)及大細胞癌(G)中檢測HS代表性的IHC染色圖譜(褐色)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B、C、D、E、F及G均
為400×)
圖9顯示腫瘤肺組織中CS的表現。藉由超敏感非生物素HRP檢測系統以抗CS抗體進行CS的IHC定位。在正常肺組織(A)、低惡性度腺癌(B)、高惡性度腺癌(C)、低惡性度鱗狀細胞癌(D)、高惡性度鱗狀細胞癌(E)、細支氣管肺泡癌(F)及大細胞癌(G)中檢測CS代表性的IHC染色圖譜(褐色)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B、C、D、E、F及G均為400×)
圖10顯示腫瘤肺組織中eGFP-GBP的靶向結果。藉由超敏感非生物素HRP檢測系統以抗eGFP抗體進行eGFP-GBP的IHC定位。在正常肺組織(A)、低惡性度腺癌(B)、高惡性度腺癌(C)、低惡性度鱗狀細胞癌(D)、高惡性度鱗狀細胞癌(E)、細支氣管肺泡癌(F)及大細胞癌(G)中檢測eGFP-GBP代表性的IHC染色圖譜(褐色)。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B、C、D、E、F及G均為400×)
圖11顯示小鼠癌症模型中eGFP-GBP的靶向結果。藉由超敏感非生物素HRP檢測系統以抗eGFP抗體進行GBP的IHC定位。於靜脈(i.v.)注射1小時後，在支氣管上皮(A)、小腸絨毛(C)、肝(E)、腎(G)及癌(I)組織中檢測eGFP-GBP代表性的IHC染色圖譜(褐色)。同時一併處理肺(B)、小腸(D)、肝(F)、腎(H)及癌(J)組織注射
eGFP的切片作為控制組。以蘇木精對比染色將細胞核染成藍色。(放大率：A、B、C、D、E、F、G、H、I、J均為400×)
圖12顯示CT-26腫瘤小鼠體內磁性奈米粒子(MNP)的分佈。於靜脈注射150 μl MNP(0.06 emu/g)30分鐘及21小時後，取得腎(A-C)及CT-26腫瘤(D-F)的核磁共振影像(MRI)軸影像(黃色箭頭)。ddH2
O作為將光亮密度標準化的正控制組(白色球)。將靜脈注射30分鐘及21小時後靶向至腎(G)及CT-26腫瘤(H)的MNP所代表的MRI信號定量。
圖13顯示MNP-GBP在體內靶向至CT-26腫瘤的結果。在靜脈注射150 μl MNP-GBP(0.06 emu/g)之前監測小鼠腎臟(A，黃箭頭)及腫瘤(D，黃箭頭)的MRI軸影像。小鼠腎臟(B，白箭頭)及腫瘤(E，白箭頭)的MRI軸影像則是在注射後0.5小時取得。同樣取得注射後24小時的小鼠腎臟(C，白箭頭)及腫瘤(F，白箭頭)影像。ddH2
O作為將光亮密度標準化的正控制組(白色球)。MNP-GBP注射後第24小時腎臟組織(G)及腫瘤(H)的MRI對比。(I)腫瘤組織經MNP-GBP注射後第24小時的顯微相片，放大率為400×，並使用普魯士藍染色檢測MNP-GBP堆積的二價鐵(黑點)。
<110> 國立清華大學
<120> 靶向胜肽用於診斷及治療癌症之套組及方法
<130> 1767-NTHU-TW
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> 人類(Homo sapiens)
<220>
<221> PEPTIDE
<222> (1)..(10)
<400> 1
Claims (5)
- 一種胜肽在製備用於將試劑特異性運輸至腺癌或鱗狀細胞癌細胞的組合物的用途，其中該胜肽包含一個胺基酸序列為序列1的癌症靶向模體並與一所欲靶向至該癌細胞之試劑連接或融合。
- 如申請專利範圍第1項所述之用途，其中該試劑係治療劑或造影劑。
- 如申請專利範圍第2項所述之用途，其中該治療劑係藥物、化療劑、放射性同位素、促凋亡劑、抗血管生成劑、存活因子、抗凋亡劑、酵素、激素、激素拮抗劑、細胞介素、細胞毒性劑、殺細胞劑、細胞生長抑制劑、生長因子、胜肽、蛋白質、抗生素、抗體、抗體的Fab片段、核酸、抗原、病毒、噬菌體、細菌、脂質體、微粒子、磁珠、微元件、酵母細胞、哺乳動物細胞、細胞或表現載體。
- 如申請專利範圍第2項所述之用途，其中該造影劑係一追蹤物質，選自由螢光標記物、化學發光蛋白、放射性同位素及磁性奈米粒子所組成之群組。
- 如申請專利範圍第1項所述之用途，其中該胜肽為序列1。
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| TWI715927B (zh) * | 2019-02-01 | 2021-01-11 | 國立清華大學 | 組合物用於製備治療癌細胞的藥物的用途 |
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| Bystrom, Jonas, Kawa Amin, and David Bishop-Bailey. "Analysing the eosinophil cationic protein-a clue to the function of the eosinophil granulocyte." Respiratory research 12.1 (2011): 10 * |
| Yip, George W., Martin Smollich, and Martin Götte. "Therapeutic value of glycosaminoglycans in cancer." Molecular cancer therapeutics 5.9 (2006): 2139-2148 * |
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