TW200407427A - Human tissue factor antibodies - Google Patents
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200407427 玖、發明說明 【發明所屬之技術領域】 本發明係關於與人類組織因子(TF)進行免疫反應以抑 制凝血因子Vila (FVIIa)結合之經單離抗體,及一種使用抗 TF之人類抗體抑制與外科手術、顯微手術、血管成形術或 創傷相關之血栓形成,或抑制發生於與諸如深層靜脈血栓 生成、彌散性血管內凝血(DIC)、冠狀動脈疾病、敗血症、 發炎、動脈粥樣硬化或癌症等疾病相關之異常止血病況中 的血栓形成及其他TF功能之免疫治療方法。本發明亦揭示 抗體製備方法以及用於製備該等人類單株抗體(Mabs)之細 胞系。 【先前技術】 血液凝固作用爲包含各種不同血液組成(或因子)間 複雜交互作用的過程,其最終會形成血纖維蛋白凝塊。通 常,參與已知稱爲凝血“級聯(cascade)”之血液組成爲酵素 或酶原,彼等係原先不具有酵素活性之蛋白質,在經由活 化劑(本身爲經活化之凝血因子)的作用後而轉化成蛋白 分解酵素。已知進行此類轉化作用之凝血因子一般稱爲“ 活化因子”並於字尾加添小寫"a 〃作爲命名(例如,因子 Vila) 〇 經活化之因子X ("Xa”)爲將凝血酶原轉化成凝血酶( 其接著將血纖維蛋白原轉化成血纖維蛋白做爲形成血纖維 蛋白凝塊之最後階段)所必需。有兩種促進因子X活化作 用之系統(或途徑)。“內源性途徑”意指該等經由利用 200407427 僅存在於血漿中之因子而促成凝血酶形成的反應。一系列 由蛋白酶所參與之活化作用最終產生因子IXa,其與因子 Villa結合而將因子X裂解生成Xa。於血液凝固之“外源 性途徑”中,相同之蛋白分解作用則係由FVIIa及其輔因 子(TF)完成。TF爲一種膜結合性蛋白且通常不會以活化形 式循環於血漿中。然而在血管破損時,TF可與FVIIa複合 而於Ca2+及磷脂質存在下催化因子X活化作用或因子IX活 化作用。而該二種凝固途徑於凝血作用上之相關重要性仍 尙未淸楚,因子VII及TF已發現在引發血液凝固作用方面 扮演著重要角色。 往往有必要選擇性阻斷患者體內的凝血級聯。諸如肝 素、香豆素、香豆寒衍生物、茚滿二酮衍生物等抗凝血劑 ,或其他藥劑可(例如)於腎臟透析時使用,或用以治療 深層靜脈血栓生成、彌散性血管內凝血(DIC)及罹患其他醫 學病症之患者。舉例而言,伴隨檸檬酸鹽離子之肝素治療 或體外治療可用於透析作用以防止治療過程中發生凝血。 肝素亦用於預防進行手術患者體內之深層靜脈血栓生成。 然而,以肝素或其他抗凝血劑進行之治療可能具有不 希望的副作用。可用之抗凝血劑通常係作用於全身,而非 特異地作用於損傷部位。例如,肝素可能導致大量出血。 而且由於肝素之半衰期爲大約80分鐘,所以會很快地從血 液淸除而需要頻繁地進行投藥。因爲肝素係作用爲抗凝血 酶III (ATIII)之輔因子,且ATIII在DIC治療中會快速被耗 盡,所以往往很難維持適當的肝素劑量,而必需持續偵測 200407427 ATIII與肝素之含量濃度。若ATIII被相當耗盡時肝素亦無 效用。此外,長期使用肝素亦可能增加血小板凝聚而減少 血小板數量,且已預期會發展成由肝素所誘發之血小板減 少症。茚滿二酮衍生物亦可能具有毒性副作用。除以上所 簡述之抗凝血劑外,已發現數種天然蛋白質亦具有抗凝血 活性。又,ATIII已被認爲係一種治療性抗凝血劑。 國際專利申請案WO 92/15686號係關於用於抑制血液 凝固之失活因子Vila。 抗體是一種由脊椎動物免疫系統在對由外來蛋白、糖 蛋白、細胞、或其他抗原性外來物質產生之刺激反應時所 製造的具特異性免疫球蛋白(Ig)。促使生物體擊敗外來細胞 入侵,或使系統淸除外來物質之事件順序至少有部份尙未 被了解。此過程之重要部份爲製造出可特異地與特定外來 物質結合的抗體。此類多肽與特定抗原之結合特異性係非 常精密的,且能夠由個別脊椎動物產生之眾多特異性在其 複雜性及多變性方面尤其受人矚目。有數以百萬計之抗原 能引發抗體反應,個別抗體幾乎排它地與引發其產生之特 定抗原反應。 現今使用兩種主要的脊椎動物抗體來源,由哺乳動物 B淋巴細胞於原位產生,及由B-細胞融合瘤於細胞培養物 中產生。抗體係因未成熟B淋巴細胞分化成漿細胞(其發 生於對由特定抗原產生之刺激的反應中)而於原位產生。 於未分化之B細胞中,編碼免疫球蛋白鏈上各種區域之 DNA部份係分開位於基因組DNA中。該等序列在表現前會 200407427 依序進行組裝。所成經排列之基因能夠於成熟B淋巴細胞 中表現而製造出所希望的抗體。然而,當特定哺乳動物即 使僅暴露於單一種抗原時,亦不會只產生單一族群抗體。 對任一種特定抗原之原位免疫反應限定於對各種呈現於該 抗原上之抗原決定基的雜合反應。各次組同源性抗體係由 單一族群B細胞所產生,因此原位產生之抗體即爲“多株 此有限但固有之異源性已在許多特殊個案中藉由使用 融合瘤技術,由B細胞融合瘤於細胞培養物中產生“單株 ”抗體而獲得解決。 於此方法中,係將得自已經注射抗原之哺乳動物的相 對較短壽命(或會死)之脾臟細胞或淋巴細胞與無限增殖 腫瘤細胞系融合,而因此產生兼具無限增殖且能夠生產B 細胞之經基因編碼抗體的雜合細胞或“融合瘤”。經此所 形成之雜合體藉由篩選、稀釋及再生而分成單一遺傳株, 且各株因而代表一個單一遺傳純系。於是彼等可製出被認 爲係均一對抗所希望抗原。此等抗體(由於其純一的遺傳 親源性)即稱爲“單株”。 具有單一-特異性之單株抗體在免疫學上具有很大影響 力,且彼等之可用性已在諸如生物學、形態學、生化學及 其他等科學中獲得證實。此類單株抗體已廣泛應用不只可 做爲診斷試劑,亦可用於治療用途(參見,例如,立茲與 許洛斯曼,血液,59:1-11,(1982))。 經由融合瘤所產生之單株抗體雖然如上所論在治療上 200407427 有效,且因爲其特異性而顯然優於多株抗體,但仍具有一 項重要的缺點。在許多應用上,當該等抗體欲用在人類時 於非人類哺乳動物所製得之單株抗體的用途即受到嚴重限 制。重複將“外來”抗體注射至人類(例如施打小鼠抗體 )可能導致有害的過敏反應。此類非由人類所衍生得之單 株抗體,當注射入人體時,會造成抗-非人類抗體反應。 抗TF之小鼠Mabs的治療用途已揭示於美國專利第 6,001,978 及 5,223,427 號。 國際專利申請案WO 99/51743號係關於抗人類TF之人 類/小鼠嵌合型單株抗體。 歐洲專利申請案83391 1號係關於抗人類TF之CDR-嫁 接抗體。 普瑞斯塔L.等人,血栓生成及凝血,卷85 (3) pp. 379-389 (2001)係關於抗TF之人源化抗體。 該項技藝中仍需要具有抗凝血活性之改良組合物,其 可以相對較低劑量進行投藥,且不會產生與傳統抗凝血組 合物相關之不希望副作用。本發明即藉由提供不具有與含 非-人類序列之傳統抗體相關之副作用的抗凝血劑而成就此 項需求,彼等可特異地作用於損傷部位並進而提供其他相 關之優點。而且本發明亦提供作用以抑制TF之細胞功能( 其包含如敗血症、發炎、動脈粥樣硬化、再狹窄或癌症等 疾病)的化合物。 【發明內容】 本發明係關於抗人類TF之非-免疫原之高親和性人類 200407427 抗體,其抑制凝血因子Vll/VIIa之結合,以及用於篩檢治 療上有效之抗人類TF之人類抗體的方法。 於第一方面,本發明關於可與呈現於人類TF上之抗原 表位進行免疫反應之經單離抗體。 術語“人類組織因子”或“人類TF”用於本文係指其 包含天然人類組織因子之胺基酸序列1-263的全長多肽受體 〇 術語“抗體”用於本文係指其具有可與一種抗原(例 如人類TF)特異結合之能力的免疫球蛋白分子及其片段。 全長抗體包含四段多肽鏈,即兩段重(H)鏈及兩段輕(L)鏈其 間以雙硫鍵相連結。每段重鏈係由一段重鏈可變區(本文 簡稱HCVR或VH)及重鏈恆定區所組成。重鏈恆定區包含 三個功能域,CtH、CH2及CH3。每段輕鏈係由一段輕鏈可 變區(本文簡稱LCVR或LH)及輕鏈恆定區所組成。重鏈 恆定區包含一個功能域,CL。VH與VL區可再進一步細分 成具有高變異性之區域(稱爲互補決定區(CDR)),其間隔 著較固有之區域(稱爲構架區(FR))。每段VH及VL係由 三段CDRs與四段FRs所組成,其從胺基末端至羧基末端之 歹!J順序爲:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4 。因此,在抗體之定義中亦包含一或多段仍保留可與一種 抗原(例如人類TF)特異結合之能力的抗體片段。已顯示 ,抗體之抗原-結合功能可藉由全長抗體之片段達成。涵括 於術語“抗體”中之結合片段實例包括⑴Fab片段,爲由 VL 、 VH 、 CL及CH1功會g域所組成之單價片段;(ii) F(ab)2 200407427 及F(ab’)2片段,爲包含兩段由雙硫鍵於鉸鏈區聯結之Fab 片段的雙價片段;(出)Fd片段其係由VH及CH1功能域組 成;(iv) Fv片段其係由單臂抗體之Vl及VH組成;(v) dAb 片段(瓦得等人(1989)自然341:544-546 ),其係由一個VH 功能域組成;及(vi)經單離之互補決定區(CDR)。而且,雖 然Fv片段之兩功能域(VL及VH)係由分開的基因所編碼 ,彼等卻可使用重組方法藉由合成型連接肽將彼等接合, 而能夠使其成爲其中該等VL與VH區配對形成單價分子之 單一蛋白質鏈(已知稱爲單鏈Fv(scFv);參見例如,博得等 人(1988)科學 242:423-426;及胡斯頓等人(1988)?1*〇〇:.他11· Acad. Sci USA 85:5879-5883 )。此類單鏈抗體亦欲包括於術 語“抗體”中。亦涵括其他形式之單鏈抗體,例如雙效抗 體(diabodies)。雙效抗體爲雙價、雙特異性抗體,其中VH 及VL功能域係表現於單一多肽鏈上,但使用一種過短而無 法令兩個位在同一鏈上之功能域互相配對之連接肽,藉此 迫使該等功能域與另一鏈之互補功能域配對而產生兩種抗 原結合部位(參見例如’胡里格’ P.等人(1993)Proc. Natl. Acad· Sci USA 90:6444-6448 ;波雅克,R·〗·等人(1994)結構 2:1121-1123)。據瞭解,人類TF可具有一或多種抗原決定 基其包含(1)由人類TF中之單一肽鏈所組成之肽抗原決定基 ,(2)由多於一個其個別氨基酸序列在人類TF多肽序列上並 不相連之空間上鄰近的肽鏈所組成之構型抗原決定基;及 (3)由於轉譯作用後共價性接附至人類TF之分子結構,例如 碳水化合物基團等(全部或部份)所組成之後-轉譯抗原決 12 200407427 定基。 術語“人類抗體”、“人類抗體(多數)”、“人類 TF抗體”及“人類TF抗體(多數)”用於本文意欲包括 具有衍生自人類種系免疫球蛋白序列之可變及恆定區的抗 體。本發明之人類抗體可例如於CDRs且特別係於CDR3中 ’包括非由人類種系免疫球蛋白序列所編碼之氨基酸殘基 (例如,藉由活體外隨機或定點致突變作用或藉由活體內 體細胞突變所導致之突變)。然而,術語“人類抗體”用 於本文不欲包括該等其中衍生自另一哺乳動物品種(例如 小鼠)之種系的CDR序列已被植入於人類構架序列上之抗 體,例如所謂的人源化抗體或人類/小鼠嵌合型抗體。 “經單離之人類抗體”用於本文意指其實質上不含其 他具有不同抗原特異性之抗體的人類抗體(例如一種特異 地與人類TF結合之經單離抗體實質上不含有可特異與除 人類TF外之抗原結合的抗體)。然而,可特異地與人類T F結合之經單離抗體對其他抗原,例如源自其他物種之丁F 分子可能具有交叉反應性(進一步詳細論述於下文)。而 且,經單離之抗體可實質上不含其他細胞物質及/或化學品 〇 術語“抗原表位”用於本文意指任何位於抗原上而抗 體與之結合的抗原決定基。抗原表位決定基通常係由分子 之具化學活性表面基團例如氨基酸或糖側鏈所組成,且通 常具有特定之三維立體結構特徵以及特殊電荷特徵。 術語“免疫反應”或“進行免疫反應”用於本文意指 13 200407427 任何抗體與其抗原表位之結合而其解離常數Kd低於10_4 Μ 。若適當,術語“免疫反應”或“進行免疫反應”可與術 語“特異地結合”交替使用。 術語“抑制”用於本文意指任何相較於參考値之減低 。例如,一種抑制人類凝血因子Vila與人類TF結合之抗 體,意即任何可使人類凝血因子Vila與人類TF之結合能 力相較於在無該抗體存在下人類凝血因子Vila與人類TF 之結合能力時被降低的抗體。 術語“親和力”用於本文意指抗體與抗原表位之結合 強度。抗體之親和力係藉由解離常數Kd測量得,其定義爲 [Ab] X [Ag]/[Ab-Ag]其中[Ab-Ag]爲抗體-抗原複合體之莫耳 濃度,[Ab]爲未結合抗體之莫耳濃度而[Ag]爲未結合抗原之 莫耳濃度。親和力常數Ka定義爲1/Kd。藉由競爭性抑制作 用測定Mabs特異性及親和性之較佳方法可見於哈洛等人, 抗體:實驗室手冊,冷泉港實驗室出版,冷泉港,N.Y. (1988),科林根等人編著,現今免疫學製程,格林奈出版社 與威利國際科學發行,N.Y. (1992,1993),及繆樂,酵素學 方法92:589-601 (1983),彼等揭示皆倂入本文作爲參考文獻 〇 於第二方面,本發明關於一種醫藥組合物,其包含治 療上有效量之可與呈現於人類TF上的抗原表位進行免疫反 應之人類抗體。 術語“治療上有效量”爲由有照開業人員可滴定劑量 以達所希望反應所測定得之有效劑量。劑量之考量因素將 14 200407427 包括效能、生物可利用性、所希望之藥物動力學/藥物動力 學槪況、治療病況(例如創傷、發炎、敗血性休克)、與 患者相關之因素(例如體重、健康、年齡等)、受否與其 他醫藥品進行共投藥、投藥時間或其他醫師已知之因素。 抗TF之人類抗體所投予患者之劑量將視欲受治療病況之種 類及嚴重性而有所變化,但一般係介於0.1-5.0毫克/公斤體 重。 術語“個體”用於本文意指任何動物,尤其是哺乳動 物,例如人類,且若適當,可與術語“患者”交替使用。 於第三方面,本發明關於一種組合物,其包含可與呈 現於人類TF上的抗原表位進行免疫反應之人類抗體。 於另一方面,本發明關於一種治療人類中與FVIIa/TF 相關之病症的方法,該方法包含將治療上有效量之可與呈 現於人類TF上的抗原表位進行免疫反應之人類抗體投藥予 該人類。 “治療”意指依預防任何病徵或病況發展之目的,或 依治癒或減緩此等已發展成之病徵或病況的目的,而投藥 有效量之本發明具治療活性的化合物。術語“治療”於是 意欲包括預防性治療。 術語“與FVIIa/TF有關之疾病”用於本文意指其中涉 及TF及FVIIa之疾病或失調症。其涵蓋與血栓形成或凝血 病相關之疾病或失調症,包括發炎反應及與血纖維蛋白形 成相關聯之慢性血栓栓塞性疾病或失調症,包括血管病例 如深層靜脈血栓生成、動脈血栓形成、手術後血栓形成、 15 200407427 冠狀動脈繞道移植物(CABG)、經皮冠狀血管成形術(PTCA) 、中風、腫瘤生長、腫瘤轉移、血管生成、血栓溶解、血 管成形術後之動脈粥樣硬化與再狹窄、急性與慢性適應症 例如發炎、敗血性休克、敗血症、高血壓、成人呼吸窘迫 症(ARDS)、彌散性血管內凝血(DIC)、肺部栓塞、血小板沈 積、心肌梗塞、或對具有發生血栓形成危險之動脈硬化血 管及其他疾病或病況之哺乳動物所施予之預防性治療。與 FVIIa/TF有關之疾病並不限定於諸如上述指名之活體內凝 血病症,而亦包括與FVIIa/TF有關之取自活體處理方法例 如可能因體外血液循環,包括於諸如透析程序過程中血液 自患者抽出、血液過濾或外科手術期間之血液繞道所造成 的凝血。 術語“因子Vila”或“FVIIa”意指藉由於Argl52-Ilel53肽鍵處之特定裂解作用而分解得之“兩鏈”經活化 凝血因子VII。FVIIa可自血液純化得或藉由重組方法製得 。顯然本文所述方法之實施與經純化因子Vila之來源無關 ,且因此本發明欲涵蓋使用任何適用於本發明之因子Vila 製劑。其較佳爲人類FVIIa。 術語"FVII”意指“單鏈”凝血因子VII。 於另一方面,本發明關於一種用於製備人類抗體之方 法,該方法包含: a) 製備抗人類TF之人類抗體, b) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此 項分析中以IC5Q値低於1 nM (例如低於500 pM,較佳係低 16 200407427 於200 pM,較佳低於100 pM,較佳低於50 pM,較佳低於 10 pM,更佳低於5 pM)抑制凝塊形成之人類抗體,或 於FXa產量分析中測試抗體並篩選出可以IC5〇値低於 100 nM (於以FVIIa濃度爲0.1 nM所進行之分析),例如 低於10 nM,較佳低於5 nM,較佳低於1 nM,更佳低於 0.1 nM,抑制FXa產生之人類抗體,或 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以 IC5Q値低於100 nM (於以FVIIa濃度爲10 nM下進行之分 析),例如低於40 nM,較佳低於20 nM,更佳低於10 nM ,抑制由TF所誘發之FVIIa醯胺分解活性之人類抗體,或 於FVIIa競爭分析中測試抗體並篩選出可與FVIIa競爭 結合之人類抗體,或 於包含TF之TF ELISA分析中測試抗體並篩選出可與 人類TF結合之人類抗體。 應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC50 値係指當使用正常人類血漿時。 於另一方面,本發明關於一種用於製備人類抗體之方 法,該方法包含: a) 製備抗人類TF之人類抗體, b) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此 項分析中以其IC5Q値低於FFR-rFVIIa之IC5G値+ 1 nM,例 如低於FFR-rFVIIa之IC5G値+ 500 pM,較佳低於FFR-rFVIIa 之 IC5。値 + 200 pM,較佳低於 FFR-rFVIIa 之 IC5〇 値 + 100 pM,較佳低於FFR-rFVIIa之IC5G値+ 50 pM,較佳低 17 200407427 於 FFR-rFVIIa 之 IC5Q 値 + 10 pM,更佳低於 FFR-rFVIIa 之 IC5Q値+ 5 pM,更佳低於FFR-rFVIIa之IC5Q値,抑制凝塊 形成之人類抗體,或 於FXa產量分析中測試抗體並篩選出可以其IC5〇値低 於FFR-rFVIIa之IC5Q値+100 nM (於該項分析中使用0.1 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5G 値 + 10 nM,較佳 低於 FFR-rFVIIa 之 IC5Q 値 + 5 nM,較佳低於 FFR-rFVIIa 之 IC5〇 値 + 1 nM,更佳低於 FFR-rFVIIa 之 IC5G 値 + 0.1 nM, 更佳低於FFR-rFVIIa之IC5()値,抑制FXa產生之人類抗體 ,或 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以其 IC5G値低於FFR-rFVIIa之IC5Q値+100 nM (於該項分析中 使用 10 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5G 値 + 40 nM,較佳地低於FFR-rFVIIa之IC5Q値+ 20 nM,較佳地低 於 FFR-rFVIIa 之 1(:5()値+10 nM,更佳地低於 FFR-rFVIIa 之IC5()値,抑制由TF所誘發之FVIIa醯胺分解活性之人類 抗體,或 於FVIIa競爭分析中測試抗體並篩選出可與FVIIa競爭 結合之人類抗體,或 於包含TF之TF ELISA分析中測試抗體並篩選出可與 人類TF結合之人類抗體。 應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC50 値係指當使用正常人類血漿時。 術語“由TF-誘發之凝塊分析”用於本文意指任何其中 18 200407427 係於包含血液血漿及TF之樣本中測量凝塊時間分析。一項 由TF-誘發之凝塊分析之實例列述於實施例1,分析7中。 術語“FXa產量分析”用於本文意指任何其中係於包 含TF、FVIIa、FX、鈣及磷脂質之樣本中測量FX之活化作 用的分析。一項FXa產量分析之實例列述於實施例1,分 析5中。 術語“FVIIa/TF醯胺分解分析”用於本文意指任何其 中係於TF存在下測量FVIIa之醯胺分解活性,亦即裂解一 小段肽受質的分析。一項FVIIa/TF醯胺分解分析之實例列 述於實施例1,分析4中。 術語“TF ELISA分析”用於本文意指任何包含TF及 抗丁F抗體的ELISA分析。TF ELISA分析之實例爲列述於 實施例1,分析1及2中之直接與間接TF ELISA分析。 術語“直接TF ELISA分析”用於本文意指任何包含固 定化TF之TF ELISA分析。直接TF ELISA分析之實例列述 於實施例1,分析1中。 術語“間接TF ELISA分析”用於本文意指任何其中 TF係存在於溶液中之TF ELISA分析。間接TF ELISA分析 之實例列述於實施例1,分析2中。 於另一方面,本發明關於一種可與存在人類TF上之抗 原表位進行免疫反應且抑制人類凝血因子Vila與人類TF 結合之經單離抗體,其可藉由包含下述步驟之方法獲得: a) 製備抗人類TF之人類抗體’ b) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此 19 200407427 項分析中以IC5〇値低於1 nM,例如低於500 pM,較佳係低 於200 pM,較佳低於100 pM,較佳低於50 pM,較佳低於 10 pM,更佳低於5 pM,抑制凝塊形成之人類抗體,或 於FXa產量分析中測試抗體並篩選出可以IC5Q値低於 100 nM (於以FVIIa濃度爲0.1 nM進行之分析中),例如 低於10 nM,較佳低於5 nM,較佳低於1 nM,更佳低於 0.1 nM,抑制FXa產生之人類抗體,或 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以 IC5Q値低於1〇〇 nM (於以FVIIa濃度爲10 nM進行之分析 中),例如低於40 nM,較佳低於20 nM,更佳低於10 nM ,抑制由TF所誘發之FVIIa醯胺分解活性之人類抗體,或 於FVIIa競爭分析中測試抗體並篩選出可與FVIIa競爭 結合之人類抗體,或 於包含TF之TF ELISA分析中測試抗體並篩選出可與 人類TF結合之人類抗體。 應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC50 値係指當使用正常人類血漿時。 於另一方面,本發明關於一種可與存在人類TF上之抗 原表位進行免疫反應且抑制人類凝血因子Vila與人類TF 結合之經單離抗體,其可藉由包含下述步驟之方法獲得: a) 製備抗人類TF之人類抗體, b) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此 項分析中以其IC5G値低於FFR-rFVIIa之IC5Q値+ 1 nM,例 如低於FFR-rFVIIa之IC5G値+ 500 pM,較佳係低於FFR- 20 200407427 rFVIIa 之 IC5。値 + 200 pM,較佳低於 FFR-rFVIIa 之 IC50 値 + 100 pM,較佳低於FFR-rFVIIa之IC5Q値+ 50 pM,較佳低 於 FFR-rFVIIa 之 IC5Q 値 + 10 pM,更佳低於 FFR-rFVIIa 之 IC5Q値+ 5 pM,更佳低於FFR-rFVIIa之IC5Q値’抑制凝塊 形成之人類抗體,或 於FXa產量分析中測試抗體並篩選出可以其IC5G値低於 FFR-rFVIIa之IC5。値+ 100 nM (於該項分析中使用0.1 nM FVIIa),例如低於FFR-rFVIIa之IC5G値+ 10 nM,較佳低 於 FFR-rFVIIa 之 IC5Q 値+ 5 nM,較佳低於 FFR-rFVIIa 之 IC5Q 値 + 1 nM,更佳低於 FFR-i*FVIIa 之 IC5Q 値 + 0.1 nM, 更佳低於FFR-rFVIIa之IC5〇値,抑制FXa產生之人類抗體 ,或 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以其IC50 値低於FFR-rFVIIa之IC5G値+ 100 nM (於該項分析中使用 10 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5G 値 + 40 nM, 較佳地低於FFR-rFVIIa之IC5Q値+ 20 nM,較佳地低於 FFR-rFVIIa 之 IC5Q 値+10 nM,更佳地低於 FFR-rFVIIa 之 IC5〇値,抑制由TF所誘發之FVIIa醯胺分解活性之人類抗 體,或 於FVIIa競爭分析中測試抗體並篩選出可與FVIIa競爭結合 之人類抗體,或 於包含TF之TF ELISA分析中測試抗體並篩選出可與人類 TF結合之人類抗體。200407427 发明 Description of the invention [Technical field to which the invention belongs] The present invention relates to an isolated antibody that performs an immune response with human tissue factor (TF) to inhibit the binding of the clotting factor Vila (FVIIa), and a human antibody inhibition using anti-TF Thrombosis associated with surgery, microsurgery, angioplasty, or trauma, or inhibition that occurs in association with factors such as deep venous thrombosis, disseminated intravascular coagulation (DIC), coronary artery disease, sepsis, inflammation, atherosclerosis Or immunotherapy for thrombosis and other TF functions in abnormal hemostatic conditions related to diseases such as cancer. The invention also discloses an antibody preparation method and a cell line for preparing the human monoclonal antibodies (Mabs). [Prior art] Blood coagulation is a process involving complex interactions between various blood components (or factors), which eventually forms a fibrin clot. In general, the blood components involved in what is known as the "cascade" of blood coagulation are enzymes or zymogens, which are proteins that did not have enzyme activity in the past through the action of activators (which are activated coagulation factors themselves). It is then converted into proteolytic enzymes. Coagulation factors known to perform such transformations are commonly referred to as "activating factors" with a lower case " a 〃 at the end of the term (eg, factor Vila). Activated factor X (" Xa ") is Necessary for the conversion of prothrombin into thrombin (which then converts fibrinogen to fibrin as the final stage in the formation of a fibrin clot). There are two systems (or pathways) that promote factor X activation. "Endogenous pathway" means those reactions that promote thrombin formation by utilizing factors that are only found in plasma in 200407427. A series of activations involving proteases ultimately produce factor IXa, which combines with factor Villa to bind factors X breaks down to form Xa. In the "exogenous pathway" of blood coagulation, the same proteolysis is accomplished by FVIIa and its cofactor (TF). TF is a membrane-bound protein and usually does not circulate in activated form In plasma. However, when the blood vessel is damaged, TF can complex with FVIIa and catalyze factor X activation or factor IX activation in the presence of Ca2 + and phospholipids, and the two kinds of coagulation Given the importance of coagulation, Factor VII and TF have been found to play important roles in triggering blood coagulation. It is often necessary to selectively block the coagulation cascade in patients. Such as heparin, fragrant Anticoagulants such as legumin, coumarin derivatives, indane dione derivatives, or other agents can be used, for example, during kidney dialysis, or to treat deep venous thrombosis, disseminated intravascular coagulation (DIC ) And patients with other medical conditions. For example, heparin therapy with citrate ions or in vitro therapy can be used for dialysis to prevent coagulation during treatment. Heparin is also used to prevent deep venous thrombosis in patients undergoing surgery However, treatments with heparin or other anticoagulants may have undesired side effects. The available anticoagulants usually work systemically, rather than specifically at the site of injury. For example, heparin may cause massive bleeding. And because the half-life of heparin is about 80 minutes, it will be quickly eliminated from the blood and needs to be performed frequently. Because heparin acts as a cofactor for antithrombin III (ATIII) and ATIII is rapidly depleted in DIC treatment, it is often difficult to maintain an appropriate heparin dose, and continuous detection of 200,407,427 ATIII and heparin is necessary Content and concentration. Heparin is also ineffective if ATIII is considerably depleted. In addition, long-term use of heparin may also increase platelet aggregation and reduce platelet number, and has been expected to develop thrombocytopenia induced by heparin. Indandione Derivatives may also have toxic side effects. In addition to the anticoagulants briefly described above, several natural proteins have also been found to have anticoagulant activity. Furthermore, ATIII has been considered a therapeutic anticoagulant. International Patent application WO 92/15686 relates to an inactivation factor Vila for inhibiting blood coagulation. Antibodies are specific immunoglobulins (Ig) produced by the vertebrate immune system in response to stimuli produced by foreign proteins, glycoproteins, cells, or other antigenic foreign substances. The sequence of events that prompt organisms to defeat the invasion of foreign cells, or cause the system to exclude foreign matter, is at least partially unknown. An important part of this process is the production of antibodies that specifically bind to specific foreign substances. The specificity of binding of such polypeptides to specific antigens is very precise, and the numerous specificities that can be produced by individual vertebrates are of particular interest for their complexity and variability. There are millions of antigens that can elicit antibody responses, and individual antibodies react almost exclusively with specific antigens that elicit their production. Two main sources of vertebrate antibodies are used today, produced in situ by mammalian B lymphocytes, and produced by B-cell fusion tumors in cell cultures. The resistance system arises in situ due to the differentiation of immature B lymphocytes into plasma cells, which occur in response to stimuli produced by specific antigens. In undifferentiated B cells, portions of the DNA encoding various regions on the immunoglobulin chain are located separately in the genomic DNA. The sequences will be assembled in sequence before presentation. The aligned genes can be expressed in mature B lymphocytes to produce the desired antibodies. However, when a particular mammal is only exposed to a single antigen, it will not produce only a single population of antibodies. The in situ immune response to any particular antigen is limited to hybrid reactions to various epitopes present on the antigen. Each group of homology resistance system is produced by a single population of B cells, so the antibody produced in situ is "multiple strains of this limited but inherent heterogeneity. In many special cases, by using fusion tumor technology, Cell fusion tumors are solved by producing "single-clone" antibodies in cell culture. In this method, relatively short-lived (or dying) spleen cells or lymphocytes from mammals that have been injected with the antigen are combined with Immortal tumor cell lines are fused, and as a result, hybrid cells or "fusion tumors" with genetically encoded antibodies capable of producing B cells that are both immortal are produced. The hybrids formed by this are divided by screening, dilution, and regeneration A single genetic strain, and each strain thus represents a single genetic pure line. They can then produce a homogeneous line against the desired antigen. These antibodies (due to their pure genetic parentage) are called "single strains" Monoclonal antibodies with single-specificity have a great impact on immunology, and their availability has been used in sciences such as biology, morphology, biochemistry and others It has been confirmed that such monoclonal antibodies have been widely used not only as diagnostic reagents but also for therapeutic purposes (see, for example, Liz and Schlossmann, Blood, 59: 1-11, (1982)). Although the single antibody produced by the fusion tumor is effective in treating 200407427 as mentioned above, and it is obviously better than multiple antibodies because of its specificity, it still has an important disadvantage. In many applications, when these antibodies are intended to be used The use of monoclonal antibodies produced in non-human mammals in humans is severely limited. Repeated injections of "foreign" antibodies into humans (such as administering mouse antibodies) can cause harmful allergic reactions. Human-derived monoclonal antibodies, when injected into humans, cause anti-non-human antibody responses. The therapeutic use of anti-TF mouse Mabs has been disclosed in US Patent Nos. 6,001,978 and 5,223,427. International Patent Applications WO 99/51743 is a human / mouse chimeric monoclonal antibody against human TF. European Patent Application No. 83391 1 is a CDR-grafted antibody against human TF. Presta L. et al. , Thrombogenesis and Coagulation, Volume 85 (3) pp. 379-389 (2001) is about humanized antibodies against TF. There is still a need for improved compositions with anticoagulant activity in this technology, which can be relatively low Dosage is administered without the undesirable side effects associated with traditional anticoagulant compositions. The present invention accomplishes this by providing anticoagulants that do not have the side effects associated with traditional antibodies containing non-human sequences As needed, they can specifically act on the injury site and further provide other related advantages. Moreover, the present invention also provides an effect to inhibit the cellular function of TF (which includes diseases such as sepsis, inflammation, atherosclerosis, restenosis or cancer, etc. [Abstract] The present invention relates to a non-immunogen high affinity human 200407427 antibody against human TF, which inhibits the binding of clotting factor Vll / VIIa, and is effective against human TF for screening treatment Of human antibodies. In a first aspect, the invention relates to an isolated antibody that can immunoreact with an antigenic epitope presented on human TF. The term "human tissue factor" or "human TF" is used herein to refer to a full-length polypeptide receptor that contains the amino acid sequence 1-263 of a natural human tissue factor. The term "antibody" is used herein to mean that it has Immunoglobulin molecules and fragments thereof capable of specifically binding to an antigen (eg, human TF). The full-length antibody contains four polypeptide chains, that is, two heavy (H) chains and two light (L) chains are linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (herein referred to as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three functional domains, CtH, CH2, and CH3. Each light chain consists of a light chain variable region (herein referred to as LCVR or LH) and a light chain constant region. The heavy chain constant region contains a functional domain, CL. The VH and VL regions can be further subdivided into regions with high variability (called complementarity determining regions (CDRs)), which are separated by more inherent regions (called framework regions (FRs)). Each segment of VH and VL is composed of three segments of CDRs and four segments of FRs. The sequence from amine terminal to carboxyl terminal is J: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Therefore, the definition of an antibody also includes one or more antibody fragments that still retain the ability to specifically bind to an antigen (such as human TF). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments included in the term "antibody" include ⑴Fab fragments, which are monovalent fragments composed of VL, VH, CL, and CH1 g domains; (ii) F (ab) 2 200407427 and F (ab ') Fragment 2 is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (out) an Fd fragment consisting of VH and CH1 functional domains; (iv) an Fv fragment consisting of a single-armed antibody Vl and VH; (v) a dAb fragment (Wade et al. (1989) Nature 341: 544-546), which consists of a VH functional domain; and (vi) a complementary complementation determining region (CDR) that is isolated. Moreover, although the two functional domains (VL and VH) of the Fv fragment are encoded by separate genes, they can use recombinant methods to join them by a synthetic linker peptide, which can make them the VL and VH The VH regions are paired to form a single protein chain of a monovalent molecule (known as a single-chain Fv (scFv); see, for example, Bode et al. (1988) Science 242: 423-426; and Huston et al. (1988)? 1 * 〇〇: he 11 · Acad. Sci USA 85: 5879-5883). Such single chain antibodies are also intended to be included in the term "antibody". Other forms of single chain antibodies are also covered, such as diabodies. Double-acting antibodies are bivalent and bispecific antibodies, in which the VH and VL functional domains are expressed on a single polypeptide chain, but a linking peptide that is too short to allow two functional domains on the same chain to pair with each other This forces these domains to be paired with complementary domains of another chain to produce two antigen-binding sites (see, eg, 'Hulig', P. et al. (1993) Proc. Natl. Acad · Sci USA 90: 6444- 6448; Boyac, R. et al. (1994) Structure 2: 1121-1123). It is understood that human TF may have one or more epitopes that include (1) a peptide epitope consisting of a single peptide chain in human TF, and (2) a human TF polypeptide sequence consisting of more than one of its individual amino acid sequences A configurational epitope composed of unconnected spatially adjacent peptide chains; and (3) a molecular structure covalently attached to human TF after translation, such as a carbohydrate group (all or a part) After the composition of the -translated antigen must be determined. The terms "human antibody", "human antibody (majority)", "human TF antibody" and "human TF antibody (majority)" are used herein to include those having variable and constant regions derived from human germline immunoglobulin sequences antibody. Human antibodies of the invention can be, for example, in CDRs and particularly in CDR3 ', including amino acid residues not encoded by human germline immunoglobulin sequences (e.g., by random or site-directed mutagenesis in vitro or by in vivo Somatic mutations). However, the term "human antibody" is used herein without intending to include such antibodies in which CDR sequences derived from a germline of another mammalian breed (eg, mouse) have been implanted on a human framework sequence, such as a so-called human Sourced antibodies or human / mouse chimeric antibodies. "Isolated human antibody" as used herein means that it is substantially free of other human antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to human TF does not substantially contain Antigen-binding antibodies other than human TF). However, isolated antibodies that specifically bind to human T F may be cross-reactive to other antigens, such as T-F molecules derived from other species (discussed in further detail below). Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. The term "epitope" as used herein means any epitope located on an antigen to which the antibody binds. An epitope determinant is usually composed of a chemically active surface group of a molecule such as an amino acid or a sugar side chain, and usually has a specific three-dimensional structural feature and a special charge characteristic. The term "immunological response" or "performing an immune response" as used herein means 13 200407427 the binding of any antibody to its epitope with a dissociation constant Kd of less than 10_4 M. Where appropriate, the terms "immune response" or "performing an immune response" may be used interchangeably with the term "specifically combining". The term "inhibition" as used herein means any reduction compared to reference VII. For example, an antibody that inhibits the binding of human clotting factor Vila to human TF, which means that any binding ability of human clotting factor Vila to human TF is compared to the ability of human clotting factor Vila to bind to human TF in the absence of the antibody Reduced antibodies. The term "affinity" as used herein means the strength of the binding of an antibody to an epitope. The affinity of an antibody is measured by the dissociation constant Kd, which is defined as [Ab] X [Ag] / [Ab-Ag] where [Ab-Ag] is the molar concentration of the antibody-antigen complex, and [Ab] is the The molar concentration of the bound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined as 1 / Kd. A better method for determining the specificity and affinity of Mabs by competitive inhibition can be found in Harlow et al., Antibodies: Laboratory Manual, Published by Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988), Edited by Collingen et al. , Today's Immunology Process, Greene Publishing and Willy International Scientific Publishing, NY (1992, 1993), and Miao Le, Enzymatic Methods 92: 589-601 (1983), their disclosures are incorporated herein by reference. In a second aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a human antibody capable of immunoreactive with an antigenic epitope presented on human TF. The term "therapeutically effective amount" is an effective dose that can be titrated by a licensed practitioner to achieve the desired response. Dosage considerations will be 14 200407427 including efficacy, bioavailability, desired pharmacokinetic / pharmacokinetic conditions, therapeutic conditions (such as trauma, inflammation, septic shock), patient-related factors (such as weight, Health, age, etc.), whether co-administration with other medicines, time of administration, or other factors known to the physician. The dose of anti-TF human antibody administered to a patient will vary depending on the type and severity of the condition to be treated, but is generally between 0.1-5.0 mg / kg body weight. The term "individual" as used herein means any animal, especially a mammal, such as a human, and may be used interchangeably with the term "patient" if appropriate. In a third aspect, the invention relates to a composition comprising a human antibody capable of immunoreactive with an antigenic epitope presented on human TF. In another aspect, the present invention relates to a method for treating a FVIIa / TF-related disorder in humans, the method comprising administering to a therapeutically effective amount of a human antibody capable of immunoreactive with an antigenic epitope present on human TF. The human. "Treatment" means administering an effective amount of a therapeutically active compound of the present invention for the purpose of preventing any signs or conditions from developing, or for the purpose of curing or slowing down those signs or conditions that have developed. The term "treatment" is therefore intended to include prophylactic treatment. The term "FVIIa / TF-related diseases" is used herein to mean diseases or disorders in which TF and FVIIa are involved. It covers diseases or disorders related to thrombosis or coagulopathy, including inflammatory reactions and chronic thromboembolic diseases or disorders associated with fibrin formation, including vascular diseases such as deep venous thrombosis, arterial thrombosis, surgery Postthrombosis, 15 200407427 Coronary Artery Bypass Graft (CABG), Percutaneous Coronary Angioplasty (PTCA), Stroke, Tumor Growth, Tumor Metastasis, Angiogenesis, Thrombosis, Atherosclerosis and Reconstruction After Angioplasty Narrow, acute and chronic indications such as inflammation, septic shock, sepsis, hypertension, adult respiratory distress (ARDS), disseminated intravascular coagulation (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or Prophylactic treatment of arteriosclerotic blood vessels and other diseases or conditions at risk of thrombosis. Illnesses related to FVIIa / TF are not limited to in vivo coagulation disorders such as those specified above, but also include in vivo treatment methods related to FVIIa / TF, which may be caused, for example, by extracorporeal blood circulation, including blood from patients during dialysis procedures. Coagulation caused by blood extraction or blood bypass of a patient during a patient's aspiration, hemofiltration. The term "factor Vila" or "FVIIa" means "two-chain" activated coagulation factor VII which is decomposed by a specific cleavage action at the Argl52-Ilel53 peptide bond. FVIIa can be purified from blood or produced by recombinant methods. Obviously the implementation of the method described herein has nothing to do with the source of the purified factor Vila, and therefore the present invention is intended to cover the use of any factor Vila formulation suitable for use in the present invention. It is preferably human FVIIa. The term " FVII " means " single-chain " coagulation factor VII. In another aspect, the invention relates to a method for preparing a human antibody, the method comprising: a) preparing a human antibody against human TF, b) Test the antibodies in TF-induced clot analysis and screen out that the IC5Q can be less than 1 nM (for example, less than 500 pM, preferably less than 16 200407427 to 200 pM, preferably less than 100 pM in this analysis, (Preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM) human antibodies that inhibit clot formation, or test the antibodies in FXa yield analysis and screen for IC50 of less than 100 nM (in Analysis with FVIIa concentration of 0.1 nM), for example, less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM, inhibit human antibodies produced by FXa, or in FVIIa / Test antibodies and screen for IC5Q 値 in TF amine decomposition analysis (analyzed at 10 nM FVIIa concentration), such as below 40 nM, preferably below 20 nM, more preferably below 10 nM, a human antibody that inhibits FVIIa amidolytic activity induced by TF, or competitive analysis in FVIIa Test antibodies and screen for human antibodies that can compete with FVIIa, or test antibodies and screen for human antibodies that can bind to human TF in a TF ELISA assay containing TF. It should be understood that this should be used for TF-induced clot analysis The specific IC50 in the description refers to when normal human plasma is used. In another aspect, the present invention relates to a method for preparing a human antibody, the method comprising: a) preparing a human antibody against human TF, b) in Test antibodies from TF-induced clot analysis and screen for IC5Q 値 IC5G 値 + 1 nM lower than FFR-rFVIIa in this analysis, such as IC5G 値 + 500 pM lower than FFR-rFVIIa, compared to Better than IC5 of FFR-rFVIIa. 値 + 200 pM, preferably lower than IC50 of FFR-rFVIIa + 100 pM, preferably lower than IC5G of FFR-rFVIIa + 50 pM, preferably lower 17 200407427 at FFR -rFVIIa IC5Q 値 + 10 pM, better than FFR-rFVIIa IC5Q 値 + 5 pM, better than FFR-rFVIIa IC5Q 値, human antibody that inhibits clot formation, or test antibody in FXa production analysis And screen out IC5Q + 100 whose IC50 is lower than FFR-rFVIIa nM (0.1 nM FVIIa is used in this analysis), for example, IC5G 低于 + 10 nM lower than FFR-rFVIIa, preferably IC5Q 低于 + 5 nM lower than FFR-rFVIIa, and preferably lower than IC5 of FFR-rFVIIa.値 + 1 nM, better than IC5G of FFR-rFVIIa 値 + 0.1 nM, better than IC5 () of FFR-rFVIIa, human antibodies that inhibit FXa production, or test in FVIIa / TF amine decomposition analysis Antibodies can be screened for IC5G 値 IC5Q 値 lower than FFR-rFVIIa + 100 nM (10 nM FVIIa used in this analysis), such as IC5G 値 +40 nM lower than FFR-rFVIIa, preferably lower than FFR -rFVIIa's IC5Q 値 + 20 nM, preferably lower than FFR-rFVIIa 1 (: 5 () 値 +10 nM, more preferably lower than FFR-rFVIIa's IC5 () 値, inhibits FVIIa induced by TF Human antibodies with amidolytic activity, or test antibodies in FVIIa competition assays and screen human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays and screen human antibodies that bind to human TF . It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. The term "TF-induced clot analysis" is used herein to mean any analysis in which clot time is measured in a sample containing blood plasma and TF. An example of a TF-induced clot analysis is shown in Example 1, Analysis 7. The term "FXa yield analysis" is used herein to mean any analysis in which the activation effect of FX is measured in a sample containing TF, FVIIa, FX, calcium, and phospholipids. An example of an FXa yield analysis is shown in Example 1, Analysis 5. The term "FVIIa / TF amidolytic analysis" is used herein to mean any analysis in which the amidolytic activity of FVIIa is measured in the presence of TF, i.e., the cleavage of a small peptide mass. An example of a FVIIa / TF amidine decomposition analysis is shown in Example 1, Analysis 4. The term "TF ELISA analysis" as used herein means any ELISA analysis that includes TF and anti-F antibody. An example of a TF ELISA analysis is the direct and indirect TF ELISA analysis listed in Example 1, Analysis 1 and 2. The term "direct TF ELISA analysis" as used herein means any TF ELISA analysis that includes immobilized TF. Examples of direct TF ELISA analysis are listed in Example 1, Analysis 1. The term "indirect TF ELISA analysis" as used herein means any TF ELISA analysis in which TF is present in solution. Examples of indirect TF ELISA analysis are listed in Example 1, Analysis 2. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) Preparation of human antibodies against human TF 'b) Testing of antibodies in TF-induced clot analysis and screening can be performed with IC50 of less than 1 nM, for example less than 500 pM in this 19 200407427 analysis, Preferably it is less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM, a human antibody that inhibits clot formation, or is used in FXa yield analysis Test the antibody and screen for IC5Q 値 below 100 nM (in analysis with FVIIa concentration of 0.1 nM), such as below 10 nM, preferably below 5 nM, preferably below 1 nM, more preferably below 0.1 nM, a human antibody that inhibits FXa production, or test the antibody in FVIIa / TF amidine decomposition analysis and screen for IC5Q <100 nM (in an analysis performed at a FVIIa concentration of 10 nM), such as low Below 40 nM, preferably below 20 nM, more preferably below 10 nM, inhibiting TF induction Human antibodies with FVIIa amidolytic activity, or test antibodies in FVIIa competition assays and screen for human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays and screen for antibodies that bind to human TF Human antibodies. It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) Preparation of human antibodies against human TF, b) Testing of antibodies in TF-induced clot analysis and screening can be performed in this analysis with IC5G 値 lower than FFR-rFVIIa IC5Q 値 + 1 nM, for example IC5G 値 +500 pM lower than FFR-rFVIIa, preferably IC5 lower than FFR-20 200407427 rFVIIa.値 + 200 pM, preferably lower than IC50 of FFR-rFVIIa 値 + 100 pM, preferably lower than IC5Q of FFR-rFVIIa + 50 pM, preferably lower than IC5Q of FFR-rFVIIa 値 + 10 pM, more preferably lower IC5Q 値 + 5 pM in FFR-rFVIIa, better than IC5Q 値 in FFR-rFVIIaa 'human antibodies that inhibit clot formation, or test antibodies in FXa yield analysis and screen out IC5G 値 lower than FFR-rFVIIa IC5.値 + 100 nM (0.1 nM FVIIa used in this analysis), such as IC5G 低于 + 10 nM lower than FFR-rFVIIa, preferably IC5Q 低于 + 5 nM lower than FFR-rFVIIa, preferably lower than FFR-rFVIIa IC5Q 値 + 1 nM, more preferably lower than FFR-i * FVIIa IC5Q 値 + 0.1 nM, more preferably lower than FFR-rFVIIa IC5〇 値, inhibit human antibodies produced by FXa, or decompose in FVIIa / TF amidine The antibody is tested during the analysis and screened for IC50 値 lower than FFR-rFVIIa IC5G 値 + 100 nM (10 nM FVIIa used in this analysis), for example, IC5G 値 + 40 nM lower than FFR-rFVIIa, preferably IC5Q 値 +20 nM lower than FFR-rFVIIa, preferably IC5Q 値 +10 nM lower than FFR-rFVIIa, more preferably IC5〇 値 lower than FFR-rFVIIa, inhibiting the decomposition of FVIIa amine induced by TF Active human antibodies, or test antibodies in FVIIa competition analysis and screen for human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays containing TF and screen for human antibodies that can bind to human TF.
應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC5Q 21 200407427 値係指當使用正常人類血漿時。 於本發明之一項具體態樣中,該於其中製造抗人類TF 之人類抗體的方法包含以人類TF免疫哺乳動物,並單離由 該受免疫哺乳動物產生之抗體。於較佳具體態樣中,該哺 乳動物爲小鼠。應瞭解,該受免疫之晡乳動物或小鼠能夠 製造人類抗體。 於另一方面,本發明關於一種用於製備人類抗體之方 法,該方法包含: a) 以人類TF免疫小鼠, b) 從該受免疫小鼠單離出抗體-生產細胞並製備可分泌 人類抗體之無限增殖細胞, c) 將包含所產生抗體之培養基與無限增殖細胞分離, d) 於包含存在溶液之TF的間接TF ELISA分析中測試 抗體,並篩選出可與溶液中之人類TF結合之人類抗體, e) 於FVIIa競爭分析中測試抗體,並篩選出可與FVIIa 競爭結合之人類抗體, f) 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以 IC5。値低於100 nM (於一項以FVIIa濃度爲10 nM進行之 分析中),例如低於40 nM,較佳低於20 nM,更佳低於10 nM,抑制由TF所誘發之FVIIa醯胺分解活性之人類抗體, g) 於FXa產量分析中測試抗體並篩選出可以IC5()値低 於100 nM (於一項以FVIIa濃度爲0.1 nM進行之分析中) ,例如低於10 nM,較佳低於5 nM,較佳低於1 nM,更佳 低於0.1 nM,抑制FXa產生之人類抗體, 22 200407427 h) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此-項分析中以IC5Q値低於1 nM,例如低於500 pM,較佳係低 於200 pM,較佳低於100 pM,較佳低於50 pM,較佳低於 10 pM,更佳低於5 pM,抑制凝塊形成之人類抗體, i) 於適宜之培養基中篩檢並培育該可生產經步驟d-h所 篩選得之抗體的無限增殖細胞, j) 從經篩檢無限增殖細胞之培養基單離出所篩選得之抗 體。 應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC5() 値係指當使用正常人類血漿時。 於另一方面,本發明關於一種用於製備人類抗體之方 法,該方法包含: a) 以人類TF免疫小鼠, b) 從該受免疫小鼠單離出抗體-生產細胞並製備可分泌 人類抗體之無限增殖細胞, c) 將包含所產生抗體之培養基與無限增殖細胞分離, d) 於包含存在溶液之TF的間接TF ELISA分析中測試 抗體,並篩選出可與溶液中之人類TF結合之人類抗體, e) 於FVIIa競爭分析中測試抗體,並篩選出可與FVIIa 競爭結合之人類抗體, f) 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以 其IC5Q値低於FFR-rFVIIa之IC5Q値+ 100 nM (於該項分析 中使用 10 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5G 値 + 40 nM,較佳低於FFR-rFVIIa之IC5Q値+ 20 nM,較佳低於 23 200407427 FFR-rFVIIa 之 IC5。値 + 10 nM,更佳低於 FFR-rFVIIa 之 IC50 値,抑制由TF所誘發之FVIIa醯胺分解活性之人類抗體, g) 於FXa產量分析中測試抗體並篩選出可以其IC5〇値 低於FFR_rFVIIa之IC5Q値+ 100 nM (於該項分析中使用0.1 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5◦値 + 10 nM,較佳 低於 FFR-rFVIIa 之 IC5Q 値 + 5 nM,較佳低於 FFR-rFVIIa 之 IC5。値 + 1 nM,更佳低於 FFR-rFVIIa 之 IC5。値 + 0.1 nM, 更佳低於FFR-rFVIIa之IC5Q値,抑制FXa產生之人類抗體 h) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此 項分析中以其IC5G値低於FFR-rFVIIa之IC5Q値+ 1 nM ’例 如低於FFR-rFVIIa之IC5G値+ 500 pM,較佳係低於FFR-rFVIIa 之 IC5G 値 + 200 pM,較佳低於 FFR-rFVIIa 之 IC50 値 + 100 pM,例如低於FFR-rFVIIa之IC5G値+ 50 pM,較佳低 於 FFR-rFVIIa 之 IC5Q 値+10 pM,更佳低於 FFR-rFVIIa 之 IC5()値+ 5 pM,更佳低於FFR-rFVIIa之IC5G値,抑制凝塊 形成之人類抗體, i) 於適宜之培養基中篩檢並培育該可生產經步驟d_h所 篩選得之抗體的無限增殖細胞, J)從經篩檢無限增殖細胞之培養基單離出所篩選得之抗 體。 應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC50 値係指當使用正常人類血漿時。 術語“抗體-生產細胞”用於本文意指任何能夠生產抗 24 200407427 體之細胞。其包括融合瘤、轉感染細胞系及得自已注射抗 原之晡乳動物的相對較短壽命(或會死)之脾臟細胞或淋 巴細胞。 於另一方面,本發明關於一種可與存在人類TF上之抗 原表位進行免疫反應且抑制人類凝血因子Vila與人類TF 結合之經單離抗體,其可藉由包含下述步驟之方法獲得: a) 以人類TF免疫小鼠, b) 從該受免疫小鼠單離出抗體-生產細胞並製備可分泌 人類抗體之無限增殖細胞, c) 將包含所產生抗體之培養基與無限增殖細胞分離, d) 於包含存在溶液之TF的間接TF ELISA分析中測試 抗體,並篩選出可與溶液中之人類TF結合之人類抗體, e) 於FVIIa競爭分析中測試抗體,並篩選出可與FVIIa 競爭結合之人類抗體, f) 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以 IC5Q値低於100 nM (於一項以FVIIa濃度爲10 nM進行之 分析中),例如低於40 nM,較佳低於20 nM,更佳低於10 nM,抑制由TF所誘發之FVIIa醯胺分解活性之人類抗體, g) 於FXa產量分析中測試抗體並篩選出可以IC5()値低 於100 nM (於一項以FVIIa濃度爲0.1 nM進行之分析中) ,例如低於10 nM,較佳低於5 nM,較佳低於1 nM,更佳 低於0.1 nM,抑制FXa產生之人類抗體, h) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此 項分析中以IC5〇値低於1 nM,例如低於500 pM,較佳係低 25 200407427 於200 PM,較佳低於100 PM,較佳低於50 pM,較佳低於 10 PM,更佳低於5 PM,抑制凝塊形成之人類抗體, i)於適宜之培養基中篩檢並培育該可生產經步驟d-h所 篩選得之抗體的無限增殖細胞, J)從經篩檢無限增殖細胞之培養基單離出所篩選得之抗 體。 應瞭解’該於由TF-誘發之凝塊分析中所述之特定IC5() 値係指當使用正常人類血漿時。 於另一方面,本發明關於一種可與存在人類TF上之抗 原表位進行免疫反應且抑制人類凝血因子Vila與人類TF 結合之經單離抗體,其可藉由包含下述步驟之方法獲得: a) 以人類TF免疫小鼠, b) 從該受免疫小鼠單離出抗體-生產細胞並製備可分泌 人類抗體之無限增殖細胞, c) 將包含所產生抗體之培養基與無限增殖細胞分離, d) 於包含存在溶液之TF的間接TF ELISA分析中測試 抗體,並篩選出可與溶液中之人類TF結合之人類抗體, e) 於FVIIa競爭分析中測試抗體,並篩選出可與FVIIa 競爭結合之人類抗體, f) 於FVIIa/TF醯胺分解分析中測試抗體並篩選出可以 其IC5G値低於FFR-rFVIIa之IC5。値+ 100 nM (於該項分析 中使用 10 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5Q 値 + 40 nM,較佳低於FFR-rFVIIa之IC5G値+ 20 nM,較佳低於 FFR-rFVIIa 之 IC5Q 値 + 10 nM,更佳低於 FFR-rFVIIa 之 IC50 26 200407427 値,抑制由TF所誘發之FVIIa醯胺分解活性之人類抗體’ g) 於FXa產量分析中測試抗體並篩選出可以其1C5〇値 低於FFR-rFVIIa之IC5Q値+ 100 nM (於該項分析中使用〇·1 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5Q 値 + 1〇 nM ’ 較佳 低於 FFR-rFVIIa 之 IC5G 値 + 5 nM,較佳低於 FFR-rFVIIa 之 IC5〇 値 + 1 nM,更佳低於 FFR-rFVIIa 之 IC5Q 値 + 〇·1 nM ’ 更佳低於FFR-rFVIIa之IC5Q値,抑制FXa產生之人類抗體 h) 於由TF-誘發之凝塊分析中測試抗體並篩選出可於此 項分析中以其IC5Q値低於FFR-rFVIIa之1<:5()値+ 1 nM ’例 如低於FFR-rFVIIa之IC5G値+ 500 pM,較佳係低於FFR-rFVIIa 之 IC5G 値+200 pM , 較佳低於 FFR-rFVIIa 之 IC50 値 + 100 pM,例如低於FFR-rFVIIa之IC5Q値+ 50 pM,較佳低 於 FFR-rFVIIa 之 IC5。値 + 10 pM,更佳低於 FFR-rFVIIa 之 IC5Q値+ 5 pM,更佳低於FFR_i*FVIIa之IC5G値,抑制凝塊 形成之人類抗體, i) 於適宜之培養基中篩檢並培育該可生產經步驟d-h所 篩選得之抗體的無限增殖細胞, j) 從經篩檢無限增殖細胞之培養基單離出所篩選得之抗 體。 應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC5〇 値係指當使用正常人類血漿時。 於本發明之一項具體態樣中,該無限增殖細胞融合瘤 細胞。 27 200407427 於另一方面,本發明關於一種生產可與存在人類TF上 之抗原表位進行免疫反應且抑制人類凝血因子Vila與人類 TF結合之人類抗體的細胞。 於本發明之一項具體態樣中,該細胞爲經單離之淋巴 細胞。 於本發明之另一項具體態樣中,該細胞係單離自小鼠 〇 於本發明之另一項具體態樣中,該細胞爲融合瘤細胞 。於本發明之一項具體態樣中該融合瘤細胞係藉由將抗體_ 生產淋巴細胞與無限增殖細胞融合而得一種抗體-生產融合 瘤細胞。 於本發明之另一項具體態樣中,該經單離之人類抗體 係抑制人類凝血因子Vila與人類TF結合。 於本發明之另一項具體態樣中,該經單離之人類抗體 爲單株抗體。 術語“單株抗體”用於本文意指同源之免疫球蛋白族 群,亦即該抗體族群之個別分子皆相同,除了自然發生之 突變。抗體正常係由衍生自骨髓中B細胞之淋巴細胞所產 生。自相同純系所衍生得之淋巴細胞製造具有單一胺基酸 序列之免疫球蛋白。淋巴細胞無法直接進行長時間培育以 製造其實質量的特異性抗體。然而,科勒等人(1975,自 然,256:495 )證實體細胞融合製程(特別是淋巴細胞與骨 髓瘤細胞間之融合)能夠產生可於培養基中生長並製造稱 爲“單株抗體”之特殊抗體的融合瘤細胞。骨髓瘤細胞爲 28 200407427 其本身常製造抗體(視細胞品系而定)之淋巴細胞腫瘤細 胞,但已知爲“非-生產性”品系。 於本發明之另一項具體態樣中,該經單離之人類抗體 爲重組型抗體。 術語“重組型抗體”用於本文欲包括所有藉由重組方 法製所備、表現、產生或單離得之人類抗體,例如使用經 轉感染入宿主細胞之重組表現載體所表現得之抗體(進一 步描述於下文第Π節中)、自重組型、組合抗體庫單離得 之抗體(進一步描述於下文第ΠΙ節中)、單離自基因轉殖 入人類免疫球蛋白基因之動物(例如小鼠)的抗體(參見 例如,泰勒,L.D·等人(1992)核酸硏究20:6287-6295 )或藉 由任何其他包含將人類免疫球蛋白基因序列剪接至其他 DNA序列之方法所製備、表現、產生或單離得之人類抗體 。此類重組型人類抗體具有衍生自人類種系免疫球蛋白序 列之可變區與恆定區。然而,於某些具體態樣中,係將此 類重組型人類抗體進行活體外致變作用(或當使用基因轉 殖入人類Ig序列之動物時,進行活體內體細胞致變作用) ,而因此該等重組型抗體之VH及VL區域的胺基酸序列爲 其雖然衍生自且與人類種系VH及VL序列相關,但可能於 活體內並不天然地存在該人類抗體種系所有組成成份中之 序列。 於本發明之另一項具體態樣中,該經單離之人類抗體 爲Fab片段。 於本發明之另一項具體態樣中,該經單離之人類抗體 29 200407427 爲F(ab)2片段。 於本發明之另一項具體態樣中,該經單離之人類抗體 爲F(abf)2片段。 於本發明之另一項具體態樣中,該經單離之人類抗體 爲單鏈Fv片段。 於本發明之另一項具體態樣中,該經單離之人類抗體 具有與人類TF之Kd値介於10·15-1(Τ8 Μ之範圍內。應瞭解 ,該人類抗體與人類TF之Kd値係於一項其中該人類抗體 爲經固定化之分析中測定得(參見分析6)。 於本發明之另一項具體態樣中,該經單離之人類抗體 具有與人類TF之Kd値介於10·15-10·1()Μ之範圍內。 於本發明之另一項具體態樣中,該經單離之人類抗體 具有與人類TF之Kd値係低於ΙΟ·8 Μ。於本發明之另一項具 體態樣中,該經單離之人類抗體具有與人類TF之Kd値係 低於1(Τ9 Μ。於本發明之另一項具體態樣中,該經單離之 人類抗體具有與人類TF之Kd値係低於ΗΤ1() Μ。於本發明 之另一項具體態樣中,該經單離之人類抗體具有與人類TF 之心値係低於10_π Μ。於本發明之另一項具體態樣中,該 經單離之人類抗體具有與人類TF之Kd値係低於1(Τ12 Μ。 於本發明之另一項具體態樣中,該經單離之人類抗體具有 與人類TF之1値係低於1(Τ13 Μ。於本發明之另一項具體 態樣中,該經單離之人類抗體具有與人類TF之Kd値係低 於HT14 Μ。於本發明之另一項具體態樣中,該經單離之人 類抗體具有與人類TF之心値係低於1(Γ15Μ。 30 200407427 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於由TF-誘發之凝塊分析中測試抗體,並篩選出 可於此項分析中以IC5()値低於1 nM抑制凝塊形成之人類抗 體。於另一項具體態樣中’該IC5Q値係低於500 pM。於另 一項具體態樣中,該IC5〇値係低於200 pM。於另一項具體 態樣中,該IC5〇値係低於100 pM。於另一項具體態樣中, 該IC5Q値係低於50 pM。於另一項具體態樣中,該IC5〇値 係低於10 PM。於另一項具體態樣中,該IC5Q値係低於5 pM 0 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於由TF-誘發之凝塊分析中測試抗體並篩選出可 於此項分析中以其IC5Q値低於FFR-rFVIIa之IC5Q値+ 1 nM ,例如低於FFR-rFVIIa之IC5G値+ 500 pM,較佳係低於 FFR-rFVIIa 之 IC5Q 値+ 200 pM,較佳低於 FFR-rFVIIa 之 IC5Q 値 + 100 pM,例如低於 FFR-rFVIIa 之 IC5G 値+ 50 pM, 較佳低於FFR-rFVIIa之IC5G値+10 pM,更佳低於FFR-rFVIIa 之 IC5Q 値 + 5 pM , 更佳低於 FFR-rFVIIa 之 IC5G 値, 抑制凝塊形成之人類抗體。 應瞭解,該於由TF-誘發之凝塊分析中所述之特定IC50 値係指當使用正常人類血漿時。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於FXa產量分析中測試抗體並篩選出可以IC50 値低於100 nM (於一項以FVIIa濃度爲0·1 nM進行之分析 中)抑制FXa產生之人類抗體。於另一項具體態樣中,該 31 200407427 IC5Q値係低於10 nM。於另一項具體態樣中,該IC5Q値係低 於5 nM。於另一項具體態樣中,該IC5。値係低於1 nM。於 另一項具體態樣中,該IC5Q値係低於0·1 nM。 於本發明之另一項具體態樣中’該用於製備人類抗體 之方法包含於FXa產量分析中測試抗體並篩選出可以iC50 値低於FFR-rFVIIa之IC5G値+ 100 nM (於該項分析中使用 0.1 nM FVIIa),例如低於 FFR-rFVIIa 之 IC5G 値 + 10 nM, 較佳低於FFR-rFVIIa之IC5G値+ 5 nM,較佳低於FFR-rFVIIa 之 IC5Q 値 + 1 nM,更佳低於 FFR-rFVIIa 之 IC5Q 値 + 0.1 nM,更佳低於FFR-rFVIIa之IC5G値,抑制FXa產生之 人類抗體。 於本發明之另一項具體態樣中’該用於製備人類抗體 之方法包含於FVIIa/TF醯胺分解分析中測試抗體並篩選出 可以其IC5Q値低於100 nM (於FVIIa濃度爲10 nM下進行 分析)抑制由TF所誘發之FVIIa醯胺分解活性之人類抗體 。於另一項具體態樣中,該IC5〇値係低於40 nM。於另一 項具體態樣中,該IC5Q値係低於20 nM。於另一項具體態 樣中,該IC5〇値係低於10 nM。於另一項具體態樣中’該 IC5Q値係低於5 nM。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於FVIIa/TF醯胺分解分析中測試抗體並篩選出 可以其IC5Q値低於FFR-rFVIIa之IC5Q値+ 100 nM (於該項 分析中使用10 nM FVIIa),例如低於FFR-rFVIIa之IC50値 + 40 nM,較佳地低於FFR-rFVIIa之IC5G値+ 20 nM,較佳 32 200407427 地低於FFR-rFVIIa之IC5Q値+ 10 nM,更佳地低於FFR-rFVIIa之IC5〇値,抑制由TF所誘發之FVIIa醯胺分解活性 之人類抗體。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於FVIIa競爭分析中測試抗體並篩選出可與 FVIIa競爭結合之人類抗體。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於TF ELISA分析中測試抗體並篩選出可與人類 TF結合之人類抗體。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於包含固定化TF之直接TF ELISA分析中測試 抗體並篩選出可與固定化人類TF結合之人類抗體。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於包含存在溶液之TF的間接TF ELISA分析中 測試抗體並篩選出可與溶液中人類TF結合之人類抗體。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於以TF表現細胞所進行之FXa產量分析中測試 抗體,並篩選出可以其IC5()値低於500 nM (於一項以 FVIIa濃度爲1 nM進行之分析中)抑制FXa產生之人類抗 體。於另一項具體態樣中,該IC5()値係低於100 nM。於另 一項具體態樣中,該IC5()値係低於50 nM。於另一項具體 態樣中,該IC5Q値係低於10 nM。於另一項具體態樣中, 該IC5〇値係低於5 nM。 於本發明之另一項具體態樣中,該用於製備人類抗體 33 200407427 之方法包含於以TF表現細胞所進行之FXa產量分析中測試 抗體,並篩選出可以IC5Q値低於FFR-rFVIIa之IC5G値+ 500 nM (於該項分析中使用1 nM FVIIa),例如低於FFR-rFVIIa 之 IC5Q 値 + 100 nM,較佳低於 FFR-rFVIIa 之 IC5〇 値 + 50 nM,較佳低於FFR-rFVIIa之IC5Q値+ 10 nM,更佳低 於 FFR-rFVIIa 之 IC5Q 値+ 5 nM,更佳低於 FFR-rFVIIa 之 IC5Q値,抑制FXa產生之人類抗體。 術語“TF表現細胞”意指任何可表現人類TF之晡乳 動物細胞。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於完整細胞TF結合分析中測試抗體,並篩選出 可與FVIIa競爭與表現於完整細胞表面上之人類TF結合的 人類抗體。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於生物傳感器分析中測試抗體,並篩選出具有 與人類TF結合之Kd値低於100 nM之人類抗體。於另一項 具體態樣中,該與人類TF結合之1値係低於10 nM。於另 一項具體態樣中,該與人類TF結合之Kd値係低於5 nM。 於另一項具體態樣中,該與人類TF結合之Kd値係低於1 nM。於另一項具體態樣中,該與人類TF結合之Kd値係低 於0·5 nM。於另一項具體態樣中,該與人類TF結合之Kd 値係低於Μ。於另一項具體態樣中,該與人類TF結合 之Kd値係低於10·11 Μ。於另一項具體態樣中,該與人類 TF結合之Kd値係低於ΙΟ·12 Μ。於另一項具體態樣中,該 34 200407427 與人類TF結合之Kd値係低於1(Τ13 Μ。於另一項具體態樣 中,該與人類TF結合之1値係低於1〇·14 Μ。於另一項具 體態樣中,該與人類TF結合之Kd値係低於Μ。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於ΜΑΡΚ信號分析中測試抗體,並篩選出可抑 制由FVIIa-所誘發ΜΑΡΚ信號活化作用之人類抗體。於一 項具體態樣中,該抗體係以98%抑制由FVIIa-所誘發之 MAPK信號活化作用。於一項具體態樣中,該抗體係以90% 抑制由FVIIa-所誘發之MAPK信號活化作用。於一項具體 態樣中,該抗體係以70%抑制由FVIIa-所誘發之MAPK信 號活化作用。於一項具體態樣中,該抗體係以50%抑制由 FVIIa-所誘發之MAPK信號活化作用。於一項具體態樣中, 該抗體係以30%抑制由FVIIa-所誘發之MAPK信號活化作 用。 術語“MAPK信號”意指其介導促***原活化蛋白激 酶(MAPK)或其同源蛋白於對各種胞外刺激之反應中所產生 之活化作用的細胞內事件級聯放大。晡乳動物細胞中已鑑 定出三群不同MAP激酶:1)經胞外調節性激酶(Erk 1/2或 P44/42),2) c-Jun N-端激酶(JNK)及 3) p38 激酶。Erkl/2 途 徑涉及Erk 1 (p44)與/或Erk 2 (p42)之磷酸化作用。經活化 之MAP激酶例如p44/42 MAPK可移位至細胞核內,於其中 彼等可進行磷酸化並活化轉錄因子包括(Elk 1)及轉錄作用 之訊號轉導物與活化物(Stat)。Erkl/2亦可於細胞質中或於 細胞核內將激酶P90RSK磷酸化,然後p90RSK可再活化數 35 200407427 種轉錄因子。 術語“蛋白激酶”意欲包括能夠將存在肽類及/或蛋白 質中之絲胺酸與/或蘇胺酸與/或酪胺酸磷酸化的酵素。 術語“由FVIIa-所誘發之MAPK信號活化作用”意指 哺乳動物細胞中FVII與TF結合並藉此誘發MAPK信號。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於基因表現分析(分析15 )中測試抗體,並篩 選出可抑制由FVIIa-所誘發獨立選自所列Fra_l、Id2及 Cyr61諸基因之增量調節作用之人類抗體。 應瞭解,可抑制TF活性之抗TF抗體可與呈現於TF之 不同抗原表位結合並可能同時抑制FVIIa或FX或FXa與人 類TF之結合。本發明之一項標的係篩選出可抑制FVIIa與 人類TF結合並藉此抑制由FVIIa所誘發之細胞內信號發生 的抗體。 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於人類癌症分析(分析13)中測試抗體’並舖 選出可抑制人類癌症成長或轉移之人類抗體° 於本發明之另一項具體態樣中,該經單離之人類抗體 可抑制由FVIIa-所誘發獨立選自所列Fra_1、Id2及Cyr61 §者 基因之增量調節作用。 _ 於本發明之另-項具體態樣單離之人類抗體 不會抑制FX或FXa與人類TF之結合° 於本發明之另一項具體態«中,該經單離之人類抗體 可抑制細胞內TF之活性。 200407427 於本發明之另一項具體態樣中,該用於製備人類抗體 之方法包含於抗原表位定位分析中測試抗體,並篩選出可 與TF上較佳抗原表位結合之人類抗體。於本發明之一項具 體態樣中,該較佳抗原表位包含殘基Trp45。於本發明之一 項具體態樣中,該較佳抗原表位包含殘基Lys46。於本發明 之一項具體態樣中,該較佳抗原表位包含殘基Tyr49。 於本發明之另一項具體態樣中,該經單離之人類抗體 可與位於TF與FVIIa之界面內的抗原表位結合。 經由X-射線結構(貝能等人,1996自然,380:41-46 ) 所測定得可進行FVIIa蛋白酶功能域與TF間之交互作用的 殘基爲:Ser39、Gly43、Trp45、Ser47、Phe50、Arg74、 Phe76、Tyr94、Pro92。此位於TF與FVIIa之界面特徵在於 其係一種含有許多能使TF與FVII間產生許多緊密接觸之 分子間氫鍵,以於FVIIa結合過程中獲得高特異性的複雜 界面區域。 本發明亦關於對TF之高親和性人類單株抗體。該含有 與FVIIa蛋白酶功能域之接觸界面之TF表面擁有易於反應 並產生蛋白質-蛋白質交互作用之特殊拓撲結構,其中另一 類型蛋白質-蛋白質交互作用係形成抗體與蛋白質配位子間 之複合體。因此,對抗此位於人類TF上之抗原表位的單株 抗體可提供高親和性Mabs。 本發明之一方面係可與對亦關於對FVIIa蛋白酶功能 域之接觸界面進行免疫反應之高親和性人類單株抗體。 本發明之人類TF抗體作用爲對由TF介導之凝血誘發 37 200407427 作用,因而抑制凝血因子FVIIa與TF結合並藉此阻斷凝血 酶產生及後續之血纖維蛋白沈積的掊抗劑。人類TF抗體尤 其可用於投藥予人類以治療各種涉及血管內凝血之病況。 如此,人類TF抗體可用於抑制TF活性而導致(例如)抑 制血液凝固、血栓形成或血小板沈積。進而,根據本發明 之人類TF抗體(作用在於抑制TF之細胞內功能,TF之信 號功能)可用於諸如敗血症、發炎、動脈粥樣硬化、再狹 窄或癌症等病況。 人類TF抗體可用於各種疾病。其涵蓋與血栓形成或凝 血病相關之疾病或失調症,包括發炎反應及與血纖維蛋白 形成相關聯之慢性血栓栓塞性疾病或失調症,包括血管病 例如深層靜脈血栓生成、動脈血栓形成、手術後血栓形成 、冠狀動脈繞道移植物(CABG)、經皮冠狀血管成形術 (PTC A)、中風、腫瘤生長、腫瘤轉移、血管生成、血栓溶 解、血管成形術後之動脈粥樣硬化與再狹窄、急性與慢性 適應症例如發炎、敗血性休克、敗血症、高血壓、成人呼 吸窘迫症(ARDS)、全身性發炎反應徵候群(SIRS)、彌散性 血管內凝血(DIC)、肺部栓塞、血小板沈積、心肌梗塞、或 對具有發生血栓形成危險之動脈硬化血管、因周邊血液祖 細胞(PBPC)移植後造成之靜脈閉塞疾病、溶血性尿毒徵候 群(HUS)、血栓性血小板減少性紫癲(TTP)及其他疾病或病 況之哺乳動物所施予之預防性治療。人類TF抗體可用於防 止經鑑定具高危險病患,例如該等進行外科手術或罹患充 血性心臟衰竭之患者中發生血栓拴塞性倂發症。人類TF抗 38 200407427 體尤其可用於治療因急性血管創傷所造成之內膜增生或再. 狹窄。急性血管創傷爲快速發生(亦即歷時數小時至數月 )之損傷,而非歷經終生發展成之慢性血管創傷(例如動 脈粥樣硬化)。急性血管創傷往往起因於外科手術程序例 如血管重建,其中使用血管成形術、動脈內膜切除術、動 脈粥樣硬化斑切除術、安置血管植入物等技術。增生可能 發生呈一種對(例如)植入物安置或器官移植反應中之延 遲反應。因爲人類TF抗體較肝素更具選擇性,其僅與已暴 露於創傷部位之TF結合,且因爲人類TF抗體不會破壞或 抑制其他凝血蛋白質,故更具功效且當用於預防深層靜脈 血栓形成時可較肝素不易造成出血倂發症。 如下列實施例所示,本發明之人類TF抗體能夠選擇性 地與細胞表面TF結合,並藉由抑制凝血FVIIa與TF之結 合而限制其功能活性。保持與TF結合之人類TF抗體可於 血管創傷部位,藉由阻斷凝血酶產生及後續之血纖維蛋白 沈積而抑制血小板累積。 由於人類TF抗體可於急性血管創傷部位阻斷凝血酶產 生及限制血小板沈積之能力,故保持與TF結合而藉此抑制 FVIIa結合之人類TF抗體可用於抑制血管再狹窄。 包含人類TF抗體之組合物當經調製成爲醫藥組合物時 尤其可用於治療患者之方法,其中彼等可投予罹患各種疾 病狀態之患者以治療與凝血相關的病況。此類能夠與TF結 合並抑制FVIIa與TF結合之人類TF抗體,當相較於其他 抗凝血劑時,可擁有較長血漿半衰期並因此具有相對較長 39 200407427 之抗凝血活性時間。於本發明標的組合物之醫藥指示中, 一般係依凝血劑用於治療,例如深層靜脈血栓生成、肺部 栓塞、中風、彌散性血管內凝血(DIC)、與敗血症相關之肺 部及腎臟血纖維蛋白沈積、抗磷脂類徵候群(APS)、動脈粥 樣硬化與心肌梗塞。該等組合物可用於抑制因機械性血管 創傷,例如由外科手術、顯微手術、氣球血管成形術、動 脈內膜切除術、還原性粥樣硬化斑切除術、固定模安置、 雷射光照治療或旋轉震動所造成之損傷,或對血管植入物 、固定模、繞道植入物或器官移植物等所發生之次級創傷 。因此該等組合物可用於抑制血小板沈積與相關之疾病。 於是,一種抑制凝血、血管再狹窄或血小板沈積之方法( 例如)包含對患者投藥一種其包含量足以有效抑制凝血、 血管再狹窄或血小板沈積之人類TF抗體的組合物。該等方 法亦發現可用於治療患者之冠狀動脈急性閉塞(例如急性 心肌梗塞),其包含將人類TF抗體與組織胞漿酶原活化劑 結合進行投藥,並可加速由tPA所誘發之溶血栓作用。人 類TF抗體係於投藥溶血栓劑(例如組織胞漿酶原活化劑) 之前、同時或稍後立即給予。 根據本發明,抗人類TF之人類單株抗體可藉由以人類 TF免疫攜帶有部份人類免疫系統而非小鼠系統之轉殖基因 小鼠(得自Medarex)而製得。如所述將得自此等轉殖基因 小鼠之脾臟細胞用於製造可分泌人類單株抗體之融合瘤( 參見,例如,伍德等人,國際專利申請案WO 91/00906,庫 契拉派提等人,PCT公開案W〇91/10741 ;隆柏格等人,國 200407427 際專利申請案WO 92/03918,凱等人,國際專利申請案W〇. 92/03917,隆柏格,N.等人,1994 自然 368:856-859 ;格林 ,L1.等人1994自然遺傳學7:13-21 ;莫理森,S.L.等人 1994,Proc. Natl. Acad. Sci USA 81:6851-6855 ;布魯吉曼等 人1993年度免疫學7:33-40 ;泰隆等人1993 PNAS 90:3720- 3724;布魯吉曼等人1991歐洲免疫學期刊21:1323-1326 ) 〇 抗人類TF之人類單株抗體亦可藉由噬菌體展示技術製 得。可從經免疫之人體構築得人類抗體文庫,並將其展示 於絲狀噬菌體之表面上。於許多個案中,已使用其中係將 所興趣抗原固定於固體表面,亦即微量滴定平盤或珠粒上 之淘選技術單離得高親和性人類單-鏈Fv (ScFv)及Fab抗體 片段(巴貝斯C.F.,III與布爾頓,D.R.生物技術趨勢1996 ,14:230-234 ;奧加美 L.等人,人類抗體 1997,8:155-68 ) 。具有大的原初文庫之噬菌體展示系統亦可能未經免疫作 用而直接單離得人類抗體(迪哈得H.L等人,生物化學期 刊 1999 , 18218-18230)。 【實施方式】 本發明進一步以下列實施例做例舉說明,然而,彼等 並非用以限定本發明之保護範圍。前述說明及下列實施例 所揭示之實質內容(個別獨立或相互組合)爲用於瞭解本 發明較佳形式之態樣。 實施例1 對人類TF具免疫特異性之人類Mab的製備 41 200407427 試劑 人類TF可如雷歐,L.V.Μ.,血栓形成硏究,51:373-384 1988所述,自人腦單離得。 亦可使用經脂質化之重組型人類TF (Dade,Innovin, 貝克斯特)做爲人類促凝血酶原激酶試劑。自腦組織製備得 大鼠、兔子、狒狒及豬促凝血酶原激酶。將二倍體積之45 °C 0.9% NaCl加至腦組織中,並將組織以人力玻璃均質器進 行均質化。待於45°C下伴隨偶爾搖晃培育30分鐘後,將樣 本於2000 Xg下離心20分鐘。將沈澱丟棄,並將上淸液等 分小量置於-80°C下保存待用。 再脂質化之TF可藉由將重組型人類全長TF (美國診 斷試劑#4500 )重新建構於磷脂質載體(PC/PS 75/25)中而獲 得。 經生物素化人類TF之製備如下:將生物素-NHS (η-琥 珀醯基生物素,西格瑪Η-1759 )溶解於DMF (二甲基甲醯 胺)中使濃度達1.7毫克/毫升。將60微升該生物素-NHS 溶液加入1毫克/毫升溶於0.1 M NaHC03緩衝液之人類TF 中並令其於室溫下反應4小時。將含有經生物素化TF之溶 液對PBS-緩衝液進行透析過夜。 所使用之FVIIa爲如提姆,L.等人Biochem 27:7785-7793,1988所述製備得之重組型人類FVIIa。 sTF :實質上如福瑞加德,P.-Ο.等人,蛋白科學5, 153M540 (1996)所述於大腸桿菌中表現及純化得之重組型 人類可溶。 42 200407427 S2288 :重建於H2〇中,濃度達17.24毫克/毫升( Chromogenix ) FX :經純化之人類血漿FX ( HFX,酵素硏究實驗室股 份有限公司) FXa :經純化之人類血漿FX以魯賽爾蛇毒活化(HFXa ,酵素硏究實驗室股份有限公司) 顯色酵素X :溶解於H20中,濃度達1.25毫克/毫升( 百靈佳英格漢公司) 125I-FVIIa係藉由標準放射標定程序製得。 FFR-rFVIIa : FVIIa 之活性部位以 D-Phe-L-Phe-L-Arg-氯 甲基酮阻斷。如索瑞森B.B.等人,生物化學期刊272:1 1863-1 1868,1997所述製備得。 免疫作用 將人類TF乳化於弗氏完全佐劑中。經由皮下注射對 HuMab小鼠或其雜交後代(Medarex)施予40微克劑量。 待經14及28天後(且實質上多次間隔14天),對小鼠以 類似注射方式追加20微克乳化於不完全弗氏佐劑之TF。 於最後一次注射後10天取樣採血並藉由TF ELISA (分析1 及2)測試血淸中是否存在人類TF特異性抗體。 融合 將自分析1-3中呈陽性血淸測試之小鼠以靜脈內注射 追加20微克人類TF並於三天後將其犧牲。無菌操作取出 脾臟並分散成單細胞懸浮液。 將狐-骨髓瘤細胞生長於CD融合瘤培養基(Gibco 43 200407427 11279-023)中。 本融合實驗係藉由PEG-方法(庫勒,G與米斯坦C· (1976),歐洲免疫學期刊,6:511-19)將脾臟細胞與骨髓瘤 細胞(P3x63 Ag8.653,ATCC CRL-1580)及 Sp2/0 (ATCC CRL-1581)進行融合。將細胞接種於微量平盤中並培育於於37°C 下。接著於兩週內換三次培養基。自每槽融合瘤細胞培養 物取出100微升上淸液並於TF ELISA (分析1及2)測試 是否存在TF特異性抗體。 實施例2 : 篩檢 各種用於篩檢血淸及培養物上淸液是否存在特定所選 抗體之分析法係描述於下: ^ 直接TF ELISA分析(分析1 ): 於4°C下將諾克-免疫平盤塗覆以1微克/毫升存於PBS 之人類sTF。將平盤以封阻劑(含5 mM CaCl2及2% BSA之 TBS)進行封阻並以TBS+0.05% Tween 20洗滌,再將取自 融合瘤細胞之上淸液加入。待於室溫下培育1小時後,將 平盤洗淨並將經山辣根過氧化酶(HRPO)標定之抗-人類IgG 加入。再另培育一小時後將平盤洗淨並以TMB-受質(Kem-EN-Tec)依廠商之操作手冊進行呈色。置於ELISA-計讀機 上於450 nm下測定吸光値。 間接TF ELISA分析(分析2): 將諾克-免疫平盤塗覆以0.5微克/毫升存於PBS之山羊 抗-人類IgG (南方生物技術協會,Cat-#2040-l)並於4°C下 44 200407427 培育過夜。將平盤以封阻緩衝液(含5 mM CaCl2& 2% BSA 之 TBS)進行封阻並於 TBS+5 mM CaCl2+0.05% Tween 20 中洗滌。將取自融合瘤細胞之上淸液加入,並將平盤於室 溫下培育1小時。其經另次洗滌後,將濃度爲1微克/毫升 之生物素化人類sTF加入,並培育1小時。待洗淨後,將 鏈霉親和素-HRPO溶液加入並培育一小時。將平盤如分析1 中所述以TMB-受質進行呈色。 FVIIa競爭分析(分析3): 於下將諾克-免疫平盤與人類STF (濃度5微克/毫 升溶於PBS)培育過夜。將平盤洗淨並於含5 mM 〇3〇:12及 2% BSA之TBS緩衝液中進行封阻。將抗-人類TF Mabs加 入並將平盤培育2小時。將平盤洗淨後再並將經生物素化 之人類FVIIa加入(1微克/毫升溶於含5 mM CaCl2及2% BSA之TBS緩衝液中)並將平盤培育1小時。再次將平盤 洗淨然後加入經HRPO標定之鏈霉親和素並培育45分鐘。 將平盤再次洗滌後以TMB-受質(Kem-EN-Tec)依廠商之操 作手冊進行呈色。 FVIIa/sTF醯胺分解活性之抑制作用(分析4): 由抗-人類TF Mabs所造成由FVIIa-TF催化之醯胺分解 活性的抑制作用係使用可溶人類TF (10 nM)、重組型人類 FVIIa (10 nM)及漸增濃度之Mabs (0.0122-50 nM)進行測試。 將各種不同濃度之抗-人類TF Mabs或FFR-rFVIIa與10 nM TF 及 10 nM FVIIa 於 BSA 緩衝液(50 mM Hepes,pH 7.4、 100 mM NaCl、5 mM 0&(:12及1毫克/毫升BSA)中於室溫 45 200407427 下進行前培養60分鐘,然後再將受質S2288 ( 1.2 mM, Chromogenix)加入。於405 nm下連續測量呈色過程達30 分鐘。醯胺分解活性係以mOD/分鐘表示。可計算得由 Mabs所致FVIia/TF醯胺分解活性抑制作用之IC5Q値。於此 項分析中 FFR-rFVIIa 之 IC50 値爲 7 +/· 3 nM。 FXa產生之抑制作用(分析5): 於添加FX (50 nM)前先將經脂質化TF (10 pM)、FVIIa (100 pM)及抗-TF Mabs 或 FFR-rFVIIa (0-50 nM)於 BAS 緩衝 液(參見分析4)中培育60分鐘。再另培育10分鐘後藉由 添加1/2體積終止緩衝液(50 mM Hepes,ρΗ7·4,100 mM NaCl,20 mM EDTA)終止反應。添加受質 S2765 ( 0.6 mM ,Chromogenix)並於405 nm下連續測量吸光値10分鐘而 測定得所產生FXa之量。可計算得Mab抑制由FVIIa/脂質 化TF-所介導FX活化作用之IC5Q値。於此項分析中FFR-rFVIIa 之 IC5。値爲 51 +/- 26 pM。 生物傳感器分析(分析6): 將抗體藉由使抗-人類TF Mab之標準溶液通過一片具 有固定化對人類IgG抗體之晶片而於Biacore儀器上進行測 試。接著通過不同濃度之sTF,其存於含有150 mM NaCl ’ 10 mM CaCl2 及 0.0003%聚山梨酯 20 之 10 mM hepes pH 7.4 中。使用統合之Biacore估算軟體自該等感應器圖譜計算得 Kd値。 TF-依賴性凝塊形成分析(分析7): 此項分析係於一種ACL300硏究凝塊形成裝置(ILS實 46 200407427 驗室)上進行。將稀釋於50 mM咪唑,pH 7·4,100 mM . NaCL· 0.1% BSA 之抗-人類 TF Mabs 溶液與 25 mM CaCl2 以 2對5之比例混合並加至該凝塊形成裝置之樣本杯中。將得 自人類、大鼠、兔子、狒狒或豬之促凝血酶原激酶以該咪 唑緩衝液稀釋,而測定得置於試劑液槽2中之不含抗體樣 本與置於試劑液槽3中之人類、大鼠、兔子、狒狒或豬血 漿的凝塊形成時間爲大約30秒。於分析期間將抗體與 CaCl2混合物轉移至25微升促凝血酶原激酶試劑中,並於 添加60微升血漿前先預培養900秒。將最大凝塊形成時間 設定爲400秒。使用該促凝血酶原激酶稀釋液做爲相對於 不含所添加抗-TF Mabs或FFR-rFVIIa之對照組用以將凝塊 形成時間轉化成TF活性之標準曲線。於此項分析中FFR-rFVIIa 之 IC5。値爲 4.4 +/- 0.4 pM。 由Mabs對FVIIa/細胞表面TF所催化FX活化之抑制作用( 分析8): 使用單層表現人類TF之細胞,例如人類肺纖維母細胞 WI-38 (ATCC No. CCL-75)、人類膽囊癌細胞系 J82 (ATCC No. HTB-1)、人類角質化細胞系 CCD 1102KerTr (ATCC no. CRL-2310)、人類成膠質細胞癌細胞系U87、或人類乳癌細 胞系MDA-MB231做爲經FVIIa/TF所催化之FX活化作用的 TF來源。將24-、48-或96-槽平盤中之鋪滿細胞單層置於緩 衝液 A ( 10 mM Hepes,pH 7.45,150 mM NaC卜 4 mM KC1 與11 mM葡萄糖)中洗滌一次並於緩衝液B (緩衝液A補 充以1毫克/毫升BSA與5 mM Ca2+)中洗滌一次。將溶於 47 200407427 緩衝液B中之FVIIa (1 nM)、FX (135 nM)及不同濃度之Mab . (或FFR-rFVIIa)同時加至該等細胞。另於添加rFVIIa及 FX前先將細胞與抗-TF Mabs或FFR-rFVIIa進行預培養15 分鐘。使Fxa形成作用於37°C下進行15分鐘。從各槽取出 50-微升等分並加至50微升終止緩衝液(緩衝液A補充以 10 mM EDTA與1毫克/毫升BSA)中。藉由將50微升上述 混合物轉移至微量滴定平盤槽中並添加25微升呈色酵素 Chromozym X (終濃度0.6 mM)至各槽而測定得所產生之 FXa總量。於405 nm下連續測量吸光値並使用FXa標準曲 線將原始呈色速率轉化成FXa濃度。於此項分析中FFR-rFVIIa 之 IC5〇 値爲 1.5 nM 。 由Mabs對125rFVIIa與細胞表面TF結合之抑制作用(分析 9): 結合硏究係使用表現人類TF之細胞,例如人類肺纖維 母細胞WI-38 (ATCC No. CCL-75)、人類膽囊癌細胞系J82 (ATCC No. HTB-1)、人類角質化細胞系 CCD 1102KerTr (ATCC no. CRL-2310)、人類成膠質細胞癌細胞系U87、或 人類乳癌細胞系MDA-MB231進行。將24-槽組織培養平盤 中之鋪滿單層以緩衝液A (參見分析8)及以緩衝液B (參 見分析8)洗滌一次。將單層與100微升冰緩衝液B進行預 培養2分鐘。將不同濃度之Mab (或FFR-rFVIIa)及經放 射標定FVIIa (0.5 nM mI-FVIIa)同時加至該等細胞(最終體 積達200微升)。將平盤於4°C下培育2小時。於培育結束 後將未結合之物質去除,將細胞以冰-冷卻之緩衝液B洗滌 48 200407427 四次並以300微升溶解緩衝液(200 mM Na〇H,1% SDS及-10 mM EDTA)溶解。於加瑪計讀機(Cobra,帕卡得儀器公 司)中測量放射活性。使用GraFit4(艾瑞卡斯軟體股份有 限公司,英國)分析結合數據及進行曲線配合。於此項分 析中 FFR-rFVIIa 之 IC5。値爲 4 nM。 由Mab對FVIIa/TF所誘發p44/42 MAPK活化之抑制作用( 分析10): 藉由從西方轉漬分析定量偵測化學發光(富士軟片 LAS-1000 )而測定得經磷酸化p44/42 MAPK與/或Akt,及/ 或P90RSK之總量。於進行實驗前先將表現人類TF之細胞 ,例如CCD 1102KerTr、NHEK P166、人類成膠質細胞癌細 胞系U87、或人類乳癌細胞系MDA-MB231培養於含有〇-0.1% FCS之培養基中24或48小時以使細胞靜止活動。於 實驗當天該等細胞必須達70-80%鋪滿。該項實驗係藉由將 細胞與過量Mab或FFR-rFVIIa於不含血淸之培養基中於37 t下進行前培養30分鐘,然後將10-100 nM FVIIa加入並 培育10分鐘。將細胞置於冰-冷卻之PBS中洗滌2次再置 於溶解緩衝液(20 mM Tris,0.1% Triton X-100,1 mM EDTA,1 mM EGTA,50 mM氟化鈉,10 mM β-甘油磷酸鈉 ,5 mM 焦磷酸鈉,150 mM NaCl,pH 7.5 含有 0.1 mM 4-(2-胺乙基)苯磺醯氟(AEBSF)及1 mM苯甲脒。於使用前添加: 1 mM原釩酸鈉,5微克/毫升亮抑蛋白酶肽,10微克/毫升 抑蛋白酶肽)溶解。將胞溶物與SDS-樣本緩衝液混合並加 樣至SDS-聚丙烯醯胺凝膠上。各片凝膠加樣一列標準生物 49 200407427 素化標記物。將於SDS-聚丙烯醯胺凝膠上分離之蛋白質藉. 由電轉漬法轉移至硝基纖維素膜,並以磷酸基特異性抗體 顯現激酶P44/42 MAPK、Akt及90RSK,並經富士軟片 LAS-1000定量化學發光量。 抗原表位圖譜分析(分析11): 可溶性TF (sTF)變體之製備 利用反向PCR ( QuikChange,史塔金公司,拉裘拉’It should be understood that the specific IC5Q 21 200407427 as described in TF-induced clot analysis refers to when normal human plasma is used. In a specific aspect of the invention, the method for producing a human antibody against human TF therein comprises immunizing a mammal with human TF and isolating the antibody produced by the immunized mammal. In a preferred embodiment, the mammal is a mouse. It is understood that the immunized lactating animals or mice are capable of producing human antibodies. In another aspect, the present invention relates to a method for preparing a human antibody, the method comprising: a) immunizing a mouse with human TF, and b) isolating an antibody-producing cell from the immunized mouse and preparing a secretable human Immortalized cells of antibodies, c) Isolate the medium containing the produced antibodies from immortalized cells, d) test the antibodies in an indirect TF ELISA assay containing TF in solution, and screen for antibodies that can bind to human TF in solution Human antibodies, e) test antibodies in FVIIa competition analysis and screen for human antibodies that can compete with FVIIa, f) test antibodies in FVIIa / TF amidation analysis and screen for IC5.値 Less than 100 nM (in an analysis performed at a concentration of FVIIa of 10 nM), for example, less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM, inhibiting FVIIa amide induced by TF Degradation activity of human antibodies, g) Test antibodies in FXa yield analysis and screen for IC5 () 値 less than 100 nM (in an analysis with FVIIa concentration of 0.1 nM), such as less than 10 nM, compared with Preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM, to inhibit human antibodies produced by FXa, 22 200407427 h) test the antibodies in the TF-induced clot analysis and screen for them- In the analysis, IC5Q 値 is less than 1 nM, such as less than 500 pM, preferably less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, and more preferably less than 5 pM, human antibody that inhibits clot formation, i) screen and grow the immortalized cells that can produce the antibodies screened in step dh in a suitable medium, j) single from the medium of the screened immortalized cells Isolate the screened antibodies. It should be understood that the specific IC5 (), described in TF-induced clot analysis, refers to when normal human plasma is used. In another aspect, the present invention relates to a method for preparing a human antibody, the method comprising: a) immunizing a mouse with human TF, and b) isolating an antibody-producing cell from the immunized mouse and preparing a secretable human Immortalized cells of antibodies, c) Isolate the medium containing the produced antibodies from immortalized cells, d) test the antibodies in an indirect TF ELISA assay containing TF in solution, and screen for antibodies that can bind to human TF in solution Human antibodies, e) test antibodies in FVIIa competition analysis and screen for human antibodies that can compete with FVIIa, f) test antibodies in FVIIa / TF scan analysis and screen for antibodies with IC5Q lower than FFR-rFVIIa IC5Q 値 + 100 nM (10 nM FVIIa is used in this analysis), for example, IC5G 値 + 40 nM lower than FFR-rFVIIa, preferably lower than IC5Q 値 + 20 nM, preferably lower than 23 for FFR-rFVIIa 200407427 IC5 of FFR-rFVIIa.値 + 10 nM, more preferably lower than the IC50 of FFR-rFVIIa 人类, a human antibody that inhibits the FVIIa amine degrading activity induced by TF, g) test the antibody in the FXa production analysis and screen out that its IC50 is lower than IC5Q 値 + 100 nM of FFR_rFVIIa (0.1 nM FVIIa is used in this analysis), for example, lower than IC5Q of FFR-rFVIIa ◦ + 10 nM, preferably lower than IC5Q of FFR-rFVIIa 値 + 5 nM, preferably lower IC5 in FFR-rFVIIa.値 + 1 nM, better than IC5 of FFR-rFVIIa.値 + 0.1 nM, better than IC5Q of FFR-rFVIIa 値, human antibodies that inhibit FXa production h) test antibodies in TF-induced clot analysis and screen for IC5G lower in this analysis IC5Q 値 + 1 nM 'at FFR-rFVIIa, such as IC5G 値 + 500 pM lower than FFR-rFVIIa, preferably IC5G 低于 + 200 pM lower than FFR-rFVIIa, preferably lower than IC50 F + of FFR-rFVIIa 100 pM, for example, IC5G 値 +50 pM lower than FFR-rFVIIa, preferably IC5Q 値 +10 pM lower than FFR-rFVIIa, more preferably IC5 () 値 + 5 pM lower than FFR-rFVIIa, more preferably lower than FFR-rFVIIa's IC5G 抑制, a human antibody that inhibits clot formation, i) Screen and grow the immortalized cells that can produce the antibodies screened in steps d_h in a suitable medium, J) Infinite proliferation from the screened The culture medium of the cells is isolated from the antibodies selected. It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. The term "antibody-producing cell" is used herein to mean any cell capable of producing an antibody. These include fusion tumors, transfected cell lines, and relatively short-lived (or dying) spleen or lymphocytes obtained from mammalian animals that have been injected with the antigen. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) immunizing mice with human TF, b) isolating antibody-producing cells from the immunized mice and preparing immortalized cells capable of secreting human antibodies, c) separating the medium containing the produced antibodies from immortalized cells, d) Testing antibodies in an indirect TF ELISA assay containing TF in solution and screening for human antibodies that can bind to human TF in solution, e) Testing antibodies in FVIIa competition analysis and screening for competitive binding to FVIIa F) human antibodies, f) test antibodies in FVIIa / TF amidolysis analysis and screen for IC5Q 値 below 100 nM (in an analysis performed at a FVIIa concentration of 10 nM), such as below 40 nM, compared to Human antibodies that inhibit FVIIa amidolytic activity induced by TF, preferably less than 20 nM, more preferably less than 10 nM, g) test antibodies in FXa yield analysis and screen for IC5 () 値 less than 100 nM ( In an analysis with FVIIa concentration of 0.1 nM), for example, less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM, human antibodies that inhibit FXa production, h) Test the antibody in the TF-induced clot analysis and screen out. In this analysis, IC50 is below 1 nM, for example below 500 pM, preferably 25 200407427 at 200 PM, preferably below 200 PM. 100 PM, preferably less than 50 pM, preferably less than 10 PM, more preferably less than 5 PM, human antibodies that inhibit clot formation, i) screen and grow in a suitable medium to produce the product that can be produced in step dh Immortalized cells of the screened antibody, J) Isolate the screened antibody from the medium of the screened immortalized cell. It should be understood that the specific IC5 () as described in TF-induced clot analysis refers to when normal human plasma is used. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) immunizing mice with human TF, b) isolating antibody-producing cells from the immunized mice and preparing immortalized cells capable of secreting human antibodies, c) separating the medium containing the produced antibodies from immortalized cells, d) Testing antibodies in an indirect TF ELISA assay containing TF in solution and screening for human antibodies that can bind to human TF in solution, e) Testing antibodies in FVIIa competition analysis and screening for competitive binding to FVIIa Human antibody, f) test the antibody in FVIIa / TF amidine decomposition analysis and screen out the IC5G which is lower than FFR-rFVIIa.値 + 100 nM (10 nM FVIIa used in this analysis), such as IC5Q 低于 + 40 nM lower than FFR-rFVIIa, preferably IC5G 値 + 20 nM lower than FFR-rFVIIa, preferably lower than FFR-rFVIIa IC5Q 値 + 10 nM, better than IC50 26 of FFR-rFVIIa 値, a human antibody that inhibits TF-induced FVIIa amine degradation activity of human antibodies' g) Test antibodies in FXa yield analysis and screen for 1C5 〇 値 IC5Q below FFR-rFVIIa + 100 nM (0.1 nM FVIIa used in this analysis), for example, IC5Q below FFR-rFVIIa 値 + 10 nM ′, preferably lower than IC5G of FFR-rFVIIa値 + 5 nM, preferably lower than ICFR0r of FFR-rFVIIa + 1 nM, more preferably lower than IC5Q of FFR-rFVIIa 値 + 〇 · 1 nM 'more preferably lower than IC5Qr of FFR-rFVIIa, inhibiting FXa production Human antibodies h) Test the antibodies in a TF-induced clot assay and screen them for an IC5Q of less than FFR-rFVIIa 1 in this assay <: 5 () 値 + 1 nM 'for example, IC5G 値 +500 pM lower than FFR-rFVIIa, preferably IC5G 値 +200 pM lower than FFR-rFVIIa, preferably lower than IC50 値 + of FFR-rFVIIa 100 pM, for example, lower than IC5Q 50 + 50 pM of FFR-rFVIIa, preferably lower than IC5 of FFR-rFVIIa.値 + 10 pM, better than IC5Q of FFR-rFVIIa 値 + 5 pM, better than IC5G of FFR_i * FVIIa 値, human antibodies that inhibit clot formation, i) screen and incubate in a suitable medium The immortalized cells that can produce the antibodies screened in step dh can be produced. J) The screened antibodies are isolated from the medium that has been screened for immortalized cells. It should be understood that the specific IC50 described in the TF-induced clot analysis refers to when normal human plasma is used. In a specific aspect of the invention, the immortal proliferating cell fusion tumor cell. 27 200407427 In another aspect, the present invention relates to a cell that produces a human antibody that immunologically reacts with an epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF. In a specific aspect of the invention, the cells are isolated lymphocytes. In another specific aspect of the present invention, the cell line is isolated from a mouse. In another specific aspect of the present invention, the cell is a fusion tumor cell. In a specific aspect of the invention, the fusion tumor cell line obtains an antibody-producing fusion tumor cell by fusing antibody-producing lymphocytes with immortalized cells. In another specific aspect of the invention, the isolated human antibody inhibits binding of human clotting factor Vila to human TF. In another specific aspect of the invention, the isolated human antibody is a monoclonal antibody. The term "single antibody" as used herein means a homologous immunoglobulin group, i.e., individual molecules of the antibody group are identical except for naturally occurring mutations. Antibodies are normally produced by lymphocytes derived from B cells in the bone marrow. Immunoglobulins with a single amino acid sequence are produced from lymphocytes derived from the same pure line. Lymphocytes cannot be cultured directly for long periods of time to make specific antibodies of the actual quality. However, Kohler et al. (1975, Nature, 256: 495) demonstrated that the somatic cell fusion process (especially the fusion between lymphocytes and myeloma cells) can produce a special product that can grow in culture and make a so-called "single antibody" Antibody for fusion tumor cells. Myeloma cells are lymphocyte tumor cells that often produce antibodies (depending on the cell strain), but are known as "non-productive" strains. In another specific aspect of the invention, the isolated human antibody is a recombinant antibody. The term "recombinant antibody" is used herein to include all human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells (further (Described in section Π below), antibodies isolated from recombinant, combinatorial antibody libraries (further described in section ΙΙΙ below), animals (eg, mice) isolated from genes transfected into human immunoglobulin genes ) Antibodies (see, for example, Taylor, L. D. et al. (1992) Nucleic Acid Research 20: 6287-6295) or any other human antibody prepared, expressed, produced, or isolated by any method that includes splicing the human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some specific aspects, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using animals transgenic with human Ig sequences), and Therefore, the amino acid sequences of the VH and VL regions of these recombinant antibodies are derived from and related to the human germline VH and VL sequences, but all components of the human antibody germline may not naturally exist in vivo. In the sequence. In another embodiment of the present invention, the isolated human antibody is a Fab fragment. In another specific aspect of the present invention, the isolated human antibody 29 200407427 is a F (ab) 2 fragment. In another specific aspect of the invention, the isolated human antibody is a F (abf) 2 fragment. In another specific aspect of the invention, the isolated human antibody is a single-chain Fv fragment. In another specific aspect of the present invention, the isolated human antibody has a Kd 値 with human TF in the range of 10 · 15-1 (T8 M. It should be understood that the human antibody and human TF Kd 値 is determined in an assay in which the human antibody is immobilized (see Analysis 6). In another specific aspect of the invention, the isolated human antibody has a Kd that is comparable to human TF値 is in the range of 10 · 15-10 · 1 () M. In another specific aspect of the present invention, the isolated human antibody has a Kd ratio of less than 10 · 8 Μ with human TF. In another specific aspect of the present invention, the isolated human antibody has a Kd 値 ratio with human TF of less than 1 (T9 M. In another specific aspect of the present invention, the isolated The isolated human antibody has a Kd ratio that is lower than that of human TF. In another specific aspect of the present invention, the isolated human antibody has a relationship with human TF that is lower than 10_π Μ. In another specific aspect of the present invention, the isolated human antibody has a Kd ratio of less than 1 (T12 Μ) with human TF. In the present invention In another specific aspect, the isolated human antibody has a ratio of less than 1 (T13 M to human TF). In another specific aspect of the invention, the isolated human antibody has The Kd ratio with human TF is lower than HT14 M. In another specific aspect of the present invention, the isolated human antibody has a heart ratio with human TF below 1 (15M. 30 200407427 in the present invention In another specific aspect, the method for preparing a human antibody includes testing the antibody in a TF-induced clot analysis, and screening can be performed in this analysis with IC5 () 値 below 1 nM inhibition A clot-forming human antibody. In another embodiment, the IC5Q is below 500 pM. In another embodiment, the IC50 is below 200 pM. In another embodiment, In the sample, the IC50 is below 100 pM. In another embodiment, the IC5Q is below 50 pM. In another embodiment, the IC50 is below 10 PM. In another specific aspect, the IC5Q is less than 5 pM 0. In another specific aspect of the present invention, the IC5Q is used for preparing human antibodies. The method includes testing antibodies in a TF-induced clot analysis and screening for IC5Q 値 + 1 nM with IC5Q 値 lower than FFR-rFVIIa in this analysis, such as IC5G 値 + 500 lower than FFR-rFVIIa pM, preferably below IC5Q 値 + 200 pM of FFR-rFVIIa, preferably below IC5Q 値 + 100 pM of FFR-rFVIIa, such as IC5G 値 + 50 pM below FFR-rFVIIa, preferably below FFR- rFVIIa's IC5G + 10 pM is better than FFR-rFVIIa's IC5QF + 5 pM, more preferably lower than FFR-rFVIIa's IC5G 値, which inhibits the formation of clot-forming human antibodies. It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. In another embodiment of the present invention, the method for preparing human antibodies includes testing antibodies in FXa yield analysis and screening for IC50 値 less than 100 nM (in one case, the FVIIa concentration is 0 · 1 nM In the analysis performed) human antibodies that inhibit FXa production. In another specific aspect, the 31 200407427 IC5Q series is below 10 nM. In another specific aspect, the IC5Q series is lower than 5 nM. In another specific aspect, the IC5. Actinide is below 1 nM. In another specific aspect, the IC5Q ratio is lower than 0 · 1 nM. In another specific aspect of the present invention, the method for preparing human antibodies includes testing the antibodies in FXa yield analysis and screening for IC5G 値 lower than FFR-rFVIIa IC50G + 100 nM (in this analysis Use 0. 1 nM FVIIa), for example, IC5G 値 + 10 nM lower than FFR-rFVIIa, preferably lower than IC5G 値 + 5 nM lower than FFR-rFVIIa, more preferably IC5Q 値 + 1 nM lower than FFR-rFVIIa, more preferably lower than IC5Q of FFR-rFVIIa 値 + 0. 1 nM, more preferably IC5G 値 than FFR-rFVIIa, inhibits human antibodies produced by FXa. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a FVIIa / TF amidolysis assay and screening the IC5Q to be less than 100 nM (at a concentration of FVIIa of 10 nM (Analyzed below) Human antibodies that inhibit FVIIa amidinolytic activity induced by TF. In another specific aspect, the IC50 is below 40 nM. In another specific aspect, the IC5Q ratio is below 20 nM. In another specific aspect, the IC50 is less than 10 nM. In another specific aspect, the IC5Q is less than 5 nM. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a FVIIa / TF amidine decomposition assay and screening for IC5Q (lower than FFR-rFVIIa) IC5Q (+100 nM). (10 nM FVIIa is used in this analysis), for example, IC50 値 +40 nM lower than FFR-rFVIIa, preferably IC5G 値 +20 nM lower than FFR-rFVIIa, preferably 32 200407427 lower than FFR-rFVIIa The IC5Q 値 +10 nM is better than the IC50 値 of the FFR-rFVIIa, which inhibits the human antibody of the FVIIa amidolytic activity induced by TF. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a FVIIa competition assay and screening for a human antibody that can compete with FVIIa for binding. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a TF ELISA assay and screening for a human antibody that can bind to human TF. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a direct TF ELISA analysis including immobilized TF and screening for a human antibody that can bind to the immobilized human TF. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in an indirect TF ELISA analysis including TF in solution and screening for a human antibody that can bind to human TF in solution. In another specific aspect of the present invention, the method for preparing a human antibody includes testing the antibody in an FXa production analysis using TF-expressing cells, and screening the IC5 () 値 below 500 nM ( Human antibodies that inhibit FXa production in an analysis performed at a FVIIa concentration of 1 nM. In another specific aspect, the IC5 () is less than 100 nM. In another specific aspect, the IC5 () ratio is less than 50 nM. In another specific aspect, the IC5Q ratio is less than 10 nM. In another specific aspect, the IC50 is less than 5 nM. In another specific aspect of the present invention, the method for preparing human antibodies 33 200407427 includes testing antibodies in an FXa production analysis performed by TF-expressing cells, and screening for IC5Q that is IC5G 値 + 500 nM (1 nM FVIIa is used in this analysis), for example, IC5Q 低于 + 100 nM lower than FFR-rFVIIa, preferably lower than IC5〇 値 + 50 nM, lower than FFR-rFVIIa, preferably lower than FFR -rFVIIa's IC5Q 値 + 10 nM, better than FFR-rFVIIa's IC5QF + 5 nM, more preferably lower than FFR-rFVIIa's IC5Q 値, inhibit human antibodies produced by FXa. The term "TF-expressing cell" means any mammalian cell that can express human TF. In another specific aspect of the present invention, the method for preparing a human antibody includes testing the antibody in a complete cell TF binding assay, and screening for those that can compete with FVIIa for binding to human TF expressed on the surface of a complete cell Human antibodies. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a biosensor analysis, and screening for a human antibody having a Kd 値 of less than 100 nM that binds to human TF. In another specific aspect, the 1 line of binding to human TF is less than 10 nM. In another specific aspect, the Kd 値 system bound to human TF is less than 5 nM. In another specific aspect, the Kd 値 of human TF binding is below 1 nM. In another specific aspect, the Kd 値 associated with human TF is lower than 0.5 nM. In another specific aspect, the Kd 値 that binds to human TF is lower than M. In another specific aspect, the Kd 値 of the human TF binding is below 10.11M. In another specific aspect, the Kd 値 binding to human TF is lower than 10 · 12M. In another specific aspect, the Kd ratio of the 34 200407427 combined with human TF is lower than 1 (T13 M. In another specific aspect, the 1 ratio of the combined human TF is lower than 10 · 14 M. In another specific aspect, the Kd 値 binding to human TF is lower than M. In another specific aspect of the present invention, the method for preparing a human antibody includes MAPK signal analysis We tested antibodies and screened human antibodies that could inhibit FVIIa-induced activation of MAPK signals. In a specific aspect, the anti-system inhibited the activation of MAPK signals induced by FVIIa- by 98%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 90%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 70%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 50%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 30%. The term "MAPK signal" means that it mediates mitogenicity Activated protein kinase (MAPK) or its homologous protein cascades in the intracellular event of activation in response to various extracellular stimuli. Three different MAP kinases have been identified in mammalian cells: 1) Extracellular regulatory kinases (Erk 1/2 or P44 / 42), 2) c-Jun N-terminal kinase (JNK) and 3) p38 kinase. The Erkl / 2 pathway involves the phosphorylation of Erk 1 (p44) and / or Erk 2 (p42). Activated MAP kinases such as p44 / 42 MAPK can be translocated into the nucleus, where they can phosphorylate and activate transcription factors including (Elk 1) and signal transductors and activators (Stat) of transcription. Erkl / 2 can also phosphorylate the kinase P90RSK in the cytoplasm or in the nucleus, and then p90RSK can reactivate a number of 35 200407427 transcription factors. The term "protein kinase" is intended to include enzymes capable of phosphorylating serine and / or threonine and / or tyrosine present in peptides and / or proteins. The term "activation of MAPK signals induced by FVIIa-" means that FVII binds to TF in mammalian cells and thereby induces MAPK signals. In another specific aspect of the present invention, the method for preparing a human antibody includes testing the antibody in a gene expression analysis (Analysis 15), and screening to inhibit the induction by FVIIa- independently selected from the listed Fra_1, Human antibodies for the up-regulating effects of Id2 and Cyr61 genes. It will be appreciated that anti-TF antibodies that inhibit TF activity can bind to different epitopes present in TF and may simultaneously inhibit the binding of FVIIa or FX or FXa to human TF. An object of the present invention is to screen antibodies that inhibit the binding of FVIIa to human TF and thereby inhibit the generation of intracellular signals induced by FVIIa. In another aspect of the present invention, the method for preparing human antibodies includes testing antibodies in a human cancer analysis (Analysis 13) and selecting human antibodies that can inhibit the growth or metastasis of human cancer. In another specific aspect, the isolated human antibody can inhibit the up-regulation of genes independently selected from the listed Fra_1, Id2 and Cyr61 § induced by FVIIa-. _ In another specific aspect of the present invention, the isolated human antibody does not inhibit the binding of FX or FXa to human TF. In another specific aspect of the present invention, the isolated human antibody can inhibit cells TF activity. 200407427 In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in an epitope mapping assay, and screening for a human antibody that can bind to a better antigenic epitope on TF. In a specific aspect of the invention, the preferred epitope comprises the residue Trp45. In a specific aspect of the invention, the preferred epitope comprises the residue Lys46. In a specific aspect of the invention, the preferred epitope comprises the residue Tyr49. In another embodiment of the present invention, the isolated human antibody can bind to an epitope located in the interface between TF and FVIIa. The residues determined by the X-ray structure (Beneng et al., 1996 Nature, 380: 41-46) that can perform the interaction between the FVIIa protease domain and TF are: Ser39, Gly43, Trp45, Ser47, Phe50, Arg74, Phe76, Tyr94, Pro92. This interface between TF and FVIIa is characterized by a complex interfacial region containing many intermolecular hydrogen bonds that can cause many intimate contacts between TF and FVII to obtain a high specificity during the FVIIa binding process. The invention also relates to high affinity human monoclonal antibodies to TF. The TF surface containing the contact interface with the FVIIa protease domain possesses a special topology that is easy to react and generate protein-protein interactions. Another type of protein-protein interactions forms a complex between an antibody and a protein ligand. Therefore, a monoclonal antibody against this antigenic epitope on human TF can provide high affinity Mabs. One aspect of the present invention is a high-affinity human monoclonal antibody capable of immunoreacting against the contact interface to the FVIIa protease domain. The human TF antibody of the present invention acts as an antagonist against TF-mediated coagulation induction 37 200407427, thereby inhibiting the binding of coagulation factor FVIIa to TF and thereby blocking thrombin production and subsequent fibrin deposition. Human TF antibodies are particularly useful for administration to humans for the treatment of various conditions involving intravascular coagulation. As such, human TF antibodies can be used to inhibit TF activity resulting in, for example, inhibition of blood coagulation, thrombosis or platelet deposition. Furthermore, the human TF antibody (acting to inhibit the intracellular function of TF, the signal function of TF) according to the present invention can be used in conditions such as sepsis, inflammation, atherosclerosis, restenosis, or cancer. Human TF antibodies are useful in a variety of diseases. It covers diseases or disorders related to thrombosis or coagulopathy, including inflammatory reactions and chronic thromboembolic diseases or disorders associated with fibrin formation, including vascular diseases such as deep venous thrombosis, arterial thrombosis, surgery Postthrombosis, coronary artery bypass graft (CABG), percutaneous coronary angioplasty (PTC A), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, atherosclerosis and restenosis after angioplasty , Acute and chronic indications such as inflammation, septic shock, sepsis, hypertension, adult respiratory distress (ARDS), systemic inflammatory response syndrome (SIRS), diffuse intravascular coagulation (DIC), pulmonary embolism, platelets Deposition, myocardial infarction, or arteriosclerotic vessels at risk of thrombosis, venous occlusive disease caused by peripheral blood progenitor cell (PBPC) transplantation, hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura ( TTP) and other diseases or conditions for the preventive treatment of mammals. Human TF antibodies can be used to prevent thrombotic thrombosis in patients identified to be at high risk, such as in those undergoing surgery or suffering from congestive heart failure. Human TF anti-38 200407427 is particularly useful for the treatment of intimal hyperplasia or regeneration caused by acute vascular trauma. narrow. Acute vascular trauma is injury that occurs quickly (that is, hours to months), rather than chronic vascular trauma (such as arterial atherosclerosis) that develops throughout life. Acute vascular trauma often results from surgical procedures such as vascular reconstruction using techniques such as angioplasty, endarterectomy, arterial atherosclerosis, and placement of vascular implants. Hyperplasia may occur as a delayed response to, for example, an implant placement or organ transplant response. Because human TF antibodies are more selective than heparin, they only bind to TF that has been exposed to the wound site, and because human TF antibodies do not destroy or inhibit other coagulation proteins, they are more effective and should be used to prevent deep vein thrombosis May be less likely to cause bleeding episodes than heparin. As shown in the following examples, the human TF antibody of the present invention can selectively bind to cell surface TF and limit its functional activity by inhibiting the binding of coagulation FVIIa and TF. Human TF antibodies that remain bound to TF can inhibit platelet accumulation at the site of vascular wounds by blocking thrombin production and subsequent fibrin deposition. Because human TF antibodies can block thrombin production and limit platelet deposition at the site of acute vascular trauma, human TF antibodies that maintain binding to TF and thereby inhibit FVIIa binding can be used to inhibit vascular restenosis. Compositions containing human TF antibodies are particularly useful in methods of treating patients when formulated into pharmaceutical compositions, where they can be administered to patients suffering from various disease states to treat coagulation-related conditions. Such human TF antibodies, which can be combined with TF to inhibit the binding of FVIIa and TF, have a longer plasma half-life and therefore have a longer anticoagulant activity time compared to other anticoagulants. In the medical instructions of the subject composition of the present invention, coagulants are generally used for treatment, such as deep venous thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), lung and kidney blood related to sepsis. Fibrin deposition, anti-phospholipid syndrome (APS), atherosclerosis and myocardial infarction. These compositions can be used to inhibit mechanical vascular trauma, such as by surgery, microsurgery, balloon angioplasty, endarterectomy, reductive atherosclerotic plaque, fixed mold placement, laser light treatment Or damage caused by rotational vibration, or secondary trauma to vascular implants, molds, bypass implants, or organ transplants. These compositions are therefore useful for inhibiting platelet deposition and related diseases. Thus, a method of inhibiting coagulation, vascular restenosis, or platelet deposition, for example, comprises administering to a patient a composition comprising a human TF antibody in an amount sufficient to effectively inhibit coagulation, vascular restenosis, or platelet deposition. These methods have also been found to be useful in the treatment of patients with acute coronary occlusion (such as acute myocardial infarction), which involves the administration of human TF antibodies in combination with histone plasminogen activator and can accelerate the thrombolytic effects induced by tPA . The human TF resistance system is administered before, at the same time or immediately after the administration of a thrombolytic agent (such as a tissue cytosinogen activator). According to the present invention, human monoclonal antibodies against human TF can be produced by immunizing transgenic mice (obtained from Medarex) carrying part of the human immune system instead of mouse system with human TF. Spleen cells obtained from these transgenic mice are used to make fusion tumors that secrete human monoclonal antibodies as described (see, for example, Wood et al., International Patent Application WO 91/00906, Kucherapie The et al., PCT Publication WO91 / 10741; Lombok et al., International Patent Application No. WO407 / 03918, 20040427, Kay et al., International Patent Application No. 92/03917, Lomberg, N. Et al. 1994 Nature 368: 856-859; Greene, L1. Et al. 1994 Natural Genetics 7: 13-21; Morrison, S. L. Et al. 1994, Proc. Natl. Acad. Sci USA 81: 6851-6855; Brugeman et al. 1993 Immunology 7: 33-40; Tyrone et al. 1993 PNAS 90: 3720-3724; Brugeman et al. 1991 European Journal of Immunology 21: 1323- 1326) o human monoclonal antibodies against human TF can also be made by phage display technology. Human antibody libraries can be constructed from immunized humans and displayed on the surface of filamentous phage. In many cases, high affinity human single-chain Fv (ScFv) and Fab antibody fragments have been isolated using panning techniques in which the antigen of interest is immobilized on a solid surface, that is, a microtiter plate or bead (Babes C. F. , III and Burton, D. R. Biotechnology Trends 1996, 14: 230-234; Et al., Human Antibodies 1997, 8: 155-68). Phage display systems with large primary libraries may also directly isolate human antibodies without immunization (Dihard H. L et al., Biochemical Journal 1999, 18218-18230). [Embodiment] The present invention is further illustrated by the following examples, however, they are not intended to limit the protection scope of the present invention. The substance (individually or in combination with each other) disclosed in the foregoing description and the following embodiments is a form for understanding the preferred form of the present invention. Example 1 Preparation of Human Mab with Immunospecificity to Human TF 41 200407427 Reagent Human TF can be Leo, L. V. Μ. , Thrombosis Research, 51: 373-384 1988, isolated from the human brain. Lipidized recombinant human TF (Dade, Innovin, Beckster) can also be used as a human thromboplastin reagent. Rat, rabbit, baboon and porcine prothrombin kinase were prepared from brain tissue. Add twice the volume at 45 ° C to 0. 9% NaCl was added to the brain tissue, and the tissue was homogenized with a human glass homogenizer. After incubating at 45 ° C for 30 minutes with occasional shaking, the samples were centrifuged at 2000 Xg for 20 minutes. Discard the pellet and store a small aliquot of the supernatant at -80 ° C until use. Relipidated TF can be obtained by reconstructing a recombinant human full-length TF (American Diagnostic # 4500) in a phospholipid carrier (PC / PS 75/25). Biotinylated human TF was prepared as follows: Biotin-NHS (η-succinyl-based biotin, Sigma-Delta-1759) was dissolved in DMF (dimethylformamide) to a concentration of 1. 7 mg / ml. Add 60 μl of this biotin-NHS solution to 1 mg / ml dissolved in 0. 1 M NaHC03 buffer in human TF and allowed to react at room temperature for 4 hours. The solution containing biotinylated TF was dialyzed against PBS-buffer overnight. The FVIIa used is such as Tim, L. Recombinant human FVIIa prepared as described in Biochem 27: 7785-7793, 1988. sTF: Substantially like Furigad, P. -Ο. Recombinant human soluble and expressed in E. coli as described in Protein Science 5, 153M540 (1996), et al. 42 200407427 S2288: Reconstituted in H2O with a concentration of 17. 24 mg / ml (Chromogenix) FX: Purified human plasma FX (HFX, Enzyme Research Laboratories Co., Ltd.) FXa: Purified human plasma FX is activated with Russell Snake Venom (HFXa, Enzyme Research Laboratories Co., Ltd.) Co., Ltd.) Chromogenic enzyme X: dissolved in H20, the concentration is 1. 25 mg / ml (Bringa Ingram) 125I-FVIIa was prepared by standard radiometric calibration procedures. FFR-rFVIIa: The active site of FVIIa is blocked with D-Phe-L-Phe-L-Arg-chloromethyl ketone. Such as Sorison B. B. Et al., Journal of Biochemistry 272: 1 1863-1 1868, 1997. Immune effect Human TF is emulsified in Freund's complete adjuvant. HuMab mice or their hybrid offspring (Medarex) were given a 40 microgram dose via subcutaneous injection. After 14 and 28 days (and a substantial interval of 14 days), mice were similarly injected with another 20 micrograms of TF emulsified in incomplete Freund's adjuvant. Blood was sampled 10 days after the last injection and tested for the presence of human TF-specific antibodies in the blood pupa by TF ELISA (Analysis 1 and 2). Fusion Mice tested positive for blood clots in Analysis 1-3 were injected intravenously with an additional 20 micrograms of human TF and sacrificed three days later. The spleen was removed aseptically and dispersed into a single cell suspension. Fox-myeloma cells were grown in CD fusion tumor medium (Gibco 43 200407427 11279-023). In this fusion experiment, spleen cells and myeloma cells (P3x63 Ag8. 653, ATCC CRL-1580) and Sp2 / 0 (ATCC CRL-1581). Cells were seeded in microplates and incubated at 37 ° C. Then change the medium three times in two weeks. One hundred microliters of supernatant was removed from each tank of fused tumor cell culture and tested for the presence of TF-specific antibodies in a TF ELISA (Analysis 1 and 2). Example 2: Screening of various analytical methods used to screen for the presence of specific selected antibodies in blood slugs and culture supernatants are described below: ^ Direct TF ELISA analysis (Analysis 1): The g-immunoplate was coated with human sTF in 1 ug / ml in PBS. The plate was blocked with a blocking agent (TBS containing 5 mM CaCl2 and 2% BSA) and TBS + 0. Wash with 05% Tween 20 and add mash from the fusion tumor cells. After incubating at room temperature for 1 hour, the plate was washed and anti-human IgG labeled with horseradish peroxidase (HRPO) was added. After another one hour of incubation, the plate was washed and colorized with TMB-substrate (Kem-EN-Tec) according to the manufacturer's operating manual. The absorbance was measured on an ELISA-reader at 450 nm. Indirect TF ELISA analysis (Analysis 2): Nock-immunoplates were coated with 0. Goat anti-human IgG (Southern Biotechnology Association, Cat- # 2040-1) stored in PBS at 5 µg / ml and incubated overnight at 4 ° C 44 200407427. The plate was blocked with blocking buffer (TBS with 5 mM CaCl2 & 2% BSA) and blocked with TBS + 5 mM CaCl2 + 0. Wash in 05% Tween 20. The mash obtained from the fusion tumor cells was added, and the plates were incubated at room temperature for 1 hour. After another washing, biotinylated human sTF was added at a concentration of 1 μg / ml and incubated for 1 hour. After washing, the streptavidin-HRPO solution was added and incubated for one hour. The plate was colored as described in Analysis 1 with TMB-substance. FVIIa competition analysis (Analysis 3): The NOCK-immunoplate was incubated with human STF (5 μg / mL in PBS) overnight. The plate was washed and blocked in TBS buffer containing 5 mM 0:30 and 2% BSA. Anti-human TF Mabs were added and the plates were incubated for 2 hours. After the plate was washed, biotinylated human FVIIa was added (1 μg / ml in TBS buffer containing 5 mM CaCl2 and 2% BSA) and the plate was incubated for 1 hour. The plate was washed again and then HRPO-calibrated streptavidin was added and incubated for 45 minutes. After washing the flat plate again, the color was developed using TMB-substrate (Kem-EN-Tec) according to the manufacturer's operation manual. Inhibitory effect of FVIIa / sTF amidolytic activity (Analysis 4): Inhibition of amidolytic activity catalyzed by FVIIa-TF caused by anti-human TF Mabs uses soluble human TF (10 nM), recombinant human FVIIa (10 nM) and increasing concentrations of Mabs (0. 0122-50 nM). Various concentrations of anti-human TF Mabs or FFR-rFVIIa with 10 nM TF and 10 nM FVIIa in BSA buffer (50 mM Hepes, pH 7. 4, 100 mM NaCl, 5 mM 0 & (: 12 and 1 mg / ml BSA) at room temperature 45 200407427 before incubation for 60 minutes, and then the substrate S2288 (1. 2 mM, Chromogenix). The color development process was continuously measured at 405 nm for 30 minutes. The amidolytic activity is expressed in mOD / min. IC5Q 値, which inhibits the inhibition of FVIia / TF amidolytic activity by Mabs. The IC50 F of FFR-rFVIIa in this analysis is 7 + / · 3 nM. Inhibition by FXa (Analysis 5): Before adding FX (50 nM), lipidated TF (10 pM), FVIIa (100 pM), and anti-TF Mabs or FFR-rFVIIa (0-50 nM) were added to Incubate in BAS buffer (see Analysis 4) for 60 minutes. After another 10 minutes of incubation, the reaction was stopped by adding 1/2 volume of stop buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 20 mM EDTA). Add substrate S2765 (0. 6 mM, Chromogenix) and continuous measurement of absorbance at 405 nm for 10 minutes to determine the amount of FXa produced. Mab can be calculated to inhibit IC5Q (R) that is activated by FVIIa / lipidated TF-. IC5 of FFR-rFVIIa in this analysis.値 is 51 +/- 26 pM. Biosensor analysis (Analysis 6): The antibody was tested on a Biacore instrument by passing a standard solution of anti-human TF Mab through a wafer with immobilized human IgG antibodies. Then pass different concentrations of sTF, which are stored in 150 mM NaCl ’10 mM CaCl2 and 0. 0003% Polysorbate 20 of 10 mM hepes pH 7. 4 in. Kd 値 was calculated from the sensor spectra using integrated Biacore estimation software. TF-dependent clot formation analysis (Analysis 7): This analysis was performed on an ACL300 research clot formation device (ILS Practice 46 200407427). Dilute to 50 mM imidazole, pH 7.4, 100 mM. NaCL. 0. 1% BSA anti-human TF Mabs solution was mixed with 25 mM CaCl2 in a ratio of 2 to 5 and added to the sample cup of the clot forming device. Thromboprokinase from humans, rats, rabbits, baboons, or pigs was diluted with the imidazole buffer, and the antibody-free samples placed in the reagent tank 2 and The clot formation time of human, rat, rabbit, baboon or pig plasma is approximately 30 seconds. During the analysis, the antibody and CaCl2 mixture was transferred to 25 microliters of prothrombin kinase reagent and precultured for 900 seconds before adding 60 microliters of plasma. The maximum clot formation time was set to 400 seconds. This thromboplastin dilution was used as a standard curve to convert clot formation time to TF activity relative to a control group without added anti-TF Mabs or FFR-rFVIIa. IC5 of FFR-rFVIIa in this analysis.値 is 4. 4 +/- 0. 4 pM. Inhibition of FX activation catalyzed by FVIIa / cell surface TF by Mabs (Analysis 8): Cells expressing human TF using a single layer, such as human lung fibroblasts WI-38 (ATCC No. CCL-75), human gallbladder cancer cell line J82 (ATCC No. HTB-1), human keratinocyte cell line CCD 1102KerTr (ATCC no. CRL-2310), human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231 serve as TF sources for FX activation catalyzed by FVIIa / TF. Place monolayers of cell-filled monolayers in 24-, 48-, or 96-well plates in buffer A (10 mM Hepes, pH 7. 45, 150 mM NaC, 4 mM KC1 and 11 mM glucose) and once in buffer B (buffer A supplemented with 1 mg / ml BSA and 5 mM Ca2 +). FVIIa (1 nM), FX (135 nM) and Mab at different concentrations were dissolved in 47 200407427 buffer B. (Or FFR-rFVIIa) to the cells at the same time. Pre-incubate cells with anti-TF Mabs or FFR-rFVIIa for 15 minutes before adding rFVIIa and FX. Fxa formation was allowed to proceed at 37 ° C for 15 minutes. Remove 50-microliter aliquots from each well and add to 50 microliters of stop buffer (Buffer A supplemented with 10 mM EDTA and 1 mg / ml BSA). By transferring 50 μl of the above mixture to a microtiter pan and adding 25 μl of the chromogenic enzyme Chromozym X (final concentration 0. 6 mM) to each well to determine the total amount of FXa produced. The absorbance was measured continuously at 405 nm and the original color rendering rate was converted to FXa concentration using the FXa standard curve. In this analysis, the IC50 of FFR-rFVIIa is 1. 5 nM. Inhibition of 125rFVIIa binding to cell surface TF by Mabs (Analysis 9): Combining research uses cells expressing human TF, such as human lung fibroblasts WI-38 (ATCC No. CCL-75), human gallbladder cancer cell line J82 (ATCC No. HTB-1), human keratinocyte cell line CCD 1102KerTr (ATCC no. CRL-2310), human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231. Monolayers in 24-well tissue culture plates were washed once with buffer A (see analysis 8) and with buffer B (see analysis 8). The monolayer was pre-incubated with 100 µl of ice buffer B for 2 minutes. Different concentrations of Mab (or FFR-rFVIIa) and FVIIa (0. 5 nM mI-FVIIa) were simultaneously added to the cells (final volume reached 200 μl). The flat plate was incubated at 4 ° C for 2 hours. After the incubation is completed, the unbound material is removed, and the cells are washed four times with ice-cooled buffer B 48 200407427 and 300 microliters of lysis buffer (200 mM NaOH, 1% SDS and -10 mM EDTA). Dissolve. Radioactivity was measured in a Garma meter (Cobra, Packard Instruments). GraFit4 (Ericas Software Limited, UK) was used to analyze the combined data and perform curve fit. IC5 of FFR-rFVIIa in this analysis.値 is 4 nM. Inhibition of p44 / 42 MAPK activation induced by FVIIa / TF by Mab (Analysis 10): Phosphorylated p44 / 42 MAPK was determined by quantitative detection of chemiluminescence (Fuji film LAS-1000) from Western blot analysis And / or Akt, and / or P90RSK. Prior to the experiment, cells expressing human TF, such as CCD 1102KerTr, NHEK P166, human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231 were cultured at 0-0. 1% FCS medium for 24 or 48 hours to allow cells to rest. The cells must be 70-80% confluent on the day of the experiment. This experiment was performed by pre-incubating the cells with excess Mab or FFR-rFVIIa in a blood-free medium at 37 t for 30 minutes, and then adding 10-100 nM FVIIa and incubating for 10 minutes. The cells were washed twice in ice-cooled PBS and then placed in lysis buffer (20 mM Tris, 0.1 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM β-glyceryl phosphate, 5 mM sodium pyrophosphate, 150 mM NaCl, pH 7. 5 contains 0. 1 mM 4- (2-aminoethyl) benzenesulfonium fluoride (AEBSF) and 1 mM benzamidine. Add before use: 1 mM sodium orthovanadate, 5 μg / ml Leprotinin, 10 μg / ml Aprotinin) to dissolve. The lysate was mixed with the SDS-sample buffer and loaded onto an SDS-polyacrylamide gel. A series of standard biological samples were loaded on each gel 49 200407427. Proteins to be separated on SDS-polyacrylamide gels. It was transferred to the nitrocellulose membrane by electroporation, and the kinases P44 / 42 MAPK, Akt, and 90RSK were visualized with phosphate-specific antibodies, and the chemiluminescence was quantified by Fujifilm LAS-1000. Epitope Mapping Analysis (Analysis 11): Preparation of Soluble TF (sTF) Variants Using Reverse PCR (QuikChange, Starkins, La Joule ’
CA,美國)使用野生型質體(福瑞加德等人,蛋白科學5 ,1531-1540,1996 )做爲模板構築得sTF變體(122C、 W45C、K46C、Y94C、F140C、W158C、K201C)。如前述 (福瑞加德等人,蛋白科學5,1531-1540,1996 )並稍作 修飾使野生型與該等變體於大腸桿菌中表現並純化。以X-壓碎(Biox,瑞典)技術於10 mM Tris-HCl緩衝液,PH 7.5 中進行細胞溶解,然後將其再懸浮於相同緩衝液中並添加1 毫克DNAse。將溶液於11000 Xg下於4°C離心20分鐘,並 將內涵體置於75毫升6 M GnHCl,0.5 M NaC卜20 mMCA, USA) Constructed sTF variants (122C, W45C, K46C, Y94C, F140C, W158C, K201C) using wild-type plastids (Frigard et al., Protein Science 5,1531-1540, 1996) as templates . As previously described (Frigard et al., Protein Science 5,1531-1540, 1996) and with minor modifications, wild-type and these variants were expressed and purified in E. coli. Cell lysis was performed using X-crush (Biox, Sweden) technology in 10 mM Tris-HCl buffer, pH 7.5, then resuspended in the same buffer and 1 mg of DNAse was added. Centrifuge the solution at 11000 Xg at 4 ° C for 20 minutes, and place the endosomes in 75 ml of 6 M GnHCl, 0.5 M NaC and 20 mM
Tris-HCl,pH 8.0中變性。於室溫下培育1小時後,藉由逐 滴將經變性之蛋白質稀釋於1升含有50 mM Tris-HCl,(U5 M NaCl,pH 8.0之溶液中並伴隨溫和攪拌約2小時而完成 再折疊。如福瑞加德等人(1996)所述,使用Q-瓊脂糖Fast Flow (法瑪西亞,優薩拉,瑞典)及FVIIa親和性層析術進 行純化。以SDS-PAGE檢視蛋白質之均質度。於^^^下測 量並使用所計算得消光係數爲37440 Minr1 (吉爾與凡希 柏,1989 )測定其濃度。 200407427 將MaxiSorp平盤(Nunc-Immuno)塗覆以溶於TBS之 野生型sTF及該等變體(1〇微克/毫升)並以封阻緩衝液( TBS含0.1%吐溫20及0.5% BSA)進行封阻。將平盤以洗 滌緩衝液(TBS及0.1%吐溫20)淸洗。加入溶於封阻緩衝 液中,濃度爲1毫微克/毫升之抗-人類TF Mabs並培育一小 時。然後將平盤使用洗滌緩衝液淸洗(6X)。接著使用存於 封阻緩衝液中,稀釋度爲1:2000之經HRP-標定抗-人類 (希利卡生物系統股份有限公司)以TMBplus-受質(凱姆科 技目錄編號4390A)偵測抗體結合作用。最終之ELISA訊 號(OD45q.62())係用做爲各抗體對所有STF變體的結合測量値 〇 凝血彈性描記法(分析12): 將人類促凝血酶原激酶(例如Innovin,Dade,貝林,最 終稀釋度50,000 乂)與〇&(:12(終濃度16.7 11^)及抗1 Mabs混合並於室溫下培育15分鐘。將經檸檬酸鹽-穩定化 之人類全血(280微升)加至r〇teg樣本杯(Pentapharm)中 並於37°C下預熱5分鐘,然後將4〇微升促凝血酶原激酶/ CaCV抗-TF Mab混合物加入。隨後於RoTEG裝置 (Pentapharm)中進行凝血彈性插記達1小時。使用c〇agPr〇 軟體™ (醫藥科學,亞薩斯,丹麥)自凝血描記圖獲得速 率分析圖。 實施例3 人類癌症分析。硏究以人類抗Mabs對小鼠模式中 人類癌症之生長及轉移的治療功效(分析13) 200407427 處理: 經由劑團注射i.v.施予人類抗-TF Mabs; 10毫克/公斤 = 0.1毫克/10克;注射體積爲〇·1毫升每克小鼠之下述 三種處理溶液中任一種: A. 載劑對照組 B. 1毫克/毫升人類FFR-rFVIIa C. 1毫克/毫升抗-TF Mabs 模式之敘述: I.結腸癌之初級生長及肝臟轉移Tris-HCl, denatured at pH 8.0. After incubation at room temperature for 1 hour, the denatured protein was diluted dropwise in 1 liter of a solution containing 50 mM Tris-HCl, (U5 M NaCl, pH 8.0 and refolded with gentle stirring for about 2 hours. Purification was performed using Q-Sepharose Fast Flow (Famacia, Eusala, Sweden) and FVIIa affinity chromatography as described by Ferrigard et al. (1996). Protein homogeneity was examined by SDS-PAGE Degree. Measured under ^^^ and determined the concentration using the calculated extinction coefficient of 37440 Minr1 (Jill and Van Hieb, 1989). 200407427 MaxiSorp flat plate (Nunc-Immuno) coated with wild type dissolved in TBS sTF and these variants (10 μg / ml) were blocked with blocking buffer (TBS containing 0.1% Tween 20 and 0.5% BSA). The plate was washed with washing buffer (TBS and 0.1% Tween). 20) Rinse. Add anti-human TF Mabs in blocking buffer at a concentration of 1 ng / ml and incubate for one hour. Then wash the plate with washing buffer (6X). Then use the HRP-calibrated anti-human in blocking buffer at a dilution of 1: 2000 (Hillika Biosystems has The company) uses TMBplus-substrate (Kemtech Catalog No. 4390A) to detect antibody binding. The final ELISA signal (OD45q.62 ()) is used to measure the binding of each antibody to all STF variants. Tracing method (Analysis 12): Mix human prothrombin protokinase (for example, Innovin, Dade, Behring, final dilution 50,000 乂) with 0 & (: 12 (final concentration 16.7 11 ^) and anti-1 Mabs and mix Incubate at room temperature for 15 minutes. Add citrate-stabilized human whole blood (280 microliters) to a roteg sample cup (Pentapharm) and preheat at 37 ° C for 5 minutes. Microliter prothrombin protokinase / CaCV anti-TF Mab mixture was added. Elastic clotting was subsequently performed in a RoTEG device (Pentapharm) for 1 hour. CoagPr0 software ™ (Medical Sciences, Assas, Denmark) A rate analysis graph was obtained from a coagulogram. Example 3 Analysis of human cancer. To investigate the therapeutic efficacy of human anti-Mabs on the growth and metastasis of human cancer in the mouse model (Analysis 13) 200407427 Treatment: administered via bolus injection iv Human anti-TF Mabs; 10 mg / kg = 0.1 mg / 10 g; the injection volume is 0.1 ml per gram of any of the following three treatment solutions: A. vehicle control group B. 1 mg / ml human FFR-rFVIIa C. 1 mg / ml anti- -TF Mabs mode description: I. Primary growth and liver metastasis of colon cancer
使用年齡7-8週之健康雌性無胸腺小鼠UwAw)。爲破壞 裸鼠對該人類細胞植入所產生之殘餘免疫抗性,遂慣常地 將小鼠於人類腫瘤移植前先置於5 Gy下照射2天(佛杰爾 等人,1997 )。如所述(李等人,人類基因療法10:3045-3053,1999 )藉由植入培養於含15%胎牛血淸(FCS)之RPMI 1640中之LS1747T人類結腸癌細胞(ATCC CCL 188)致免小 鼠。簡言之,以胰蛋白酶-EDTA採收細胞,洗滌兩次,並 再懸浮於補充以肝素化鈉溶液(1 U/毫升)之無血淸RPMI 中。然後於麻醉下切出一小段肋骨下切口並將存於50毫升 磷酸鹽緩衝食鹽水之3 3 106 LS174T細胞注射入脾臟中。 經3至5分鐘後,將脾臟血管結紮並將脾臟以手術取出。 此項程序將造成穩定發生肝臟轉移(超過95%)。於植入 後立即開始處理以抗-TF Mabs並持續至硏究全期。於腫瘤 細胞接種第15及30天將小鼠犧牲,取出肝臟並稱重,及 計數位於肝臟表面之可目測腫瘤結數目。將肝樣本於AFA 52 200407427 (5%醋酸,75%乙醇,2%福瑪林,18%水)中固定過夜,轉 移至100%乙醇,並包埋於石蠟中,製備5-毫米切片供組織 學定量轉移結瘤,供進行免疫組織學及細胞凋亡定量分析 〇 硏究1-1 : 目的:爲硏究所施予劑團注射i.v. ; 10毫克/公斤抗-TF Mabs對LS174T結腸腫瘤於裸鼠中之巨觀生長及肝臟轉移 的影響。 小鼠:60純合子nu/nu 6週齡NMRI雄性。 分組:將小鼠隨機分成各含15隻小鼠之四小組並處理 以溶液A、B或C。 終結:於體重減少>20%或有其他嚴重中毒之明顯徵兆 出現時即將小鼠終結。 參數:於生長階段期間每日以兩直角直徑記錄腫瘤大 小。開始即記錄體重並於每週記錄2-3次。屍體解剖測定 肝臟中之轉移結瘤形成。 II.乳癌之初級生長及肺臟轉移 將人類乳癌細胞MDA-MB-231 (ATCC HTB26)培養於補 充以10%胎牛血淸(FCS)的杜氏改進之英格氏培養基(DMEM) 中。以皮下注射將MDA-MB-231細胞(3 3 106)注入裸鼠(7-或8-週-齡雌性小鼠)中。如先前所述(李等人,人類基因 療法12:515-526,2001 )評估初級腫瘤生長及轉移。 硏究II-1 :Healthy female athymic mice (UwAw) aged 7-8 weeks were used. In order to destroy the residual immune resistance generated by the implantation of human cells in nude mice, the mice were routinely irradiated with 5 Gy for 2 days before human tumor transplantation (Foger et al., 1997). As described (Li et al., Human Gene Therapy 10: 3045-3053, 1999) by implantation of LS1747T human colon cancer cells (ATCC CCL 188) cultured in RPMI 1640 containing 15% fetal bovine blood pupa (FCS) Free of mice. Briefly, cells were harvested with trypsin-EDTA, washed twice, and resuspended in bloodless RPMI supplemented with heparinized sodium solution (1 U / ml). A small subcostal incision was then made under anesthesia and 3 3 106 LS174T cells in 50 ml of phosphate buffered saline were injected into the spleen. After 3 to 5 minutes, the spleen blood vessels were ligated and the spleen was surgically removed. This procedure will cause stable liver metastases (more than 95%). Treatment was started immediately after implantation with anti-TF Mabs and continued until the full phase of investigation. Mice were sacrificed on days 15 and 30 of tumor cell inoculation, the liver was removed and weighed, and the number of visually detectable tumor nodes on the surface of the liver was counted. Liver samples were fixed in AFA 52 200407427 (5% acetic acid, 75% ethanol, 2% formalin, 18% water) overnight, transferred to 100% ethanol, and embedded in paraffin to prepare 5-mm sections for tissue Quantitative metastasis of nodules for quantitative analysis of immunohistology and apoptosis. Investigate 1-1: Objective: To inject iv injection of iv; 10 mg / kg anti-TF Mabs for LS174T colon tumors. The effect of macroscopic growth and liver metastasis in nude mice. Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Termination: Mice will be terminated when weight loss > 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and 2-3 times a week. Autopsy measures Metastatic nodule formation in the liver. II. Primary Growth and Lung Metastasis of Breast Cancer Human breast cancer cells MDA-MB-231 (ATCC HTB26) were cultured in Duchenne's Modified Inger's Medium (DMEM) supplemented with 10% fetal bovine blood pupa (FCS). MDA-MB-231 cells (3 106) were injected subcutaneously into nude mice (7- or 8-week-old female mice). Primary tumor growth and metastasis were assessed as previously described (Li et al., Human Gene Therapy 12: 515-526, 2001). Study II-1:
目的:爲硏究所施予劑團注射i.v. ; 10毫克/公斤抗-TF 53 200407427Purpose: to inject i.v .; 10 mg / kg anti-TF 53 200407427
Mabs對MDA-MB-231乳腫瘤於裸鼠中之巨觀生長及肝臟轉 移的影響。 小鼠:60純合子nu/nu 6週齡NMRI雄性。 分組:將小鼠隨機分成各含15隻小鼠之四小組並處理 以溶液A、B或C。 終結:於體重減少>20%或有其他嚴重中毒之明顯徵兆 出現時即將小鼠終結。 參數:於生長階段期間每日以兩直角直徑記錄腫瘤大 小。開始即記錄體重並於每週記錄2-3次。屍體解剖測定 肺臟中之轉移結瘤形成。 III.成膠細胞癌腫瘤異種移植物之初級生長 腫瘤細胞系MG U373爲人類成膠質母細胞多形細胞系 ,於裸鼠中具有高血管生成活性,高血管密度且生長快速 。將腫瘤接種於肋腹,接著進行標準程序(參見實驗計畫 所揭示之提案)。每天兩次觀察小鼠是否出現中毒徵兆並 每日以兩垂直直徑測量腫瘤大小。 將腫瘤移植到具有NMRI背景之nu/nu純合子裸鼠的肋 腹中。小鼠係得自M&B (Ry,丹麥)之7-週-齡雄性小鼠 。將動物餵養於悉生生物環境中並使其無限量接受無菌食 物團塊及飮用水。 以該成膠細胞癌腫瘤模式進行三項不同硏究: 硏究III-1 : 目的:爲硏究所施予劑團注射i.v. ; 10毫克/公斤抗-TF Mabs對U373腫瘤於裸鼠中之巨觀生長的影響。 54 200407427 小鼠:60純合子nu/nu 6週齡NMRI雄性。 分組:將小鼠隨機分成各含15隻小鼠之四小組並處理 以溶液A、B或C。 終結:於體重減少>20%或有其他嚴重中毒之明顯徵兆 出現時即將動物終結。 參數:於生長階段期間每日以兩直角直徑記錄腫瘤大 小。開始即記錄體重並於每週記錄2-3次。 硏究III-2 : 目的:爲硏究所施予劑團注射i.v. ; 10毫克/公斤抗_TF Mabs對U373腫瘤於裸鼠中之巨觀生長的影響。於預先治 療性腫瘤生長已確立後。 小鼠:60純合子nu/nu 6週齡NMRI雄性。 分組:將小鼠隨機分成各含15隻小鼠之四小組並處理 以溶液A、B或C。當6次連續(每日)測量顯示岡柏茲生 長時即開始進行處理。此相當於100-200 mm3 終結:持續處理直到腫瘤生長超過約1.0 cm3之最大體 積,亦即,無腫瘤直徑大於15 mm,或直到經6次連續測 量已確立達岡柏茲再生長。於終結時將得自各組之腫瘤切 片以進行組織學及免疫化學評估。於體重減少>20%或有其 他嚴重中毒之明顯徵兆出現時即將動物終結。 參數:於生長階段期間每日以兩直角直徑記錄腫瘤大 小。開始即記錄體重並於每週記錄2次。 硏究III-3 : 目的:爲硏究抗-TF Mabs對顱內U373腫瘤於裸鼠中之 55 200407427 生長的影響。 小鼠:60純合子nu/nu 6週齢NMRI雄性。 腫瘤:將U373依照標準程序經眼部植入右半球。 分組:將小鼠隨機分成各含15隻小鼠之四小組並處理 以溶液A、B或C。 終結:將呈現慢性神經損傷徵兆之小鼠安樂死。 數據:經由卡普藍-麥爾統計學定量存活率(亦即時間 對神經損傷)。 實施例4 (分析14) 將一塊0.5毫升基質凝膠活塞以皮下方式置於其中其 TF基因已被剔除且***人類TF基因之小鼠(mTF-KO/hTF-KI小鼠)的腹部皮膚下。該基質凝膠中併入b-FGF (5毫微 克)且於一週後藉由測量血紅蛋白含量來定量該凝膠中新顯 明之血管形成(血管生成)。人類抗-TF Mabs之抑制能力 (血紅蛋白之抑制百分比% )可於單一或重複非經腸道投藥 該等蛋白質後進行評估。 實施例5 (分析15) 用以判別可防止FVIIa與TF結合之抗體及可防止FX 與TF結合之抗體的基因表現分析 於cDNA微列陣分析法中已觀察到經FVIIa處理之 BHK-TF細胞中有三種基因受到特異性增量調節。此等包括 :Fra-Ι (編碼Fos相關抗原1之基因)、Id2 (編碼螺旋-環 -螺旋類蛋白質之一成員的基因)及編碼胞外基質訊號蛋白 之Cyr61。下述分析係經設計用於篩檢防止經FVIIa所誘發 56 200407427The effect of Mabs on the macroscopic growth and liver metastasis of MDA-MB-231 breast tumors in nude mice. Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Termination: Mice will be terminated when weight loss > 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and 2-3 times a week. Autopsy measures Metastatic nodule formation in the lungs. III. Primary Growth of Glioblastoma Tumor Xenografts The tumor cell line MG U373 is a human glioblast polymorphic cell line with high angiogenic activity, high vascular density, and rapid growth in nude mice. Tumors are inoculated into the flank, followed by standard procedures (see proposals disclosed in experimental plans). Mice were observed twice daily for signs of poisoning and tumor size was measured daily with two vertical diameters. Tumors were transplanted into the ribs of nu / nu homozygous nude mice with an NMRI background. Mice were obtained from 7-week-old male mice from M & B (Ry, Denmark). Animals are fed in a living organism environment and are allowed to receive an unlimited amount of sterile food pellets and water in water. Three different studies were performed using this glioblastoma tumor model: Study III-1: Objective: To inject iv to the study group administered iv; 10 mg / kg of anti-TF Mabs against U373 tumors in nude mice The effect of macroscopic growth. 54 200407427 Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Termination: Animals will be terminated when weight loss > 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and 2-3 times a week. Study III-2: Objective: To study the effect of injection of i.v .; 10 mg / kg anti-TF Mabs on the macroscopic growth of U373 tumors in nude mice. After pre-treatment tumor growth has been established. Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Processing begins when 6 consecutive (daily) measurements indicate that Gambertz is growing. This is equivalent to the end of 100-200 mm3: the treatment is continued until the tumor grows to a maximum volume of about 1.0 cm3, that is, the tumor-free diameter is greater than 15 mm, or until 6 consecutive measurements have been established to regenerate Dagonbates. Tumor sections from each group will be obtained at the end for histological and immunochemical evaluation. Animals are about to end when weight loss > 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and twice a week. Study III-3: Objective: To study the effect of anti-TF Mabs on the growth of intracranial U373 tumors in nude mice. Mice: 60 homozygous nu / nu 6 weeks 齢 NMRI male. Tumor: U373 was implanted through the eye into the right hemisphere according to standard procedures. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. End: euthanize mice that show signs of chronic nerve injury. Data: Quantitative survival (i.e. time versus nerve damage) via Kaplan-Meier statistics. Example 4 (Analysis 14) A 0.5 ml matrix gel plunger was placed subcutaneously under the abdominal skin of a mouse (mTF-KO / hTF-KI mouse) in which the TF gene has been knocked out and inserted into the human TF gene . B-FGF (5 ng) was incorporated into the matrix gel and the newly manifested angiogenesis (angiogenesis) in the gel was quantified by measuring the hemoglobin content after one week. Human anti-TF Mabs' inhibitory ability (inhibition percentage of hemoglobin) can be evaluated after single or repeated parenteral administration of these proteins. Example 5 (Analysis 15) To identify the gene expression of antibodies that prevent FVIIa from binding to TF and antibodies that prevent FX from binding to TF Analysis of FHKa-treated BHK-TF cells has been observed in cDNA microarray analysis Three genes are specifically upregulated. These include: Fra-1 (the gene encoding Fos-associated antigen 1), Id2 (the gene encoding a member of the helix-loop-helix protein), and Cyr61, which encodes an extracellular matrix signaling protein. The following analysis is designed for screening to prevent induction by FVIIa 56 200407427
Fra-1、Id2 或 Cyr61 增量調節之抗-TF Mabs。 細胞培養 除非另加標示,否則所用試劑係購自GIBCO-BRL生命 技術公司。 將BHK-TF細胞(如普爾森L.K.等人,J. Biol. Chem. 273,6228-6232,1998所述製得)生長於含有10% FCS、 100 IU/毫升青黴素及100微克/毫升鏈黴素的杜氏改進之英 格氏培養基中以得到95-100%鋪滿,將其洗滌並再於不含 FCS之培養基中培養16-18小時。將細胞再次進行洗滌並暴 露至含有100 nM FVIIa之無FCS培養基。 爲選殖供北方轉漬分析之片段,遂將細胞以下述方法 處理。將BHK_TF細胞生長於含有10% FCS、100 IU/毫升青 黴素及100微克/毫升鏈黴素的杜氏改進之英格氏培養基中 以得到95-100%鋪滿,將其洗滌並再於不含FCS之培養基 中培養16-18小時。將細胞再次進行洗滌並暴露至含有100 nM FVIIa之無FCS培養基1小時。將CRL2091細胞(ATCC) 生長於含有10% FBS、100 U/毫升青黴素及100微克/毫升 鏈黴素的艾氏改進之英格氏培養基中以得到95-100%鋪滿。 接著,將細胞進行血淸飢餓達16-18小時並處理以含有100 nM FVIIa之無FBS培養基6小時。將鼠類3T3-L1細胞 (ATCC)維持於補充以10%胎牛血淸、100 U/毫升青黴素及 100微克/毫升鏈黴素的杜氏改進之英格氏培養基中。使細 胞生長到鋪滿並以含有1 μΜ***(西格瑪)、10微 克/毫升人類胰島素(諾佛諾地斯克A/S )及1 μΜ 57 200407427 BRL49653 (諾佛諾地斯克A/S)之培養基進行誘發1小時· 〇 選殖供北方轉漬分析之片段 使用superscript II套組(生命科技公司)根據廠商之 操作手冊,藉由反向轉錄PCR從單離自經***、胰島 素及BRL49653處理1小時之3T3-L1細胞的RNA選殖得 Fra-Ι。再藉由反向轉錄PCR從分別單離自經FVIIa處理1 小時之BHK-TF細胞及自經FVIIa處理6小時之CRL2091細 胞的RNA選殖得Id2與Cyr61。上游及下游引子爲:5’-GCGGCCGCCATGTACCGAGACTACGGGGAACCG-3,及 5’-GCGGCCGCTCACAAAGCCAGGAGTGTAGG-3’ 用於 Fra-Ι , 55- CAGCATGAAAGCCTTCAGTC-3’ 及 5’-CTCTGGTGATGCAGGCTGAC-3’ 用於 M2,5,-CGTCACCCTOTCCACTOA-3,及 5,-CTTGGTCTOCTGCATTTCT-3, 用於Cyr61。進行PCR之參數爲:於94°C下變性10秒、於65°C 下接合15秒及於72t下延展1.5分鐘之一次循環,於94t 下變性10秒、於64°C下接合15秒及於72°C下延展1.5分 鐘之一次循環,於94°C下變性10秒、於63°C下接合15秒 及於72°C下延展1.5分鐘之一次循環,於94t下變性10秒 、於62°C下接合15秒及於72°C下延展1.5分鐘之一次循環 ,於94°C下變性10秒、於61°C下接合15秒及於72°C下延 展1.5分鐘之一次循環,於94°C下變性10秒、於60°C下接 合15秒及於72°C下延展1.5分鐘之一次循環,於94°C下變 性10秒、於55°C下接合15秒及於72°C下延展1·5分鐘之 四十次循環。將所有片段選殖入ΤΟΡΟ 2.1 (因維托金)中 58 200407427 並使用Megabase定序儀進行定序。 北方轉漬分析 使用TriZol遵循廠商之操作指示自經與FVIIa、FX、 ASIS、1F44A1或TF8-5G9培養之BHK-TF細胞單離得總體 RNA。將20微克RNA置於含有1%瓊脂糖、20 mM MOPS、 5 mM NaOAc、6%福馬林及1 mM EDTA之變性凝膠中進行 大小分級,藉由毛細管轉漬法轉移至Hybond N+膜(艾默遜 )上並以UV交聯作用固定。使用[a-32P] dATP (艾默遜) 將編碼Fra-卜Id2或Cyr61之cDNA以Prime It套組(史塔 金)標定並使用Express Hyb (克隆科技)依照製造商指示 進行雜交且以放射自動顯影術呈像。 實施例6 (分析16) 經由Elkl轉錄因子/螢光素酶報告基因(PathDetect)進行 之MAPK分析 於進行轉染前一天將HeLa細胞接種於T-80培養瓶中 達40%鋪滿。如手冊所述使用36微升FuGene (洛奇)將細 胞轉染以150毫微克pFA-Elkl (史塔金)、3微克人類 TF/pcDNA3及3微克小鼠經蛋白酶活化之受器2/pcDNA3,l + 。次日以VerseneTM (因維托金)使細胞脫附並以細胞密度 爲20,000細胞每槽接種於黑色96槽視察平盤(Packard)中。 待細胞已再吸附平盤後,將培養基置換以160毫升每槽之 無血淸杜氏改進的英格氏培養基(因維托金)並培養16小 時。 將細胞與20微升無血淸培養基(對照組)、20微升 59 200407427 2.5 μΜ FFR-rFVIIa (對照組)、20 微升 2.5 μΜ 抗 TF Mab B 或 20 微升 2.5 μΜ 抗 TF Mab A。將 20 微升 0·5 μΜ FVIIa 加 至槽之一半並將培養基加至另一半。經4小時培育後將細 胞進行螢光素酶基因分析。如製造商指示將LucLlte (Packard)試劑加至該等細胞中。於TopCount微量平盤聞燦 計讀器(Packard)上讀取螢光素酶表現量。 【圖式簡單說明】 本發明將於各實施例伴隨參考所附之圖式做進$ 述,其中 圖1.圖示說明所例舉用於篩選出抗TF之人類單株胃 親和性抗體的篩檢分析。 圖2.如實施例1所述篩檢分析1-3之詳細圖示說明° 圖3.如實施例1所述篩檢分析4-7之詳細圖示說明° 圖4.如實施例1所述篩檢分析8-10之詳細圖示說明° 圖5. —項藉由第4號分析篩檢抗體之實例。由FFR_ rFVIIa (實心圓)及由人類抗-TF單株抗體HuTF-31F2 ( € 心圓)所導致之sTF/FVIIa醯胺分解活性的抑制作用。 圖6. —項藉由第5號分析篩檢抗體之實例。由FFR-rFVIIa (實心圓)及由人類抗-TF單株抗體HuTF-31F2 (空 心圓)所導致之因子Xa產生的抑制作用。 圖].一項藉由第7號分析篩檢抗體之實例。由FFR-rFVIIa (實心圓)及由人類抗-TF單株抗體HuTF-31F2 (空 心圓)所導致之人類TF-誘發凝塊生成的抑制作用。 圖8. —項藉由第10號分析篩檢抗體之實例。僅有防 200407427 止FVIIa結合之抗-TF單株抗體可抑制由TF/FVIIa-所介導之· 信號發生作用。 圖9.人類抗TF Mab抑制由FVIIa所誘發之P44/42 MAPK磷酸化作用(分析1〇)。將經TF轉染之BHK細胞 進行血淸飢餓2小時使細胞靜止活動。於添加FVIIa (15 nN)前15分鐘時先將抗體HuMab 30F5 (500 nM)及HuMab 31F2 (500 nM)加至該細胞中。將細胞溶解並將蛋白質置於 SDS-PAGE上分離再藉由電轉漬法轉移到硝基纖維素膜上。 使用對P44/42 MAPK之多株磷酸基-特異性抗體完成西方轉 漬分析。次級抗體爲與山辣根過氧化酶共軛之抗-兔子IgG 。使用經冷卻之CCD4目機進行化學發光。將位於數位化相 片上之條帶定量並將以FVIIa測定得之條帶設定爲100%。 當將細胞以HuMab 30F5 (500 nM)進行預培養時觀察到磷酸 化條帶量減少50%,而當將細胞以HuMab 31F2 (500 nM)進 行預培養時觀察到量減少25%。結論,此實驗顯示抗TF之 人類抗體(30F5及31F2)可部份抑制由FVIIa所誘發之 P44/42 MAPK磷酸化作用。使用50 nM FVIIa觀察到類似的 結果。 圖10· —項藉由第16號分析篩檢抗體之實例。該圖證 明可藉由抗TF單株抗體抑制TF表現細胞中之TF細胞內活 性。抗TF Mab B抑制TF細胞內活性,而抗-TF Mab A則否。 圖11· 一項藉由第12號分析篩檢抗體之實例。以0.5 nM之FFR-rFVIIa及人類抗-TF抗體HuTF-31F2所測得之凝 血彈性描記之速率圖譜。 61Fra-1, Id2, or Cyr61 up-regulated anti-TF Mabs. Cell cultures Unless otherwise specified, reagents used were purchased from GIBCO-BRL Life Technologies. BHK-TF cells (produced as described by Purson LK et al., J. Biol. Chem. 273, 6228-6232, 1998) are grown in cells containing 10% FCS, 100 IU / ml penicillin, and 100 μg / ml streptomyces In Duchenne's modified Inger's medium to obtain a 95-100% confluence, it was washed and cultured in FCS-free medium for 16-18 hours. The cells were washed again and exposed to FCS-free medium containing 100 nM FVIIa. To select fragments for northern transfection analysis, the cells were treated as described below. BHK_TF cells were grown in Duchenne modified Ingrid medium containing 10% FCS, 100 IU / ml penicillin and 100 μg / ml streptomycin to obtain a 95-100% confluence, washed and re-fCS-free The medium was cultured for 16-18 hours. The cells were washed again and exposed to FCS-free medium containing 100 nM FVIIa for 1 hour. CRL2091 cells (ATCC) were grown in Ehrlich's modified Ingrid medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin to obtain 95-100% confluence. Next, the cells were starved for 16-18 hours and treated with FBS-free medium containing 100 nM FVIIa for 6 hours. Murine 3T3-L1 cells (ATCC) were maintained in Dugar's modified Ingrid medium supplemented with 10% fetal bovine blood pupa, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells were grown to confluence and contained 1 μM dexamethasone (sigma), 10 μg / ml human insulin (Novo Nordisk A / S) and 1 μM 57 200407427 BRL49653 (Novo Nordisk A / S) The medium was induced for 1 hour. 〇Selected fragments for northern transfection analysis. Using the superscript II kit (Life Technology) according to the manufacturer's manual, reverse transcription PCR was performed from dexamethasone, insulin and The RNA of 3T3-L1 cells treated with BRL49653 for 1 hour was selected to produce Fra-1. Id2 and Cyr61 were cloned from RNA isolated from BHK-TF cells treated with FVIIa for 1 hour and CRL2091 cells treated with FVIIa for 6 hours by reverse transcription PCR. Upstream and downstream primers are: 5'-GCGGCCGCCATGTACCGAGACTACGGGGAACCG-3, and 5'-GCGGCCGCTCACAAAGCCAGGAGTGTAGG-3 'for Fra-1, 55- CAGCATGAAAGCCTTCAGAG-3-3 and 5'-CTCTGGTGATGCAGGCTGAC-3' for M2,5, -CGTCACCCT 3, and 5, -CTTGGTCTOCTGCATTTCT-3 for Cyr61. The parameters for PCR are: a cycle of denaturation at 94 ° C for 10 seconds, bonding at 65 ° C for 15 seconds, and extension at 72t for 1.5 minutes, denaturation at 94t for 10 seconds, bonding at 64 ° C for 15 seconds, and One cycle of extension for 1.5 minutes at 72 ° C, 10 seconds of denaturation at 94 ° C, 15 seconds of bonding at 63 ° C, and one cycle of 1.5 minutes extension at 72 ° C, 10 seconds of denaturation at 94t One cycle of bonding for 15 seconds at 62 ° C and extension of 1.5 minutes at 72 ° C, one cycle of denaturation at 94 ° C for 10 seconds, 15 seconds of bonding at 61 ° C, and 1.5 minute extension at 72 ° C, One cycle of denaturation at 94 ° C for 10 seconds, bonding at 60 ° C for 15 seconds and extension of 1.5 minutes at 72 ° C, denaturation at 94 ° C for 10 seconds, bonding at 55 ° C for 15 seconds and 72 Forty cycles of 1.5 minutes at ° C. All fragments were cloned into TOPO 2.1 (Invitokine) 58 200407427 and sequenced using a Megabase sequencer. Northern Transfection Analysis Use TriZol to follow the manufacturer's instructions to isolate total RNA from BHK-TF cells cultured with FVIIa, FX, ASIS, 1F44A1, or TF8-5G9. 20 micrograms of RNA were placed in a denaturing gel containing 1% agarose, 20 mM MOPS, 5 mM NaOAc, 6% formalin, and 1 mM EDTA for size fractionation, and transferred to a Hybond N + membrane (Ai (Mersen) and fixed by UV crosslinking. [A-32P] dATP (Emerson) The cDNA encoding Fra-Bu Id2 or Cyr61 was calibrated with Prime It kit (Starkin) and hybridized using Express Hyb (cloning technology) according to the manufacturer's instructions and radiated Autoradiography. Example 6 (Analysis 16) MAPK analysis via Elkl transcription factor / luciferase reporter gene (PathDetect) HeLa cells were seeded in T-80 culture flasks up to 40% confluence one day before transfection. Cells were transfected with 150 μg of pFA-Elkl (Starkin), 3 μg of human TF / pcDNA3, and 3 μg of mouse-activated receptor 2 / pcDNA3 as described in the manual using FuGene (Rocky). , L +. The next day the cells were detached with VerseneTM and seeded in black 96-slot inspection plates (Packard) at a cell density of 20,000 cells per well. After the cells had re-adsorbed the plate, the medium was replaced with 160 ml of blood-free Duchenne's Modified Inger's Medium (Invitokine) and cultured for 16 hours. Cells were treated with 20 μl of blood-free 淸 media (control group), 20 μl 59 200407427 2.5 μM FFR-rFVIIa (control group), 20 μl of 2.5 μM anti-TF Mab B or 20 μl of 2.5 μM anti-TF Mab A. Add 20 μl of 0.5 μM FVIIa to one half of the tank and medium to the other half. After 4 hours of incubation, cells were analyzed for luciferase gene. LucLlte (Packard) reagent was added to the cells as instructed by the manufacturer. Read the luciferase expression on a TopCount microplate reader (Packard). [Brief description of the drawings] The present invention will be described with reference to the accompanying drawings in each of the embodiments, where FIG. 1. illustrates the illustrated example of screening for a single human gastric affinity antibody against TF Screening analysis. Figure 2. Detailed illustration of screening analysis 1-3 as described in Example 1 ° Figure 3. Detailed illustration of screening analysis 4-7 as described in Example 1 ° Figure 4. As described in Example 1 The detailed graphic description of the screening analysis 8-10 is described in Figure 5.-Example of screening antibody by No. 4 analysis. Inhibition of sTF / FVIIa amidolytic activity by FFR_ rFVIIa (filled circle) and human anti-TF monoclonal antibody HuTF-31F2 (€ circle). Figure 6.-Example of antibody screening by assay No. 5. Inhibition of factor Xa by FFR-rFVIIa (filled circles) and human anti-TF monoclonal antibody HuTF-31F2 (filled circles). Figure]. An example of screening antibodies by analysis No. 7. Inhibition of human TF-induced clot formation by FFR-rFVIIa (filled circles) and human anti-TF monoclonal antibody HuTF-31F2 (filled circles). Figure 8. —Example of screening antibodies by analysis No. 10. Only anti-TF monoclonal antibodies that prevent 200407427 from blocking FVIIa binding can inhibit TF / FVIIa-mediated signaling. Figure 9. Human anti-TF Mab inhibits P44 / 42 MAPK phosphorylation induced by FVIIa (Analysis 10). BHK cells transfected with TF were subjected to blood starvation for 2 hours to quiescent cells. The antibodies HuMab 30F5 (500 nM) and HuMab 31F2 (500 nM) were added to the cells 15 minutes before FVIIa (15 nN) was added. The cells were lysed and the proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane by electroporation. Western blot analysis was performed using multiple phosphate-specific antibodies to P44 / 42 MAPK. The secondary antibody was an anti-rabbit IgG conjugated to horseradish peroxidase. Chemiluminescence was performed using a cooled CCD4 eyepiece. The band on the digitized film was quantified and the band measured by FVIIa was set to 100%. A 50% reduction in phosphorylated bands was observed when cells were precultured with HuMab 30F5 (500 nM), and a 25% reduction was observed when cells were precultured with HuMab 31F2 (500 nM). In conclusion, this experiment shows that anti-TF human antibodies (30F5 and 31F2) can partially inhibit the phosphorylation of P44 / 42 MAPK induced by FVIIa. Similar results were observed with 50 nM FVIIa. Figure 10 · —Example of antibody screening by No. 16 analysis. This figure demonstrates that the intracellular activity of TF in TF expressing cells can be inhibited by an anti-TF monoclonal antibody. Anti-TF Mab B inhibits TF intracellular activity, while anti-TF Mab A does not. Figure 11. An example of screening antibodies by analysis No. 12. Rate profile of hemagglutination measured with 0.5 nM of FFR-rFVIIa and human anti-TF antibody HuTF-31F2. 61
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