TW200813088A

TW200813088A - Antibodies to EGFL7 and methods for their use

Info

Publication number: TW200813088A
Application number: TW96109167A
Authority: TW
Inventors: Weilan Ye; Maike Schmidt; Jo-Anne Hongo; Yan Wu
Original assignee: Genentech Inc
Priority date: 2006-03-16
Filing date: 2007-03-16
Publication date: 2008-03-16
Also published as: TWI429655B

Abstract

The invention provides anti-EGFL7 antibodies, and compositions comprising and methods of using these antibodies.

Description

200813088 九、發明說明：【發明所屬之技術領域】本發明大體係關於用於調節血管發育之組合物及方法。特定言之，本發明係關於與表皮生長因子樣結構域7 (EGFL7)多肽結合之抗體。本發明另外係關於與血管生成相關之病況及疾病的診斷及治療。【先前技術】血管聯結之發育為許多生理及病理過程之基本需要。諸 f' 如胚胎及腫瘤之活躍生長組織需要適當之血液供應。它們藉由產生促進新血管經由稱為血管生成之過程形成的促血管生成因子來滿足此需求。血管形成是一個複雜但有序之生物事件，其涉及全部或多個以下步驟：a)内皮細胞（EC) 由現存EC增殖或由祖細胞分化；b)EC遷移並聚結形成束狀結構；c)接著血管束經歷管生成（tubulogenesis)形成具有中央腔之血管；d)現存管束或血管伸出新的分枝形成二級血管；e)原始血管叢經歷進一步重塑及再成形；及f)募集 ^ ' 外内皮細胞嵌入内皮管中，從而向血管提供維持及調節功能；此等細胞包括小毛細管之周細胞、較大血管之平滑肌細胞及心臟中之心肌細胞。Hanahan，277:48_50 (1997); Hogan 及 Kolodziej，3:513-23 (2002); Lubarsky及 Krasnow，CW/ 112:19-28 (2003) ο 現已充分確定多種病症之發病機理均涉及血管生成。此等病症包括實體腫瘤及轉移瘤；動脈粥樣硬化；晶狀體後纖維組織增生；血管瘤；慢性炎症；眼内新生血管疾病， 119234.doc 200813088 諸如增生性視網膜病變（例如糖尿病性視網膜病變）、年齡相關之黃斑變性（AMD)、新生血管性青光眼；移植角膜組織及其他組織之免疫排斥反應；類風濕性關節炎；及牛皮癣0 Folkman等人，Biol. Chem. 267:10931-34 (1992)； Klagsbrun等人，53:217-39 (1991)及 Garner A·，Pathobiology of Ocular Disease 中之’’Vascular diseases’’。A Dynamic Approach，Garner A.，Klintworth GK 編輯，第 2版（Marcel Dekker，NY，1994)，第 1625-1710 頁。在腫瘤生長之情況下，血管生成傯乎對於增生轉變成瘤形成及向腫瘤生長及轉移提供營養至關重要。F〇lkman等人’ 339:58 (1989)。新血管生成允許腫瘤細胞相比正常細胞獲取生長優勢及增殖自主性。腫瘤通常以單一異常細胞起始，該異常細胞可因與可用毛細血管床之距離而僅增生至幾立方毫米之尺寸，且腫瘤可在一段較長時間段内保持”休眠’’而不進一步生長及散佈。接著某些腫瘤細胞轉換成血管生成表型而活化内皮細胞，其增殖並成熟成為新毛細企管。此等新形成之血管不僅允許初生腫瘤持續生長’且亦允許轉移腫瘤細胞散佈及再移生。相應地，在乳癌以及數種其他腫瘤中已觀察到腫瘤切片中之微血管密度與患者存活率之間的相關性。Weidner等人，见以g/·丄200813088 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to compositions and methods for regulating vascular development. In particular, the present invention relates to antibodies that bind to epidermal growth factor-like domain 7 (EGFL7) polypeptides. The invention further relates to the diagnosis and treatment of conditions and diseases associated with angiogenesis. [Prior Art] The development of vascular connections is a basic requirement for many physiological and pathological processes. The f's such as embryos and tumors that actively grow tissue require an appropriate blood supply. They address this need by creating a pro-angiogenic factor that promotes the formation of new blood vessels via a process called angiogenesis. Angiogenesis is a complex but ordered biological event involving all or more of the following steps: a) endothelial cells (EC) are proliferated by existing EC or differentiated by progenitor cells; b) EC migrates and coalesces to form a bundle structure; c) then the vascular bundle undergoes tubulogenesis to form a vessel with a central lumen; d) the existing bundle or vessel protrudes into a new branch to form a secondary vessel; e) the primitive vascular bundle undergoes further remodeling and reshaping; Recruitment ^ 'External endothelial cells are embedded in the inner tube to provide maintenance and regulation to the blood vessels; these cells include pericytes of small capillaries, smooth muscle cells of larger blood vessels, and cardiomyocytes in the heart. Hanahan, 277:48_50 (1997); Hogan and Kolodziej, 3:513-23 (2002); Lubarsky and Krasnow, CW/112:19-28 (2003) ο It is now well established that the pathogenesis of various disorders involves angiogenesis . Such conditions include solid tumors and metastases; atherosclerosis; post-lens fibrous tissue hyperplasia; hemangioma; chronic inflammation; intraocular neovascular disease, 119234.doc 200813088 such as proliferative retinopathy (eg diabetic retinopathy), Age-related macular degeneration (AMD), neovascular glaucoma; immune rejection of transplanted corneal tissue and other tissues; rheumatoid arthritis; and psoriasis 0 Folkman et al., Biol. Chem. 267:10931-34 (1992) Klagsbrun et al., 53: 217-39 (1991) and Garner A., ''Vascular diseases'' in Pathobiology of Ocular Disease. A Dynamic Approach, Garner A., Klintworth GK, 2nd ed. (Marcel Dekker, NY, 1994), pp. 1625-1710. In the case of tumor growth, angiogenesis is essential for the conversion of hyperplasia into neoplasia and nutrition for tumor growth and metastasis. F〇lkman et al. 339:58 (1989). Neovascularization allows tumor cells to gain growth advantage and proliferation autonomy compared to normal cells. Tumors usually start with a single abnormal cell that can only proliferate to a few cubic millimeters due to the distance from the available capillary bed, and the tumor can remain "sleeping" for a longer period of time without further growth. And then spread. Some tumor cells convert to an angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillaries. These newly formed blood vessels not only allow primary tumors to continue to grow, but also allow metastatic tumor cells to spread and re-transfer. Immigration. Correspondingly, the correlation between microvessel density in tumor sections and patient survival has been observed in breast cancer and several other tumors. Weidner et al., see g/·丄

Mel 324:1-6 (1991) ; Horak 等人，厶⑽⑽ 340:1120-24 (1992) ; Macchiarini等人，icmcei 340:145-46 (1992)。控制血管生成轉換之確切機制尚未完全瞭解，但據信腫瘤塊之新血管生成係由大量Α管生成刺激劑與抑制劑之淨差引 119234.doc 200813088 起（Folkman，Med 1(1):27-31 (1995))。血管發育之過程受到嚴格調控。截至目前，已顯示大量分子（主要是由周圍細胞產生之分泌因子）調控EC之分化、增殖、遷移及聚結成為束狀結構。舉例而言，已鑑別血管内皮生長因子（VEGF)為刺激血管生成及誘導血管滲透性所涉及之關鍵因子。Ferrara等人，五7^ν· 18:4_25Mel 324:1-6 (1991); Horak et al., 厶(10)(10) 340:1120-24 (1992); Macchiarini et al., icmcei 340:145-46 (1992). The exact mechanism for controlling angiogenesis switching is not fully understood, but it is believed that the neovascularization of the tumor mass is caused by a large number of fistulas generating a net difference between the stimulant and the inhibitor. 119234.doc 200813088 (Folkman, Med 1(1):27- 31 (1995)). The process of vascular development is strictly regulated. Up to now, it has been shown that a large number of molecules (mainly secreted factors produced by surrounding cells) regulate the differentiation, proliferation, migration and coalescence of EC into a bundle structure. For example, vascular endothelial growth factor (VEGF) has been identified as a key factor involved in stimulating angiogenesis and inducing vascular permeability. Ferrara et al., 5 7^ν· 18:4_25

(1997)。有關甚至單一 VEGF等位基因缺失亦導致胚胎死亡之發現指出此因子在血管系統發育及分化中所起到的不可替代之作用。此外’已顯不VEGF係與腫瘤及眼内病症相關之新血管生成之關鍵介體。Ferrara等人，五?同上文。所檢查之大部分人類腫瘤均過度表現VEGF mRNA。Berkman等人，/· C/h. /πναί. 91:153-59 (1993); Βτον/η 專尺，Human Pathol· 26:86-91 (1995) ； Brown 等人，Cancer 及53:4727-35 (1993) ; Mattern等人， J. Career 73:931-34 (1996) ; Dvorak等人，dm· «/· Ραί/ζο/· 146:1029-39 (1995)。另外，眼液中之VEGF濃度值與患有糖尿病及其他局部缺血相關視網膜病變之患者體内存在血管活躍增生高度相關。Aiello等人，见五叹/· J. Med. 331:1480-87 (1994)。此外，研究已證實VEGF定位於身受AMD之患者之脈絡膜新生血管膜中。Lopez 等人，m 心厂 37:855-68 (1996) 〇抗VEGF中和抗體抑制多種人類腫瘤細胞株於裸小鼠體内之生長（Kim 等人，iVaiwre 362:841-44 (1993) ; Warren等 119234.doc 200813088(1997). The discovery that even single VEGF allele deletions also lead to embryonic death indicates an irreplaceable role for this factor in vascular system development and differentiation. In addition, it has been shown that VEGF is a key mediator of neovascularization associated with tumors and intraocular disorders. Ferrara et al., the same as above. Most of the human tumors examined showed excessive expression of VEGF mRNA. Berkman et al., /· C/h. /πναί. 91:153-59 (1993); Βτον/η Special Rule, Human Pathol· 26:86-91 (1995); Brown et al., Cancer and 53:4727- 35 (1993); Mattern et al., J. Career 73:931-34 (1996); Dvorak et al., dm·«/· Ραί/ζο/· 146:1029-39 (1995). In addition, the VEGF concentration in the eye fluid is highly correlated with the presence of vascular active hyperplasia in patients with diabetes and other ischemic retinopathy. Aiello et al., see the five sighs / J. Med. 331:1480-87 (1994). In addition, studies have demonstrated that VEGF is localized in the choroidal neovascular membrane of patients with AMD. Lopez et al, m Heart Factory 37: 855-68 (1996) 〇 anti-VEGF neutralizing antibodies inhibit the growth of various human tumor cell lines in nude mice (Kim et al, iVaiwre 362:841-44 (1993); Warren et al 119234.doc 200813088

人，J. C7z·”· /πναί· 95:1789-97 (1995) ; Borgstrdm等人， Cancer Res. 56:4032-39 (1996) ； Melnyk^ A 5 Cancer Res, 5 6:921_24 (1996))，且亦抑制局部缺血視網膜病症模型中之眼内血管生成（Adamis 等人，drc/z. Ophthalmol. 114:66-71 (1996))。因此，抗VEGF單株抗體或VEGF作用之其他抑制劑係治療腫瘤及各種眼内新生血管病症之值得期望之候選物。此等抗體描述於（例如）1998年1月14日公開之EP 817,648 及 1998 年 10 月 15 日公開之 WO 98/4533 1 及 WO -X w j w | rjy 〇 ▼ 丄 la HiL· 少 S |-^ - j v xxx cv cy y l—」由FDA批准與化學治療方案組合用於治療轉移結腸直腸癌 (CRC)。在許多正在進行之臨床試驗中亦在研究用於治療各種癌症指征之貝伐單抗。已知細胞外基質（ECM)在血管生成過程中起到重要作用0 Madri，rraw〆· 5:179-83 (1997)。EC在其遷移期間由臨時ECM包圍，且在形成内腔後黏附至新合成之血管基底膜。除在毛細血管形態形成過程中提供支架外，已亦顯示ECM對EC之功能起到複雜之局部控制作用。舉例而言，ECM能夠調控可溶血管生成介體對於EC之可用性且規定與整合素及細胞黏附分子相互作用之性質及類型。亦已表明EC之存活受生長因子受體與整合素間之協作調控，其又受局部ECM之組成控制。Stupack及Cheresh， 22:9022-29 (2003) 〇儘管血管生成領域已取得許多進展，但血管形成過程中之某些步驟仍有待清晰瞭解。特定言之，有關管形成如何 119234.doc 200813088 調控一血管束如何進展成為管及何種因子調控此轉變知之甚少。鑒於血管生成在許多疾病及病症中之作用，需要一種降低或抑制一或多種引起此等過程之生物作用的方法。亦需要一種檢定正常及疾病條件且尤其是癌症中致病多肽之存在的方法。亦存在對於可增強現存抗血管生成治療之功效之組合物及方法的需求。【發明内容】本發明部分上係基於鑑別具有指示其特別有益於治療之特性的抗EGFL7抗體。在一態樣中，本發明提供由融合瘤抗EGFL7 mumab 4F11.1.8、抗 EGFL7 mumab 10G9.1.6 及抗 EGFL7 mumab 18F7.1.8產生之抗體。在一態樣中，本發明提供一種抗EGFL7抗體，其包含一或多個選自由以下序列組成之群之互補判定區（CDR) : (a) 4F11 CDR-L1 序列 KASQSVDYDGDSYMS (SEQ ID NO: 5) ; (b) 4F11 CDR-L2序列 GASNLES (SEQ ID NO: 6) ; (c) 4F11 CDR-L3序列 QQNNEDPYT (SEQ ID NO: 7) ; (d) 4F11 CDR-H1 序列 TYGMS (SEQ ID NO: 8) ; (e) 4F11 CDR-H2序列 WINTHSGVPTYADDFKG (SEQ ID NO: 9);及（f) 4F11 CDR-H3 序列 LGSSA (SEQ ID NO: 10)。在一些實施例中，該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：KASQSVDYDGDSYMS (SEQ ID NO: 5)、GASNLES (SEQ ID NO: 6)及 QQNNEDPYT (SEQ ID NO: 7)。在一些實施例中，該抗體之重鏈包含至少一個、 119234.doc •10· 200813088 至少兩個或所有三個選自以下序列之CDR序列：TYGMS (SEQ ID NO: 8)、WINTHSGVPTYADDFKG (SEQ ID NO: 9)及LGSSA (SEQ ID NO: 10)。在一些實施例中，該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之 CDR序列：KASQSVDYDGDSYMS (SEQ ID NO: 5)、 GASNLES (SEQ ID NO: 6)及 QQNNEDPYT (SEQ ID NO: 7);且該抗體之重鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：TYGMS (SEQ ID NO: 8)、 WINTHSGVPTYADDFKG (SEQ ID NO: 9)及LGSSA (SEQ ID NO: 10)。在一些實施例中，該抗體之輕鏈包含以下序列： DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMSWY QQKPGQPPKLLIYGASNLESGIPARFSGSGSGTDFTLNIHPV EEEDAATYYCQQNNEDPYTFGGGTKVEIKR (SEQ ID NO: I) 。在一些實施例中，該抗體之重鏈包含以下序列： QIQLVQSGPELKKPGETVKISCKASGHTFTTYGMSWVKQA PGKGLKWMGWINTHSGVPTYADDFKGRFAFSLETSASTAH LQINNLKNEDTATYFCARLGSSAVDYWGQGTTVTVSS (SEQ ID NO: 2)。在一態樣中，本發明提供一種抗EGFL7抗體，其包含一或多個選自由以下序列組成之群之互補判定區（CDR) : (a) 10G9 CDR-L1 序列 RSSQSLVHTNGITYLH (SEQ ID NO: II) ; (b) 10G9 CDR-L2序列 KVSNRFS (SEQ ID NO: 12); (c) 10G9 CDR-L3序列 SQSTHVPLT (SEQ ID NO: 13) ; (d) 10G9 CDR-H1 序列 DYYMNSDYYMN (SEQ ID NO: 14) ; (e) 119234.doc -11 - 200813088 10G9 CDR-H2序列 DINPKNGGTTYNQKFKG (SEQ ID NO: 15);及（f) 10G9 CDR-H3序列（SEQ ID NO: 16)。在一些實施例中，該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：RSSQSLVHTNGITYLH (SEQ ID NO: 11)、KVSNRFS (SEQ ID NO: 12)及 SQSTHVPLT (SEQ ID NO: 13)。在一些實施例中，該抗體之重鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列： DYYMNSDYYMN (SEQ ID NO: 14)、DINPKNGGTTYNQKFKG fSFO ΤΓ> ΧΓΠ· 1 泠 AT GVFFIV fSFn τη ΜΠ· 1 。太一 4b 眘， ▲，· A. V J ▼ ▲ 一在、—一 Jk. m， ▲，· Λ. j I - 施例中，該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：RSSQSLVHTNGITYLH (SEQ ID NO: 11)、KVSNRFS (SEQ ID NO: 12)及 SQSTHVPLT (SEQ ID NO: 13);且該抗體之重鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：DYYMNSDYYMN (SEQ ID NO: 14) ^ DINPKNGGTTYNQKFKG (SEQ ID NO: 15) 及ALGVFDY (SEQ ID NO: 16)。在一些實施例中，該抗體之輕鏈包含以下序列：DIVMTQTPLSLPVSLGDQASISCR SSQSLVHTNGITYLHWYLQKPGQSPKLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGA GTKVEIKR (SEQ ID NO: 3)。在一些實施例中，該抗體之重鏈包含以下序列：EVQLQQSGPELVKPGASVKISCKASG YTFSDYYMNSDYYMNWVKQSHGKSLEWIGDINPKNGGTT YNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE ADYDPIYYAMDYWGQGTTLTVSA (SEQ ID NO: 4)。 119234.doc -12- 200813088 在二實靶例中，本發明提供與包含以下胺基酸序列之之夕肽特異性結合的抗EGFL7抗體：CCP、TIY及ACS。在一 μ施例中，本發明提供與本發明之其他抗體結合於人類 EGFT 7 ϊ» 、上之相同抗原決定基的經分離抗體。在一些實 *例中本發明提供與本發明之其他抗體競爭與Egfl7結合之經分離抗體。在一些實施例中，本發明之抗體為單株抗體。在一些實施例中本發明之抗體為嵌合抗體、人化抗體、親和力成 …^ 人類^體或雙特異性抗體。在一些實施例中，該抗體為抗體片段。在一些實施例中，本發明提供一種包含本發明之抗 EGFL7抗體之醫藥組合物。在一些實施例中，該醫藥組合物另外包含抗血管生成劑。在一些實施例中，該抗血管生成劑為貝伐單抗或雷尼株單抗（ranibizumab)。在一些實施例中，本發明提供一種編碼本發明之抗體之聚核苷酸。在一些實施例中，本發明提供包含此等聚核苷酸之載體。在一些實施例中，該載體為表現載體。在一些實施例中，本發明提供包含此等載體之宿主細胞，包括原核及真核細胞（包括哺乳動物細胞）。在一些實施例中，本發明提供一種製造抗EGFL7抗體之方法，其包含（a)使表現載體在適當宿主細胞中表現；及（b)回收抗體。在一些實施例中，本發明提供一種降低或抑制患有與血管生成相關之病理病況之受檢者體内血管生成之方法，其包含向該受檢者投與有效量之本發明之抗EGFL7抗體或包 119234.doc -13· 200813088 含本發明之抗EGFL7抗體之醫藥組合物。在一些實施例中，該病理病況為贅瘤，例如癌瘤。在一些實施例中，該病理病況與眼有關，例如眼内新生血管疾病。在一些實施例中，除本發明之抗EGFL7抗體外，亦將抗血管生成劑投與受檢者。在一些實施例中，抗血管生成劑為血管内皮生長因子（VEGF)之拮抗劑，例如抗VEGF抗體（包括貝伐單抗及雷尼株單抗）。在一些實施例中，抗血管生成劑係在投與抗EGFL7抗體之前或之後投與。在一些實施例中，抗血管生成劑係與抗EGFL7抗體同時投與。在一些實施例中，本發明提供一種增強患有與血管生成相關之病理病況之受檢者體内抗血管生成劑之功效的方法’其包含向該受檢者投與本發明之抗EGFL7抗體或包含本發明之抗EGFL7抗體之醫藥組合物。在一些實施例中，該病理病況為贅瘤，例如癌瘤。在一些實施例中，該病理病況與眼有關，例如眼内新生血管疾病。在一些實施例中’除本發明之抗EGFL7抗體外，亦將抗血管生成劑投與受檢者。在一些實施例中，抗血管生成劑為血管内皮生長因子（VEGF)之拮抗劑，例如抗VEGF抗體（包括貝伐單抗及雷尼株單抗）。在一些實施例中，抗血管生成劑係在投與抗EGFL7抗體之前或之後投與。在一些實施例中，抗血管生成劑係與抗EGFL7抗體同時投與。在一些實施例中，亦投與其他治療，例如皮質類固醇或光動力療法。【實施方式】本發明提供抗EGFL7抗體，其可用於（例如）治療或預防 119234.doc -14· 200813088 與EGFL7之表現及/或活性（諸如表現及/或活性增強或不合需要之表現及/或活性）相關之疾病狀態。在一些實施例中，本發明之抗體可用於治療腫瘤、癌症及/或細胞增生性病症。在另一態樣中，本發明之抗EGFL7抗體可用作偵測及/ 或分離EGFL7(諸如偵測各種組織及細胞類型中之EGFL7) 之試劑。本發明另外提供製造抗EGFL7抗體、編碼抗EGFL7抗體之聚核苷酸及包含編碼抗EGFL7抗體之聚核苷酸之細胞的方法。通用技術熟習此項技術者一般已充分瞭解且通常使用常規方法來使用本文描述或提及之技術及程序，諸如Sambrook等人’ Molecular Cloning: A Laboratory Manual 第 3版（2001) Cold Spring Harbor Laboratory Press，Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel等人編輯，（2003)) ; METHODS IN ENZYMOLOGY 系列（Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson，B. D. Hames及 G. R. Taylor 編輯（1995))，Harlow 及 Lane 編輯（1988) ANTIBODIES，A LABORATORY MANUAL及 ANIMAL CELL CULTURE (R· I. Freshneyb編輯（1987))中所述之廣泛使用之方法。定義 "經分離’’抗體為已經鑑別且與其天然環境之組份分離及/ 119234.doc -15· 200813088 或自天然環境之έ日々乂士，，兄之料中回收之抗體。其天然環境份為將干擾抗體之診 . 研口黡用途之物質，且可包括酵素、激素及其他蛋白或非疋 ― 攻非蛋白〉谷貝。在一些實施例中，體將·（ 1)經純化至如葬.篓稭由勞立法（Lowry method)所測定大Human, J. C7z·”· /πναί· 95:1789-97 (1995); Borgstrdm et al, Cancer Res. 56:4032-39 (1996); Melnyk^ A 5 Cancer Res, 5 6:921_24 (1996) And also inhibit intraocular angiogenesis in a model of ischemic retinopathy (Adamis et al, drc/z. Ophthalmol. 114: 66-71 (1996)). Thus, anti-VEGF monoclonal antibodies or other VEGF effects Inhibitors are desirable candidates for the treatment of tumors and various intraocular neovascular disorders. Such antibodies are described, for example, in EP 817,648, published January 14, 1998, and WO 98/4533, published on October 15, 1998. 1 and WO -X wjw | rjy 〇▼ 丄la HiL· Less S |-^ - jv xxx cv cy yl-" is approved by the FDA in combination with a chemotherapy regimen for the treatment of metastatic colorectal cancer (CRC). Bevacizumab, which is used to treat various cancer indications, is also being studied in a number of ongoing clinical trials. Extracellular matrix (ECM) is known to play an important role in angiogenesis 0 Madri, rraw〆 5:179-83 (1997). The EC is surrounded by a temporary ECM during its migration and adheres to the newly synthesized vascular basement membrane after formation of the lumen. In addition to providing stents during capillary morphogenesis, ECM has also been shown to have complex local control of EC function. For example, ECM can regulate the availability of soluble angiogenic mediators for EC and define the nature and type of interaction with integrins and cell adhesion molecules. It has also been shown that the survival of EC is regulated by the synergy between growth factor receptors and integrins, which in turn is controlled by the composition of local ECM. Stupack and Cheresh, 22:9022-29 (2003) 〇 Despite many advances in the field of angiogenesis, some steps in the process of angiogenesis remain to be understood. In particular, how the tube formation is 119234.doc 200813088 Little is known about how the regulation of a vascular bundle progresses into a tube and what factors regulate this transformation. In view of the role of angiogenesis in many diseases and conditions, there is a need for a method of reducing or inhibiting one or more of the biological effects that cause such processes. There is also a need for a method of determining the presence of pathogenic polypeptides in normal and disease conditions, and particularly in cancer. There is also a need for compositions and methods that enhance the efficacy of existing anti-angiogenic therapies. SUMMARY OF THE INVENTION The present invention is based, in part, on identifying an anti-EGFL7 antibody having properties indicative of its particular benefit to treatment. In one aspect, the invention provides antibodies produced by fusion tumor anti-EGFL7 mumab 4F11.1.8, anti-EGFL7 mumab 10G9.1.6, and anti-EGFL7 mumab 18F7.1.8. In one aspect, the invention provides an anti-EGFL7 antibody comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) 4F11 CDR-L1 sequence KASQSVDYDGDSYMS (SEQ ID NO: 5 (b) 4F11 CDR-L2 sequence GASNLES (SEQ ID NO: 6); (c) 4F11 CDR-L3 sequence QQNNEDPYT (SEQ ID NO: 7); (d) 4F11 CDR-H1 sequence TYGMS (SEQ ID NO: 8); (e) 4F11 CDR-H2 sequence WINTHSGVPTYADDFKG (SEQ ID NO: 9); and (f) 4F11 CDR-H3 sequence LGSSA (SEQ ID NO: 10). In some embodiments, the light chain of the antibody comprises at least one, at least two, or all three CDR sequences selected from the group consisting of: KASQSVDYDGDSYMS (SEQ ID NO: 5), GASNLES (SEQ ID NO: 6), and QQNNEDPYT ( SEQ ID NO: 7). In some embodiments, the heavy chain of the antibody comprises at least one, 119234.doc •10·200813088 at least two or all three CDR sequences selected from the group consisting of: TYGMS (SEQ ID NO: 8), WINTHSGVPTYADDFKG (SEQ ID NO: 9) and LGSSA (SEQ ID NO: 10). In some embodiments, the light chain of the antibody comprises at least one, at least two, or all three CDR sequences selected from the group consisting of: KASQSVDYDGDSYMS (SEQ ID NO: 5), GASNLES (SEQ ID NO: 6), and QQNNEDPYT ( SEQ ID NO: 7); and the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of TYGMS (SEQ ID NO: 8), WINTHSGVPTYADDFKG (SEQ ID NO: 9) and LGSSA (SEQ ID NO: 10). In some embodiments, the light chain of the antibody comprises the sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMSWY QQKPGQPPKLLIYGASNLESGIPARFSGSGSGTDFTLNIHPV EEEDAATYYCQQNNEDPYTFGGGTKVEIKR (SEQ ID NO: I). In some embodiments, the heavy chain of the antibody comprises the sequence: QIQLVQSGPELKKPGETVKISCKASGHTFTTYGMSWVKQA PGKGLKWMGWINTHSGVPTYADDFKGRFAFSLETSASTAH LQINNLKNEDTATYFCARLGSSAVDYWGQGTTVTVSS (SEQ ID NO: 2). In one aspect, the invention provides an anti-EGFL7 antibody comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) 10G9 CDR-L1 sequence RSSQSLVHTNGITYLH (SEQ ID NO: II (b) 10G9 CDR-L2 sequence KVSNRFS (SEQ ID NO: 12); (c) 10G9 CDR-L3 sequence SQSTHVPLT (SEQ ID NO: 13); (d) 10G9 CDR-H1 sequence DYYMNSDYYMN (SEQ ID NO: 14); (e) 119234.doc -11 - 200813088 10G9 CDR-H2 sequence DINPKNGGTTYNQKFKG (SEQ ID NO: 15); and (f) 10G9 CDR-H3 sequence (SEQ ID NO: 16). In some embodiments, the light chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: RSSQSLVHTNGITYLH (SEQ ID NO: 11), KVSNRFS (SEQ ID NO: 12), and SQSTHVPLT ( SEQ ID NO: 13). In some embodiments, the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: DYYMNSDYYMN (SEQ ID NO: 14), DINPKNGGTTYNQKFKG fSFO ΤΓ> ΧΓΠ· 1 泠AT GVFFIV fSFn Τη ΜΠ· 1 .太一4b Caution, ▲,· A. VJ ▼ ▲ 一在,—一Jk. m, ▲,· Λ. j I - In the example, the light chain of the antibody contains at least one, at least two or all three CDR sequences from the following sequences: RSSQSLVHTNGITYLH (SEQ ID NO: 11), KVSNRFS (SEQ ID NO: 12), and SQSTHVPLT (SEQ ID NO: 13); and the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of DYYMNSDYYMN (SEQ ID NO: 14) ^ DINPKNGGTTYNQKFKG (SEQ ID NO: 15) and ALGVFDY (SEQ ID NO: 16). In some embodiments, the light chain of the antibody comprises the sequence: DIVMTQTPLSLPVSLGDQASISCR SSQSLVHTNGITYLHWYLQKPGQSPKLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGA GTKVEIKR (SEQ ID NO: 3). In some embodiments, the heavy chain of the antibody comprises the sequence: EVQLQQSGPELVKPGASVKISCKASG YTFSDYYMNSDYYMNWVKQSHGKSLEWIGDINPKNGGTT YNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARE ADYDPIYYAMDYWGQGTTLTVSA (SEQ ID NO: 4). 119234.doc -12- 200813088 In the second embodiment, the present invention provides anti-EGFL7 antibodies that specifically bind to the compound peptide comprising the following amino acid sequence: CCP, TIY and ACS. In one embodiment, the invention provides an isolated antibody that binds to the same epitope on human EGFT 7 ϊ», with other antibodies of the invention. In some embodiments, the invention provides isolated antibodies that compete with other antibodies of the invention for binding to Egfl7. In some embodiments, the antibodies of the invention are monoclonal antibodies. In some embodiments, the antibodies of the invention are chimeric antibodies, humanized antibodies, affinity human or bispecific antibodies. In some embodiments, the antibody is an antibody fragment. In some embodiments, the invention provides a pharmaceutical composition comprising an anti-EGFL7 antibody of the invention. In some embodiments, the pharmaceutical composition additionally comprises an anti-angiogenic agent. In some embodiments, the anti-angiogenic agent is bevacizumab or ranibizumab. In some embodiments, the invention provides a polynucleotide encoding an antibody of the invention. In some embodiments, the invention provides vectors comprising such polynucleotides. In some embodiments, the vector is a performance vector. In some embodiments, the invention provides host cells comprising such vectors, including prokaryotic and eukaryotic cells (including mammalian cells). In some embodiments, the invention provides a method of making an anti-EGFL7 antibody comprising (a) rendering a performance vector in a suitable host cell; and (b) recovering the antibody. In some embodiments, the invention provides a method of reducing or inhibiting angiogenesis in a subject having a pathological condition associated with angiogenesis, comprising administering to the subject an effective amount of an anti-EGFL7 of the invention Antibody or kit 119234.doc -13· 200813088 A pharmaceutical composition comprising an anti-EGFL7 antibody of the invention. In some embodiments, the pathological condition is a neoplasm, such as a carcinoma. In some embodiments, the pathological condition is associated with the eye, such as an intraocular neovascular disorder. In some embodiments, an anti-angiogenic agent is administered to a subject in addition to the anti-EGFL7 antibody of the present invention. In some embodiments, the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF), such as an anti-VEGF antibody (including bevacizumab and Raney strain). In some embodiments, the anti-angiogenic agent is administered before or after administration of the anti-EGFL7 antibody. In some embodiments, the anti-angiogenic agent is administered concurrently with an anti-EGFL7 antibody. In some embodiments, the invention provides a method of enhancing the efficacy of an anti-angiogenic agent in a subject having a pathological condition associated with angiogenesis comprising administering to the subject an anti-EGFL7 antibody of the invention Or a pharmaceutical composition comprising an anti-EGFL7 antibody of the invention. In some embodiments, the pathological condition is a neoplasm, such as a carcinoma. In some embodiments, the pathological condition is associated with the eye, such as an intraocular neovascular disorder. In some embodiments, an anti-angiogenic agent is also administered to a subject in addition to the anti-EGFL7 antibody of the present invention. In some embodiments, the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF), such as an anti-VEGF antibody (including bevacizumab and Raney strain monoclonal antibody). In some embodiments, the anti-angiogenic agent is administered before or after administration of the anti-EGFL7 antibody. In some embodiments, the anti-angiogenic agent is administered concurrently with an anti-EGFL7 antibody. In some embodiments, other treatments, such as corticosteroids or photodynamic therapy, are also administered. [Embodiment] The present invention provides an anti-EGFL7 antibody which can be used, for example, to treat or prevent the performance and/or activity of 119234.doc -14. 200813088 and EGFL7 (such as performance and/or activity enhancement or undesirable performance and / Or active) related disease state. In some embodiments, the antibodies of the invention are useful for treating tumors, cancer, and/or cell proliferative disorders. In another aspect, the anti-EGFL7 antibodies of the invention are useful as reagents for detecting and/or isolating EGFL7, such as EGFL7 in various tissues and cell types. The invention further provides methods of making an anti-EGFL7 antibody, a polynucleotide encoding an anti-EGFL7 antibody, and a cell comprising a polynucleotide encoding an anti-EGFL7 antibody. General Techniques Those skilled in the art are generally well aware of and generally use conventional techniques to use the techniques and procedures described or referenced herein, such as Sambrook et al. 'Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., (2003)); METHODS IN ENZYMOLOGY Series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor Edit (1995)), Harlow and Lane Editor (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R. I. Freshneyb, eds. (1987)). The "isolated' antibody is an antibody that has been identified and isolated from its natural environment and/or recovered from 119234.doc -15.200813088 or from the natural environment. Its natural environment is a substance that will interfere with the diagnosis of antibodies. It may include enzymes, hormones, and other proteins or non-proteins. In some embodiments, the body is purified to such a burial. The stalk is determined by the Lowry method.

於95重量％抗體且有時大於99重量％，·⑺經純化至足以藉由使用方疋轉杯式疋序儀獲得N末端或内部胺基酸序列之至少15個殘基之程度，·或⑺藉由在還原或非還原條件下使用考馬斯亮藍（c〇〇massie blue)或銀染色藉由sds_page法經純化至均質。由於抗體天然環境艾至少一種組份將不存在，故經分離之抗體包括原位處於重組細胞内之抗體。然而，經分離抗體通常藉由至少一個純化步驟製備。 π經分離”核酸分子為經鑑別且與至少一種通常在抗體核酸之天然來源中與其締合之污染物核酸分子分離的核酸分子。經分離核酸分子之形式或配置不同於其在自然中所見之形式或配置。由此使經分離核酸分子與存在於天然細胞中時之核酸分子相區別。然而，經分離核酸分子包括通常表現抗體之細胞中所含有的核酸分子，其中例如該核酸分子與天然細胞之核酸分子處於不同染色體位置中。術語π根據Kabat編號之可變域殘基”或”根據Kabat編號之胺基酸位”或其變體係指Kabat等人，Sequences of Proteins of Immunological Interest 5 第 5 版 Public Health Service， National Institutes of Health，Bethesda，MD· (1991)中用於抗體編碼之重鏈可變域或輕鏈可變域之編號系統。使用此編號系統，實際線性胺基酸序列可含有與可變域之FR或 119234.doc -16- 200813088 CDR縮短或***相對應之較少或額外胺基酸。舉例而言，重鏈可變域可包括H2殘基52後之單一胺基酸***（根據 Kabat之殘基52a)及重鏈FR殘基82後之***殘基（例如，根據Kabat之殘基82a、82b及82c等）。可藉由將抗體序列之同源區與’’標準"Kabat編號序列對準來確定既定抗體中殘基之Kabat編號。如本文所使用之短語，，實質上類似”或"實質上相同”表示 (、兩個數字值（一般一個數字值與本發明之抗體相關且另一個與參考/比較抗體相關）之間具有足夠高之相似度，從而使熟習此項技術者可認為在由該等值（例如&(1值）量測之生物學特徵範圍内該兩個值之間具有極小差異或不具有生物及/或統計學顯著差異。隨參考/比較抗體之值而變化的該兩個值之間的差異一般小於約5〇%、約4〇%、約3〇()/。、約 20%或約 1〇%。 ’’結合親和力” 一般係指分子（例如抗體）之單一結合位點 ( 與其結合搭配物（例如抗原）間之非共價相互作用的總強度。除非另作指示，否則如本文所使用之，，結合親和力，，係指反映結合對（例如抗體與抗原）成員之間1:1相互作用之固有、、々a親和力。分子X對其搭配物γ之親和力一般可由解離常數（Kd)表示。可藉由此項技術中已知之常用方法（包括本文所述之方法）量測親和力。低親和力抗體一般與抗原緩k結合且傾向於容易解離，而高親和力抗體一般與抗 f Q 口車又快且傾向於保持較長時間結合。此項技術中已知夕種里測結合親和力之方法，該等方法中之任一者均可用 H9234.doc -17- 200813088 於達成本發明之目的。以下描述特定例示性實施例。在一實施例中，如以下檢定所述藉由以所關注之抗體之 Fab型式及其抗原進行之放射性標記抗原結合檢定（RIA)來量測本發明之"Kd”或"Kd值”：在未經標記抗原之連續滴定存在下以最小濃度之（1251)標記抗原平衡Fab，接著捕捉與經抗Fab抗體塗覆之培養盤結合之抗原，藉此來量測Fab對抗原之溶液結合親和力（Chen等人，乂 Mo/· J5b/· 293:865-81 (1999))。為確立檢定條件，用50 mM碳酸鈉（pH 9·6)中之5 {ig/ml捕捉抗Fab抗體（Cappel Labs)塗覆徼量滴定盤 (Dynex)隔夜，且隨後在室溫（約23°C)下以PBS中之2% (w/v)牛血清白蛋白阻斷2至5小時。在無吸附劑培養盤 (Nunc #269620)中，將 100 pM或26 pM [1251]抗原與連續稀釋之所關注之Fab混合（例如與抗VEGF抗體Fab-12之評定相一致，Presta等人，57:4593-99 (1997))。接著培育所關注之Fab隔夜；然而，培育可持續一段較長之時間（例如65小時）以確保達到平衡。此後，在室溫下將混合物轉移至捕捉培養盤中以用於培育（例如歷時1小時）。接著移除溶液並用PBS中之0.1% Tween-20洗滌培養盤8次。當培養盤已經乾燥時，每孔添加150 μΐ閃燦體（MicroScint· 20; Packard)，且於Topcount γ計數器（Packard)上對培養盤計數10分鐘。選擇提供小於或等於20%最大結合之各Fab 濃度用於競爭性結合檢定。根據另一實施例，藉由在25°C 下使用表面電漿共振檢定使用BIAcoreTM-2000或 BIAcoreTM-3000 (BIAcore，Inc·，Piscataway，NJ)以約 10個反 119234.doc -18 - 200813088 應單元（RU)之固定抗原CM5晶片量測Kd或Kd值。簡而言之，根據供應商之說明書用N-乙基·Ν’-(3-二甲基胺基丙基）碳化二醯亞胺鹽酸鹽（EDC)及Ν-羥基琥珀醯亞胺（NHS) 活化羧曱基化葡聚糖生物感應器晶片（CM5，BIAcore Inc)。用10 mM乙酸鈉（pH 4·8)將抗原稀釋至5 pg/ml(約0·2 μΜ)，接著以每分鐘5 μΐ之流動速率進行注射以達成約10 個反應單元（RU)之偶聯蛋白。注射抗原後，注射1 Μ乙醇胺以阻斷未反應之基團。對於動力學量測而言，在25°C下以約25 μΐ/min冬流動速率注射在具有〇_〇5% Tween 20之 PBS (PBST)中兩倍連續稀釋之Fab (0.78 nM至500 nM)。使用簡單的一對一 Langmuir結合模型（BIAcore評估軟體3.2 版）藉由同時擬合締合與解離感應譜來計算締合速率（U 及解離速率（koff)。根據koff/k。!!之比率來計算平衡解離常數 (Kd)。例如參看 Chen，Y.等人，J· Mo/· 5b/. 293:865-881 (1999)。若由上述表面電漿共振檢定獲得之締合速率超過 106 NT1 S — 1，則可在如光譜儀（諸如裝備停流裝置之分光光度計（Aviv Instruments)或具有攪拌比色管之8000系列SLM-Aminco光譜光度計（ThermoSpectronic))所量測濃度遞增之抗原存在下，藉由使用於25t：下量測PBS (pH 7.2)中之20 nM抗抗原抗體（Fab形式）之螢光發射強度之增加或降低（激發=295 nm ;發射=340 nm，16 nm帶通）的螢光淬滅技術來測定締合速率。根據本發明，亦可在25 C下使用BIAcoreTM-2000或 BIAcoreTM_3 000 (BIAcore，Inc·，Piscataway，NJ)以與上述相 119234.doc -19- 200813088 同之表面電漿共振技術使用約10個反應單元（RU)之固定抗原CM5晶片測定”締合速率”或”kon’’。簡而言之，根據供應商之說明書用N-乙基-N’-(3-二甲基胺基丙基）碳化二醯亞胺鹽酸鹽（EDC)及N-羥基琥珀醯亞胺（NHS)活化羧甲基化葡聚糖生物感應器晶片（CM5, BIAcore Inc)。用10 mM乙酸鈉（pH 4.8)將抗原稀釋至5 pg/ml(約0·2 μΜ)，接著以每分鐘5 μΐ之流動速率進行注射以達成約10個反應單元（RU)之偶聯蛋白。注射抗原後，注射1 Μ乙醇胺以阻斷未反應之其囿。縣於私七恩蔷泪丨丨而古，Α 〇ς°Γ -ΤΓ ρ； ,,1 /min ^ U-I 'V、 /V -4 —，”V ^ I ，/、 V，w WV X / XXX X XX /7It. 動速率注射在具有0.05% Tween 20之PBS (PBST)中兩倍連續稀釋之Fab (0·78 nM至500 nM)。使用簡單的一對一 Langmuir結合模型（BIAcore評估軟體3.2版）藉由同時擬合締合與解離感應譜來計算締合速率及解離速率。根據k^f/kcn之比率來計算平衡解離常數（Kd)。例如參看 Chen，Y·等人，丄 Mo/· BzW· 293:865-81 (1999)。然而，若由上述表面電聚共振檢定獲得之締合速率超過1〇6 M-1 S·1，則一般在如光譜儀（諸如裝備停流裝置之分光光度計（Aviv Instruments)或具有攪拌比色管之8000系列SLM-Aminco光譜光度計（ThermoSpectronic))所量測濃度遞增之抗原存在下，藉由使用於25°C下量測PBS (pH 7·2)中之20 nM抗抗原抗體（Fab形式）之螢光發射強度之增加或降低（激發=295 nm;發射=340 rnn，16 nm帶通）的螢光淬滅技術來測定締合速率。如本文所使用之術語”載體"意指能夠轉運所連接之另一 119234.doc -20- 200813088 核酸分子之核酸分子。一類載體為”質體"，其係指可接合額外DNA區段之環形雙鏈DNA環。另一類載體為噬菌體載體。另一類載體為病毒載體，其中可將額外DNA區段接合至病毒基因組中。某些載體能夠在引入該等載體之宿主細胞中自主複製（例如具有細菌複製起點之細菌載體及游離型嗜乳動物載體）。其他載體（例如非游離型哺乳動物載體）可在引入宿主細胞之後整合至宿主細胞之基因組中，且藉此與宿主基因組一起複製。此外，某些載體能夠指導與其以可％作方式連接之基因之表現。在本文中將此等載辦稱為π重組表現載體π(或簡稱為”重組載體”）。一般而言，可用於重組DNA技術中之表現載體通常為質體之形式。在本次明書中’由於負體係最常用之載體形式，故”質體"盘，，載體π可互換使用。如本文可互換使用之"聚核苷酸"或”核酸”係指任何長度之核苦酸之聚合物，且包括DNA及RNA。核普酸可為去氧核糖核苷酸、核糖核苷酸、經修飾之核苷酸或驗基及/或其類似物’或可藉由DNA或RNA聚合酶或合成反應併入聚合物中之任何基質。聚核苷酸可包含經修飾之核皆酸，諸如甲基化核苷酸及其類似物。若存在對核苷酸結構之修飾’則可在組裝成聚合物之前或之後進行修飾。核苦酸序列可由非核苷酸組份間斷。可在合成後（諸如）藉由與標記共耗對聚核苷酸進行進一步修飾。其他類型之修都包括 (例如），，封端”；一或多個天然存在之核苷酸經類似物取代；核苷酸間修飾，諸如具有不帶電鍵結（例如，膦酸甲 H9234.doc -21- 200813088 酯、磷酸三酯、磷醯胺酸、胺基甲酸酯等）及帶電鍵結（例如，硫代磷酸酯、二硫代磷酸酯等）之聚核苷酸，含有諸如蛋白質（例如核酸酶、毒素、抗體、信號肽、聚_L_離胺酸等）之側位部分之聚核苷酸，具有嵌入劑（例如吖啶、補骨脂素（psoralen)等）之聚核苷酸，含有螯合劑（例如金屬、放射性金屬、硼、氧化性金屬等）之聚核苷酸，含有烷化劑之聚核苷酸，具有經修飾鍵結之聚核苷酸（例如α變旋異構核酸等）以及未經修飾形式之聚核苷酸。另外，通常存在於糖中之任何羥基均可（例如）經膦酸酯基、磷酸酯基置換’經標準保護基團保護或經活化以製備與額外核苦酸之額外鍵結；或可與固體或半固體支撐物共軛。5，及3,末端 ΟΗ可經磷酸化或經胺或具有1至2〇個碳原子之有機封端基團部分取代。其他羥基亦可衍生成為標準保護基團。聚核苷酸亦可含有此項技術中一般已知之核糖或去氧核糖之類似形式，包括（例如）2，-0-曱基-、2，-0-烯丙基、2，-氟-或 2’-疊氮基-核糖；碳環糖類似物；α_變旋異構糖；差向異構糖，諸如阿缸伯糠、木糖或來蘇糖；哌喃糖；呋喃糖；景天庚酮糖；非環狀類似物；及基本核苷類似物，諸如甲基核糖苷。一或多個磷酸二酯鍵可經替代性連接基團置換。此等替代性連接基團包括（但不限於）磷酸酯經P(〇)S (’’硫醇酯”）、P(S)S (，，二硫醇酯”）、（〇)NR2 (”醯胺化物，，）、 P(〇)R、P(0)0R’、CO或CH2 (”甲縮醛”）置換之實施例，其中各R或R’獨立地為Η或視情況含有醚（-〇-)鍵之經取代或未經取代之烷基（1-20個C)、芳基、烯基、環烷基、環烯基 119234.doc -22- 200813088 或芳烷基。聚核苷酸中之所有鍵結無需相同。上文之描述適用於本文所提及之所有聚核苷酸，包括RNA及DNA。如本文所使用之”寡核苷酸”一般係指長度一般（但非必需）小於約200個核苷酸之短的、一般為單鏈、一般為合成之聚核苷酸。術語，，寡核苷酸，，及，，聚核苷酸"並不相互排除。上文有關聚核苷酸之描述同樣且完全適用於寡核苦酸。除非另外明確指示或於上下文中另作指示，否則如本文所使用t術語"EGFL7”（可互換稱為，，表皮生長因子樣7")係指如（例如）WO 2005/117968中所述之任何天然或變異體 (天然或合成）EGFL7多肽，該專利之揭示内容以全文引用之方式併入本文中用於所有目的。術語"天然序列”特別涵蓋天然存在之截斷或分泌形式（例如胞外域序列）、天然存在之變異體形式（例如替代性剪接形式）及天然存在之等位基因變異體。術語"野生型EGFL7”一般係指包含天然存在之EGFL7蛋白之胺基酸序列的多肽。術語"野生型£(}1^7序列一般係指於天然存在之EGFL7中發現之胺基酸序列。術語"抗體”與”免疫球蛋白"在廣義上可互換使用，且包括單株抗體（例如全長或完整單株抗體）、多株抗體、多價抗體、多特異性抗體（例如雙特異性抗體，只要其展現出所需之生物活性即可），且亦可包括某些抗體片段（如本文更詳細地描述）。抗體可為人類抗體、人化抗體及/或親和力成熟抗體。術語"可變"係指抗體間可變域之某些部分的序列普遍不 119234.doc •23- 200813088 同且該等部分用於各特定抗體對其特定抗原之結合及特異性的事實。然而，變異性並非均勻分佈於整個抗體可變域中。其集中於輕鏈與重鏈可變域中稱為互補判定區（CDR) 或高變區之三個區段。可變域中高度保守之部分稱為構架 (FR)。天然重鏈及輕鏈之可變域各自包含主要採用β_折疊構型的由三個CDR連接之四個FR區，其形成環連接，且在一些情況下形成β-折疊結構之部分。各鏈中之CDR由FR區以緊密接近之方式固持在一起，且與其他鏈之CDR—起有助於形成抗體之抗原結合位點（參看Kabat等人，Sequences of Proteins of Immunological Interest，第 5版，National95% by weight of antibody and sometimes more than 99% by weight, (7) purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a square-turned cup formater, or (7) Purification to homogeneity by sds_page method by using coomassie blue or silver staining under reducing or non-reducing conditions. Since at least one component of the antibody natural environment will not exist, the isolated antibody includes an antibody that is in situ in a recombinant cell. However, isolated antibodies are typically prepared by at least one purification step. A π-isolated nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule that is normally associated with it in the natural source of the antibody nucleic acid. The isolated nucleic acid molecule is in a form or configuration different from that found in nature. Form or configuration whereby the isolated nucleic acid molecule is distinguished from the nucleic acid molecule present in the native cell. However, the isolated nucleic acid molecule comprises a nucleic acid molecule contained in a cell that typically exhibits an antibody, wherein, for example, the nucleic acid molecule is The nucleic acid molecule of the cell is in a different chromosomal location. The term π is a variable domain residue according to Kabat numbering or "amino acid acid number according to Kabat number" or its variant system refers to Kabat et al., Sequences of Proteins of Immunological Interest 5 Numbering system for antibody-encoded heavy chain variable domains or light chain variable domains in the 5th edition of Public Health Service, National Institutes of Health, Bethesda, MD (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the FR of the variable domain or the CDR shortening or insertion of the 119234.doc-16-200813088 CDR. For example, the heavy chain variable domain can include a single amino acid insertion following H2 residue 52 (residue 52a according to Kabat) and an insertion residue following heavy chain FR residue 82 (eg, residues according to Kabat) 82a, 82b, 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by aligning the homologous region of the antibody sequence to the ''standard"Kabat numbering sequence. As used herein, the phrase substantially similar to "or "substantially identical" means (between two numerical values (generally one numerical value associated with an antibody of the invention and another associated with a reference/comparative antibody) Having a sufficiently high degree of similarity that one skilled in the art can recognize that there is little or no difference between the two values within the range of biological characteristics measured by the equivalent (eg, & 1 value) And/or statistically significant difference. The difference between the two values that varies with the value of the reference/comparison antibody is generally less than about 5%, about 4%, about 3 〇 () /, about 20% or Approximately 1%. ''Binding Affinity' generally refers to the total strength of a non-covalent interaction between a single binding site of a molecule (eg, an antibody) (with respect to its binding partner (eg, an antigen), unless otherwise indicated) As used herein, binding affinity refers to the intrinsic, 々a affinity of a 1:1 interaction between a binding pair (eg, an antibody and an antigen) member. The affinity of the molecule X for its conjugate is generally determined by the dissociation constant. (Kd) said. Affinity can be measured by common methods known in the art, including the methods described herein. Low affinity antibodies generally bind to the antigen slow k and tend to dissociate easily, while high affinity antibodies are generally associated with anti-f Q It is fast and tends to remain for a longer period of time. A method for measuring binding affinity is known in the art, and any of these methods can be used for the purpose of the present invention by H9234.doc -17-200813088. Specific exemplary embodiments are described. In one embodiment, the "Kd of the present invention is measured by radiolabeled antigen binding assay (RIA) with a Fab version of the antibody of interest and its antigen as described below "or "Kd value": labeling the antigen at a minimum concentration (1251) in the presence of a continuous titration of unlabeled antigen to equilibrate the Fab, followed by capture of the antigen bound to the culture plate coated with the anti-Fab antibody, thereby The binding affinity of Fab to the antigen is measured (Chen et al., 乂Mo/·J5b/· 293:865-81 (1999)). To establish the assay conditions, 5 of 50 mM sodium carbonate (pH 9·6) is used. Ig/ml capture anti-Fab antibody (Cappel Labs) coated with a titration plate (Dynex) overnight and then blocked with 2% (w/v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (about 23 ° C). In a non-sorbent culture plate (Nunc #269620), 100 pM or 26 pM [1251] antigen was mixed with serially diluted Fab of interest (eg, consistent with evaluation of anti-VEGF antibody Fab-12, Presta et al., 57). :4593-99 (1997)). Then raise the Fab of interest overnight; however, the cultivation lasts for a longer period of time (eg 65 hours) to ensure equilibrium. Thereafter, the mixture is transferred to a capture culture dish at room temperature for incubation (e.g., for 1 hour). The solution was then removed and the plate was washed 8 times with 0.1% Tween-20 in PBS. When the plate was dry, 150 μM of blister (MicroScint·20; Packard) was added to each well, and the plate was counted on a Topcount γ counter (Packard) for 10 minutes. Each Fab concentration providing a maximum binding of less than or equal to 20% is selected for competitive binding assays. According to another embodiment, BIAcoreTM-2000 or BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) is used with about 10 counters 119234.doc -18 - 200813088 by using a surface plasma resonance assay at 25 °C. The fixed antigen CM5 wafer of the unit (RU) measures the Kd or Kd value. Briefly, N-ethyl Ν'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and hydrazine-hydroxysuccinimide (Nicong) were used according to the supplier's instructions. NHS) Activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc). The antigen was diluted to 5 pg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4·8), followed by injection at a flow rate of 5 μM per minute to achieve about 10 reaction units (RU). Connexin. After the antigen was injected, 1 Μ ethanolamine was injected to block the unreacted group. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected at 25 ° C at a winter flow rate of about 25 μΐ/min in PBS with 〇〇 5% Tween 20 (PBST). ). The association rate (U and dissociation rate (koff) was calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2) by simultaneously fitting the association and dissociation induction spectra. According to the ratio of koff/k.!! To calculate the equilibrium dissociation constant (Kd), for example, see Chen, Y. et al., J. Mo/. 5b/. 293: 865-881 (1999). If the association rate obtained by the above surface plasma resonance test exceeds 106 NT1 S — 1, an increasing concentration of antigen can be measured in a spectrometer such as a spectrophotometer equipped with a flow stop (Aviv Instruments or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirring colorimetric tube) In the presence of an increase or decrease in fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25t: excitation = 295 nm; emission = 340 nm, 16 nm Fluorescence quenching technique to determine the association rate. According to the invention, BIAcoreTM-2000 or BIAcoreTM_3 000 (BIAcore, Inc., Piscataway, NJ) can also be used at 25 C to interact with the above phase 119234.doc - 19- 200813088 Same use of surface plasma resonance technology Approximately 10 reaction units (RU) of immobilized antigen CM5 wafers were measured for "association rate" or "kon". Briefly, N-ethyl-N'-(3-dimethyl) was used according to the supplier's instructions. Aminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc) with 10 mM acetic acid Sodium (pH 4.8) was diluted to 5 pg/ml (about 0.2 μM), followed by injection at a flow rate of 5 μM per minute to achieve about 10 reaction units (RU) of coupled protein. Inject 1 Μ ethanolamine to block the unreacted 囿. The county is in the private seven 蔷蔷 tears and ancient, Α 〇ς °Γ -ΤΓ ρ; ,, 1 / min ^ UI 'V, /V -4 — ,"V ^ I , /, V, w WV X / XXX X XX /7It. Dynamically injected Fab (0·78 nM to 500 nM) in two-fold serial dilutions in PBS (PBST) with 0.05% Tween 20 Calculate the association rate and dissociation rate by simultaneously fitting the association and dissociation induction spectra using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software Version 3.2). Calculate the ratio based on the ratio of k^f/kcn The dissociation constant (Kd). See, for example, Chen, Y. et al., 丄 Mo/· BzW. 293:865-81 (1999). However, if the association rate obtained by the above surface electropolymerization resonance test exceeds 1〇6 M-1 S·1, it is generally in a spectrometer such as a spectrophotometer equipped with a flow stop device (Aviv Instruments) or with a stirring colorimeter. 20 nM anti-antibody antibody (Fab in PBS (pH 7.2) was measured at 25 ° C in the presence of increasing concentrations of antigen in a 8000 Series SLM-Aminco Spectrophotometer (ThermoSpectronic) Fluorescence quenching techniques for increasing or decreasing the intensity of fluorescence emission (excitation = 295 nm; emission = 340 rnn, 16 nm bandpass) to determine the association rate. The term "vector" as used herein, refers to a nucleic acid molecule capable of transporting another 119234.doc -20-200813088 nucleic acid molecule to which it is linked. One type of vector is "plastid", which refers to an additional DNA segment that can be ligated. A circular double-stranded DNA loop. Another type of vector is a phage vector. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which such vectors are introduced (e.g., a bacterial vector having a bacterial origin of replication and a free mammalian vector). Other vectors (e. g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the performance of genes that are linked in a % manner. These vectors are referred to herein as π recombinant expression vectors π (or simply "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are typically in the form of plastids. In this book, 'the most commonly used carrier form of the negative system, so the plastid "disk, the carrier π is used interchangeably. As used herein, the "polynucleotide" or "nucleic acid" system Refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotide can be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or a test group, and/or an analog thereof. Or any matrix that can be incorporated into the polymer by DNA or RNA polymerase or synthetic reaction. The polynucleotide may comprise a modified nucleocapamic acid, such as a methylated nucleotide and analogs thereof. The modification of the structure of the nucleotide structure can be modified before or after assembly into a polymer. The nucleotide sequence can be interrupted by a non-nucleotide component. The polynucleotide can be synthesized after synthesis, such as by co-consumption with the label. Further modifications. Other types of modifications include, for example, "capping"; one or more naturally occurring nucleotides are substituted by analogs; internucleotide modifications, such as having an uncharged bond (eg, phosphonic acid) A H9234.doc -21- 200813088 ester, phosphate triester, phosphorus Polynucleotides such as lysine, urethane, etc., and charged linkages (eg, phosphorothioates, phosphorodithioates, etc.), such as proteins (eg, nucleases, toxins, antibodies, signal peptides) a polynucleotide of a lateral portion of poly(L_isoamine, etc.), a polynucleotide having an intercalating agent (for example, acridine, psoralen, etc.), and a chelating agent (for example, metal, a polynucleotide of a radioactive metal, boron, an oxidizing metal, or the like, a polynucleotide containing an alkylating agent, a modified-bonded polynucleotide (for example, a α-helical heteromeric nucleic acid, etc.), and unmodified Form of a polynucleotide. In addition, any of the hydroxyl groups normally present in the sugar may, for example, be replaced by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages with additional nucleotides; or The solid or semi-solid support is conjugated. 5, and 3, terminal hydrazine may be substituted by phosphorylation or by an amine or an organic capping group having 1 to 2 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2,-0-mercapto-, 2,-0-allyl, 2,-fluoro- Or 2'-azido-ribose; carbocyclic sugar analogue; alpha-spin isomeric sugar; epimeric sugar, such as Acacia, xylose or lyxose; palladium; furanose; Sedum heptanose; acyclic analog; and a basic nucleoside analog such as methyl riboside. One or more phosphodiester linkages can be replaced by an alternative linking group. Such alternative linking groups include, but are not limited to, phosphate esters via P(〇)S (''thiol esters)), P(S)S (,, dithiol esters), (〇)NR2 ( Examples of substitutions of "ammonium,", P(〇)R, P(0)0R', CO or CH2 ("methylal"), wherein each R or R' is independently Η or optionally A substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl 119234.doc-22-200813088 or aralkyl group of an ether (-〇-) linkage. All linkages in a polynucleotide need not be the same. The above description applies to all polynucleotides referred to herein, including RNA and DNA. As used herein, "oligonucleotide" generally refers to a length generally (but not necessarily) a short, generally single-stranded, generally synthetic polynucleotide of less than about 200 nucleotides. The term, oligonucleotide, and, polynucleotide " Mutual exclusion. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise indicated or indicated otherwise in the context, the term t "EGFL7" In other words, epidermal growth factor-like 7" refers to any natural or variant (natural or synthetic) EGFL7 polypeptide as described, for example, in WO 2005/117968, the disclosure of which is incorporated by reference in its entirety Used in this article for all purposes. The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternative splicing forms), and naturally occurring allelic variants. The term "wild type EGFL7" generally refers to a polypeptide comprising an amino acid sequence of a naturally occurring EGFL7 protein. The term "wild type £(1) sequence generally refers to the amino acid sequence found in naturally occurring EGFL7. The terms "antibody" and "immunoglobulin" are used interchangeably and include Strain antibodies (eg full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg bispecific antibodies, as long as they exhibit the desired biological activity), and may also include certain An antibody fragment (as described in more detail herein). The antibody can be a human antibody, a humanized antibody, and/or an affinity matured antibody. The term "variable" refers to the sequence of certain portions of the variable domain between antibodies that are generally not 119234 .doc •23- 200813088 The same fact that these components are used for the binding and specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. It is concentrated in the light chain and heavy The variable region of the chain is called the complementarity determining region (CDR) or the three regions of the hypervariable region. The highly conserved portion of the variable domain is called the framework (FR). The variable domains of the native heavy and light chains each contain Mainly adopt β_ fold a configuration of four FR regions joined by three CDRs that form a loop junction and, in some cases, form part of a beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and Together with the CDRs of other chains, it helps to form antigen binding sites for antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, National)

Institute of Health，Bethesda，MD (1991))。抗體與抗原之結合並不直接涉及恆定域，但其展現出各種效應功能，諸如使抗體參與抗體依賴性細胞毒性。木瓜酵素消化抗體會產生兩個各自具有單一抗原結合位點稱為Fab’’片段的相同抗原結合片段，及剩餘"Fc"片段，其名稱反映其易於結晶之能力。胃蛋白酶處理會得到具有兩個抗原組合位點且仍能夠交聯抗原之F(ab，）2片段。 n F v ”為含有完整抗原識別位點及結合位點之最小抗體片段。在雙鏈Fv種類中，此區域係由緊密、非共價締合之一個重鏈可變域與一個輕鏈可變域之二聚體組成。在單鏈& 種類中，一個重鏈可變域與一個輕鏈可變域可經彈性肽連接子共價連接，從而使輕鏈與重鏈可以與兩鏈以種類中之結構類似之"二聚"結構締合。各可變域之三個cdr即係以此構型相互作用而界定VH_VL二聚體表面上之抗原結合位 119234.doc -24- 200813088 點。總起來說，六個CDR賦予抗體抗原結合特異性。然而，甚至單一可變域（或僅包含三個抗原特異性CDR之一半Fv)亦具有識別並結合抗原之能力，但其親和力低於完整結合位點。Institute of Health, Bethesda, MD (1991)). The binding of an antibody to an antigen does not directly involve the constant domain, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cellular cytotoxicity. Papaya enzyme digestion of the antibody produces two identical antigen-binding fragments each having a single antigen-binding site called a Fab'' fragment, and the remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment results in an F(ab,)2 fragment that has two antigen combining sites and is still capable of cross-linking the antigen. n F v ” is the smallest antibody fragment containing the entire antigen recognition site and binding site. In the double-stranded Fv species, this region is composed of a tight, non-covalent association of a heavy chain variable domain and a light chain. Dimorphic dimer composition. In the single-stranded & species, one heavy chain variable domain and one light chain variable domain can be covalently linked via an elastic peptide linker, such that the light and heavy chains can be linked to both chains A structurally similar "dimeric" structure association in the species. The three cdr of each variable domain define the antigen binding site on the surface of the VH_VL dimer by this configuration interaction 119234.doc -24 - 200813088 points. In total, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or only one of the three antigen-specific CDRs) will have the ability to recognize and bind antigen, but Affinity is lower than the intact binding site.

Fab片段亦含有輕鍵怪定域及重鍵第一恆定域（chi)。 Fab’片段因在重鏈CH1結構域之羧基末端添加有少量殘基 (包括一或多個來自抗體鉸鏈區之半胱胺酸）而不同於Fab片段。Fab’-SH在本文中係對恆定域之半胱胺酸殘基帶有游離硫醇基之Fab，的命名。最初F(ab，)2抗體片段經製造為其間具有鉸鏈半胱胺酸之Fab’片段對。亦已知抗體片段之其他化學偶聯。可基於恆定域之胺基酸序列將任何脊椎動物物種之抗體 (免疫球蛋白）的”輕鏈”分為兩種截然不同之類型（稱為尺及 λ)中的·一種。The Fab fragment also contains a light bond domain and a first constant domain (chi). The Fab' fragment differs from the Fab fragment by the addition of a small number of residues (including one or more of the cysteine from the antibody hinge region) at the carboxy terminus of the heavy chain CH1 domain. Fab'-SH is herein referred to as the Fab of a constant domain cysteine residue with a free thiol group. The original F(ab,)2 antibody fragment was made into a Fab' fragment pair with hinged cysteine. Other chemical couplings of antibody fragments are also known. The "light chain" of antibodies (immunoglobulins) of any vertebrate species can be divided into one of two distinct types (referred to as the ruler and λ) based on the amino acid sequence of the constant domain.

視重鏈恆定域之胺基酸序列而定，可將免疫球蛋白分為不同種類。存在五種主要的免疫球蛋白類別：lgA、igD、 IgE、IgG及IgM，且此等類別之數種可進一步細分成子類 (同型）：例如 IgGl、IgG2、lgG3、IgG4、IgA1 及 IgA2。與不同類別之免疫球蛋白相對應之重鏈怪定域分別稱為α、 δ ε γ及μ。不同類別之免疫球蛋白之次單元結構及三維構型係熟知的。 "抗體片段"僅包含完整抗體之一部分，#中該部分較佳保留至4 -種’較佳大部分或所有當存在於完整抗體中時通常與該部分相關之功能。抗體片段之實例包括㈣、 119234.doc -25- 200813088Immunoglobulins can be classified into different classes depending on the amino acid sequence of the heavy chain constant domain. There are five major classes of immunoglobulins: lgA, igD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes): for example, IgG1, IgG2, lgG3, IgG4, IgA1, and IgA2. The heavy chain strange domains corresponding to different classes of immunoglobulins are called α, δ ε γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. "antibody fragment" contains only one part of the intact antibody, and this part of # is preferably retained to a 4-type 'better' or a majority of all functions normally associated with the part when present in the intact antibody. Examples of antibody fragments include (iv), 119234.doc -25- 200813088

Fab，、F(ab，)2及Fv片段，雙功能抗體，線性抗體，單鏈抗體分子及由抗體片段形成之多特異性抗體。在一實施例中，抗體片段包含完整抗體之抗原結合位點且因此保留結合抗原之能力。在另一實施例中，抗體片段（例如包含Fc 區之片段）保留至少一種當存在於完整抗體中時通常與以區相關之生物功能’諸如FcRn結合、抗體半衰期調節、 ADCC功能及補體結合。在一實施例中，抗體片段為具有實質上與完整抗體類似之活體内半衰期的單價抗體。舉例而3 ’此k體片#又可包含連接至f c序列能夠賦予該片段活體内穩定性之抗原結合臂。當用於本文中時’術语"面.變區，’、"Hvr"或”HV，，係指序列高變且/或形成結構上確定之環之抗體可變域的區域。大體而a ’抗體包含/、個面變區，三個在VH (HI、H2、 H3)中且三個在VL (L1、L2、L3)中。本文中使用且涵蓋多種有關雨變區之描繪。Kabat互補判定區（CDR)係基於序列變異性且最常使用（Kabat等人，Sequences 〇f Pr〇teins of Immunological Interest，第 5 版，Public Health Service，Fab, F(ab,)2 and Fv fragments, bifunctional antibodies, linear antibodies, single-stranded antibody molecules and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen binding site of the intact antibody and thus retains the ability to bind to the antigen. In another embodiment, an antibody fragment (e.g., a fragment comprising an Fc region) retains at least one, when present in an intact antibody, typically associated with a region-associated biological function such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to an intact antibody. For example, 3' this k-body fragment # may further comprise an antigen-binding arm linked to the fc sequence to confer stability to the fragment in vivo. As used herein, the term "face.variant,", "Hvr" or "HV," refers to the region of the antibody variable domain that is hypervariable and/or forms a structurally defined loop. Whereas a 'antibody contains /, a facet variable region, three in VH (HI, H2, H3) and three in VL (L1, L2, L3). This article uses and covers a variety of depictions of rain zones. The Kabat complementarity determining region (CDR) is based on sequence variability and is most commonly used (Kabat et al., Sequences 〇f Pr〇teins of Immunological Interest, 5th edition, Public Health Service,

National Institutes of Health, Bethesda，MD. (1991)) 〇而National Institutes of Health, Bethesda, MD. (1991))

Chothia涉及結構環之位置（Chothia 及 Lesk J. Mo/. 196:901-17 (1987))。AbM高變區表示Kabat CDR 與 Chothia 結構環間之折衷，且藉由Oxford Molecular’s AbM抗體模型化軟體加以使用。’’接觸’’高變區係基於對可用複雜晶體結構之分析。高變區可包含如下"延長高變區”：乂1^中之24-36 (1^1)、 119234.doc -26- 200813088 46-56 (L2)及 89-97 (L3);及 VH 中之 26-35 (H1)、49_65 或 50至65 (H2)及93-102 (H3)。關於此等定義中之各者可根據Kabat等人（同上文）對可變域殘基進行編號。 π構架’’或nFR”殘基為除如本文所定義之高變區殘基外的彼等可變域殘基。 π人化’’形式之非人類（例如鼠科）抗體為含有自非人類免疫球蛋白獲得之最小序列的嵌合抗體。在極大程度上，人化抗體為人類免疫球蛋白（接受者抗體），其中接受者高變區之殘基經具有所需特異性、親和力及能力之非人類物種 (供體抗體）（諸如小鼠、大鼠、兔或非人類靈長類動物）高變區之殘基置換。在一些情況下，人類免疫球蛋白之構架 £ (FR)殘基經相應之非人類殘基置換。此外，人化抗體可包含未見於接受者抗體或供體抗體中之殘基。進行此等修飾以進一步改進抗體之效能。一般而言，人化抗體將包含實質上所有至少一個且通常兩個可變域，其中所有或實質上所有南變環對應於非人類免疫球蛋白之高變環，且所有或實質上所有FR為人類免疫球蛋白序列之Fr。人化抗體視情況亦將包含至少一部分免疫球蛋白恆定區（Fc)，通常為人類免疫球蛋白之恆定區。有關其他細節，參看J〇nes 等人 ’ 狀e 321:522-25 (1986); Riechmann等人， 332:323-29 (1988)及 Presta，Cwrr. Op. Sirwc，· 5ζ·ο/· 2:593-96 (1992)。亦可參看以下評論論文及其中所引用之參考文獻· Vaswani及 Hamilton，J⑽所α ά /所所⑽ο/· 1.105-15 (1998); Harris, Biochem. Soc. Transactions 119234.doc -27- 200813088 23:1035-38 (1995); Hurle 及 Gross，Cwrr. 0户· 5:428-33 (1994)〇 n嵌合”抗體（免疫球蛋白）具有一部分與自特定物種或屬於特定抗體類別或子類獲得之抗體中的相應序列相同或同源的重鏈及/或輕鏈，而鏈之剩餘部分與自另一物種或屬於另一抗體類別或子類獲得之抗體以及此等抗體之片段中的相應序列相同或同源，只要其展現出所需之生物活性即可（美國專利第4,816,567號及Morris on等人，Pro c. ^cad.心?·. ？7以81:6851-6855 (1984))。如本文所使用之人化抗體為嵌合抗體之子集。 π單鏈Fvn或nscFv”抗體片段包含抗體之VH及VL結構域，其中此等結構域係存在於單一多肽鏈中。一般而言， scFv多肽另外包含VH與VL結構域之間使scFv能夠形成抗原結合所需結構之多肽連接子。有關svFv之評論，參看 Pluckthun, The Pharmacology 〇f Monoclonal Antibodies » 第 113 卷，Rosenburg 及 Moore 編輯，Springer-Verlag，NewChothia relates to the position of the structural ring (Chothia and Lesk J. Mo/. 196:901-17 (1987)). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the Oxford Molecular's AbM antibody modeling software. The 'contact' hypervariable zone is based on an analysis of the available complex crystal structures. The hypervariable region may include the following "extended hypervariable regions": 24-36 (1^1), 119234.doc -26-200813088 46-56 (L2) and 89-97 (L3); 26-35 (H1), 49_65 or 50 to 65 (H2) and 93-102 (H3) in VH. Each of these definitions may be based on Kabat et al. (supra) for variable domain residues. The π-framework ''or nFR') residues are those variable domain residues other than the hypervariable region residues as defined herein. A non-human (e.g., murine) antibody of the π humanized '' form is a chimeric antibody containing the smallest sequence obtained from a non-human immunoglobulin. To a large extent, humanized antibodies are human immunoglobulins (recipient antibodies) in which the residues of the hypervariable region of the recipient are passed through a non-human species (donor antibody) having the desired specificity, affinity and ability (such as small Residue replacement in the hypervariable region of murine, rat, rabbit or non-human primate. In some cases, the framework of the human immunoglobulin £ (FR) residue is replaced by a corresponding non-human residue. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the efficacy of the antibody. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the south variable loop corresponds to a hypervariable loop of a non-human immunoglobulin, and all or substantially all of the FR It is the Fr of the human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For additional details, see J〇nes et al.' s e 321:522-25 (1986); Riechmann et al., 332:323-29 (1988) and Presta, Cwrr. Op. Sirwc, · 5ζ·ο/· 2 :593-96 (1992). See also the following review papers and references cited therein. Vaswani and Hamilton, J(10), α ά / Institute (10) ο/· 1.105-15 (1998); Harris, Biochem. Soc. Transactions 119234.doc -27- 200813088 23 :1035-38 (1995); Hurle and Gross, Cwrr. 0 household · 5:428-33 (1994) 嵌合n chimeric antibodies (immunoglobulins) have a part of a specific species or belong to a specific antibody class or subclass The heavy or/and light chain of the same sequence or homologous in the obtained antibody, and the remainder of the strand and the antibody obtained from another species or belonging to another antibody class or subclass and fragments of such antibodies The corresponding sequences are identical or homologous as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morris on et al, Pro c. ^cad. heart?..7 to 81:6851-6855 (1984) The humanized antibody as used herein is a subset of chimeric antibodies. The π single-chain Fvn or nscFv" antibody fragment comprises the VH and VL domains of the antibody, wherein such domains are present in a single polypeptide chain. In general, the scFv polypeptide additionally comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of svFv, see Pluckthun, The Pharmacology 〇f Monoclonal Antibodies » Volume 113, Rosenburg and Moore Editor, Springer-Verlag, New

York ，第 269-315頁（1994)。 n抗原”為抗體可選擇性結合之預定抗原。目標抗原可為多肽、醣、核酸、脂質、半抗原或其他天然存在或合成之化合物。一般而言，目標抗原為多肽。 ”抗原決定基”為抗體選擇性結合之抗原部分。對於多狀抗原而言，抗原決定基一般為具有約4-1 0個胺基酸之肽部分。術# ”雙功能抗體”係指具有兩個抗原結合位點之小抗體 119234.doc -28- 200813088York, pp. 269-315 (1994). An n antigen is a predetermined antigen to which an antibody can selectively bind. The antigen of interest may be a polypeptide, a sugar, a nucleic acid, a lipid, a hapten or other naturally occurring or synthetic compound. Generally, the antigen of interest is a polypeptide. "Antigenic determinant" An antigenic moiety that selectively binds to an antibody. For a polymorphic antigen, the epitope is typically a peptide moiety having about 4 to 10 amino acids. [#] "Bifunctional antibody" refers to having two antigen binding sites. Small antibody 119234.doc -28- 200813088

片段’該等片段包含與相同多肽鏈（VH-VL)中之輕鏈可變域（VL)連接之重鏈可變域（VH)。藉由使用過短而使相同鏈上之兩個結構域之間無法配對之連接子，迫使該等結構域與另一鏈之互補結構域配對並產生兩個抗原結合位點。雙功能抗體更詳細地描述於（例如）EP 404,097、WO 93/11161 及 Hollinger 等人，Procr. Wi/· dcd 5W. t/U， 90:6444-48 (1993)中。 π人類抗體，，為具有與人類所產生之抗體之胺基酸序列相對應的胺基酸序列且/或使用本文所揭示之任何製造人類抗體之技術製得的抗體。此有關人類抗體之定義特別排除包含非人類抗原結合殘基之人化抗體。 Μ親和力成熟”抗體為在一或多個CDR中具有一或多種變化之抗體，與不具有彼等變化之親本抗體相比較，彼等變化使得抗體對抗原之親和力得以改良。較佳之親和力成熟抗體將對目標抗原具有奈莫耳或甚至皮莫耳親和力。親和力成熟抗體可藉由此項技術中已知之程序製得。Marks等人10:779-83 (1992)描述藉由VH及VL結構域改組達成親和力成熟。CDR及/或構架殘基之隨機突變描述於以下文獻中：Barbas等人，Proe dead· *SW· USA 91:3809-13 (1994); Schier 等人 Gwe 169:147-55 (1995); Yelton等人，J. 155:1994-2004 (1995);Fragments 'The fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen-binding sites. Bifunctional antibodies are described in more detail in, for example, EP 404,097, WO 93/11161 and Hollinger et al, Procr. Wi/.dcd 5W.t/U, 90:6444-48 (1993). A π human antibody, which is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or using any of the techniques for producing human antibodies disclosed herein. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. ΜAffinity matured antibodies are antibodies that have one or more changes in one or more CDRs, and such changes result in improved affinity of the antibody for the antigen compared to parental antibodies that do not have such changes. Preferred affinity maturation The antibody will have a nanomolar or even picomolar affinity for the antigen of interest. Affinity matured antibodies can be made by procedures known in the art. Marks et al. 10:779-83 (1992) describe the structure by VH and VL Domain shuffling achieves affinity maturation. Random mutations in CDR and/or framework residues are described in Barbas et al., Proe dead. *SW. USA 91:3809-13 (1994); Schier et al. Gwe 169:147- 55 (1995); Yelton et al., J. 155: 1994-2004 (1995);

Jackson 等人，乂 154(7):33 10-19 (1995)及Jackson et al., 154 154(7): 33 10-19 (1995) and

Hawkins^ A 5 Mol, Biol. 226:889-96 (1992) ° 抗體’’效應功能"係指由抗體之Fc區（天然序列Fc區或胺基 119234.doc -29- 200813088 酸序列變異體Fc區）引起之彼等生物活性，且其隨抗體同型而變化。抗體效應功能之實例包括：Clq結合及補體依賴性細胞毒性、Fc受體結合、抗體依賴性細胞介導之細胞毒性（ADCC)、吞噬作用、細胞表面受體（例如B細胞受體）下調及B細胞活化。 ”抗體依賴性細胞介導之細胞毒性π或nADCC"係指一種細胞毒性形式，其中與存在於某些細胞毒性細胞（例如天然殺傷（NK)細胞、嗜中性白血球及巨噬細胞）上之Fc受體 (FcR〇結合的分泌Ig使此等細胞毒效應細胞能夠與帶有抗原之目標細胞特異性結合且隨後以細胞毒素殺傷目標細胞。抗體”配備π細胞毒細胞且完全為此殺傷所需。介導ADCC 初級細胞（ΝΚ細胞）僅表現FcyRIII，而單核細胞表現 FcyRI、FcyRII及FcyRIII。造血細胞上之FcR表現概述於 Ravetch及 Kinet，乂/?㈣· 9:457-92 (1991)中第 464頁之表3中。為評定所關注之分子的ADCC活性，可進行活體外ADCC檢定，諸如美國專利第5,500,362號或第 5,821，337號或卩^81&美國專利第6,737,056號中所述。用於此等檢定中之效應細胞包括周邊血液單核細胞（PBMC)及天然殺傷（NK)細胞。另外或其他，可在活體内，例如在動物模型（諸如 Clynes 等人，/Voc· #(2//. dead. Scz·. 95:652-56 (1998)中所揭示之動物模型）中評定所關注之分子的ADCC活性。 ’’人類效應細胞’’係表現一或多個FcR且執行效應功能之白血球。該等細胞較佳至少表現FcyRIII並執行ADCC效應 119234.doc -30- 200813088 功能。介導ADCC之人類白血球之實例包括周邊血液單核細胞（PBMC)、天然殺傷（NK)細胞、單核細胞、細胞毒性T 細胞及嗜中性白血球，其中PBMC及NK細胞較佳。效應細胞可自天然來源（例如血液）分離。受體”或"FcR”描述與抗體Fc區結合之受體。較佳之 FcR為天然序列人類FcR。此外，較佳之FcR為結合IgG抗體之FcR (γ受體）且包括FcyRI、FcyRII及FcyRIII子類之受體，包括此等受體之等位基因變異體及另外剪接形式。 FcyRJI受體包括FcyRIIA (’’活化受體”）及FcyRIIB (”抑制受體Ί，其具有主要在細胞質域中不同之類似胺基酸序列。活化受體FcyRIIA之細胞質域中含有免疫受體酪胺酸基活化基元（ITAM)。抑制受體FcyRIIB之細胞質域中含有免疫受體路胺酸基抑制基序（ITIM)(參看評論M· DaSron，d⑽w. Rev· Immunol· 15:203-34 (1997))。FcR論述於 Ravetch 及 Kinet，jwww· /mmwwo/· 9:457-92 (1991); Capel等人，Hawkins^ A 5 Mol, Biol. 226:889-96 (1992) ° Antibody ''effector function' refers to the Fc region of an antibody (native sequence Fc region or amino group 119234.doc -29-200813088 acid sequence variant) The Fc region) causes their biological activity and it varies with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (eg, B cell receptors), and B cell activation. "Antibody-dependent cell-mediated cytotoxicity π or nADCC" refers to a cytotoxic form in which it is present on certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages). The Fc receptor (FcR〇-binding secretory Ig enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen and subsequently kill the target cell with cytotoxin. The antibody is equipped with π cytotoxic cells and is completely killed by this. Needed. mediated ADCC primary cells (ΝΚ cells) only show FcyRIII, while monocytes show FcyRI, FcyRII and FcyRIII. The FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, 乂/?(4)·9:457-92 (1991 In Table 3 on page 464. In order to assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337 or U.S. Patent No. 6,737,056. The effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Additionally or alternatively, may be in vivo, for example in animal models. 'Assessing the ADCC activity of the molecule of interest in Clynes et al., /Voc. #(2//. dead. Scz. 95:652-56 (1998)). ''Human effector cells '' is a white blood cell that exhibits one or more FcRs and performs effector functions. These cells preferably exhibit at least FcyRIII and perform ADCC effects 119234.doc -30- 200813088. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear Cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, of which PBMC and NK cells are preferred. Effector cells can be isolated from natural sources (eg, blood). "FcR" describes a receptor that binds to the Fc region of an antibody. Preferably, the FcR is a native sequence human FcR. Further, preferably, the FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes FcyRI, FcyRII, and FcyRIII subclasses. , including allelic variants of these receptors and additional spliced forms. FcyRJI receptors include FcyRIIA (''activating receptors') and FcyRIIB ("inhibiting receptor sputums, which have similarities mainly in the cytoplasmic domain Amine The cytoplasmic domain of the activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM), which contains an immunoreceptor-glycosylation-inhibiting motif (ITIM) in the cytoplasmic domain of the receptor FcyRIIB (see comments) M. DaSron, d(10)w. Rev. Immunol. 15:203-34 (1997)). FcR is discussed in Ravetch and Kinet, jwww· /mmwwo/· 9:457-92 (1991); Capel et al.

Immunomethods 4:25-34 (1994)及 de Haas 等人，J, Lab· C/z>2. Mel 126:330-41 (1995)中。本文之術語”FcRn中涵蓋其他FcR ’包括日後鑑別之FcR。此術語亦包括新生兒受體FcRn，其負責將母體IgG轉移至胎兒體内（Guyer等人， J· Immunol· 117:587 (1976)及 Kim 等人，《/· Immunol· 24:249 (1994))並調控免疫球蛋白之動態穩定。 WO 00/42072(Pi*esta)描述與FcR之結合經改良或經降低之抗體變異體。該專利公開案之内容以引用方式明確併入本文中。亦參看 Shields 等人·/· 9(2): 6591-6604 119234.doc -31- 200813088 (2001)。量測與FcRn結合之方法係已知的（例如參看Ghetie及 Ward, /mm㈣〇/· 7¾心;；18:592-8 (1997))。可（例如）在表現人類FcRn之轉殖基因小鼠或經轉染人類細胞株中或已投與 Fc變異體多肽之靈長類動物體内檢定活體内與人類FcRn之結合及人類FcRn高親和力結合多肽之血清半衰期。 π補體依賴性細胞毒性，，或"CDC”係指在補體存在下目標細胞之溶解。經典補體路徑之活化係藉由補體系統之第一組伶（C 1 q)結合至與其同源抗原結合之（適當子類之）抗體而起始。為評定補體活化，可進行CDC檢定，例如Gazzano-Immunomethods 4:25-34 (1994) and de Haas et al, J, Lab. C/z> 2. Mel 126: 330-41 (1995). The term "FcRn encompasses other FcRs" includes FcRs that are identified in the future. This term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG into the fetus (Guyer et al, J. Immunol. 117:587 (1976) And Kim et al., // Immunol 24:249 (1994) and regulate the dynamic stability of immunoglobulins. WO 00/42072 (Pi*esta) describes improved or reduced antibody variants that bind to FcR The contents of this patent publication are expressly incorporated herein by reference. See also Shields et al. / / 9(2): 6591-6604 119234.doc -31- 200813088 (2001). Method for measuring binding to FcRn It is known (for example, see Ghetie and Ward, /mm(4)〇/·73⁄4心;; 18:592-8 (1997)). It can be, for example, in a transgenic mouse or a transfected human cell that expresses human FcRn. The serum half-life of in vivo binding to human FcRn and human FcRn high affinity binding polypeptide in a primate or a primate that has been administered an Fc variant polypeptide. π complement dependent cytotoxicity, or "CDC" Refers to the dissolution of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first set of 伶 (C 1 q) of the complement system to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, such as Gazzano-

Santoro 等人，汄 Μ廳⑽/. 202:163 (1996)中所述。具有已變化之Fc區胺基酸序列及增強或降低之ciq結合Santoro et al., 汄 Μ (10)/. 202:163 (1996). Amino acid sequence with altered Fc region and enhanced or reduced ciq binding

月色力的多肽變異體描述於美國專利第6，ΐ94,551Β1號及WO 99/5 1642中。彼等專利公開案之内容以引用方式特別併入本文中。亦參看Idusogie等人，乂 /济所以⑽/· 164: 4178-84 (2000) 〇阻斷抗體或拮抗劑”抗體為抑制或降低所結合之抗原之生物活性的抗體。較佳之阻斷抗體或拮抗劑抗體實質或完全抑制抗原之生物活性。 ’’病症"或’’疾病”為將自經本發明之物質/分子或方法進行之治療獲益的任何病況。此包括慢性及急性病症或疾病， i括使哺乳動物易患所述病症之彼等病理病況。本文中待治療之病症的非限制性實例包括惡性及良性腫瘤、癌瘤、 119234.doc -32- 200813088 母細胞瘤及肉瘤。術語"細胞增生性病症’’及”增生性病症，，係指與某種程度之異常細胞增殖相關之病症。在一實施例中，細胞增生性病症為癌症。如本文所使用之”腫瘤"係指所有贅生性細胞（惡性或良性）之生長及增殖，及所有癌前及癌細胞及組織。如本文所提及之術語，，癌症"、"癌性”、"細胞增生性病症"、"增生性病症’’及”腫瘤”並不互相排除。術語"癌症，，及"癌性"係指或描述哺:乳動物體内通常以不受調控之細胞生長/增瘦為特徵之生理病況。癌症之實例包括(但不限於)癌瘤、淋巴瘤、母細胞瘤、肉瘤及白血病。此等癌症更特定之實例包括鱗狀細胞癌、小細胞肺癌、垂體癌、食營痛、jg# 4 & Κ星形細胞瘤、軟組織肉瘤、非小細胞肺癌、肺腺癌、肺鱗狀癌、腹膜癌、肝細胞癌、胃腸癌、胰腺癌、神經膠母έ眙胗母、、、田胞瘤、子宮頸癌、_巢癌、肝癌、膀胱癌、肝腫瘤、乳癌、姓 ^ ^ ^ 展、、、口腸癌、結腸直腸癌、子宮内膜癌或子宮癌、唾液腺癌、 ^ ^ m ‘月癌、肝癌、***癌、外陰癌、甲狀腺癌、肝癌、腦管癌、膽囊癌、胃癌、心二呂内膜癌、華丸癌、膽 ”、…、瘤及各種類型之頭頸部癌。血吕生成力月匕失調可引起恭> &、广』由本發明之組合物及方法治、、/正。此等病症包括非贅生性盘款^ 病況包括（但不限於）上文所述之病/ =病況°贊生性 (但不限於)不當或異常肥大、關r广生性病症包括购、牛㈣、牛皮癬斑塊、類肉W類風濕性關節炎類肉瘤病、動脈粥樣硬化、 119234.doc -33 - 200813088 動脈粥樣硬化斑、糖尿病性及其他增生性視網膜病變（包括早產兒視網膜病變）、晶狀體後纖維組織增生、新生血管性青光眼、年齡相關之黃斑變性、糖尿病性黃斑水腫、角膜新血管生成、角膜移植物新血管生成、角膜移植物排斥反應、視網膜/脈絡膜新血管生成、隅角新血管生成（虹膜紅變）、眼内新生血管疾病、血管再狹窄、動靜脈畸形 (AVM)、腦膜瘤、血管瘤、血管纖維瘤、甲狀腺增生（包括格雷夫氏病（Grave’s disease))、角膜及其他組織移植、慢 •生火症、肺炎、急性肺損傷/Ap、DS、敗血症、原發性肺循環血壓過高、惡性肺部積液、腦水腫（例如與急性中風/閉合性頭部損傷/外傷有關）、滑膜炎、RA中之血管翳形成、骨化性肌炎、肥厚性骨形成、骨關節炎（〇A)、難治性腹水、多囊卵巢病、子宮内膜異位、流體第三間隔疾病 spacing 〇f fluid diseases)(胰腺炎、間隔症候群、燒傷、腸病）、子宮纖維瘤、早產、諸如IBD之慢性炎症（克隆氏病 (Crohn’s disease)及潰瘍性結腸炎）、腎同種異體移植排斥反應乂症性腸病、腎病症候群、不當或異常組織腫塊生長（非癌症）、血友病性關節、肥厚性瘢痕、頭髮生長之抑制、奥羊氏症候群（Osier-Weber syndrome)、化腹性肉芽腫晶狀體後纖維組織增生、硬皮病、沙眼、血管黏附、滑膜炎、皮炎、先兆子癇、腹水、心包積液（諸如與心包炎相關之心包積液）及胸腔積液。術語’’消耗性”病症（例如消耗性症候群、惡病質、肌肉減少症）係指由不當及/或不健康之體重減輕或體細胞質量減 119234.doc -34- 200813088 輕所引起之病症。在老年人以及AIDS及癌症患者中，消耗性疾病可導致不當之體重減輕，包括脂肪與無脂肪間隔之體重減輕。消耗性疾病可由不恰當之食物攝入及/或與疾病及/或衰老過程相關之代謝改：侧患者以及廣泛手術後或患有慢性感染、免=及甲狀腺，月匕几進、克隆氏病、精神性疾病、慢性心臟衰竭或八他嚴重外傷之患者通常身受消耗性疾病，消耗性疾病有時亦稱為惡病質、代謝病症且有時稱為進食障礙。另外，惡病質以代諸充進及分解代謝過度為特徵。儘管通常互換使用惡病質與消耗性病症來指代消耗性病況，但存在至夕種體研究根據去脂體重且尤其是體細胞質量之減輕字心病貝與，肖耗性症候群區別開來（Mayer，j· N咖^ s ^ 6S 59S (1999))。肌肉減少症（另一種可影響衰老個體之病症）通常係以肌肉質量減輕為特徵。如上文所述肖耗〖生疾病可在身受惡病質或肌肉減少症之個體體内發展。如本文所使用之”治療”係指試圖改變所治療之個體或細胞之自然過程的臨床干預，且可出於預防之目的或臨床病 —予中進行。所需之治療作用包括預防疾病之發生或復發冑解症狀、減小疾病之任何直接或間接病理結果、降低疾病進展速率、改善或減輕疾病狀態及病情好轉或改良預後。在一些實施例中，使用本發明之抗體延緩疾病或病症之發展。個體文檢者’’或’’患者，，為脊椎動物，例如哺乳動 119234.doc -35- 200813088 物，尤其包括人類。哺乳動物包括（但不限於）人類、家畜及農畜，及動物園動物、競技類動物或寵物，諸如犬、馬、貓、奶牛等。 "有效量”係指有效達成所需治療或預防結果所必需之劑量及時間量。本發明之物質/分子、激動劑或拮抗劑之"治療有效量，，可根據以下因素而變化：諸如個體之疾病狀態、年齡、性別广及體重，及物質/分子、激動劑或拮抗劑於個體體内誘發所需反應之能力。治療有效量亦係伤質/分子、激動劑或拮抗劑之治療有益作用超過其任何有毒或有害作用的量。預防有效l’’係指有效達成所需預防結果所必需之劑量及時間量。由於預防劑量係在疾病之前或疾病早期用於受檢者，故預防有效量通常（但非必需）將小於治療有效量。如本文所使用之”細胞毒性劑”係指抑制或阻礙細胞功能且/或引起細胞破壞之物質。此術語意欲包括放射性同位〆素（例如 At211、I131、I125、Y90、Re186、Rel88、Sml53、Polymorphic polypeptide variants are described in U.S. Patent No. 6, ΐ94,551, and WO 99/5 1642. The contents of their patent publications are specifically incorporated herein by reference. See also Idusogie et al., 乂/吉和(10)/· 164: 4178-84 (2000) 〇 Blocking antibodies or antagonists. Antibodies are antibodies that inhibit or reduce the biological activity of the antigen to which it is bound. Preferably blocking the antibody or An antagonist antibody substantially or completely inhibits the biological activity of an antigen. A 'disease' or ''disease' is any condition that would benefit from treatment with a substance/molecule or method of the invention. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose a mammal to the condition. Non-limiting examples of conditions to be treated herein include malignant and benign tumors, carcinomas, 119234.doc-32-200813088 blastomas and sarcomas. The term "cell proliferative disorder" and "proliferative disorder", refers to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. As used herein, "tumor " refers to the growth and proliferation of all neoplastic cells (malignant or benign), and all precancerous and cancerous cells and tissues. As the term is mentioned herein, cancer ""cancerous", "cell proliferative disorders", "proliferative disorders" and "tumor" are not mutually exclusive. The term "cancer, And "cancerous" refers to or describes a physiological condition in which a mammal is usually characterized by unregulated cell growth/slimming. Examples of cancer include, but are not limited to, carcinoma, lymphoma, and mother. Cell tumors, sarcomas, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, food camp pain, jg# 4 & astrocytoma, soft tissue sarcoma, non-small cell lung cancer, Lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, gynecological mother, dysentery, cervical cancer, cervical cancer, _ nest cancer, liver cancer, bladder cancer, liver Tumor, breast cancer, surname ^ ^ ^ exhibition, , orectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, ^ ^ 'month cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer , cerebral canal cancer, gallbladder cancer, gastric cancer, heart two inner membrane Cancer, Huamao cancer, gallbladder, ..., tumors and various types of head and neck cancer. The blood stagnation force stagnation can cause Gong >&&>, and the composition and method of the present invention are treated, and/or positive. Such conditions include non-neoplastic discs. Conditions include, but are not limited to, the above mentioned conditions/=conditions. • Praise (but not limited to) inappropriate or abnormal hypertrophy, or genus, including purchase, cattle (4), Psoriasis plaque, meat type W rheumatoid arthritis atherosclerosis, atherosclerosis, 119234.doc -33 - 200813088 Atherosclerotic plaque, diabetic and other proliferative retinopathy (including retinopathy of prematurity), Post-lens fibrous tissue hyperplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal/choroidal neovascularization, hornbeam new Angiogenesis (iris redness), intraocular neovascular disease, vascular restenosis, arteriovenous malformation (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasia (including Grave's disease), cornea And other tissue transplantation, slow • fire, pneumonia, acute lung injury / Ap, DS, sepsis, primary pulmonary circulation, high blood pressure, Pulmonary effusion, cerebral edema (eg associated with acute stroke/closed head injury/trauma), synovitis, vasospasm formation in RA, myositis ossificans, hypertrophic bone formation, osteoarthritis ( 〇A), refractory ascites, polycystic ovary disease, endometriosis, fluid sf fluid diseases (pancreatitis, septal syndrome, burns, bowel disease), uterine fibroids, premature delivery, such as Chronic inflammation of IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, septic bowel disease, renal disease, inappropriate or abnormal tissue mass growth (non-cancer), hemophilic joint Hypertrophic scar, inhibition of hair growth, Osier-Weber syndrome, pelvic granuloma, posterior fibrous tissue hyperplasia, scleroderma, trachoma, vascular adhesion, synovitis, dermatitis, pre-eclampsia, Ascites, pericardial effusion (such as pericardial effusion associated with pericarditis) and pleural effusion. The term 'consumptive' condition (eg, wasting syndrome, cachexia, sarcopenia) refers to a condition caused by inappropriate and/or unhealthy weight loss or somatic mass loss 119234.doc -34-200813088. In humans, as well as in AIDS and cancer patients, wasting disease can lead to inappropriate weight loss, including weight loss between fat and fat-free compartments. Consumable diseases can be caused by inappropriate food intake and/or associated with disease and/or aging processes. Metabolic changes: Patients with side-effects and after extensive surgery or suffering from chronic infection, exemption = and thyroid, menstrual cramps, Crohn's disease, mental illness, chronic heart failure or severe trauma are usually suffering from wasting disease, consumption Sexually transmitted diseases are sometimes also referred to as cachexia, metabolic disorders and sometimes referred to as eating disorders. In addition, cachexia is characterized by substituting and catabolism. Although cachexia and wasting disorders are often used interchangeably to refer to consumptive conditions, However, there is a study on the growth of the body and the quality of the somatic cells. Groups are distinguished (Mayer, j·N coffee^ s ^ 6S 59S (1999)). Muscle reduction (another condition that affects aging individuals) is usually characterized by muscle mass loss. The disease can develop in an individual suffering from cachexia or sarcopenia. As used herein, "treatment" refers to a clinical intervention that attempts to alter the natural course of the individual or cell being treated, and may be for preventive purposes or clinical purposes. The disease is prescribed. The therapeutic effects required include preventing the occurrence or recurrence of the disease, reducing any direct or indirect pathological results of the disease, reducing the rate of disease progression, improving or reducing the disease state, and improving the condition or improving the prognosis. In some embodiments, the use of an antibody of the invention delays the progression of a disease or condition. An individual examiner ''or'' patient, is a vertebrate, such as a mammal, 119234.doc-35-200813088, including in particular humans. Mammals include, but are not limited to, humans, livestock and farm animals, and zoo animals, competitive animals or pets such as dogs, horses, cats, and cows. . &Quot; the amount of the agent and the amount of time an effective amount "means the necessary effective to achieve the desired therapeutic or prophylactic result. The "therapeutically effective amount of a substance/molecule, agonist or antagonist of the present invention may vary depending on factors such as the disease state of the individual, age, sex and body weight, and substance/molecule, agonist or antagonist The ability to induce a desired response in an individual. A therapeutically effective amount is also one in which the therapeutic benefit of the wound/molecule, agonist or antagonist exceeds any of its toxic or detrimental effects. Prophylactically effective l'' refers to the amount and amount of time necessary to effectively achieve the desired prophylactic result. Since the prophylactic dose is administered to the subject prior to or prior to the disease, the prophylactically effective amount will generally (but not necessarily) be less than the therapeutically effective amount. "Cytotoxic agent" as used herein refers to a substance that inhibits or impairs cellular function and/or causes cell destruction. This term is intended to include radioisotopes (eg, At211, I131, I125, Y90, Re186, Rel88, Sml53,

Bi212、P32及Lu之放射性同位素）；化學治療劑，例如甲胺喋呤（methotrexate)、阿黴素（adriamicin)、長春鹼類（vinca alkaloids)(長春新鹼（vincristine)、長春鹼（vinblastine)、依託泊苷（etoposide))、多柔比星（d〇xorubicin)、美法侖 (melphalan)、絲裂黴素 C (mitomycin C)、苯丁酸氮芥 (chlorambucil)、柔紅黴素（daunorubicin)或其他嵌入劑；酵素及其片段，諸如核分解酶；抗生素；及毒素，諸如細菌、真菌、植物或動物來源之小分子毒素或酶活性毒素 119234.doc -36 - 200813088 (包括其片段及/或變異體）；及下文所揭示之各種抗腫瘤或抗癌劑。其他細胞毒性劑描述於下文中。殺腫瘤劑引起腫瘤細胞之破壞。 ”化學治療劑π係可用於治療癌症之化合物。化學治療劑之實例包括烧基化劑，諸如σ塞替旅（thiotepa)及 CYTOXAN®環填醯胺（cyclosphosphamide);石黃酸烧酯，諸如白消安（busulfan)、英丙舒凡（improsulfan)及旅泊舒凡 (piposulfan);氮丙咬，諸如苯并多巴（benzodopa)、卡波酉昆 (carboquone)、麥西多巴（meturedopa)及尤利多巴 (uredopa);伸乙基亞胺及甲基三聚氰胺，包括六甲密胺 (altretamine)、曲他胺（triethylenemelamine)、三伸乙基構醯胺（trietylenephosphoramide)、三伸乙基硫代填醯胺 (triethiylenethiophosphoramide)及三經甲基三聚氰胺 (trimethylolomelamine)；乙醢精寧（acetogenin)(尤其是布拉他辛（bullatacin)及布拉他辛酮（bullatacinone)) ; δ-9-四氫***酴（曲***盼（dronabinol)，MARINOL®) ; β-拉帕酮 (beta-lapachone);拉帕醇（lapachol);秋水仙驗 (colchicine);樺木酸（betulinic acid);喜樹鹼 (camptothecin)(包括合成類似物拓撲替康 (topotecan)(HYCAMTIN®)、CPT-11(伊立替康（irinotecan) CAMPTOSAR®)、乙醯喜樹驗、斯可波萊丁（scopolectin) 及9-胺基喜樹驗）；苔蘚蟲素（bryostatin);卡利他汀 (cally statin) ; CC-1065(包括其阿多來新（adozelesin)、卡折來新（carzelesin)及比折來新（bizelesin)合成類似物）；鬼臼 119234.doc -37- 200813088 毒素（podophyllotoxin);足葉草酸（podophyllinic acid);替尼泊甙（teniposide);念珠藻環狀（cryptophycin)(尤其是念珠藻環肽1及念珠藻環肽8);海兔毒素（dolastatin);多卡黴素（duocarmycin)(包括合成類似物，KW-21 89及CB1-TM1)；艾權素（eleutherobin)；潘卡替他汀 (pancratistatin);沙科地辛（sarcodictyin);斯潘他、;丁 (spongistatin);氮芥末（nitrogen mustard)，諸如苯丁酸氮芥、萘氮芥（chlornaphazine)、膽碟酿胺 (cholophosphamide)、雌莫司汀（estramustine)、異環填酸胺（ifosfamide)、氮芥（mechlorethamine)、氧化氮芥鹽酸鹽、美法侖、新恩比興（novembichin)、苯芥膽甾醇 (phenesterine)、潑尼莫司汀（prednimustine)、曲填胺 (trofosfamide)、尿嘴唆芬末（uracil mustard);亞石肖基脲 (nitrosurea)，諸如卡莫司汀（carmustine)、氣脲黴素 (chlorozotocin)、福莫司汀（fotemustine)、洛莫司汀 (lomustine)、尼莫司汀（nimustine)及雷莫司汀 (ranimnustine);抗生素，諸如烯二炔類抗生素（enediyne antibiotic)(例如刺孢黴素（calicheamicin)，尤其是刺孢黴素 γΐΐ及刺孢黴素ΩΙ1 (例如參看」gwew, CTzem Ed·五叹/·， 33: 183-186 (1994))，地尼黴素（dynemicin)(包括地尼黴素 A(dynemicin A))，伊斯帕黴素（esperamicin)以及新製癌菌素發色團（neocarzinostatin chromophore)及相關色蛋白烯二快類抗生素發色團）、阿克萊諾黴素（aclacinomysin)、放線菌素（actinomycin)、奥瑟黴素（authramycin)、偶氮絲胺酸 119234.doc -38- 200813088 (azaserine)、博萊黴素（bleomycin)、放線菌素 C(cactinomycin)、卡拉比星（carabicin)、洋紅黴素 (carminomycin)、嗜癌菌素（carzinophilin)、色黴素 (chromomycinis)、放線菌素 D(dactinomycin)、柔紅黴素 (daunorubicin)、地托比星（detorubicin)、6 -重氮-5-側氧基-L-正白胺酸、ADRIAMYCIN®多柔比星（包括嗎啉基多柔比星、氰基嗎啉基-多柔比星、2-吡咯啉基-多柔比星及去氧多柔比星）、表柔比星（epirubicin)、依索比星 (esorubicin)、伊達比星（idarubicin)、蘇西羅徽素 (marcellomycin)、絲裂黴素（mitomycin)(諸如絲裂黴素 C)、黴紛酸（mycophenolic acid)、諾拉黴素（nogalamycin)、橄欖黴素（olivomycin)、培洛黴素（peplomycin)、博替羅黴素 (potfiromycin)、嗓羅黴素（puromycin)、奎拉黴素 (quelamycin)、羅多比星（rodorubicin)、鏈黑黴素 (streptonigrin)、鏈佐星（streptozocin)、殺結核菌素 (tubercidin)、烏苯美司（ubenimex)、淨司他 ί丁 (zinostatin)、左柔比星（zorubicin);抗代謝物，諸如曱胺喋呤及5_氟尿嘧啶（5-FU);葉酸類似物，諸如迪諾特寧 (denopterin)、甲胺嗓呤、蝶羅呤（pteropterin)、三曱曲沙 (trimetrexate) ； σ票呤類似物，諸如氟達拉賓 (fludarabine)、6-魏基 11票呤、硫口米嗓呤（thiamiprine)、硫鳥嘌吟（thioguanine);喷唆類似物，諸如安西他濱 (ancitabine)、阿紮胞皆（azacitidine)、6_氮尿普、卡莫氟 (carmofur)、阿糖胞皆（cytarabine)、雙去氧尿普、去氧氟 119234.doc -39- 200813088 尿普（doxifluridine)、依諾他濱（enocitabine)、氮尿芽 (floxuridine);雄激素，諸如卡普睾酮（calusterone)、丙酸屈他雄酮（dromostanolone propionate)、環硫雄醇 (epitiostanol)、美雄烧(mepitiostane)、睾内赂 (testolactone)；抗腎.上腺劑，諸如胺魯米特 (aminoglutethimide)、米托坦（mitotane)、曲洛司坦 (trilostane);葉酸補充劑，諸如葉酸；醋葡駿内酯 (aceglatone)；酸構醯胺糖普（aldophosphamideBi212, P32 and Lu radioisotopes; chemotherapeutic agents such as methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine) , etoposide, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin Daunorubicin) or other intercalating agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin 119234.doc -36 - 200813088 (including fragments thereof) And/or variants; and various anti-tumor or anti-cancer agents disclosed below. Other cytotoxic agents are described below. Tumor killing agents cause destruction of tumor cells. The chemotherapeutic agent π is a compound useful for treating cancer. Examples of chemotherapeutic agents include an alkylating agent such as thiotepa and CYTOXAN® cyclosphosphamide; pyruvate, such as Busulfan, improsulfan, and piposulfan; azepine, such as benzodopa, carboquone, meturedopa And uredopa; acetamimine and methyl melamine, including altretamine, triethylenemelamine, trietylenephosphoramide, tri-ethyl sulphide Triethiylenethiophosphoramide and trimethylolomelamine; acetogenin (especially bullatacin and bullatacinone); δ-9-four Hydrogen cannabis (dronabinol, MARINOl®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (camptothecin) (including synthetic Topotecan (HYCAMTIN®), CPT-11 (irinotecan CAMPTOSAR®), acetaminophen, scopolectin, and 9-amino-based assays; Bryostatin; calally statin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogues);臼 119234.doc -37- 200813088 toxin (podophyllotoxin); podophyllinic acid; teniposide; cryptophycin (especially Nostoccal cyclic peptide 1 and Nostoccal cyclic peptide 8 ); dolastatin; duocarmycin (including synthetic analogues, KW-21 89 and CB1-TM1); eleutherobin; pancastatin; shaco Sarcodictyin; spanistatin; nitrogen mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine ), isoosfamide, mechlorethamine, nitric oxide Mustard hydrochloride, melphalan, neombibichin, phenesterine, prednimustine, trofosfamide, uracil mustard ; nitrosurea, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and thunder Ranimnustine; antibiotics, such as enediyne antibiotics (eg calicheamicin), especially calicheamicin gamma and calicheamicin ΩΙ1 (see eg gwew, CTzem Ed) · Wu sigh / ·, 33: 183-186 (1994)), dynemicin (including dynemicin A), esperamicin (esperamicin) and new carcinogen A chromophore (neocarzinostatin chromophore) and a related chromoprotein bisphenol antibiotic chromophore), aclacinomysin, actinomycin, authramycin, azosilamine Acid 119234.doc -38- 200813088 (azaserine), bleomycin (bleo) Proxy), actinomycin C, caracycline, carminomycin, carzinophilin, chromomycinis, dactinomycin, soft red Daunorubicin, detorubicin, 6-diazo-5-oxo-L-positive leucine, ADRIAMYCIN® doxorubicin (including morpholinyl doxorubicin, cyano Morpholinyl-doxorubicin, 2-pyrrolyl-doxorubicin and deoxydoxonol), epirubicin, esorubicin, idarubicin , marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, culture Peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin Star (streptozocin), tuberculosis, ubenimex, zinostatin, left soft Zorubicin; antimetabolites such as amidoxime and 5-fluorouracil (5-FU); folic acid analogues such as denoptinin, methotrexate, pteropterin, three Trimetrexate; σ 呤呤 analogs, such as fludarabine (fludarabine), 6-Weiji 11 votes, thiamiprine, thioguanine; sneeze similar Such as ancitabine, azacitidine, 6-nitrogen, carmofur, cytarabine, double deoxyuridine, deoxyfluoride 119234. Doc -39- 200813088 Doxfluridine, enocitabine, floxuridine; androgens, such as calutosterone, dromostanolone propionate, cyclosulphur Epitiostanol, mepitiostane, testolactone; anti-renal adrenal agents, such as aminoglutethimide, mitotane, trilostane; Folic acid supplements, such as folic acid; aceglatone; acid glutamine aldophosphamide

σ 1 v r 〇 q ί Η ρΛ * a J ^ ^ j · t—ij_i_ « _ 、w w' ▲ w · a 森秦泰▲ ▼ w ▼ — / / Z、，，4 · 。密唆（eniluracil);安 σ定（amsacrine);貝曲布辛 (bestrabucil);比生群（bisantrene);伊達曲仙 (edatraxate)；德弗法明（defofamine);秋水仙胺 (demecol cine);地 °丫酿(diaziquone)；伊弗尼辛 (elfornithine);依利醋铵（elliptinium acetate);艾普塞隆 (epothilone);依託格魯（etoglucid);石肖酸鎵；經基尿素 (hydroxyurea);香蒜多糖（lentinan)；洛尼達寧 (lonidainine);美登素類（maytansinoids)，諸如美登素 (maytansine)及安絲菌素（ansamitocin)；米托脈腙 (mitoguazone)；米托蒽酉昆（mitoxantrone);莫比丹莫 (mopidanmol);尼曲伊寧（nitraerine);喷司他、汀 (pentostatin)；蛋胺氮芥（phenamet)；比柔比星 (pirarubicin);洛索蒽 (losoxantrone) ; 2_ 乙基醯肼；丙卡巴肼（procarbazine) ; PSK® 多醋複合物（JHS Natural Products，Eugene，OR);雷佐生（razoxane);根瘤菌素 119234.doc -40- 200813088 (rhizoxin)；西佐喃（sizofiran);錯螺胺（spirogermanium); 細交鏈孢菌酮酸（tenuazonic acid); 三亞胺酉昆 (tdaziquone) ; 2,2’，2"-三氣三乙胺；單端孢黴毒素 (trichothecene)(尤其是 T-2 毒素、維拉箭毒 A(verracurin A)、漆斑菌素A(roridin A)及安吉毒素（anguidine));烏拉坦（urethan);長春地辛（vindesine)(ELDISINE® 、 FILDESIN®);達卡巴嗪（dacarbazine);甘露莫司汀 (mannomustine)；二漠甘露糖醇（mitobronitol);二漠衛矛醇（mitolactol); 略泊漠烧（pipobroman): 瓜西托辛 (gacytosine);阿糖皆（arabinoside)(’’Ara-C”）； σ塞替派；紫杉醇類（taxoids)，例如 TAXOL® 紫杉醇（卩&〇川&乂61)(81^1〇1· Myers Squibb Oncology, Princeton, N.J.) ^ ABRAXANE™ 無十六醇聚氧乙烯醚之紫杉醇之白蛋白工程設計奈米顆粒調酉己物（American Pharmaceutical Partners，Schaumberg， Illinois)及 TAXOTERE® 多西他賽（(1〇乂61&父61)(111101^-Poulenc Rorer，Antony，France);氯蘭布辛（chloranbucil); 吉西他賓（gemcitabine)(GEMZAR®) ; 6·硫鳥嗓呤；魏基口票呤；甲胺嗓呤；始類似物，諸如順顧（cisplatin)及卡始 (carboplatin);長春鹼（VELBAN®);鉑；依託泊苷（VP-16);異環磷醯胺；米托蒽醌；長春新鹼（ONCOVIN®);奥賽力始（oxaliplatin);甲醢四氫葉酸（leucovovin);長春瑞賓（vinorelbine)(NAVELBINE®);諾凡特龍（novantrone); 依達曲沙（edatrexate);道諾黴素（daunomycin);胺基嗓呤 (aminopterin);伊班膦酸鹽（ibandronate);拓撲異構酶抑 119234.doc -41- 200813088 制劑 RFS 2000 ;二 I 甲基鳥胺酸（difluoromethylornithine (DMFO));類視色素（retinoid)，諸如視黃酸；卡西他賓 (capecitabine)(XELODA®);上述任何物質的醫藥學上可接受之鹽、酸或衍生物；以及兩種或兩種以上上述物質之組合，諸如CHOP(環磷醯胺、多柔比星、長春新鹼及潑尼松龍（prednisolone)之組合療法的縮寫）及FOLFOX(奥赛力鉑（ELOXATIN™)與5-FU及甲醯四氫葉酸組合之治療方案的縮寫）。此定義中亦包括抗激素劑，其用於調控、降低、阻斷或抑制可促進癌症生長之激素的作用且通常為全身性或整體治療之形式。其可為激素本身。實例包括抗***及選擇性***受體調節劑（SERM)，包括（例如）他莫昔芬 (tamoxifen)(包括 NOLVADEX® 他莫昔芬）、EVISTA® 雷洛昔芬（raloxifene)、屈洛昔芬（droloxifene)、4-經基他莫昔芬 '曲沃昔芬（trioxifene)、雷洛昔芬鹽酸鹽（keoxifene)、 LY117018、奥那司酮（onapristone)及 FARESTON® 托瑞米芬（toremifene);抗孕酮；***受體下調劑（ERD);用於抑制或阻止卵巢之藥劑，例如黃體生成激素釋放激素 (LHRH)促效劑，諸如LUPRON®及ELIGARD®乙酸亮丙立德（leuprolide acetate)、乙酸戈舍瑞林（goserelin acetate)、乙酸布舍瑞林（buserelin acetate)及三蝶瑞林（tripterelin); 其他抗雄激素，諸如氟他胺（flutamide)、尼魯胺 (nilutamide)及比卡魯胺（bicalutamide);及抑制調節腎上腺中***產生之芳香酶的芳香酶抑制劑，諸如4(5)-咪唑、 119234.doc -42- 200813088 胺魯米特、MEGASE®乙酸甲地孕明（megestrol acetate)、 AROMASIN®依西美坦（exemestane)、福美司坦 (formestanie)、法屈唾（fadrozole)、RIVISOR® 伏羅吐 (vorozole)、FEMARA® 來曲唑（letrozole)及 ARIMIDEX® 安美達錠（anastrozole)。此外，此化學治療劑定義亦包括雙膦酸鹽，諸如氯屈膦酸鹽（clodronate)(例如BONEFOS®或 OSTAC®)、DIDROCAL® 依替膦酸鹽（etidronate)、NE-58095、ZOMETA®嗤來膦酸/嗤來膦酸鹽（zoledronic acid/zoledronate)、FOSAMAX⑧阿命膦酸鹽（alendronate)、 AREDIA® 帕米膦酸鹽（pamidronate)、SKELID® 替魯膦酸鹽（tiludronate)或 ACTONEL® 利塞膦酸鹽（risedronate);以及曲沙他濱（troxacitabine)(l，3-二氧戊環胞嘧唆核苷類似物）；反義募核苷酸，尤其是抑制與異常細胞增殖相關之信號路徑中之基因表現之反義寡核苷酸，諸如PKC-α、 Raf、H-Ras及表皮生長因子受體（EGF-R);疫苗，諸如 THERATOPE®疫苗及基因療法疫苗（例如ALLOVECTIN®疫苗、LEUVECTIN®疫苗及 VAXID®疫苗）；LURTOTECAN® 拓撲異構酶1抑制劑；ABARELIX® rmRH ;拉帕替尼二曱苯磺酸鹽（lapatinib ditosylate)(—種 ErbB-2 及 EGFR雙重酪胺酸激酶小分子抑制劑，亦稱為GW572016);及上述物質中之任一者的醫藥學上可接受之鹽、酸或衍生物。當用於本文中時，”生長抑制劑"係指抑制細胞（諸如表現 EGFL7之細胞）活體外或活體内生長之化合物或組合物。因此，生長抑制劑可為顯著降低S期中細胞（諸如表現 119234.doc -43- 200813088 EGFL7之細胞）百分比之藥劑。生長抑制劑之實例包括阻斷細胞週期進程（除S期外之其他位置）之藥劑，諸如誘導 G1停滯及Μ期停滞之藥劑。經典馗期阻斷劑包括長春鹼類 (長春新鹼及長春鹼）、紫杉烷（taxane)及拓撲異構酶π抑制劑（諸如多柔比星、表柔比星、柔紅黴素、依託泊苷及博萊黴素）。彼等使G1停滞之藥劑亦伴隨產生s期停滯，例如 DNA烷基化劑，諸如他莫昔芬、潑尼松（prednis〇ne)、達卡巴嗪、氮芬、順鉑、甲胺喋呤、5-氟尿嘧啶及ara_c。其他資訊可見於Murakami等人之The Molecular Basis 〇f Cancer，Mendelsohn 及 Israel編輯，第 1章，標題為”CeU cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders: Philadelphia，1995)，尤其第 13頁中。紫杉烷（紫杉醇及多西他赛）為源自紫杉樹之抗癌藥物。源自歐洲紫杉之夕西他賽（TAXOTERE®，Rhone-Poulenc Rorer)為紫杉醇（TAXOL㊣，Bristol-Myers Squibb)之半合成類似物。紫杉醇及多西他赛促進自微管蛋白二聚體組裝成微管且藉由防止解聚作用而使微管穩定，此將導致細胞有絲***抑制。 π多柔比星"係一種蒽環黴素類抗生素。多柔比星之完整化學名稱為(8S_順）_1〇_[(3-胺基_2,3,6•三去氧_a_L-來蘇_己 °比喃糖基）氧基]-7,8,9，1〇_四氫·6,8，i卜三羥基_8•(羥基乙醯基甲氧基-5，12-并四苯二酮。術浯包含Fc區之多肽”係指包含以區之多肽，諸如抗體或免疫黏附素（參看本文之定義）。可（例如）在純化多肽期間或藉由重組工程設計編碼多肽之核酸來移除Fc區之c末 119234.doc -44- 200813088 端離胺酸（根據EU編3虎糸統之殘基44 7)。因此，根據本發明包含具有Fc區之多肽之組合物可包含具有K447、已移除所有K447之多肽或具有及不具有K447殘基之多肽之混合物。本發明之組合物及其製造方法本發明涵蓋包含抗EGFL7抗體及包含編碼抗EGFL7抗體之序列之聚核苷酸的組合物（包括醫藥組合物）。如本文所使用，組合物包含一或多種與EGFL7結合之抗體及/或一或多種包含編碼一或多種與EGFL7結合之抗體之序列的聚核苷酸。此等組合物可另外包含此項技術中熟知之適當載劑，諸如醫藥學上可接受之賦形劑（包括緩衝液）。本發明亦涵蓋經分離之抗體及聚核苷酸之實施例。本發明亦涵蓋實質上純的抗體及聚核苷酸之實施例。本發明之抗EGFL7抗體較佳為單株抗體。本發明之範轉内亦涵蓋本文所提供之抗EGFL7抗體之Fab、Fab，、Fab，-SH及F(ab’)2片段。此等抗體片段可藉由諸如酶促消化之傳統方式產生，或可藉由重組技術產生。此等抗體片段可為嵌合或人化抗體片段。此等片段可用於達成下文所述之診斷及治療目的。單株抗體可自實質上均質之抗體群體（亦即包含個別抗體之群體除可能天然存在可以少量存在之突變外其餘均相同）獲得。因此’修飾語，，單株”表示並非離散抗體之混合物形式之抗體的特徵。本發明之抗EGFL7單株抗體可使用最先由Kohler等人， 119234.doc -45- 200813088 仏re 256:495 (1975)描述之融合瘤方法製得，或可藉由重組DNA方法（美國專利第4,816,567號）製得。在融合瘤方法中，對小鼠或其他適當宿主動物（諸如倉鼠）免疫以誘出產生或能夠產生將與用於免疫之蛋白質特異性結合之抗體的淋巴細胞。一般藉由多次經皮下（sc)或腹膜内（ip)注射EGFL7及佐劑而在動物體内產生抗EGFL7 抗體。可使用此項技術中熟知之方法製備EGFL7，某些方法將於本文中進一步描述。舉例而言，下文將描述EGFL7 夕旁細制；告。名一宭絲，你I Φ，闲冬右鼬合$备疝Bf? I白亩 ▼ _·_ ^I— ^s 9 , ·， u# / y v-^ — ^ ，钃，f~ 鏈Fc部分之EGFL7之細胞外域（ECD)的EGFL7衍生物免疫動物。在一實施例中，用EGFL7-IgGl融合蛋白免疫動物。通常使動物對EGFL7與單磷醯脂質A (MPL)/海藻糖二棒狀黴菌酸醋（TDM)(Ribi Immunochem. Research, Inc.5 Hamilton，MT)之免疫原性共輛物或衍生物產生免疫且在多個部位皮層内注射溶液。兩週後對動物加強免疫。7至14 天對動物取血且檢定血清之抗EGFL7力價。對動物加強免疫直至力價達到平穩階段。或者，可於活體外免疫淋巴細胞。接著使用適當融合劑 (諸如聚乙二醇）使淋巴細胞與骨髓瘤細胞融合以形成融合瘤細胞（Goding，Monoclonal Antibodies: Principles and Practice，第 59-103 頁（Academic Press，1986))。接種由此製備之融合瘤，並使其在較佳含有一或多種抑制未融合之親代骨髓瘤細胞生長或存活之物質的適當培養基中生長。舉例而言，若親代骨髓瘤細胞缺乏酵素次黃嘌 119234.doc -46- 200813088 呤-鳥嘌呤磷酸核糖轉移酶（HGPRT或HPRT)，則用於融合瘤之培養基中通常將包括次黃嘌呤、胺基蝶呤及胸苷（HAT 培養基），該等物質會阻止HGPRT缺陷細胞之生長。較佳之骨髓瘤細胞為有效融合，使所選抗體產生細胞穩定高量產生抗體且對諸如HAT培養基之培養基敏感之骨髓瘤細胞。其中，較佳之骨髓瘤細胞株為鼠科骨髓瘤株，諸如自購自 Salk Institute Cell Distribution Center，San Diego, California USA之MOPC-21及MPC-11小鼠腫瘤及購自σ 1 v r 〇 q ί Η ρΛ * a J ^ ^ j · t_ij_i_ « _ , w w' ▲ w · a Sen Qintai ▲ ▼ ▼ ▼ / / / Z, ,, 4 · . Eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecol cine ;diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium silicate; hydroxyurea ); lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; Mitoxantrone; mopidanmol; nitraerine; pentastatin, pentostatin; phenamet; pirarubicin; Los (losoxantrone); 2_ ethyl hydrazine; procarbazine; PSK® multi-vine complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin 119234.doc -40- 200813088 (rhizoxin); sizofiran; spirogermanium; fine oxysporum acid (t Enuazonic acid); triammine tdaziquone; 2,2',2"-three-gas triethylamine; trichothecene (especially T-2 toxin, veracurin A) ), roridin A and anguidine (inguidine); urethane (vinthan) (vindisine) (ELDISINE®, FILDESIN®); dacarbazine (dacarbazine); mannimostatin (mannomustine); mitobronitol; mitolactol; pipobroman: gacytosine; arabinoside (''Ara-C ); σ 塞派; taxoids, such as TAXOL® paclitaxel (卩 & 〇川 & 乂 61) (81^1〇1· Myers Squibb Oncology, Princeton, NJ) ^ ABRAXANETM No sixteen Alcohol polyoxyethylene ether paclitaxel albumin engineering nanoparticle syrup (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® docetaxel ((1〇乂61 & father 61) (111101^-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6 · thioguanine; Wei Kekou 呤; methotrexate; initial analogues, such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; guanidine tetrahydrofolate (leucovovin); vinorelbine (NAVELBINE®); Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase 119234.doc - 41- 200813088 Formulation RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); medicine of any of the above substances a chemically acceptable salt, acid or derivative; and a combination of two or more of the foregoing, such as a combination of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) Abbreviation of therapy) and the treatment of FOLFOX (ELOXATINTM) combined with 5-FU and formazan tetrahydrofolate ). Anti-hormonal agents are also included in this definition for regulating, reducing, blocking or inhibiting the action of hormones that promote cancer growth and are typically in the form of systemic or holistic treatment. It can be the hormone itself. Examples include anti-estrogen and selective estrogen receptor modulators (SERMs) including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, and Droloxifene, 4-amino tamoxifen, trioxifene, leroxifene hydrochloride, LY117018, onapristone, and FARESTON® Toremifene; antiprogesterone; estrogen receptor down-regulator (ERD); an agent used to inhibit or prevent ovaries, such as luteinizing hormone releasing hormone (LHRH) agonists, such as LUPRON® and ELIGARD® Leuprolide acetate, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide, niru An amine (nilutamide) and bicalutamide; and an aromatase inhibitor that inhibits the aromatase produced by estrogen in the adrenal gland, such as 4(5)-imidazole, 119234.doc-42-200813088 amine ubmet, MEGASE® acetate megestrol (megestrol Acetate), AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole and ARIMIDEX® (anastrozole). In addition, this chemotherapeutic definition also includes bisphosphonates such as clodronate (eg BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA®嗤Zoledronic acid/zoledronate, FOSAMAX8 adendronate, AREDIA® pamidronate, SKELID® tiludronate or ACTONOL® Risedronate; and troxacitabine (l,3-dioxolan cytosine nucleoside analog); antisense nucleotides, especially inhibition associated with abnormal cell proliferation Genes in the signal pathway represent antisense oligonucleotides such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccines and gene therapy vaccines (eg ALLOVECTIN) ® vaccine, LEUVECTIN® vaccine and VAXID® vaccine); LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (-ErbB-2 and EGFR double cheese) Amino acid kinase small molecule inhibitor, also known as Is pharmaceutically acceptable salt, acid or derivative of GW572016); and any of the above. As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells, such as cells expressing EGFL7, in vitro or in vivo. Thus, growth inhibitors can significantly reduce cells in the S phase (such as Agents 119234.doc -43- 200813088 cells of EGFL7 percentage. Examples of growth inhibitors include agents that block cell cycle progression (other than S phase), such as agents that induce G1 arrest and stagnation. Classical sputum blockers include vinblastine (vincristine and vinblastine), taxanes, and topoisomerase π inhibitors (such as doxorubicin, epirubicin, daunorubicin, Etoposide and bleomycin). These agents that arrest G1 are also associated with s-stagnation, such as DNA alkylating agents such as tamoxifen, prednis〇ne, dacarbazine, Nitrogen, cisplatin, methotrexate, 5-fluorouracil, and ara_c. Additional information can be found in Murakami et al., The Molecular Basis 〇f Cancer, edited by Mendelsohn and Israel, Chapter 1, entitled "CeU cycle regulation, oncogene s, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), especially on page 13. Taxanes (taxol and docetaxel) are anticancer drugs derived from yew trees. TAXOTERE® (Rhone-Poulenc Rorer) is a semi-synthetic analog of paclitaxel (TAXOL, Bristol-Myers Squibb). Taxol and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to cell mitosis inhibition. π Doxorubicin " is an anthracycline antibiotic. The complete chemical name of doxorubicin is (8S_cis)_1〇_[(3-amino-2,3,6•three deoxy_a_L-laisu_hexosepyranosyl)oxy]-7 ,8,9,1〇_tetrahydro·6,8,i-trihydroxy-8•(hydroxyethyl methoxy-5,12-tetracylenedione. The polypeptide containing the Fc region) Refers to a polypeptide comprising a region, such as an antibody or immunoadhesin (as defined herein). The Fc region can be removed, for example, during purification of the polypeptide or by recombinant engineering of the nucleic acid encoding the polypeptide 119234.doc - 44-200813088 A terminally-acidic acid (residue 44 7 according to EU code 3). Thus, a composition comprising a polypeptide having an Fc region according to the invention may comprise a polypeptide having K447, having all K447 removed or having And mixtures of polypeptides having no K447 residues. Compositions of the invention and methods of making the same The invention encompasses compositions (including pharmaceutical compositions) comprising an anti-EGFL7 antibody and a polynucleotide comprising a sequence encoding an anti-EGFL7 antibody. As used herein, a composition comprising one or more antibodies that bind to EGFL7 and/or one or more comprising one or more of the antibodies that bind to EGFL7 Polynucleotides of the sequence of the antibody. Such compositions may additionally comprise suitable carriers well known in the art, such as pharmaceutically acceptable excipients (including buffers). The present invention also encompasses isolated antibodies. Examples of polynucleotides. The invention also encompasses examples of substantially pure antibodies and polynucleotides. The anti-EGFL7 antibody of the present invention is preferably a monoclonal antibody. The present invention also covers the present invention. Fab, Fab, Fab, -SH and F(ab')2 fragments of anti-EGFL7 antibodies are provided. These antibody fragments can be produced by conventional means such as enzymatic digestion, or can be produced by recombinant techniques. Antibody fragments can be chimeric or humanized antibody fragments. These fragments can be used for the diagnostic and therapeutic purposes described below. Monoclonal antibodies can be derived from a substantially homogeneous population of antibodies (ie, a population comprising individual antibodies, except for the possibility of naturally occurring It can be obtained in the same manner as the mutations which are present in a small amount. Therefore, 'modifications, single plants' means the characteristics of antibodies which are not in the form of a mixture of discrete antibodies. The anti-EGFL7 monoclonal antibody of the present invention can be used most. It is produced by the fusion tumor method described by Kohler et al., 119234. doc-45-200813088 仏re 256:495 (1975), or can be produced by recombinant DNA method (U.S. Patent No. 4,816,567). Immunizing a mouse or other appropriate host animal, such as a hamster, to induce the production or production of lymphocytes that will specifically bind to the protein used for immunization. Typically by multiple subcutaneous (sc) or intraperitoneal (ip) EGFL7 and an adjuvant are administered to produce an anti-EGFL7 antibody in the animal. EGFL7 can be prepared using methods well known in the art, and some methods are further described herein. For example, the EGFL7 eve will be described below; Name one silk, you I Φ, leisure winter right $ $ 疝 Bf? I white mu ▼ _·_ ^I- ^s 9 , ·, u# / y v-^ — ^ , 钃, f~ chain The EGFL7 derivative of the extracellular domain (ECD) of EGFL7 of the Fc portion was immunized to animals. In one embodiment, the animal is immunized with an EGFL7-IgG1 fusion protein. Animals are typically produced against immunogenic co-arms or derivatives of EGFL7 and monophosphorus lipid A (MPL)/trehalose bismuth mold vinegar (TDM) (Ribi Immunochem. Research, Inc. 5 Hamilton, MT). Immunize and inject the solution into the cortex at multiple sites. The animals were boosted two weeks later. Animals were bled for 7 to 14 days and serum anti-EGFL7 strength was determined. Animals are strengthened from immunization until the price reaches a stable stage. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form a fusion tumor cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The thus prepared fusion tumor is inoculated and grown in a suitable medium preferably containing one or more substances which inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine 119234.doc -46-200813088 呤-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium used for fusion tumors will usually include hypoxanthine. , aminopterin and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells. Preferably, the myeloma cells are fused efficiently, allowing the selected antibody to produce cells that stably and in high amounts produce antibodies and are susceptible to myeloma cells such as HAT medium. Among them, the preferred myeloma cell line is a murine myeloma strain, such as MOPC-21 and MPC-11 mouse tumors purchased from Salk Institute Cell Distribution Center, San Diego, California USA, and purchased from

American Type Culture Collection，Rockville，Maryland USA之SP-2或X63-Ag8-653細胞獲得之細胞株。亦已描述人類骨髓瘤及小鼠-人類雜交骨髓瘤細胞株用於產生人類單株抗體（Kozbor，J· Immunol·，133:3001 (1984); Brodeur 等人，Monoclonal Antibody Production Techniques and Applications，第 51-63 頁（Marcel Dekker，Inc·，New York， 1987))。檢定融合瘤細胞生長於其中之培養基中抗EGFL7之單株抗體的產生。較佳藉由免疫沉澱或藉由活體外結合檢定 (諸如放射免疫檢定（RIA)或酶聯結免疫吸附檢定（ELIS 測定由融合瘤細胞產生之單株抗體之結合特異性。舉例而言，可藉由 Munson等人，dwa/· 5107:220 (1980)之Scatchard分析測定單株抗體之結合親和力。鑑別產生具有所需特異性、親和力及/或活性之抗體之融合瘤細胞後，可藉由有限稀釋程序次選殖純系並藉由標準方法（Goding，Monoclonal Antibodies: Principles an(j 119234.doc -47- 200813088American Type Culture Collection, cell line obtained from SP-2 or X63-Ag8-653 cells of Rockville, Maryland USA. Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Pages 51-63 (Marcel Dekker, Inc., New York, 1987)). The production of a monoclonal antibody against EGFL7 in a medium in which the fusion tumor cells were grown was assayed. Preferably, the binding specificity of the monoclonal antibodies produced by the fusion tumor cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELIS). For example, The binding affinity of a single antibody is determined by the Scatchard analysis of Munson et al., dwa/. 5107: 220 (1980). Identification of fusion tumor cells that produce antibodies with the desired specificity, affinity and/or activity can be limited by Dilution procedures are used to select pure lines and by standard methods (Goding, Monoclonal Antibodies: Principles an (j 119234.doc -47- 200813088

Practice’ 弟 59-103 頁（Academic Press，1986))使其生長。適用於此目的之培養基包括（例如）D_MEM或RPMM640培養基。此外’可使融合瘤細胞於活體内以動物體内之腹水腫瘤形式生長。藉由習知免疫球蛋白純化程序使由次純系分泌之單株抗體與培養基、腹水或血清適當分離，該等純化程序諸如蛋白A-瓊脂糖、羥磷灰石層析、凝膠電泳、透析或親和層析。可藉由使用組合文庫篩選具有所需活性之合成抗體純系製得本發明之抗EGFL7抗體。大體上，藉由篩選含有呈現融合噬菌體鞘蛋白之抗體可變區（Fv)之各種片段之噬菌體的噬菌體文庫來選擇合成抗體純系。藉由對抗所欲抗原之親和層析來篩檢此等噬菌體文庫。使表現能夠與所欲抗原結合之Fv片段之純系吸附至該抗原且因此使其與文庫中非結合純系分離。接著將結合純系自該抗原溶離，且可藉由額外之抗原吸附/溶離循環進一步富集。可藉由設計適當之抗原篩選程序選擇所關注之嗟菌體純系，隨後使用來自所關注之噬菌體純系之Fv序列及Kabat等人，Sequences of Proteins of Immunological Interest ，第 5 版，NIH Publication 91-3242，Bethesda MD (1991)，第 1-3卷中所述之適當恆定區（Fc)序列構築全長抗EGFL7抗體純系來獲得本發明之某些抗EGFL7抗體。抗體之抗原結合域係由兩個具有約110個胺基酸之可變 (V)區形成，兩個可變區分別來自輕鏈（VL)及重鏈（VH)， 119234.doc -48- 200813088 兩者均存在三個高變環或互補判定區（CDR)。如Winter等人，Ann. Rev. Immunol· 12: 433-55 (1994)中所述，可使可變域以其中VH與VL經由短的彈性肽共價連接之單鏈 Fv(scFv)片段形式或以其中其各自融合至恆定域且非共價相互作用之Fab片段形式功能性地呈現於噬菌體上。如本文所使用，scFv編碼性噬菌體純系及Fab編碼性噬菌體純共同稱為”Fv噬菌體純系”或”Fv純系”。可藉由聚合酶鏈反應（PCR)單獨選殖VH及VL基因譜系，且將其隨機重組於噬菌體文庫中，接著可如Winter等人， dnw· /mmwmo/· 12: 433-55 (1994)中所述自其中搜索抗原結合純系。來自經免疫來源之文庫提供抗免疫原之高親和力抗體而無需構築融合瘤。或者，可選殖天然譜系以在無任何免疫之情況下提供對抗廣泛範圍之非自體以及自體抗原之人類抗體的單一來源，如Griffiths等人，五MBO 乂 12: 725-34 (1993)所述。最後，亦可藉由自幹細胞選殖未經重排之V基因區段且使用含有隨機序列之PCR引子編碼南可變CDR3區並實現活體外重排而合成得到天然文庫，如 Hoogenboom及 Winter，乂 Mo/·出〇/· 227:381-88 (1992)所述0 使用絲狀噬菌體藉由與少量鞘蛋白？111融合來呈現抗體片段。抗體片段可以單鏈Fv片段之形式呈現，其中¥11與 VL結構域經彈性多肽間隔子於相同多肽鏈上連接，（例如）如 Marks 等人，乂 Μο/·价〇/· 222:581_597 (1991)所述；或以Fab片段形式呈現，其中一條鏈與ρΙΠ融合而另一鏈分泌 119234.doc -49- 200813088 至細菌宿主細胞周質中，在該細胞周質中組裝Fab-鞘蛋白結構，其經由某些野生型鞘蛋白移位而呈現於噬菌體表面上，（例如）如 Hoogenboom等人，TVwc/· 19:4133- 37 (1991)所述。大體而言，編碼抗體基因片段之核酸可自由人類或動物採集之免疫細胞獲得。若需要傾向於抗EGFL7純系之文庫，則用EGFL7免疫受檢者來產生抗體反應，且回收脾細胞及/或循環Β細胞或其他周邊血液淋巴細胞（PBL)以用於文庫構築。在一較佳實施例中，其藉由在具有功能性人類免疫球蛋白基因陣列（且缺乏功能性内源抗體產生系統）之轉殖基因小鼠體内產生抗人類EGFL7抗體反應，以致於該 EGFL7免疫作用造成產生抗EGFL7人類抗體之Β細胞，因而獲得傾向於抗人類EGFL7純系之人類抗體基因片段文庫。下文將描述產生人類抗體之轉殖基因小鼠的產生。可藉由使用適當篩選程序分離表現EGFL7特異性膜結合抗體之Β細胞（例如藉由用EGFL7親和層析分離細胞或使細胞吸附至經螢光染料標記之EGFL7隨後進行流式活化細胞分選（FACS))來獲得抗EGFL7反應性細胞群體之額外富集。或者’使用未經免疫之供體之脾細胞及/或B細胞或其他 PBL提供可能抗體譜系之更佳展示，且亦允許使用EGFL7 不具抗原性之任何動物（人類或非人類）物種構築抗體文庫。對於併入活體外抗體基因構築之文庫而言，自受檢者採集幹細胞以提供編碼未經重排之抗體基因區段之核酸。 119234.doc -50- 200813088 所關注之免疫細胞可自多種動物物種獲得，諸如人類、小鼠、大鼠、兔類、狼、犬科、貓科、豬、牛、馬及鳥類物種等。自所關注之細胞中回收編碼抗體可變基因區段（包括VH 及VL區段）之核酸並使其擴增。在經重排之vh及VL基因文庫之情況下，可藉由自淋巴細胞中分離基因組DNA或 mRNAP遠後用與經重排Vh及VL基因之5,及3,端匹配的引子進行聚合酶鏈反應（PCR)(如Orlandi等人，Pn Wi/. dad· 86:3833-37 (1989)所述），由此製得用於表現之多種V基因譜系，從而獲得所需DNA。可由cDNA及基因組DNA擴增V基因，其中後置引子在編碼成熟v結構域之外顯子的5’端且正向引子在J區段内，如〇riandi等人 (1989)及 Ward 等人，341:544-46 (1989)中所述。然而’對於由cDNA進行擴增’後置引子亦可如jones等人， 9:88-89 (1991)中所述在前導序列之外顯子中，且正向引子如Sastry等人，尸⑺匕#如/. 心/. [/μ 86:5728-32 (1989)中所述在恆定區内。為使互補性最大化，可如Orlandi等人（1989)或Sastry等人（1989)所述將簡並性併入引子中。較佳藉由使用以用以擴增免疫細胞核酸樣本中所存在之所有可用VH及VL排列之各V基因家族為目標之PCR引子使文庫多樣性最大化，（例如）如Marks等人， J. Mo/· 5b/· 222:581-97 (1991)之方法中所述或如〇rum 等人，TVwc/· 乂以心及以· 21:4491-4498 (1993)之方法中所述。對於將經擴增DNA選殖至表現載體中，可如〇riandi等人 119234.doc -51 - 200813088 (1989)中所述在PCR引子内引入稀有限制性位點作為一端之標籤，或如 Clackson等人，TVa/wre 352:624-628 (1991)中所述藉由用經標記引子進一步進行PCR擴增引入。合成性重排之V基因譜系可於活體外自V基因區段獲得。已對大部分人類VH基因區段進行選殖並測序（報導於 Tomlinson等人，Mo/. 5/〇/· 227:776-98 (1992)中），且進行定位（報導於 Matsuda等人，Gewei· 3:88-94 (1993) 中）；可使用此等經選殖區段（包括HI及H2環之所有主要構型）用編碼具有多種序列及長度之H3環的PCR引子產生多種 VH 基因譜系，如 Hoogenboom 及 Winter，/. Mol· Biol· 227:381-88 (1992)中所述。亦可製得所有序列多樣性集中於單一長度之長H3環中的VH譜系，如Barbas等人，尸roc· Natl. Acad· Sci. USA 89:4457-61 (1992)中所述。已對人類 Vk及νλ區段進行選殖並測序（報導於Williams及Winter，五wr. J. 23:1456-61 (1993)中）且可用其製造合成輕鏈譜系。基於一定範圍之VH與VL折疊及L3與H3長度之合成V基因譜系將編碼具有大量結構多樣性之抗體。擴增 V基因編碼 DNA後，可根據Hoogenboom及 Winter, Mo/· 5沁/. 227:381-88 (1992)之方法活體外重排生殖系V基因區段。一可以數種方式藉由將VH與VL基因譜系組合在一起來構築抗體片段譜系。各譜系可在不同載體中產生，且該等載體係於活體外（例如，如Hogrefe等人，Gene 128:119-26 (1993)中所述）或於活體内藉由組合感染（例如，如 119234.doc -52- 200813088Practice's page 59-103 (Academic Press, 1986)) makes it grow. Media suitable for this purpose include, for example, D_MEM or RPMM 640 medium. Furthermore, the fusion tumor cells can be grown in vivo as ascites tumors in animals. The monoclonal antibodies secreted by the sub-pure lines are appropriately separated from the culture medium, ascites or serum by a conventional immunoglobulin purification program such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis. Or affinity chromatography. The anti-EGFL7 antibody of the present invention can be produced by screening a synthetic library for the desired activity using a combinatorial library. In general, a synthetic antibody-derived line is selected by screening a phage library containing phage displaying various fragments of the antibody variable region (Fv) of the fusion phage sheath protein. These phage libraries are screened by affinity chromatography against the desired antigen. A pure line that expresses an Fv fragment capable of binding to the desired antigen is adsorbed to the antigen and thus is separated from the non-binding pure line in the library. The bound pure line is then eluted from the antigen and further enriched by additional antigen adsorption/dissolution cycles. The pure strain of the bacterium can be selected by designing an appropriate antigen screening program, followed by the Fv sequence from the pure phage line of interest and Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242 The appropriate constant region (Fc) sequences described in Bethesda MD (1991), Volumes 1-3 construct a full length anti-EGFL7 antibody pure line to obtain certain anti-EGFL7 antibodies of the invention. The antigen binding domain of the antibody is formed by two variable (V) regions having about 110 amino acids, the two variable regions are from the light chain (VL) and the heavy chain (VH), respectively, 119234.doc -48- 200813088 There are three hypervariable loops or complementary decision regions (CDRs) in both. As described in Winter et al, Ann. Rev. Immunol. 12: 433-55 (1994), the variable domain can be in the form of a single-chain Fv (scFv) fragment in which VH and VL are covalently linked via a short elastin peptide. Or functionally presented on phage in the form of Fab fragments in which each is fused to a constant domain and non-covalently interacted. As used herein, the scFv-encoding phage-pure line and the Fab-encoding phage are collectively referred to as "Fv phage pure line" or "Fv pure line". The VH and VL gene lineages can be individually cloned by polymerase chain reaction (PCR) and randomly recombined into a phage library, followed by Winter et al., dnw. /mmwmo/. 12: 433-55 (1994) The antigen-binding pure line is searched for from it. Libraries from immunized sources provide high affinity antibodies against the immunogen without the need to construct fusion tumors. Alternatively, the natural lineage can be selected to provide a single source of human antibodies against a wide range of non-autologous and autoantigens without any immunization, such as Griffiths et al., V. MBO 乂 12: 725-34 (1993) Said. Finally, natural libraries such as Hoogenboom and Winter can also be synthesized by selecting the unrearranged V gene segment from stem cells and encoding the southern variable CDR3 region using a PCR primer containing a random sequence and realizing in vitro rearrangement.乂Mo/·出〇/· 227:381-88 (1992) 0 using filamentous phage by using a small amount of sheath protein? 111 is fused to present antibody fragments. The antibody fragment can be presented as a single-chain Fv fragment, wherein the ¥11 and VL domains are joined to the same polypeptide chain via an elastomeric polypeptide spacer, for example, as Marks et al., 乂Μο/·价〇/· 222:581_597 ( Said in 1991); or in the form of a Fab fragment in which one strand is fused to ρΙΠ and the other chain secretes 119234.doc -49-200813088 into the periplasm of the bacterial host cell, in which the Fab-sheath protein structure is assembled, via a certain These wild-type sheath proteins are translocated and presented on the surface of the phage, for example as described by Hoogenboom et al., TVwc/19:4133-37 (1991). In general, nucleic acids encoding antibody gene fragments are obtained freely from immune cells harvested by humans or animals. If a library that favors the EGFL7 pure line is required, the subject is immunized with EGFL7 to generate an antibody response, and spleen cells and/or circulating sputum cells or other peripheral blood lymphocytes (PBL) are recovered for library construction. In a preferred embodiment, the anti-human EGFL7 antibody response is produced in a transgenic mouse having a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that EGFL7 immunization results in the production of a sputum cell against the EGFL7 human antibody, thus obtaining a library of human antibody gene fragments that are prone to anti-human EGFL7 pure lineage. The production of a transgenic mouse producing a human antibody will be described below. Purine cells expressing EGFL7-specific membrane-bound antibodies can be isolated by using appropriate screening procedures (eg, by isolating cells with EGFL7 affinity chromatography or by adsorbing cells to fluorescent dye-labeled EGFL7 followed by flow-activated cell sorting ( FACS)) to obtain additional enrichment of the anti-EGFL7 reactive cell population. Or 'use spleen cells and/or B cells or other PBLs that are not immunized donors to provide a better display of possible antibody lineages, and also allow the construction of antibody libraries using any animal (human or non-human) species that are not antigenic in EGFL7 . For libraries incorporating in vivo antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding the unrearranged antibody gene segments. 119234.doc -50- 200813088 Immune cells of interest may be obtained from a variety of animal species, such as humans, mice, rats, rabbits, wolves, canines, felines, pigs, cattle, horses, and birds. Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged vh and VL gene libraries, the polymerase can be isolated by isolating genomic DNA or mRNAP from lymphocytes and then using primers that match the 5 and 3 ends of the rearranged Vh and VL genes. Chain reaction (PCR) (as described by Orlandi et al., Pn Wi/. dad. 86:3833-37 (1989)), thereby producing a variety of V gene lineages for expression to obtain the desired DNA. The V gene can be amplified from cDNA and genomic DNA, wherein the post-primer encodes the 5' end of the mature v domain and the forward primer is in the J segment, such as 〇riandi et al. (1989) and Ward et al. , 341: 544-46 (1989). However, the 'for amplification by cDNA' post-introduction can also be found in the exon of the leader sequence as described in Jones et al., 9:88-89 (1991), and positive primers such as Sastry et al., corpse (7)匕# as in /. Heart /. [/μ 86: 5728-32 (1989) in the constant zone. To maximize complementarity, degeneracy can be incorporated into the primers as described by Orlandi et al. (1989) or Sastry et al. (1989). Library diversity is preferably maximized by using PCR primers targeted to amplify each V gene family of all available VH and VL sequences present in the nucleic acid nucleic acid sample, for example, as Marks et al., J Mo/· 5b/· 222:581-97 (1991) as described in the method or as 〇rum et al., TVwc/· 乂及 and in the method of 21: 4491-4498 (1993). For the selection of the amplified DNA into the expression vector, a rare restriction site can be introduced into the PCR primer as a label at one end as described in 〇riandi et al. 119234.doc-51 - 200813088 (1989), or as Clackson It is introduced by further PCR amplification with a labeled primer as described in TVa/wre 352:624-628 (1991). A synthetic rearranged V gene lineage can be obtained from a V gene segment in vitro. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al, Mo/. 5/〇/. 227:776-98 (1992)) and localized (reported in Matsuda et al., Gewei·3:88-94 (1993); can be used to generate a variety of VHs using PCR primers encoding H3 loops of various sequences and lengths using these selected segments (including all major configurations of HI and H2 loops) Gene lineages are described in Hoogenboom and Winter, /. Mol. Biol. 227:381-88 (1992). All VH lineages in which sequence diversity is concentrated in a single long length H3 loop can also be made, as described in Barbas et al., Corpus Roc. Natl. Acad. Sci. USA 89: 4457-61 (1992). Human Vk and νλ segments have been cloned and sequenced (reported in Williams and Winter, 5 wr. J. 23: 1456-61 (1993)) and can be used to make synthetic light chain lineages. A synthetic V gene lineage based on a range of VH and VL folds and L3 and H3 lengths will encode antibodies with substantial structural diversity. After amplification of the V gene encoding DNA, the germline V gene segment can be rearranged in vitro according to Hoogenboom and Winter, Mo/. 5沁/. 227:381-88 (1992). There are several ways to construct an antibody fragment lineage by combining the VH and VL gene lineages. Each lineage can be produced in a different vector, and the vectors are in vitro (for example, as described in Hogrefe et al, Gene 128: 119-26 (1993)) or by in vivo infection (eg, as 119234.doc -52- 200813088

Waterhouse等人，wc/·々油及以· 21:2265-66 (1993)中所述之MxP系統）重組。活體内重組方法利用Fab片段之雙鏈性夤來克服大腸桿菌（五轉化功率所強加之對於文庫大小之限制。單獨選殖天然VH及VL譜系，將一個選殖至噬菌粒中而另一個選殖至噬菌體載體中。接著藉由含有噬菌粒之細菌的噬菌體感染來組合兩個文庫，從而使各細胞含有不同組合且使文庫大小僅受所存在細胞數量（約1〇!2個純系）限制。兩載體均含有活體内重組信號，從而使VH與 VL基因重組於早一複製子上並共包裝於嗟菌體病毒粒子中。此等大型文庫提供大量具有良好親和力（Kd-i為約1〇·8 M)之多種抗體。或者，可相繼將譜系選殖至相同載體中，（例如）如Waterhouse et al., WC/·Oyster Oil and MxP Systems as described in 21:2265-66 (1993) are reorganized. The in vivo recombination method utilizes the double-stranded sputum of the Fab fragment to overcome E. coli (the five transformation power imposes limitations on the size of the library. The natural VH and VL lineages are separately selected, one is selected into the phagemid and the other is Colonization into phage vectors. The two libraries are then combined by phage infection of bacteria containing phagemids, so that each cell contains different combinations and the size of the library is only affected by the number of cells present (about 1 〇! 2 pure lines) Limitation. Both vectors contain an in vivo recombination signal, such that the VH and VL genes are recombined on the early replicon and co-packaged in the bacteriophage virions. These large libraries provide a large number of good affinities (Kd-i is a plurality of antibodies of about 1 〇 8 M). Alternatively, the lineage can be successively selected into the same vector, for example, as

Barbas等人，[/5^4 88:7978-82 (1991) 中所述’或可精由PCR將譜糸組裝在一起且接著選殖，（例如）如 Clackson等人，352:624-28 (1991)中所述。亦可使用PCR組裝將VH及VL DNA與編碼彈性肽間隔子之 DNA連接在一起以形成單鏈Fv (scFv)譜系。在另一種技術中，使用”細胞内PCR組裝’’藉由PCR將VH與VL基因在淋巴細胞内組合且接著選殖已連接基因之譜系，如Embleton等人，#1^/.^(以心心5>.20:3831-37 (1992)中所述。由天然文庫產生之抗體（天然或合成）可具有中等親和力 (Kd-1為約106至1〇7 M-1)，但亦可藉由自次級文庫進行構築並重選而於活體外模擬親和力成熟，如Winter等人 (1994)，同上文中所述。舉例而言，可以Hawkins等人，/· 119234.doc -53- 200813088Barbas et al., [/5/4 88:7978-82 (1991) described in '/ can be used to assemble the putatives by PCR and then colonize, for example, as Clackson et al., 352: 624-28 (1991). The VH and VL DNA can also be ligated together with the DNA encoding the elastin spacer using PCR assembly to form a single-chain Fv (scFv) lineage. In another technique, the "intracellular PCR assembly" is used to combine the VH and VL genes in lymphocytes by PCR and then to select the lineage of the linked genes, such as Embleton et al., #1^/.^ Hearts 5 >. 20:3831-37 (1992). Antibodies produced by natural libraries (natural or synthetic) may have moderate affinity (Kd-1 is about 106 to 1 〇 7 M-1), but may also Affinity maturation is simulated in vitro by constructing and reselecting from a secondary library, as described by Winter et al. (1994), as described above. For example, it can be obtained by Hawkins et al., 119234.doc-53-200813088

Mol· 226:889-96 (1992)之方法或 Gram 等人，尸Mol. 226: 889-96 (1992) or Gram et al.

Sc/ 89:3576-80 (1992)之方法藉由使用易誤聚合酶（報導於Leung等人，1:11-15 (1989)中）於活體外隨機引入突變。此外，可藉由（例如）用具有跨越所關注之CDR之隨機序列的引子使用pcr在所選個別Fv純系中使一或多個CDR隨機突變並篩選較高親和力純系來進行親和力成熟。WO 96/07754 (1996年3月14日公開）描述一種誘導免疫球蛋白輕鏈互補判定區突變以建立輕鏈基因文庫的方法。另一有效方法為將由噬菌體呈現所選之VH或 VL結構域與自未經免疫之供體獲得的天然存在之V結構域變異體譜系重組，且以數輪鏈重配置篩選較高親和力者’ 如 Marks等人，10:779-83 (1992)中所述。此技術允許製造親和力在1(Γ9 Μ範圍内之抗體及抗體片段。可使用EGFL7之所需區域中之胺基酸序列設計編碼 EGFL7之核酸序列。或者，可使用cDNA序列（或其片段）。額外EGFL7序列另外揭示於（例如）NM_022963及Xie等人，加11:729-35 (1999)中。可藉由此項技術中已知之多種方法製備編碼EGFL7之DNA。此等方法包括（但不限於）由 Engels等人，dgwew· Chem. Int. Ed. EngL 28:716-34 (1989)中所述之任何方法進行之化學合成，諸如三酯、亞磷酸酯、胺基磷酸酯及H-膦酸酯方法。在一實施例中，使用表現宿主細胞優選之密碼子設計EGFL7編碼DNA。或者，可自基因組或cDNA文庫分離編碼EGFL7之DNA。構築編碼EGFL7之DNA分子後，使DNA分子以可操作方 119234.doc -54- 200813088 式連接至表現載體（諸如質體）中之表現控制序列，其中控制序列由經載體轉化之宿主細胞識別。一般而言，質體載體含有自可與宿主細胞相容之物種獲得的複製及控制序列。載體通常具有複製位點以及編碼能夠在經轉化細胞中提供表型選擇之蛋白質的序列。此項技術中已知用於在原核及真核宿主細胞中表現之適當載體，且某些載體將在本文中進一步描述。可使用真核生物體（諸如酵母）或自多細胞生物體（諸如哺乳動物）獲得之細胞。視情況使編碼EGFL7之DNA以可操作方式連接至分泌箭導序列而導致宿主細胞將表現產物分泌至培養基中。分泌剷導序列之實例包括stn、ec〇tin、lamB、疮療GD、lpp、驗性鱗酸酶、轉化酶及α因子。蛋白A之3 6胺基酸前導序列 (Abrahmsen等人，五M50 乂 4:3901 (1985))亦適用於本文中。用上文所述之本發明之表現或選殖載體轉染且較佳轉化宿主細胞，並將其培養於經改質以適於誘導啟動子、選擇轉化體或擴增編碼所需序列之基因的習知營養培養基中。轉係心實際表現或不表現任何編碼序列之宿主細胞對表現載體之吸收。一般熟習此項技術者已知多種轉染方法，例如CaPCU沉澱及電穿孔。一般當有關此載體操作之任何跡象出現於宿主細胞内時識別轉染成功。此項技術中熟知轉染方法，且某些方法將於本文中進一步描述。轉化意謂將DNA引入生物體中使得DNA可作為染色體外元件或由染色體成份複製。視所使用之宿主細胞而定，使 119234.doc -55- 200813088 用適於此等細胞之標準技術進行轉化。此項技術中熟知轉化方法，且某些方法將於本文中進一步描述。可將用於產生EGFL7之原核宿主細胞如Sambrook等人 (同上文）中大體描述進行培養。可將用於產生EGFL7之哺乳動物宿主細胞培養於此項技術中熟知之多種培養基中，且某些培養基描述於本文中。本揭示内容中所提及之宿主細胞涵蓋活體外培養物中之細胞以及宿主動物體内之細胞。 f、 " 可使用技術公認之方法實現對EGFL7之純化。可使經純化EGFL7附著於用於親和層析分離噬菌體呈現純系之適當基質上，諸如瓊脂糖微珠、丙烯醯胺微珠、玻璃微珠、纖維素、各種丙烯酸系共聚物、甲基丙烯酸羥基酯凝膠、聚丙烯酸及聚甲基丙烯酸系共聚物、耐侖 (nylon)、中性及離子性載體及其類似物。可藉由 Enzymo/·第44卷（1976)中所述之方法實現EGFL7蛋白附著 / 於基質上。使蛋白配位體附著於多醣基質（例如瓊脂糖、葡聚糖或纖維素）上之常用技術涉及用_化氰活化載體且隨後使肽配位體之初級脂族或芳族胺與經活化基質偶聯。或者，可將EGFL7用於塗覆吸附培養盤之孔，表現於固定至吸附培養盤上之宿主細胞上或用於細胞分選，或與生物素共軛以用塗覆抗生蛋白鏈菌素之微珠捕捉，或用於篩檢嗟菌體呈現文庫之任何其他此項技術中已知之方法中。使噬菌體文庫樣本與經固定之EGFL7在適於使至少一部分嗟菌體顆粒與吸附劑結合之條件下接觸。一般而言，對 119234.doc -56· 200813088 包括pH值、離子強度、溫度及其類似條件之條件進行選擇以模擬生理條件。洗滌與固相結合之噬菌體，且接著用酸溶離（例如，如Barbas等人，仍」 88:7978-82 (1991)中所述）或用鹼溶離（例如，如乂訂匕等人，乂 Mo/·万沁/· 222:581_97 (1991)中所述）或藉由EGFL抗原競爭溶離（例如，以與clacks〇n等人，Λn 352:624_28 (1991)之抗原競爭方法類似之程序）。可在單輪選擇中富集 20_1，000倍之噬菌體。此外，可使所富集之噬菌體在細菌培養物中生長益經歷另'輪選擇。選擇效率視許多因素而定，包括洗滌過程中之解離動力學及單一嗟菌體上之多個抗體片段能否同時與抗原接合。具有快速解離動力學（及弱結合親和力）之抗體可藉由使用短時間洗滌、多價噬菌體呈現及抗原於固相中之高塗覆密度予以保留。高密度不僅經由多價相互作用使噬菌體穩定，且亦有利於使已解離之噬菌體再結合。可藉由使用長時間洗滌及單價噬菌體呈現（如Bass等人，户⑺…^ 8:3〇9_ 14 (1990)及WO 92/09690中所述）及低抗原塗覆密度（如 Marks等人，万沁化〜似/· 10:779-83 (1992)中所述）促進對於具有緩慢解離動力學（及良好結合親和力）之抗體的選擇。可在對EGFL7具有不同親和力，甚至具有略微不同之親和力之噬菌體抗體之間進行選擇。然而，所選抗體之隨機突變（例如，如上文所述之某些親和成熟技術中進行）可能產生許多突變體，其中大部分與抗原結合而少數具有較高親和力。使用限制性EGFL7，可競爭淘汰極少之高親和力 119234.doc -57- 200813088 噬菌體。為保留所有具有較高親和力之突變體，可用過量經結合生物素之EGFL7培育噬菌體，但經結合生物素之 EGFL7具有低於EGFL7之恆定目標莫耳親和力之莫耳濃度的濃度。接著可以經抗生蛋白鏈菌素塗覆之順磁微珠捕捉高親和力結合之噬菌體。此”平衡捕捉”使得能夠根據抗體之結合親和力以敏感性選擇抗體，該敏感性允許使具有僅兩倍高親和力之突變體純系與具有較低親和力之大量過量噬菌體分離。亦可操控洗滌與固相結合之噬菌體中所用The method of Sc/89: 3576-80 (1992) randomly introduces mutations in vitro by using an error-prone polymerase (reported in Leung et al., 1:11-15 (1989)). In addition, affinity maturation can be performed by, for example, using a primer having a random sequence spanning the CDRs of interest, using PCR to randomly mutate one or more CDRs in a selected individual Fv line and screening for a higher affinity line. WO 96/07754 (published Mar. 14, 1996) describes a method of inducing mutations in the immunoglobulin light chain complementarity determining region to establish a light chain gene library. Another effective method is to recombine the VH or VL domain selected by the phage to a naturally occurring V domain variant lineage obtained from an unimmunized donor and to screen for higher affinity in a number of rounds of reconfiguration. As described in Marks et al., 10:779-83 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the range of 1 (Γ9 。. The nucleic acid sequence encoding EGFL7 can be designed using the amino acid sequence in the desired region of EGFL7. Alternatively, a cDNA sequence (or a fragment thereof) can be used. Additional EGFL7 sequences are additionally disclosed, for example, in NM_022963 and Xie et al., 11: 729-35 (1999). DNA encoding EGFL7 can be prepared by a variety of methods known in the art. Limited to) chemical synthesis by any of the methods described by Engels et al., dgwew. Chem. Int. Ed. Eng L 28: 716-34 (1989), such as triesters, phosphites, amino phosphates and H- Phosphonate method. In one embodiment, the EGFL7 encoding DNA is designed using a preferred codon for the host cell. Alternatively, the DNA encoding EGFL7 can be isolated from a genomic or cDNA library. After constructing a DNA molecule encoding EGFL7, the DNA molecule is Operable 119234.doc -54- 200813088 is linked to a expression control sequence in a performance vector, such as a plastid, wherein the control sequence is recognized by a host cell transformed with the vector. In general, the plastid vector contains There are replication and control sequences obtained from species compatible with the host cell. Vectors typically have a replication site and a sequence encoding a protein capable of providing phenotypic selection in transformed cells. This technique is known in the art for pronuclei. And suitable vectors for expression in eukaryotic host cells, and certain vectors will be further described herein. Cells obtained from eukaryotic organisms (such as yeast) or from multicellular organisms (such as mammals) can be used. The DNA encoding EGFL7 is operably linked to the secretory leader sequence resulting in the host cell secreting the expression product into the culture medium. Examples of secretion scribing sequences include stn, ec〇tin, lamB, sore GD, lpp, test scale Acidase, invertase and alpha factor. The amino acid amino acid leader sequence of Protein A (Abrahmsen et al., V. M50 4:3901 (1985)) is also suitable for use herein. Or the selection vector is transfected and preferably transformed into a host cell and cultured in a conventional nutrient culture modified to be suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence. The transfer of the host cell to the expression vector, which is actually or does not exhibit any coding sequence. A variety of transfection methods are known to those skilled in the art, such as CaPCU precipitation and electroporation. Transfection is recognized by any indication when it appears in the host cell. Transfection methods are well known in the art, and some methods are further described herein. Transformation means the introduction of DNA into an organism such that the DNA can serve as an extrachromosomal element or Replicated by chromosome components. 119234.doc -55- 200813088 is transformed with standard techniques appropriate for such cells, depending on the host cell used. Transformation methods are well known in the art and some methods are further described herein. Prokaryotic host cells for the production of EGFL7 can be cultured as generally described in Sambrook et al. (supra). Mammalian host cells for the production of EGFL7 can be cultured in a variety of media well known in the art, and certain media are described herein. Host cells referred to in the present disclosure encompass cells in in vitro culture as well as cells in host animals. f, " Purification of EGFL7 can be achieved using technically accepted methods. The purified EGFL7 can be attached to a suitable substrate for affinity chromatography to isolate the phage from a pure line, such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, methacrylic acid hydroxyl groups. Ester gels, polyacrylic acid and polymethacrylic copolymers, nylon, neutral and ionic carriers and the like. The attachment of EGFL7 protein to the substrate can be achieved by the method described in Enzymo/.Vol. 44 (1976). A common technique for attaching a protein ligand to a polysaccharide matrix, such as agarose, dextran or cellulose, involves activating the carrier with cyanohydrin and subsequently activating the primary aliphatic or aromatic amine of the peptide ligand. Matrix coupling. Alternatively, EGFL7 can be used to coat the wells of the adsorption plate, either on the host cells immobilized on the adsorption plate or for cell sorting, or conjugated with biotin to coat the streptavidin. Microbead capture, or any other method known in the art for screening a bacterial display library. The phage library sample is contacted with immobilized EGFL7 under conditions suitable for binding at least a portion of the sputum bacterial particles to the adsorbent. In general, the conditions including pH, ionic strength, temperature, and the like are selected to simulate physiological conditions for 119234.doc -56· 200813088. The phage bound to the solid phase is washed and then eluted with acid (for example, as described in Barbas et al., still 88:7978-82 (1991)) or dissolved with an alkali (for example, such as 乂匕匕 et al, 乂Mo/· Wan沁/· 222:581_97 (1991)) or competitively dissociated by EGFL antigen (for example, a procedure similar to that of clacks〇n et al., Λn 352:624_28 (1991)) . The phage can be enriched 20-1,000 times in a single round of selection. In addition, the enriched phage can be grown in bacterial culture for a further round of selection. The efficiency of selection depends on a number of factors, including the dissociation kinetics during the washing process and whether multiple antibody fragments on a single bacterium can simultaneously bind to the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinities) can be retained by using short washes, multivalent phage presentation, and high coating density of the antigen in the solid phase. High density not only stabilizes the phage via multivalent interactions, but also facilitates recombination of the dissociated phage. Can be presented by using long-term washing and monovalent phage display (as described in Bass et al., households (7)...^8:3〇9_14 (1990) and WO 92/09690) and low antigen coating density (eg Marks et al. The selection of antibodies with slow dissociation kinetics (and good binding affinity) is promoted by Wanhuahua~10/779 (83). Selection can be made between phage antibodies that have different affinities for EGFL7, even with slightly different affinities. However, random mutations in selected antibodies (e. g., as performed in certain affinity maturation techniques as described above) may result in a number of mutants, most of which bind to the antigen and a few have a higher affinity. Use of restricted EGFL7 can compete for the elimination of very low affinities. 119234.doc -57- 200813088 Phage. To retain all mutants with higher affinity, phage can be incubated with excess EGFL7 bound to biotin, but EGFL7 bound to biotin has a concentration of molar concentration below the constant target molar affinity of EGFL7. High affinity binding phage can then be captured by streptavidin coated paramagnetic beads. This "equilibrium capture" enables the selection of antibodies with sensitivity based on the binding affinity of the antibody, which allows for the isolation of a mutant line with only twice the high affinity from a large excess of phage with a lower affinity. Can also be used in the control of phage combined with washing and solid phase

I I 條件以基於解離動力學進行區分。抗ECtFL7純系亦可根據活性加選擇。可谷易地分離編碼本發明之源自融合瘤之單株抗體或嗟菌體呈現Fv純系之DNA，並使用習知程序（例如藉由使用經設計以由融合瘤或噬菌體DNA模板特異性擴增所關注之重鏈及輕鏈編碼區之募核苷酸引子）進行測序。分離後可將DNA置於表現載體内，接著將表現載體轉染至不會另外 & ' 產生免疫球蛋白之宿主細胞（諸如大腸桿菌細胞、猿COS細胞、中國倉鼠卵巢（CHO)細胞或骨髓瘤細胞）中以於重組宿主細胞中獲得所需單株抗體之合成。有關於抗體編碼Dna 在細菌中之重組表現之評論論文包括Skeira等人，Cwrr.I I conditions are distinguished based on dissociation kinetics. Anti-ECtFL7 pure lines can also be selected based on activity. The monoclonal antibody derived from the fusion tumor of the present invention or the sputum cell can be isolated from the Fv-pure DNA and used in a conventional procedure (for example, by using a template designed to specifically expand from a fusion tumor or phage DNA template). The nucleotide primers of the heavy and light chain coding regions of interest are added for sequencing. After isolation, the DNA can be placed in an expression vector, and then the expression vector can be transfected into host cells (such as E. coli cells, 猿COS cells, Chinese hamster ovary (CHO) cells, or bone marrow) that do not produce additional immunoglobulins. In tumor cells, the synthesis of the desired monoclonal antibody is obtained in a recombinant host cell. Review papers on the recombination performance of antibody-encoded DNA in bacteria include Skeira et al., Cwrr.

OpWon h 5·· 256 (1993)及 Pltickthim，/mm⑽〇/· i?ev· 130:151 (1992)。可將編碼本發明Fv純系之DNA與已知編碼重鏈及/或輕鏈恆定區之DNA序列（例如適當之DNA序列可由Kabat等人 (同上文）獲得）組合以形成編碼全長或部分長度重鏈及/或 119234.doc -58- 200813088 輕鏈之純系。應瞭解為達成此目的可使用任何同型之恆定區，包括IgG、IgM、IgA、IgD及IgE恆定區，且此等恆定區可由任何人類或動物物種獲得。如本文所使用之定義，，嵌合”抗體及’’雜交”抗體中包括自一種動物（諸如人類）物種之可變域DNA獲得且接著融合至另一動物物種之恆定區 DNA中以形成雜父π編碼序列（全長重鍵及/或輕鍵）的ρ v純系。在一較佳實施例中，自人類可變DNA獲得之Fv純系係融合至人類恆定區DNA中以形成全人類編碼序列，即全長或部分長度重鏈及/或輕鏈。舉例而言，亦可藉由以人類重鏈及輕鏈恆定域之編碼序列取代自融合瘤純系獲得之同源鼠科序列（例如，如OpWon h 5·· 256 (1993) and Pltickthim, /mm(10)〇/· i?ev· 130:151 (1992). A DNA encoding an Fv pure line of the invention can be combined with a DNA sequence known to encode a constant region of a heavy chain and/or a light chain (e.g., a suitable DNA sequence can be obtained from Kabat et al. (supra)) to form a full-length or partial length Chain and / or 119234.doc -58- 200813088 pure chain of light chain. It will be appreciated that any isotype constant region can be used to achieve this, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. As used herein, a chimeric antibody and a 'hybridization' antibody are obtained from variable domain DNA of an animal (such as a human) species and then fused to constant region DNA of another animal species to form a hybrid The ρ v pure line of the parent π coding sequence (full length heavy key and/or light key). In a preferred embodiment, the Fv pure line obtained from human variable DNA is fused to human constant region DNA to form a fully human coding sequence, i.e., a full length or partial length heavy and/or light chain. For example, a homologous murine sequence obtained by substituting a pure line of a fusion tumor with a coding sequence of a human heavy and light chain constant domain (for example,

Morrison等人，81:685 1-55 (1984)之方法）來修飾編碼本發明之自融合瘤獲得之抗 EGFL7抗體的DNA。可藉由使非免疫球蛋白多肽之所有或部分編碼序列與免疫球蛋白編碼序列共價連接來進一步修飾編碼自融合瘤或Fv純系獲得之抗體或片段的DNA。以此方式製備具有本發明之自Fv純系或融合瘤純系獲得之抗體之結合特異性的M嵌合，，或，，雜交"抗體。抗體片段本發明涵蓋抗體片段。在某些情形下，使用抗體片段而非使用整個抗體具有優點。較小尺寸之片段允許迅速清除且可導致對進入實體腫瘤之改良。已研發出製造抗體片段之多種技術。傳統上，經由蛋白水解消化完整抗體得到此等片段（例如參看M〇rim〇t〇等 119234.doc -59- 200813088 尺，J· Biochem. Biophys· Meth· 2Α·Λ0Ί-\Ί {\992、反 Brennan等人，229:81 (1985))。然而，現可藉由重組宿主細胞直接產生此等片段。Fab、Fv及ScFv抗體片段均可表現於大腸桿菌中且由大腸桿菌分泌，由此可容易地產生大量此等片段。可自上文所討論之抗體噬菌體文庫中分離抗體片段。或者，可直接自大腸桿菌回收Fab，-SH片段且使其化學偶聯以形成F(ab，）2片段（Carter等人，幻；10:163-67 (1992))。根據另一方法，可直接自重組宿主細胞培養物中分離F(ab，）2片段。具有增加之活體内半衰期的包含補救受體結合抗原決定基殘基之Fab 及F(ab’)2片段描述於美國專利第5,869,046號中。其他用於產生抗體片段之技術對於熟習此項技術者而言應為顯而易見的。在其他實施例中，所選擇之抗體為單鏈Fv片段 (scFv)。參看WO 93/16185、美國專利第5,571，894號及第 5,587,458號。Fv及sFv為僅有的缺少恆定區但具有完整組合位點之種類；因此，其適於在活體内使用期間達成降低之非特異性結合。可構築sFv融合蛋白來獲得效應蛋白在 sFv胺基或羧基末端之融合。參看AnUb〇dy以“以⑷叫，編輯 Borrebaeck，W.H· Freeman and c〇mpany (i992)。抗體片段亦可為”線性抗體”，（例如）如美國專利第MW·號中所述。此等線性抗體片段可為單特異性或雙特異性。人化抗體 ~ 本發明涵蓋人化抗體。人類抗體之方法。舉例而此項技術中已知多種用於人化非 Q，人化抗體可具有一或多個由 119234.doc •60- 200813088 非人類來源引入其中之胺基酸殘基。此等非人類胺基酸殘基通常稱為”輸入，，殘基，其通常係自’’輸入”可變域取得。可基本上根據Winter及其同事（Jones等人，321:522-25; Riechmann等人，TVaiwre 332:323-27 (1988); Verhoeyen 等人，239:1534-36 (1988))之方法藉由以高變區序列取代人類抗體之相應序列進行人化。因此，此等，，人化” 抗體為欲合抗體（美國專利第4,816,567號），其中實質上小於完整人類可變域之序列已經來自非人類物種之相應序列取代。實際上，人化抗體通常為某些高變區殘基及可能某些FR殘基經來自齧齒動物抗體類似位點之殘基取代的人類抗體。對用於製造人化抗體之人類可變域（輕鍵與重鍵）的選擇對於降低抗原性而言極為重要的。根據所謂之，，最佳擬合，，方法，對照已知人類可變域序列之完整文庫_選齧齒動物抗體之可變域序列。接著將與齧齒動物序列最接近之人類序列接受為人化抗體之人類構架（Sims等人，j. Immun〇1 151:2296 (1993); Chothia等人，j. Μ〇1· Bi〇1 196:9〇1 (1987))。另一方法使用自具有特定輕鏈或重鏈子群之所有人類抗體之一致序列獲得的特定構架。相同構架可用於數種不同之人化抗體（Carter等人，Pr〇c. Natl. Aead. Sei. USA，89:4285 (1992); Presta等人，j Im_〇i，i5i 2623 (1993)) 〇另外，抗體經人化而保持對抗原之高親和力及其他有利生物學特性極為重要。為達成此目標，根據_種方法，藉 119234.doc -61- 200813088 由使用親本序列及人化序列之三維模型分析親本序列及各種概念上之人化產物的方法來製備人化抗體。通常可用= 維免疫球蛋白模型且其為熟習此項技術者所熟悉。存在描述且呈現所選擇之候選免疫球蛋白序列之大致三維構形= 構的電腦程式。此等呈現之檢驗允許分析殘基對候選免疫球蛋白序列之功能可能起到的作用，亦即分析影響候選免疫球蛋白與其抗原結合之能力的殘基。以此方式，可自接受者及輸入序列選擇FR殘基並使其組合，從而達成所需抗體特徵（諸如對目標抗原增加之親和力）。一般而言，對於抗原結合之影響直接且最實質上涉及高變區殘基。人類抗體本發明之人類抗EGFL7抗體可藉由將選自源自人類之嗟菌體呈現文庫之Fv純系可變域序列與如上所述之已知人類怪定域序列組合來構築。或者，可藉由融合瘤方法製得本發明之人類單株抗EGFL7抗體。用於產生人類單株抗體之人類骨髓瘤及小鼠-人類雜交骨髓瘤細胞株已由以下文獻描述：例如Kozbor J· /m/ww加/·，133:3001 (1984); Brodeur 等人，Monoclonal Antibody Production Techniques and Applications’ 第 51-63 頁（Marcel Dekker，Inc.，New York， 1987)及 Boerner 等人，J· /mw⑽〇/·，147: 86 (1991) 〇現在可產生免疫後能夠在不產生内源免疫球蛋白之情況下產生全人類抗體谱糸的轉殖基因動物（例如小鼠）。舉例而言’已描述嵌合及生殖系突變體小鼠中抗體重鏈連接區 (JH)基因的純合子缺失導致對於内源抗體產生之完全抑 119234.doc -62- 200813088 制。將人類生殖系免疫球蛋白基因陣列轉移至此等生殖系突變體小鼠體内將會於抗原激發後引起人類抗體之產生。例如參看 Jakobovits 等人，Λ^/. 心/ t/M， 90:2551 (1993); Jakobovits等人，362: 255 (1993);Morrison et al, 81:685 1-55 (method) (1984) to modify the DNA encoding the anti-EGFL7 antibody obtained from the self-fused tumor of the present invention. DNA encoding an antibody or fragment obtained from a fusion tumor or Fv pure line can be further modified by covalently linking all or a portion of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, an M chimeric, or, hybrid "antibody having the binding specificity of the antibody obtained from the Fv pure line or the fusion line of the present invention is prepared. Antibody Fragments The present invention encompasses antibody fragments. In some cases, the use of antibody fragments rather than the use of whole antibodies has advantages. Fragments of smaller size allow for rapid clearance and can result in improvements in access to solid tumors. A variety of techniques for making antibody fragments have been developed. Traditionally, these fragments have been obtained by proteolytic digestion of intact antibodies (see, for example, M〇rim〇t〇 et al. 119234.doc -59-200813088 ft, J. Biochem. Biophys· Meth· 2Α·Λ0Ί-\Ί {\992, Anti Brennan et al., 229:81 (1985)). However, such fragments can now be produced directly by reconstituting the host cell. Fab, Fv and ScFv antibody fragments can all be expressed in E. coli and secreted by E. coli, whereby a large number of such fragments can be easily produced. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, the Fab, -SH fragment can be directly recovered from E. coli and chemically coupled to form an F(ab,)2 fragment (Carter et al., Magic; 10: 163-67 (1992)). According to another approach, the F(ab,)2 fragment can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments comprising a salvage receptor binding epitope residue having an increased in vivo half-life are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments should be apparent to those skilled in the art. In other embodiments, the antibody selected is a single chain Fv fragment (scFv). See WO 93/16185, U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and sFv are the only species that lack a constant region but have a complete combinatorial site; therefore, they are suitable for achieving reduced non-specific binding during in vivo use. The sFv fusion protein can be constructed to obtain fusion of the effector protein at the sFv amine or carboxy terminus. See AnUb〇dy for "calling (4), editing Borrebaeck, WH Freeman and c〇mpany (i992). Antibody fragments can also be "linear antibodies", as described, for example, in U.S. Patent No. MW. Linear antibody fragments can be monospecific or bispecific. Humanized antibodies ~ The invention encompasses humanized antibodies. Methods of human antibodies. For example, various techniques are known in the art for humanized non-Q, humanized antibodies can have One or more amino acid residues introduced by 119234.doc • 60- 200813088 from a non-human source. These non-human amino acid residues are commonly referred to as "inputs, residues, which are usually derived from '' Variable domain acquisition. Basically according to Winter and colleagues (Jones et al., 321:522-25; Riechmann et al., TVaiwre 332:323-27 (1988); Verhoeyen et al., 239: 1534-36 (1988) The method of human) is humanized by substituting the corresponding sequence of the human antibody with a hypervariable region sequence. Thus, such a humanized antibody is an antibody (U.S. Patent No. 4,816,567), which is substantially smaller than the intact human. The sequence of the variable domain has come from non-human species Substituted by the corresponding sequence. In fact, humanized antibodies are typically human antibodies that have certain hypervariable region residues and possibly some FR residues that have been replaced by residues from analogous sites in rodent antibodies. The selection of human variable domains (light and heavy) for the production of humanized antibodies is extremely important for reducing antigenicity. According to the so-called, best fit, method, control the complete library of known human variable domain sequences - the variable domain sequence of the rodent antibody. The human sequence closest to the rodent sequence is then accepted as the human framework for humanized antibodies (Sims et al, j. Immun〇 1 151:2296 (1993); Chothia et al., j. Μ〇1· Bi〇1 196 :9〇1 (1987)). Another method uses a specific framework obtained from a consensus sequence of all human antibodies with a particular light chain or heavy chain subpopulation. The same framework can be used for several different humanized antibodies (Carter et al, Pr.c. Natl. Aead. Sei. USA, 89: 4285 (1992); Presta et al, j Im_〇i, i5i 2623 (1993) In addition, it is extremely important that antibodies are humanized to maintain high affinity for antigens and other beneficial biological properties. To achieve this goal, humanized antibodies were prepared according to the method of 119234.doc -61-200813088 by analyzing the parental sequence and various conceptual humanized products using a three-dimensional model of the parental sequence and the humanized sequence. The immunoglobulin model is commonly available and is familiar to those skilled in the art. There is a computer program that describes and presents a roughly three-dimensional configuration of the selected candidate immunoglobulin sequence. Examination of such presentations allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this manner, FR residues can be selected and combined from the recipient and the input sequence to achieve the desired antibody characteristics (such as increased affinity for the target antigen). In general, the effects on antigen binding directly and most substantially involve hypervariable region residues. Human antibody The human anti-EGFL7 antibody of the present invention can be constructed by combining an Fv pure line variable domain sequence selected from a human-derived bacterium-producing library with a known human genomic sequence as described above. Alternatively, the human monoclonal anti-EGFL7 antibody of the present invention can be produced by the fusion tumor method. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described by, for example, Kozbor J. /m/ww plus /, 133:3001 (1984); Brodeur et al. Monoclonal Antibody Production Techniques and Applications' Pages 51-63 (Marcel Dekker, Inc., New York, 1987) and Boerner et al, J. /mw(10)〇/·, 147: 86 (1991) 〇 can now produce immunity A transgenic animal (e.g., a mouse) that produces a full human antibody profile without producing endogenous immunoglobulin. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production 119234.doc-62-200813088. Transfer of human germline immunoglobulin gene arrays into these germline mutant mice will result in the production of human antibodies following antigen challenge. See, for example, Jakobovits et al., Λ^/. Heart/t/M, 90:2551 (1993); Jakobovits et al., 362: 255 (1993);

Bruggermann等人，Year in Immunol., 7: 33 (1993) ° 亦可使用基因改組自非人類（例如齧齒動物）抗體獲得人類抗體，其中人類抗體具有與初始非人類抗體類似之親和力及特異性。根據此方法（亦稱為”抗原決定基印模”），以人類V結構域基因譜系置換藉由如上文所述乞噬菌體呈現技術獲得的非人類抗體片段之重鏈或輕鏈可變區，從而產生非人類鏈/人類鏈scFv或Fab嵌合體群體。以抗原進行選擇可導致非人類鍵/人類鍵嵌合scFv或Fab之分離，其中人類鏈恢復移除初級嗤菌體呈現純系中之相應非人類鏈時損壞的抗原結合位點，亦即抗原決定基決定（印模）對於人類鏈搭配物之選擇。當重複此過程以置換剩餘非人類鏈時，獲得人類抗體（參看1993年4月1日公開之pct W0 93/06213)。與傳統藉由CDR移植人化非人類抗體不同，此技術提供不具有非人類來源之FR或CDR殘基的完整人類抗體。雙特異性抗體雙特異性抗體為對至少兩種不同抗原具有結合特異性之單株抗體，較佳為人類或人化抗體。在本案中，結合特異性中之一係對於EGFL7而言且另一者係對於任何其他抗原而言。例示性雙特異性抗體可與EGFL7蛋白之兩種不同抗 119234.doc -63 - 200813088 原決定基結合。雙特異性抗體亦可用於將細胞毒性劑定位於表現EGFL7之細胞。此等抗體具有EGFL7結合臂及結合細胞毒性劑（例如沙伯甯（saporin)、抗干擾素-α、長春驗、蓖麻毒素-Α鏈、甲胺喋呤或放射性同位素半抗原）之臂。可製備全長抗體或抗體片段形式之雙特異性抗體（例如 F(ab’）2雙特異性抗體）。用於製造雙特異性抗體之方法已為此項技術中所知。傳統上，重組產生雙特異性抗體係基於兩個免疫球蛋白重鏈-輕鏈對之共同表現，其中兩條重鏈具有不同特異性 (Milstein 及 Cuello，Nature，305:537 (1983))。由於免疫球蛋白重鏈及輕鏈之隨機分配，此等融合瘤（四源雜交瘤）產生10種不同抗體分子之潛在混合物，其中僅一種具有正碟雙特異性結構。通常藉由親和層析步驟進行之正確分子之純化相當繁瑣’且產物產率較低。類似程序揭示於丨993年 5 月 13 日公開之 WO 93/08829及 Traunecker 等人，五J·, 10:3655 (1991)中〇根據不同且更佳之方法，將具有所需結合特異性（抗體_ 抗原組合位點）之抗體可變域與免疫球蛋白恆定域序列融合。融合較佳係與包含至少部分鉸鏈區、CH2及CH3區之免疫球蛋白重鏈恒定域之融合。較佳為在至少一種融合中存在含有輕鏈結合所必需之位點之第一重鏈恆定區 (CH1)。將編碼免疫球蛋白重鏈融合體及（若需要）免疫球蛋白輕鏈之DNA***單獨表現載體中，且將其共轉染至適當之宿主生物體中。在構築時所用三條多肽鏈之不等比率 119234.doc -64- 200813088 提供最佳產率之實施例中，此使對於三種多肽片段相互比例之調節具有極大靈活性。然而，當至少兩條多肽鏈以相等比率表現產生高產率或當此等比率並非特別重要時，可 ;兩條戈所有二條多肤鍵之編碼序列***一種表現載體中。在此方法之一些實施例中，雙特異性抗體係由一臂中具有第結合特異性之雜交免疫球蛋白重鏈及另一臂中之雜交免疫球蛋白重鏈-輕鏈對（提供第二結合特異性）構成。已發現由於免疫球蛋白輕鏈僅存在於一半雙特異性分子中提供種簡便之分離方式，故此不對稱結構促進所需雙特異 f生化合物與非吾人所要之免疫球蛋白鏈組合之分離。此方法揭示於WO 94/04690中。有關產生雙特異性抗體之其他細即’例如參看SUresh等人，施汍心砂咖A mho (1986) 〇根據另一方法，可工程設計一對抗體分子間之界面以使 I 由重組細胞培養物回收的雜二聚體之百分比最大化。較佳界面包含抗體恆定域之至少一部分CH3結構域。以此方法使第抗體分子界面之一或多條小胺基酸側鏈經較大侧鏈（例如，酪胺酸或色胺酸）置換。藉由用較小胺基酸側鏈（例如丙胺酸或蘇胺酸）置換較大胺基酸側鏈而於第二抗體分子之界面上產生具有與較大側鏈相同或類似尺寸之補仞空穴”。此提供一種使雜二聚體之產率增加超過其他非吾人所要之終產物（諸如均二聚體）的機制。雙特異性抗體包括交聯或"雜共輛"抗體。舉例而言，雜 119234.doc •65- 200813088 共軛物中之一抗體可與抗生蛋白偶聯，其他抗體與生物素偶聯。舉例而言，已提出此等抗體以針對非吾人所要細胞之免疫系統細胞為目標（美國專利第4,676,98〇號），且用於治療ΗΙν感染（WO 91/00360及WO 92/00373)。可使用任何適宜之交聯方法製造雜共軛抗體。適當之交聯劑已為此項技術中所热知，且揭示於美國專利第4,676,98〇號以及大量交聯技術中。自抗體片段產生雙特異性抗體之技術已描述於文獻中。舉例而言’可使用化學鍵聯製備雙特異性抗體。BreilIlan 等人，似e，229: 81 (1985)描述一種蛋白水解裂解完整抗體以產生F(ab’）2片段之程序。在二硫醇錯合劑亞砷酸鈉存在下還原此等片段以穩定鄰近二硫醇且防止分子間二硫鍵形成。接著將所產生之Fab，片段轉化為硫代硝基苯甲酸酉旨（TNB)衍生物。接著藉由用巯基乙胺還原將Fab，-TNB衍生物之一再轉化為Fab’-硫醇，且將其與等莫耳量之另一 Fab’-TNB衍生物混合以形成雙特異性抗體。所產生之雙特異性抗體可用作用於選擇性固定酵素之試劑。近期之發展已促進自大腸桿菌直接回收Fab，-SH片段，該等片段可經化學偶聯而形成雙特異性抗體。Shalaby等人，汄五印.Med· 175: 217-25 (1992)描述完全人化雙特異性抗體F(ab，）2分子之產生。大腸桿菌單獨分泌各Fab，片段且使其經歷活體外定向化學偶聯以形成雙特異性抗體。由此形成之雙特異性抗體能夠與過度表現HER2受體之細胞及正常人類T細胞結合，且亦觸發人類細胞毒性淋巴細胞 119234.doc -66- 200813088 對人類***腫瘤目標之溶解活性。亦已描述直接由重組細胞培養物製造並分離雙特異性抗體片段之多種技術。舉例而t，已使用白胺酸拉鏈產生雙特異性抗體。K〇Stelny#A 心 7讀咖/ i48(5) 1547 53 (1992)。藉由基因融合將來自—及—蛋白之白胺酸拉鏈肽與兩種不同抗體之Fab，部分連接。在较鏈區還原抗體均二聚體以形成單體’且接著使其再氧化以形成抗體雜二聚體。此方法亦可用於產生抗體均二聚體。H〇mnger等人，Bruggermann et al, Year in Immunol., 7: 33 (1993) ° Human antibodies can also be obtained by gene shuffling from non-human (e.g., rodent) antibodies, wherein human antibodies have similar affinities and specificities as the original non-human antibodies. According to this method (also referred to as "epitope impression"), the human V domain gene lineage is substituted for the heavy or light chain variable region of a non-human antibody fragment obtained by the phage display technology as described above, Thereby a non-human chain/human chain scFv or Fab chimera population is produced. Selection with an antigen can result in the isolation of a non-human bond/human bond chimeric scFv or Fab, wherein the human chain restores the antigen binding site that is disrupted when the primary sputum cell exhibits a corresponding non-human chain in the pure line, ie, antigenic determination Base decision (impression) for the choice of human chain collocation. When this process is repeated to replace the remaining non-human strands, human antibodies are obtained (see pct W0 93/06213, published on April 1, 1993). Unlike traditional transplantation of humanized non-human antibodies by CDRs, this technique provides intact human antibodies that do not have FR or CDR residues of non-human origin. Bispecific Antibodies Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens, preferably human or humanized antibodies. In the present case, one of the binding specificities is for EGFL7 and the other for any other antigen. An exemplary bispecific antibody can bind to two different resistances of the EGFL7 protein, 119234.doc-63 - 200813088. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing EGFL7. Such antibodies have an EGFL7 binding arm and an arm that binds to a cytotoxic agent (e.g., saporin, anti-interferon-alpha, vintagine, ricin-purine chain, methotrexate or radioisotope hapten). A bispecific antibody (e.g., F(ab')2 bispecific antibody) in the form of a full length antibody or antibody fragment can be prepared. Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of a bispecific antibody system is based on the common expression of two immunoglobulin heavy chain-light chain pairs, two of which have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these fusion tumors (quaternary hybridomas) produce a potential mixture of 10 different antibody molecules, of which only one has a positive dish bispecific structure. Purification of the correct molecule, usually by affinity chromatography steps, is quite cumbersome' and the product yield is low. A similar procedure is disclosed in WO 93/08829, published May 13, 993, and in Traunecker et al., JJ, 10:3655 (1991). The desired binding specificity (antibody) will be based on different and better methods. The antibody variable domain of the antigen binding site is fused to the immunoglobulin constant domain sequence. The fusion is preferably fused to an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, the CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into the appropriate host organism. The unequal ratio of the three polypeptide chains used in the construction 119234.doc -64- 200813088 In the examples providing the best yield, this gives great flexibility for the adjustment of the ratio of the three polypeptide fragments to each other. However, when at least two polypeptide chains are expressed in equal ratios to produce high yields or when such ratios are not of particular importance, the coding sequences for all two of the two polypeptide bonds can be inserted into an expression vector. In some embodiments of the method, the bispecific anti-system consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (providing a second Binding specificity constitutes. It has been found that since the immunoglobulin light chain is present only in half of the bispecific molecule to provide a convenient means of isolation, the asymmetric structure promotes the separation of the desired bispecific f-biosynthesis from the non-human immunoglobulin chain combination. This method is disclosed in WO 94/04690. For other details on the production of bispecific antibodies, see, for example, SUresh et al., Shimock Amo (1986). According to another method, an interface between antibody molecules can be engineered to allow I to be cultured by recombinant cells. The percentage of heterodimers recovered is maximized. Preferably, the interface comprises at least a portion of the CH3 domain of the antibody constant domain. In this way, one or more of the small amino acid side chains of the first antibody molecule interface are replaced by a larger side chain (e.g., tyrosine or tryptophan). Reducing the same or similar size to the larger side chain at the interface of the second antibody molecule by replacing the larger amino acid side chain with a smaller amino acid side chain (eg, alanine or threonine) Holes. This provides a mechanism to increase the yield of heterodimers over other non-human end products such as homodimers. Bispecific antibodies include cross-linking or "hybrid" antibodies For example, one of the antibodies in the conjugate of 119234.doc •65-200813088 can be conjugated to an antibiotic protein, and the other antibodies are conjugated to biotin. For example, such antibodies have been proposed to target non-human cells. The immune system cells are targeted (U.S. Patent No. 4,676,98) and are used to treat ΗΙν infection (WO 91/00360 and WO 92/00373). Any suitable cross-linking method can be used to make the heteroconjugate antibody. The cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,98, the disclosure of which is incorporated herein by reference in its entirety in the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire all Words can use chemistry A bispecific antibody is prepared. Breil Ilan et al., e., 229: 81 (1985) describes a procedure for proteolytic cleavage of intact antibodies to produce F(ab')2 fragments. In the presence of the dithiol miscicide sodium arsenite These fragments are reduced to stabilize the adjacent dithiol and prevent the formation of intermolecular disulfide bonds. The resulting Fab, fragment is then converted to a thionitrobenzoic acid (TNB) derivative, followed by thiol Amine reduction re-converts one of the Fab,-TNB derivatives to a Fab'-thiol and mixes it with another molar amount of another Fab'-TNB derivative to form a bispecific antibody. Antibodies can be used as reagents for selective immobilization of enzymes. Recent developments have facilitated the direct recovery of Fab, -SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., 汄五印. Med 175: 217-25 (1992) describes the production of a fully humanized bispecific antibody F(ab,)2 molecule. E. coli secretes each Fab, fragments and subjects them to undergo in vitro directed chemical coupling to form bispecific Sexual antibody. Specific antibodies bind to cells that overexpress HER2 receptors and normal human T cells, and also trigger the lytic activity of human cytotoxic lymphocytes 119234.doc-66-200813088 on human breast tumor targets. Also described directly by recombinant cells Various techniques for the manufacture and isolation of bispecific antibody fragments by cultures. For example, t-specific antibodies have been generated using leucine zippers. K〇Stelny#A Heart 7 Read Coffee / i48 (5) 1547 53 (1992). The leucine zipper peptide from - and - protein is partially linked to the Fab of two different antibodies by gene fusion. The antibody homodimer is reduced in the chain region to form a monomer' and then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. H〇mnger et al,

Proe’ 5W·乙90:6444-48 (1993)所述雙功能抗體’’技術已提供一種用於製造雙特異性抗體片段之替代機制。該等片段包含經連接子連接至輕鏈可變域（VL)之重鏈可變域（VH)，該連接子過短而使相同鏈上之兩個結構域之間無法配對。因此，迫使一個片段之VH& VL結構域與另一片段之互補VL及VH結構域配對，藉此形成兩個抗原結合位點。亦已報導另一種藉由使用單鏈Fv(sFv)二聚體來製造雙特異性抗體片段之策略。參看Gruber等人，义 Immunol· 152:5368 (1994) ° 本發明亦涵蓋大於二價之抗體。舉例而言，可製備三特異性抗體0 Tutt等人 J· /mmwwo/· 147:60 (1991) 〇多價抗體與二價抗體相比表現與抗體結合之抗原之細胞可更快地内化（及/或異化）多價抗體。本發明之抗體可為具有三個或三個以上抗原結合位點之多價抗體（IgM類別之抗體除外）（例如四價抗體），多價抗體可容易地藉由重組表現編碼 119234.doc -67- 200813088 抗體多肽鏈之核酸來製得。多價抗體可包含二聚化結構域及三個或三個以上抗原結合位點。較佳之二聚化結構域包含Fc區或鉸鏈區（或由其組成）。在此情形下，抗體將包含 Fc區及二個或二個以上在卜區胺基末端之抗原結合位點。本文中之較佳多價抗體包含三至約八個，但較佳四個抗原結合位點（或由其組成）。多價抗體包含至少一條多肽鏈（且較佳兩條多肽鏈），其中多肽鏈包含兩個或兩個以上可變域。舉例而言，多肽鏈可包含VD1_(xl)n_VD2_(X2)n_Fc，其中VD1為第一可變域，VD2為第二可變域，以為以區之一條多肽鏈，XI及X2表示胺基酸或多肽，且n為。舉例而言，多肽鏈可包含VH_CH1-彈性連接子^^(：111_^區The dual function antibody '' technology described in Proe' 5W. B 90:6444-48 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) joined to the light chain variable domain (VL) via a linker which is too short to allow pairing between the two domains on the same strand. Thus, the VH& VL domain of one fragment is forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., Immunol. 152: 5368 (1994) ° The present invention also encompasses antibodies that are greater than bivalent. For example, a trispecific antibody can be prepared. 0 Tutt et al. J. /mmwwo/. 147:60 (1991) A multivalent antibody can be internalized more rapidly than a cell exhibiting an antibody-bound antigen compared to a bivalent antibody ( And/or catabolism) multivalent antibodies. The antibody of the present invention may be a multivalent antibody (except for an antibody of the IgM class) (for example, a tetravalent antibody) having three or more antigen-binding sites, and the multivalent antibody can be easily encoded by a recombinant expression 119234.doc - 67- 200813088 A nucleic acid of an antibody polypeptide chain is produced. A multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. Preferably, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and two or more antigen binding sites at the amino terminus of the region. Preferred multivalent antibodies herein comprise from (or consist of) from three to about eight, but preferably four, antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain may comprise VD1_(xl)n_VD2_(X2)n_Fc, wherein VD1 is the first variable domain and VD2 is the second variable domain, such that one of the polypeptide chains is in the region, and XI and X2 represent the amino acid. Or a polypeptide, and n is. For example, a polypeptide chain may comprise a VH_CH1-elastic linker ^(:111_^ region

包含至少兩條（且較佳四條）輕鏈可變域多肽。本文之多價抗體可（例如）包含約兩條至約八條輕鏈可變域多肽。本文所涵蓋之輕鏈可變域多肽包含輕鏈可變域且視情形另外包含CL結構域。抗體變異體在二實她例中，本發明涵蓋本文所述之抗體的胺基酸序列修飾。舉例而言，可需要改良抗體之結合親和力及/At least two (and preferably four) light chain variable domain polypeptides are included. A multivalent antibody herein can, for example, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides encompassed herein comprise a light chain variable domain and, where appropriate, additionally comprise a CL domain. Antibody Variants In the second example, the invention encompasses amino acid sequence modifications of the antibodies described herein. For example, it may be desirable to improve the binding affinity of the antibody and/or

及/或取代。可進行缺失、***及取代之任何組合達成最終構築體，只要最終構築體具有所需特徵即可。可在製造 119234.doc -68 - 200813088 序列時將胺基酸變化引入標的抗體胺基酸序列中。可用於鑑別作為較佳突變位置之抗體之某些殘基或區域的方法稱為”丙胺酸掃描突變，，，如Cunningham及Weiis，And / or replace. Any combination of deletions, insertions, and substitutions can be made to achieve the final structure, as long as the final structure has the desired characteristics. Amino acid changes can be introduced into the target antibody amino acid sequence during the manufacture of the 119234.doc -68 - 200813088 sequence. A method that can be used to identify certain residues or regions of an antibody that is a preferred location of mutation is referred to as an "alanine scanning mutation," such as Cunningham and Weiis.

Sc/πα，244:1081_85 (1989)所述。對此，鑑別殘基或目標殘基之群（例如帶電殘基，諸如arg、asp、his、丨”及以匀且將其以中性或負電胺基酸（最佳為丙胺酸或聚丙胺酸）置換以影響胺基酸與抗原的相互作用。接著藉由亨取代位點處引入另外或其他變異體來改進彼等對取代展^出功能敏感性之胺基酸位置。因此，儘管已預定引入胺基酸序列變化之位點，但突變本身之性質無需預定。舉例而言，為分析既定位點處突變之效能，在目標密碼子或區域處進行“a掃描或隨機突變並針對所需活性篩選經表現之免疫球蛋白。胺基酸序列***包括長度在一個殘基至含有一百個或更多殘基之多肽之範圍内的胺基末端及/或羧基末端融合以及單一或多個胺基酸殘基之序列内***。末端***之實例包括具有N-末端甲硫胺醯基殘基之抗體或融合至細胞毒性多肽之抗體。抗體分子之其他***變異體包括抗體之N末端或c末端與酵素（例如用於ADEpT)或增加抗體血清半衰期之多肽之融合。另一類型之抗體胺基酸變異體變化抗體之原始糖基化模式。此變化包括缺失抗體中所存在之一或多個醣部分及/ 或添加一或多個抗體中不存在之糖基化位點。多肽之糖基化通常為N連接或〇連接。N連接係指醣部分附著於天；ϋ胺酸殘基之侧鍵。三狀序列天冬酿胺酸-X- 119234.doc -69- 200813088 絲胺酸及天冬醯胺酸-X-蘇胺酸（其中X為除脯胺酸外之任何胺基酸）為醣部分與天冬醯胺酸側鏈酶促附著之識別序列。因此，多肽中此等三肽序列任一者之存在產生潛在之糖基化位點。Ο連接糖基化係指糖N-乙醯半乳糖胺、半乳糖或木糖之一附著至羥基胺基酸，最通常為絲胺酸或蘇胺酸，但亦可使用5-羥基脯胺酸或5-羥基離胺酸。藉由變化胺基酸序列從而使其含有上述三肽序列中之一或多者來便利地實現在抗體中添加糠基化位點（對於N連接糖基化位點而言）。亦可藉由將一或多個絲胺酸或蘇胺酸殘基添加至或取代原始抗體之序列來進行變化（對於0連接糖基化位點而言）。在抗體包含Fc區之情況下，可變化附著於其上之醣。舉例而言，具有缺乏附著至抗體Fc區之岩藻糖之成熟醣結構的抗體已描述於美國專利申請案第US 2003/0157108號 (Presta，L.)中。亦參看 US 2004/0093621 (Kyowa Hakko Kogyo Co·，Ltd)。附著至抗體Fc區之醣中具有平分型N-乙醯葡糖胺（GlcNAc)之抗體在 WO 2003/011878、Jean-Mairet 等人及美國專利第6,602,684號Umafta等人中有所提及。附著至抗體Fc區之寡醣中具有至少一個半乳糖殘基之抗體報導WO 97/3 0087，Patel等人中。有關具有附著至抗體Fc區之經變化之醣的抗體，亦可參看WO 98/58964 (Raju，S·)及 WO 99/22764 (Raju，S.)。有關具有經修飾糖基化作用之抗原結合分子，亦可參看US 2005/0123546 (Umafia等人）。本文之較佳糖基化變異體包含Fc區，其中附著至Fc區之 119234.doc -70- 200813088 醣結構缺乏岩藻糖。此等變異體具有經改良之ADCC功能。視情況，Fc區中另外包含一或多處進一步改良ADCC 之胺基酸取代，例如Fc區298、333及/或334位（殘基之EU 編號）之取代。有關"去岩藻糖化”或”岩藻糖缺陷”抗體之公開案之實例包括：US 2003/0157108、WO 2000/61739、 WO 2001/29246、US 2003/0115614、US 2002/0164328、 US 2004/0093621、US 2004/0132140、US 2004/0110704、 US 2004/0110282、US 2004/0109865、WO 2003/085119、 WO 2003/084570、WO 2005/035586、WO 2005/035778、 W02005/053742 ^ Okazaki# A ^ J. Mol Biol. 336:1239-49 (2004); Yamane-Ohnuki 等人，87: 614 (2004)。產生去海藻糖化抗體之細胞株之實例包括在蛋白質岩藻糖化方面有缺陷之Lecl3 CHO細胞（Ripka等人， 5/oc/zew· 5/(9/7办>^· 249:533-45 (1986);美國專利申請案第 US 2003/0157108 A1 號，Presta，L及 WO 2004/056312 Al，Adams等人，尤其是實例11)及基因剔除細胞株，諸如 α-1，6-岩藻糖基轉移酶基因（FUT8)基因剔除CHO細胞 (Yamane-Ohnuki等人87:614 (2004)) 〇另一類型之變異體為胺基酸取代變異體。此等變異體在抗體分子冲具有至少一種經不同殘基置換之胺基酸殘基。最引人關注之取代突變位點包括高變區，但亦涵蓋FR變化。表1中標題”較佳取代”下展示保守型取代。若此等取代導致生物活性改變，則可引入表1中命名為"例示性取代π 或如下文參考胺基酸類別進一步描述之更具實質性之改 119234.doc -71- 200813088 變且篩選產物。表1 原始殘基例示性取代較佳取代 Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp; Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His H) Asn; Gin; Lys; Arg Arg Tie m ---\~/ Leu; Val; Met; Ala; Phe ;正白胺酸 Leu Leu (L) 正白胺酸；lie; Val; Met; Ala; Phe Ile Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr⑺ Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala ;正白胺酸 Leu 對抗體生物學特性之實質修飾可藉由選擇取代來實現，該等取代在其維持以下各物之作用方面顯著不同：（a)取代區域中多肽骨架之結構，例如折疊或螺旋構形；（b)分子目標位點處之電荷或疏水性；或（c)侧鏈體積。可基於共同側鏈特性將天然存在之殘基分組： (1) 疏水性：正白胺酸、met、ala、val、leu、ile ; (2) 中性親水性：Cys、Ser、Thr、Asn、Gin ; 119234.doc -72- 200813088 (3) 酸性·· asp、glu ; (4) 驗性：his、lys、arg ; (5) 影響鍵定向之殘基：giy、pro ;及 (6) 方族：trp、tyr、phe。非保守型取代將需要此等類別之一之成員與另一類別交換0 一種類型之取代變異體涉及取代親本抗Sc/πα, 244: 1081_85 (1989). In this regard, identify residues or groups of target residues (eg, charged residues such as arg, asp, his, 丨) and evenly neutralize them with neutral or negatively charged amino acids (preferably alanine or polypropylamine) Acid) substitution to affect the interaction of the amino acid with the antigen. The introduction of additional or other variants at the hen substitution site to improve their amino acid position sensitivity to the substitution function is thus improved. It is intended to introduce a site in which the amino acid sequence changes, but the nature of the mutation itself need not be predetermined. For example, to analyze the efficiency of the mutation at the localization point, "a scan or random mutation is performed at the target codon or region and Active screening of expressed immunoglobulins. Amino acid sequence insertions include amine-terminal and/or carboxy-terminal fusions ranging from one residue to a polypeptide containing one hundred or more residues and single or multiple Insertion of a sequence of amino acid residues. Examples of terminal insertions include antibodies having N-terminal methionine residues or antibodies fused to cytotoxic polypeptides. Other insertion variants of antibody molecules Fusion of the N-terminus or c-terminus of an antibody to an enzyme (eg, for ADEpT) or a polypeptide that increases the serum half-life of the antibody. Another type of antibody amino acid variant alters the original glycosylation pattern of the antibody. This change includes deletion of the antibody. One or more sugar moieties are present and/or a glycosylation site that is not present in one or more antibodies is added. The glycosylation of the polypeptide is typically an N-linked or hydrazone linkage. The N-linked system refers to the attachment of the sugar moiety to the day. ; side bond of a proline residue. Tri-sequence aspartic acid-X- 119234.doc -69- 200813088 Serine and aspartic acid-X-threonine (where X is in addition to decylamine Any amino acid other than an acid is a recognition sequence for the enzymatic attachment of a sugar moiety to the aspartic acid side chain. Thus, the presence of any of these tripeptide sequences in the polypeptide creates a potential glycosylation site. Linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly seric acid or threonine, but 5-hydroxyproline may also be used. Or 5-hydroxy lysine. By changing the amino acid sequence to contain one of the above-described tripeptide sequences Or more conveniently to add a thiolation site to the antibody (for N-linked glycosylation sites). Also by adding one or more serine or threonine residues to or Substituting the sequence of the original antibody for the change (for a 0-linked glycosylation site). In the case where the antibody comprises an Fc region, the sugar attached thereto may be altered. For example, there is a lack of attachment to the antibody Fc region. An antibody to the mature sugar structure of fucose has been described in US Patent Application No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.) attached to an antibody. An antibody having a bismuth-type N-acetylglucosamine (GlcNAc) in the Fc region is mentioned in WO 2003/011878, Jean-Mairet et al., and U.S. Patent No. 6,602,684 to Umafta et al. Antibody having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody is reported in WO 97/3 0087, Patel et al. For antibodies having altered sugars attached to the Fc region of an antibody, see also WO 98/58964 (Raju, S.) and WO 99/22764 (Raju, S.). For antigen binding molecules having modified glycosylation, see also US 2005/0123546 (Umafia et al.). Preferred glycosylation variants herein comprise an Fc region in which the 190234.doc-70-200813088 glycostructure is attached to the Fc region lacking fucose. These variants have improved ADCC capabilities. Optionally, the Fc region additionally comprises one or more amino acid substitutions that further improve ADCC, such as substitution of the 298 region 298, 333 and/or 334 (EU numbering of residues). Examples of publications relating to "defucosylation" or "fucose-deficient" antibodies include: US 2003/0157108, WO 2000/61739, WO 2001/29246, US 2003/0115614, US 2002/0164328, US 2004 /0093621, US 2004/0132140, US 2004/0110704, US 2004/0110282, US 2004/0109865, WO 2003/085119, WO 2003/084570, WO 2005/035586, WO 2005/035778, W02005/053742 ^ Okazaki# A ^ J. Mol Biol. 336:1239-49 (2004); Yamane-Ohnuki et al., 87: 614 (2004). Examples of cell lines that produce de-alginylated antibodies include Lecl3 CHO, which is defective in protein fucosylation. Cells (Ripka et al., 5/oc/zew. 5/(9/7 Office>^. 249:533-45 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L and WO 2004) /056312 Al, Adams et al., especially Example 11) and gene knockout cell lines, such as the α-1,6-fucosyltransferase gene (FUT8) gene knockout CHO cells (Yamane-Ohnuki et al. 87:614 ( 2004)) Another type of variant is an amino acid substitution variant. These variants have at least one different residue set in the antibody molecule. Amino acid residues. The most interesting substitution sites include hypervariable regions, but also encompass FR changes. Conservative substitutions are shown under the heading "Preferred substitutions" in Table 1. If such substitutions result in changes in biological activity Further, a more substantial modification of 119234.doc-71-200813088, which is further described in the reference amino acid class as described below, can be introduced in Table 1 and the product is screened. Table 1 Illustrative of the original residues Sex substitution is preferably substituted for Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp; Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His H) Asn; Gin; Lys; Arg Arg Tie m ---\~/ Leu; Met; Ala; Phe; ortho-leucine Leu Leu (L) ortho-amine; lie; Val; Met; Ala; Phe Ile Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(7) Trp; Phe; Thr Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Substantial modification of the biological properties of an antibody by acid Leu can be achieved by selective substitution which differs significantly in its role in maintaining the following: (a) the structure of the polypeptide backbone in the substitution region, such as a folded or helical configuration (b) the charge or hydrophobicity at the molecular target site; or (c) the side chain volume. Naturally occurring residues can be grouped based on common side chain properties: (1) Hydrophobic: positive leucine, met, ala, val, leu, ile; (2) neutral hydrophilicity: Cys, Ser, Thr, Asn Gin; 119234.doc -72- 200813088 (3) Acid ·· asp, glu; (4) Testability: his, lys, arg; (5) Residues affecting bond orientation: gyi, pro; and (6) Square: trp, tyr, phe. Non-conservative substitutions will require members of one of these categories to exchange with another category. 0 One type of substitution variant involves substitution of the parental resistance

體或人類抗體）之一或多個高變區殘基。一般而言，選擇用於進一步研究t所得變異體應相對於產生其之親本抗體具有經改良之生物學特性。一種用於產生此等取代變異體之適宜方式涉及使用噬菌體呈現進行之親和力成熟。簡而言之，使數個高變區位點（例如6-7個位點）突變，於各位點處產生所有可能之胺基酸取代。當融合至包裝於各絲狀噬菌體顆粒内之M13之基因ΙΠ產物時，由絲狀噬菌體顆粒呈現由此產生之抗體。接著針對如本文所揭示之抗體之生物學活性（例如結合親和力）篩選噬菌體呈現之變異體。為鑑別用於修飾之候it高變區位點，可進行丙胺酸掃描突變以鑑別顯著有助於抗原結合之高變區殘基。另外或其他，其可有益於分析抗原-抗體複合物之晶體結構以鑑：抗體與抗原之間的接觸點。此等接觸殘基及相鄰殘基為根據本文詳述之技術進行取代之候選物。在產生此等變異體後，如本文所述使變異體組合經歷篩選，且』在一或多個相關檢定中選擇具有優良特性之抗體用於進一步研究。編碼抗體胺基酸序列變異體之核酸刀卞了糟由此項技術 119234.doc -73- 200813088 中已知之多種方法製備。此等方法包括（但不限於）自天然來源分離（在天然存在之胺基酸序列變異體之情況下）或藉由先前製備之抗體之變異體或非變異體型式的募核苷酸介導（或定點）突變、PCR突變及盒式突變來製備。可需要將一或多處胺基酸修_引入本發明之免疫球蛋白多肽之Fc區中，藉此產生Fc區變異體。FC區變異體可包含在一或多個胺基酸位置（包括鉸鏈半胱胺酸位置）處包含胺基酸修飾（例如取代）之人類Fc區序列（例如人類IgG1、 IgG2、IgG3 或 IgG4 Fc 區）。根據本說明及此項技術之教示，預期在一些實施例中與野生型對應物抗體相比本發明之方法中所使用之抗體可包含一或多處（例如）Fc區中之變化。然而，此等抗體將保留與其野生型對應物相比實質上相同之治療效用所需特徵。舉例而έ，據認為可於Fc區中進行將引起c lq結合及/或補體依賴性細胞毒性（CDC)變化（亦即改良或降低）之某些變化’（例如）如WO 99/51642中所述。有關pc區變異體之其他實例，亦可參看 Duncan 及 Winter 322:738_40 (1988);美國專利第5,648,260號、美國專利第5,624,821號及 WO 94/29351。WO 00/42072 (Presta)及 WO 2004/056312 (Lowman)描述與FcR之結合經改良或經降低之抗體變異體。此等專利公開案之内容以引用之方式明確地併入本文中。亦參看 Shields 等人 J. 5ζ·ο/· C7z 隱 9(2):6591-6604 (2001)。US2005/0014934A1 (Hinton等人）中描述具有增加之半衰期及經改良之與新生兒Fc受體（FcRn)之結合的抗 119234.doc -74- 200813088 體，其中FcRn負責將母體IgG轉移至胎兒體内（Guyer等 k，J· Immunol· ]ΛΊ··5 名 Ί (19Ί6、ΑΚ·ηη專尺，J· jmmun〇i 24:249 (1994))。此等抗體包含具有一或多處改良Fc區與 FcRn之結合之取代的Fc區。具有變化之Fc區胺基酸序列及增加或降低之Clq結合能力之多肽變異體描述於美國專利第6，194,55^1號、|〇 99/51642中。彼等專利公開案之内谷以引用之方式明確地併入本文中。亦參看Uus〇gie等人 J. Immunol. 164:4178-84 (2000)。抗體衍生物可進一步修飾本發明之抗體使其含有此項技術中已知且可易於獲得之額外非蛋白部分。適於衍生抗體之部分較佳為水溶性聚合物。水溶性聚合物之非限制性實例包括（但不限於）聚乙二醇（PEG)、乙二醇/丙二醇共聚物、缓甲基纖維素、葡.聚糖、聚乙烯醇、聚乙烯，比嘻、聚_以二氧戊環、聚-1，3,6_三钱、乙烯/順丁烯二酸酐共聚物、聚胺基Μ均聚物或無規共聚物)及葡聚糖或聚(N乙烯吡咯啶綱）聚乙—醇、丙二醇均聚物、聚氧化丙稀/氧化乙烯共聚物、聚氧乙稀多元醇(例如甘油)、聚乙烯醇及其混合物。聚乙二龄丙駿因其於水中之穩定性而可於製造時具有優點。聚合物可具有任钿八工3 有任何分子置，且可具支鏈或不具支鏈。附著於抗體之聚合物mi y口物的數目可變化，且若附著一合物，則它們可為相π + 百裡U上聚 ^ Λ入或不同分子。一般而言，用於衍生作用之聚合物之數目丁生或類型可基於包括（但不限於彳彳ij： & 良抗體之特殊特性或劢处^ 个限於）待改一力此，抗體衍生物是否將用於指定條 119234.doc -75- 200813088 件下之療法等考慮來確定。篩選具有所需特性之抗艎本發明之抗體與EGFL7結合，且在一些實施例中可調節一或多個與EGFL7相關作用之態樣，包括（但不限於）破壞任何生物學相關之EGFL7生物路徑，及/或治療及/或預防腫瘤、細胞增生性病症或癌症，及/或治療或預防與EGFL7 表現及/或活性（諸如增加之EGFL7表現及/或活性）相關之病症。舉例而言，如本文所述可針對抗體阻斷HUVEC細胞與EGFL7黏附及HUVEC於經EGFL7蛋白塗覆之培養盤上遷移之能力筛選本發明之抗體。可將經純化之抗體另外藉由一系列檢定表徵，該等檢定包括（但不限於）N末端測序、胺基酸分析、非變性尺寸排除高壓液相層析（HPLC)、質譜法、離子交換層析及木瓜酵素消化。在本發明之某些實施例中，分析本文所製造之抗體之生物活性。在一些實施例中，測試本發明抗體之抗原結合活性。此項技術中已知且可用於本文中之抗原結合檢定包括 (但不限於）使用諸如西方墨點法之技術進行的任何直接或競爭性結合檢定、放射免疫檢定、ELISA(酶聯結免疫吸附劑檢定）、’’夾層’’免疫檢定、免疫沉殿檢定、螢光免疫檢定及蛋白A免疫檢定。下文之實例部分中提供說明性抗原結合檢定。在一些實施例中，本發明提供一種與包含如下輕鏈可變域及如下重鏈可變域之抗體競爭與EGFL7結合的抗EGFL7 119234.doc -76- 200813088 抗體，該輕鏈可變域包含選自SEQ ID NO: 1及SEQ ID NO: 3之序列且該重鏈可變域包含選自SEQ ID NO: 2及SEQ ID NO: 4之序列。可藉由針對與包含如下輕鏈可變域及如下重鏈可變域之經標記抗體競爭與固定EGFL7之結合篩選抗 EGFL7融合瘤上清液來獲得此等競爭抗體，該輕鏈可變域包含選自SEQ ID NO: 1及SEQ ID NO: 3之序列且該重鏈可變域包含選自SEQ ID NO: 2及SEQ ID NO: 4之序列。此等競爭抗體包括識別與該抗體所識別之EGFL7抗原決定基相同或與其重疊之EGFL7抗原決定基的抗體。與含有不相關 (或無）抗體之對照結合混合物中所偵測之已結合、經標記抗體的量相比較，含有競爭劑抗體之融合瘤上清液將降低標的競爭結合混合物中所偵測之已結合、經標記抗體的量。本文所述之任何競爭性結合檢定均適用於前述程序。可以任何適宜之方法藉由針對所需特性篩選抗EGFL7融合瘤純系來獲得具有本文所述之特性的本發明之抗EGFL7 抗體。舉例而言，若需要與包含如下輕鏈可變域及如下重鏈可變域之抗體競爭或不與其競爭與EGFL7結合之抗 EGFL7單株抗體，其中該輕鏈可變域包含選自SEQ ID NO: 1及SEQ ID NO: 3之序列且該重鏈可變域包含選自SEQ ID NO: 2及SEQ ID NO: 4之序列，則可於結合競爭性檢定中測試候選抗體。此項技術中熟知競爭性檢定。此項技術中已知測定抗EGFL7抗體之抑制能力的其他功能性檢定，某些檢定將在本文中例示說明。在一些實施例中，本發明涵蓋已變化之抗體，其具有某 119234.doc -77- 200813088 些但並非所有效應功能，此使其成為抗體在活體内之半衰期極為重要而某些效應功能（諸如補體及ADCC)卻並非必需或有害之多種應用中的合乎需要之候選物。在某些實施例中，量測所產生之免疫球蛋白之Fc活性以確保僅所需特性得以保持。可進行活體外及/或活體内細胞毒性檢定以確認CDC及/或ADCC活性之降低/衰竭。舉例而言，可進行 Fc受體（FcR)結合檢定以確保抗體缺乏Fc^R結合（由此可能缺乏ADCC活性）但仍保留FcRn結合能力。介導ADCC之初妨知*始f N iC知1始A /蓄矣ΐ目！r* R T T T，而誤士衣知始矣招F p R T、 ’ 、Π \ ▲，美赢、’ 一》 J ▲擎▲▲▲▲a * * U t ▲One or more hypervariable region residues of a human or human antibody). In general, variants selected for further study t should have improved biological properties relative to the parent antibody from which they are produced. One suitable way to generate such substitution variants involves affinity maturation using phage display. In short, several hypervariable region sites (e. g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each point. The antibody thus produced is expressed from filamentous phage particles when fused to the gene product of M13 packaged in each filamentous phage particle. Phage-presented variants are then screened for the biological activity (e.g., binding affinity) of the antibodies as disclosed herein. To identify potential hypervariable regions for modification, alanine scanning mutations can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Additionally or alternatively, it may be useful to analyze the crystal structure of the antigen-antibody complex to account for the point of contact between the antibody and the antigen. These contact residues and adjacent residues are candidates that are substituted according to the techniques detailed herein. After the generation of such variants, the variant combinations are subjected to screening as described herein, and antibodies having superior properties are selected for further study in one or more relevant assays. Nucleic acids encoding antibody amino acid sequence variants are prepared by a variety of methods known in the art 119234.doc-73-200813088. Such methods include, but are not limited to, isolation from a natural source (in the case of a naturally occurring amino acid sequence variant) or mediated by a variant of a previously prepared antibody or a non-variant type of nucleotide. (or site-directed) mutations, PCR mutations and cassette mutagenesis were prepared. It may be desirable to introduce one or more amino acid residues into the Fc region of an immunoglobulin polypeptide of the invention, thereby producing an Fc region variant. The FC region variant may comprise a human Fc region sequence comprising an amino acid modification (eg, a substitution) at one or more amino acid positions (including the hinge cysteine position) (eg, human IgGl, IgG2, IgG3, or IgG4 Fc) Area). In accordance with the teachings of this specification and the teachings of the art, it is contemplated that in some embodiments the antibodies used in the methods of the invention may comprise one or more, for example, changes in the Fc region, as compared to wild-type counterpart antibodies. However, such antibodies will retain substantially the same therapeutic utility characteristics as their wild-type counterparts. By way of example, it is believed that certain changes that will cause c lq binding and/or complement dependent cytotoxicity (CDC) changes (ie, improvement or reduction) can be made in the Fc region (eg, as in WO 99/51642) Said. See also, Duncan and Winter 322:738_40 (1988); U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94/29,351. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe improved or reduced antibody variants that bind to FcR. The contents of these patent publications are expressly incorporated herein by reference. See also Shields et al. J. 5ζ·ο/· C7z Hidden 9(2): 6591-6604 (2001). An anti-119234.doc-74-200813088 body having an increased half-life and improved binding to a neonatal Fc receptor (FcRn) is described in US 2005/0014934 A1 (Hinton et al.), wherein FcRn is responsible for the transfer of maternal IgG to the fetal body. (Guyer et al, J. Immunol· ]ΛΊ··5 Ί (19Ί6, ΑΚ·ηη special rule, J. jmmun〇i 24:249 (1994)). These antibodies contain one or more modified Fc Substituted Fc regions that bind to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described in U.S. Patent No. 6,194,55^1, |〇99/ In U.S. Patent Publication No. 5,642, the entire disclosure of which is hereby incorporated by reference in its entirety in its entirety in its entirety in its entirety in its entirety in its entirety the disclosure of the disclosure of U.S. The antibody is such that it contains additional non-protein portions known in the art and readily available. The portion suitable for derivatizing the antibody is preferably a water soluble polymer. Non-limiting examples of water soluble polymers include, but are not limited to, Polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, slow methylation Cellulose, glucomannan, polyvinyl alcohol, polyethylene, bismuth, poly-dioxolane, poly-1,3,6_three money, ethylene/maleic anhydride copolymer, polyamine ΜHomopolymer or random copolymer) and dextran or poly(N-vinylpyrrolidine) polyethyl alcohol, propylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylene polyol ( For example, glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene Erneng Bing Jun has advantages in manufacturing due to its stability in water. The polymer may have any molecular group and may be branched or unbranched. The number of polymeric mi y mouthpieces attached to the antibody can vary, and if a compound is attached, they can be polypyrrole or different molecules in the phase π + 百里. In general, the number or type of polymer used for derivatization may be based on, but not limited to, the specific properties of 彳彳ij: & Whether the substance will be used for the treatment of the specified article 119234.doc -75- 200813088 is considered. Screening for antibodies having the desired properties The antibodies of the invention bind to EGFL7 and, in some embodiments, can modulate one or more aspects associated with EGFL7, including, but not limited to, disrupting any biologically relevant EGFL7 organism. Route, and/or treat and/or prevent tumors, cell proliferative disorders or cancer, and/or treat or prevent conditions associated with EGFL7 expression and/or activity, such as increased EGFL7 expression and/or activity. For example, antibodies of the invention can be screened for antibodies that block the adhesion of HUVEC cells to EGFL7 adhesion and HUVECs to EGFL7 protein coated plates as described herein. The purified antibody can additionally be characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange. Chromatography and digestion of papaya enzymes. In certain embodiments of the invention, the biological activity of the antibodies made herein is analyzed. In some embodiments, the antigen binding activity of an antibody of the invention is tested. Antigen binding assays known in the art and useful herein include, but are not limited to, any direct or competitive binding assays, such as Western blotting techniques, radioimmunoassays, ELISA (enzyme-linked immunosorbent) Verification), ''Mezzanine'' immunoassay, immunosuppression test, fluorescent immunoassay, and protein A immunoassay. An illustrative antigen binding assay is provided in the Examples section below. In some embodiments, the invention provides an anti-EGFL7 119234.doc-76-200813088 antibody that competes with an antibody comprising a light chain variable domain and a heavy chain variable domain that binds to EGFL7, the light chain variable domain comprising A sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3 and the heavy chain variable domain comprises a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. Such competing antibodies can be obtained by screening anti-EGFL7 fusion tumor supernatants for binding to a labeled antibody that comprises a light chain variable domain and a heavy chain variable domain that competes with the immobilized EGFL7, the light chain variable domain A sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3 and the heavy chain variable domain comprises a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. Such competing antibodies include antibodies that recognize the EGFL7 epitope that is the same as or overlaps with the EGFL7 epitope recognized by the antibody. The fusion cell supernatant containing the competitor antibody will reduce the detectable binding in the target competitive binding mixture as compared to the amount of bound, labeled antibody detected in the control binding mixture containing the irrelevant (or no) antibody. The amount of bound, labeled antibody. Any of the competitive binding assays described herein are applicable to the foregoing procedures. The anti-EGFL7 antibodies of the invention having the properties described herein can be obtained by any suitable method by screening anti-EGFL7 fusion tumor lines for the desired properties. For example, an anti-EGFL7 monoclonal antibody that competes with or does not compete with EGFL7 for binding to an antibody comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises a SEQ ID NO: 1 and the sequence of SEQ ID NO: 3 and the heavy chain variable domain comprises a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4, and the candidate antibody can be tested in a binding competition assay. Competitive assays are well known in the art. Other functional assays for determining the ability of anti-EGFL7 antibodies to inhibit are known in the art, and certain assays will be exemplified herein. In some embodiments, the invention encompasses altered antibodies having some, but not all, of the effector functions of 119234.doc-77-200813088, which makes it important for the half-life of the antibody in vivo to be important and some effector functions (such as Complement and ADCC) are not desirable or desirable candidates for a variety of applications. In certain embodiments, the Fc activity of the produced immunoglobulin is measured to ensure that only the desired characteristics are maintained. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks Fc^R binding (and thus may lack ADCC activity) but still retains FcRn binding ability. Mediated ADCC at the beginning of the game * Start f N iC know 1 start A / charge! r* R T T T, and the misunderstood clothing knows that F p R T, ’ 、 Π \ ▲, mei win, ’ ’ J ▲ ▲ ▲ ▲ ▲ a * * U t ▲

FcRII及FcRIII。造血細胞上之FcR表現概述於Ravetch及 Kinet，/mmwno/. 9:457-92 (1991)第 464 頁之表 3 中。評定所關注分子之ADCC活性之活體外檢定之實例描述於美國專利第5,500,362號或第5,821，337號中。可用於此等檢定之效應細胞包括周邊血液單核細胞（PBMC)及天然殺傷（NK)細胞。另外或其他，可（例如）在動物模型（諸如 Clynes# A ^ Proc. NatL Acad. Sci. USA 95:652-656 (1998) 中所揭示之動物模型）中於活體内評定所關注分子之ADCC 活性。亦可進行Clq結合檢定以確認抗體無法與Clq結合且因此缺乏CDC活性。對於評定補體活化，可進行CDC檢定，（例如）如 Gazzano-Santoro 等人，J· /mmwno/· 202:163 (1996)中所述。亦可使用此項技術中已知之方法進行FcRn結合及活體内清除/半衰期測定。在一些實施例中，本發明提供具有增強之效應功能及/ 或增加之半衰期之經變化抗體。 119234.doc -78- 200813088 載艘、宿主細胞及重組方法為重組產生本發明之抗體，分離編碼該抗體之核酸且將其***可複製之載體中用於進一步選殖（DNA之擴增）或表現。谷易地分離編碼抗體之DNA且使用習知程序（例如藉由使用能夠與編碼抗體重鏈及輕鏈之基因特異性結合的寡核苦酸探針）進行測序。多種載體均可用分上視所使用之宿主細胞而定一般而言，較佳之= 胞為原核或真核（一般為哺乳動物）來源。應瞭解為達成此目的可使用任何同型之恆定1，包括IgG、！gM、私、FcRII and FcRIII. The FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, /mmwno/. 9:457-92 (1991), Table 464, page 464. An example of an in vitro assay for assessing the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Effector cells that can be assayed for this include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Additionally or alternatively, the ADCC of the molecule of interest can be assessed in vivo, for example, in an animal model such as the animal model disclosed in Clynes # A ^ Proc. NatL Acad. Sci. USA 95:652-656 (1998) active. A Clq binding assay can also be performed to confirm that the antibody is unable to bind to Clq and thus lacks CDC activity. For assessing complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. /mmwno/. 202:163 (1996). FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art. In some embodiments, the invention provides a modified antibody having enhanced effector function and/or increased half-life. 119234.doc -78- 200813088 Carrier, host cell and recombinant method for recombinant production of an antibody of the invention, isolating the nucleic acid encoding the antibody and inserting it into a replicable vector for further selection (amplification of DNA) or which performed. The DNA encoding the antibody is isolated from the valley and sequenced using a conventional procedure (e.g., by using an oligonucleotide probe capable of specifically binding to a gene encoding the heavy and light chains of the antibody). A variety of vectors may be used depending on the host cell used. Generally, the cells are of prokaryotic or eukaryotic (generally mammalian) origin. It should be understood that any constant of the same type can be used for this purpose, including IgG,! gM, private,

IgD及IgE恆$區’ 1此等恆定區可由任何人類或動物物種獲得。 a·使用原核宿主細胞產生抗艘 i·載體構築可使用標準重組技術獲得編碼本發明抗體之多肽組份的聚核苷&L序列。可自產生抗體之細胞（諸如融合瘤細胞）中分離所需聚核苷酸序列並進行測序。或者，可使用核苷酸 cr成器或PCR技術合成聚核_酸。在獲得編碼多肽之序列後’將其插人⑥夠在原核宿主中複製並表現異源聚核皆酸之重組載體中。對於本發明而言可使用可用且為此項技術中所知之多種載體。對於適當載體之選擇將主要視***載體中之核酸的大小及經載體轉化之特定宿主細胞而定。各載體視其功能（異源聚核苷酸擴增或表現或兩者）及其與其所滯留之特定宿主細胞的相容性而^含有多種組份。載體組份一般包括（但不限於）複製起點、選擇標記基因、啟動 119234.doc -79- 200813088 子、核糖體結合位點（RBS)、信號序列、異源核酸***物及轉錄終止序列。一般而言，與此等宿主一起使用含有自與宿主細胞相容的物種獲得之複製子及控制序列的質體載體。載體通常具有複製位點以及能夠於經轉化細胞中提供表型選擇之標記序列。舉例而言，通常使用pBR322(一種自大腸桿菌物種獲得之質體）轉化大腸桿菌。pBR322含有編碼安比西林 (ampicillin)(Amp)及四環素（tetracycline)(Tet)抗性之基因’且因此提供簡便的鑑別經轉化細胞之方式。 PBR322、其衍生物或其他微生物質體或噬菌體亦可含有或經修飾而含有可由微生物生物體用於表現内源蛋白之啟動子。用於表現特定抗體之pBR322衍生物之實例詳細描述於Carter等人之美國專利第5,648,237號中。此外，可與此等宿主一起使用含有與宿主微生物相容之複製子及控制序列的噬菌體載體作為轉化載體。舉例而言，諸如λΟΕΜΤΜ_η之噬菌體可用於製造可用以轉化易感宿主細胞（諸如大腸桿菌LE392)之重組載體。本發明之表現載體可包含兩種或兩種以上編碼各多肽組伤之啟動子順反子對。啟動子為位於調節其表現之順反子上游（5’）之未經轉譯之調控序列。原核啟動子通常分為兩類，亦即料型及組成型。誘導型啟動子為在其對培養條件之改變（例如養分存在與否或溫度改變）作出回靡制下起始順反子以增加量轉錄之啟動子。 ^ 大量可由多g潛在帛主細胞所別之啟動+已為吾人所熟 119234.doc 200813088 知。可藉由經限制酶消化將所選擇之啟動子自移除且將所分離之啟動子序列***本發明之載體中而使該啟動子以了操作方式連接至編碼輕鏈或重鏈之順反子Dna。可使用天然啟動子序列與多種異源啟動子指導目標基因之擴增及/或表現。在一些實施例中，因與天然目標多肽啟動子相比，異源啟動子一般允許經表現之目標基因達成較快轉錄及較高產率，故而使用異源啟動子。適用於原核宿主之啟動子包括助以啟動子、卜半乳聚糖酶及乳糖啟動子系統、色胺酸^”)啟動子系統及雜合啟動子（諸如（心或啟動子）。然而，在細菌中具有功能性之其他啟動子（諸如其他已知之細菌或噬菌體啟動子）亦適用。其核苷酸序列已經公佈，藉此熟習此項技術者能夠使用提供任何所需限制位點之連接子或轉接子以可操作方式將其與編碼目標輕鏈及重鏈之順反子接合（SiebenHst等人， Ce// 20:269 (1980)) 〇在本發明之一態樣中，重組載體内之各順反子均包含引導經表現之多肽跨膜移位之分泌信號序列組份。一般而曰，彳§號序列可為載體之組份，或其可為***載體中之目標多肽DNA之部分。出於本發明之目的所選擇之信號序列應為可由宿主細胞識別及處理（亦即由信號肽酶裂解）之信號序列。對於不識別及處理異源多肽之天然信號序列的原核宿主細胞而言，信號序列經（例如）選自由以下各物組成之群之原核信號序列取代：鹼性磷酸酶、青黴素酶、bp 或熱穩定性腸毒素II (STII)前導序列、LamB、ph〇E、 119234.doc -81- 200813088IgD and IgE constant $regions 1 These constant regions can be obtained from any human or animal species. a. Generation of anti-occupation using prokaryotic host cells i. Vector construction A polynucleoside & L sequence encoding a polypeptide component of an antibody of the invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from the antibody-producing cells (such as fusion tumor cells) and sequenced. Alternatively, the polynuclear acid can be synthesized using a nucleotide crinder or PCR technique. After obtaining the sequence encoding the polypeptide, it is inserted into a recombinant vector capable of replicating in a prokaryotic host and expressing a heterologous polynucleocapamic acid. A wide variety of vectors that are available and are known in the art can be used in the present invention. The choice of appropriate vector will depend primarily on the size of the nucleic acid inserted into the vector and the particular host cell transformed by the vector. Each vector contains a plurality of components depending on its function (a heterologous polynucleotide amplification or expression or both) and its compatibility with the particular host cell in which it is retained. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter 119234.doc-79-200813088, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence. Generally, plastid vectors containing replicons and control sequences obtained from species compatible with the host cell are used with such hosts. Vectors typically have a replication site and a marker sequence capable of providing phenotypic selection in transformed cells. For example, pBR322, a plastid obtained from E. coli species, is typically used to transform E. coli. pBR322 contains a gene encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a convenient means of identifying transformed cells. PBR322, its derivatives or other microbial plastids or phages may also contain or be modified to contain promoters which can be used by microorganism organisms to express endogenous proteins. An example of a pBR 322 derivative for the expression of a specific antibody is described in detail in U.S. Patent No. 5,648,237 to the name of PCT. In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors with such hosts. For example, phage such as λΟΕΜΤΜ_η can be used to make recombinant vectors that can be used to transform susceptible host cells, such as E. coli LE392. The expression vector of the present invention may comprise two or more promoter cistron pairs encoding each polypeptide group. The promoter is an untranslated regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are usually divided into two categories, namely, material type and constitutive type. An inducible promoter is a promoter that initiates a cistron to increase transcription in response to changes in culture conditions (e.g., presence or absence of nutrients or temperature changes). ^ A large number can be activated by many g potential 帛 main cells + already cooked for us 119234.doc 200813088 know. The promoter can be operably linked to the coding light or heavy chain by self-removal of the selected promoter by restriction enzyme digestion and insertion of the isolated promoter sequence into the vector of the present invention. Child Dna. Natural promoter sequences and a variety of heterologous promoters can be used to guide the expansion and/or expression of the target gene. In some embodiments, a heterologous promoter is typically used to allow for faster transcription and higher yields of the expressed target gene as compared to the native target polypeptide promoter. Promoters suitable for use in prokaryotic hosts include promoters, galactanase and lactose promoter systems, tryptonic acid promoters, and heterozygous promoters such as (hearts or promoters). Other promoters that are functional in bacteria, such as other known bacterial or bacteriophage promoters, are also suitable. Nucleotide sequences have been published, whereby those skilled in the art can use linkages that provide any desired restriction sites. The sub or adaptor is operably linked to the cistron encoding the target light and heavy chains (Sieben Hst et al, Ce/20:269 (1980)) in one aspect of the invention, recombined Each cistron in the vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptide across the membrane. Typically, the 彳§ sequence can be a component of the vector, or it can be a polypeptide of interest inserted into the vector. Part of DNA. The signal sequence selected for the purposes of the present invention should be a signal sequence that can be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For natural signal sequences that do not recognize and handle heterologous polypeptides. In the case of a prokaryotic host cell, the signal sequence is replaced, for example, by a prokaryotic signal sequence selected from the group consisting of: alkaline phosphatase, penicillinase, bp or thermostable enterotoxin II (STII) leader sequence, LamB, ph〇E, 119234.doc -81- 200813088

PelB、OmpA及MBP。在本發明之一實施例中，用於表現系統之兩種順反子中之信號序列為STII信號序列或其變異體。在另一悲樣中’本發明之免疫球蛋白的產生可於宿主細胞之細胞質中發生，且因此無需各順反子内均存在分泌信號序列。就此而言，免疫球蛋白之輕鏈及重鏈皆係在細胞質内表現、折疊並組裝形成功能性免疫球蛋白。某些宿主菌株（例如大腸桿菌irW菌株）提供有利於二硫鍵形成之細〇、胞貝條件’精此允峰適當折豎及纪裝經表現之蛋白次單元。Proba及 Pltickthun，Gene 159:203 (1995)。適於表現本發明抗體之原核宿主細胞包括古細菌 (Archaebacteria)及真細菌（Eubacteria)，諸如革蘭氏陰性 (Gram-negative)或革蘭氏陽性生物體。適用細菌之實例包括埃希氏菌例如大腸桿菌）、桿菌 (5⑽·///)(諸如枯草桿菌（及 μ如、腸内菌 ( (五、假單胞菌而mo⑽s)物種（例如綠膿桿菌（P. wri/g—μ))、鼠傷寒沙門桿菌加//α 、黏質沙雷氏菌（Serraik 似）、克雷伯氏菌（iaeh/’e/⑷、變形桿菌（tvw⑽4、志贺桿菌 (別如/⑷、根瘤菌⑷、透明顫菌（阶⑷或副球菌（ParacMcw)。在一些實施例中，使用革蘭氏陰性細胞。在一些實施例中，將大腸桿菌細胞用作本發明之宿主。大腸桿菌菌株之實例包括W3110菌株（Bachmann， Cellular and Molecular Biology，第 2 卷（Washingt〇n， 119234.doc -82· 200813088 D.C.:American Society for Microbiology, 1987)，第 1190-1219頁；ATCC⑧寄存號27,325)及其衍生物，包括具有基因型 W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA (nmpc-fepE) degP41 kanR 之 33D3 菌株（美國專利第 5,639,635號）。其他菌株及其衍生物，諸如大腸桿菌294 (ATCC⑧31，446)、大腸桿菌B、大腸桿菌λ 1776 (ATCC® 31，5 3 7)及大腸桿菌1^3 08 (八丁<：€©31，608)亦適用。此等實例係出於說明而非限制之目的。用於構築任何具有指定基因型之上文所述細菌之衍生物的方法已為此項技術中所知且描述於（例如）Bass等人，iVWe/似，8:309-14 (1990)中。一般需要考慮複製子在細菌細胞中之可複製性來選擇適當之細菌。舉例而言，當使用熟知之質體（諸如PBR322、 pBR325、pACYC177或pKN410)提供複製子時，大腸桿菌、沙雷氏菌或沙門氏菌物種可適於用作宿主。一般而言，宿主細胞應分泌最低量蛋白水解酶，且可適宜地將額外之蛋白酶抑制劑併入細胞培養物中〇 U.抗體產生用上述表現載體轉化宿主細胞，並將其培養於經改質以適於誘導啟動子、選擇轉化體或擴增編碼所需序列之基因的習知營養培養基中。轉化意謂將DNA引入原核宿主中使得DNA可作為染色體外元件或由染色體成份複製。視所使用之宿主細胞而定，使用適於此等細胞之標準技術進行轉化。使用氯化鈣進行 119234.doc • 83 - 200813088 鈣處理一般用於含有實質細胞壁障壁之細菌細胞。另一轉化方法採用聚乙二醇/DMS0。所使用之另一技術為電穿孔。使用於產生本發明多肽之原核細胞在此項技術中已知且適於培養所選擇之宿主細胞的培養基中生長。適當培養基之實例包括加有必需養分補充物之Luria肉湯（LB)。在一些實施例中，培養基亦含有基於表現載體之構造而選擇之選擇劑，其用以選擇性允許含有表現載體之原核細胞生長。舉例而言’將安比西林添加至培養基中以使表現安比西林抗性基因之細胞生長。亦可包括單獨或以與另一種補充物或培養基之混合物 (諸如複合氮源）之形式以適當濃度引入的除碳、氮及無機磷酸鹽來源外之任何必需補充物。視情況，培養基可含有一或多種選自由以下各物組成之群的還原劑：麵胱甘肽、半胱胺酸、胱胺、巯基乙酸酯、二硫赤藻糖醇及二硫蘇糖醇。將原核宿主細胞在適當溫度下培養。對於大腸桿菌之生長而言，例如一般使用在約2(rc至約39t範圍内之溫度，通常為約2rc至約m，例如約3(rc。培養基之pH:;主要視宿主生物體而定為約5至約9範圍内之任何？11值。對於大腸桿菌而言’ pH值-般為約6.8至約74，且通常為約 7.0。若在本發明之表現載體中使用誘導型啟動子該啟動子活化之條件下誘導蛋白表現則在適於在本發明之一態樣 119234.doc -84 - 200813088 中’使用啟動子控制多肽之轉錄。因此，將經轉化之宿主細胞培養於磷酸鹽限制性培養基中以用於誘導。填酸鹽限制性培養基一般為C.R.A.P培養基（例如參看Simm〇ns 荨人’乂 Mei/z· 263:133-47 (2002))。如此項技術中已知，可根據所使用之載體構築體使用多種其他誘導物。在一實施例中，本發明之經表現多肽經分泌至宿主細胞周質中且自周質將其回收。蛋白回收通常涉及一般藉由諸如滲壓震擾、超音波處理或溶解之方式***徵生物。*** 細胞後，可藉由離心或過濾移除細胞碎片或整個細胞。可進一步（例如）藉由親和樹脂層析純化蛋白質。或者，可將蛋白質轉移至培養基中並於其中進行分離。可將細胞自培養物中移除，並過濾培養物上清液且濃縮以進一步純化所產生之蛋白質。可進一步分離經表現之多肽且使用諸如聚丙烯醯胺凝膠電泳（PAGE)及西方墨點檢定之通常已知的方法加以鐘別。在本發明之一態樣中，藉由醱酵方法來進行大量抗體之製造。可使用多種大規模饋料分批醱酵程序製造重組蛋白。大規模醱酵具有至少1，000公升之容量，較佳約^000 至100，_公升之容量。此等醱酵罐使用攪拌器葉輪分配氧氣及養分，尤其是葡萄糖(常用碳/能量源小規模酸酵一般係指在不大於約100公升之容量且可在約丨公升至約 100公升範圍内之醱酵罐中進行的醱酵。、在酸酵過程中，通常在細胞已於適t條件下生長至所需 119234.doc -85- 200813088 禮、度（例如od^q為約180至220)後起始對於蛋白表現之誘導，在該階段細胞係處於穩定期早期。如此項技術中已知且如上文所述，可根據所使用之載體構築體使用多種誘導物。可於誘導前使細胞生長一段較短之時間。儘管可使用車乂長或較短之誘導時間，但通常誘導細胞歷經約12至5〇個小時。為改良本發明多肽之產率及品質，可改變多種醱酵條件。舉例而言’為改良經分泌抗體多肽之適當組裝及折疊’可使用過度表現伴隨蛋白之額外載體共轉化宿主原核細胞’該等伴隨蛋白諸如Dsb蛋白（DsbA、DsbB、DsbC、 DsbD及/或DsbG)或FkpA(具有伴隨活性之肽基脯胺醯基順反異構酶）。已證實伴隨蛋白促進細菌宿主細胞中所產生之異源蛋白之適當折疊及溶解性。Chen等人，J C/2em 274:19601-05 (1999); Georgiou等人，美國專利第 6,083,715 號；Georgiou等人，美國專利第6,〇27,888號；Bothmann及 Pltickthun J· Bb/. CTzem. 275:17100-05 (2000); Ramm及 Pliickthun J. 5b/· CTzem. 275:17106-13 (2000); Arie等人， Mo/· 39:199-210 (2001)。為使經表現異源蛋白（尤其對於蛋白水解敏感之蛋白）之蛋白水解降至最低，可將某些蛋白水解酶缺陷性宿主菌株用於本發明中。舉例而言，可修飾宿主細胞菌株以實現編碼已知細菌蛋白酶（諸如蛋白酶ΠΙ、OmpT、DegP、Tsp、蛋白酶I、蛋白酶Mi、蛋白酶V、蛋白酶VI及其組合）之基因的基因突變。可使用某些大腸桿菌蛋白酶缺陷菌株且其 119234.doc -86- 200813088 描述於（例如）Joly等人’（1998)’同上文；Georgiou等人，美國專利第5,264,365號；Georgiou等人，美國專利第 5,508，192號；Hara等人，Drwg 及以以？⑽ce，2:63-72 (1996)中。在一實施例中，使用經過度表現一或多種伴隨蛋白之質體轉化的蛋白水解酶缺陷性大腸桿菌菌株作為本發明之表現系統中的宿主細胞。 iii.抗體純化可使用此項技術中已知之標準蛋白純化方法。下述程序為例示性適當純化程序：免疫親和管柱或離子交換管柱分級分離、乙醇沉澱、逆相HPLC、矽石或陽離子交換樹脂 (諸如DEAE)層析、層析聚焦、SDS-PAGE、硫酸銨沉澱及使用（例如）Sephadex G-75之凝膠過濾。在一態樣中’將固定於固相上之蛋白A用於本發明之全長抗體產物的免疫親和純化。蛋白A為來自金黃色葡萄球菌（57叩仙^^似）之41 kD細胞壁蛋白，其以高親和力與抗體之Fc區結合。Lindmark等人，J· /所顧⑽厂 62:1-13 (1983)。固定蛋白A之固相較佳為包含玻璃或矽石表面之管柱，更佳為受控微孔玻璃管柱或矽酸管柱在一些應用中，管柱經諸如甘油之試劑塗覆以力圖防止污染物之非特異性黏附。乍為、、、屯化之第—步驟’可將如上文所述自細胞培養物獲得之製備物塗覆於^蛋白人之固相上以使所關注抗體與蛋白Α特異性結合。接著洗務固相以移除與固相非特異性 119234.doc -87 - 200813088 結合之 >可染物。最後，藉由溶離自固相回收所關注之抗體。 b·使用真核宿主細胞產生抗體載體組份一般包括（但不限於）一或多種以下組份：信號序列、複製起點、一或多個標記基因、增強子元件、啟動子及轉錄終止序列。 (Θ信號序列組份用於真核伯主細胞中之載體亦可含有信號序列或在所關注之成熟蛋白或多肽末端具有特異性裂解位點的其他夕狀所選擇之異源信號序列較佳為可由宿主細胞識別及處理（亦即由信號肽酶裂解）之信號序列。在哺乳動物細胞表現中’可用哺乳動物信號序列以及病毒分泌前導序列，例如單純疱疹gD信號。此前驅區之DNA在閱讀框架中與編碼抗體之〇ΝΑ接合。 (U)複製起點一般而言，哺乳動物表現載體無需複製起點組份。舉例而言，通常可使用SV40起點僅係因為其含有早期啟動子。 (ΗΌ選擇基因組份表現及選殖載體可含有選擇基因，亦稱為可選標記物。典型之選擇基因編碼具有以下功能之蛋白質（a)賦予對於抗生素或其他毒素（例如安比西林、新黴素（ne〇mycin)、甲胺嗓吟或四環素）之抗性；（b)補充營養缺陷（在相關時）；或 (C)提供不能自複合培養基獲得之關鍵養分。選擇流程之一實例係利用藥物使宿主細胞之生長停滞。 H9234.doc -88 - 200813088 經異源基因成功轉化之彼等細胞可產生呈現藥物抗性之蛋白質且因此在該選擇方案中存活。此顯性選擇之實例使用為物新锨素、彳致齡酸及濕黴素（hygromycin)。適用於哺乳動物細胞之可選標記物的另一實例為使得能夠鐘別能夠吸收抗體核酸之細胞的可選標記物，諸如 DHFR、胸苷激酶、金屬硫蛋白^及π(較佳為靈長類動物金屬硫蛋白基因）、腺苷脫胺酶、鳥胺酸脫羧酶等。舉例而言，首先藉由在含有甲胺喋呤（Mtx)(DHFR之競 f性拮抗劑）之培養基中培養所有轉化體來鑑別經DHFR選擇基因轉化的細胞。當使用野生型DHFR時，其中一種適當之宿主細胞為缺失DHFR活性之中國倉鼠卵巢（CHO)細胞株（例如 ATCC® CRL_9096)。或者’可藉由在含有針對可選標記物之選擇劑（諸如胺基糖苷抗生素’例如康黴素（kanamycin)、新黴素或G418) 的培養基中進行細胞生長來選擇經編碼抗體、野生型 DHFR蛋白及另一可選標記物（諸如胺基糖苷3，_填酸轉移酶 (ΑΡΗ))之DNA序列轉化或共轉化之宿主細胞（尤其是含有内源DHFR之野生型宿主）。參看美國專利第4,965，199號。 (iv)啟動子組份表現及選殖載體通常含有由宿主生物體識別且以可操作方式連接至抗體多肽核酸之啟動子。已知用於真核生物之啟動子序列。實際上所有真核基因均具有位於轉錄起始位點上游大約25至30個鹼基處之富AT區。另一可見於多種基因之轉錄起始點上游70至80個驗基處之序列為CNC A AT區 119234.doc -89- 200813088 (SEQ ID N〇: 19)，其巾N可為任何核㈣。在大部分直枝基因之3,端為AATAAA序列（SEQ ID N〇: 2〇)，其可為將聚 A尾添加至編碼序列3,端之信號。所有此等序列均適於插入至真核表現載體中。PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence in the two cistrons used to express the system is the STII signal sequence or a variant thereof. In another sad case, the production of the immunoglobulin of the present invention can occur in the cytoplasm of the host cell, and thus there is no need for a secretory signal sequence to be present in each cistron. In this regard, the light and heavy chains of immunoglobulins are expressed, folded, and assembled in the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., E. coli irW strain) provide a fine, stimulating, and stimulating condition for disulfide bond formation. Proba and Pltickthun, Gene 159: 203 (1995). Prokaryotic host cells suitable for expression of the antibodies of the invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of suitable bacteria include Escherichia coli such as Escherichia coli, Bacillus (5(10)·///) (such as Bacillus subtilis (and μ, eg, enterobacteria (5, Pseudomonas and mo(10)s) species (eg, green pus) Bacillus (P. wri/g-μ)), Salmonella typhimurium plus / / α, Serratia marcescens (Serraik-like), Klebsiella (iaeh / 'e / (4), Proteus (tvw (10) 4, Shigella (such as / (4), Rhizobium (4), Vitreoscilla (Grade (4) or ParacMcw). In some embodiments, Gram-negative cells are used. In some embodiments, E. coli cells are used As a host of the present invention, examples of Escherichia coli strains include W3110 strain (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washingt〇n, 119234.doc-82·200813088 DC: American Society for Microbiology, 1987), 1190- Page 1219; ATCC8 Accession No. 27,325) and its derivatives, including the 33D3 strain (G. No. 5,639,635) having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA (nmpc-fepE) degP41 kanR. Other strains and their derivatives Object, such as the large intestine 294 (ATCC 831,446), E. coli B, E. coli λ 1776 (ATCC® 31, 5 3 7) and E. coli 1^3 08 (eight butyl <: €© 31, 608) are also applicable. For purposes of illustration and not limitation, methods for constructing any of the above-described derivatives of bacteria having the specified genotype are known in the art and are described, for example, in Bass et al., iVWe/like, 8 :309-14 (1990). It is generally necessary to consider the replicability of replicons in bacterial cells to select appropriate bacteria. For example, when using well-known plastids (such as PBR322, pBR325, pACYC177 or pKN410) to provide replication In case of E. coli, Serratia or Salmonella, the host cell may be suitable for use as a host. In general, the host cell should secrete a minimal amount of proteolytic enzyme, and may suitably incorporate additional protease inhibitors into the cell culture. 〇U. Antibody Production The host cell is transformed with the above expression vector and cultured in a conventional nutrient medium modified to be suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence. Introducing DNA The prokaryotic host allows the DNA to be replicated as an extrachromosomal element or by a chromosomal component, depending on the host cell used, using standard techniques appropriate for such cells. Calcium Chloride 119234.doc • 83 - 200813088 Calcium treatment is generally used for bacterial cells containing parenchymal cell wall barriers. Another conversion method uses polyethylene glycol/DMS0. Another technique used is electrical vias. Prokaryotic cells used to produce the polypeptides of the invention are grown in a medium known in the art and suitable for culturing selected host cells. Examples of suitable media include Luria Broth (LB) supplemented with essential nutrient supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing the ampicillin resistance gene. Any necessary supplements other than carbon, nitrogen and inorganic phosphate sources, introduced alone or in a mixture with another supplement or medium, such as a complex nitrogen source, may also be included. Optionally, the medium may contain one or more reducing agents selected from the group consisting of: glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreose. alcohol. Prokaryotic host cells are cultured at appropriate temperatures. For the growth of E. coli, for example, a temperature in the range of about 2 (rc to about 39 t is generally used, usually about 2 rc to about m, for example about 3 (rc. pH of the medium: mainly depending on the host organism) Any value of from about 5 to about 9 is in the range of from about 5 to about 9. For pH E. coli, the pH is generally from about 6.8 to about 74, and usually about 7.0. If an inducible promoter is used in the expression vector of the present invention The expression of the induced protein under the conditions of activation of the promoter is adapted to control the transcription of the polypeptide using a promoter in an aspect of the invention 119234.doc-84 - 200813088. Therefore, the transformed host cell is cultured in phosphate. The restriction medium is used for induction. The acid-binding medium is generally CRAP medium (see, for example, Simm〇ns 荨人'乂Mei/z. 263:133-47 (2002)). As is known in the art, A variety of other inducers can be used depending on the vector construct used. In one embodiment, the expressed polypeptide of the invention is secreted into the host cell periplasm and recovered from the periplasm. Protein recovery typically involves, for example, osmotic pressure. Shock Ultrasonic treatment or lysis means cleavage. After dividing the cells, the cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transferred to The medium is separated from the culture. The cells can be removed from the culture, and the culture supernatant is filtered and concentrated to further purify the produced protein. The expressed polypeptide can be further isolated and condensed using, for example, polyamine A commonly known method of gel electrophoresis (PAGE) and Western ink dot assay is used to carry out the method. In one aspect of the invention, a large number of antibodies are produced by a fermentation method. A plurality of large-scale feed batches can be used. The fermentation process produces recombinant proteins. Large-scale fermentation has a capacity of at least 1,000 liters, preferably about 2,000 to 100 liters. These fermenters use a blender impeller to distribute oxygen and nutrients, especially glucose. (Common carbon/energy source Small-scale acid fermentation generally refers to fermentation in a volume of no more than about 100 liters and can range from about liters to about 100 liters. In the acid fermentation process, usually after the cells have been grown under suitable conditions to the desired 119234.doc -85-200813088 etiquette (such as od^q is about 180 to 220) For the induction of protein expression, the cell line is in the early stationary phase at this stage. As is known in the art and as described above, various inducers can be used depending on the vector construct used. The cells can be grown before induction. In a short period of time, although the induction time of the rut or the shorter one can be used, the cells are usually induced for about 12 to 5 hours. To improve the yield and quality of the polypeptide of the present invention, various fermentation conditions can be changed. For example, 'to improve the proper assembly and folding of a secreted antibody polypeptide' can be used to co-transform host prokaryotic cells with an additional vector that overexpresses the accompanying protein, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG). Or FkpA (peptidyl amidoxime cis-trans isomerase with concomitant activity). The accompanying protein has been shown to promote proper folding and solubility of the heterologous protein produced in the bacterial host cell. Chen et al., JC/2em 274:19601-05 (1999); Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6, 〇27,888; Bothmann and Pltickthun J.Bb/. CTzem. 275 :17100-05 (2000); Ramm and Pliickthun J. 5b/· CTzem. 275:17106-13 (2000); Arie et al., Mo/. 39:199-210 (2001). In order to minimize proteolysis of heterologous proteins (especially proteins susceptible to proteolysis), certain proteolytic enzyme-deficient host strains can be used in the present invention. For example, host cell strains can be modified to effect gene mutations encoding genes for known bacterial proteases such as protease, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Certain E. coli protease deficient strains can be used and are described in, for example, Joly et al. (1998)' supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5, 508, 192; Hara et al., Drwg and I? (10) ce, 2: 63-72 (1996). In one embodiment, a proteolytic enzyme-deficient E. coli strain exhibiting one or more plastid transformations with accompanying proteins is used as a host cell in the expression system of the present invention. Iii. Antibody Purification Standard protein purification methods known in the art can be used. The following procedure is an exemplary appropriate purification procedure: immunoaffinity column or ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, vermiculite or cation exchange resin (such as DEAE) chromatography, chromatofocusing, SDS-PAGE, Ammonium sulfate precipitation and gel filtration using, for example, Sephadex G-75. In one aspect, protein A immobilized on a solid phase is used for immunoaffinity purification of the full length antibody product of the present invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureus (57 叩 ^ ^^) which binds to the Fc region of the antibody with high affinity. Lindmark et al., J. / 顾 (10) Factory 62:1-13 (1983). The solid phase of immobilized protein A is preferably a column comprising a glass or vermiculite surface, more preferably a controlled microporous glass column or a citrate column. In some applications, the column is coated with a reagent such as glycerol in an effort to Prevent non-specific adhesion of contaminants. The first step-step of 乍, , 屯, can be applied to the solid phase of the protein human as described above to specifically bind the antibody of interest to the peptone. The solid phase is then washed to remove > dyeables in combination with solid phase non-specific 119234.doc -87 - 200813088. Finally, the antibody of interest is recovered from the solid phase by dissolution. b. Production of antibodies using eukaryotic host cells Vector components generally include, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. (The vector of the Θ signal sequence component used in the eukaryotic primary cell may also contain a signal sequence or other heterologous signal sequence selected at the other end of the mature protein or polypeptide end of interest having a specific cleavage site. A signal sequence that can be recognized and processed by a host cell (ie, cleaved by a signal peptidase). In mammalian cell expression, 'a mammalian signal sequence can be used as well as a viral secretion leader sequence, such as the herpes simplex gD signal. In the reading frame, it is conjugated to the coding antibody. (U) Origin of replication Generally, the mammalian expression vector does not require replication of the starting component. For example, the SV40 origin can usually be used only because it contains an early promoter. The selection of the genomic expression and selection vector may contain a selection gene, also referred to as a selectable marker. A typical selection gene encodes a protein having the following functions (a) conferring antibiotics or other toxins (eg, ampicillin, neomycin (ne) Resistance to 〇mycin), methotrexate or tetracycline; (b) supplemental auxotrophy (when relevant); or (C) A key nutrient that cannot be obtained from a complex medium. An example of a selection process is the use of drugs to arrest the growth of host cells. H9234.doc -88 - 200813088 These cells, which have been successfully transformed by a heterologous gene, produce a protein that exhibits drug resistance and It is therefore surviving in this alternative. Examples of this dominant selection use neomycin, sputum acid and hygromycin. Another example of a selectable marker for mammalian cells is to enable A selectable marker for cells that are capable of absorbing antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein and π (preferably primate metallothionein genes), adenosine deaminase, ornithine Decarboxylase, etc. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx) (DHFR). In DHFR, one of the appropriate host cells is a Chinese hamster ovary (CHO) cell line (eg, ATCC® CRL_9096) lacking DHFR activity. Or 'can be used with optional markers Cell growth is performed in a medium such as an aminoglycoside antibiotic such as kanamycin, neomycin or G418 to select an encoded antibody, a wild-type DHFR protein, and another selectable marker (such as Host cells transformed or co-transformed with a DNA sequence of aminoglycoside 3,_acid transferase (indole) (especially a wild-type host containing endogenous DHFR). See U.S. Patent No. 4,965,199. Subcomponent performance and selection vectors typically contain a promoter that is recognized by the host organism and is operably linked to the antibody polypeptide nucleic acid. Known promoter sequences for eukaryotes. Virtually all eukaryotic genes are located An AT-rich region approximately 25 to 30 bases upstream of the transcription start site. Another sequence that can be found at 70 to 80 upstream of the transcription initiation site of a plurality of genes is the CNC A AT region 119234.doc-89-200813088 (SEQ ID N〇: 19), and the towel N can be any core (four) . At the 3' end of most of the stalk genes is the AATAAA sequence (SEQ ID N 〇: 2 〇), which may be the signal that adds the poly A tail to the coding sequence 3 end. All of these sequences are suitable for insertion into eukaryotic expression vectors.

哺乳動物宿主細胞中自載體之抗體多肽轉錄（例如）受自病毒（諸如多瘤病毒、禽痘病毒、腺病毒（諸如腺病毒2)、牛乳頭狀瘤病毒、禽肉瘤病毒、巨細胞病毒、反轉錄病毒、B型肝炎病毒及猿病毒4〇(SV4〇))基因組，自異源哺Z 動物啟動子（例如肌動蛋白啟動子或免疫球蛋白啟動子），自熱休克啟動子獲得的啟動子控制，其限制條件為此等啟動子與宿主細胞系統相容。 SV40病毒之早期及晚期啟動子係以亦含有sv4〇病毒複製起點之SV40限制片段形式便利獲得。人類巨細胞病毒之立即早期啟動子係以历Τ^ΙΠ E限制片段形式便利獲得。使用牛乳頭狀瘤病毒作為載體於哺乳動物宿主中表現dna之系統揭示於美國專利第4,419,446號中。對於此系統之修飾描述於美國專利第4,601，978號中。或者，可使用勞斯肉瘤病毒（Rous Sarcoma Virus)之長末端重複序列作為啟動子。 (4増強子元件組份由較高等真核生物對編碼本發明抗體多肽之DNA的轉錄通常可藉由將增強子序列***載體中來增加。現已知多種來自哺乳動物基因（血球蛋白、彈性蛋白酶、白蛋白、月σ蛋白及胰島素）之增強子序列。然而，吾人通常將使用來自真核細胞病毒之增強子。實例包括複製起點（bp 100- H9234.doc -90- 200813088 270)後側之SV40增強子、巨細胞病毒早期啟動子增強子、複製起點後側之多瘤病毒增強子及腺病毒增強子。關於用於活化真核啟動子之增強元件，亦可參看Yaniv, 297:17-18 (1982)。可將增強子在抗體多肽編碼序列之5’或 3’位置處剪接至載體中，但其較佳位於啟動子之5’位置處。 (vi) 轉錄終止組份用於真核宿主細胞中之表現載體通常亦含有終止轉錄及穩定mRNA所必需乞序列。此等序列通常可自真核或病毒 DNA或cDNA之5’且有時3’非轉譯區獲得。此等區含有轉錄為編碼抗體之mRNA之非轉譯部分中之多聚腺嘌呤化片段的核苷酸區段。一種適用之轉錄終止組份為牛生長激素多聚腺嘌呤化區。參看WO 94/11026及其中所揭示之表現載體。 (vii) 宿主細胞之選擇及轉化適用於選殖或表現本文載體中之DNA之宿主細胞包括本文所述之較高等真核生物細胞，包括脊椎動物宿主細胞。使脊椎動物細胞在培養物（組織培養物）中繁殖已成為一種常規程序。適用之哺乳動物宿主細胞株之實例為經SV40轉化之猴腎CV1細胞株（COS-7，ATCC® CRL 1651)、人類胚腎細胞株（293細胞或經次選殖在懸浮液培養物中生長之 293細胞，Graham等人，J· Gw Wro/· 36:59 (1977))、幼倉鼠腎細胞（BHK，ATCC® CCL 10)、中國倉鼠卵巢細胞/-DHFR (CHO，Urlaub 等人，Natl. Acad, Sci. USA 77: 4216 119234.doc -91- 200813088 (1980))、小鼠塞利特氏細胞（mouse sertoli cell)(TM4, 万紿/· 23:243-251 (1980))、猴腎細胞（CV1 ATCC® CCL 70)、非洲綠猴腎細胞（VERO-76，ATCC® CRL-1587)、人類宮頸癌細胞（HELA，ATCC⑧CCL 2)、犬科腎細胞（MDCK，ATCC® CCL 34)、水牛鼠肝細胞（buffalo rat liver cell)(BRL 3A，ATCC® CRL 1442)、人類肺細胞 (W138，ATCC® CCL 75)、人類肝細胞（Hep G2，HB 8065)、小鼠乳腺腫瘤（MMT 060562, ATCC⑧ CCL51)、TRI 細跑（Mather 等人，Annals Ν·Υ· Acad. Sci· 3 83:44 68 (1982))、MRC 5細胞、FS4細胞及人類肝腫瘤細胞株（Hep G2)。用上述用於產生抗體之表現或選殖載體轉化宿主細胞，並將其培養於經改質以適於誘導啟動子、選擇轉化體或擴增編碼所需序列之基因的習知營養培養基中。 (viii)培養宿主細胞可將用於產生本發明抗體之宿主細胞培養於多種培養基中。諸如Ham’s F10 (Sigma)、最低必需培養基 (MEM)(Sigma)、RPMI-1640 (Sigma)及杜貝卡氏經改質伊格氏培養基（Dulbecco’s Modified Eagle’s Medium (DMEM)，Sigma)之市售培養基均適於培養宿主細胞。此外，可使用以下文獻中所述之任何培養基作為宿主細胞之培養基：Ham等人，Mei/z. 58:44 (1979); Barnes等人，Anal· J5bc/^m.l02:255 (1980);美國專利第 4,767,704 號、第 4,657,866號、第 4,927,762號、第 4,560,655 號或第 119234.doc -92- 200813088 5，122,469號 ’ W0 9_343()、w〇 87/G()195或美國專利參考30,985。任何此等培養基均可視需要補充激素及/或其他生長因子（諸如胰島素、運鐵蛋白或表皮生長因子）、鹽（諸如氯化鈉、鈣、鎂及磷酸鹽）、緩衝液（諸如HEpEs)、核苷酸（諸如腺苷及胸苷）、抗生素（諸如gentamycintm藥物）、示蹤元素（定義為無機化合物，通常以在微莫耳範圍内之最終濃度存在）及葡萄糖或等效能源。亦可包括將為熟習此項技術者所知之適當濃度的任何其他必需補充物。 ( 培養條件（諸如温度、PH值及其類似條件）為選擇用於表現之宿主細胞先前所使用之條件且對於一般技術者為顯而易見的。 (ix)抗體純化當使用重組技術時，抗體可於細胞内產生或經直接分泌至培養基中。若抗體於細胞内產生，則作為第一步驟，例如藉由離心或超濾移除宿主細胞或已溶解片段之顆粒碎片。在抗體經分泌至培養基中之情形下，通常首先使用市售蛋白濃縮過濾器（例如Amicon或Millipore Pellicon超遽裝置）濃縮來自此等表現系統之上清液。任何前述步驟中均可包括蛋白酶抑制劑（諸如PMSF)以抑制蛋白水解且可包括抗生素以阻止外來污染物之生長。可使用（例如）羥磷灰石層析、凝膠電泳、透析及親和層析純化由細胞製備之抗體組合物，其中親和層析為較佳純化技術。蛋白A作為親和配位體之適用性視物種及存在於抗體中之任何免疫球蛋白Fc結構域的同型而定。蛋白A可 119234.doc -93- 200813088 用於純化基於人類γΐ、γ2或γ4重鏈之抗體（Lindmark等人， J· /mm ι/π ο厂Μ以/2. 62:1-13 (1983)) 〇蛋白G推薦用於所有小鼠同型及人類y3(Guss等人，五ΜΒΟ丄5:1567-75 (1986))。親和配位體所附著之基質最常見為瓊脂糖，但亦可使用其他基質。與使用瓊脂糖可達成之流動速率及處理時間相比，諸如受控微孔玻璃或聚（苯乙烯二乙烯基）苯之機械穩定性基質允許較快的流動速率及較短的處理時間。當抗體包含CH3結構域時，可使用Bakerbond ABXTM樹脂（1.丁· Baker，Phi 11 ipsburg，NJ)周於純化。視待回收之抗體面定，亦可使用其他用於蛋白純化之技術，諸如離子交換管柱分級分離、乙醇沉澱、逆相HPLC、矽石層析、肝素 SEPHAROSETM層析、陰離子或陽離子交換樹脂（諸如聚天冬胺酸管柱）層析、層析聚焦、SDS-PAGE及硫酸銨沉澱。在任何初步純化步驟後，可使包含所關注抗體及污染物之混合物經歷使用pH值在約2.5-4.5之間的溶離緩衝液較佳在低鹽濃度（例如約〇-〇_25 Μ鹽）下進行的低pH值疏水性相互作用層析。免疫共輥物本發明亦提供免疫共軛物（亦互換稱為”抗體-藥物共軛物’’ 或ADC)，其包含與細胞毒性劑，諸如化學治療劑、藥物、生長抑制劑、毒素（例如細菌、真菌、植物或動物來源之酶活性毒素或其片段）或放射性同位素（亦即放射性共輛物）共軛之本文所述之任何抗EGFL7抗體。使用抗體-藥物共軛物局部傳遞細胞毒性劑或細胞生長 119234.doc -94- 200813088 抑制劑（亦即在癌症治療中用於殺傷或抑制腫瘤細胞之藥物）（Syrigos 及 Epenetos，Anticancer Research 19:605-14 (1999); Niculescu-Duvaz 及 Springer，j(iv. Drg De/. 26:151-72 (1997);美國專利第4,975,278號）允許將藥物部分目標傳遞至腫瘤且於其中實現細胞内積累，其中此等非共軛藥劑之全身投藥可產生對正常細胞以及意欲消除之腫瘤細胞不可接受之毒性含量（Baldwin等人，Z⑽cei，第 (1986 年 3 月 15 日）：603-05 頁（1986); Thorpe，"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review’’，The antibody polypeptide from a vector in a mammalian host cell is transcribed (for example) from a virus (such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus). , retrovirus, hepatitis B virus and prion 4〇 (SV4〇)) genome, derived from a heterologous Z animal promoter (eg actin promoter or immunoglobulin promoter), obtained from a heat shock promoter The promoter is controlled, and its constraints are such that the promoter is compatible with the host cell system. The early and late promoters of the SV40 virus are conveniently obtained in the form of SV40 restriction fragments that also contain the sv4 prion replication origin. The immediate early promoter of human cytomegalovirus is conveniently obtained in the form of a restriction fragment. A system for expressing dna in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications to this system are described in U.S. Patent No. 4,601,978. Alternatively, the long terminal repeat of Rous Sarcoma Virus can be used as a promoter. (4. The transcription of the DNA encoding the antibody polypeptide of the present invention by higher eukaryotes by higher eukaryotes can generally be increased by inserting the enhancer sequence into the vector. A variety of mammalian genes (hemoglobulin, Enhancer sequences for elastase, albumin, erythropoietin, and insulin. However, we will generally use enhancers from eukaryotic viruses. Examples include the origin of replication (bp 100-H9234.doc-90-200813088 270) The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the replication origin, and the adenovirus enhancer. For enhancement elements for activation of eukaryotic promoters, see also Yaniv, 297: 17-18 (1982). The enhancer can be spliced into the vector at the 5' or 3' position of the antibody polypeptide coding sequence, but is preferably located 5' to the promoter. (vi) Transcription termination component Expression vectors in eukaryotic host cells usually also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are typically obtained from 5' and sometimes 3' non-translated regions of eukaryotic or viral DNA or cDNA. These regions contain a nucleotide segment transcribed as a polyadenylation fragment in the untranslated portion of the mRNA encoding the antibody. One suitable transcription termination component is the bovine growth hormone polyadenylation region. See WO 94 /11026 and the expression vector disclosed therein. (vii) Selection and transformation of host cells Host cells suitable for use in the selection or expression of DNA in the vectors herein include higher eukaryotic cells as described herein, including vertebrate host cells. It has become a routine procedure to propagate vertebrate cells in culture (tissue culture). An example of a suitable mammalian host cell strain is SV40 transformed monkey kidney CV1 cell line (COS-7, ATCC® CRL 1651). Human embryonic kidney cell line (293 cells or 293 cells that have been subcultured in suspension culture, Graham et al, J. Gw Wro/36:59 (1977)), baby hamster kidney cells (BHK, ATCC® CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Natl. Acad, Sci. USA 77: 4216 119234.doc -91- 200813088 (1980)), mouse Celite cells ( Mouse sertoli cell)(TM4, Wan Hao /· 23:243-251 (1980)), monkey kidney cells (CV1 ATCC® CCL 70), African green monkey kidney cells (VERO-76, ATCC® CRL-1587), human cervical cancer cells (HELA, ATCC8CCL 2) , canine kidney cells (MDCK, ATCC® CCL 34), buffalo rat liver cells (BRL 3A, ATCC® CRL 1442), human lung cells (W138, ATCC® CCL 75), human hepatocytes ( Hep G2, HB 8065), mouse mammary gland tumor (MMT 060562, ATCC8 CCL51), TRI run (Mather et al., Annals Ν·Υ·Acad. Sci·3 83:44 68 (1982)), MRC 5 cells, FS4 cells and human liver tumor cell lines (Hep G2). The host cell is transformed with the above-described expression or selection vector for producing an antibody, and cultured in a conventional nutrient medium modified to be suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence. (viii) Culturing host cells Host cells for producing the antibodies of the present invention can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma Both are suitable for culturing host cells. Furthermore, any of the media described in the following literature can be used as a medium for host cells: Ham et al, Mei/z. 58:44 (1979); Barnes et al., Anal J5bc/^m.l02:255 (1980) U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 119,234.doc-92-200813088 5,122,469 'W0 9_343(), w〇87/G() 195 or US Patent Reference 30,985 . Any such medium may optionally be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEpEs), Nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycintm drugs), trace elements (defined as inorganic compounds, usually present in the final concentration in the micromolar range), and glucose or equivalent energy. Any other necessary supplements that will be of a suitable concentration known to those skilled in the art may also be included. (Culture conditions such as temperature, pH, and the like) are conditions previously used for selection of host cells for expression and will be apparent to those of ordinary skill. (ix) Antibody purification When recombinant techniques are employed, antibodies can be used Intracellular production or direct secretion into the medium. If the antibody is produced in the cell, as a first step, for example, by centrifugation or ultrafiltration to remove the host cell or the fragmented particles of the fragment. The antibody is secreted into the medium. In this case, a supernatant from such performance systems is typically first concentrated using a commercially available protein concentration filter (eg, Amicon or Millipore Pellicon Ultrasonic device). Protease inhibitors (such as PMSF) may be included in any of the foregoing steps to inhibit Proteolytic and may include antibiotics to prevent the growth of foreign contaminants. Antibody compositions prepared from cells may be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is Good Purification Technology. Applicability of Protein A as an Affinity Ligand Depending on the species and any immunity present in the antibody The protein Fc domain is isotyped. Protein A 119234.doc -93- 200813088 is used to purify antibodies based on human γΐ, γ2 or γ4 heavy chains (Lindmark et al., J· /mm ι/π ο厂Μ / 2. 62:1-13 (1983)) Prion G is recommended for all mouse isotypes and human y3 (Guss et al., 5:1567-75 (1986)). Matrix to which the affinity ligand is attached. The most common is agarose, but other matrices can be used. Mechanically stable matrices such as controlled microporous glass or poly(styrenedivinyl)benzene allow for flow rates and processing times achievable with agarose. Faster flow rate and shorter processing time. When the antibody contains the CH3 domain, it can be purified using Bakerbond ABXTM resin (1. D. Baker, Phi 11 ipsburg, NJ) depending on the antibody to be recovered. Other techniques for protein purification, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, vermiculite chromatography, heparin SEPHAROSETM chromatography, anion or cation exchange resins (such as polyaspartic acid column), can also be used. Chromatography, chromatography focusing, SDS-PAGE and sulfur Ammonium precipitation. After any preliminary purification step, the mixture comprising the antibody of interest and the contaminant can be subjected to a dissolution buffer having a pH between about 2.5 and 4.5, preferably at a low salt concentration (eg, about 〇-〇_25). Low pH hydrophobic interaction chromatography performed under guanidinium salt. Immune co-rollers The invention also provides immunoconjugates (also referred to interchangeably as "antibody-drug conjugates" or ADCs), which comprise cells Toxic agents, such as chemotherapeutic agents, drugs, growth inhibitors, toxins (eg, enzymatically active toxins or fragments thereof of bacterial, fungal, plant or animal origin) or radioisotopes (ie, radioactive co-hosts) are conjugated as described herein. Any anti-EGFL7 antibody. Local delivery of cytotoxic agents or cell growth using antibody-drug conjugates 119234.doc -94- 200813088 Inhibitors (ie drugs used to kill or inhibit tumor cells in cancer therapy) (Syrigos and Epenetos, Anticancer Research 19: 605-14 (1999); Niculescu-Duvaz and Springer, j (iv. Drg De/. 26:151-72 (1997); U.S. Patent No. 4,975,278), allowing the delivery of a drug moiety to a tumor and achieving intracellular therein Accumulation, wherein systemic administration of such non-conjugated agents produces unacceptable levels of toxicity to normal cells and tumor cells that are intended to be eliminated (Baldwin et al., Z(10)cei, pp. (March 15, 1986): 603-05 ( 1986); Thorpe,"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review'',

Monoclonal Antibodies ’84: Biological And Clinical Applications, A. Pinchera 等人（編輯），第 475-506 頁 (1985))。藉此同時尋求最大功效與最小毒性。已報導多株抗體與單株抗體均適用於此等策略（Rowland等人， /mm· 2 1:1 83-87 (1986)) 〇此等方法中戶斤使用之藥物包括道諾黴素、多柔比星、曱胺喋呤及長春地辛 (Rowland等人，（1986)同上文）。抗體-毒素共軛物中所使用之毒素包括細菌毒素，諸如白喉毒素；植物毒素，諸如蓖麻毒素；小分子毒素，諸如格爾德黴素 (geldanamycin)(Mandler 等人，《/· Nat· Cancer Inst. 92(19):1573-81 (2000); Mandler等人，ά Me凌 Chem. Letters 10:1025-28 (2000); Mandler 等人，Monoclonal Antibodies ’84: Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506 (1985)). This also seeks maximum efficacy and minimal toxicity. It has been reported that multiple antibodies and monoclonal antibodies are suitable for such strategies (Rowland et al., /mm. 2 1:1 83-87 (1986)). The drugs used in these methods include daunorubicin, Doxorubicin, amidoxime and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin; phytotoxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al., /. Nat. Cancer Inst. 92(19): 1573-81 (2000); Mandler et al., ά Me Ling Chem. Letters 10:1025-28 (2000); Mandler et al.

C/7em· 13:786-91 (2002))、美登素類（EP 1391213; Liu等人，尸dead. Scz·· 93:8618- 8623 (1996))及刺孢黴素（Lode等人，Cancer 58:2928 119234.doc -95- 200813088 (1998); Hinman等人，Cawcer 53:3336-3342 (1993)) 〇毒素可藉由包括微管蛋白結合、DNA結合或拓撲異構酶抑制之機制實現其細胞毒性及細胞生長抑制作用。某些細胞毒性藥物在與大抗體或蛋白受體配位體共軛時傾向於不具活性或具有較低活性。 ZEVALIN™ (替伊莫單抗（ibritumomab tiuxetan)， Biogen/Idec)係一種抗體-放射性同位素共輛物，其係由經硫脲連接子-螯合劑結合之針對可見於正常及惡性B淋巴細跑表面上之CD20抗原之鼠科IgGlK單株抗體輿mIn或9GY放射性同位素構成（Wiseman等人，五wr. Jour· Nucl. Med. 27(7):766-77 (2000); Wiseman等人，99(12):4336-42 (2002); Witzig 等人，《/. C7M. Ομο/· 20(10):2453-63 (2002); Witzig 等人，J. C7M. Omo/· 20(15):3262-69 (2002))。儘管ZEVALIN具有對抗B細胞非霍奇金氏淋巴瘤 (Hodgkin’s Lymphoma，NHL)之活性，但投藥在大多數患者體内引起嚴重且長期之血球減少。MYLOTARG™ (吉妥單抗（gemtuzumab ozogamicin)，Wyeth Pharmaceuticals)係一種由連接至刺胞黴素之hu CD33抗體構成的抗體藥物共軛物，已於2000年經批准用於注射治療急性骨髓白血病 (jDrwgs 〇/ Fz/iwre 25(7):686 (2000);美國專利第 4970198號、第 5079233號、第 5585089號、第 5606040號、第 5693762號、第 5739116 號、第 5767285 號、第 5773001 號）。康妥單抗（Cantuzumab mertansine，Immunogen，Inc.) 係一種由經由二硫化物連接子SPP連接至美登素類藥物部 119234.doc -96- 200813088 分DM1之huC242抗體構成的抗體藥物共輛物，已進入用於治療表現CanAg之癌症（諸如結腸癌、胰腺癌、胃癌及其他癌症）之 II期試驗。MLN-2704 (Millennium Pharm·，BZL Biologies，Immunogen Inc·)係一種由連接至美登素類藥物部分DM1之抗***特異性膜抗原（PSMA)單株抗體構成的抗體藥物共軛物，其對***腫瘤之潛在治療正在研究中。使奥瑞他汀肽（auristatin peptide)，奥瑞他汀E(AE)及單甲基奥瑞他汀（MMAE)(海兔毒素之合成類似物）與嵌合葶株抗體cBR96(對癌瘤之Lewis Y具特異性）及cACIO(對血液科惡性疾病之CD30具特異性）共輛（Doronina等人， 21(7):778-784 (2003))且正在對其進行治療研究。可用於產生免疫共軛物之化學治療劑已描述於本文（例如上文）中。可使用之酶活性毒素及其片段包括白喉毒素A 鏈、白喉毒素之非結合活性片段、外毒素A鏈（來自綠膿桿菌）、蓖麻毒素A鏈、相思子毒素A鏈、蒴蓮根毒蛋白 (modeccin) A 鏈、帚魏菌素（α-sarcin)、桐油（Aleurites fordii)蛋白、康乃馨（dianthin)蛋白、洋商陸 α所azeawa)蛋白（ΡΑΡΙ、ΡΑΡΙΙ 及 PAP-S)、苦瓜 c/zar⑽ίζ·α)抑制劑、麻楓樹蛋白（curcin)、巴豆毒素 (crotin)、肥息草（sapaonaria officinalis)抑制劑、天堂果蛋白（gelonin)、有絲***素（mitogellin)、侷限麴菌素 (restrictocin)、酚黴素（phenomycin)、伊諾黴素（enomycin) 及黴菌毒素（tricothecene)。例如參看1993年10月28日公開 119234.doc -97- 200813088 之WO 93/H232。多種放射性核種可用於製造放射性共軛抗體。實例包括212Bi、131I、131ln、9GY及186Re。抗體與細胞毒性劑之共軛物可使用多種雙功能蛋白偶聯劑製得，該等蛋白偶聯劑諸如3-(2-σ比啶基二硫基）丙酸N-琥珀醯亞胺酯（SPDP)、亞胺基硫雑環戊烷（ΙΤ)、醯亞胺酯之雙功能衍生物（諸如己二亞胺酸二甲酯鹽酸鹽）、活性酯（諸如辛二酸一琥珀醯亞胺酯）、酸類（諸如戊二酸）、雙-疊氮基化合物 (諸如雙-(對疊氮基苯曱醯基）己二胺）、雙-重氮鹽衍生物 (諸如雙-(對重氮鹽笨甲醯基）-乙二胺）、二異氰酸酯（諸如曱本2,6- 一異鼠酸醋）及雙活性氟化合物（諸如1，5_二氟_2,4· 二硝基苯）。舉例而言，蓖麻毒素免疫毒素可如Vitetta等人，238:1098 (1987)中所述來製備。碳14標記之 1-異硫來氧基本甲基-3-甲基二伸乙三胺五乙酸（mx_dtpa) 為一種用於使放射性核苷酸與抗體共軛之例示性f合劑。參看 WO 94/11026。本文亦涵蓋抗體與一或多種小分子毒素（諸如刺胞黴素、美登素類、海兔毒素、奥瑞他汀、單端孢黴毒素及 CC1065以及此等毒素之具有毒素活性之衍生物）之共扼物。 i·美登素及美登素類化合物在一些實施例中，免疫共軛物包含與一或多個美登素類分子共之本發明之抗體（全長或片段）。美登素類化合物為藉由抑制微管蛋白聚合發揮作用之有絲***抑制劑。美登素最先係自東非灌木齒葉美登木 119234.doc -98 - 200813088 ⑽s πααία)分離（美國專利第3,896,111號）。隨後，發現某些微生物類亦產生美登素類化合物，諸如美登醇及 C-3美登醇酯（美國專利第4，151，〇42號）。合成美登醇及其衍生物及類似物揭示於（例如）美國專利第4,137,230號、第 4,248,870 號、第 4,256,746 號、第 4,260,608 號、第 4,265,814號、第 4,294,757號、第 4,307,016號、第 4,308,268 號、第 4,308,269號、第 4,309,428 號、第 4,313,946號、第 4,315,929 號、第 4,317,821 號、第 4,322,348 號、第 4,331，598 號、第 4,361，650 號、第 4,364,866 號、第 4,424,219 號、第 4,450,254 號、第 4,362,663 號及第 4,371，533號中。美登素類藥物部分為抗體藥物共輛物中最引人關注之藥物部分，此係因為其：⑴相對易於藉由醱酵或化學改質、酸酵產物之衍生作用來製備；（ii)易於經適於經由非二硫化物連接子與抗體共軛之官能基衍生；（iii)在血漿中穩定；及（iv)有效對抗多種腫瘤細胞株。含有美登素類化合物之免疫共軛物、其製造方法及其治療用途揭示於（例如）美國專利第5,208,〇2〇號、第5,416,〇64 號及歐洲專利EP 0 425 235 B1中，該等專利之揭示内容以引用之方式明確地併入本文中。Liu等人，勤" Ad· 5W· [/以93:8618-23 (1996)描述包含連接至針對人類結腸直腸癌之單株抗體C242之美登素類化合物（命名為 DM1)的免疫共軛物。已發現該共軛物對所培養之結腸癌細胞具有高細胞毒性且在活體内腫瘤生長檢定中展示出抗 H9234.doc -99- 200813088 腫瘤活性。Chari等人，Cawar 52:127 31 (1992) 描述免疫共輕物’其中美登素類化合物經由二硫化物連接子與結合至人類結腸癌細胞株上之抗原的氣科抗體A 7丘軛，或與結合HER-2/neu致癌基因之另_鼠科單株抗體 ΤΑ· 1共輕。在活體外’於每個細胞表現3 X 1 個her_2表面抗原之人類乳癌細胞株SK-BR-3上測試ΤΑ·Κ美登素類化人物共軛物之細胞毒性。藥物共軛物達成與游離美登素類藥物類似之細胞毒性程度，該細胞毒性程度可藉由增加每個抗體分子美登素類化合物分子之數目西增加。A?·致总素類化合物共軛物在小鼠體内展示出低全身細胞毒性。可藉由在不顯著降低抗體或美登素類化合物分子之生物學活性的情況下將抗體與美登素類化合物分子化學連接來製備抗體-美登素類化合物共軛物。例如參看美國專利第 5,208,020號（其揭示内容係以引用之方式明確地併入本文中）。平均每個抗體分子共軛3_4個美登素類化合物分子已展示出增強目標細胞之細胞毒性之功效而對抗體功能或溶解性並無不利影響，但預期甚至丨分子毒素/抗體即可增強細胞毒性超過使用裸抗體。美登素類化合物已為此項技術中所热知，且可藉由已知技術合成或自天然來源分離。適田之美登素類化合物揭示於（例如）美國專利第5,2〇8,〇2〇號及上文提及之其他專利及非專利公開案中。較佳之美登素類化口物為美登醇及在美登素醇分子之芳環中或其他位置處經改質之美登醇類似物（諸如各種美登醇酯）。此項技術中已知多種用於製造抗體·美登素類化合物共 119234.doc -100- 200813088 輛物之連接基，包括（例如）以下文獻中所揭示之連接基··美國專利第5,208,020號或EP專利0 425 235 Bl; Chari等人， Omari?以wrc/z 52:127-131 (1992)及US2005/0169933A1，該等文獻之揭示内容係以引用之方式明確地併入本文中。可如 US2005/0169933 A1中所揭示來製備包含連接子組份SMCC 之抗體-美登素類化合物共軛物。連接基包括二硫基團、硫醚基團 '酸不穩定性基團、光不穩定性基團、肽酶不穩定性基團或酯酶不穩定性基團，如上文識別之專利中所揭示二硫基團及硫醚基團較佳。本文中描述及例示說明額外連接基。抗體與美登素類化合物之共軛物可使用多種雙功能蛋白偶聯劑製得，該等蛋白偶聯劑諸如3-(2-吡啶基二硫基）丙酸…琥珀醯亞胺酯（SPDP)、4-(N-馬來醯亞胺基甲基）環己烷-1-甲酸琥珀醯亞胺酯（SMCC)、亞胺基硫雑環戊烧 (IT)、醯亞胺酯之雙功能衍生物（諸如己二亞胺酸二甲醋鹽酸鹽）、活性酯（諸如辛二酸二琥珀醯亞胺酯）、醛類（諸如戊二醛）、雙_疊氮基化合物（諸如雙_(對疊氮基苯甲醯基）己二胺）、雙_重氮鹽衍生物（諸如雙_(對重氮鹽苯甲醯基）_乙二胺）、二異氰酸酯（諸如甲苯2,6-二異氰酸酯）及雙活性氟化合物（諸如1，5-二氟-2,4-二硝基苯）。特別較佳之偶聯劑包括提供二硫鍵之3-(2_吡啶基二硫基）丙酸N•琥珀醯亞胺酉旨（SPDP)(CarlSSOn等人，細仏隱人 173:723 37 (1978))及 4-(2-吼咬基硫基）戊酸]SU琥珀醯亞胺酯（spp)。可視連接類型而定將連接子附著於美登素類化合物分子 119234.doc -101 - 200813088 之各種位置處。舉例而言，可藉由使用習知偶聯技術與羥基反應形成酯鍵。反應可發生於具有羥基之C-3位置、經羥甲基改質之C-14位置、經羥基改質之C-15位置及具有羥基之C-20位置處。在一較佳實施例中，鍵結形成於美登醇或美登醇類似物之C-3位置處。 ii.奥瑞他汀及海兔毒素在一些實施例中，免疫共軛物包含與海兔毒素或海兔毒素之肽類似物及衍生物奥瑞他汀共軛之本發明抗體（美國直别繁、坌S7R0S8R聽、。海备I去泠盔搜拙. ，一，•疇，·，一，一，·▼ ·*"，* w，一、j ,77'人I、，ι"ν 汀已展示干擾微管動力學、GTP水解及核及細胞*** (Woyke等人，」wi/m/crW. jgeWs 45(12):3580- 3584 (2〇01))且具有抗癌（US 5,663，149)及抗真菌活性 (Pettit 等人，Agents Chemother· 42:2961-2965 (1998))。可經由肽藥物部分之N(胺基）末端或C(羧基）末端使海兔毒素或奥瑞他汀藥物部分附著於抗體（WO 02/88172) 〇例示性奥瑞他汀實施例包括N末端連接之單甲基奥瑞他汀藥物部分DE 及 DF，其揭示於’’Monomethylvaline Compounds Capable of Conjugation to Ligands’’，US2005/0238649 中，該文獻之揭示内容以全文引用之方式明確地併入本文中。一般而言，肽基藥物部分可藉由在兩個或兩個以上胺基酸及/或肽片段之間形成肽鍵而製備。此等肽鍵可（例如）根據肽化學領域中熟知之液相合成方法（參看E. Schr6der及K. Ltibke，，，The Peptides，，，第 1 卷，第 76-136 頁，1965, 119234.doc -102- 200813088C/7em· 13:786-91 (2002)), maytansinoids (EP 1391213; Liu et al., corpse dead. Scz·93: 8618-8623 (1996)) and calicheamicin (Lode et al. , Cancer 58: 2928 119234.doc -95- 200813088 (1998); Hinman et al, Cawcer 53: 3336-3342 (1993)) Scorpion toxin can be inhibited by including tubulin binding, DNA binding or topoisomerase The mechanism achieves its cytotoxicity and cell growth inhibition. Certain cytotoxic drugs tend to be inactive or less active when conjugated to large antibody or protein receptor ligands. ZEVALINTM (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope co-host that is bound by a thiourea linker-chelating agent for normal and malignant B lymphatic runs. Surface composition of the CD20 antigen of murine IgG1K monoclonal antibody 舆mIn or 9GY radioisotope (Wiseman et al., V. Wour. Jour. Nucl. Med. 27(7): 766-77 (2000); Wiseman et al., 99 (12): 4336-42 (2002); Witzig et al., // C7M. Ομο/· 20(10):2453-63 (2002); Witzig et al., J. C7M. Omo/· 20(15) :3262-69 (2002)). Although ZEVALIN is active against B-cell Hodgkin's Lymphoma (NHL), administration causes severe and long-term blood loss in most patients. MYLOTARGTM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals) is an antibody drug conjugate consisting of hu CD33 antibody linked to echinomycin, which was approved for injection in 2000 to treat acute myeloid leukemia ( jDrwgs 〇/ Fz/iwre 25(7): 686 (2000); US Patent Nos. 4970198, 5079233, 5558089, 5606060, 5693762, 5739116, 5767285, 5773001) . Cantuzumab mertansine (Immunogen, Inc.) is an antibody drug co-complex consisting of huC242 antibody linked to the maytansinoids unit 119234.doc-96-200813088 by DM1 via a disulfide linker SPP. Phase II trials for the treatment of cancers that exhibit CanAg, such as colon, pancreas, stomach, and other cancers. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.) is an antibody drug conjugate consisting of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid moiety DM1. The potential treatment of prostate tumors is under investigation. Auristatin peptide, auristatin E (AE) and monomethyl auristatin (MMAE) (synthetic analog of dolastatin) and chimeric strain antibody cBR96 (Lewis Y for cancer) Specific) and cACIO (specific for CD30 for hematological malignancies) (Doronina et al, 21 (7): 778-784 (2003)) and are undergoing therapeutic studies. Chemotherapeutic agents that can be used to generate immunoconjugates are described herein (examples above). The enzyme-active toxins and fragments thereof can be used, including the diphtheria toxin A chain, the non-binding active fragment of diphtheria toxin, the exotoxin A chain (from Pseudomonas aeruginosa), the ricin A chain, the abrin toxin A chain, and the lotus root toxic protein. (modeccin) A chain, α-sarcin, Aleurites fordii protein, dianthin protein, azeawa protein (ΡΑΡΙ, ΡΑΡΙΙ and PAP-S), bitter gourd c/ Zar(10)ίζ·α) inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogen (mitogellin), constricted sputum (restrictocin) ), phenomycin, enomycin and trichothecene. See, for example, WO 93/H232, published October 28, 1993, 119, 234, doc-97-200813088. A variety of radionuclides can be used to make radioconjugated antibodies. Examples include 212Bi, 131I, 131ln, 9GY, and 186Re. The conjugate of the antibody and the cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-σ-pyridyldithio)propionic acid N-succinimide. (SPDP), iminothiolane cyclopentane (ΙΤ), bifunctional derivative of quinone imide (such as dimethyl dimethyl imidate hydrochloride), active ester (such as suberic acid amber bismuth) Imines), acids (such as glutaric acid), bis-azido compounds (such as bis-(p-azidophenyl) hexamethylenediamine), bis-diazonium derivatives (such as bis-( a diazonium salt of a carbamide)-ethylenediamine), a diisocyanate (such as guanidine 2,6-iso-iso citrate) and a double active fluorine compound (such as 1,5-difluoro-2,4·2 Nitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., 238: 1098 (1987). Carbon 14-labeled 1-isothioxanylmethyl-3-methyldiethylenetriaminepentaacetic acid (mx_dtpa) is an exemplary f-mixer for conjugating radionucleotides to antibodies. See WO 94/11026. Also encompassed herein are antibodies and one or more small molecule toxins (such as echinomycin, maytansinoids, dolastatin, auristatin, trichothecene, and CC1065, and derivatives of toxins of such toxins) The common object. i. Maytansine and Maytansinoid Compounds In some embodiments, an immunoconjugate comprises an antibody (full length or fragment) of the invention in combination with one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansin was firstly isolated from the East African shrub tooth meiden wood 119234.doc -98 - 200813088 (10)s πααία) separation (US Patent No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinoids such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,151, No. 42). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268. Nos. 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663 No. 4,371,533. The maytansin moiety is the most interesting part of the drug drug family because it is: (1) relatively easy to prepare by fermentation or chemical modification, derivatization of acid fermentation products; (ii) Easily adapted to be derived from functional groups conjugated to the antibody via a non-disulfide linker; (iii) stable in plasma; and (iv) effective against a variety of tumor cell lines. An immunoconjugate comprising a maytansinoid compound, a process for its manufacture, and a therapeutic use thereof are disclosed, for example, in U.S. Patent Nos. 5,208, 2, 5, 416, 〇 64, and European Patent EP 0 425 235 B1. The disclosures of these patents are expressly incorporated herein by reference. Liu et al., et al., Ad. 5W. [/93:8618-23 (1996) describes immunoconjugation of a maytansinoid compound (designated DM1) comprising a monoclonal antibody C242 linked to human colorectal cancer. Things. This conjugate has been found to be highly cytotoxic to cultured colon cancer cells and exhibits anti-H9234.doc-99-200813088 tumor activity in an in vivo tumor growth assay. Chari et al., Cawar 52: 127 31 (1992) describe immuno-co-lights, in which the maytansinoids pass through a disulfide linker and a gas-based antibody A 7 yoke that binds to an antigen on a human colon cancer cell line, Or lightly combined with another mouse monoclonal antibody ΤΑ·1 that binds to the HER-2/neu oncogene. The cytotoxicity of the conjugated human conjugates was tested on human breast cancer cell line SK-BR-3, which showed 3 X 1 of the her 2 surface antigen per cell. The drug conjugate achieves a degree of cytotoxicity similar to that of the free maytansinoid, which can be increased by increasing the number of molecules of each of the antibody molecule maytansinoids. A?·Toxin-like compound conjugates exhibit low systemic cytotoxicity in mice. The antibody-maytansinoid conjugate can be prepared by chemically linking the antibody to the maytansinoid molecule without significantly reducing the biological activity of the antibody or maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020, the disclosure of which is expressly incorporated herein by reference. On average, conjugated 3 to 4 maytansinoid molecules per antibody molecule have been shown to enhance the cytotoxicity of target cells without adversely affecting antibody function or solubility, but it is expected that even sputum molecular toxins/antibodies can enhance cells. Toxicity exceeds the use of naked antibodies. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Phytosanitary compounds of the genus are disclosed in, for example, U.S. Patent No. 5,2,8, 〇 2 及 and other patents and non- patent publications mentioned above. A preferred meta-salt is a maytansinol and a modified meta-alcohol analog (such as various maytansinol) in the aromatic ring of the maytansinol molecule or elsewhere. A variety of linkers for the manufacture of antibody-maytansin compounds 119234.doc-100-200813088 are known in the art, including, for example, the linkers disclosed in the following documents: U.S. Patent No. 5,208,020 Or EP Patent 0 425 235 Bl; Chari et al., Omari®, Wr/z 52: 127-131 (1992) and US 2005/0169933 A1, the disclosures of each of which are expressly incorporated herein by reference. An antibody-maytansinoid conjugate comprising a linker component SMCC can be prepared as disclosed in US 2005/0169933 A1. The linker includes a disulfide group, a thioether group 'acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group, as described in the above identified patents. It is preferred to disclose disulfide groups and thioether groups. Additional linkers are described and exemplified herein. The conjugate of the antibody and the maytansinoid compound can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propionic acid...amber imidate ( SPDP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid amber succinimide (SMCC), imidothioindole cyclopentane (IT), quinone Bifunctional derivatives (such as dimethyl imidate hydrochloride), active esters (such as disuccinimide suberate), aldehydes (such as glutaraldehyde), bis-azido compounds ( Such as bis(p-azidobenzylidene) hexamethylenediamine), bis-diazonium salt derivatives (such as bis-(p-diazonium benzylidene)-ethylenediamine), diisocyanate (such as toluene) 2,6-diisocyanate) and a double active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include 3-(2-pyridyldithio)propionic acid N•succinimide (SPDP) which provides a disulfide bond (CarlSSOn et al., 仏隐 173: 723 37 ( 1978)) and 4-(2-carbylthio)pentanoic acid] SU amber imidate (spp). The linker is attached to various positions of the maytansinoid molecule 119234.doc-101 - 200813088 depending on the type of visual connection. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at a C-3 position having a hydroxyl group, a C-14 position modified by a hydroxymethyl group, a C-15 position modified by a hydroxy group, and a C-20 position having a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of the maytansinol or maytansinol analog. Ii. Auristatin and Dolastatin In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to a peptide analog of a dolastatin or a rabbit toxin and a derivative of auristatin (American坌S7R0S8R listen,. Haibei I go to the helmet search., one, • domain, ·, one, one, ·▼ ·*", * w, one, j, 77' person I, , ι" ν 汀Interfering microtubule dynamics, GTP hydrolysis, and nuclear and cell division have been demonstrated (Woyke et al., "wi/m/crW. jgeWs 45(12): 3580-3584 (2〇01)) and have anticancer (US 5,663, 149) and antifungal activity (Pettit et al, Agents Chemother 42: 2961-2965 (1998)). Canine toxin or auristatin can be obtained via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety Drug moiety attached to antibody (WO 02/88172) 〇 An exemplary auristatin example includes an N-terminally linked monomethyl auristatin drug moiety DE and DF, which is disclosed in ''Monomethylvaline Compounds Capable of Conjugation to Ligands'' The disclosure of this document is expressly incorporated herein by reference in its entirety. In contrast, a peptidyl drug moiety can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can, for example, be based on liquid phases well known in the art of peptide chemistry. Synthetic methods (see E. Schr6der and K. Ltibke, , The Peptides,,, Vol. 1, pp. 76-136, 1965, 119234.doc -102- 200813088

Academic Press)製備。奥瑞他汀/海兔毒素藥物部分可根據以下文獻之方法製備：US 5,635,483、US 5,780,588，Pettit 等人，dw. C/zem.夕％· 1 11:5463-65 (1989); Pettit等人， Anti-Cancer 13:243-77 (1998); Pettit 等人，办719-25 (1996)及 Pettit 等人，J· C/zem· iSoc. 7>a則· 1 5:859-863 (1996) 〇亦參看Doronina，TVaiwre 21(7):778-84 (2003); "Monomethylvaline Compounds Capable of Conjugation to Ligands”，US2005/0238649，其以其全文引用之方式併入本文中（揭示（例如）連接子及製備與連接子共軛之諸如MMAE及MMAF之單甲基纈胺酸化合物的方法）。 iii.刺胞黴素在其他實施例中，免疫共輛物包含與一或多個刺胞黴素分子共軛之本發明抗體。刺胞黴素家族之抗生素能夠在亞皮莫耳濃度下產生雙鏈DNA斷裂。有關刺胞黴素家族共軛物之製備，參看美國專利第5,712,374號、第5,714,586號、第 5,739，116 號、第 5,767,285 號、第 5,770,701 號、第 5,770,710 號、第 5,773,001 號及第 5,877,296 號（均頒予 American Cyanamid Company)。可使用之刺胞黴素之結構類似物包括（但不限於）γΐΐ、α2Ι、（x3I、N-乙醯基-γΐΐ、 PSAG 及 ΘΙ1 (Hinman 等人，Cancer 53:3336-42 (1993); Lode等人，Career 58:2925-28 (1998)及上文所提及之頒予American Cyanamid之美國專利）。可與抗體共軛之另一抗腫瘤藥物為QFA，其為抗葉酸劑。刺胞黴素與QFA皆具有細胞内作用位點且不易於跨過質膜。因 119234.doc -103 - 200813088 此，細胞經由抗體介導之内化作用攝取此等藥劑顯著增強其細胞毒性作用。 iy、其他細胞毒性劑可與本發明抗體共軛之其他抗腫瘤劑包括BCNU、鏈脲佐菌素（streptozoticin)、長春新鹼及5_氟尿嘧啶，美國專利第5,053,394號、第5,770,710號中所述之共同稱為LL- E33288複合物的藥劑家族以及伊斯帕黴素（美國專利第 5,877,296號）。Academic Press) Preparation. The auristatin/conine toxin drug moiety can be prepared according to the following literature: US 5,635,483, US 5,780,588, Pettit et al, dw. C/zem. eve % 1 11:5463-65 (1989); Pettit et al, Anti-Cancer 13: 243-77 (1998); Pettit et al., 719-25 (1996) and Pettit et al., J. C/zem·iSoc. 7>a, 1 5: 859-863 (1996) 〇 See also Doronina, TVaiwre 21(7): 778-84 (2003); "Monomethylvaline Compounds Capable of Conjugation to Ligands", US 2005/0238649, which is incorporated herein by reference in its entirety herein in And methods for preparing a monomethylproline compound such as MMAE and MMAF conjugated to a linker. iii. Citricin In other embodiments, the immunocomplex comprises one or more bacillus An antibody of the invention conjugated to a molecule. The antibiotic of the echinomycin family is capable of producing double-stranded DNA breaks at a concentration of the sub-picmole. For the preparation of a conjugate of the echinomycin family, see U.S. Patent No. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5, 770,710, 5,773,001 and 5,877,296 (both awarded to American Cyanamid Company). Structural analogs of echinomycin that can be used include, but are not limited to, γΐΐ, α2Ι, (x3I, N-ethylidene-γΐΐ) , PSAG and ΘΙ1 (Hinman et al, Cancer 53: 3336-42 (1993); Lode et al, Career 58: 2925-28 (1998) and the above-mentioned US patent granted to American Cyanamid). Another antitumor drug conjugated by the antibody is QFA, which is an antifolate. Both echinomycin and QFA have an intracellular site of action and are not easily translocated across the plasma membrane. 119234.doc -103 - 200813088 Uptake of these agents via antibody-mediated internalization significantly enhances their cytotoxic effects. iy, other cytotoxic agents Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoticin, Changchun New bases and 5-fluorouracils, a family of agents commonly referred to as LL-E33288 complexes as described in U.S. Patent Nos. 5,053,394 and 5,770,710, and ispamycin (U.S. Patent No. 5,877,296).

可使用之蜂活性毒素及其片段包括白喉毒素A鏈、白喉毒素之非結合活性片段、外毒素A鏈（來自綠膿桿菌）、蓖麻毒素A鏈、相思子毒素人鏈、蒴蓮根毒蛋白人鏈、帚麴菌素、桐油蛋白、康乃馨蛋白、洋商陸蛋白（ΡΑρι、ρΑριι& PAP-S) '苦瓜抑制劑、麻楓樹蛋白、巴豆毒素、肥皂草抑制劑、天堂果蛋白、有絲***素、侷限麴菌素、酚黴素、伊諾黴素及黴菌毒素。例如參看丨993年丨〇月28日公開之 WO 93/21232 〇本發明另外涵蓋抗體與具有核分解 '口丨工< 1C* _〜π切（例，核糖核酸酶或DNA内切酶，諸如去氧核糖核酸酶；酶）之間所形成的免疫共軛物。 ’ 為選擇性破壞腫瘤’抗體可包含高度放射性原子。用多種放射性同位素產生放射性共輛抗： A' R〜〜ml53Bee active toxins and fragments thereof may include diphtheria toxin A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, acacia toxin human chain, and lotus root toxic protein Human chain, bacteriocin, tung oil protein, carnation protein, foreign commercial protein (ΡΑρι, ρΑριι& PAP-S) 'Bitter melon inhibitor, jatropha, croton toxin, saponin inhibitor, paradise fruit protein, mitosis , limited to bacteriocin, phenolic acid, INOmycin and mycotoxins. See, for example, WO 93/21232, published on May 28, 993. The present invention additionally encompasses antibodies with nuclear breakdown & sputum < 1C* _~π cleavage (eg, ribonuclease or endonuclease, An immunoconjugate formed between such as a ribonuclease; an enzyme). Antibodies that selectively destroy tumors can contain highly radioactive atoms. Radioactive co-arming with a variety of radioisotopes: A' R~~ml53

Pb⑴及Lu之放射性同位素。當使用共㈣進行可句会用协戸弓時’，閃爍攝衫研究之放射性原子，例如tc”m: 119234.doc 200813088 i ;或用於核磁共振⑺難）成像（亦稱為磁共振成像， md)之自旋標記，諸如碘_123及碘_131、銦-iu、氣_19、碳-13、氮-15、氧-17、釓、錳或鐵。可以已知方式將放射性標記或其他標記併入共軛物中。舉例而言，肽可生物合成，或可藉由使用適當胺基酸前驅物進行化學胺基酸合成（例如涉及以氟_丨9胺基酸替代氫）來合成。可經由肽中之半胱胺酸殘基附著諸如一^或I!23、 Re186、Re188及In111之標記。可經由離胺酸殘基附著釔_ 9〇。可使用TODOGEN方法（Fraker等人，心如所价叩㈣ C謂 m 亂 80: 49_57 (1978))併入碘 _123。”MonoclonalRadioisotopes of Pb(1) and Lu. When using a total of (four) for a sentence can be used with a bow, 'flashing the radioactive atom of the study, such as tc" m: 119234.doc 200813088 i; or for nuclear magnetic resonance (7) difficult imaging (also known as magnetic resonance imaging, Spin labeling of md), such as iodine-123 and iodine-131, indium-iu, gas-19, carbon-13, nitrogen-15, oxygen-17, cesium, manganese or iron. Radioactive labeling or Other labels are incorporated into the conjugate. For example, the peptide can be biosynthesized, or chemical amino acid synthesis can be performed by using an appropriate amino acid precursor (eg, involving the replacement of hydrogen with a fluorine-丨9 amino acid). Synthesis. Labels such as I or I!23, Re186, Re188, and In111 may be attached via a cysteine residue in the peptide. The 钇_9〇 may be attached via an amine acid residue. The TODOGEN method may be used (Fraker et al. People, the heart is as good as the price (four) C said m chaos 80: 49_57 (1978)) incorporated iodine _123." Monoclonal

Antibodies in Immunoscintigraphy”（Chatal，CRC Press 1989)詳細描述其他方法。抗體與細胞毒性劑之共軛物可使用多種雙功能蛋白偶聯劑製付亥專蛋白偶聯劑諸如3-(2-吼唆基二硫基）丙酸琥轴醯亞胺酯（SPDP)、4-(N-馬來醯亞胺基甲基）環己烷-；[_ 甲酸琥拍醯亞胺酯（SMCC)、亞胺基硫雑環戊烷（it)、醯亞胺酯之雙功能衍生物（諸如己二亞胺酸二甲酯鹽酸鹽）、活性酯（諸如辛二酸二琥珀醯亞胺酯）、醛類（諸如戊二醛）、雙-疊氮基化合物（諸如雙-(對疊氮基苯甲醯基）己二胺）、雙-重氮鹽衍生物（諸如雙-(對重氮鹽苯甲醯基）-乙二胺）、二異氰酸酯（諸如甲苯2,6-二異氰酸酯）及雙活性氟化合物 (諸如1，5-—氟-2,4-二硝基苯）。舉例而言，萬麻毒素免疫毒素可如Vitetta等人，238:1098 (1987)中所述來製備。碳14標記之1-異硫氰氧基苯甲基_3_甲基二伸乙三胺 119234.doc •105- 200813088 五乙酸（ΜΧ-DTPA)為一種用於使放射性核苷酸與抗體共軛之例示性螯合劑。參看WO 94/11026。連接子可為促進細胞毒性藥物在細胞中釋放之’’可裂解連接子’’。舉例而言，可使用酸不穩定性連接子、肽酶敏感性連接子、光不穩定性連接子、二甲基連接子或含二硫鍵連接子（Chari等人， C ⑽ 52:127-31 (1992);美國專利第 5,208,020 號）。本發明之化合物特別涵蓋（但不限於）用如下交聯劑試劑製備之 ADC : BMPS、EMCS、GMBS、HBVS、LC-SMCC、MBS、ΜΡΒΗ、SBAP、SIA、SIAB、SMCC、 SMPB、SMPH、磺酸基-EMCS、磺酸基-GMBS、磺酸基-KMUS、磺酸基-MBS、磺酸基-SIAB、磺酸基-SMCC及磺酸基-SMPB及SVSB ((4-乙烯基砜）苯甲酸琥珀醯亞胺酯），該等交聯劑試劑為可購得的（例如購自Pierce Biotechnology，Inc.，Rockford，IL·，U.S.A)。參看 2003· 2004 Applications Handbook and Catalog，第 467-498 頁。 v.抗體藥物共辄物之製備在本發明之抗體藥物共輛物（ADC)中，抗體（Ab)經由連接子（L)與一或多個藥物部分（D)共軛，例如每個抗體約1至約20個藥物部分。可使用熟習此項技術者已知之有機化學反應、條件及試劑，藉由數種途徑來製備式I之ADC，該等途徑包括：（1)使抗體之親核基團與二價連接子試劑反應以經由共價鍵形成Ab-L，隨後與藥物部分D反應；及（2)使藥物部分之親核基團與二價連接子試劑反應以經由共價鍵 119234.doc -106- 200813088 形成D-L，隨後與抗體之親核基團反應。本文描述用於製備ADC之額外方法。Other methods are described in detail in Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989). Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-indole). Acryl iodide propionate (SPDP), 4-(N-maleimidomethyl)cyclohexane-; [_ succinimide (SMCC), sub a bifunctional derivative of anthracene sulfonium cyclopentane (it), sulfilimine (such as dimethyl dimethyl imidate hydrochloride), an active ester (such as diamyl succinimide), An aldehyde (such as glutaraldehyde), a bis-azido compound (such as bis-(p-azidobenzylidene) hexamethylenediamine), a bis-diazonium salt derivative (such as a bis-(p-diazonium salt) Benzomethylene)-ethylenediamine), diisocyanate (such as toluene 2,6-diisocyanate) and bis-active fluorine compound (such as 1,5-fluoro-2,4-dinitrobenzene). The cannabinoid immunotoxin can be prepared as described in Vitetta et al., 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-3-methyldiethylenetriamine 119234. Doc 105- 200813088 Pentaacetic acid (ΜΧ-DTPA) is an exemplary chelating agent for conjugated radionucleotides to antibodies. See WO 94/11026. Linkers can be used to promote the release of cytotoxic drugs in cells. A cleavable linker'. For example, an acid labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker can be used (Chari et al. , C (10) 52: 127-31 (1992); U.S. Patent No. 5,208,020. The compounds of the present invention specifically encompass, but are not limited to, ADCs prepared using the following crosslinker reagents: BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, ΜΡΒΗ, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfonate-EMCS, sulfonate-GMBS, sulfonate-KMUS, sulfonate-MBS, sulfonate-SIAB, sulfonic acid Base-SMCC and sulfonate-SMPB and SVSB ((4-vinylsulfone)benzoic acid amber ylide), such crosslinker reagents are commercially available (eg, available from Pierce Biotechnology, Inc., Rockford) , IL·, USA). See 2003. 2004 Applications Handbook and Catalog, pages 467-498 v. Preparation of antibody drug conjugates In the antibody drug complex (ADC) of the present invention, the antibody (Ab) is conjugated to one or more drug moieties (D) via a linker (L), for example, each antibody About 1 to about 20 drug parts. The ADC of Formula I can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) nucleophilic groups of antibodies and divalent linker reagents Reaction to form Ab-L via a covalent bond, followed by reaction with drug moiety D; and (2) reacting a nucleophilic group of the drug moiety with a divalent linker reagent to form via a covalent bond 119234.doc-106-200813088 DL, followed by reaction with the nucleophilic group of the antibody. This article describes additional methods for preparing an ADC.

Ab(LD)p I 連接子可由一或多種連接子組份構成。例示性連接子組份包括6-馬來醯亞胺基己醯基、馬來醯亞胺基丙醯基（MP )、顯胺酸-瓜胺酸（"vai-cit”）、丙胺酸-***酸 (nala-phe”）、對胺基苯甲氧基羰基（，，PaB”）、4_(2_吡啶基硫基）戊酸N·琥珀醯亞胺酯（”SPP”）、4_(斗馬來醯亞胺基甲基）環己烷-1-甲酸N·琥珀醯亞胺酯（"SMCC，，）及（4-碘-乙醯基）胺基苯甲酸N-琥珀醯亞胺酯（"SIAB")。額外之連接子組份已為此項技術中所知且某些描述於本文中。亦參看 ,fMonomethylvaline Compounds Capable of Conjugation to Ligands”，US2005/0238649，其内容以全文引用之方式併入本文中。在一些實施例中，連接子可包含胺基酸殘基。例示性胺基酸連接子組份包括二肽、三肽、四肽或五肽。例示性二肽包括顯胺酸_瓜胺酸（vc或val-cit)、丙胺酸-***酸（af 或ala-phe)。例示性三肽包括甘胺酸纈胺酸-瓜胺酸（gly_ val-cit)及甘胺酸-甘胺酸-甘胺酸（giy_giy_giy)。包含胺基酉文連接子組份之胺基酸殘基包括天然存在之胺基酸以及次要胺基酸及非天然存在之胺基酸類似物，諸如瓜胺酸。可對胺基酸連接子組份進行設計且優化其對特定酵素（例如腫瘤相關蛋白酶、組織蛋白酶B、C及D或纖溶蛋白酶）之 119234.doc -107- 200813088 酶促裂解之選擇性。抗體上之親核基團包括（但不限於）：（i)N•末端胺基；（π) 側鏈胺基，例如離胺酸；（in)側鏈硫醇基，例如半胱胺酸；及（IV)糖羥基或胺基，其中抗體經糖基化。胺基、硫醇基及羥基為親核基團且能夠與連接子部分及連接子試劑上之親電子基團反應形成共價鍵，該等親電子體包括：⑴ 活性酯，諸如NHS酯、HOBt酯、鹵代曱酸酯及酸鹵化物； (11)烷基及苯甲基鹵化物，諸如鹵代乙醯胺；（iii)醛、酮、 Γ% 、 ’ 羧基及馬來醯亞胺基。某些抗體具有可還原之鏈問二硫鍵’亦即半胱胺酸橋。可藉由用諸如DTT(二硫蘇糖醇）之還原劑進行處理而使抗體具有與連接子試劑共軛之反應性。由此’理論上各半胱胺酸橋將形成兩個反應性硫醇親核體。可經由離胺酸與2-亞胺基硫雑環戊烷（Traut試劑）之反應使胺轉化為硫醇而將額外親核基團引入抗體中。可藉由引入一個、兩個、三個、四個或四個以上半胱胺酸殘基 ◎(例如製備包含一或多個非天然半胱胺酸胺基酸殘基之突變體抗體）而將反應性硫醇基引入抗體（或其片段）中。本發明之抗體藥物共軛物亦可藉由修飾抗體以引入可與連接子試劑或藥物上之親核取代基反應之親電子部分而製得。可將糖基化抗體之糖以（例如）過碘酸鹽氧化試劑氧化以形成可與連接子試劑或藥物部分之胺基反應的醛或酮基。所得亞胺希夫鹼（Schiff base)基團可形成穩定鍵，或可經（例如）硼氫化物試劑還原而形成穩定胺鍵。在一實施例中，糖基化抗體之醣部分與半乳糖氧化酶或偏過碘酸鈉 119234.doc •108· 200813088 之反應可於蛋白質中產生可與藥物上之適當基團反應的羰基（酸及酮基團）（Hermanson，Bioconjugate Techniques)。在另一實施例中，可使含有N-末端絲胺酸或蘇胺酸殘基之蛋白質與偏過碘酸鈉反應而導致產生替代第一胺基酸之醛 (Geoghegan及 Stroh， Bioconjugate Chem· 3:138-46， (1992) U.S· 5,362,852)。此醛可與藥物部分或連接子親核體反應。同樣，藥物部分上之親核基團包括（但不限於）：胺、硫醇、·基、醯肼、將、肼、硫半卡（thiosemicarbazone)、羧酸肼及芳基醯胼基團，其能夠與連接子部分及連接子試劑上之親電子基團反應形成共價鍵，該等親電子體包括： ⑴活性酯，諸如NHS酯、HOBt酯、鹵代甲酸酯及酸鹵化物；（11)烧基及苯甲基鹵化物，諸如鹵代乙醯胺；（丨丨丨）駿、酮、羧基及馬來醯亞胺基。或者，可（例如）藉由重組技術或肽合成製造包含抗體及細胞毒性劑之融合蛋白。DNA之長度可包含編碼共軛物之兩個部分的各別區域，該等區域可彼此相鄰或由編碼不破壞共輛物所需特性之連接子肽之區域分離。在另一實施例中，可使抗體與"受體”（諸如抗生蛋白鏈菌素）共輛以用於預靶向腫瘤，其中將抗體_受體共軛物投與患者，隨後使用清除劑自循環中移除未結合之共軛物，且接著投用與細胞毒性劑（例如放射性核苷酸）共軛之"配位體π (例如抗生物素蛋白）。 - 醫藥調配物藉由將具有所需純度之抗體與生理學上于工搔又之可選载 119234.doc -109- 200813088 劑、賦形劑或穩定劑混合（Remingt〇n: The Seienee and Practice of Pharmac}^ 20版（2〇〇〇))來製備呈水溶液凍乾或其他乾燥調配物形式之包含本發明抗體之治療調配物以供儲存。可接受之載劑、賦形劑或穩定劑在所使用之劑量及濃度下對接受者無毒性，且包括緩衝液，諸如磷酸鹽、才丁檬fee鹽、組胺酸及其他有機酸；抗氧化劑，包括抗壞血酸及甲硫胺酸；防腐劑（諸如十八烷基二甲基苯甲基氯化銨、氣化六羥季銨、氯化苯甲烴銨、苄索氣銨、苯酚、丁西-f或苯甲醇、對羥基苯曱酸烷酯（諸如對羥审醅审The Ab(LD)p I linker can be composed of one or more linker components. Exemplary linker components include 6-maleimido hexamethylene, maleimide propyl propyl (MP), leucine- citrulline ("vai-cit", alanine -nala-phe", p-aminobenzyloxycarbonyl (,,PaB"), 4-(2-pyridylthio)pentanoic acid N.succinimide ("SPP"), 4_ (Doraminide iminomethyl)cyclohexane-1-carboxylic acid N.succinimide ("SMCC,,) and (4-iodo-ethenyl)aminobenzoic acid N-amber Imidites ("SIAB"). Additional linker components are known in the art and some are described herein. See also, fMonomethylvaline Compounds Capable of Conjugation to Ligands", US 2005/0238649, the content of which This is incorporated herein by reference in its entirety. In some embodiments, the linker can comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides or pentapeptides. Exemplary dipeptides include leucine- citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine citrate-glycine (gly_val-cit) and glycine-glycine-glycine (giy_giy_giy). Amino acid residues comprising an amine-based linker component include naturally occurring amino acids as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. The amino acid linker component can be designed and optimized for the selectivity of enzymatic cleavage of specific enzymes (e.g., tumor associated proteases, cathepsins B, C and D or plasmin) 119234.doc -107-200813088. Nucleophilic groups on the antibody include, but are not limited to, (i) N• terminal amine groups; (π) side chain amine groups such as lysine; (in) side chain thiol groups such as cysteine And (IV) a saccharide hydroxyl or amine group in which the antibody is glycosylated. The amine group, the thiol group and the hydroxyl group are nucleophilic groups and are capable of reacting with the electrophilic group on the linker moiety and the linker reagent to form a covalent bond, the electrophiles comprising: (1) an active ester such as an NHS ester, HOBt ester, halogenated decanoate and acid halide; (11) alkyl and benzyl halide, such as halogenated acetamide; (iii) aldehyde, ketone, hydrazine, 'carboxyl and maleimide base. Some antibodies have a reducible chain of disulfide bonds, i.e., a cysteine bridge. The antibody can be reacted with a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, in theory each cysteine bridge will form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody by conversion of the amine to the thiol by reaction of the amine acid with 2-iminothioindole cyclopentane (Traut reagent). By introducing one, two, three, four or more cysteine residues ◎ (for example, preparing a mutant antibody comprising one or more non-natural cysteine amino acid residues) A reactive thiol group is introduced into the antibody (or a fragment thereof). The antibody drug conjugate of the present invention can also be produced by modifying an antibody to introduce an electrophilic moiety reactive with a nucleophilic substituent on a linker reagent or drug. The sugar of the glycosylated antibody can be oxidized, for example, with a periodate oxidizing reagent to form an aldehyde or ketone group reactive with the linker reagent or the amine group of the drug moiety. The resulting imine Schiff base group can form a stable bond or can be reduced by, for example, a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of the sugar moiety of the glycosylated antibody with galactose oxidase or sodium metaperiodate 119234.doc •108·200813088 produces a carbonyl group in the protein that reacts with a suitable pharmaceutically acceptable group ( Acid and ketone groups) (Hermanson, Bioconjugate Techniques). In another embodiment, a protein containing an N-terminal serine acid or a threonine residue can be reacted with sodium metaperiodate to produce an aldehyde that replaces the first amino acid (Geoghegan and Stroh, Bioconjugate Chem. 3: 138-46, (1992) US 5,362,852). This aldehyde can be reacted with a drug moiety or a linker nucleophile. Likewise, nucleophilic groups on the drug moiety include, but are not limited to, amines, thiols, amides, hydrazines, hydrazines, thiosemicarbazones, bismuth carboxylates, and aryl hydrazine groups, It is capable of reacting with a linker moiety and an electrophilic group on a linker reagent to form a covalent bond, the electrophiles comprising: (1) an active ester such as an NHS ester, a HOBt ester, a haloformate, and an acid halide; (11) a base and a benzyl halide such as a halogenated acetamide; a hydrazine, a ketone, a carboxyl group and a maleimine group. Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be made, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise separate regions encoding the two portions of the conjugate which may be adjacent to each other or separated by a region encoding a linker peptide which does not destroy the desired properties of the consensus. In another embodiment, the antibody can be co-imported with a "receptor" (such as streptavidin) for pretargeting the tumor, wherein the antibody-receptor conjugate is administered to the patient, followed by clearance The agent removes the unbound conjugate from the circulation and then administers a "ligand π (eg, avidin) conjugated to a cytotoxic agent (eg, a radioactive nucleotide). - Pharmaceutical Formulation Lever Mixing an antibody of the desired purity with a physiologically acceptable additive, 119234.doc-109-200813088, excipient or stabilizer (Remingt〇n: The Seienee and Practice of Pharmac}^ 20 Edition (2)) to prepare a therapeutic formulation comprising an antibody of the invention in the form of an aqueous solution lyophilized or other dry formulation for storage. Acceptable carriers, excipients or stabilizers at the dosages employed And the concentration is not toxic to the recipient, and includes buffers such as phosphate, butyl gallate, histidine and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives such as octadecane Dimethylbenzyl ammonium chloride Gasification hexahydric quaternary ammonium, benzalkonium chloride, benzethonium ammonium gas, phenol, butyl or benzyl alcohol -f West, paraben Yue acid alkyl esters (such as p-hydroxyphenyl grains Unexamined Laid

τ I I 或對羥基苯甲酸丙酯）、兒茶酚、間苯二酚、環己醇、弘戊醇及間甲酚低分子量（小於約1〇個殘基）多肽；蛋白質，諸如血清白蛋白、明膠或免疫球蛋白；親水性聚合物，諸如聚乙烯吼洛咬胺基酸，諸如甘胺酸、麵醯胺酸、天冬醯胺酸、組胺酸、精胺酸或離胺酸；單醣、雙醣及其他醣，包括葡萄糖、甘露糖或糊精；螯合劑，諸如edta ; 糖，諸如蔗糖、甘露糖醇、海藻糖或山梨糖醇；成鹽抗衡離子，諸如鈉；金屬錯合物（例如Zn-蛋白錯合物）；及/或非離子界面活性劑，諸如TWEENTM、pLlJR〇NICSTM或聚乙二醇（PEG)。本文之調配物亦可視需要含有一種以上用於所治療之特定指征的活性化合物，較佳為具有彼此無不利影響之互補 /舌性之活性化合物。此等分子適於以有效用於預定目的之 1存在於組合中。亦可將活性成份截留於膠狀藥物傳遞系統（例如脂質 119234.doc -110- 200813088 體、白蛋白微球體、微乳液、奈米顆粒及奈米膠囊）或巨乳液中（例如）藉由凝聚技術或界面聚合而製備之微膠囊（例如’刀別為經甲基纖維素或明膠_微膠囊及聚（甲基丙烯酸甲醋）微膠囊）中。此等技術揭示於Remington: The Science and Practice of Pharmacy 第 20版（2000)中。待用於活體内投藥之調配物必須無菌。此易於藉由經無菌濾膜過濾、來實現。τ II or propyl paraben), catechol, resorcinol, cyclohexanol, pentaerythritol and m-cresol low molecular weight (less than about 1 residue) polypeptide; protein, such as serum albumin , gelatin or immunoglobulin; a hydrophilic polymer such as polyvinyl guanidine amino acid, such as glycine, isphoric acid, aspartic acid, histidine, arginine or lysine; Monosaccharides, disaccharides and other sugars, including glucose, mannose or dextrin; chelating agents such as edta; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; Compounds (eg, Zn-protein complexes); and/or nonionic surfactants such as TWEENTM, pLlJR〇NICSTM or polyethylene glycol (PEG). The formulations herein may also optionally contain more than one active compound for the particular indication being treated, preferably a complementary/tongue active compound which has no adverse effects on each other. These molecules are suitable for being present in the combination in an effective manner for the intended purpose. The active ingredient may also be retained in a gelled drug delivery system (eg, lipid 119234.doc-110-200813088, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions (eg, by coacervation) Microcapsules prepared by technical or interfacial polymerization (eg, 'Knife is methylcellulose or gelatin_microcapsules and poly(methyl methacrylate) microcapsules). Such techniques are disclosed in Remington: The Science and Practice of Pharmacy 20th Edition (2000). Formulations to be administered in vivo must be sterile. This is easily accomplished by filtration through a sterile filter.

可製備持續釋放製劑。持續釋放製劑之適當實例包括含 ’>，a月免疫球蛋白之固體疏水性聚合物的半參透性基貝°亥專基質為成形物品之形式，例如薄膜或微膠囊。持績釋放基質之實例包括聚酯、水凝膠（例如聚（2_羥基乙基_ 甲基丙烯酸酯）或聚（乙烯醇））、聚乳酸交酯（美國專利第 3,773,919號）、L-麵胺酸與γ乙基_L_麩胺酸酯之共聚物、不降解乙烯-乙酸乙烯酯、可降解乳酸_乙醇酸共聚物（諸如 LUPR〇NDEPOTTM(由乳酸·乙醇酸共聚物及乙酸亮丙瑞林構成的可注射微球體））及聚•羥基丁酸。諸如乙烯_ 乙酸乙烯酯及乳酸-乙醇酸之聚合物使得分子能夠釋放超過100天’而某些水凝膠釋放蛋白f持續較短時間。當經封裝免疫球蛋白在體内保持一段較長時間時，其可因在 37°C下暴露至濕氣而變性或凝集，從而導致生物學活性之喪失及免疫原性之可能改變。可視所涉及之機制而定設計合理的穩定策略。舉例而言，若發現凝集機制為經由硫基-一硫化物互換形成分子問又刀于間S-S鍵，則可藉由修飾氫硫基殘基、由酸性溶㈣乾、_濕氣含量、使用適當添加劑 119234.doc -111. 200813088 及形成特定聚合物基質組合物來達成穩定。用途本發明之抗體可用於（例如）活體外、離體及活體内治療方法。在一些實施例中，本發明提供降低或抑制患有與血管生成相關之病理病況之受檢者體内血管生成的方法，其包含向該受檢者投與有效量之本發明之抗£(}1^7抗體。此等病況包括（例如）贅瘤（包括癌瘤）及某些眼病。可由本發明之治療改善之癌症包括（但不限於）癌瘤、淋巴瘤、母細胞瘤、肉瘤及白血病或淋巴惡性疾病。更特定言之，此等癌症之實例包括乳癌、結腸癌、直腸癌、結腸直腸癌、腎癌、肺癌（包括小細胞肺癌、非小細胞肺癌、肺腺癌及肺鱗狀癌）、鱗狀細胞癌（例如上皮鱗狀細胞癌）、子宮頸癌、卵巢癌、***癌'肝癌、膀胱癌、腹膜癌、肝細胞癌、胃癌（包括胃腸癌）、胰腺癌、頭頸部癌、神經膠母細胞瘤、視網膜母細胞瘤、星形細胞瘤、泡膜細胞瘤、卵巢男性細胞瘤、肝腫瘤、血液科惡性疾病（包括非霍奇金氏淋巴瘤（NHL)、多發性骨髓瘤及急性血液科吳性疾病）、子宮内膜癌或子宮癌、子宮内膜異位、纖維肉瘤、絨膜癌、唾液腺癌、外陰癌、甲狀腺癌、食管癌、肝癌、肛門癌、陰莖癌、鼻咽癌、喉癌、卡波希氏肉瘤 (Kaposi’s sarcoma)、黑素瘤、皮膚癌、神經鞘瘤、少突神經膠質瘤、神經母細胞瘤、橫紋肌肉瘤、骨源性肉瘤、平滑肌肉瘤、尿道癌、甲狀腺癌、威爾姆氏瘤 119234.doc •112- 200813088 tumor)以及與母斑病相關之異常血管增生、水腫（諸如與腦腫瘤相關之水腫）及米格氏症候群（Meigs’ syndrome)。癌症較么係選自由乳癌、結腸直腸癌、非小細胞肺癌、非霍奇金氏淋巴瘤（NHL)、腎癌、***癌、肝癌、頭頸部癌、黑素瘤、卵巢瘤、間皮瘤及多發性骨髓瘤組成之群。癌症更佳為結腸直腸癌。可由本發明之治療改善之癌病況包括轉移性癌症。本發明尤其適於治療血管形成腫瘤。、可由本發明之治療改善之眼病包括眼内新生血管疾病， f 包括（但不限於）年齡相關乞黃斑變性、糖尿病性黃斑水腫、增生性糖尿病性視網膜病變、視網膜中央靜脈阻塞併發黃斑囊樣水腫、視網膜分枝靜脈阻塞併發黃斑囊樣水腫、虹膜紅變、病理性近視/CNV、逢希伯·林道症候群 (Von Hippel Lindau Syndrome)、翼狀胬肉、p〇hs(組織漿菌病）/CNV、脈絡膜血管瘤、早產兒視網膜病（R〇p)、放射性視網膜病變、眼内腫瘤（例如黑素瘤、視網膜母細胞 ( 瘤、轉移瘤及眼眶海綿狀血管瘤）、息肉狀脈絡膜病變、特發性近中心凹型毛細管擴張、伊爾斯氏病（Eales， Disease)眼眶海綿狀血管瘤、眼眶***瘤、眼臉毛細血管瘤、角膜移植物血管形成、角膜移植物新血管生成、滲出性視網膜病（Coats Disease)及與青光眼手術相關之創傷癒合問題。此外，至少某些本發明之抗體可結合來自其他物種之抗原。因此，本發明之抗體可用於（例如）在含有抗原之細胞培養物中、人類受檢者體内或具有與本發明之抗體交叉反 119234.doc -113- 200813088 應之抗原的其他哺乳動物受檢者（例如黑猩猩、狒狒、織猴、獼猴及恆河猴、豬或小鼠）體内結合特異性抗原活性。在一些實施例中，可藉由使本發明之抗體與抗原接觸從而抑制抗原活性而將抗體用於抑制抗原活性。抗原較佳為人類蛋白分子。在一些實施例中，本發明之抗體可用於結合身受與抗原表現及/或活性增加相關之病症之受檢者體内之抗原的方法中，該方法包含向受檢者投與本發明之抗體從而與受檢者體内t &原結合。抗原較佳為人類蛋白分子且受檢者為人類受檢者。或者，受檢者可為表現與本發明抗體結合之抗原的哺乳動物。另外，受檢者可為已引入抗原（例如藉由投與抗原或藉由表現抗原轉殖基因）之哺乳動物。可將本發明之抗體投與人類受檢者用於治療目的。此外，可將本發明之&體投與表現與It免疫球蛋白交χ反應之抗原的非人類哺乳動物(例如靈長類動物、豬或小鼠)用於獸醫目的或將其作為人類疾病之動物模型。就後種情況而言，此等動物板$可用於評估本發明抗體之治療功效（例如測試投藥劑量及時程）。本發明之抗體可用於治療、抑制、延緩與一或多種抗原分子之表現及/或活性相關之疾病、病症或病況之進程、防止/延緩其復發、改善或預防該等錢、病症或病況。在某些實施例中，將包含與—或多種細胞毒性劑共輛之抗體的免疫共輛物投與患者。在-些實施例中，免疫丘輛物及/或與其結合之抗原經細胞内化，從而引起免疫：輛 119234.doc -114 - 200813088 物殺傷其所結合之目標細胞之治療功效增加。在一實施例中，細胞毒性劑靶向或干擾目標細胞中之核酸。在一實施例中，細胞秦性劑靶向或干擾微管聚合。此等細胞毒性劑之實例包括本文所述之任何化學治療劑（諸如美登素類化合物、奥瑞他汀、海兔毒素或刺胞黴素）、放射性同位素或核糖核酸酶或DNA核酸内切酶。 aSustained release formulations can be prepared. Suitable examples of sustained release formulations include those containing ', a semi-permeable matrix of a solid hydrophobic polymer of a month of immunoglobulin in the form of a shaped article, such as a film or microcapsule. Examples of performance release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl) methacrylate or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L- Copolymer of facial acid with γ-ethyl_L_glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (such as LUPR〇NDEPOTTM (lighted by lactic acid·glycolic acid copolymer and acetic acid) Injectable microspheres composed of cyprenine) and polyhydroxybutyrate. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable molecules to be released for more than 100 days' while certain hydrogels release protein f for a short period of time. When the encapsulated immunoglobulin is maintained in the body for a prolonged period of time, it can be denatured or agglomerated by exposure to moisture at 37 ° C, resulting in loss of biological activity and possible alteration of immunogenicity. A reasonable stability strategy can be designed depending on the mechanism involved. For example, if it is found that the agglutination mechanism is to form a molecule via a thio-monosulfide interchange, the sulfonate residue can be modified by an acidic solution (tetra), _molyb content, and use. Suitable additives 119234.doc -111. 200813088 and the formation of specific polymer matrix compositions to achieve stabilization. Uses The antibodies of the invention are useful, for example, in in vitro, ex vivo and in vivo therapeutic methods. In some embodiments, the invention provides a method of reducing or inhibiting angiogenesis in a subject having a pathological condition associated with angiogenesis, comprising administering to the subject an effective amount of the invention of the invention ( }1^7 antibodies. Such conditions include, for example, neoplasms (including cancers) and certain ocular diseases. Cancers that can be ameliorated by the treatment of the invention include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas And leukemia or lymphoid malignant diseases. More specifically, examples of such cancers include breast cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung). Squamous cell carcinoma, squamous cell carcinoma (eg, epithelial squamous cell carcinoma), cervical cancer, ovarian cancer, prostate cancer 'liver cancer, bladder cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, Head and neck cancer, glioblastoma, retinoblastoma, astrocytoma, vesicular cell tumor, ovarian male cell tumor, liver tumor, hematological malignancies (including non-Hodgkin's lymphoma (N HL), multiple myeloma and acute hematological disease), endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, liver cancer , anal cancer, penile cancer, nasopharyngeal carcinoma, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, schwannomas, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, bone Derived sarcoma, leiomyosarcoma, urethral cancer, thyroid cancer, Wilm's tumor 119234.doc •112- 200813088 tumor) and abnormal vascular hyperplasia, edema associated with maternal disease (such as edema associated with brain tumors) and rice Meigs' syndrome. Cancer is selected from breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian tumor, mesothelioma And a group of multiple myeloma. Cancer is better for colorectal cancer. Cancer conditions that can be ameliorated by the treatment of the present invention include metastatic cancer. The invention is particularly suitable for treating angioed tumors. Eye diseases which can be improved by the treatment of the present invention include intraocular neovascular diseases, f including but not limited to age-related macular degeneration, diabetic macular edema, proliferative diabetic retinopathy, central retinal vein occlusion and macular cystic edema Retinal branch vein obstruction complicated with cystoid macular edema, iris reddenation, pathological myopia/CNV, Von Hippel Lindau Syndrome, pterygium, p〇hs (tissue bacteremia)/ CNV, choroidal hemangioma, retinopathy of prematurity (R〇p), radiation-induced retinopathy, intraocular tumors (eg melanoma, retinoblastoma (tumor, metastases and orbital cavernous hemangioma), polypoid choroidal lesions, Idiopathic near-concave telangiectasia, Eales disease, orbital cavernous hemangioma, orbital lymphangioma, ocular surface capillary hemangioma, corneal graft angiogenesis, corneal graft neovascularization, exudation Coats Disease and wound healing problems associated with glaucoma surgery. Furthermore, at least some of the present invention The antibody can bind to antigens from other species. Thus, the antibodies of the invention can be used, for example, in cell cultures containing antigen, in human subjects, or with antibodies against the invention 119234.doc-113 - 200813088 Other mammalian subjects (eg, chimpanzees, baboons, gems, macaques, rhesus monkeys, pigs or mice) that bind antigens bind to specific antigenic activity in vivo. In some embodiments, The antibody of the present invention is contacted with an antigen to inhibit antigen activity and the antibody is used to inhibit antigen activity. The antigen is preferably a human protein molecule. In some embodiments, the antibody of the present invention can be used for binding and antigen expression and/or activity increase. In the method of the antigen in the subject of the related condition, the method comprises administering to the subject an antibody of the present invention to bind to the original t & the antigen is preferably a human protein molecule and The examiner is a human subject. Alternatively, the subject may be a mammal exhibiting an antigen that binds to the antibody of the present invention. In addition, the subject may be an introduced antigen (for example, A mammal which is administered with an antigen or by expressing an antigen-transforming gene. The antibody of the present invention can be administered to a human subject for therapeutic purposes. Further, the & body administration and expression of the present invention can be immunized with It Non-human mammals (eg, primates, pigs, or mice) that are antigens of the globulin-crossing reaction are used for veterinary purposes or as animal models of human disease. In the latter case, such animal boards are $ It can be used to assess the therapeutic efficacy of the antibodies of the invention (e.g., to test the dosage of the agent). The antibodies of the invention can be used to treat, inhibit, delay the progression of a disease, disorder or condition associated with the performance and/or activity of one or more antigenic molecules. Prevent/slow recurrence, improve or prevent such money, illness or condition. In certain embodiments, an immunoconjugate comprising an antibody co-located with - or a plurality of cytotoxic agents is administered to a patient. In some embodiments, the immune hills and/or antigens associated therewith are internalized by the cells to cause immunity: the therapeutic efficacy of the 119234.doc-114 - 200813088 agent killing the target cells to which it binds is increased. In one embodiment, the cytotoxic agent targets or interferes with nucleic acids in the target cell. In one embodiment, the cellular agent targets or interferes with microtubule polymerization. Examples of such cytotoxic agents include any of the chemotherapeutic agents described herein (such as maytansinoids, auristatin, dolastatin or echinomycin), radioisotopes or ribonucleases or DNA endonucleases . a

本發明之抗體可單獨或與其他組合物組合用於治療。舉例而言’本發明之抗體可與化學治療劑（包括化學治療劑之混合液）、其他細胞毒性劑、抗抗原劑、細跑因子及/或生長抑制劑共投藥。上文所述之此等組合療法包括組合投藥（其中兩種或兩種以上藥劑包括於相同或單獨調配物中）及單獨投藥，在後種情況下投與本發明之抗體可在投與辅助療法之前及/或之後進行。本發明之抗體（及輔助治療劑）可藉由任何適當之方式投與’包括非經腸、皮下、腹膜内、肺内及鼻内，且視需要在病灶内投藥用於局部治療。非經腸輸注包括肌肉内、靜脈内、動脈内、腹膜内或皮下投藥。此外，抗體亦適於夢 =脈衝輸注投與，尤其以遞減劑量投與抗體。部分上視^ 藥之短期或長期性定，又、、主射，钱“ & J *由任何適當途徑(例如藉由 /射啫如靜脈内或皮下注射）給藥。本發明之抗體組合物可以與良配、給藥及投率。在bp土子醫療只踐相符之方式調糸在此方面需考慮之因辛定病症、所治療之特定哺乳㈣〃 h括H療之特病症之起因、傳㈣個體患者之臨床情況、傳遞樂劑之部位、投藥方法、投藥•及專 119234.doc -115· 200813088 業醫師已知之其他因素。抗體無需但視情況可與一或多種 f前用於預防或治療所述病症之藥劑—起調配。此等其他樂劑之有效量視存在於調配物中之本發明之抗體的量、病症或治療之類型及上文所討論之其他因素而定。此等藥劑 -般係以與上文所用相同之劑量及投藥途徑使用，或以上文所用劑量之約1%至99%之劑量使用。對於預防或治療疾病，本發明抗體之適當劑量（當單獨使用或與其他藥劑組合使用時）將視待治療之疾病類型、抗體類型、疾病之嚴重程度及病程、出於預防抑或治療目的投與抗體、先前療法、患者臨床病史及對抗體之反應以及主治醫師之判斷而定。抗體適於一次性或經一系列治療投與至患者。視疾病類型及嚴重程度而定，約1 4^0至15 mg/kg (例如〇」mg/kg_1〇 mg/kg)之抗體可為（例如）藉由一或多次單獨投藥或藉由連續輸注投與至患者之初始候選劑量。一種典型曰劑量可在約i叫/]^至100 mg/kg*更高劑里之範圍内’此視上文所提及之因素而定。對於經數天戍更長時間重複投藥而言，視病況而定，將持續進行治療直至出現所需疾病症狀抑制。抗體之一例示性劑量將在約 0·05 rng/kg至約10 mg/kg之範圍内。因此，可以約〇 5 mg/kg、2.0 mg/kg、4.0 mg/kg或 10 mg/kg (或其任何組合）中之一或多種劑量向患者投藥。此等劑量可間歇投與，例如每週或每三週（例如使患者接收約兩次劑量至約二十次劑量’例如約六次劑量之抗體）。可以較高之初始負荷劑篁’隨後以一或多次較低劑量投藥。例示性給藥方案包含 119234.doc -116- 200813088 投與約4 mg/kg之初始負荷劑量之抗體，隨後投與約2 mg/kg之每週維持劑量之抗體。然而，其他劑量方案亦可用。此療法之進展易於藉由習知技術及檢定監測。本發明之抗EGFL7抗體可用於偵測特定細胞或組織中 EGFL7之表現的檢定（諸如診斷或預後檢定）中，其中抗體如下文所述經標記且/或經固定於不可溶基質上。本發明提供偵測EGFL7之方法，該等方法包含偵測樣本中之EGFL7-抗_EGFL7抗體複合物。如本文所使用之術語”偵測”白.括泉老赤X來老掛昭橹況下之宗神及/戒宗晉俏沏I ί詈 -,、III， 1 / V ^ ,’，， « / ^ ”擎 « _· ，_ _ _ , ， ν _ ·, ，，、 - — 測含量）。本發明提供診斷與EGFL7表現及/或活性相關之病症的方法，該等方法包含偵測來自患有或懷疑患有該病症之患者的生物樣本中之EGFL7-抗-EGFL7抗體複合物。在一些實施例中，EGFL7表現為增加之表現或異常（不合需要）表現。本發明提供本文所述之任何抗EGFL7抗體，其中該抗 EGFL7抗體包含可偵測之標記。本發明提供一種本文所述之任何抗EGFL7抗體與EGFL7 之複合物。在一些實施例中，該複合物為活體内或活體外複合物。在一些實施例中，該複合物包含癌細胞。在一些實施例中，抗EGFL7抗體經可偵測地標記。可以多種熟知之偵測檢定方法中之任一種使用抗EGFL7 抗體偵測EGFL7。舉例而言，可藉由自所需來源獲得樣本，將樣本與抗EGFL7抗體混合以使抗體與混合物中存在 119234.doc -117- 200813088 之任何EGFL7形成抗體/EGFL7複合物，並偵測混合物中存在之任何抗體/EGFL7複合物來檢定生物樣本中之EGFL7。可藉由此項技術中已知之適於特定樣本之方法製備生物樣本用於檢定。根據所使用之檢定類型選擇將樣本與抗體混合之方法及偵測抗體/EGFL7複合物之方法。此等檢定包括免疫組織化學、競爭及夾層檢定及空間抑制檢定。用於EGFL7之分析方法均使用一或多種以下試劑：經標記之EGFL7類似物、經固定之EGFL7類似物、經標記之抗 EGFL7抗體、經固定冬抗EGFL7抗體及空間共軛物。經標記之試劑亦稱為’’示蹤劑"。所使用之標記為不干擾EGFL7與抗EGFL7抗體之結合的任何可偵測之官能基。已知多種用於免疫檢定之標記，實例包括可直接偵測之部分，諸如螢光染料、化學發光劑及放射性標記；以及必須經反應或衍生化而得以偵測之部分，諸如酵素。所使用之標記為不干擾EGFL7與抗EGFL7抗體之結合的任何可偵測之官能基。已知多種用於免疫檢定之標記，實例包括可直接偵測之部分，諸如螢光染料、化學發光劑及放射性標記；以及必須經反應或衍生化而得以偵測之部分，諸如酵素。此等標記之實例包括放射性同位素32p、 14C、1251、3H及1311 ;螢光團，諸如稀土螯合劑或螢光素及其衍生物、若丹明（rhodamine)及其衍生物、丹醯基、繳嗣；螢光素酶，例如螢火蟲螢光素酶及細菌螢光素酶（美國專利第4,737,456號）；蟲螢光素；2,3_二氫酞嗪二酮；辣 119234.doc -118- 200813088 根過氧化物酶（HRP);驗性碗酸酶；β -半乳糖苷酶；葡萄糖澱粉酶；溶菌酶；醣氧化酶，例如葡萄糖氧化酶、半乳糖氧化酶及葡萄糖-6-填酸脫氫酶；雜環氣化酶，諸如尿酸酶及黃嘌呤氧化酶，其與使用過氧化氫氣化染料前驅物之酵素（諸如HRP、乳過氧化物酶或微過氧化物酶）偶聯；生物素/抗生物素蛋白，自旋標記，嗤函體標記；穩定自由基及其類似物。可使用習知方法使此等標記與蛋白質或多肽共價結合。舉例而言，可使用偶聯劑將抗體與上述螢光、化學發光及酵素標記連接，該等偶聯劑諸如二醛、碳化二醯亞胺、二馬來醯亞胺、雙醢亞胺酯、雙重氮化聯笨胺及其類似物。例如參看美國專利第3,940,475號（螢光測定法）及第 3,645,090 號（酵素法）；Hunter 等人，144: 945 (1962); David等人，13: 1014-21 (1974); Pain 等人，J· /mww ⑽/· 40: 219-30 (1981)及 Nygren，J. and 30: 407-12 (1982)。本文中之較佳標記為諸如辣根過氧化物酶及鹼性磷酸酶之酵素。此標記（包括酵素）與抗體之共軛對於一般熟習免疫檢定技術者而言為標準操作程序。例如參看O’Sullivan等人，’’Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay,ff Methods Enzymol. ^、編輯 J. J. Langone 及 H. Van Vunakis，第 73 卷（Academic Press，New York， New York，1981)，第 147-166 頁。某些檢定方法中需要固定試劑。固定使抗EGFL7抗體與 119234.doc -119- 200813088 在溶液中保持游離之任何EGFL7分離。通常藉由在檢定程序之如藉由吸附至水不溶性基質或表面（Bennich等人， U-S· 3,720,760)，藉由共價偶聯（例如使用戊二醛交聯）使抗EGFL7抗體或EGFL7類似物不溶解化或藉由在檢定程序之後（例如）藉由免疫沉澱使抗EGFL7抗體或EGFL7類似物不溶解化來實現此目的。可使用免疫組織化學及染色實驗程序檢測樣本中蛋白質 _ 之表現。已顯示組織切片之免疫組織化學染色係一種評定或偵測樣本中蛋白質之存在的可靠方法。免疫組織化學 ("IHC”）技術一般藉由發色或螢光方法利用抗體原位探查並觀測細胞抗原。對於樣本之製備而言，可使用來自哺乳動物（通常為人類患者）之組織或細胞樣本。可藉由此項技術中已知之夕種私序獲得樣本，該等程序包括（但不限於）手術切除、抽吸或活組織檢查。組織可為新鮮或冷凍組織。在一實施例中，將樣本固定並嵌埋至石蠟或其類似物 t 中。可藉由習知方法固定（亦即保存）組織樣本。一般熟習 ^此項技術者應瞭解固定劑之選擇由待經組織學染色或另外分析樣本之目的確定。一般熟習此項技術者亦將瞭解固定長度視組織樣本之大小及所使用之固定劑而定。 IHC可與諸如形怨染色及/或原位螢光雜交之額外技術組口進行。存在兩種IHC之通用方法，即直接與間接檢定。根據第一檢定，直接測定抗體與目標抗原（例如egfl7)之結合。此直接檢定使用可在無進一步抗體相1作用之情況下觀測之經標記試劑，諸如螢光標籤或酵素標記之一級抗 119234.doc -120- 200813088 體。在典型間接檢定中’未共輛之一級抗體與抗原結合，且接者經彳示§己之二級抗體與^一級抗體結合。在二級抗體鱼酵素標記共軛之情況下，添加發色或螢光底物以便觀測抗原。由於數種二級抗體可與一級抗體上之不同抗原決定基反應，故而出現信號擴增。用於免疫組織化學之一級及/或二級抗體通常可經可偵測部分標記。可使用多種標記，其在文中的其他部份描述。除上文所討論之樣本製備程序外，可需要在IHC之前、期間或之後進一步處理組織切片。舉例而言，可進行抗原決定基修復方法，諸如檸檬酸鹽緩衝液中加熱組織樣本 (例如參看Leong等人却〆4(3):201 (1996)) 〇在可選阻斷步驟後，在適當條件下使組織切片暴露至一級抗體歷時一段足夠之時間段，從而使一級抗體與組織樣本中之目標蛋白抗原結合。用於達成此目的之適當條件可藉由常規實驗確定。藉由使用上文所討論之可偵測標記中之任一種測定抗體與樣本之結合程度。標記較佳為催化發色底物（諸如3,3’-二胺基聯苯胺色原體）化學變化之酶標記 (例如HPRQ)。較佳使酶標記與特異性結合至—級抗體之抗體共軛（例如一級抗體為兔多株抗體且二級抗體為山羊抗兔抗體）。可女裝由此製備之試樣並以蓋玻片覆蓋。接著（例如）使用顯微鏡測《載片評估，且可使用此項技術常規使用之染 119234.doc -121. 200813088 色強度標準。稱為競爭或夾層檢定之其他檢定方法已良好建立且廣泛用於商業診斷產業中。競爭檢定係基於示蹤劑EGFL7類似物與測試樣本EGFL7 競爭有限量之抗EGFL7抗體抗原結合位點之能力。一般在競爭之前或之後使抗EGFL7抗體不溶解，且接著使與抗 EGFL7抗體結合之示蹤劑及EGFL7與未結合之示蹤劑及 EGFL7分離。此分離可藉由傾析（在使結合搭配物預先不溶解之情況下）或藉由離心（在競爭反應後使結合搭配物沉澱之情況下）實現。測試樣本EGFL7之量與如由標記物質之量所量測的結合示蹤劑之量成反比。製備已知EGFL7量之劑量反應曲線並將其與測試結果相比較以定量測定測試樣本中所存在之EGFL7量。當將使用酵素作為可偵測標記物時，此等檢定稱為ELISA系統。稱為’’均相π檢定之另一種競爭檢定不需要相分離。對此，製備並使用酵素與EGFL7之共軛物，從而當抗EGFL7 抗體與EGFL7結合時，抗EGFL7抗體之存在將改變酵素活性。在此情況下，使EGFL7或其免疫活性片段經雙功能有機橋與酵素（諸如過氧化物酶）共軛。選擇用於抗EGFL7抗體之共軛物使得抗EGFL7抗體之結合將抑制或加強標記之酵素活性。此方法本身已以EMIT之名稱經廣泛實踐。將空間共軛物用於均相檢定之位阻方法中。藉由使低分子量半抗原與小EGFL7片段共價連接合成此等共軛物，從而使針對半抗原之抗體實質上無法與抗EGFL7抗體同時與 119234.doc -122- 200813088 共輛物結合。在此檢定程序中，存在於測試樣本中之 EGFL7將與抗EGFL7抗體結合，藉此使抗半抗原與共軛物結合，從而導致共軛物半抗原之特徵改變，例如當半抗原為螢光團時螢光性改變。夾層檢定尤其可用於測定EGFL7或抗EGFL7抗體。在連續夾層檢定中，使用經固定抗EGFL7抗體吸附測試樣本 EGFL7，如藉由洗滌移除測試樣本，使用已結合之EGFL7 吸附第二、經標記抗EGFL7抗體，且接著使已結合之物質溆兹钕壬總劍公雜。妹各士總满丨1夕吾&泪彳諸媒太FGFT.7 成比例。在”同時”夾層檢定中，在添加經標記抗EGFL7之前不分離測試樣本。使用抗EGFL7單株抗體作為一種抗體且使用多株抗EGFL7抗體作為另一種抗體的連續夾層檢定可用於測試樣本中之EGFL7。上述檢定僅為有關EGFL7之例示性偵測檢定。本發明之範疇内包括現在或以後所研發之使用抗EGFL7抗體測定 EGFL7的其他方法，包括本文所述之生物檢定。製造物品在本發明之另一態樣中，提供一種含有可用於治療、預防及/或診斷上文所述病症之物質的製造物品。該製造物品包含容器及容器上或與容器相聯之標籤或包裝插頁。適當之容器包括（例如）瓶子、小瓶、注射器等。容器可由多種材料（諸如玻璃或塑料）形成。容器容納單獨或與有效治療、預防及/或診斷病況之另一種組合物組合之組合物，且可具有無菌接取口（例如容器可為靜脈内溶液袋或具有 119234.doc -123 - 200813088 可由皮下注射針刺穿之塞子的小瓶）。組合物中之至少一種活性劑為本發明之抗體。標籤或包裝插頁指示組合物係用於治療所選擇之病況，諸如哮喘。此外，製造物品亦可包含（a)其中含有組合物之第一容器，其中該組合物包含本發明之抗體；及（b)其中含有組合物之第二容器。在本發明之此實施例中，製造物品可另外包含應指示第一及第二抗體組合物可用於治療特定病況（諸如哮喘）之包裝插頁。另外或其他，製造物品亦可另外包含第二（或第三）容器，其白.合嫛鏟璺卜矸接辱夕鳐俺该，諸如如餡汴射闲永一 — , I、 ^ ▼ ^ ，|,、 1-4 ，| r▼ — , Ί， , ，磚 _ (BWFI)、經磷酸鹽緩衝之生理食鹽水、林格氏溶液 (Ringer’s solution)及右旋糖溶液。其可另外包括自商業及使用者之角度而言合乎需要之其他物質，包括其他緩衝液、稀釋劑、過濾器、針及注射器。以下為本發明之方法及組合物之實例。應瞭解可基於上文所提供之基本描述實施各種其他實施例。實例實例1抗EGFL7單株抗體之製造及表徵單株抗體之製造鑑別並選殖EGFL7力圖發現新穎的人類分泌及跨膜蛋白，尤其是血管發育調控所涉及之蛋白。有關選殖及表現人類EGFL7之詳細内容描述於（例如）專利申請案 US2003/0224948A1 (其中將 EGFL7稱為 PRO 1449)中。人類 EGFL7 之 GenBank® 寄存編號為 NM_016215。一週兩次（八次劑量）使用經Ribi佐劑（Corixia，Hamilton， 119234.doc -124- 200813088 MT)稀釋的大腸桿菌產生之經His6標記之重組人類及小鼠 EGFL7蛋白經由足墊免疫Egfl7純合子基因易，J除小鼠（在〇61^1^6〇]1產生）。自1〇隻展現高血清力價之小鼠之淋巴結採集B細胞，並使其與小鼠骨髓瘤細胞（X63.Ag8.653;The antibodies of the invention may be used alone or in combination with other compositions for treatment. For example, the antibody of the present invention can be administered together with a chemotherapeutic agent (including a mixture of chemotherapeutic agents), other cytotoxic agents, anti-antigen agents, sprint factors, and/or growth inhibitors. Such combination therapies described above include combination administration (in which two or more agents are included in the same or separate formulations) and administration alone, and in the latter case administration of the antibody of the present invention may be administered in the administration Perform before and/or after the therapy. The antibodies (and adjunctive therapeutic agents) of the invention may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and administered as needed in the lesion for topical treatment. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, antibodies are also suitable for dream-pulse infusion administration, especially in decreasing doses. In some cases, the short-term or long-term nature of the drug, and the main shot, the money "& J* is administered by any appropriate route (for example, by injection/injection such as intravenous or subcutaneous injection). The antibody combination of the present invention The substance can be matched with, matched, and administered. In the way of bp soil medical practice, it is necessary to consider the specific diseases that need to be considered in this regard, the specific breast-feeding treatment (4) 〃 h including the special treatment of H Causes, transmissions (4) Clinical conditions of individual patients, parts of the agent, methods of administration, administration, and other factors known to physicians. Antibodies are not required but can be used with one or more types as appropriate. The agent for preventing or treating the condition is formulated. The effective amount of such other agents depends on the amount of the antibody of the present invention present in the formulation, the type of the condition or treatment, and other factors discussed above. These agents are generally used in the same dosages and routes of administration as used above, or in dosages from about 1% to 99% of the dosages used above. For the prevention or treatment of diseases, the appropriate dose of the antibody of the invention (when Separately Or when used in combination with other agents, depending on the type of disease to be treated, the type of antibody, the severity and duration of the disease, the administration of antibodies for prophylactic or therapeutic purposes, prior therapies, clinical history of the patient and response to antibodies, and the attending physician Depending on the judgment, the antibody is suitable for administration to a patient once or in a series of treatments, depending on the type and severity of the disease, about 1 4^0 to 15 mg/kg (eg 〇mg/kg_1〇mg/kg) The antibody can be, for example, an initial candidate dose administered to the patient by one or more separate administrations or by continuous infusion. A typical sputum dose can range from about i//^ to 100 mg/kg* higher agent' depending on the factors mentioned above. For repeated administrations over several days, depending on the condition, treatment will continue until the desired symptoms of the disease are inhibited. An exemplary dosage of one of the antibodies will range from about 0.05 rng/kg to about 10 mg/kg. Thus, one or more doses of about 5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., subjecting the patient to about two doses to about twenty doses', e.g., about six doses of the antibody). The higher initial loading agent 篁' can then be administered in one or more lower doses. An exemplary dosing regimen comprising 119234.doc-116-200813088 is administered with an initial loading dose of about 4 mg/kg of antibody followed by a weekly maintenance dose of about 2 mg/kg of antibody. However, other dosage regimens are also available. The progress of this therapy is easily monitored by conventional techniques and assays. The anti-EGFL7 antibodies of the invention can be used in assays (e. g., diagnostic or prognostic assays) for detecting the expression of EGFL7 in a particular cell or tissue, wherein the antibody is labeled and/or immobilized on an insoluble substrate as described below. The invention provides a method of detecting EGFL7, the method comprising detecting an EGFL7-anti-EGFL7 antibody complex in a sample. As used in this article, the term "detection" is white. Including the old red X, the old god and the ancestors of the ancestors and the ancestors of the ancestors I, 、,, III, 1 / V ^ , ',, « / ^ "Qing « _· , _ _ _ , , ν _ , , , , - - Measure content) The present invention provides methods for diagnosing a condition associated with EGFL7 performance and/or activity, including detection An EGFL7-anti-EGFL7 antibody complex in a biological sample from a patient having or suspected of having the condition. In some embodiments, EGFL7 exhibits an increased performance or an abnormal (undesirable) performance. Any anti-EGFL7 antibody, wherein the anti-EGFL7 antibody comprises a detectable label. The invention provides a complex of any of the anti-EGFL7 antibodies described herein and EGFL7. In some embodiments, the complex is in vivo or In vitro complex. In some embodiments, the complex comprises cancer cells. In some embodiments, the anti-EGFL7 antibody is detectably labeled. Anti-EGFL7 antibody can be used in any of a variety of well-known detection assays. Detect EGFL7. For example, The sample is obtained from the desired source, and the sample is mixed with an anti-EGFL7 antibody to form an antibody/EGFL7 complex with any EGFL7 present in the mixture with 119234.doc-117-200813088, and any antibody/EGFL7 present in the mixture is detected. The complex is used to assay EGFL7 in a biological sample. The biological sample can be prepared for verification by a method suitable for a specific sample known in the art. The method of mixing the sample with the antibody and the detecting antibody are selected according to the type of assay used. /EGFL7 complex method. These assays include immunohistochemistry, competition, and sandwich assays and steric suppression assays. The assays used for EGFL7 use one or more of the following reagents: labeled EGFL7 analog, immobilized EGFL7 , labeled anti-EGFL7 antibody, immobilized winter anti-EGFL7 antibody and steric conjugate. The labeled reagent is also known as ''tracer'". The label used does not interfere with the binding of EGFL7 to the anti-EGFL7 antibody. Any detectable functional group. A variety of markers for immunoassays are known, examples include directly detectable parts such as fluorescent Materials, chemiluminescent agents, and radioactive labels; and parts that must be detected by reaction or derivatization, such as enzymes. The label used is any detectable functional group that does not interfere with the binding of EGFL7 to the anti-EGFL7 antibody. A wide variety of labels for immunoassays are known, examples of which are directly detectable, such as fluorescent dyes, chemiluminescent agents, and radioactive labels; and moieties that must be detected by reaction or derivatization, such as enzymes. Examples include radioisotopes 32p, 14C, 1251, 3H, and 1311; fluorophores such as rare earth chelating agents or luciferins and their derivatives, rhodamine and its derivatives, tanshinyl, and valence; Luciferases, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456); luciferin; 2,3-dihydropyridazinedione; Spicy 119234.doc-118-200813088 Peroxidase (HRP); testicular acidase; β-galactosidase; glucoamylase; lysozyme; sugar oxidase, such as glucose oxidase, galactose oxidase and glucose-6-acid dehydrogenation Enzyme a heterocyclic gasification enzyme, such as uricase and xanthine oxidase, coupled to an enzyme (such as HRP, lactoperoxidase or microperoxidase) using a hydrogen peroxide dye precursor; biotin/antibiotic Biotin protein, spin labeling, purine ligand labeling; stable free radicals and their analogues. These labels can be covalently bound to a protein or polypeptide using conventional methods. For example, a coupling agent can be used to link the antibody to the above-described fluorescent, chemiluminescent, and enzymatic labels, such as dialdehyde, carbodiimide, dimaleimide, bismuth imidate. , double nitriding linked amide and its analogues. See, for example, U.S. Patent Nos. 3,940,475 (Fluorescence) and 3,645,090 (Enzyme Method); Hunter et al., 144: 945 (1962); David et al., 13: 1014-21 (1974); Pain et al. J· /mww (10)/· 40: 219-30 (1981) and Nygren, J. and 30: 407-12 (1982). Preferred markers herein are enzymes such as horseradish peroxidase and alkaline phosphatase. The conjugation of this marker (including enzymes) to antibodies is a standard procedure for those who are generally familiar with immunoassays. See, for example, O'Sullivan et al., ''Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay, ff Methods Enzymol. ^, ed. JJ Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166. Fixative reagents are required in some assay methods. Immobilization of the anti-EGFL7 antibody was separated from any EGFL7 that remained free in solution with 119234.doc-119-200813088. Anti-EGFL7 antibodies or EGFL7 analogs are typically made by covalent coupling (eg, cross-linking with glutaraldehyde) by adsorption to a water insoluble matrix or surface (Bennich et al., US 3,720,760) during assay procedures. This is achieved by insolubilization or by insolubilization of the anti-EGFL7 antibody or EGFL7 analog by immunoprecipitation after the assay procedure. Immunohistochemistry and staining assay procedures can be used to detect the expression of protein _ in the sample. Immunohistochemical staining of tissue sections has been shown to be a reliable method for assessing or detecting the presence of proteins in a sample. Immunohistochemistry ("IHC") techniques typically use in situ detection and observation of cellular antigens by chromogenic or fluorescent methods. For the preparation of samples, tissues from mammals (usually human patients) or Cell samples. Samples can be obtained by the private sequence known in the art, including but not limited to surgical resection, aspiration, or biopsy. The tissue can be fresh or frozen tissue. In one embodiment The sample is fixed and embedded in paraffin or its analog t. The tissue sample can be fixed (ie, preserved) by a conventional method. Generally, those skilled in the art should understand that the selection of the fixative is to be organized. The purpose of staining or otherwise analyzing the sample is determined. Those skilled in the art will also understand that the fixed length depends on the size of the tissue sample and the fixative used. IHC can be correlated with, for example, staining and/or in situ fluorescence hybridization. Additional technology group implementation. There are two general methods of IHC, direct and indirect assays. According to the first assay, directly determine the junction of the antibody and the target antigen (eg egfl7) This direct assay uses a labeled reagent that can be observed without the action of further antibody phase 1, such as a fluorescent label or an enzyme label, one of the grades 119234.doc-120-200813088. In a typical indirect assay, 'not a total The primary antibody binds to the antigen, and the conjugated secondary antibody binds to the primary antibody. In the case of the secondary antibody fish enzyme label conjugate, a chromogenic or fluorescent substrate is added to observe the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody. Primary and/or secondary antibodies for immunohistochemistry can usually be labeled with detectable moieties. Marking, which is described elsewhere in the text. In addition to the sample preparation procedures discussed above, tissue sections may need to be further processed before, during or after IHC. For example, epitope-repairing methods such as lemons may be performed. Heating the tissue sample in the acid salt buffer (see, for example, Leong et al., 〆 4(3): 201 (1996)). After the optional blocking step, the tissue is cut under appropriate conditions. Exposure to the primary antibody for a sufficient period of time to allow binding of the primary antibody to the target protein antigen in the tissue sample. Suitable conditions for accomplishing this can be determined by routine experimentation by using the detectables discussed above. Any one of the test labels determines the extent of binding of the antibody to the sample. The label is preferably an enzyme label (e.g., HPRQ) that catalyzes the chemical change of a chromogenic substrate such as 3,3'-diaminobenzidine chromogen. The preferred enzyme label is conjugated to an antibody that specifically binds to the -level antibody (eg, the primary antibody is a rabbit polyclonal antibody and the secondary antibody is a goat anti-rabbit antibody). The sample prepared therefrom can be covered with a cover slip. Coverage. Then, for example, the slide evaluation is performed using a microscope, and the dyeing 119234.doc-121. 200813088 color strength standard conventionally used in the art can be used. Other assays known as competition or mezzanine assays have been well established and widely used in the commercial diagnostic industry. The competition assay is based on the ability of the tracer EGFL7 analog to compete with the test sample EGFL7 for a limited amount of anti-EGFL7 antibody antigen binding site. The anti-EGFL7 antibody is generally insoluble before or after competition, and then the tracer and EGFL7 bound to the anti-EGFL7 antibody are separated from the unbound tracer and EGFL7. This separation can be achieved by decantation (in the case where the binding partner is not dissolved in advance) or by centrifugation (in the case where the binding partner is precipitated after the competitive reaction). The amount of test sample EGFL7 is inversely proportional to the amount of bound tracer as measured by the amount of labeling material. A dose response curve of the known amount of EGFL7 was prepared and compared to the test results to quantify the amount of EGFL7 present in the test sample. When an enzyme is used as a detectable label, such assays are referred to as ELISA systems. Another competitive assay called the ''homogeneous π assay does not require phase separation. For this, a conjugate of the enzyme and EGFL7 was prepared and used, so that when the anti-EGFL7 antibody binds to EGFL7, the presence of the anti-EGFL7 antibody will change the enzyme activity. In this case, EGFL7 or an immunologically active fragment thereof is conjugated to an enzyme (such as a peroxidase) via a bifunctional bridge. The conjugate for the anti-EGFL7 antibody is selected such that binding of the anti-EGFL7 antibody will inhibit or potentiate the enzymatic activity of the label. This method itself has been widely practiced under the name EMIT. The spatial conjugate is used in a steric hindrance method for homogeneous assay. These conjugates are synthesized by covalently linking the low molecular weight hapten to the small EGFL7 fragment, such that the antibody against the hapten is substantially incapable of binding to the anti-EGFL7 antibody simultaneously with the 119234.doc-122-200813088. In this assay procedure, EGFL7 present in the test sample will bind to the anti-EGFL7 antibody, thereby binding the anti-hapten to the conjugate, resulting in a change in the characteristics of the conjugate hapten, for example when the hapten is fluorescent Fluorescent changes in the group. Sandwich assays are especially useful for determining EGFL7 or anti-EGFL7 antibodies. In a continuous sandwich assay, the test sample EGFL7 is adsorbed using a immobilized anti-EGFL7 antibody, such as by removing the test sample by washing, using the bound EGFL7 to adsorb the second, labeled anti-EGFL7 antibody, and then subjecting the bound material to The general sword is mixed. Sisters and sisters are always full of 夕吾吾 && tears, the media is too FGFT.7 proportional. In the "simultaneous" sandwich assay, the test sample is not separated prior to the addition of the labeled anti-EGFL7. A continuous sandwich assay using anti-EGFL7 monoclonal antibody as one antibody and multiple anti-EGFL7 antibodies as another antibody can be used to test EGFL7 in a sample. The above verification is only an exemplary detection test for EGFL7. Other methods for determining EGFL7 using anti-EGFL7 antibodies, now or later developed, including the bioassays described herein, are included within the scope of the invention. Manufacture of Articles In another aspect of the invention, an article of manufacture comprising a substance useful for treating, preventing, and/or diagnosing the conditions described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials such as glass or plastic. The container holds a composition alone or in combination with another composition effective to treat, prevent and/or diagnose the condition, and may have a sterile access port (eg, the container may be an intravenous solution bag or have 119234.doc -123 - 200813088 A vial of a stopper pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a condition selected, such as asthma. Further, the article of manufacture may also comprise (a) a first container comprising the composition, wherein the composition comprises an antibody of the invention; and (b) a second container comprising the composition. In this embodiment of the invention, the article of manufacture may additionally comprise a package insert that should indicate that the first and second antibody compositions are useful for treating a particular condition, such as asthma. In addition or in addition, the manufactured article may additionally comprise a second (or third) container, the white, the shovel, the shovel, the sputum, the sorrow, the sorrow, such as, for example, the stuffing, the eternal life - I, ^ ▼ ^ ,|,, 1-4 , | r▼ — , Ί, , , Brick _ (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may additionally include other materials that are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments can be implemented based on the basic description provided above. EXAMPLES Example 1 Production and characterization of anti-EGFL7 monoclonal antibodies Production of monoclonal antibodies Identification and colonization of EGFL7 sought to discover novel human secretory and transmembrane proteins, particularly those involved in the regulation of vascular development. Details regarding the selection and performance of human EGFL7 are described, for example, in the patent application US 2003/0224948 A1 (where EGFL 7 is referred to as PRO 1449). The GenBank® registration number for human EGFL7 is NM_016215. Two-weekly (eight doses) of His6-tagged recombinant human and mouse EGFL7 proteins produced by E. coli diluted with Ribi adjuvant (Corixia, Hamilton, 119234.doc-124-200813088 MT) were immunized with Egfl7 pure via the footpad. The zygote gene is easy, and J is divided into mice (produced in 〇61^1^6〇]1). B cells were collected from lymph nodes of mice showing only high serum valence, and made into mouse myeloma cells (X63.Ag8.653;

American Type Culture Collection® (ATCC®))融合。1〇_14 天後，藉由使用重組EGFL7蛋白進行直接ELISA來針對抗體產生篩選上清液。次選殖陽性組兩次以達成單株性質。對於大規模製造經純化抗體而言，將融合瘤細胞腹膜内注射至經姥鲛烷預致敏之BALB/c小鼠體内，或將其培養於 Integra生物反應器中。藉由蛋白A親和層析（Pharmacia Fast Protein Liquid Chromatography; Pharmacia, Uppsala, Sweden)純化腹水液或培養物上清液。選擇此等单株抗體中命名為4F11、10G9及18F7之三種抗體用於進一步分析。 2006年1月將此等單株抗體寄存於ATCC®。單株抗體阻斷HUVEC細胞黏附及遷移先前已顯示塗覆於培養盤上之EGFL7促進人類臍靜脈内皮細胞（HUVEC)黏附，但黏附之強度顯著弱於其他細胞黏附分子，諸如纖維結合蛋白及膠原蛋白（Parker等人， 428:754-58 (2004))。因此，進行實驗確定 Mab 4F11、10G9及18F7能否阻斷細胞黏附至經EGFL7塗覆之培養盤。用5 pg/ml纖維結合蛋白（Roche)及大腸桿菌中產生之重組人類EGFL7塗覆培養盤。PBS沖洗後，以EGM2培養基（Cambrex)中 5xl05/cm2之密度塗覆 HUVEC (Cambrex)，並以140 g離心5分鐘以使細胞附著同步，且接著進行培 119234.doc -125- 200813088 育。為分析抗體活性，在塗覆前用50 mM Tris/125 mM NaCl (pH 8.6)中0·5、5或50 pg/ml濃度之抗體預先培育 EGM2培養基中之HUVEG。Mab 4F11、10G9及18F7各自均以濃度依賴性方式阻斷細胞黏附至經人類或小鼠EGFL7蛋白塗覆之培養盤且Mab均未阻斷細胞黏附至經纖維結合蛋白塗覆之培養盤，從而證實阻斷對EGFL7具特異性。亦檢測此等抗體能否阻斷HUVEC於經EGFL7塗覆之培養盤上遷移。用5 pg/ml以下蛋白質之一塗覆培養盤：BSA (Sgima)、膠原蛋白（Upstate)、纖维結合蛋白（Roche)及重組人類EGFL7(在Genentech於大腸桿菌中產生）。PBS沖洗後，以EGM2培養基（Cambrex)中5 X 105/cm2之密度塗覆 HUVEC (Cambrex)。允許細胞附著2小時且使用移液管尖單層劃痕。用EGM2將孔洗滌兩次，並分別添加含有50 pg/ml對照Mab或4F11、10G9、18F7之新鮮培養基。24小時内於數個時間間隔拍取孔之相片以監測劃痕閉合。Mab 4F11、10G9及18F7各自均阻斷經EGFL7蛋白塗覆之培養盤上之細胞遷移，但經其他蛋白質塗覆之培養盤上無阻斷。對照Mab對任何蛋白質均無阻斷活性。此等結果表明所有三種抗EGFL7 mab均特異性阻斷MUVEC於EGFL7上之遷移。有趣的是觀察到Mab 4F11之阻斷視調配物而定。特定言之，當在50 mM Tris/125 mM NaCl中製備抗體儲備液時，抗體對於阻斷HUVEC細胞黏附非常有效，但當在PBS製備時顯示出極小之功效。對於其他兩種Mab未觀察到此差 119234.doc -126- 200813088 異。American Type Culture Collection® (ATCC®)) fusion. After 1 〇 14 days, the supernatant was screened for antibody production by direct ELISA using recombinant EGFL7 protein. The subcultured positive group was twice to achieve individual plant properties. For large scale production of purified antibodies, the fusion tumor cells were injected intraperitoneally into decane presensitized BALB/c mice or cultured in an Integra bioreactor. The ascites fluid or culture supernatant was purified by Protein A affinity chromatography (Pharmacia Fast Protein Liquid Chromatography; Pharmacia, Uppsala, Sweden). Three antibodies designated 4F11, 10G9 and 18F7 in these monoclonal antibodies were selected for further analysis. In January 2006, these individual antibodies were deposited in ATCC®. Monoclonal antibodies block HUVEC cell adhesion and migration. EGFL7 coated on culture plates has been shown to promote adhesion of human umbilical vein endothelial cells (HUVEC), but the adhesion is significantly weaker than other cell adhesion molecules such as fibronectin and collagen. Protein (Parker et al, 428:754-58 (2004)). Therefore, experiments were conducted to determine whether Mab 4F11, 10G9, and 18F7 could block cell adhesion to EGFL7 coated plates. Plates were coated with 5 pg/ml of fibronectin (Roche) and recombinant human EGFL7 produced in E. coli. After PBS washing, HUVEC (Cambrex) was coated at a density of 5 x 105/cm2 in an EGM2 medium (Cambrex), and centrifuged at 140 g for 5 minutes to synchronize cell attachment, and then cultured 119234.doc-125-200813088. To analyze antibody activity, HUVEG in EGM2 medium was pre-incubated with antibodies at a concentration of 0.5, 5 or 50 pg/ml in 50 mM Tris/125 mM NaCl (pH 8.6) prior to coating. Mab 4F11, 10G9, and 18F7 each blocked cell adhesion to a culture plate coated with human or mouse EGFL7 protein in a concentration-dependent manner and none of the Mabs blocked cell adhesion to the fibronectin-coated plate. Blocking was confirmed to be specific for EGFL7. It was also tested whether these antibodies could block HUVEC migration on EGFL7 coated plates. Plates were coated with one of 5 pg/ml of protein: BSA (Sgima), collagen (Upstate), fibronectin (Roche), and recombinant human EGFL7 (produced in Genentech in E. coli). After rinsing with PBS, HUVEC (Cambrex) was coated at a density of 5 X 105/cm2 in EGM2 medium (Cambrex). The cells were allowed to adhere for 2 hours and a single layer of scratches was used using a pipette tip. The wells were washed twice with EGM2 and fresh medium containing 50 pg/ml of control Mab or 4F11, 10G9, 18F7 was added separately. Photographs of the holes were taken at intervals of 24 hours to monitor the scratch closure. Mab 4F11, 10G9, and 18F7 each blocked cell migration on EGFL7 protein coated plates, but there was no blockage on other protein coated plates. Control Mab has no blocking activity on any protein. These results indicate that all three anti-EGFL7 mabs specifically block the migration of MUCEC on EGFL7. Interestingly, it was observed that Mab 4F11 blocked the visual modulator. In particular, when antibody stocks were prepared in 50 mM Tris/125 mM NaCl, the antibodies were very effective at blocking HUVEC cell adhesion, but showed minimal efficacy when prepared in PBS. This difference was not observed for the other two Mabs 119234.doc -126- 200813088.

Mab 4F11及10G9之序列測定使用RNeasy®小型套組（Qiagen，Germany)自產生小鼠抗人類EGFL7單株抗體4F11及10G9之融合瘤細胞中提取全 RNA。以如下簡並引子使用RT-PCR擴增4F11及10g9之可變輕鏈（VL)及可變重鏈（VH)結構域：對於4F11而言：輕鏈（LC)正向：5f-GTCAGATATCGTKCTSACMCARTCT O CCWGC-V rSFO m NO- 9n a a. i '%aa^ , mmm ▲ j 重鏈（HC)正向：5，-GATCGACGTACGCTCAGATHCARYT GGTGCARTCTGGGATCGACGTACGCTCAGATHCARYTGG TGCARTCTGG-31 (SEQ ID NO: 22) 對於10G9而言：輕鏈（LC)正/向：5，-GATCGATATCGTGATGACBCARACT CCACT-3f (SEQ ID NO: 23)Sequence analysis of Mab 4F11 and 10G9 Total RNA was extracted from fusion tumor cells producing mouse anti-human EGFL7 monoclonal antibodies 4F11 and 10G9 using the RNeasy® Mini Kit (Qiagen, Germany). The variable light chain (VL) and variable heavy (VH) domains of 4F11 and 10g9 were amplified by RT-PCR using the following degenerate primers: For 4F11: Light chain (LC) forward: 5f-GTCAGATATCGTKCTSACMCARTCT O CCWGC-V rSFO m NO- 9n a a. i '%aa^ , mmm ▲ j Heavy chain (HC) forward: 5,-GATCGACGTACGCTCAGATHCARYT GGTGCARTCTGGGATCGACGTACGCTCAGATHCARYTGG TGCARTCTGG-31 (SEQ ID NO: 22) For 10G9: light chain (LC) Forward/Direction: 5,-GATCGATATCGTGATGACBCARACT CCACT-3f (SEQ ID NO: 23)

重鏈（HC)正向：5’-GATCGACGTACGCTGAGGTYCAGC u j TSCAGCAGTCTGG-3f (SEQ ID NO: 24) 對於4F11與10G9而言：輕鏈反向：S^TTTDAKYTCCAGCTTGGTACCJ (SEQ ID NO: 25) 重鏈反向：5，-ACAGTGGGCCCTTGGTGGAGGCTGMRG AGACDGTGASHRDRGT-3’（SEQ ID NO: 26)。正向引子對兩種抗體之VL及VH區N末端胺基酸序列具有特異性。分別設計LC及HC反向引子以使其與恆定輕鏈 119234.doc -127- 200813088 域（CL)及恆定重鏈域（CH1)中之區域黏接，該區域對於兩種抗體係相同的且跨物種高度保守。將經擴增之VL選殖至pRK哺乳動物細胞表現載體（Shields等人，/· Ckm. 276:659-04 (2000))中。將經擴增之VH***pRK哺乳動物細胞表現載體中。使用常規測序方法測定***物之聚核苷酸序列。4F11輕鏈與重鏈（分別為SEQ ID NO: 1及2)及 10G9輕鏈與重鏈（分別為SEQ ID NO: 3及4)之序列展示於圖1至4中。因刑淛Μ β Μ各Μ知六使用標準方法測定Mab 4F11、10G9及18F7為同型 IgG2b ° 在室溫下，藉由使用 Pharmacia BIAcore⑧ 3000 (BIAcore AB，Uppsala，Sweden)進行表面電漿共振來測定Mab對人類及小鼠EGFL7之結合親和力（例如參看Morton等人，MeA· £/7叮所〇/· 295:268-94 (1998))。將抗EGFL7抗體經由一級胺基固定於感應器晶片（CM5)上。藉由以5 μΐ/min注射20 μΐ 0.025 Μ Ν-羥基琥珀醯亞胺與0.1 Μ Ν-乙基-Ν，（二甲基胺基丙基）碳化二醯亞胺之混合物來活化經羧曱基化之感應器晶片表面基質。以5 μΐ/min注射5· 10 μΐ 10 pg/ml之重組人類或小鼠EGFL7蛋白於10 mM乙酸鈉（pH 4.5)中之溶液。偶聯後，藉由注射20 μΐ 1 Μ乙醇胺（pH 8.5)阻斷晶片上未經佔據之位點。電泳緩衝液為含有0.05%聚山梨醇酯20之 PBS。對於動力學量測而言，將於電泳缓衝液中經兩倍連續稀釋之經聚-His標記之EGFL7以30 μΐ/min之流動速率注 119234.doc -128- 200813088 射經過流槽歷時3分鐘，且使已結合之經polyhis標記之 EGFL7解離20分鐘。藉由注射20 μΐ 10 mM甘胺酸鹽酸鹽 (pH 1.5)使結合表面再生。使用經活化但不具有固定抗體之一號流槽作為參考槽。經聚His標記之EGFL7與一號流槽不存在顯著之非特異性結合。為計算表觀結合親和力，使用1:1結合模型使用整體擬合分析資料。同時擬合締合及解離速率常數（BIAevaluation軟體）。此等使用Mab於50 mM Tris/125 mM NaCl中之儲備液進行之實驗的結果展示於表2中。Heavy chain (HC) forward: 5'-GATCGACGTACGCTGAGGTYCAGC uj TSCAGCAGTCTGG-3f (SEQ ID NO: 24) For 4F11 and 10G9: Light chain reversal: S^TTTDAKYTCCAGCTTGGTACCJ (SEQ ID NO: 25) Heavy chain reversal: 5, -ACAGTGGGCCCTTGGTGGAGGCTGMRG AGACDGTGASHRDRGT-3' (SEQ ID NO: 26). The forward primer is specific for the VL and VH region N-terminal amino acid sequences of the two antibodies. LC and HC reverse primers were designed to bind to regions in the constant light chain 119234.doc -127- 200813088 domain (CL) and constant heavy chain domain (CH1), which are identical for both resistance systems and Cross-species is highly conserved. The amplified VL was cloned into a pRK mammalian cell expression vector (Shields et al., /. Ckm. 276:659-04 (2000)). The expanded VH is inserted into a pRK mammalian cell expression vector. The polynucleotide sequence of the insert is determined using conventional sequencing methods. The sequences of the 4F11 light and heavy chains (SEQ ID NOS: 1 and 2, respectively) and the 10G9 light and heavy chains (SEQ ID NOS: 3 and 4, respectively) are shown in Figures 1 to 4. Determination of Mab 4F11, 10G9 and 18F7 as isotype IgG2b ° by standard methods for the determination of surface plasmon resonance by using Pharmacia BIAcore 8 3000 (BIAcore AB, Uppsala, Sweden) The binding affinity of Mab to human and mouse EGFL7 (see, for example, Morton et al, MeA. £/7 叮 · 295:268-94 (1998)). The anti-EGFL7 antibody was immobilized on a sensor wafer (CM5) via a primary amine group. Activate carboxyindole by injecting a mixture of 20 μΐ 0.025 Ν 羟基-hydroxysuccinimide with 0.1 Μ Ν-ethyl-hydrazine, (dimethylaminopropyl) carbodiimide at 5 μΐ/min The base of the sensor wafer surface. A solution of 5·10 μΐ 10 pg/ml of recombinant human or mouse EGFL7 protein in 10 mM sodium acetate (pH 4.5) was injected at 5 μΐ/min. After coupling, the unoccupied sites on the wafer were blocked by injection of 20 μΐ 1 Μ ethanolamine (pH 8.5). The running buffer was PBS containing 0.05% polysorbate 20. For kinetic measurements, poly-His-labeled EGFL7, which was double-diluted in running buffer, was injected at a flow rate of 30 μΐ/min. 119234.doc -128- 200813088 shot through the flow cell for 3 minutes And the combined polyhis-labeled EGFL7 was dissociated for 20 minutes. The binding surface was regenerated by injection of 20 μΐ 10 mM glycine hydrochloride (pH 1.5). A flow cell that is activated but does not have a fixed antibody is used as a reference cell. There was no significant non-specific binding of the poly-His-labeled EGFL7 to the No. 1 flow cell. To calculate the apparent binding affinity, a 1:1 binding model was used to analyze the data using global fit. At the same time, the association and dissociation rate constants (BIAevaluation software) were fitted. The results of these experiments using a stock solution of Mab in 50 mM Tris/125 mM NaCl are shown in Table 2.

表2 Mab對人類及小鼠EGFL7之KDTable 2 Mab to KD of human and mouse EGFL7

Mab KD (hEGFL7) KD (mEGFL7) 4F11 0.473 nM 0.756 nM 10G9 Ι.ΙΟηΜ 1.83 nM 18F7 0.411 nM 0.191 nM 由Mab識別之EGFL7抗原決定基之測定測定由单株抗體識別之抗原決定基’首先定位各Mab所結合之EGFL7區域。用包含如圖5中所示之全長EGFL7或截斷形式蛋白質之表現載體轉染293細胞。接著用Mab 4F11、10G9及18F7探查經轉染細胞之細胞溶解物的西方墨點。觀察到各Mab均與EGFL7之EMI部分結合。為縮小由各Mab識別之特異性抗原決定基之範圍，合成跨越一部分EMI結構域之重疊多肽並測試其與全長EGFL7 競爭與Mab結合之能力。舉例而言，用於Mab 4F11之多肽的序列如下： 119234.doc -129- 200813088Mab KD (hEGFL7) KD (mEGFL7) 4F11 0.473 nM 0.756 nM 10G9 Ι.ΙΟηΜ 1.83 nM 18F7 0.411 nM 0.191 nM Determination of EGFL7 epitope recognized by Mab Determination of epitopes recognized by monoclonal antibodies' First localization of each Mab The EGFL7 area is combined. 293 cells were transfected with a expression vector containing the full length EGFL7 or truncated form of the protein as shown in Figure 5. Western blots of cell lysates of transfected cells were then probed with Mab 4F11, 10G9 and 18F7. Each Mab was observed to bind to the EMI portion of EGFL7. To narrow the range of specific epitopes recognized by each Mab, overlapping polypeptides spanning a portion of the EMI domain were synthesized and tested for their ability to compete with full-length EGFL7 for binding to Mab. For example, the sequence of the polypeptide for Mab 4F11 is as follows: 119234.doc -129- 200813088

Pl RSPGLAPARPRYA (SEQ ID NO: 27) p2 RPRYACCPGWKRT (SEQ ID NO: 28) p3 GWKRTSGLPGACG (SEQ ID NO: 29) 將Mab 4F11與EGFL7蛋白及10倍莫耳過量之多肽pl、p2Pl RSPGLAPARPRYA (SEQ ID NO: 27) p2 RPRYACCPGWKRT (SEQ ID NO: 28) p3 GWKRTSGLPGACG (SEQ ID NO: 29) Mab 4F11 and EGFL7 protein and 10-fold molar excess of polypeptide pl, p2

及p3之各者混合，免疫沉澱所得複合物並藉由SDS-PAGE 對其進行觀測。對於Mab 4F11而言，觀察到僅p2與全長 EGFL7競爭與Mab 4F11之結合。結果表明Mab 4F11識別包含序列CCP之EGFL7抗原決定基。使用具有以下序列之多肽對其他兩種Mab進行類似實〇驗： p4 LTTCDGHRACSTY (SEQ ID NO: 30) P5 RACSTYRTIYRTA (SEQ ID NO: 31) p6 RTAYRRSPGVTPA (SEQ ID NO: 32)The mixture was mixed with p3, and the resulting complex was immunoprecipitated and observed by SDS-PAGE. For Mab 4F11, only p2 was observed to compete with full-length EGFL7 for binding to Mab 4F11. The results indicate that Mab 4F11 recognizes the EGFL7 epitope comprising the sequence CCP. A similar assay was performed on the other two Mabs using a polypeptide having the sequence: p4 LTTCDGHRACSTY (SEQ ID NO: 30) P5 RACSTYRTIYRTA (SEQ ID NO: 31) p6 RTAYRRSPGVTPA (SEQ ID NO: 32)

測定Mab 10G9與18F7均識別包含序列RTIY (SEQ ID NO 33)之抗原決定基。實例2抗EGFL7 Mab活體内抑制腫瘤生長在此實例中，測試抗EGFL7 Mab活體内抑制數種模型中 I 腫瘤生長之能力。首先在C〇1〇205模型（人類結腸直腸癌）及 A673模型（人類橫紋肌肉瘤模型）中測試PBS中之Mab。並未觀察到作為單一藥劑之PBS中之Mab在此等模型中之作用。接著測試單獨Mab及/或Mab與抗EGFL7抗體Β20·4·1(描述於WO 2005/012359中）之組合。在以下三種模型中測試抗體·· Her2人類乳癌模型（"Fo5模型"）、人類肺癌（NSCLC) 模型（，Ή1299Π)及另一人類乳癌模型（"MDA-MB231”）。此 119234.doc -130- 200813088 等腫瘤模型已經良好建立且描述於（例如）Lee等人，C/h Ca加er 11(16):6065-74 (2005); Cameron等人， Ο// 5:23 (2005); Finkle等人，C/zWm/ Cancer 及 10:2499-25 1(2004)中。以抗豚草Mab(對照）、單獨 B20.4、單獨18F7，或Β20·4與4F11、10G9或18F7處理各動物。簡而言之，對於Η1299模型而言，將lxlO7 Η1299腫瘤細胞皮下注射於HRLN雌性nu/nu小鼠側腹；且對於MDA-MB231 模型而言，將5xl06 MDA-MB231腫瘤細胞皮下注射於 HRLN雖性nu/mi小鼠側腹。對於各模型而言，當平均腫瘤尺寸達到100 mm3(對應於圖6-9中之第0天）時開始抗體治療。每週一次以10 mg/kg投與抗豚草對照Mab及B20.4，且每週兩次以10 mg/kg投與4F11、10G9及18F7(在圖6、8及9 中以X軸下之箭頭表示）。在Fo5模型中未觀察到作用。在其他兩個模型中觀察到顯著腫瘤抑制作用。如圖6-7中所示，在H1299模型中，與 B20.4.1組合之Mab 4F11比對照或單獨B20.4.1顯著有效。如圖8中所示，在MDA-MB231模型中，與B20.4.1組合之 Mab 4F11或Mab 10G9比對照或單獨B20.4.1有效。如圖9中所示，在MDA-MB231模型中，單獨M18F7比對照有效，且與B20.4.1組合之Mab 18F7比單獨Mab 18F7或單獨 B20.184.1顯著有效。有趣的是亦觀察到在H1299模型中Mab 4F11治療阻礙停止抗VEGF治療（Β20·4·1)後之完全血管恢復。當停止治療後，將經單獨Β20.4.1治療之腫瘤與經Β20.4.1及4F11治療 119234.doc -131- 200813088 之腫瘤的腫瘤血管模式相比較，觀察到腫瘤血管再生成顯著延遲。此等結果明確表明抗EGFL7療法在與抗VEGF療法組合時可提供附加或甚至協同之功效。亦使用此項領域中可用之其他腫瘤模型測試抗EGFL7抗體之抗腫瘤活性。此等模型包括（但不限於）：LSI74T(結腸）、BXPC3(***）、HCT116(結腸）、MV-522 (NSCLC)、SKMES (NSCLC)、Colon26(結腸）、MDA㈣ MB231(***）、MCF7(***）、H1299 (NSCLC)、SW620(結 (’ 腸）、L,L(肺）、Fo5(***）、4T1(***）、HT29(結腸）、 SW480(結腸）、786-0(直腸）。以下融合瘤已寄存於American Type Culture Collection®，PO Box 1549，Manassas，VA，20108，USA (ATCC®): 細胞株 ATCC®寄存編號寄存曰期抗EGFL7 mumab 4F11.1.8 PTA-7343 2006年2月 1 日抗EGFL7 mumab 10G9.1.6 PTA-7344 2006年2月 1 日 v 抗EGFL7 mumab 18F7.1.8 PTA-7345 2006年2月 1 日此等寄存係遵國際認可用於專利程序之循微生物寄存布達佩斯條約及相關細則（Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder)(布達佩斯條約）的規定進行。此確保自寄存之曰起維持能活寄存物30年。此等細胞株可由ATCC遵循布 119234.doc -132- 200813088 達佩斯條約之條款使用，且服從Genentech，Inc與ATCC達成之協定，其確保當有關美國專利頒佈時或任何美國或國外專利申請案向公眾公開（無論何種情況首先出現）時公眾對此等細胞株永久及不受限制之使用，且確保由經授權之美國專利及商標委員會委員（U.S· Commissioner of Patents and Trademarks)根據35 USC §122及依照其之委員會規則 (包括37 CFR §1.14及對886 OG 638之特別參考）所確定的人員對此等細胞株之使用。 f、本申請案之受讓人已同意若所寄存之細胞株在適當條件下培養時毀損或破壞，則應迅速發出通告以相同細胞株之試樣將其置換。不應將所寄存之細胞株之可用性解釋為違反任何政府當局根據其專利法律授予之權利實踐本發明之許可。儘管上文已出於清楚理解之目的藉助說明及實例略為詳細地描述本發明，但不應將說明及實例解釋為限制本發明之範轉的限制。【圖式簡單說明】圖1展示Mab 4F11輕鏈可變域（SEQ ID NO: 1)及HuKI (SEQ ID NO: 17)之胺基酸序列。圖2展示Mab 4F11重鏈可變域（SEQ ID NO: 2)及HuIII (SEQ ID NO: 18)之胺基酸序列。圖3展示Mab 10G9輕鏈可變域（SEQ ID NO: 3)及HuKI (SEQ ID NOM 7)之胺基酸序列。Both Mab 10G9 and 18F7 were determined to recognize the epitope comprising the sequence RTIY (SEQ ID NO 33). Example 2 Anti-EGFL7 Mab Inhibits Tumor Growth in Vitro In this example, the ability of anti-EGFL7 Mab to inhibit tumor growth in several models in vivo was tested in vivo. Mab in PBS was first tested in the C〇1〇205 model (human colorectal cancer) and the A673 model (human rhabdomyosarcoma model). The role of Mab in PBS as a single agent in these models was not observed. The combination of Mab and/or Mab alone with anti-EGFL7 antibody Β20·4·1 (described in WO 2005/012359) was then tested. The antibody was tested in the following three models: the Her2 human breast cancer model ("Fo5 model"), the human lung cancer (NSCLC) model (, Ή1299Π), and another human breast cancer model ("MDA-MB231"). This 119234. Tumor models such as doc-130-200813088 have been well established and described, for example, in Lee et al., C/h Ca plus er 11(16): 6065-74 (2005); Cameron et al., Ο// 5:23 ( 2005); Finkle et al., C/zWm/Female and 10:2499-25 1 (2004). Anti-ragweed Mab (control), B20.4 alone, 18F7 alone, or Β20·4 with 4F11, 10G9 or 18F7 treatment of each animal. Briefly, for the Η1299 model, lxlO7 Η1299 tumor cells were injected subcutaneously into the flanks of HRLN female nu/nu mice; and for the MDA-MB231 model, 5xl06 MDA-MB231 tumor cells were Subcutaneous injection of HRLN in the flank of nu/mi mice. For each model, antibody treatment was started when the average tumor size reached 100 mm3 (corresponding to day 0 in Figure 6-9). Mg/kg was administered against ragweed control Mab and B20.4, and 4F11, 10G9 and 18F7 were administered at 10 mg/kg twice a week (in Figures 6, 8 and 9 by X) The arrow below indicates) No effect observed in the Fo5 model. Significant tumor inhibition was observed in the other two models. As shown in Figure 6-7, in the H1299 model, Mab 4F11 combined with B20.4.1 It is significantly more effective than the control or B20.4.1 alone. As shown in Figure 8, in the MDA-MB231 model, Mab 4F11 or Mab 10G9 combined with B20.4.1 is more effective than the control or B20.4.1 alone. In the MDA-MB231 model, M18F7 alone was more effective than the control, and Mab 18F7 combined with B20.4.1 was significantly more effective than Mab 18F7 alone or B20.184.1 alone. Interestingly, Mab 4F11 treatment was also observed to stop in the H1299 model. Complete vascular recovery after anti-VEGF treatment (Β20·4·1). When the treatment was stopped, tumors treated with sputum 20.4.1 alone and tumors of 119234.doc-131-200813088 treated with sputum 20.4.1 and 4F11 were treated. A significant delay in tumor revascularization was observed when compared to the pattern. These results clearly indicate that anti-EGFL7 therapy can provide additional or even synergistic effects when combined with anti-VEGF therapies. The anti-tumor activity of anti-EGFL7 antibodies is also tested using other tumor models available in the art. These models include (but are not limited to): LSI74T (colon), BXPC3 (prostate), HCT116 (colon), MV-522 (NSCLC), SKMES (NSCLC), Colon26 (colon), MDA (four) MB231 (breast), MCF7 ( Breast), H1299 (NSCLC), SW620 (knot ('intestine), L, L (lung), Fo5 (breast), 4T1 (breast), HT29 (colon), SW480 (colon), 786-0 (rectal). The following fusion tumors have been deposited with American Type Culture Collection®, PO Box 1549, Manassas, VA, 20108, USA (ATCC®): Cell line ATCC® deposit number registered flood season anti-EGFL7 mumab 4F11.1.8 PTA-7343 February 2006 1st anti-EGFL7 mumab 10G9.1.6 PTA-7344 February 1, 2006 v anti-EGFL7 mumab 18F7.1.8 PTA-7345 February 1, 2006 These deposits are in accordance with the internationally recognized Budapest Treaty on Microbiological Hosting for Patent Procedures The provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (the Budapest Treaty) ensure that the active deposits are maintained for 30 years from the time of registration. The cell line can be used by the ATCC in accordance with the provisions of the Dapps Treaty of 119234.doc-132-200813088 and is subject to an agreement between Genentech, Inc and the ATCC, which ensures that when the relevant US patent is issued or any US or foreign patent application The public is open to the public (regardless of the circumstances), and the public is permanently and unrestricted in their use of such cell lines, and is ensured by the authorized US Patent and Trade Commissions (US Commissioner of Patents and Trademarks) under 35 USC § 122 and the use of such cell lines by persons identified in accordance with its committee rules (including 37 CFR § 1.14 and special reference to 886 OG 638) f. The assignee of this application has agreed to the cells deposited If the strain is damaged or destroyed when cultured under appropriate conditions, it should be promptly issued to replace it with a sample of the same cell line. The availability of the deposited cell line should not be interpreted as a violation of any government authority granted under its patent law. Practice the license of the present invention. Although the present invention has been described in detail by way of illustration and example, BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the amino acid sequence of the Mab 4F11 light chain variable domain (SEQ ID NO: 1) and HuKI (SEQ ID NO: 17). Figure 2 shows the amino acid sequence of the Mab 4F11 heavy chain variable domain (SEQ ID NO: 2) and HuIII (SEQ ID NO: 18). Figure 3 shows the amino acid sequence of the Mab 10G9 light chain variable domain (SEQ ID NO: 3) and HuKI (SEQ ID NOM 7).

圖4展示Mab 10G9重鏈可變域（SEQ ID NO: 4)及HuIII 119234.doc -133 - 200813088 (SEQ ID NO: 18)之胺基酸序歹》J。圖5描述用於定位抗體結合位點之全長EGFL7及其截斷形式之結構域。圖6展示在用抗VEGF抗體及本發明之抗EGFL7抗體治療之過程中經人類肺癌轉染之基因轉殖小鼠模型（NSCLC; H1299)之活體内腫瘤體積。圖7展示在用抗VEGF抗體及本發明之抗EGFL7抗體治療之過程中經人類肺癌轉染之基因轉殖小鼠活體内模型 (NSCLC;H1299)之存活率。圖8展示在用抗VEGF抗體及本發明之抗EGFL7抗體治療之過程中經人類乳癌轉染之基因轉殖小鼠模型（MDA_ MB231)之活體内腫瘤體積。圖9展示在用抗VEGF抗體及本發明之抗EGFL7抗體Mab 1 8F7治療之過程中經人類乳癌轉染之基因轉殖小鼠模型 (MDA_MB231)之活體内腫瘤體積。Figure 4 shows the Mab 10G9 heavy chain variable domain (SEQ ID NO: 4) and the amino acid sequence of HuIII 119234. doc-133 - 200813088 (SEQ ID NO: 18). Figure 5 depicts the full length EGFL7 and its truncated form of the domain used to localize the antibody binding site. Figure 6 shows the in vivo tumor volume of a gene-transferred mouse model (NSCLC; H1299) transfected with human lung cancer during treatment with an anti-VEGF antibody and an anti-EGFL7 antibody of the present invention. Figure 7 shows the survival rate of an in vivo model of transgenic mice transfected with human lung cancer (NSCLC; H1299) during treatment with an anti-VEGF antibody and an anti-EGFL7 antibody of the present invention. Figure 8 shows in vivo tumor volumes of a gene transfer mouse model (MDA_MB231) transfected with human breast cancer during treatment with an anti-VEGF antibody and an anti-EGFL7 antibody of the present invention. Figure 9 shows in vivo tumor volumes of a gene transfer mouse model (MDA_MB231) transfected with human breast cancer during treatment with an anti-VEGF antibody and the anti-EGFL7 antibody Mab 1 8F7 of the present invention.

119234.doc -134- 200813088 序列表〈110>美商建南德克公司 <120〉抗EGFL7之抗體及其使用方法 <130> P2327R1 <140〉 096109167 <141〉 2007-03-16 <150> US 60/783,686 <151〉 2006-03-16119234.doc -134- 200813088 Sequence Listing <110> US-based Nandek Corporation <120> Anti-EGFL7 antibody and method of use <130> P2327R1 <140> 096109167 <141> 2007-03-16 <150> US 60/783,686 <151〉 2006-03-16

<150*> US 60/812,569 <Ί ςιn οηη«-η«-ηα 、丄 v·/ 丄, ua vy >-/ \y <160〉 33 <210〉 1 <211〉 112 <212〉 PRT <213〉小家鼠 <400> 1<150*> US 60/812,569 <Ί ςιn οηη«-η«-ηα , 丄v·/ 丄, ua vy >-/ \y <160〉 33 <210〉 1 <211〉 112 <212> PRT <213> Mus musculus <400> 1

Asp lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu 15 10 15 oAsp lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu 15 10 15 o

Gly Gin Arg Ala Thr lie Ser Cys Lys Ala Ser Gin Ser Val Asp 20 25 30Gly Gin Arg Ala Thr lie Ser Cys Lys Ala Ser Gin Ser Val Asp 20 25 30

Tyr Asp Gly Asp Ser Tyr Met Ser Trp Tyr Gin Gin Lys Pro Gly 35 40 45Tyr Asp Gly Asp Ser Tyr Met Ser Trp Tyr Gin Gin Lys Pro Gly 35 40 45

Gin Pro Pro Lys Leu Leu lie Tyr Gly Ala Ser Asn Leu Glu Ser 50 55 60Gin Pro Pro Lys Leu Leu lie Tyr Gly Ala Ser Asn Leu Glu Ser 50 55 60

Gly lie Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75Gly lie Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75

Thr Leu Asn He His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr 80 85 90Thr Leu Asn He His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr 80 85 90

Tyr Cys Gin Gin Asn Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly 95 100 105 119234-序列表.doc 200813088Tyr Cys Gin Gin Asn Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly 95 100 105 119234 - Sequence Listing.doc 200813088

Thr Lys Val Glu lie Lys Arg 110 <210〉 2 <211〉 117 〈212〉 PRT 〈213〉小家鼠〈400〉 2Thr Lys Val Glu lie Lys Arg 110 <210〉 2 <211> 117 <212> PRT <213> Mus musculus <400> 2

Gin He Gin Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly 15 10 15Gin He Gin Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly 15 10 15

Glu Thr Val Lys lie Ser Cys Lys Ala Ser Gly His Thr Phe Thr 20 25 30Glu Thr Val Lys lie Ser Cys Lys Ala Ser Gly His Thr Phe Thr 20 25 30

Thr* Tyr Gly Met Ser Trp Val Lys Gin Ala Pro Gly Lys Gly Leu 35 40 45Thr* Tyr Gly Met Ser Trp Val Lys Gin Ala Pro Gly Lys Gly Leu 35 40 45

Lys Trp Met Gly Trp He Asn Thr His Ser Gly Val Pro Thr Tyr 50 55 60Lys Trp Met Gly Trp He Asn Thr His Ser Gly Val Pro Thr Tyr 50 55 60

Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser 65 70 75Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser 65 70 75

Ala Ser Thr Ala His Leu Gin He Asn Asn Leu Lys Asn Glu Asp 80 85 90Ala Ser Thr Ala His Leu Gin He Asn Asn Leu Lys Asn Glu Asp 80 85 90

Thr Ala Thr Tyr Phe Cys Ala Arg Leu Gly Ser Ser Ala Val Asp 95 100 105Thr Ala Thr Tyr Phe Cys Ala Arg Leu Gly Ser Ser Ala Val Asp 95 100 105

Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 110 115 <210〉 3 <211〉 113 <212〉 PRT 〈213>小家鼠〈400〉 3Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 110 115 <210> 3 <211> 113 <212> PRT <213> Mus musculus <400> 3

Asp lie Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu 15 10 15Asp lie Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu 15 10 15

Gly Asp Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val 20 25 30 119234-序列表.doc 200813088Gly Asp Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val 20 25 30 119234 - Sequence Listing.doc 200813088

His Thr Asn Gly He Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro 35 40 45His Thr Asn Gly He Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro 35 40 45

Gly Gin Ser Pro Lys Leu Leu lie Tyr Lys Val Ser Asn Arg Phe 50 55 60Gly Gin Ser Pro Lys Leu Leu lie Tyr Lys Val Ser Asn Arg Phe 50 55 60

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 65 70 75Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 65 70 75

Phe Thr Leu Lys He Ser Arg Val Glu Ala Glu Asp Leu Gly Val 80 85 90Phe Thr Leu Lys He Ser Arg Val Glu Ala Glu Asp Leu Gly Val 80 85 90

Tyr Phe Cys Ser Gin Ser Thr His Val Pro Leu Thr Phe Gly Ala 95 100 105Tyr Phe Cys Ser Gin Ser Thr His Val Pro Leu Thr Phe Gly Ala 95 100 105

Gly Thr Lys Val Glu lie Lys Arg 110 〈210〉 4 <211〉 128 <212> PRT <213〉小家鼠〈400〉 4Gly Thr Lys Val Glu lie Lys Arg 110 <210> 4 <211> 128 <212> PRT <213> Mus musculus <400> 4

Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly 15 10 15Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly 15 10 15

Ala Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser 20 25 30Ala Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser 20 25 30

Asp Tyr Tyr Met Asn Ser Asp Tyr Tyr Met Asn Trp Val Lys Gin 35 40 45Asp Tyr Tyr Met Asn Ser Asp Tyr Tyr Met Asn Trp Val Lys Gin 35 40 45

Ser His Gly Lys Ser Leu Glu Trp lie Gly Asp lie Asn Pro Lys 50 55 60Ser His Gly Lys Ser Leu Glu Trp lie Gly Asp lie Asn Pro Lys 50 55 60

Asn Gly Gly Thr Thr Tyr Asn Gin Lys Phe Lys Gly Lys Ala Thr 65 70 75Asn Gly Gly Thr Thr Tyr Asn Gin Lys Phe Lys Gly Lys Ala Thr 65 70 75

Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg 80 85 90Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg 80 85 90

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu 95 100 105Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu 95 100 105

Ala Asp Tyr Asp Pro He Tyr Tyr Ala Met Asp Tyr Trp Gly Gin 119234-序列表.doc 200813088 110 115 120Ala Asp Tyr Asp Pro He Tyr Tyr Ala Met Asp Tyr Trp Gly Gin 119234 - Sequence Listing.doc 200813088 110 115 120

Gly Thr Thr Leu Thr Val Ser Ala 125 <210〉 5 <211〉 15 〈212〉 PRT <213〉小家鼠 <400〉 5Gly Thr Thr Leu Thr Val Ser Ala 125 <210> 5 <211> 15 <212> PRT <213> Mus musculus <400> 5

Lys Ala Ser Gin Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Ser 15 10 15Lys Ala Ser Gin Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Ser 15 10 15

〈210〉 6 <211〉 7<210> 6 <211〉 7

/91 ON PPT/91 ON PPT

、LJ i imd， X XV X 〈213〉小家鼠 <400〉 6, LJ i imd, X XV X <213> Mus musculus <400> 6

Gly Ala Ser Asn Leu Glu Ser 5 <210〉 7 <211〉 9 <212〉 PRT <213〉小家鼠 <400〉 7Gly Ala Ser Asn Leu Glu Ser 5 <210〉 7 <211> 9 <212〉 PRT <213> Mus musculus <400〉 7

Gin Gin Asn Asn Glu Asp Pro Tyr Thr 5 〈210〉 8 〈211〉 5 <212> PRT <213〉小家鼠〈400〉 8Gin Gin Asn Asn Glu Asp Pro Tyr Thr 5 <210> 8 <211> 5 <212> PRT <213> Mus musculus <400> 8

Thr Tyr Gly Met Ser <210〉 9 〈211〉 17 <212〉 PRT 〈213>小家鼠 119234-序列表.doc 200813088 <400> 9Thr Tyr Gly Met Ser <210> 9 <211> 17 <212> PRT <213> Mus musculus 119234 - Sequence Listing.doc 200813088 <400> 9

Trp lie Asn Thr His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 15 10 15Trp lie Asn Thr His Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 15 10 15

Lys Gly <210〉 10 〈211〉 5 〈212〉 PRT <213〉小家鼠 <400> 10Lys Gly <210> 10 <211> 5 <212> PRT <213> Mus musculus <400> 10

Leu Gly Ser Ser Ala 5Leu Gly Ser Ser Ala 5

<211〉 16 <212〉 PRT <213〉小家鼠〈400〉 11<211> 16 <212> PRT <213> Mus musculus <400> 11

Arg Ser Ser Gin Ser Leu Val His Thr Asn Gly lie Thr Tyr Leu 15 10 15Arg Ser Ser Gin Ser Leu Val His Thr Asn Gly lie Thr Tyr Leu 15 10 15

His <210〉 12 <211〉 7 〈212〉 PRT 〈213〉小家鼠 <400〉 12His <210> 12 <211> 7 <212> PRT <213> Mus musculus <400> 12

Lys Val Ser Asn Arg Phe Ser 5 <210〉 13 〈211〉 9 <212> PRT <213〉小家鼠〈400〉 13Lys Val Ser Asn Arg Phe Ser 5 <210> 13 <211> 9 <212> PRT <213> Mus musculus <400> 13

Ser Gin Ser Thr His Val Pro Leu Thr 119234-序列表.doc 200813088 <210〉 14 <211〉 11 <212〉 PRT <213〉小家鼠 <400> 14Ser Gin Ser Thr His Val Pro Leu Thr 119234 - Sequence Listing.doc 200813088 <210> 14 <211> 11 <212> PRT <213> Mus musculus <400>

Asp Tyr Tyr Met Asn Ser Asp Tyr Tyr Met Asn 5 10 〈210〉 15 <211〉 17 <212〉 PRT <213〉小家鼠 <400〉 15Asp Tyr Tyr Met Asn Ser Asp Tyr Tyr Met Asn 5 10 <210> 15 <211> 17 <212> PRT <213> Mus musculus <400> 15

Asp He Asn Pro Lys Asn Gly Gly Thr Thr Tyr Asn Gin Lys Phe 15 l〇 15Asp He Asn Pro Lys Asn Gly Gly Thr Thr Tyr Asn Gin Lys Phe 15 l〇 15

Lys Gly 〈210〉 16 <211〉 7 <212〉 PRT <213〉小家鼠 <400〉 16Lys Gly <210> 16 <211> 7 <212> PRT <213> Mus musculus <400> 16

Ala Leu Gly Val Phe Asp Tyr <210〉 17 <211〉 108 <212〉 PRT <213〉人類 <400〉 17Ala Leu Gly Val Phe Asp Tyr <210> 17 <211> 108 <212> PRT <213>human <400> 17

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 15 10 15Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 15 10 15

Gly Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Ser He Ser 20 25 30Gly Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Ser He Ser 20 25 30

Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 35 40 45Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 35 40 45

Leu Leu lie Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser -6- 119234-序列表.doc 200813088 50 55 60Leu Leu lie Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser -6- 119234 - Sequence Listing.doc 200813088 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 65 70 75Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 65 70 75

Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin 80 85 90Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin 80 85 90

Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105

He Lys ArgHe Lys Arg

〈210〉 18 <211〉 112 <212> PRT <213〉人類 <400〉 18<210> 18 <211> 112 <212> PRT <213>human <400> 18

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 15 10 15Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 15 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 35 40 45Ser Tyr Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 35 40 45

Glu Trp Val Ser Val lie Ser Gly Asp Gly Gly Ser Thr Tyr Tyr 50 55 60Glu Trp Val Ser Val lie Ser Gly Asp Gly Gly Ser Thr Tyr Tyr 50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser 65 70 75Ala Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser 65 70 75

Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp 80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asp Tyr Trp Gly Gin Gly 95 100 105Thr Ala Val Tyr Tyr Cys Ala Arg Phe Asp Tyr Trp Gly Gin Gly 95 100 105

Thr Leu Val Thr Val Ser Ala 110 〈210〉 19 <211〉 6 119234-序列表.doc 200813088 <212〉 DNA <213〉人造序列 <220〉 <223〉序列係合成的 <220〉〈221> modified一base <222> 2 〈223>驗基為g、a、t或c <220> <221〉 CAAT_signal modified_base <222> full <223〉CAAT 盒Thr Leu Val Thr Val Ser Ala 110 <210> 19 <211> 6 119234 - Sequence Listing.doc 200813088 <212> DNA <213>Artificial Sequence <220> <223>Sequence Synthesis <220 〉 <221> modified-base <222> 2 <223> The base is g, a, t or c <220><221> CAAT_signal modified_base <222> full <223>CAAT box

cncaat 6 <210〉 20 <211> 6 <212> DNA <213〉人造序列 <220〉 <223〉序列係合成的 <220〉 <221> polyA_signal <222> full 〈223>多聚腺嘌呤化信號 <400〉 20 aataaa 6 <210〉 21 <211〉 30 <212〉 DNA <213〉人造序列 <220〉 <223〉序列係合成的 <220> 〈221> Modified—base <222> full 119234-序列表.doc 200813088 <223〉擴增抗體序列之引子 <220〉 <221> modified_base <222〉 13 <223〉鹼基為g或t <220〉 <221> modified_base <222〉 16 <223〉鹼基為g或c <220〉Cncata 6 <210> 20 <211> 6 <212> DNA <213>artificial sequence<220><223><220>>221> polyA_signal <222> 223 > polyadenylation signal <400> 20 aataaa 6 <210> 21 <211> 30 <212> DNA <213>artificial sequence <220> <223>Sequence synthesis<220> <221> Modified-base <222> full 119234 - Sequence Listing.doc 200813088 <223>Introduction of amplified antibody sequence <220> <221> modified_base <222> 13 <223> Is g or t <220> <221> modified_base <222> 16 <223> base is g or c < 220〉

<221> modified一base <222〉 19 <223〉鹼基為a或c <220> 〈221> modified—base <222〉 22 〈223>鹼基為g或a <220〉 <221> modified_base <222〉 28 <223〉鹼基為ait <400〉 21 gtcagatatc gtkctsacmc artctccwgc 30 <210〉 22 <211> 74 <212〉 DNA <213〉人造序列 <220〉 <223〉序列係合成的 <220〉 <221〉 Modified_base <222> full <223〉擴增抗體序列之引子 <220〉 <221> modified_base <222〉 20, 57 119234-序列表.doc 200813088 <223〉鹼基為a或c或t <220〉 <221> modified_base <222〉23, 32, 60, 69 <223〉鹼基為g或a <220〉 <221> modified一base <222〉 24， 61 〈223>鹼基為t或c <400〉 22 gatcgacgta cgctcagath carytggtgc artctgggat cgacgtacgc 50 (、 tcagathcar ytggtgcart ctgg 74 <210〉 23 <211> 30 <212〉 DNA <213〉人造序列 <220〉〈223>序列係合成的 <220〉 <221〉 Modified_base <222〉 full <223〉擴增抗體序列之引子 I, <220> <221> modified_base <222> 19 <223〉鹼基為g或c或t <220〉 <221> modified_base <222〉 22 <223〉鹼基為g或a <400> 23 gatcgatatc gtgatgacbc aractccact 30<221> modified-base <222> 19 <223>base is a or c <220><221> modified-base <222> 22 <223> base is g or a <220> <221> modified_base <222> 28 <223>base is ait <400> 21 gtcagatatc gtkctsacmc artctccwgc 30 <210> 22 <211> 74 <212> DNA <213>artificial sequence< 220> <223>Sequence synthesis <220> <221> Modified_base <222> full <223>Initiation of amplified antibody sequence<220><221> modified_base <222> 20, 57 119234 - Sequence Listing.doc 200813088 <223> The base is a or c or t < 220 < 221 > modified_base < 222 > 222 > 32, 60, 69 < 223 > base is g or a <;220><221> modified-base <222> 24, 61 <223> base is t or c <400> 22 gatcgacgg cgctcagath carytggtgc artctgggat cgacgtacgc 50 (, tcagathcar ytggtgcart ctgg 74 <210> 23 < 211 > 30 <212> DNA < 213 > artificial sequence < 220 < 220 > 223 > sequence synthesis < 220 < 221 > Modified_ba Se <222> full <223> primer A of the amplified antibody sequence, <220><221> modified_base <222> 19 <223> 223> base is g or c or t < 220> <221> modified_base <222> 22 <223> base is g or a <400> 23 gatcgatatc gtgatgacbc aractccact 30

<210〉 24 <211〉 37 <212> DNA 10- 119234·序列表.doc 200813088 <213〉人造序列 <220〉 <223〉序列係合成的 <220> 〈221〉 Modified_base <222> full 〈223>擴增抗體序列之引子 <220〉 <221> modified—base <222〉 20 <223〉鹼基為t或c 〇 <220> <221 > modifi pd_ba.9R <222〉 26 〈223>鹼基為g或c <400〉 24 gatcgacgta cgctgaggty cagctscagc <210〉 25 <211〉 21 <212> DNA 〈213〉人造序列 agtctgg 37<210> 24 <211> 37 <212> DNA 10-119234· Sequence Listing.doc 200813088 <213>Artificial Sequence<220> <223>Sequence Synthesis <220> <221> Modified_base <222> full <223> The primer for amplifying the antibody sequence <220><221> modified-base <222> 20 <223> base is t or c 〇 <220><221> Modifi pd_ba.9R <222> 26 <223> The base is g or c <400> 24 gatcgacgta cgctgaggty cagctscagc <210> 25 <211> 21 <212> DNA <213> artificial sequence agtctgg 37

U <220〉 <223〉序列係合成的 <220> <221> modified_base <222〉 4 〈223>鹼基為a或g或t <220〉 <221> modified—base <222〉 6 〈223>鹼基為g或t <220〉 <221> modified—base <222〉 7 <223〉鹼基為t或c 119234-序列表.doc -11 - 200813088 <220> <221〉 Modified_base <222> full 〈223>擴增抗體序列之引子 <400〉 25 tttdakytcc agcttggtac c 21 <210〉 26 <211〉 43 <212> DNA 〈213>人造序列 <220〉 <223〉序列係合成的 <220〉 <221〉 Modified一base <222> full <223〉擴增抗體序列之引子 <220〉 <221> modified_base <222〉 25 <223〉鹼基為a或c <220> <221> modified_base <222〉26, 39 <223〉鹼基為g或aU < 220 > < 223 > sequence synthesis <220><221> modified_base <222> 4 <223> base is a or g or t < 220> <221> modified-base <;222> 6 <223> The base is g or t < 220> <221> modified-base <222> 7 <223> The base is t or c 119234 - Sequence Listing. doc -11 - 200813088 <;220><221〉 Modified_base <222> full <223> primer for amplifying antibody sequence <400> 25 tttdakytcc agcttggtac c 21 <210> 26 <211> 43 <212> DNA <213> Sequence <220> <223>Sequence Synthesis <220> <221> Modified-base <222> Full <223>Initiation of Amplified Antibody Sequence<220> <221> modified_base < 222> 25 <223> base is a or c <220><221> modified_base <222>26, 39 <223> base is g or a

<220〉 <221> modified一base <222〉 32， 40 <223〉鹼基為a或g或t <220〉 <221> modified—base <222> 37 <223〉鹼基為g或c <220> <221> modified—base <222〉 38 <223〉鹼基為a或c或t -12 119234-序列表.doc 200813088 <400〉 26 acagtgggcc cttggtggag gctgmrgaga cdgtgashrd rgt 43 <210〉 27 <211〉 13 〈212〉 PRT <213〉人類 <400〉 27<220> <221> modified-base <222> 32, 40 < 223 > 223 > base is a or g or t < 220 < 221 > 221 > modified - base < 222 > 37 < 223 > The base is g or c <220><221> modified-base <222> 38 <223> base is a or c or t -12 119234-sequence table.doc 200813088 <400> 26 acagtgggcc cttggtggag Gctgmrgaga cdgtgashrd rgt 43 <210〉 27 <211> 13 <212> PRT <213>human <400〉 27

Arg Ser Pro Gly Leu Ala Pro Ala Arg Pro Arg Tyr Ala 5 10 o <210〉 28 <211〉 13 <212> PRT 〈213〉人類 <400〉 28Arg Ser Pro Gly Leu Ala Pro Ala Arg Pro Arg Tyr Ala 5 10 o <210> 28 <211> 13 <212> PRT <213> Human <400> 28

Arg Pro Arg Tyr Ala Cys Cys Pro Gly Trp Lys Arg Thr 5 10 〈210〉 29 <211〉 13 〈212〉 PRT 〈213〉人類〈400〉 29Arg Pro Arg Tyr Ala Cys Cys Pro Gly Trp Lys Arg Thr 5 10 <210> 29 <211> 13 <212> PRT <213> Human <400> 29

Gly Trp Lys Arg Thr Ser Gly Leu Pro Gly Ala Cys Gly 5 10 <210〉 30 〈211〉 13 <212〉 PRT 〈213>人類 <400〉 30Gly Trp Lys Arg Thr Ser Gly Leu Pro Gly Ala Cys Gly 5 10 <210> 30 <211> 13 <212> PRT <213> Human <400> 30

Leu Thr Thr Cys Asp Gly His Arg Ala Cys Ser Thr Tyr 5 10 <210〉 31 〈211〉 13 <212> PRT <213〉人類〈400〉 31Leu Thr Thr Cys Asp Gly His Arg Ala Cys Ser Thr Tyr 5 10 <210> 31 <211> 13 <212> PRT <213>Human <400> 31

Arg Ala Cys Ser Thr Tyr Arg Thr lie Tyr Arg Thr Ala -13 - 119234-序列表.doc 10 200813088 <210〉 32 <211〉 13 <212> PRT <213〉人類 <400〉 32Arg Ala Cys Ser Thr Tyr Arg Thr lie Tyr Arg Thr Ala -13 - 119234 - Sequence Listing.doc 10 200813088 <210> 32 <211> 13 <212> PRT <213> Human <400> 32

Arg Thr Ala Tyr Arg Arg Ser Pro Gly Val Thr Pro Ala 5 10 <210〉 33 <211〉 4 <212〉 PRT 〈213〉人類 o <400> 33 Arg Thr lie TyrArg Thr Ala Tyr Arg Arg Ser Pro Gly Val Thr Pro Ala 5 10 <210> 33 <211> 4 <212> PRT <213>Human o <400> 33 Arg Thr lie Tyr

14- 119234·序列表.doc14- 119234· Sequence Listing.doc

Claims

200813088 十、申請專利範圍： 1. 一種抗體，其係由一選自由以下各物組成之群之融合瘤產生：抗 EGFL7 mumab 4F11 · 1.8、抗 EGFL7 mumab 10G9.1.6 及抗 EGFL7 mumab 18F7.1.8。 2. 一種抗EGFL7抗體，其包含一或多個選自由以下序列組成之群之互補判定區（CDR): (a) 4F11 CDR-L1 序列 KASQSVDYDGDSYMS (SEQ ID NO: 5)；〇 (b) 4F11 CDR-L2序列 GASNLES (SEQ ID NO: 6); (c) 4F11 CDR-L3序列 QQNNEDPYT (SEQ ID NO: 7); (d) 4F11 CDR-H1 序列 TYGMS (SEQ ID NO: 8); (e) 4F11 CDR-H2序列 WINTHSGVPTYADDFKG (SEQ ID NO: 9);及 (f) 4F11 CDR-H3序列 LGSSA (SEQ ID NO: 10)。 3. 如請求項2之抗EGFL7抗體，其中該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：〇 KASQSVDYDGDSYMS (SEQ ID NO: 5)、GASNLES (SEQ ID NO: 6)及 QQNNEDPYT (SEQ ID NCh 7)。 4. 如請求項2之抗EGFL7抗體，其中該抗體之重鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列： TYGMS (SEQ ID NO: 8)、WINTHSGVPTYADDFKG (SEQ ID NO: 9)及 LGSSA (SEQ ID NO: 10)。 5. 如請求項2之抗EGFL7抗體，其中該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列： 119234.doc 200813088 KASQSVDYDGDSYMS (SEQ ID NO: 5)、GASNLES (SEQ ID NO: 6)及 QQNNEDPYT (SEQ ID NO: 7);且其中該抗體之重鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：TYGMS (SEQ ID NO: 8)、 WINTHSGVPTYADDFKG (SEQ ID NO: 9)及 LGSSA (SEQ ID NO: 10)。 6. 如請求項2之抗五0?1^7抗體，其中該抗體之輕鏈包含以下序列：DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDS ymswvqqkpgqppklliygasnlesgiparfsgsgsgtd FTLNIHPVEEEDAATYYCQQNNEDPYTFGGGTKVEIKR (SEQ ID NO: 1)。 7. 如請求項2之抗EGFL7抗體，其中該抗體之重鏈包含以下序列：QIQLVQSGPELKKPGETVKISCKASGHTFTTYGM SWVKQAPGKGLKWMGWINTHSGVPTYADDFKGRFAFS LETSASTAHLQINNLKNEDTATYFCARLGSSAVDYWGQG TTVTVSS (SEQ ID NO: 2)。 8. 一種抗EGFL7抗體，其包含一或多個選自由以下序列組成之群之互補判定區（CDR): (a) 10G9 CDR-L1 序列 RSSQSLVHTNGITYLH (SEQ ID NO: 11)； (b) 10G9 CDR-L2序列 KVSNRFS (SEQ ID NO: 12); (c) 10G9 CDR-L3 序列 SQSTHVPLT (SEQ ID NO: 13)； (d) 10G9 CDR-H1 序列 DYYMNSDYYMN (SEQ ID 119234.doc -2- 200813088 NO: 14)； (e) 10G9 CDR-H2序列 DINPKNGGTTYNQKFKG (SEQ ID NO: 15);及 (f) 10G9 CDR-H3序列 ALGVFDY (SEQ ID NO: 16)。 9.如請求項8之抗EGFL7抗體，其中該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列： RSSQSLVHTNGITYLH (SEQ ID NO: 11)、KVSNRFS (SEQ ID NO: 12)及 SQSTHVPLT (SEQ ID NO: 13)。 P 10.如請求項8之抗EGFL7抗體，其中該抗體之重鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列： DYYMNSDYYMN (SEQ ID NO: 14)、DINPKNGGTTYNQ KFKG (SEQ ID NO: 15)及 ALGVFDY (SEQ ID NO: 16)。 11. 如請求項8之抗EGFL7抗體，其中該抗體之輕鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列： RSSQSLVHTNGITYLH (SEQ ID NO: 11)、KVSNRFS (SEQ ID NO: 12)及 SQSTHVPLT (SEQ ID NO: 13);且 Q 其中該抗體之重鏈包含至少一個、至少兩個或所有三個選自以下序列之CDR序列：DYYMNSDYYMN (SEQ ID NO: 14)、DINPKNGGTTYNQKFKG (SEQ ID NO·· 15)及 ALGVFDY (SEQ ID NO: 16) 〇 12. 如請求項8之抗EGFL7抗體，其中該抗體之輕鏈包含以下序歹，J : DIVMTQTPLSLPVSLGDQASISCRSSQSLVHTNGI TYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKVEIKR 119234.doc 200813088 (SEQ ID NO: 3) 〇 13. 如請求項8之抗EGFL7抗體，其中該抗體之重鏈包含以下序列：EVQLQQSGPELVKPGASVKISCKASGYTFSDYYM NSDYYMNWVKQSHGKSLEWIGDINPKNGGTTYNQKFK GKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREADYDP IYYAMDYWGQGTTLTVSA (SEQ ID NO: 4)。 14. 一種抗EGFL7抗體，其與一包含一選自由以下序列組成之群之胺基酸序列之多肽特異性結合：CCP、RTIY (SEQ ID NO 33)。 15. —種經分離抗體，其與如請求項1至14中任一項之抗體結合於人類EGFL7上之相同抗原決定基。 16. —種經分離抗體，其與如請求項1至14中任一項之抗體競爭與EGFL7結合。 17. 如請求項1至16中任一項之抗體，其中該抗體為單株抗體。 1 8.如請求項1至1 6中任一項之抗體，其中該抗體係選自由嵌合抗體、人化抗體、親和力成熟抗體、人類抗體及雙特異性抗體組成之群。 19. 如請求項1至16中任一項之抗體，其中該抗體為一抗體片段。 20. —種醫藥組合物，其包含如請求項1至19中任一項之抗 EGFL7抗體。 2 1 ·如請求項20之醫藥組合物，其另外包含抗血管生成劑。 22.如請求項21之醫藥組合物，其中該抗血管生成劑係選自 119234.doc 200813088 由貝伐單抗（bevacizumab)及雷尼株單抗（ranibizumab)組成之群。 23_ —種聚核苷酸，其編碼如請求項1至丨9中任一項之抗體。 24· —種載體，其包含如請求項23之聚核苷酸。 25·如請求項24之載體，其中該載體為一表現載體。 26· —種宿主細胞，包含如請求項24或25之載體。 27.如請求項26之宿主細胞，其中該宿主細胞為原核細胞。〇 28·如請求項26之宿主細胞，其中該宿主細胞為真核細胞。 29·如請求項26之宿主細胞，其中該宿主細胞為哺乳動物細胞。 30· —種製造抗EGFL7抗體之方法，該方法包含（a)使如請求項25之載體在一適當宿主細胞中表現；及（b)回收該抗體。 31.如請求項30之方法，其中該宿主細胞為原核細胞。 ◎ 32.如請求項30之方法，其中該宿主細胞為真核細胞。 33· —種如請求項1至19中任一項之抗£〇17]^7抗體或如請求項 2〇之醫藥組合物之用途，其係用於製造降低或抑制一患有與血答生成相關之病理病況之受檢者體内血管生成之藥物。 34. 35. 36. 37. 如請求項33之用途，其中該病理病況為贅瘤。如請求項34之用途，其中該贅瘤為癌瘤。如請求項33之用途，其中該病理病況係與眼有關。如請求項36之用途，其中該病理病況為眼内新生血管疾 119234.doc 200813088 病。 38·如請求項33至35中任一項之用途，其另外包含向該受檢者投與抗血管生成劑。 39·如請求項38之用途，其中該抗血管生成劑為血管内皮生長因子（VEGF)之拮抗劑。 40·如請求項39之用途，其中該拮抗劑為抗VEGF抗體。 41·如請求項40之用途，其中該抗VEGF抗體為貝伐單抗。 42.如明求項36或37之用途，其另外包含向該受檢者投與抗血管生成劑。 43·如請求項42之用途，其中該抗血管生成劑為血管内皮生長因子（VEGF)之拮抗劑。 44.如請求項43之用途，其中該拮抗劑為抗vegF抗體。 45·如請求項44之用途，其中該抗VEGF抗體為雷尼株單抗。 46·如請求項38之用途，其中該抗血管生成劑係在投與該抗 EGFL7抗體之前或之後投與。 47·如印求項38之用途，其中該抗血管生成劑係與該抗 EGFL7抗體同時投與。 48· —種如請求項1至19中任一項之抗體或如請求項汕或^ 之醫藥組合物之用途，其係用於製造增強一患有與血管生成相關之病理病況之受檢者體内抗血管生成劑之功效之藥物。 49·如請求項48之用途，其中該病理病況為贅瘤。 50·如請求項49之用途，其中該贅瘤為癌瘤。 119234.doc 200813088 51·如請求項48至50中任一項之用途，其中該抗血管生成劑為貝伐單抗。 52. 如請求項48至5〇中任一項之用途，其另外包含投與化學治療劑。 53. 如請求項48之用途，其中該病理病況係與眼有關。 54·如請求項53之用途，其中該病理病況為眼内新生血管疾病0200813088 X. Patent Application Range: 1. An antibody produced by a fusion tumor selected from the group consisting of anti-EGFL7 mumab 4F11 · 1.8, anti-EGFL7 mumab 10G9.1.6 and anti-EGFL7 mumab 18F7.1.8. 2. An anti-EGFL7 antibody comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) 4F11 CDR-L1 sequence KASQSVDYDGDSYMS (SEQ ID NO: 5); 〇(b) 4F11 CDR-L2 sequence GASNLES (SEQ ID NO: 6); (c) 4F11 CDR-L3 sequence QQNNEDPYT (SEQ ID NO: 7); (d) 4F11 CDR-H1 sequence TYGMS (SEQ ID NO: 8); (e) 4F11 CDR-H2 sequence WINTHSGVPTYADDFKG (SEQ ID NO: 9); and (f) 4F11 CDR-H3 sequence LGSSA (SEQ ID NO: 10). 3. The anti-EGFL7 antibody of claim 2, wherein the light chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: 〇KASQSVDYDGDSYMS (SEQ ID NO: 5), GASNLES (SEQ ID NO: 6) and QQNNEDPYT (SEQ ID NCh 7). 4. The anti-EGFL7 antibody of claim 2, wherein the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: TYGMS (SEQ ID NO: 8), WINTHSGVPTYADDFKG (SEQ ID NO) : 9) and LGSSA (SEQ ID NO: 10). 5. The anti-EGFL7 antibody of claim 2, wherein the light chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: 119234.doc 200813088 KASQSVDYDGDSYMS (SEQ ID NO: 5), GASNLES (SEQ ID NO: 6) and QQNNEDPYT (SEQ ID NO: 7); and wherein the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of TYGMS (SEQ ID NO: 8 ), WINTHSGVPTYADDFKG (SEQ ID NO: 9) and LGSSA (SEQ ID NO: 10). 6. The antibody according to claim 2, wherein the light chain of the antibody comprises the following sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDS ymswvqqkpgqppklliygasnlesgiparfsgsgsgtd FTLNIHPVEEEDAATYYCQQNNEDPYTFGGGTKVEIKR (SEQ ID NO: 1). 7. The anti-EGFL7 antibody of claim 2, wherein the heavy chain of the antibody comprises the sequence: QIQLVQSGPELKKPGETVKISCKASGHTFTTYGM SWVKQAPGKGLKWMGWINTHSGVPTYADDFKGRFAFS LETSASTAHLQINNLKNEDTATYFCARLGSSAVDYWGQG TTVTVSS (SEQ ID NO: 2). 8. An anti-EGFL7 antibody comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) 10G9 CDR-L1 sequence RSSQSLVHTNGITYLH (SEQ ID NO: 11); (b) 10G9 CDR -L2 sequence KVSNRFS (SEQ ID NO: 12); (c) 10G9 CDR-L3 sequence SQSTHVPLT (SEQ ID NO: 13); (d) 10G9 CDR-H1 sequence DYYMNSDYYMN (SEQ ID 119234.doc -2- 200813088 NO: 14); (e) 10G9 CDR-H2 sequence DINPKNGGTTYNQKFKG (SEQ ID NO: 15); and (f) 10G9 CDR-H3 sequence ALGVFDY (SEQ ID NO: 16). 9. The anti-EGFL7 antibody of claim 8, wherein the light chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: RSSQSLVHTNGITYLH (SEQ ID NO: 11), KVSNRFS (SEQ ID NO) : 12) and SQSTHVPLT (SEQ ID NO: 13). P 10. The anti-EGFL7 antibody of claim 8, wherein the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: DYYMNSDYYMN (SEQ ID NO: 14), DINPKNGGTTYNQ KFKG (SEQ. ID NO: 15) and ALGVFDY (SEQ ID NO: 16). 11. The anti-EGFL7 antibody of claim 8, wherein the light chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of: RSSQSLVHTNGITYLH (SEQ ID NO: 11), KVSNRFS (SEQ ID NO) : 12) and SQSTHVPLT (SEQ ID NO: 13); and Q wherein the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of DYYMNSDYYMN (SEQ ID NO: 14), DINPKNGGTTYNQKFKG (SEQ ID NO. 15) and ALGVFDY (SEQ ID NO: 16) 〇12. The anti-EGFL7 antibody of claim 8, wherein the light chain of the antibody comprises the following sequence: J: DIVMTQTPLSLPVSLGDQASISCRSSQSLVHTNGI TYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKVEIKR 119234.doc 200813088 ( SEQ ID NO: 3) The anti-EGFL7 antibody of claim 8, wherein the heavy chain of the antibody comprises the sequence: EVQLQQSGPELVKPGASVKISCKASGYTFSDYYM NSDYYMNWVKQSHGKSLEWIGDINPKNGGTTYNQKFK GKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREADYDP IYYAMDYWGQGTTLTVSA (SEQ ID NO: 4). An anti-EGFL7 antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of CCP, RTIY (SEQ ID NO 33). 15. An isolated antibody which binds to the same epitope on human EGFL7 as the antibody of any one of claims 1 to 14. 16. An isolated antibody which competes with EGFL7 for competition with an antibody according to any one of claims 1 to 14. The antibody of any one of claims 1 to 16, wherein the antibody is a monoclonal antibody. The antibody of any one of claims 1 to 16, wherein the anti-system is selected from the group consisting of a chimeric antibody, a humanized antibody, an affinity matured antibody, a human antibody, and a bispecific antibody. The antibody of any one of claims 1 to 16, wherein the antibody is an antibody fragment. 20. A pharmaceutical composition comprising the anti-EGFL7 antibody of any one of claims 1 to 19. A pharmaceutical composition according to claim 20, which additionally comprises an anti-angiogenic agent. The pharmaceutical composition according to claim 21, wherein the anti-angiogenic agent is selected from the group consisting of bevacizumab and ranibizumab, 119234.doc 200813088. A polynucleotide which encodes an antibody according to any one of claims 1 to 9. 24. A vector comprising the polynucleotide of claim 23. 25. The vector of claim 24, wherein the vector is a performance vector. 26. A host cell comprising the vector of claim 24 or 25. 27. The host cell of claim 26, wherein the host cell is a prokaryotic cell. The host cell of claim 26, wherein the host cell is a eukaryotic cell. 29. The host cell of claim 26, wherein the host cell is a mammalian cell. 30. A method of making an anti-EGFL7 antibody, the method comprising (a) rendering the vector of claim 25 in a suitable host cell; and (b) recovering the antibody. The method of claim 30, wherein the host cell is a prokaryotic cell. The method of claim 30, wherein the host cell is a eukaryotic cell. 33. The use of a pharmaceutical composition according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 2, for use in the manufacture of a reduced or inhibited A drug that produces angiogenesis in a subject in a related pathological condition. 34. 35. 36. 37. The use of claim 33, wherein the pathological condition is a tumor. The use of claim 34, wherein the tumor is a carcinoma. The use of claim 33, wherein the pathological condition is associated with the eye. The use of claim 36, wherein the pathological condition is intraocular neovascular disease 119234.doc 200813088 disease. The use of any one of claims 33 to 35, which additionally comprises administering an anti-angiogenic agent to the subject. 39. The use of claim 38, wherein the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF). 40. The use of claim 39, wherein the antagonist is an anti-VEGF antibody. 41. The use of claim 40, wherein the anti-VEGF antibody is bevacizumab. 42. The use of claim 36 or 37, which additionally comprises administering an anti-angiogenic agent to the subject. 43. The use of claim 42, wherein the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF). 44. The use of claim 43, wherein the antagonist is an anti-vegF antibody. 45. The use of claim 44, wherein the anti-VEGF antibody is a Raney strain monoclonal antibody. 46. The use of claim 38, wherein the anti-angiogenic agent is administered before or after administration of the anti-EGFL7 antibody. 47. The use of claim 38, wherein the anti-angiogenic agent is administered concurrently with the anti-EGFL7 antibody. 48. The use of an antibody according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 汕 or ^, for the manufacture of a subject enhancing a pathological condition associated with angiogenesis A drug that acts as an anti-angiogenic agent in the body. 49. The use of claim 48, wherein the pathological condition is a tumor. 50. The use of claim 49, wherein the tumor is a carcinoma. The use of any one of claims 48 to 50, wherein the anti-angiogenic agent is bevacizumab. 52. The use of any one of claims 48 to 5, which additionally comprises administering a chemotherapeutic agent. 53. The use of claim 48, wherein the pathological condition is associated with the eye. 54. The use of claim 53, wherein the pathological condition is intraocular neovascular disease.

55.如請求項53或54之用途，其中該抗血管生成劑為雷尼株 I w 56·如請求項53或54之用途 57·如請求項53或54之用途其另外包含投與皮質類固醇。其另外包含投與光動力療法。〇 119234.doc55. The use of claim 53 or 54, wherein the anti-angiogenic agent is Raney strain I w 56. The use of claim 53 or 54. 57. The use of claim 53 or 54 additionally comprising administering a corticosteroid . It additionally includes administration of photodynamic therapy. 〇 119234.doc