TW201106972A - Combination treatments - Google Patents

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TW201106972A
TW201106972A TW099124592A TW99124592A TW201106972A TW 201106972 A TW201106972 A TW 201106972A TW 099124592 A TW099124592 A TW 099124592A TW 99124592 A TW99124592 A TW 99124592A TW 201106972 A TW201106972 A TW 201106972A
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antibody
axl
cancer
antibodies
vegf
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TW099124592A
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Chinese (zh)
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Suzana Couto
Jo-Anne S Hongo
Dara Y Kallop
yong-jun Li
Jin-Feng Liu
Mary Ludlam
Lin Pei
Christine Tan
Xiao-Fen Ye
Jiping Zha
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Genentech Inc
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Abstract

The invention provides Axl antagonists and methods of using the same.

Description

201106972 「、發明說明： 【發明所屬之技術領域】 本發明大體上係關於分子生物學領域。更明確地說，本 發明係關於Axl拮抗劑及其使用方法。 本案主張2009年7月27曰所申請之美國專利申請案第 61/228,915號及2010年6月18日所申請之第61/3 56,498號之 優先權，該等案之内容以引用方式併入本文中。 【先前技術】201106972 ", the description of the invention: [Technical field to which the invention pertains] The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to an Axl antagonist and a method of using the same. The present invention claims 27 July 2009 Priority is claimed on U.S. Patent Application Serial No. 61/228,915, filed on Jun.

Axl屬於ΤΑΜ受體酪胺酸激酶（RTK)亞家族，該亞家族亦 包括 Tyro3 及 Mer(0，Bryan J.P_等人，（1991) Mo/. Ce// 5ζ_ο/. 1 1:5016-5031 ; Lai C.及 G. Lemke (1991) TVewrow 6:691-704)。TAM受體之特徵為以下之組合：位於胞外區中之兩 個免疫血球蛋白樣結構域及雙重纖維結合蛋白III型重複， 以及細胞質激酶域。ΤΑΜ受體之配位體為Gas6(生長抑制 特異性6)及蛋白質S(兩種顯示43%胺基酸序列一致性且共 有類似域結構的維生素K依賴性蛋白質）（Varnum B.C.等 人，（1995) iVaiwre 373:623-626 ; Stitt T.N.等人，（1995) Ce// 80:661-670)。各蛋白質具有：含有11個γ-羧基麩胺酸 殘基的Ν端Gla域，後接四個表皮生長因子（EGF)樣模組， 及由兩個串聯層黏連蛋白G域組成之C端性激素結合球蛋 白（SHBG)樣結構。SHBG域對於ΤΑΜ受體結合及活化為必 要且充分的，而Gla域結合帶負電的膜磷脂且在ΤΑΜ介導 之凋亡細胞吞噬作用中起重要作用（Sasaki Τ.等人，（2006) EMBO J. 25:80-87 ； Hasanbasic I.f A » (2005) J. Thromb. 149799.doc 201106972Axl belongs to the subunit of the purine receptor tyrosine kinase (RTK), which also includes Tyro3 and Mer (0, Bryan J. P_ et al., (1991) Mo/. Ce// 5ζ_ο/. 1 1:5016 -5031; Lai C. and G. Lemke (1991) TVewrow 6:691-704). The TAM receptor is characterized by a combination of two immunoglobulin-like domains and a dual fibronectin type III repeat located in the extracellular region, and a cytoplasmic kinase domain. The ligands for purine receptors are Gas6 (growth inhibition specificity 6) and protein S (two vitamin K-dependent proteins showing a 43% amino acid sequence identity and sharing a similar domain structure) (Varnum BC et al., ( 1995) iVaiwre 373:623-626; Stitt TN et al. (1995) Ce// 80:661-670). Each protein has: a g-terminal Gla domain containing 11 γ-carboxy glutamic acid residues, followed by four epidermal growth factor (EGF)-like modules, and a C-terminal consisting of two tandem laminin G domains. Sex hormone binding globulin (SHBG)-like structure. The SHBG domain is necessary and sufficient for purine receptor binding and activation, while the Gla domain binds to negatively charged membrane phospholipids and plays an important role in ΤΑΜ-mediated apoptotic cell phagocytosis (Sasaki Τ. et al., (2006) EMBO J. 25:80-87; Hasanbasic If A » (2005) J. Thromb. 149799.doc 201106972

Haemost. 3'.1Ί90-2Ί9Ί、。 ΤΑΜ活化及信號傳導已牽涉多種細胞反應，包括細胞存 活、增殖、遷移及黏附（Hafizi S.及B. Dahlback (2006) 273:5231-5244)。ΤΑΜ受體信號傳導已顯示可調節 血管平滑肌穩定（Melaragno M.G.等人，（1999) Cardiovasc. Med. 9:250-253 ； Korshunov V.A.^ A 5 (2006) Circ. 98:1446-1452 ; Korshunov V.A.等人，（2007) 50:1057-1062)、血小板功能、企栓穩定性 (Angelillo-Scherrer A.等人，（2001) Met/· 7:215-221 ; Gould W.R.等人，（2005) ·/_ TTzromZ?· //aemosi. 3:733-741) 及紅血球生成（Angelillo-Scherrer A.等人，（2008) ·/. C7i«. 7«vesί_ 1 18:583-596)。TAM受體亦牽涉募樹突神經勝質細 胞存活之控制（Shankar S.L.等人，（2006) X iVewroMi·. 26:5638-5648)及蝕骨細胞功能之調節（Katagiri Μ.等人， (2001) «/·价〇/· C//ew. 276:7376-73 82)。對基因剔除小氣之 最新研究表明ΤΑΜ受體在先天免疫中起重要作用（Lemke G.及 C.V.，Rothlin (2008) _/Vai. /mmwwo/· 8:327-336)。 TAM抑制巨噬細胞及樹突狀細胞之炎症（Rothlin C.V.等 人，（2007) Ce// 131:1124-1136 ; Sharif M.N.等人，（2006) «/.五:>Φ· Med. 203:1891-1901)、促進凋亡細胞吞嗤作用 (Prasad D.等人，（2Ό06) Mo/· CW/· iVewrosc/·. 33:96-108 ; 1^().等人，（1999)7\/«〜?^ 398:723-728)且刺激自然殺手細 胞之分化（Caraux A.等人，（2006) Α/α/· /wmwwo/. 7:747-754)。在許多此等情況下，主要下游ΤΑΜ信號傳導路徑似 149799.doc 201106972 乎為 PI3K/AKT 路徑（Angelillo-Scherrer Α·等人，（2001) iVai· Med. 7:215-221 ; Shankar S.L.等人，（2006) ·/. 26:5638-5648 ; Keating A.K.等人，（2006) 0« ⑶gene 25: 6092-6100);然而，傑納斯激酶（Janus kinase)- STAT路徑為 TAM介導之免疫反應所必需（Rothlin C.V.等人，（2007) Ce// 131:1124-1136)。此外，許多由ΤΑΜ調節之生物功能 需要ΤΑΜ受體與細胞激素受體信號傳導網路之間的協同交 互作用（Rothlin C.V.等人，（2007) Ce// 131:1124-1136; Budagian V. # A > (2005) EMBO J. 24:4260-4270) °Haemost. 3'.1Ί90-2Ί9Ί,. Indole activation and signaling have been implicated in a variety of cellular responses, including cell survival, proliferation, migration, and adhesion (Hafizi S. and B. Dahlback (2006) 273:5231-5244). Purine receptor signaling has been shown to modulate vascular smooth muscle stabilization (Melaragno MG et al., (1999) Cardiovasc. Med. 9: 250-253; Korshunov VA^ A 5 (2006) Circ. 98: 1446-1452; Korshunov VA et al. Human, (2007) 50: 1057-1062), platelet function, stability of the plug (Angelillo-Scherrer A. et al., (2001) Met/. 7:215-221; Gould WR et al., (2005) ·/ _ TTzromZ?· //aemosi. 3:733-741) and red blood cell formation (Angelillo-Scherrer A. et al., (2008) ·/. C7i«. 7«vesί_ 1 18:583-596). TAM receptors are also involved in the control of dendritic neuronal survival (Shankar SL et al. (2006) X iVewroMi. 26:5638-5648) and regulation of osteoclast function (Katagiri Μ. et al., 2001 «/·Price〇/· C//ew. 276:7376-73 82). Recent studies on gene knockout have shown that purine receptors play an important role in innate immunity (Lemke G. and C.V., Rothlin (2008) _/Vai. /mmwwo/. 8:327-336). TAM inhibits inflammation of macrophages and dendritic cells (Rothlin CV et al., (2007) Ce// 131: 1124-1136; Sharif MN et al. (2006) «/. V: > Φ· Med. 203 :1891-1901), promotes apoptotic cell swallowing (Prasad D. et al., (2Ό06) Mo/· CW/· iVewrosc/·. 33:96-108; 1^(). et al., (1999) 7\/«~?^ 398:723-728) and stimulates differentiation of natural killer cells (Caraux A. et al., (2006) Α/α/· /wmwwo/. 7:747-754). In many of these cases, the main downstream ΤΑΜ signal transduction path is like the 149799.doc 201106972 is the PI3K/AKT path (Angelillo-Scherrer Α et al., (2001) iVai· Med. 7:215-221; Shankar SL et al. (2006) ·/. 26:5638-5648 ; Keating AK et al., (2006) 0« (3)gene 25: 6092-6100); however, the Janus kinase-STAT pathway is TAM-mediated immunity. Required for the reaction (Rothlin CV et al. (2007) Ce//131: 1124-1136). In addition, many of the biological functions regulated by sputum require a synergistic interaction between the sputum receptor and the cytokine receptor signaling network (Rothlin CV et al., (2007) Ce// 131: 1124-1136; Budagian V. # A > (2005) EMBO J. 24:4260-4270) °

Axl最初自慢性骨髓性白血病患者選殖且在過度表現時 呈現轉型潛能（O’Bryan J.P.等人，（1991) Mo/· Ce// 11:5016-5031 ; Janssen J.W.等人，（1991) Owcogewe 6: 2113-2120)。已報導在多種人類癌症中存在Axl過度表現 (Berclaz G.等人，（2001) dm C>«co/. 12:819-824 ; Craven R.J.等人，（1995) /«ί· J. Cancer 60:791-797 ; Shieh Y.S.等 人，（2005) iVeop/aha 7:1058-1064 ; Sun W.等人，（2004) 66:450-457 ; Ito T.等人，（19 9 9 ) 9:563- 567)，且Axl過度表現與以下癌之侵襲及轉移有關：肺癌 (Shieh Y.S.等人，（2005) 7:1058-1064)、*** 癌（Sainaghi P.P.等人，（2005) «/· Cell. Physiol. 204:36-44)、乳癌（Meric F.等人，（2002) Ties. 8:361- 367)及胃癌（Wu C.W.等人，（2002) 22: 1071-1078)以及腎細胞癌瘤（Chung B.I.等人，（2003) DAM Ce//· 22:533-540)及神經膠母細胞瘤（glioblastoma 149799.doc 201106972 (HuttererM.等人，（2008) C7/1 C⑽14:130-138)。 最新研究表明，Axl經由「酪胺酸激酶開關」過度表現引 起胃腸道受質腫瘤對伊馬替尼（imatinib)之抗性（Mahadevan D.等人 ’（2007) 26:3909-3919)。Axl 表現受化學 療法藥物誘導且Axl過度表現引起急性骨髓性白血病之抗 藥性（Hong C_C.等人，（2008) C⑽268:314-324)。Axl was originally selected from patients with chronic myelogenous leukemia and exhibits transformational potential in overexpression (O'Bryan JP et al., (1991) Mo/· Ce// 11:5016-5031; Janssen JW et al., (1991) Owcogewe 6: 2113-2120). Overexpression of Axl has been reported in a variety of human cancers (Berclaz G. et al., (2001) dm C> «co/. 12:819-824; Craven RJ et al., (1995) / «ί· J. Cancer 60 :791-797 ; Shieh YS et al., (2005) iVeop/aha 7:1058-1064; Sun W. et al., (2004) 66:450-457; Ito T. et al., (19 9 9 ) 9: 563-567), and Axl overexpression is associated with invasion and metastasis of the following cancers: lung cancer (Shieh YS et al, (2005) 7: 1058-1064), prostate cancer (Sainaghi PP et al, (2005) «/· Cell Physiol. 204:36-44), breast cancer (Meric F. et al., (2002) Ties. 8:361-367) and gastric cancer (Wu CW et al. (2002) 22: 1071-1078) and renal cell carcinoma Tumors (Chung BI et al. (2003) DAM Ce//. 22: 533-540) and glioblastoma (glioblastoma 149799.doc 201106972 (Hutterer M. et al., (2008) C7/1 C(10) 14: 130-138 Recent studies have shown that Axl over-expresses via the "tyrosine kinase switch" causes resistance to imatinib in gastrointestinal tumors (Mahadevan D. et al. (2007) 26: 3909-3919). Axl performance by chemotherapy drugs Guide and Axl overexpression cause drug resistance of acute myeloid leukemia (Hong C_C et al., (2008) C⑽268:. 314-324).

Ax〗亦顯示可調節内皮細胞遷移及及血管形成（Holland S.J. 等人 ’（2005) C ⑽ cer 65:9294-9303)。此等發現表 明’ Axl可能涉及腫瘤形成之多個方面之調節，包括腫瘤 生長、襲及血管生成。與Axl及抗Axl抗體相關的其他公 開案包括 WO 2004/039955、WO 2009/063965 ;共同擁有 之同在申請中的美國專利申請案61/356,5〇8(2〇1〇年6月18 曰申請）；共同擁有之同在申請中的美國專利申請案 61/356,514(2010 年 6 月 18 日申請）；WO 2009/062690 ; WO 2004/008147 ;及 5,468,634。 也管系統之發育為許多生理及病理過程之基本需要。諸 如胚胎及腫瘤之活躍生長組織需要足夠之血液供應。其藉 由產生促進新血管經由稱為企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：勾内皮細胞（ec)由 現有EC增殖或由袓細胞分化；b)EC遷移並聚結以形成束 狀結構；c)接著血管束經歷管生成（tubul〇genesis)a形成具 有中央腔之血管；d)現有管束或血管長出新枝而形成二級 血官；e)原始血管叢經歷進一步重塑及再成形；及〇募集 149799.doc 201106972 外皮内皮細胞包圍内皮管，從而向血管提供維持及調節功 能；該等細胞包括小毛細血管之外被細胞、較大灰管之平 滑肌細胞及心臟中之心肌細胞。Hanahan，D. Science 277:48-50 (1997) ； Hogan, B. L.AKolodziej, P. A. NatureAx has also been shown to modulate endothelial cell migration and angiogenesis (Holland S. J. et al. (2005) C (10) cer 65: 9294-9303). These findings indicate that 'Axl may be involved in the regulation of multiple aspects of tumor formation, including tumor growth, invasion, and angiogenesis. Other publications relating to Axl and anti-Axl antibodies include WO 2004/039955, WO 2009/063965; commonly owned U.S. Patent Application Serial No. 61/356, filed on Jun. 18, 2011.曰Applications; co-owned U.S. Patent Application Serial No. 61/356,514, filed on Jun. 18, 2010; WO 2009/062690; WO 2004/008147; and 5,468,634. It also governs the development of the system as a basic need for many physiological and pathological processes. Active growth tissues such as embryos and tumors require adequate blood supply. It addresses this need by generating pro-angiogenic factors that promote the formation of new blood vessels through a process known as angiogenesis. Angiogenesis is a complex but ordered biological event involving all or more of the following steps: hook endothelial cells (ec) are proliferated by existing EC or differentiated by sputum cells; b) EC migrates and coalesces to form a bundle structure; c) then the vascular bundle undergoes tubul〇genesis a to form a blood vessel having a central lumen; d) the existing bundle or blood vessel grows new branches to form a secondary blood donor; e) the original vascular bundle undergoes further remodeling and reshaping; 〇 〇 149799.doc 201106972 The dermal endothelial cells surround the endothelial tube to provide maintenance and regulation to the blood vessels; these cells include cells outside the small capillaries, smooth muscle cells of the larger gray tube, and cardiomyocytes in the heart. Hanahan, D. Science 277:48-50 (1997) ; Hogan, B. L.AKolodziej, P. A. Nature

Reviews Genetics· 3:513-23 (2002) ; Lubarsky，B 及 * Krasnow，M. A. Cell. 112:19-28 (2003)。 現已充分確定，血管生成牽涉多種病症之發病機理。此 〇 等病症包括實體腫瘤及轉移；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，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 癬。Folkman 等人，J. Biol. Chem., 267:10931-10934 (1992)，Klagsbrun等人，Annu. Rev. Physiol. 53:217-239 (1991);及 Garner A”「Vascular diseases」，於：Pathobiology Q Ocular Disease. A Dynamic Approach, Garner A.,Reviews Genetics·3:513-23 (2002); Lubarsky, B and * Krasnow, M. A. Cell. 112:19-28 (2003). It has been well established that angiogenesis involves the pathogenesis of a variety of conditions. Such symptoms include solid tumors and metastases; atherosclerosis; post-lens fibrous tissue hyperplasia; hemangioma; chronic inflammation; intraocular neovascular diseases such as proliferative retinopathy (eg, diabetic retinopathy), age-related macula Degeneration (AMD), neovascular glaucoma; immune rejection of transplanted corneal tissue and other tissues; rheumatoid arthritis; and psoriasis. Folkman et al, J. Biol. Chem., 267: 10931-10934 (1992), Klagsbrun et al, Annu. Rev. Physiol. 53:217-239 (1991); and Garner A" "Vascular diseases", at: Pathobiology Q Ocular Disease. A Dynamic Approach, Garner A.,

Klintworth GK編，第 2版（Marcel Dekker, NY, 1994)，第 1625-1710頁。 在腫瘤生長情況下’血管生成對於增生轉變為贅瘤形成 及向腫瘤生長及轉移提供營養似乎至關重要。F〇lknlan等 人’ Nature 339:58 (1989)。與正常細胞相比，新管生成 允§午腫瘤細胞獲取生長優勢及增殖自主性。腫瘤通常始於 單個異常細胞，其因與可利用之毛細A管床距離遠而可僅 增生至幾立方毫米之大小，且腫瘤可長期保持「休眠」而 149799.doc 201106972 不進一步生長及散播。接著一些腫瘤細胞轉變成血管生成 表型以活化内皮細胞，其增殖並成熟成為新毛細血管。此 等新形成之血管不僅允許原發性腫瘤持續生長，而且允許 轉移的腫瘤細胞散播及再定殖。相應地，已在乳癌及多種 其他腫瘤中觀測到腫瘤切片中之微血管密度與患者存活率 之間的相互關係。Weidner等人，N· Engl. j. Med 324:1-6 (1991) ’ Horak 等人 ’ Lancet 340:1120-1124 (1992). Macchiarini等人，Lancet 340:145-146 (1992)。尚未清楚 瞭解控制血管生成開關之準確機制，但咸信腫瘤塊之新血 &生成係由大量血官生成刺激物及抑制物之淨平衡引起 (Folkman, 1995, Nat Med 1(1):27-31) ° 血管發育過程受到嚴格調節。迄今，大量分子（主要為 由周圍細胞產生之分泌因子）已顯示可調節EC分化、增 殖、遷移及聚結成為束狀結構。舉例而言，血管内皮生長 因子（VEGF)已鑑別為涉及刺激血管生成及誘導血管滲透性 之關鍵因子。Ferrara等人，Endocr. Rev. 18:4-25 (1997)。 喪失甚至單一 VEGF對偶基因亦導致胚胎死亡之發現表 明’此因子在血管系統形成及分化中起到不可替代之作 用。此外’ VEGF已顯示為與腫瘤及眼内病症相關之新血 管生成之關鍵介體。Ferrara等人，Endocr. Rev.同上。所 檢查之人類腫瘤大部分過度表現VEGF mRNA。Berkman等 人，J· Clin. Invest. 91:153-159 (1993) ; Brown等人， Human Pathol. 26:86-91 (1995) ; Brown等人，Cancer Res. 53:4727-4735 (1993) ; Mattern等人，Brit. J. Cancer 73: 149799.doc 201106972 931-934 (1996) ; Dvorak等人，Am. J. Pathol. 146:1029-1039 (1995)。 抗VEGF中和抗體抑制裸小鼠中多種人類腫瘤細胞株之 生長（Kim 等人，Nature 362:841-844 (1993) ; Warren 等 人，J· Clin. Invest· 95:1789-1797 (1995) ; Borgstr0m 等 人，Cancer Res. 56:4032-4039 (1996) ; Melnyk 等人， Cancer Res. 56:921-924 (1996))且亦抑制缺血性視網膜病 0 症模型中眼内血管生成。Adamis等人，Arch. Ophthalmol. 114:66-71 (1996)。因此，抗VEGF單株抗體或其他VEGF作 用抑制劑為有望用於治療腫瘤及各種眼内新生血管病症之 候選藥物。該等抗體描述於例WEp 817,648(1998年14 日公開）；及WO 98/45331 與WO 98/45332(均於 1998年10月 1 5曰公開）中。 癌症為對人類健康之最致命威脅之一。僅在美國，癌症 每年衫響近一百二十萬新患者且為心血管疾病之後的第二 ❹^]主要死亡原因，在死亡中占約1/4。實體腫瘤是造成大 刀彼等死亡的原因。儘管某些癌症之醫療已顯著進步， 但在過去2〇年中所有癌症之總體5年存活率僅提高約 10%。儘管癌症治療顯著進步，但仍然在尋求改良療法。 本文中引用之所有參考文獻，包括專利申請案及公開 案，均以全文引用的方式併入本文中。 【發明内容】 本發明提供治療諸如癌症之病理性病狀之組合療法，其 中將AX1拮抗劑與VEGF拮抗劑組合。 149799.doc 201106972 在一態樣中，本發明提供治療個體癌症之方法，其包含 向個體投與治療有效量之Axl拮抗劑及VEGF拮抗劑。 在另一態樣中，本發明提供抑制個體癌轉移之方法，其 包含向個體投與治療有效量之Axl拮抗劑及VEGF抬抗劑。 在另一態樣中，本發明提供抑制個體企管生成之方法， 其包含向個體投與治療有效量之Axl拮抗劑及VEGF拮抗 劑。 在另一態樣中，本發明提供抑制個體細胞增殖之方法， 其包含向個體投與治療有效量之Axl拮抗劑及VEGF拮抗 劑。 在某些實施例中，VEGF拮抗劑為阻礙VEGF與細胞受體 結合之化合物。該等VEGF阻斷拮抗劑之實例包括（但不限 於）可溶性VEGF受體、對VEGF具有特異性之適體或肽 體，及抗VEGF抗體。在一實施例中，抗VEGF抗體為貝伐 珠單抗（bevacizumab)。 在某些實施例中，當組合使用時，貝伐珠單抗的投與量 範圍為約0.05 mg/kg至約15 mg/kg。在一實施例中，可向 個體投與約 0.5 mg/kg、1.0 mg/kg、2.0 mg/kg、3.0 mg/kg ' 4.0 mg/kg、5.0 mg/kg、6.0 mg/kg ' 7.0 mg/kg、 7.5 mg/kg、8.0 mg/kg、9.0 mg/kg、10 mg/kg或 15 mg/kg 中 之一或多種劑量（或其任何組合）。該等劑量可間歇地投 與，例如每天、每三天、每週或每兩至三週。Klintworth GK, 2nd ed. (Marcel Dekker, NY, 1994), pp. 1625-1710. In the case of tumor growth, angiogenesis appears to be crucial for the conversion of hyperplasia to neoplasia and to providing nutrients for tumor growth and metastasis. F〇lknlan et al. Nature 339:58 (1989). Compared with normal cells, new tube formation allows tumor cells to gain growth advantage and proliferation autonomy. Tumors usually start with a single abnormal cell, which can only proliferate to a few cubic millimeters due to the distance from the available capillary A tube bed, and the tumor can remain "dormant" for a long time. 149799.doc 201106972 No further growth and dissemination. Some tumor cells then transform into an angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillaries. These newly formed blood vessels not only allow the primary tumor to continue to grow, but also allow the metastatic tumor cells to spread and re-colonize. Accordingly, the correlation between microvessel density in tumor sections and patient survival has been observed in breast cancer and various other tumors. Weidner et al., N. Engl. j. Med 324: 1-6 (1991) ‘ Horak et al.’ Lancet 340: 1120-1124 (1992). Macchiarini et al., Lancet 340: 145-146 (1992). The exact mechanism for controlling angiogenic switches is not well understood, but the new blood & generation line of the tumor block is caused by a large balance of blood-stimulated stimuli and inhibitors (Folkman, 1995, Nat Med 1(1):27 -31) ° The vascular development process is strictly regulated. To date, a large number of molecules (mainly secreted factors produced by surrounding cells) have been shown to modulate EC differentiation, proliferation, migration and coalescence into bundle structures. 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., Endocr. Rev. 18:4-25 (1997). The discovery that loss of even a single VEGF-pair gene also causes embryonic death indicates that this factor plays an irreplaceable role in vascular system formation and differentiation. In addition, VEGF has been shown to be a key mediator of new angiogenesis associated with tumors and intraocular disorders. Ferrara et al., Endocr. Rev. Ibid. Most of the human tumors examined showed excessive expression of VEGF mRNA. Berkman et al, J. Clin. Invest. 91: 153-159 (1993); Brown et al, Human Pathol. 26: 86-91 (1995); Brown et al, Cancer Res. 53: 4727-4735 (1993) Mattern et al, Brit. J. Cancer 73: 149799.doc 201106972 931-934 (1996); Dvorak et al, Am. J. Pathol. 146:1029-1039 (1995). Anti-VEGF neutralizing antibodies inhibit the growth of a variety of human tumor cell lines in nude mice (Kim et al, Nature 362: 841-844 (1993); Warren et al, J. Clin. Invest 95: 1789-1797 (1995) Borgstr0m et al, Cancer Res. 56:4032-4039 (1996); Melnyk et al, Cancer Res. 56:921-924 (1996)) and also inhibits intraocular angiogenesis in a model of ischemic retinopathy. Adamis et al., Arch. Ophthalmol. 114: 66-71 (1996). Therefore, anti-VEGF monoclonal antibodies or other VEGF inhibitors are promising candidates for the treatment of tumors and various intraocular neovascular disorders. Such antibodies are described in, for example, WEp 817, 648 (published on Jun. 14, 1998); and WO 98/45331 and WO 98/45332 (both published on Oct. 15, 1998). Cancer is one of the most deadly threats to human health. In the United States alone, cancer accounts for nearly 1.2 million new patients per year and is the second leading cause of death after cardiovascular disease, accounting for about a quarter of deaths. Solid tumors are the cause of the death of the surgeon. Although the medical treatment of certain cancers has made significant progress, the overall 5-year survival rate of all cancers in the past 2 years has only increased by about 10%. Despite significant advances in cancer treatment, improved therapies are still being sought. All references, including patent applications and publications, cited herein are hereby incorporated by reference in their entirety. SUMMARY OF THE INVENTION The present invention provides combination therapies for the treatment of pathological conditions such as cancer, wherein an AX1 antagonist is combined with a VEGF antagonist. In one aspect, the invention provides a method of treating cancer in a subject comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. In another aspect, the invention provides a method of inhibiting cancer metastasis in a subject comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. In another aspect, the invention provides a method of inhibiting the production of an individual, comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. In another aspect, the invention provides a method of inhibiting cell proliferation in an individual comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. In certain embodiments, the VEGF antagonist is a compound that blocks the binding of VEGF to a cellular receptor. Examples of such VEGF blocking antagonists include, but are not limited to, soluble VEGF receptors, aptamers or peptides specific for VEGF, and anti-VEGF antibodies. In one embodiment, the anti-VEGF antibody is bevacizumab. In certain embodiments, bevacizumab is administered in an amount ranging from about 0.05 mg/kg to about 15 mg/kg when used in combination. In one embodiment, the subject may be administered about 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg '4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg' 7.0 mg/ One or more doses (or any combination thereof) of kg, 7.5 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10 mg/kg, or 15 mg/kg. These doses can be administered intermittently, for example daily, every three days, every week or every two to three weeks.

Axl拮抗劑之實例包括（但不限於）可溶性Axl受體、可溶 性Axl配位體變異體、對Axl或Axl配位體具有特異性之適 149799.doc -10- 201106972 體或肽體、Axl小分子、抗Axl抗體及抗Axl配位體抗體。 在一些實施例中，Axl拮抗劑為抗Axl抗體。在一些實施例 中，Axl拮抗劑不為華法林（warfarin)。 在一些實施例中，抗Axl抗體包含含有序列EVQLVESG GGLVQPGGSLRLSCAASGFSLSGSWIHWVRQAPGKGLEW VGWINPYRGYAYYADSVKGRFTISADTSKNTAYLQMNSLR AEDTAVYYCAREYSGWGGSSVGYAMDYWGQGTLV(SEQ ID ΝΟ:1)的重鏈可變區及含有序列DIQMTQSPSSLSASV GDRVTITCRASQDVSTAYAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTF GQGTKVEIKR(SEQIDNO:2)的輕鏈可變區。 在一些實施例中，Axl拮抗劑為R428(Rigel; Holland等 人，Cancer Research 70，1544 (2010))或 PF02341066。 在某些實施例中，該方法進一步包含投與EGFR拮抗 劑。在某些實施例中，EGFR拮抗劑為埃羅替尼 (erlotinib)。在某些實施例中，可以每天150 mg之劑量或 以每天100 mg之劑量投與埃羅替尼。 本發明之方法可用於改變任何適合的病理狀態。舉例而 言，本發明之方法可用於治療不同癌症（實體腫瘤與液體 腫瘤以及相似的軟組織腫瘤）。可依照本發明之療法治療 的癌症之非限制性實例包括乳癌、結腸直腸癌、直腸癌、 非小細胞肺癌、非霍奇金氏淋巴瘤（non-Hodgkins lymphoma，NHL)、腎細胞癌、***癌、肝癌（諸如肝細 胞癌）、胰臟癌、軟組織肉瘤、卡波西氏肉瘤（kaPosi's 149799.doc 11 201106972 ma)類癌瘤（carcinoid carcinoma)、頭頸癌、署色素 瘤、卵巢癌、胃癌、間皮瘤及多發性骨髓瘤。在某些態樣 中癌症為轉移性癌。在其他態樣中，癌症為非轉移性 癌。 在一些實施例中，抗Αχ1抗體及抗VEGF抗體（諸如貝伐 珠單抗）用於諸如非小細胞肺癌之癌症之組合療法中。 視待治療之特定適應症而定，可將本發明之組合療法與 諸如化學治療劑之其他治療劑或諸如放射線療法或手術之 其他療法組合。許多已知化學治療劑可用於本發明之組合 療法中。在某些實施例中’本發明之組合療法可與一種以 上化學治療劑組合。在某些實施例中，所用的彼等化學治 療劑為用於治療特定適應症之標準藥劑。在另一實施例 中’組合使用之各種治療劑之劑量或頻率等同於或小於不 使用其他治療劑時相應治療劑之劑量或頻率。 在某些實施例中，癌症顯示Axl表現（諸如Axl過度表 現）、擴增或活化。 在某些實施例中，個體經診斷患有表現Axl之癌症。 本發明亦提供抑制組成性Axl活化之方法，其包含使細 胞與下調Axi表現（降低例如細胞表面（諸如腫瘤細胞表面） 上之Axl表現及/或降低細胞（諸如腫瘤細胞）中之總體Αχί表 現）之抗體（或其抗原結合片段）接觸。 本發明亦提供治療個體癌症之方法，其包含向個體投與 下調Axl表現（降低例如細胞表面（諸如腫瘤細胞表面）上之 Axl表現及/或降低細胞（諸如腫瘤細胞）中之總體Axl表現） 149799.doc -12- 201106972 的抗Axl抗體。 本發明亦提供阻斷配位體與Axl結合之方法，其包含使 表現Axl之細胞與結合Axl且抑制配位體與Axl結合之抗體 或其抗原結合片段接觸，其中該抗體識別人類Axl而非小 % 鼠Axl上之抗原決定基。單株抗體可為例如嵌合抗體、人 ’ 類化抗體、單鏈抗體或雙特異性抗體。在一些實施例中， 抗體結合的抗原決定基與選自由3G9、8B5、12A11及4F8 〇 組成之群之單株抗體所結合之抗原決定基相同。在一些實 施例中，抗體抑制配位體與Axl結合。在一些實施例中， 抗體下調Axl表現。在一些實施例中，抗體識別Αχί上之變 性抗原決定基’如可由西方墨點法（Western blot)偵測。在 一些實施例中’抗體之IC5〇為至少約1〇〇 ng/ml。在一些實 施例中’抗體抑制Gas6誘導之Axl磷酸化。 在一態樣中’本發明提供由融合瘤細胞株3G9· 19.7產生 之抗體，融合瘤細胞株3G9.19.7具有美國菌種保存中心 ❹ （ATCC)編號_ ，寄存於_。 在一態樣中’本發明提供分離抗體，其包含由具有美國 菌種保存中心（ATCC)編號_______之融合瘤細胞株 • 3G9.19·7所產生之抗體之重鏈及/或輕鏈可變域，其中該分 • 離抗體特異性結合人類Axl。 在一態樣中，本發明提供包含至少1個（至少2個、至少3 個 '至少4個、至少5個、及/或6個）高變序列（Hvr)之分離 抗體’該（等）高變序列包含選自由具有美國菌種保存中心 (ATCC)編號--^融合瘤細胞株3G9.19.7所產生之 149799.doc 13 201106972 抗體之HVR-Ll、HVR.L2、HVR-L3、HYR-Hl、HVH-m 及/或HVR-H3組成之群的序列，其中該分離抗體特異性結 合人類Axl。 在一態樣中，本發明提供所結合之人類Αχ1上之抗原決 定基與由具有美國菌種保存中心（ATCC)編號一______之 融合瘤細胞株3G9.19.7產生之抗體所結合之人類Αχ1上之 抗原決定基相同的分離抗體。 在一態樣中’本發明提供與由具有美國菌種保存中心 (ATCC)編號--——之融合瘤細胞株3G9.19.7所產生之 抗體競爭結合人類Axl的分離抗體。 本發明亦提供下調細胞中Axl表現之方法，其包含使該 細胞與引起該細胞中Axl表現降低之抗體接觸。抗體可為 單株抗體。抗體可為例如嵌合抗體、人類化抗體、單鏈抗 體或雙特異性抗體。在一些實施例中，抗體結合的抗原決 定基與選自由3G9、8B5、12A11及4F8組成之群之單株抗 體所結合之抗原決定基相同。在一些實施例中，抗體抑制 配位體與Axl結合。在一些實施例中，抗體識別Αχί上之變 性抗原決定基’如可由西方墨點法彳貞測。在一些實施例 中，抗體之IC5〇為至少約1 00 ng/ml。在一些實施例中，抗 體抑制Gas6誘導之Axl填酸化。 本發明亦提供偵測樣品中Axl之方法，其包含使該樣品 與特異性結合於Axl上之抗原決定基且不與該樣品中其他 蛋白質交叉反應的抗體接觸。在一些實施例中，抗體結合 的抗原決定基與選自由3G9、8B5、12A11及4F8組成之群 149799.doc • 14- 201106972 之單株抗體所結合之抗原決定基相同。在一些實施例中， 抗體識別Axl上之變性抗原決定基，如可由西方墨點法摘 測。在一些實施例中，抗體之ICS()為至少約丨⑼叩“卜 纟I月亦提（、0斷患有以Αχι表現（在—些實施例中，為 過度表現）為特徵之病狀之患者的方法，其包含自該患者 獲得樣品且針對Axl表現分析該樣品，其中該分析法包含 使用所結合之抗原決定基與選自由3G9、8B5、丨2A11及 Q 8、’且成之群之抗體或其抗原結合片段所結合之抗原決定 基相同的杬體。在一些實施例中，抗體為3G9、8B5、 12A11、4F8，其抗原結合片段，或其組合。抗體可為單株 抗體、嵌合抗體、人類化抗體、單鏈抗體或其抗原結合片 段。在一些實施例中，抗體與可偵測標記（諸如（但不限於） 放射性同位素、螢光化合物或酶標記）結合。 本發明亦提供治療患者癌症之方法，其包含：（a)使用第 一抗體偵測該患者之樣品中Axl之過度表現；及（b)向該患 〇 者投與包含第二抗體及醫藥學上可接受之載劑的組合物， 其中該第一抗體特異性結合於人類Axl上之抗原決定基且 該第二抗體抑制配位體與Axl結合、Axl磷酸化，或下調 • Axl表現。癌症可為選自由以下組成之群的癌症：骨髓性 . 白血病、肺癌（例如非小細胞肺癌（NSCLC))、胃癌、乳 癌、***癌、腎細胞癌、胰臟癌及神經膠母細胞瘤。在 一些實施例中，第一抗體結合的Axl抗原決定基與選自由 3G9、8B5、12A11及4F8組成之群之單株抗體或其抗原結 合片段所結合之Axl抗原決定基相同。在一些實施例中， 149799.doc -15- 201106972 第二抗體結合的Axl抗原決定基與選自由3G9、8B5、 12A11及4F8組成之群之單株抗體或其抗原結合片段所結合 之Axl抗原決定基相同。在一些實施例中，第一抗體及第 二抗體為單株抗體、嵌合抗體、人類化抗體、單鏈抗體或 其抗原結合片段。在一些實施例中，第一抗體及/或第二 抗體與細胞毒性劑（諸如（但不限於）化學治療劑、毒素或放 射性同位素）結合。在一些實施例中，該方法進一步包含 在投與結合Axl之抗體之前、之後或同時投與抗VEGF抗 體。 本發明亦提供抑制過度表現Axl之細胞之血管生成之方 法’其包含向該等細胞投與抗體，該抗體特異性結合人類 Axl上之抗原決定基且抑制配位體與Αχί結合、Axl填酸 化’或下調Axl表現。癌症可為選自由以下組成之群的癌 症：骨髓性白血病、肺癌（例如非小細胞肺癌（NSCLC))、 胃癌、乳癌、***癌、腎細胞癌、胰臟癌及神經膠母細 胞瘤。在一些實施例中，抗體結合的抗原決定基與選自由 3G9、8B5、12A11及4F8組成之群之抗體或其抗原結合片 段所結合之抗原決定基相同。在一些實施例中，抗體為單 株抗體、嵌合抗體、人類化抗體、單鏈抗體或其抗原結合 片段。在一些實施例中，抗體與細胞毒性劑（諸如（但不限 於）化學治療劑、毒素或放射性同位素）結合。在一些實施 例中，該方法進一步包含在投與結合Ax丨之抗體之前、之 後或同時投與抗VEGF抗體。抗Axl抗體及抗VEGF抗體可 呈多特異性抗體形式，諸如（但不限於）雙特異性抗體。 149799.doc -16· 201106972 在一態樣中，本發明提供抗Axl抗體及其使用方法。 在一些實施例中’本發明提供特異性結合人類Axl而非 小鼠Axl上之抗原決定基的單株抗體，或該抗體之抗原結 合片段。單株抗體可為例如嵌合抗體、人類化抗體、單鏈 抗體或雙特異性抗體。在一些實施例中，抗體結合的抗原 決定基與選自由3G9、8B5、12A11及4F8組成之群之單株 抗體所結合之抗原決定基相同。在一些實施例中，抗體抑 q 制配位體與Axl結合。在一些實施例中，抗體下調Axl表 現。在一些實施例中，抗體識別Axl上之變性抗原決定 基，如可由西方墨點法偵測。在一些實施例中，抗體之 ICso為至少約100 ng/ml。在一些實施例中，抗體抑制Gas6 誘導之Axl磷酸化。 本發明亦提供醫藥組合物，其包含如前述段落中所述之 單株抗體及醫藥學上可接受之載劑。在一些實施例中，醫 藥組合物進一步包含抗VEGF抗體。 〇 本發明進一步提供包含容器、容器中所含組合物及藥品 §兒明書之製品，其中該組合物包含所結合之Αχ1抗原決定 基與選自由3G9、8B5、12A11及4F8組成之群之單株抗體 或其抗原結合片段所結合之Αχ1抗原決定基相同的單株抗 • 體，且該藥品說明書包含該組合物之使用說明。在一些實 轭例中’抗體為選自由3G9、8B5、12A11及4F8組成之群 之單株抗體。 在一態樣中，提供編碼任一種上述抗體之聚核苷酸。在 一實施例中，提供包含聚核苷酸之載體。在一實施例中， 149799.doc -17- 201106972 挺供包含載體之宿主細胞。在一實施例中，宿主細胞為真 核細胞。在一實施例中，宿主細胞為CHO細胞。在一實施 例中，提供製備抗Axl抗體之方法，其中該方法包含在適 於表現編碼抗體之聚核苷酸的條件下培養宿主細胞、及分 離抗體。 在一實施例中，任一種上述抗體均為單株抗體。在一實 施例中’抗體為選自Fab、Fab，-SH、Fv、scFv或（Fab，）2片 段之抗體片段。在一實施例中，抗體為人類化抗體。在一 實施例中，抗體為人類抗體。 【實施方式】 本發明提供以組合療法使用Axl拮抗劑及VEGF拮抗劑治 療諸如癌症之病理病狀之方法。 本發明亦提供結合Axl之分離抗體及使用其之方法，例 如使用其診斷或治療諸如以下之癌症的方法：骨髓性白血 病、肺癌（例如非小細胞肺癌（NSCLC))、胃癌、乳癌、前 列腺癌、腎細胞癌、胰臟癌及神經膠母細胞瘤。 I. 一般技術Examples of Axl antagonists include, but are not limited to, soluble Axl receptors, soluble Axl ligand variants, specific for Axl or Axl ligands, 149799.doc -10- 201106972some or peptibosome, Axl small Molecule, anti-Axl antibody and anti-Axl ligand antibody. In some embodiments, the Axl antagonist is an anti-Axl antibody. In some embodiments, the Axl antagonist is not warfarin. In some embodiments, the anti-Axl antibody comprises a sequence comprising EVQLVESG GGLVQPGGSLRLSCAASGFSLSGSWIHWVRQAPGKGLEW VGWINPYRGYAYYADSVKGRFTISADTSKNTAYLQMNSLR AEDTAVYYCAREYSGWGGSSVGYAMDYWGQGTLV (SEQ ID ΝΟ: 1) and a heavy chain variable region comprising the sequence DIQMTQSPSSLSASV GDRVTITCRASQDVSTAYAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTF GQGTKVEIKR (SEQIDNO: 2) light chain variable region. In some embodiments, the Axl antagonist is R428 (Rigel; Holland et al, Cancer Research 70, 1544 (2010)) or PF02341066. In certain embodiments, the method further comprises administering an EGFR antagonist. In certain embodiments, the EGFR antagonist is erlotinib. In certain embodiments, erlotinib can be administered at a dose of 150 mg per day or at a dose of 100 mg per day. The methods of the invention can be used to alter any suitable pathological condition. For example, the methods of the invention can be used to treat different cancers (solid and liquid tumors and similar soft tissue tumors). Non-limiting examples of cancers that can be treated in accordance with the therapies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cell carcinoma, prostate Cancer, liver cancer (such as hepatocellular carcinoma), pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma (kaPosi's 149799.doc 11 201106972 ma) carcinoid carcinoma, head and neck cancer, dysplasia, ovarian cancer, stomach cancer , mesothelioma and multiple myeloma. In some cases, the cancer is metastatic cancer. In other aspects, the cancer is non-metastatic cancer. In some embodiments, an anti-Αχ1 antibody and an anti-VEGF antibody (such as bevacizumab) are used in combination therapies such as cancer of non-small cell lung cancer. The combination therapies of the invention may be combined with other therapeutic agents such as chemotherapeutic agents or other therapies such as radiation therapy or surgery, depending on the particular indication being treated. Many known chemotherapeutic agents are useful in the combination therapies of the present invention. In certain embodiments, the combination therapies of the invention can be combined with one or more chemotherapeutic agents. In certain embodiments, the chemotherapeutic agents used are standard agents for the treatment of a particular indication. In another embodiment, the dosage or frequency of the various therapeutic agents used in combination is equal to or less than the dose or frequency of the respective therapeutic agent when no other therapeutic agent is used. In certain embodiments, the cancer exhibits Axl expression (such as Axl overexpression), amplification or activation. In certain embodiments, the individual is diagnosed with a cancer that exhibits Axl. The invention also provides a method of inhibiting constitutive Axl activation comprising rendering cells with down-regulated Axi expression (reducing, for example, Axl expression on a cell surface (such as a tumor cell surface) and/or reducing overall expression in cells (such as tumor cells) The antibody (or antigen-binding fragment thereof) is contacted. The invention also provides a method of treating cancer in a subject comprising administering to the individual a down-regulated Axl expression (reducing, for example, Axl expression on a cell surface (such as a tumor cell surface) and/or reducing overall Axl expression in a cell (such as a tumor cell)) 149799.doc -12- 201106972 anti-Axl antibody. The invention also provides a method for blocking binding of a ligand to Axl, comprising contacting a cell expressing Axl with an antibody that binds to Axl and inhibits binding of the ligand to Axl, or an antigen-binding fragment thereof, wherein the antibody recognizes human Axl instead of Small % of the epitope on mouse Axl. The monoclonal antibody may be, for example, a chimeric antibody, a humanized antibody, a single chain antibody or a bispecific antibody. In some embodiments, the epitope to which the antibody binds is the same as the epitope bound to the monoclonal antibody selected from the group consisting of 3G9, 8B5, 12A11, and 4F8(R). In some embodiments, the antibody inhibits binding of the ligand to Axl. In some embodiments, the antibody downregulates Axl expression. In some embodiments, the antibody recognizes a variable epitope on Αχ ί as detected by Western blot. In some embodiments, the antibody has an IC5 〇 of at least about 1 ng/ml. In some embodiments, the antibody inhibits Gas6-induced Axl phosphorylation. In one aspect, the present invention provides an antibody produced by the fusion tumor cell line 3G9· 19.7, and the fusion tumor cell line 3G9.19.7 has the American Type Culture Collection Center (ATCC) No. _, deposited in _. In one aspect, the invention provides an isolated antibody comprising a heavy chain and/or light of an antibody produced by a fusion cell line of the American Type Culture Collection (ATCC) number _______, 3G9.19·7 A chain variable domain in which the antibody specifically binds to human Axl. In one aspect, the invention provides an isolated antibody comprising at least one (at least 2, at least 3 'at least 4, at least 5, and/or 6) hypervariable sequences (Hvr) 'this (etc.) The hypervariable sequence comprises HVR-L1, HVR.L2, HVR-L3, HYR- selected from the group 149799.doc 13 201106972 antibody produced by the American Type Culture Collection Center (ATCC) number--- fusion cell line 3G9.19.7 A sequence of a population consisting of H1, HVH-m and/or HVR-H3, wherein the isolated antibody specifically binds to human Axl. In one aspect, the invention provides a human that binds to an epitope on human Αχ1 in combination with an antibody produced by a fusion cell cell line 3G9.19.7 having the American Type Culture Collection (ATCC) number ______ The isolated antibody with the same epitope on Αχ1. In one aspect, the present invention provides an isolated antibody that competes with human Axl for competition with an antibody produced by the fusion cell line 3G9.19.7 having the American Type Culture Collection (ATCC) number. The invention also provides a method of downregulating Axl expression in a cell comprising contacting the cell with an antibody that causes a decrease in Axl expression in the cell. The antibody can be a monoclonal antibody. The antibody may be, for example, a chimeric antibody, a humanized antibody, a single chain antibody or a bispecific antibody. In some embodiments, the antibody-binding antigenic determinant is the same as the epitope conjugated to a monoclonal antibody selected from the group consisting of 3G9, 8B5, 12A11, and 4F8. In some embodiments, the antibody inhibits binding of the ligand to Axl. In some embodiments, the antibody recognizes a variability epitope on Αχ ί as determined by Western blotting. In some embodiments, the antibody has an IC5 〇 of at least about 100 ng/ml. In some embodiments, the antibody inhibits Gas6-induced Axl acidification. The invention also provides a method of detecting Axl in a sample comprising contacting the sample with an antibody that specifically binds to an epitope on Axl and does not cross-react with other proteins in the sample. In some embodiments, the antibody-binding epitope is the same as the epitope bound by a monoclonal antibody selected from the group consisting of 3G9, 8B5, 12A11, and 4F8, 149799.doc • 14-201106972. In some embodiments, the antibody recognizes a denatured epitope on Axl, as can be measured by Western blotting. In some embodiments, the ICS(s) of the antibody is at least about 丨 (9) 叩 "different 纟 月 月 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( A method of a patient comprising obtaining a sample from the patient and analyzing the sample for Axl expression, wherein the assay comprises using the bound epitope and a group selected from 3G9, 8B5, 丨2A11, and Q8, The antibody or antigen-binding fragment thereof binds to the same steroid of the epitope. In some embodiments, the antibody is 3G9, 8B5, 12A11, 4F8, an antigen-binding fragment thereof, or a combination thereof. The antibody may be a monoclonal antibody, A chimeric antibody, a humanized antibody, a single chain antibody, or an antigen binding fragment thereof. In some embodiments, the antibody is associated with a detectable label such as, but not limited to, a radioisotope, a fluorescent compound, or an enzyme label. Also provided is a method of treating cancer in a patient comprising: (a) detecting an overexpression of Axl in a sample of the patient using the first antibody; and (b) administering to the subject a second antibody and pharmaceutically acceptable accept A carrier composition, wherein the first antibody specifically binds to an epitope on human Axl and the second antibody inhibits binding of the ligand to Axl, phosphorylation of Axl, or downregulation of Axl. Cancer may be selected Free cancer of the following group: myeloid. Leukemia, lung cancer (eg, non-small cell lung cancer (NSCLC)), gastric cancer, breast cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, and glioblastoma. In some embodiments Wherein the Axl epitope bound by the first antibody is identical to the Axl epitope bound to the monoclonal antibody or antigen-binding fragment thereof selected from the group consisting of 3G9, 8B5, 12A11 and 4F8. In some embodiments, 149799. Doc -15- 201106972 The second antibody-binding Axl epitope is identical to the Axl epitope bound by a monoclonal antibody or antigen-binding fragment thereof selected from the group consisting of 3G9, 8B5, 12A11 and 4F8. In some embodiments The first antibody and the second antibody are monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies or antigen-binding fragments thereof. In some embodiments, the first antibody and/or the second antibody are A cytotoxic agent such as, but not limited to, a chemotherapeutic agent, a toxin or a radioisotope is combined. In some embodiments, the method further comprises administering an anti-VEGF antibody before, after or simultaneously with administration of the antibody that binds Axl. The invention also provides a method of inhibiting angiogenesis in cells overexpressing Axl, which comprises administering to such cells an antibody that specifically binds to an epitope on human Axl and inhibits binding of the ligand to Αχ, Axl acidation Or down-regulating Axl performance. Cancer may be a cancer selected from the group consisting of myeloid leukemia, lung cancer (eg, non-small cell lung cancer (NSCLC)), gastric cancer, breast cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, and nerve glue. Maternal tumor. In some embodiments, the epitope to which the antibody binds is the same as the epitope bound to the antibody or antigen-binding fragment thereof selected from the group consisting of 3G9, 8B5, 12A11 and 4F8. In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a single chain antibody, or an antigen binding fragment thereof. In some embodiments, the antibody is combined with a cytotoxic agent such as, but not limited to, a chemotherapeutic agent, a toxin or a radioisotope. In some embodiments, the method further comprises administering an anti-VEGF antibody before, after or simultaneously with administering the antibody that binds to Ax. The anti-Axl antibody and the anti-VEGF antibody can be in the form of multispecific antibodies such as, but not limited to, bispecific antibodies. 149799.doc -16· 201106972 In one aspect, the invention provides an anti-Axl antibody and methods of use thereof. In some embodiments, the invention provides a monoclonal antibody that specifically binds to an epitope of human Axl but not mouse Axl, or an antigen-binding fragment of the antibody. The monoclonal antibody may be, for example, a chimeric antibody, a humanized antibody, a single chain antibody or a bispecific antibody. In some embodiments, the antibody-binding epitope is the same as the epitope bound to a monoclonal antibody selected from the group consisting of 3G9, 8B5, 12A11, and 4F8. In some embodiments, the antibody inhibits binding of the ligand to Axl. In some embodiments, the antibody downregulates Axl expression. In some embodiments, the antibody recognizes a denatured epitope on Axl, as detected by Western blotting. In some embodiments, the antibody has an ICso of at least about 100 ng/ml. In some embodiments, the antibody inhibits Gas6-induced Axl phosphorylation. The invention also provides a pharmaceutical composition comprising a monoclonal antibody as described in the preceding paragraph and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises an anti-VEGF antibody. The present invention further provides an article comprising a container, a composition contained in the container, and a pharmaceutical product, wherein the composition comprises a combined Αχ1 epitope and a group selected from the group consisting of 3G9, 8B5, 12A11, and 4F8. The monoclonal antibody or the antigen-binding fragment thereof binds to the same monoclonal antibody of the Αχ1 epitope, and the pharmaceutical manufacturer's instructions include instructions for use of the composition. In some conjugates, the antibody is a monoclonal antibody selected from the group consisting of 3G9, 8B5, 12A11 and 4F8. In one aspect, a polynucleotide encoding any of the above antibodies is provided. In one embodiment, a vector comprising a polynucleotide is provided. In one embodiment, 149799.doc -17- 201106972 is available for host cells containing the vector. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a CHO cell. In one embodiment, a method of making an anti-Axl antibody, wherein the method comprises culturing a host cell, and isolating the antibody under conditions suitable for displaying the polynucleotide encoding the antibody. In one embodiment, any of the above antibodies are monoclonal antibodies. In one embodiment the antibody is an antibody fragment selected from the group consisting of Fab, Fab, -SH, Fv, scFv or (Fab,). In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a human antibody. [Embodiment] The present invention provides a method of treating a pathological condition such as cancer using an Axl antagonist and a VEGF antagonist in combination therapy. The present invention also provides an isolated antibody that binds to Axl and a method of using the same, for example, a method for diagnosing or treating cancer such as: myeloid leukemia, lung cancer (for example, non-small cell lung cancer (NSCLC)), gastric cancer, breast cancer, prostate cancer , renal cell carcinoma, pancreatic cancer, and glioblastoma. I. General technology

本文中所述或所參考之技術及程序為熟習該項技術者已 廣泛瞭解且共同使用的習知方法，諸如描述於以下文獻中 之廣泛使用方法·· Sambrook等人，Mo/ecw/ar JThe techniques and procedures described or referenced herein are well-known methods that are widely known and commonly used by those skilled in the art, such as the widely used methods described in the following documents: Sambrook et al., Mo/ecw/ar J

Laboratory Manual 第 3 版（2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. ； Current Proioco/s /n Mo/ecw/ar (F. M· Ausubel，等人編， (2003));叢刊 Enz少wo/〇g_y (Academic Press, 149799.doc •18· 201106972Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Proioco/s /n Mo/ecw/ar (F. M. Ausubel, et al., (2003)); Series Enz less Wo/〇g_y (Academic Press, 149799.doc •18· 201106972

Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames及 G· R. Taylor編（1995)), Harlow及 Larie編（1988) Antibodies, A Laboratory Manual^ Animal Cell Culture (R. I. Freshney 編（1987)) ; (M. J.Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and G. R. Taylor (1995)), Harlow and Larie (1988) Antibodies, A Laboratory Manual^ Animal Cell Culture (RI Freshney ed. (1987) )) ; (MJ

Gait編，1984) ; /·« Mo/ecw/ar HumanaEdited by Gait, 1984) ; /·« Mo/ecw/ar Humana

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Freshney)編，1987) ; Introduction to Cellj and Tissue Cw/iwre (J. P. Mather及 P. E. Roberts, 1998) Plenum Press ;Freshney), 1987); Introduction to Cellj and Tissue Cw/iwre (J. P. Mather and P. E. Roberts, 1998) Plenum Press ;

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Sons ； Handbook of Experimental Immunology (D. M. Weir 及 C. C. Blackwell編）；Fecior·?/or Mamwa"a« Cells (J. M. Miller AM. P. Calos 編，1987) ; PCi?: 77ie Po/ywerc^e C/zaz'w jReaciz'on，（Mullis等人編，1994) ; CwrreW /Woco/s k /www«o/og_y (J. E. Coligan等人編，1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999) ; /ww㈣oZ?z'o/og_y (C. A. Janeway 及 P· Travers， 1997) ; Antibodies (P. Finch, 1997) ; Antibodies: A Prac"ca/ (D. Catty.編，IRL Press, 1988-1989);Sons ; Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell); Fecior·?/or Mamwa"a« Cells (edited by JM Miller AM. P. Calos, 1987); PCi?: 77ie Po/ywerc^e C/zaz 'w jReaciz'on, (Mullis et al., ed., 1994); CwrreW /Woco/sk /www«o/og_y (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999) ; Ww (iv) oZ?z'o/og_y (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Prac"ca/ (D. Catty. ed., IRL Press, 1988-1989);

Monoclonal Antibodies: A Practical Approach (P. Shepherd 及 C. Dean 編，Oxford University Press, 2000) ； Using J Mawwa/ (E. Harlow 及 D. Lane (Cold Spring Harbor Laboratory Press, 1999) ； The 149799.doc •19- 201106972 山'es (M. Zanetti及 J. D. Capra編，Harwood AcademicMonoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000); Using J Mawwa/ (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The 149799.doc • 19- 201106972 山'es (M. Zanetti and JD Capra, Harwood Academic

Publishers, 1995);及 Ccmcer.. Practice 〇/Publishers, 1995); and Ccmcer.. Practice 〇/

Owco/og_y (V. T. DeVita等人編 ’ J.B. Lippincott Company, 1993)〇 II.定義 為解釋本說明書之目的，以下定義將適用，且只要適合 時’以單數所用之術語亦將包括複數，反之亦然。若下述 任何定義與以引用的方式併入本文中之任何文獻相悖，則 以下述定義為準。 多肽「變異體」意謂與原生序列多肽具有至少約80〇/〇胺 基酸序列一致性之生物學活性多肽。該等變異體包括例如 在多肽之N或C端添加或缺失一或多個胺基酸殘基之多 狀。通常’變異體與原生序列多肽將具有至少約8〇%胺基 酸序列一致性、更佳至少約9〇%胺基酸序列一致性且甚至 更佳至少約95%胺基酸序列一致性。 「原生序列」多肽包含與源自自然界之多肽具有相同胺 基酸序列之多肽。因此，原生序列多肽可具有任何哺乳動 物之天然存在多肽的胺基酸序列。該原生序列多肽可自自 然界分離，或可由重組或合成方法製備。術語「原生序 列」多肽特定包涵天然存在之截短型或分泌型多肽（例如 胞外域序列）、天然存在之變異型（例如選擇性剪接型）多肽 及天然存在之對偶基因變異體多肽。 抗Axl抗體」為以足夠親和力及特異性結合Αχι之抗 體。所選抗體通常對Axl具有足夠強之結合親和力，例如 149799.doc -20· 201106972 抗體可以100 nM-l pM之間的Kd值結合人類Axl。在某些實 施例中，結合Axl之抗體具有$1 μΜ、£100 nM、$10 nM、 SI nM或£0.1 nM之解離常數（Kd)。舉例而言，抗體親和力 可藉由例如基於表面電漿子共振之分析法（諸如描述於PCT 申請公開案第WO 2005/012359號中之BIAcore分析法）、酶 聯免疫吸附分析法（ELISA)及競爭分析法（例如RIA)測定。 較佳地，抗Axl抗體與不相關非Axl蛋白之結合程度小於該 抗體與Axl之結合程度之約10%，如藉由例如放射免疫分 析法（RIA)所量測。在某些實施例中，抗Axl抗體可用作靶 向及干預涉及Axl活性之疾病或病狀的治療劑。此外，可 對抗體進行其他生物活性分析法，例如以評估其作為治療 劑之有效性。該等分析法在此項技術中已知且視標靶抗原 及抗體之預定用途而定。 「Axl拮抗劑」（可互換地稱為「Axl抑制劑」）為干擾 Axl活化或功能之藥劑。Axl抑制劑之實例包括Axl抗體； Axl配位體抗體；小分子Axl拮抗劑；Axl酪胺酸激酶抑制 劑；反義及抑制性RNA(例如shRNA或siRNA)分子（參見例 如WO 2004/87207)。較佳地，Axl抑制劑為結合Axl之抗體 或小分子。在一特定實施例中，Axl抑制劑對Axl具有約 1,000 nM或小於1,000 nM之結合親和力（解離常數）。在另 一實施例中，Axl抑制劑對Axl具有約100 nM或小於100 nM 之結合親和力。在另一實施例中，Axl抑制劑對Axl具有約 50 nM或小於50 nM之結合親和力。在一特定實施例中， Axl抑制劑與Axl共價結合。在一特定實施例中，Axl抑制 149799.doc -21 201106972 劑以1,000 nM或小於1,000 nM之IC5〇抑制Axl信號傳導。在 另一實施例中，Axl抑制劑以500 nM或小於500 nM之IC50 抑制Axl信號傳導。在另一實施例中，Axl抑制劑以50 nM 或小於50 nM之IC5〇抑制Axl信號傳導。 「Axl活化」係指Axl受體之活化或麟酸化。通常，Αχί 活化引起信號轉導（例如由Axl受體之胞内激酶域使Axl或 受質多肽内之酪胺酸殘基磷酸化所引起）。Axl活化可由 Axl配位體（例如Gas6)結合相關Axl受體介導。Axl配位體 結合Axl可活化Axl之激酶域且由此引起Axl中之酷胺酸殘 基磷酸化及/或其他受質多肽中之酪胺酸殘基磷酸化。 如本文中所用之術語「配位體非依賴性」，例如應用於 受體信號傳導活性時，係指信號傳導活性不依賴於配位體 之存在。舉例而言，EGFR信號傳導可由與HER家族之其 他成員（諸如HER2)二聚所引起。受體具有配位體非依賴性 激酶活性不一定會妨礙配位體與該受體之結合，從而產生 激酶活性之其他活化。 如本文中所用之術語「組成性」，例如應用於受體激酶 活性時’係指受體之連續信號傳導活性不依賴於配位體或 八他活化分子之存在。舉例而言，常見於多形性神經膠母 細胞瘤中之EGFR變異體m(EGFRvIII)已缺失其大部分胞 外域。儘管配位體不能結合£(}1?以111，然而其持續具有活 性且與異常增殖及存活相關。視受體性質而定，所有活性 可為、’且成性的或受體之活性可由其他分子（例如配位體）結 〇進步活化。一般熟習此項技術者熟知引起受體活化之 149799.doc -22- 201106972 細胞事件。舉例而言，活化可包括寡聚（例如二聚、三聚 等）成高級受體複合物。複合物可包含單一物種之蛋白 質，亦即均聚複合物。或者，複合物可包含至少兩個不同 物種之蛋白質，亦即雜聚複合物。複合物形成可由例如正 常或突變型受體在細胞表面上過度表現而引起。複合物形 成亦可由受體之一或多個特異性突變而引起。 術語「VEGF」或「VEGF-Α」用於指165-胺基酸人類血 管内皮細胞生長因子及相關的121-胺基酸、189-胺基酸及 206-胺基酸人類血管内皮細胞生長因子，如Leung等人， Science, 246:13 06 (1989)及 Houck 等人，Mol. Endocrin., 5:1806 (1991)所述，以及其天然存在之對偶基因形式及經 處理形式。VEGF-A為包括 VEGF-B、VEGF-C、VEGF-D、 VEGF-E、VEGF-F及P1GF之基因家族之一部分。VEGF-A 主要結合兩種高親和力受體酪胺酸激酶VEGFR-1 (Fit-1)及 VEGFR-2(Flk-l/KDR)，VEGFR-2(Flk-l/KDR)為 VEGF-A之 血管内皮細胞致有絲***信號傳導之主要傳遞質。此外， 神經纖毛蛋白-1已鑑別為結合肝素之VEGF-A同功異型物 之受體且可在血管發育中起重要作用。術語「VEGF」或 「VEGF-A」亦指非人類物種（諸如小鼠、大鼠或靈長類動 物）之VEGF。特定物種之VEGF有時用諸如hVEGF(對於人 類VEGF)或mVEGF(對於鼠類VEGF)之術語表示。術語 「VEGF」亦用於指包含165-胺基酸人類血管内皮細胞生 長因子之胺基酸8至109或1至109之多肽之截短形式或片 段。在本案中，提及任何該等形式之VEGF均可用例如 149799.doc -23- 201106972 「VEGF(8-109)」、「VEGF(1-109)」或「VEGF165」標識。 「截短」型原生VEGF之胺基酸位置如原生VEGF序列中所 示進行編號。舉例而言，截短型原生VEGF中之胺基酸位 置17(曱硫胺酸）亦為原生VEGF中之位置17(曱硫胺酸）。截 短型原生VEGF對KDR及Flt-Ι受體之結合親和力與原生 VEGF相當。 如本文中所用之術語「VEGF變異體」係指在原生VEGF 序列中包括一或多個胺基酸突變的VEGF多肽。一或多個 胺基酸突變視情況包括胺基酸取代。為簡寫本文中所述之 VEGF變異體名稱之目的，應注意編號係指沿公認原生 VEGF之胺基酸序列之胺基酸殘基位置（Leung等人（同上）及 Houck等人（同上）提供）。 「VEGF生物活性」包括與任何VEGF受體結合或任何 VEGF信號傳導活性，諸如調節正常與異常血管生成及血 小管生成（Ferrara 及 Davis-Smyth (1997) 18:4-25 ； Ferrara (1999) J. Mol. Med. 77:527-543)；促進胚 胎jk小管生成及血管生成（Carmeliet等人，（1996) TVaiwre 380:435-439 ; Ferrara等人，（1996) iVai㈣ 380:439-442); 及調節雌性生殖道中環狀血管增生及骨骼生長及軟骨形成 (Ferrara 等人，（1998) iVaiwT-e Met/· 4:336-340 ; Gerber 等 人，（1999) Mei 5:623-628)。除作為血管生成及血 小管生成中之血管生成因子之外，作為多效性生長因子之 VEGF亦在其他生理學過程（諸如内皮細胞存活、血管滲透 性及血管擴張、單核細胞趨化性及鈣流入）中展現多重生 149799.doc -24- 201106972 物效應（Ferrara及Davis-Smyth (1997),同上及Cebe-Suarez 等人，Ο//· Mo/· Zz/e &ζ· 63:601-615 (2006))。此外，最 近研究已報導VEGF對少數非内皮細胞類型（諸如視網膜色 素上皮細胞、胰管細胞及許旺氏細胞（Schwann cell))的促 有絲***作用。Guerrin等人，（1995) ·/. Ce// 164: 385-394 ； Oberg-Welsh^ A 5 (1997) Mol. Cell. Endocrinol. 126:125-132 ; Sondell等人，（1999) /_ 19:5731- 5740 ° 「血管生成抑制劑」或「抗血管生成劑」係指直接或間 接抑制血管生成、血小管生成或非所要之血管通透性的小 分子量物質、聚核苷酸、多肽、分離蛋白、重組蛋白、抗 體、或其結合物或融合蛋白。應瞭解，抗血管生成劑包括 結合血管生成因子或其受體且阻斷血管生成因子或其受體 之血管生成活性的抗血·管生成劑。舉例而言，抗血管生成 劑為如上文所定義之抗體或其他血管生成劑拮抗劑，例如 抗VEGF-A或VEGF-A受體（例如KDR受體或Flt-Ι受體）之抗 體、抗PDGFR抑制劑，諸如GLEEVEC®(甲磺酸伊馬替尼 (Imatinib Mesylate))。抗血管生成劑亦包括原生血管生成 抑制劑’例如血管抑制素、内皮抑制素等。參見例如 Klagsbrun 及 D'Amore，心仙.如ν ρ/υΛ5ζ·〇/ , 53:217_39 (1991)，Streit及 Detmar, 〇„c〇_e, 22·3172 3179 (2〇〇3) (例如表3列出惡性黑色素瘤之抗血管生成療法）；Owco/og_y (VT DeVita et al., eds. JB Lippincott Company, 1993) 〇 II. Definitions For the purposes of this specification, the following definitions will apply, and where appropriate, the terms used in the singular will also include the plural and vice versa. . In the event that any of the following definitions are contrary to any of the documents incorporated by reference, the following definitions will control. A polypeptide "variant" means a biologically active polypeptide having at least about 80 〇/〇 amino acid sequence identity to a native sequence polypeptide. Such variants include, for example, the addition or deletion of one or more amino acid residues at the N or C terminus of the polypeptide. Typically, the 'variant and native sequence polypeptide will have at least about 8% amino acid sequence identity, more preferably at least about 9% amino acid sequence identity and even more preferably at least about 95% amino acid sequence identity. A "native sequence" polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide derived from nature. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring polypeptide of any mammal. The native sequence polypeptide can be isolated from the natural boundaries or can be prepared by recombinant or synthetic methods. The term "primary sequence" polypeptide specifically encompasses naturally occurring truncated or secreted polypeptides (e.g., extracellular domain sequences), naturally occurring variant (e.g., alternatively spliced) polypeptides, and naturally occurring dual gene variant polypeptides. The anti-Axl antibody" is an antibody that binds to Αχι with sufficient affinity and specificity. The selected antibody typically has a sufficiently strong binding affinity for Axl, for example 149799.doc-20-201106972 The antibody binds to human Axl with a Kd value between 100 nM and 1 pM. In certain embodiments, the antibody that binds Axl has a dissociation constant (Kd) of $1 μΜ, £100 nM, $10 nM, SI nM, or £0.1 nM. For example, antibody affinity can be determined, for example, by surface plasmon resonance based analysis (such as the BIAcore assay described in PCT Application Publication No. WO 2005/012359), enzyme-linked immunosorbent assay (ELISA), and Competitive analysis (eg RIA) determination. Preferably, the degree of binding of the anti-Axl antibody to the unrelated non-Axl protein is less than about 10% of the degree of binding of the antibody to Axl, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an anti-Axl antibody can be used as a therapeutic agent for targeting and interfering with a disease or condition involving Axl activity. In addition, other biological activity assays can be performed on the antibody, for example to assess its effectiveness as a therapeutic. Such assays are known in the art and depend on the intended use of the target antigen and antibody. "Axl antagonists" (interchangeably referred to as "Axl inhibitors") are agents that interfere with Axl activation or function. Examples of Axl inhibitors include Axl antibodies; Axl ligand antibodies; small molecule Axl antagonists; Axl tyrosine kinase inhibitors; antisense and inhibitory RNA (eg, shRNA or siRNA) molecules (see, eg, WO 2004/87207) . Preferably, the Axl inhibitor is an antibody or small molecule that binds to Axl. In a particular embodiment, the Axl inhibitor has a binding affinity (dissociation constant) of about 1,000 nM or less for Axl. In another embodiment, the Axl inhibitor has a binding affinity for Axl of about 100 nM or less. In another embodiment, the Axl inhibitor has a binding affinity for Axl of about 50 nM or less than 50 nM. In a specific embodiment, the Axl inhibitor is covalently bound to Axl. In a specific embodiment, Axl inhibits 149799.doc -21 201106972 to inhibit Axl signaling at an IC5 of 1,000 nM or less. In another embodiment, the Axl inhibitor inhibits Axl signaling with an IC50 of 500 nM or less. In another embodiment, the Axl inhibitor inhibits Axl signaling at 50 nM or less than 50 nM of IC5. "Axl activation" refers to the activation or linonication of the Axl receptor. Typically, Αχί activation causes signal transduction (e. g., caused by phosphorylation of Axl or a tyrosine residue in a polypeptide of interest by the intracellular kinase domain of the Axl receptor). Axl activation can be mediated by binding of an Axl ligand (e.g., Gas6) to a related Axl receptor. Axl ligand binding to Axl activates the kinase domain of Axl and thereby causes phosphorylation of valine residues in Axl and/or phosphorylation of tyrosine residues in other receptor polypeptides. The term "ligand-independent" as used herein, for example, when applied to receptor signaling activity, means that signaling activity is independent of the presence of a ligand. For example, EGFR signaling can be caused by dimerization with other members of the HER family, such as HER2. Receptor-having ligand-independent kinase activity does not necessarily interfere with the binding of the ligand to the receptor, resulting in additional activation of kinase activity. The term "constitutive" as used herein, for example when applied to a receptor kinase activity, refers to the continuous signaling activity of a receptor independent of the presence of a ligand or an octa-activated molecule. For example, the EGFR variant m (EGFRvIII), which is commonly found in pleomorphic glioblastoma, has lost most of its extracellular domain. Although the ligand does not bind £(}1? to 111, it is continuously active and is associated with abnormal proliferation and survival. Depending on the nature of the receptor, all activities may be, 'and adult or receptor activity may be Other molecules (e.g., ligands) are progressively activated. The 149799.doc -22-201106972 cellular events that cause receptor activation are well known to those skilled in the art. For example, activation can include oligomerization (e.g., dimerization, tristimulus). The compound may be a high-level receptor complex. The complex may comprise a protein of a single species, that is, a homo-poly complex. Alternatively, the complex may comprise at least two proteins of different species, that is, a hetero-poly complex. It can be caused by, for example, excessive expression on the cell surface by normal or mutant receptors. Complex formation can also be caused by one or more specific mutations in the receptor. The term "VEGF" or "VEGF-Α" is used to refer to 165- Amino acid human vascular endothelial growth factor and related 121-amino acid, 189-amino acid and 206-amino acid human vascular endothelial growth factor, such as Leung et al, Science, 246:13 06 (1989) And Houck et al., Mol. Endocrin., 5:1806 (1991), and its naturally occurring dual gene form and treated form. VEGF-A includes VEGF-B, VEGF-C, VEGF-D, VEGF- Part of the gene family of E, VEGF-F and P1GF. VEGF-A mainly binds to two high-affinity receptors, tyrosine kinases VEGFR-1 (Fit-1) and VEGFR-2 (Flk-1/KDR), VEGFR- 2 (Flk-l/KDR) is the major mediator of mitogenic signaling induced by vascular endothelial cells of VEGF-A. In addition, neuropilin-1 has been identified as a receptor for heparin-binding VEGF-A isoforms and It plays an important role in vascular development. The term "VEGF" or "VEGF-A" also refers to VEGF of non-human species (such as mice, rats or primates). VEGF of specific species is sometimes used such as hVEGF (for The term "human VEGF) or mVEGF (for murine VEGF) is also used. The term "VEGF" is also used to refer to a polypeptide comprising amino acid 8 to 109 or 1 to 109 of 165-amino acid human vascular endothelial growth factor. Short form or fragment. In the present case, reference to any of these forms of VEGF can be used, for example, 149799.doc -23- 201106972 "VEGF (8-1) 09)", "VEGF(1-109)" or "VEGF165". The amino acid position of the "truncated" type of native VEGF is numbered as shown in the native VEGF sequence. For example, truncated native VEGF The amino acid position 17 (anthracene thioglycolate) is also position 17 (anthracene thiocyanate) in native VEGF. The binding affinity of the truncated native VEGF to KDR and Flt-Ι receptors is comparable to that of native VEGF. The term "VEGF variant" as used herein refers to a VEGF polypeptide comprising one or more amino acid mutations in the native VEGF sequence. One or more amino acid mutations optionally include amino acid substitutions. For the purposes of abbreviating the names of the VEGF variants described herein, it should be noted that the numbering refers to the position of the amino acid residue along the amino acid sequence of the recognized native VEGF (Leung et al. (supra) and Houck et al. (supra). ). "VEGF biological activity" includes binding to any VEGF receptor or any VEGF signaling activity, such as regulation of normal and abnormal angiogenesis and tubule formation (Ferrara and Davis-Smyth (1997) 18:4-25; Ferrara (1999) J Mol. Med. 77: 527-543); promotes embryonic jk tubule formation and angiogenesis (Carmeliet et al., (1996) TVaiwre 380: 435-439; Ferrara et al., (1996) iVai (iv) 380: 439-442); And regulation of circular vascular proliferation and bone growth and cartilage formation in the female reproductive tract (Ferrara et al., (1998) iVaiw T-e Met/. 4: 336-340; Gerber et al., (1999) Mei 5: 623-628). In addition to angiogenic factors in angiogenesis and tubule formation, VEGF as a pleiotropic growth factor is also involved in other physiological processes such as endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis, and Influx of calcium in the presence of multiple births 149799.doc -24- 201106972 effects (Ferrara and Davis-Smyth (1997), ibid. and Cebe-Suarez et al., Ο//· Mo/· Zz/e & ζ· 63: 601-615 (2006)). In addition, recent studies have reported mitogenic effects of VEGF on a few non-endothelial cell types such as retinal pigment epithelial cells, pancreatic duct cells, and Schwann cells. Guerrin et al. (1995) ·/. Ce// 164: 385-394; Oberg-Welsh^ A 5 (1997) Mol. Cell. Endocrinol. 126:125-132; Sondell et al., (1999) /_ 19 :5731- 5740 ° "Angiogenesis inhibitor" or "anti-angiogenic agent" refers to small molecular weight substances, polynucleotides, peptides that directly or indirectly inhibit angiogenesis, tubule formation or undesired vascular permeability. An isolated protein, recombinant protein, antibody, or a combination thereof or a fusion protein. It is to be understood that the anti-angiogenic agent includes an anti-blood agent which binds to an angiogenic factor or a receptor thereof and blocks angiogenic activity of an angiogenic factor or a receptor thereof. For example, an anti-angiogenic agent is an antibody or other angiogenic agent antagonist as defined above, eg, an antibody against VEGF-A or a VEGF-A receptor (eg, a KDR receptor or a Flt-Ι receptor), an antibody PDGFR inhibitors such as GLEEVEC® (Imatinib Mesylate). Anti-angiogenic agents also include pro-angiogenic inhibitors such as angiostatin, endostatin and the like. See, for example, Klagsbrun and D'Amore, Hearts. For example, ν ρ/υΛ5ζ·〇/ , 53:217_39 (1991), Streit and Detmar, 〇„c〇_e, 22·3172 3179 (2〇〇3) (eg Table 3 lists anti-angiogenic therapies for malignant melanoma);

Alitalo, TtoMre Me山cz«e 5:1359-1364 (1999) ; Tonini 等 人，22:6549-6556 (2003)(例如表2列出已知的抗 149799.doc -25- 201106972 血管生成因子）；及 Sato. /«，.《/. C//«. 0«co/·，8:200-206 (2003)(例如表1列出臨床試驗中所使用的抗血管生成劑）。 「VEGF拮抗劑」係指能夠中和、阻斷、抑制、中止、 減少或干擾VEGF活性（包括其與一或多個VEGF受體之結 合）的分子。在某些實施例中，VEGF拮抗劑將VEGF之表 現量或生物活性減少或抑制至少10%、20%、30%、40%、 50%、60%、70%、80%、90°/。或 90% 以上。在一實施例 中，受VEGF拮抗劑抑制之VEGF為VEGF(8-109)、 VEGF(1-109)或VEGF165。適用於本發明方法中之veGF拮 抗劑包括特異性結合VEGF之肽基或非肽基化合物，諸如 抗VEGF抗體及其抗原結合片段；特異性結合VEGF之多肽 或其片段；及特異結合VEGF、藉此隔絕其與一或多種受 體（例如可溶性VEGF受體蛋白或其VEGF結合片段，或嵌 合VEGF受體蛋白）結合之受體分子及衍生物；與編碼 VEGF多肽之核酸分子之至少一個片段互補的反義核苷鹼 基券聚物，與編碼VEGF多狀之核酸分子之至少一個片段 互補的小RNA ;靶向VEGF之核糖酶；抗VEGF之肽體；及 VEGF適體。 「抗VEGF抗體」為以足夠親和力及特異性結合VEGF之 抗體。所選抗體對VEGF通常具有足夠強之結合親和力， 例如抗體可以100 nM-1 pM之間的Kd值結合hVEGF。抗體 親和力可藉由例如基於表面電漿子共振之分析法（諸如描 述於PCT申請公開案第WO 2005/012359號中之BIAcore分 析法）、酶聯免疫吸附分析法（ELISA)及競爭分析法（例如 149799.doc -26- 201106972 RIA)測定。在某些實施例中，本發明之抗VEGF抗體可用 作把向及干預涉及VEGF活性之疾病或病狀的治療劑。此 外，可對抗體進行其他生物活性分析法，例如以評估其作 為治療劑之有效性。該等分析法在此項技術中已知且視標 靶抗原及抗體之預定用途而定。實例包括HUVEC抑制分 析法（如以下實例中所述）；腫瘤細胞生長抑制分析法（如 (例如）WO 89/06692中所述）；抗體依賴性細胞介導之細胞 毒性（ADCC)及補體介導之細胞毒性（CDC)分析法（美國專 利5,500,362);及拮抗活性或血細胞生成分析法（參見WO 95/27062) 〇抗VEGF抗體通常不會結合其他VEGF同源物， 諸如VEGF-B或VEGF-C，亦不會結合其他生長因子，諸如 P1GF、PDGF 或 bFGF。 在某些實施例中，抗VEGF抗體包括單株抗體，該單株 抗體所結合的抗原決定基與由融合瘤ATCC HB 10709所產 生之單株抗VEGF抗體A4.6.1(根據Presta等人，Cawcer/Je·?. 57:4593-4599 (1997)所產生之重組人類化抗VEGF單株抗 體）所結合之抗原決定基相同。在一實施例中，抗VEGF抗 體為「貝伐珠單抗（BV)」，亦稱為「rhuMAb VEGF」或 「AVASTIN®」。貝伐珠單抗包含突變型人類igGl構架區及 抗原結合互補決定區，抗原結合互補決定區來自阻斷人類 VEGF與其受體結合之鼠類抗hVEGF單株抗體A.4.6.1。貝 伐珠單抗（包括大部分構架區）之胺基酸序列中約93%來源 於人類IgGl ’且約7%序列來源於鼠類抗體A4.6.1。貝伐珠 單抗具有約149,000道爾頓（dalton)之分子量且經糖基化。 149799.doc •27- 201106972 貝伐珠單抗已由FDA批准與化學治療方案組合用於治療轉 移性結腸直腸癌（CRC)及非小細胞肺癌（NSCLC)。Hurwitz 等人 ’ N. Engl. J. Med. 350:2335-42 (2004)，Sandler 專 人，Ν· Engl. J. Med. 355:2542-50 (2006)。目前許多臨床 試驗正在對貝伐珠單抗治療各種癌症適應症進行研究。 Kerbel, 乂 19:45S-51S (2001) ; De Vore等人’ 尸roc. *Soc. C/M. 0加〇/· 19:485a. (2000) ; Hurwitz 等 人，Co/orecia/ C⑽cer 6:66-69 (2006) ; Johnson 等 人，Proc. dm. Soc. C7i«. 厂 20:315a (2001) ; Kabbinavar 等人，J. C"«_ 21:60-65 (2003) ; Miller 等人，Alitalo, TtoMre Me Mountain cz«e 5:1359-1364 (1999); Tonini et al., 22:6549-6556 (2003) (eg Table 2 lists known anti-149799.doc -25- 201106972 angiogenic factors) And Sato. / «,. "/. C / / «. 0 « co / ·, 8: 200-206 (2003) (for example, Table 1 lists the anti-angiogenic agents used in clinical trials). "VEGF antagonist" refers to a molecule that is capable of neutralizing, blocking, inhibiting, halting, reducing or interfering with VEGF activity, including its association with one or more VEGF receptors. In certain embodiments, the VEGF antagonist reduces or inhibits the performance or biological activity of VEGF by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90°/. Or more than 90%. In one embodiment, the VEGF that is inhibited by the VEGF antagonist is VEGF (8-109), VEGF (1-109) or VEGF165. VELGF antagonists suitable for use in the methods of the invention include peptidyl or non-peptidyl compounds that specifically bind to VEGF, such as anti-VEGF antibodies and antigen-binding fragments thereof; polypeptides or fragments thereof that specifically bind VEGF; and specifically bind to VEGF, This isolates a receptor molecule and derivative thereof that binds to one or more receptors (eg, a soluble VEGF receptor protein or a VEGF-binding fragment thereof, or a chimeric VEGF receptor protein); and at least one fragment of a nucleic acid molecule encoding a VEGF polypeptide A complementary antisense nucleobase conjugate, a small RNA complementary to at least one fragment encoding a VEGF polymorphic nucleic acid molecule; a VEGF ribozyme; an anti-VEGF peptibody; and a VEGF aptamer. An "anti-VEGF antibody" is an antibody that binds VEGF with sufficient affinity and specificity. The selected antibody typically has a sufficiently strong binding affinity for VEGF, for example, the antibody can bind to hVEGF with a Kd value between 100 nM-1 pM. The antibody affinity can be determined, for example, by surface plasmon resonance based analysis (such as the BIAcore assay described in PCT Application Publication No. WO 2005/012359), enzyme-linked immunosorbent assay (ELISA), and competition assay ( For example, 149799.doc -26- 201106972 RIA) Determination. In certain embodiments, an anti-VEGF antibody of the invention can be used as a therapeutic agent to target and interfere with a disease or condition involving VEGF activity. In addition, other biological activity assays can be performed on the antibody, for example to assess its effectiveness as a therapeutic. Such assays are known in the art and depend on the intended use of the target antigen and antibody. Examples include HUVEC inhibition assays (as described in the Examples below); tumor cell growth inhibition assays (as described, for example, in WO 89/06692); antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated Guided cytotoxicity (CDC) assay (U.S. Patent 5,500,362); and antagonistic activity or hematopoietic assays (see WO 95/27062). Anti-VEGF antibodies typically do not bind to other VEGF homologs, such as VEGF-B or VEGF. -C, also does not bind to other growth factors such as P1GF, PDGF or bFGF. In certain embodiments, the anti-VEGF antibody comprises a monoclonal antibody, the epitope bound by the monoclonal antibody and the monoclonal anti-VEGF antibody A4.6.1 produced by the fusion tumor ATCC HB 10709 (according to Presta et al., Cawcer /Je·?. 57:4593-4599 (1997) The recombinant humanized anti-VEGF monoclonal antibody produced by the antibody has the same antigenic determinant. In one embodiment, the anti-VEGF antibody is "bevacizumab (BV)", also known as "rhuMAb VEGF" or "AVASTIN®". Bevacizumab comprises a mutant human igG1 framework region and an antigen binding complementarity determining region, and the antigen binding complementarity determining region is derived from a murine anti-hVEGF monoclonal antibody A.4.6.1 which blocks binding of human VEGF to its receptor. Approximately 93% of the amino acid sequence of bevacizumab (including most of the framework regions) is derived from human IgG1' and approximately 7% of the sequence is derived from murine antibody A4.6.1. Bevacizumab has a molecular weight of about 149,000 daltons and is glycosylated. 149799.doc •27- 201106972 Bevacizumab has been approved by the FDA in combination with a chemotherapy regimen for the treatment of metastatic colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). Hurwitz et al. 'N. Engl. J. Med. 350: 2335-42 (2004), Sandler, Ν Engl. J. Med. 355: 2542-50 (2006). Many clinical trials are currently investigating the treatment of various cancer indications with bevacizumab. Kerbel, 乂19:45S-51S (2001); De Vore et al.' corpse roc. *Soc. C/M. 0 〇/· 19:485a. (2000) ; Hurwitz et al., Co/orecia/ C(10)cer 6 :66-69 (2006) ; Johnson et al., Proc. dm. Soc. C7i«. Plant 20:315a (2001); Kabbinavar et al., J. C"«_ 21:60-65 (2003); Miller et al. people,

Caw. TVeai. 94:Suppl 1:S6 (2005)。 貝伐珠單抗及其他人類化抗VEGF抗體進一步描述於 2005年2月26日頒予之美國專利第6,884,879號中。其他抗 體包括G6或B20系列抗體（例如G6-31，B20-4.1)，如PCT公 開案第 WO 2005/012359號、PCT公開案第 WO 2005/044853 號及美國專利申請案60/991,302中所述，此等專利申請案 之内容以引用方式明確併入本文中。關於其他抗體，參見 美國專利第 7,060,269 號、第 6,582,959 號、第 6,703,020 號 ' 第 6,054,297 號；WO 98/45332 ； WO 96/30046 ； WO 94/10202 ; EP 0666868B1 ;美國專利申請公開案第 2006009360號、第 20050186208號、第 20030206899號、第 20030190317號、第 20030203409 號及第 20050112126號； 及 Popkov 等人，Journal of Immunological Methods 288:149-164 (2004)。其他抗體包括可與包含殘基FI 7、 149799.doc -28- 201106972 M18、D19、Y21、Y25、Q89、191、K101、E103 及 C104 或者包含殘基 F17、Y21、Q22、Y25、D63、183 及 Q89 之 人類VEGF上之功能性抗原決定基結合的抗體。 本發明之「G6系列抗體」為來源於PCT公開案第WO 2005/012359號之圖7、24-26及34-35中任一者之G6抗體或 G6衍生抗體之序列的抗VEGF抗體，該案全部内容以引用 方式明確併入本文中。亦參見PCT公開案第WO 2005/044853號，該案全部内容以引用方式明確併入本文 中。在一實施例中，G6系列抗體與包含殘基F17、Y21、 Q22、Y25、D63、183及Q89之人類VEGF上之功能性抗原 決定基結合。 本發明之「B20系列抗體」為來源於PCT公開案第WO 2005/012359號之圖27-29中任一者之B20抗體或B20衍生抗 體之序列的抗VEGF抗體，該案全部内容以引用方式明確 併入本文中。亦參見PCT公開案第WO 2005/044853號及美 國專利申請案60/991，302，此等專利申請案之内容以引用 方式明確併入本文中。在一實施例中，B20系列抗體與包 含殘基 F17、M18、D19、Y21、Y25、Q89、191、K101、 E103及C104之人類VEGF上之功能性抗原決定基結合。 本發明之「功能性抗原決定基」係指抗原中之對抗體結 合起積極作用的胺基酸殘基。任一個起積極作用之抗原殘 基之突變（例如由丙胺酸或同系物突變引起之野生型VEGF 突變）將中斷抗體之結合使得抗體之相對親和力比率（IC50 突變型VEGF/IC50野生型VEGF)大於5(參見WO 2005/ 149799.doc -29- 201106972 012359之實例2)。在一實施例中，相對親和力比率係利用 溶液結合噬菌體呈現ELISA測定。簡言之，在4°C下用2 ug/ml濃度（含於PBS中）之待測試抗體之Fab形式塗佈96孔 Maxisorp免疫培養盤（NUNC)隔夜，且在室溫下用PBS、 0.5% BSA及0.05% Tween20(PBT)阻斷2小時。噬菌體呈現 hVEGF丙胺酸點突變體（殘基8-109形式）或野生型 hVEGF(8-109)於PBT中之連續稀釋物首先在室溫下、在 Fab塗佈之培養盤上培育15分鐘，且用PBS、0.05% Tween20(PBST)洗務培養盤。所結合噬菌體用1:5000於 PBT中稀釋之抗M13單株抗體辣根過氧化酶（Amersham Pharmacia) 4貞測，且用3,3'，5,5'-四曱基聯苯胺（TMB, Kirkegaard & Perry Labs, Gaithersburg, MD)受質顯色約5 分鐘，用1·0 M H3P〇4淬滅，且在450 nm下以分光光度計 讀取。IC5〇值之比率（IC5Q,ala/IC5〇，wt)表示結合親和力之降 低倍數（相對結合親和力）。 「酪胺酸激酶抑制劑」為在某種程度上抑制酪胺酸激酶 之酷胺酸激酶活性的分子，諸如Axl受體。 「嵌合VEGF受體蛋白」為具有來源於至少兩種不同蛋 白質之胺基酸序列的VEGF受體分子，其中至少一者為 VEGF受體蛋白。在某些實施例中，嵌合VEGF受體蛋白能 夠結合VEGF且抑制VEGF之生物活性。 具有指定抗體的「生物學特徵」之抗體為具有彼抗體之 一或多種生物學特徵的抗體，該等生物學特徵將其與結合 相同抗原之其他抗體相區分。 149799.doc -30- 201106972 片段」意謂一種多肽或核酸分子之一部分，該部分較 佳含有參考核酸分子或多肽之全長的至少1 〇%、20%、 30%、40%、50%、60%、70%、80%、90%、95%或 95%以 上。片段可含有 10、20、30、40、50、60、70、80、90或 100、200、3 00、400、5 00、000 或 000個以上核苷酸，或 10、20、30、40、50、60、70、80、90、1〇〇、120、 140、160、180、190、200或200個以上胺基酸。 Q 「治療」係指治療性處理及防治性或預防性措施。需要 /α療者包括已患有良性、癌前期或非轉移性腫瘤者，以及 預防癌症發生或復發者。 術語「治療有效量」係指治療或預防哺乳動物疾病或病 症之治療劑的量。在癌症情況下，治療劑之治療有效量可 減少癌細胞數目；降低原發性腫瘤尺寸；抑制（亦即在某 種程度上減緩且較佳終止）癌細胞浸潤至周邊器官中；抑 制（亦即在某種程度上減緩且較佳終止）腫瘤轉移；在某種 Ο 程度上抑制腫瘤生長；及/或在某種程度上減輕與病症相 關的一或多種症狀。就藥物可防止生長及/或殺死現有癌 細胞而言，藥物可為細胞生長抑制劑及/或細胞毒性劑。 ·· :癌症治療之活體内功效可藉由例如評估存活持續時間、疾 錢展時間（ΤΤΡ)、反應率_、反應持續時間及/或生活 品質來量測。 術語「癌症」及「癌性」係指或描㈣乳動物體内通常 以細胞生長調節異常為特徵之生理病況。此定義包括良性 及惡性癌症。「早期癌症」力「早期腫瘤」意謂非侵襲性 149799.doc •31 · 201106972 或轉移性或分類為〇、I或π期癌症之癌症。癌症之實例包 括（但不限於）癌瘤、淋巴瘤、母細胞瘤（包括神經管母細胞 瘤及視網膜母細胞瘤）、肉瘤（包括脂肪肉瘤及滑膜細胞肉 瘤）、神經内分泌腫瘤（包括類癌腫瘤、胃泌素瘤及胰島細 胞癌）、間皮瘤、神經鞘瘤（包括聽神經瘤）、腦膜瘤、腺 癌、黑色素瘤及白血病或淋巴惡性疾病。該等癌症之更特 疋貫例包括鱗狀細胞癌（例如上皮鱗狀細胞癌）、肺癌（包括 小細胞肺癌（SCLC)、非小細胞肺癌（NSCLC)、肺腺癌及肺 鱗狀癌）、腹膜癌、肝細胞癌、胃癌（包括胃腸癌）、胰臟 癌、神經膠母細胞瘤、子宮頸癌、卵巢癌、肝癌（Hver cancer)、膀胱癌、肝腫瘤（hepat〇ma)、乳癌（包括轉移性乳 癌）、結腸癌、直腸癌、結腸直腸癌、子宮内膜或子宮 癌、唾液腺癌、腎癌、***癌、陰門癌 '曱狀腺癌、肝 癌（hepatic carcinoma)、肛門癌、陰莖癌、睾丸癌、食道 癌、膽道腫瘤以及頭頸癌。 「轉移」意謂癌症自其原發性位點擴散至體内其他位 置。癌細胞可脫離原發性腫瘤，滲入淋巴及血管中，經由 血流循環且在體内其他位置處之正常組織内的遠端病灶中 生長（轉移）。轉移可為局部或遠端轉移。視腫瘤細胞脫離 原發性腫瘤、經由血流行進且停留在遠端位點而定，轉移 為連續過程。在新位點，細胞確立血液供給且可生長而形 成威脅生命之團塊。 「降低或抑制」意謂引起20%、 60%、70%、75%、80%、85%、90%、 3 0%、40% ' 50% 95%或大於95%之 總 149799.doc -32- 201106972 體降幅之能力。降低或抑制可涉及所治療病症之症狀、轉 移之存在或尺寸、原發性腫瘤尺寸、或血管生成病症中血 管之尺寸或數目。 本文中之術語「抗體」以最廣泛意義使用且尤其涵蓋單 株抗體、多株抗體、由至少兩個完整抗體形成之多特異性 抗體（例如雙特異性抗體），及抗體片段，只要其展現所要 生物活性即可。Caw. TVeai. 94: Suppl 1: S6 (2005). Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Patent No. 6,884,879, issued Feb. 26, 2005. Other antibodies include the G6 or B20 series of antibodies (e.g., G6-31, B20-4.1) as described in PCT Publication No. WO 2005/012359, PCT Publication No. WO 2005/044853, and U.S. Patent Application Serial No. 60/991,302. The contents of these patent applications are expressly incorporated herein by reference. For other antibodies, see U.S. Patent Nos. 7,060,269, 6,582,959, 6, 703, 020, No. 6,054, 297, WO 98/45332, WO 96/30046, WO 94/10202, EP 0 666 868 B1, and U.S. Patent Application Publication No. 2006009360, No. 20050186208, No. 20030206899, No. 20030190317, No. 20030203409, and No. 20050112126; and Popkov et al., Journal of Immunological Methods 288: 149-164 (2004). Other antibodies include and may comprise residues FI 7, 149799.doc -28- 201106972 M18, D19, Y21, Y25, Q89, 191, K101, E103 and C104 or residues W17, Y21, Q22, Y25, D63, 183 An antibody that binds to a functional epitope on human VEGF of Q89. The "G6 series antibody" of the present invention is an anti-VEGF antibody derived from the sequence of the G6 antibody or the G6-derived antibody of any of Figures 7, 24-26 and 34-35 of PCT Publication No. WO 2005/012359, which is The entire contents of this application are expressly incorporated herein by reference. See also PCT Publication No. WO 2005/044853, the entire disclosure of which is expressly incorporated by reference. In one embodiment, the G6 series antibody binds to a functional epitope on human VEGF comprising residues F17, Y21, Q22, Y25, D63, 183 and Q89. The "B20 series antibody" of the present invention is an anti-VEGF antibody derived from the sequence of the B20 antibody or the B20-derived antibody of any of Figures 27-29 of PCT Publication No. WO 2005/012359, the entire contents of which is incorporated by reference. It is expressly incorporated herein. See also PCT Publication No. WO 2005/044853 and U.S. Patent Application Ser. In one embodiment, the B20 series antibody binds to a functional epitope on human VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103 and C104. The "functional epitope" of the present invention refers to an amino acid residue in the antigen which plays a positive role in the binding of the antibody. Mutation of any active antigenic residue (eg, a wild-type VEGF mutation caused by a mutation in alanine or a homolog) will disrupt the binding of the antibody such that the relative affinity ratio of the antibody (IC50 mutant VEGF/IC50 wild-type VEGF) is greater than 5 (see Example 2 of WO 2005/149799.doc -29-201106972 012359). In one embodiment, the relative affinity ratio is presented using a solution-bound phage display ELISA assay. Briefly, a 96-well Maxisorp immunoculture dish (NUNC) was coated overnight at 4 °C with a Fab format of the antibody to be tested at a concentration of 2 ug/ml (containing in PBS), and at room temperature with PBS, 0.5 % BSA and 0.05% Tween 20 (PBT) were blocked for 2 hours. Phage displaying a serial dilution of hVEGF alanine point mutant (residue 8-109 form) or wild type hVEGF (8-109) in PBT was first incubated on a Fab coated plate for 15 minutes at room temperature. The plates were washed with PBS, 0.05% Tween 20 (PBST). The bound phage was assayed with 1:5000 anti-M13 monoclonal antibody horseradish peroxidase (Amersham Pharmacia) diluted in PBT, and 3,3',5,5'-tetradecylbenzidine (TMB, Kirkegaard & Perry Labs, Gaithersburg, MD) was subjected to color development for approximately 5 minutes, quenched with 1·0 M H3P〇4, and read spectrophotometrically at 450 nm. The ratio of IC5 depreciation (IC5Q, ala/IC5〇, wt) indicates a decrease in binding affinity (relative binding affinity). A "tyramine kinase inhibitor" is a molecule that inhibits the tyrosine kinase activity of tyrosine kinase to some extent, such as the Axl receptor. A "chimeric VEGF receptor protein" is a VEGF receptor molecule having an amino acid sequence derived from at least two different proteins, at least one of which is a VEGF receptor protein. In certain embodiments, a chimeric VEGF receptor protein is capable of binding to VEGF and inhibiting the biological activity of VEGF. An antibody having a "biological characteristic" of a specified antibody is an antibody having one or more of the biological characteristics of the antibody, which distinguishes it from other antibodies that bind to the same antigen. 149799.doc -30-201106972 Fragment means a portion of a polypeptide or nucleic acid molecule which preferably contains at least 1%, 20%, 30%, 40%, 50%, 60 of the full length of the reference nucleic acid molecule or polypeptide. %, 70%, 80%, 90%, 95% or 95% or more. Fragments may contain 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, 200, 3 00, 400, 5 00, 000 or more nucleotides, or 10, 20, 30, 40 50, 60, 70, 80, 90, 1 〇〇, 120, 140, 160, 180, 190, 200 or more than 200 amino acids. Q "Treatment" means therapeutic treatment and preventive or preventive measures. Those who need /alpha therapy include those who have already had benign, precancerous or non-metastatic tumors, as well as those who prevent cancer from developing or recurring. The term "therapeutically effective amount" refers to an amount of a therapeutic agent that treats or prevents a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of the therapeutic agent reduces the number of cancer cells; reduces the size of the primary tumor; inhibits (ie, somewhat slows and preferably terminates) the infiltration of cancer cells into peripheral organs; That is, to some extent slow and preferably terminate) tumor metastasis; inhibit tumor growth to some extent; and/or to some extent alleviate one or more symptoms associated with the condition. The drug may be a cytostatic agent and/or a cytotoxic agent insofar as the drug prevents growth and/or kills existing cancer cells. • The in vivo efficacy of cancer treatment can be measured, for example, by assessing the duration of survival, time of onset (ΤΤΡ), response rate, duration of response, and/or quality of life. The terms "cancer" and "cancerous" refer to or describe (4) physiological conditions in milk animals that are usually characterized by abnormal cell growth regulation. This definition includes benign and malignant cancers. "Early cancer" force "early tumor" means non-invasive 149799.doc •31 · 201106972 or a cancer that is metastatic or classified as a sputum, I or π cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including neuroblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including classes) Cancer, gastrin and islet cell carcinoma, mesothelioma, schwannomas (including acoustic neuroma), meningiomas, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, and lung squamous cell carcinoma). , peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (Hver cancer), bladder cancer, liver cancer (hepat〇ma), breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, squamous cell carcinoma, squamous adenocarcinoma, hepatic carcinoma, anal cancer, Penile cancer, testicular cancer, esophageal cancer, biliary tract cancer, and head and neck cancer. "Transfer" means that the cancer spreads from its primary site to other locations in the body. Cancer cells can detach from primary tumors, infiltrate lymph and blood vessels, and circulate through the bloodstream and grow (metastasize) in distant lesions in normal tissues at other locations in the body. Transfer can be local or remote. Depending on whether the tumor cells are detached from the primary tumor, proceed through the blood episode and stay at the distal site, the metastasis is a continuous process. At new sites, cells establish a blood supply and can grow to form a life-threatening mass. "Reducing or suppressing" means causing 20%, 60%, 70%, 75%, 80%, 85%, 90%, 30%, 40% '50% 95% or greater than 95% of the total 149799.doc - 32- 201106972 The ability to reduce the body. Decreasing or inhibiting may relate to the symptoms of the condition being treated, the presence or size of the metastasis, the size of the primary tumor, or the size or number of blood vessels in the angiogenic condition. The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, as long as they exhibit The desired biological activity can be.

「分離」抗體為已鑑別且與其天然環境之組分分離及/ 或自其天然環境之組分中回收的抗體。其天然環境之污染 物組分為干擾抗體之研究、診斷性或治療性使用的物質， 且可包括酵素、激素及其他蛋白性溶質或非蛋白性溶質。 在一些實施例中，抗體純化至（1)大於95重量％之抗體且在 一些實施例中大於99重量％(如利用例如洛瑞法 method)所測定）；（2)足以獲得N端或内部胺基酸序列之至 少15個殘基的程度（使用例如旋杯式定序儀（spinning cup sequenator));或（3)均質之程度（在還原或非還原性條件下 使用例如庫馬斯藍（Coomassie blue)或銀染料進行观· PAGE)。由於抗體之天然環境中之至少—種組分將不存 在：因此分離抗體包括重組細胞内原位之抗體。然而，分 離抗體通常藉由至少一個純化步驟來製備。 「原生抗體」通常為約150,000道爾頓之雜四聚糖蛋 白，其由兩條相同輕鏈⑹及兩條相同重鏈（H)構成。各輕 鏈經由-個共價雙硫鍵與重鏈連接，而在不同免疫球蛋白 同型之重鏈間，雙硫鍵之數目不同。各重鏈及輕鏈亦具有 149799.doc -33- 201106972 律〖門隔之鏈内雙硫橋。各重鏈在-端具有可變域 (H) 〃後》大置恆定域。各輕鏈在一端具有可變域 且在另一端具有以域；輕鏈之‘以域與重鍵之第-怪定 域對準’且輕鏈可變域與重鏈可變域對準。咸信特定胺基 酸殘基形成輕鏈與重鏈可變域之間的界面。 i體之可變區」或「可變域」係指抗體重鏈或輕鏈之 胺基Uf域。重鏈之可變域可稱為「」。輕鏈之可變 域可稱為VL」。此等結構域通常為抗體之最易變部分且 含有抗原結合位點。 術語「可變」係、指可變域之某些部分的序列在各種抗體 之間廣泛不同且該等部分用於各種特定抗體對其特定抗原 之'、’α 〇及特異性。然而，可變性在整個抗體可變域中並非 均勻分佈。其集中於輕鏈可變域及重鏈可變域中三個稱為 问變區（HVR)之區段中。可變域之較高度保守部分稱為構 架區（FR)。原生重鏈及輕鏈之可變域各包含四個區，該 4 FR區主要採用β片構形、經由三個hvr連接，從而形成 連接β片結構的環，且在一些情況下該等環形成ρ片結構之 一部分。各鏈中之HVR經由FR區緊密結合在一起，且與另 一鏈之HVR—起促使形成抗體之抗原結合位點（參見Kabat 專 k ’ Sequences of Proteins of Immunological Interest，第 5版，National Institute of Health, Bethesda，MD (1991))。 怪定域不直接涉及抗體與抗原之結合，但展現各種效應功 能’諸如抗體參與抗體依賴性細胞毒性。 來自任何脊椎動物種之抗體（免疫球蛋白）之「輕鏈」基 149799.doc -34- 201106972 於其恆定域之胺基酸序列可歸類為兩種明顯不同類型（稱 為κ及λ)之一。 抗體（免疫球蛋白）視其重鏈之恆定域之胺基酸序列而定 可歸為不同類。存在五種主要類別之免疫球蛋白：IgA、 IgD、IgE、IgG及IgM，且此等免疫球蛋白中之若干者可進 一步分為亞類（同型）’例如 IgGi、IgG2、IgG3、IgG4、IgAi 及IgA2。對應於不同類免疫球蛋白的重鏈怪定域分別稱為 〇 α、β、ε、γ及μ。不同類免疫球蛋白之次單位結構及三維 構形已熟知且大體描述於例如Abbas等人，Ce//w/ar Mo/. ，第 4版（W.B. Saunders, Co” 2000)中。抗 體可為由該抗體與一或多種其他蛋白質或肽共價或非共價 結合所形成之較大融合分子的一部分。 術語「全長抗體」、「完整抗體」及「整個抗體」在本文 中可互換使用以指呈實質上完整形式之抗體，而非如下所 定義之抗體片段。該等術語尤其指具有包含Fc區之重鏈的 抗體。 用於本文目的之「裸抗體」為未與細胞毒性部分或放射 性標記結合之抗體。 • 「抗體片段」包含完整抗體之一部分，較佳包含其抗原 ； 結合區。抗體片段之實例包括Fab、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。 木瓜蛋白酶消化抗體產生兩個稱為「Fab」片段之相同 抗原結合片段’各具有單一抗原結合位點；及一殘餘 149799.doc -35- 201106972 「Fc」片段，其名稱反映其易於結晶之能力。胃蛋白酶處 理產生具有兩個抗原結合位點且仍能夠與抗原交聯之 F(ab')2片段。 「F”為含有完整抗原結合位點之最小抗體片段。在一 實施例中’雙州物質由緊密、非共價結合之—個重鍵可 變域與一個輕鏈可變域之二聚體組成。在單鏈卜^^幻物 質中，-個重鏈可變域與一個輕鏈可變域可經由柔性狀連 接子共價連接，使得輕鏈與重鏈可以類似於雙鏈&物質結 構之「二聚」結構結合。在此構形中，各可變域之三個 HVR相互作用以設定抓几二聚體表面上之抗原結合位 點。總而言之，六個HVR使抗體具有抗原結合特異性。，欲 而，即使單個可變域（或僅包含三個對抗原具有特異性之 HVR之Fv的一半）亦能夠識別及結合抗原但是親和力低 於完整結合位點。An "isolated" antibody is an antibody that has been identified and separated from components of its natural environment and/or recovered from components of its natural environment. The contaminant component of its natural environment is a substance that interferes with the research, diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous solutes or non-proteinaceous solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight of the antibody and, in some embodiments, greater than 99% by weight (as determined using, for example, the Lori method); (2) sufficient to obtain the N-terminus or internal The extent of at least 15 residues of the amino acid sequence (using, for example, a spinning cup sequenator); or (3) the degree of homogenization (using, for example, Coomass Blue under reducing or non-reducing conditions) (Coomassie blue) or silver dye for PAGE). Since at least one of the components of the antibody's natural environment will not be present: thus isolating antibodies includes antibodies in situ in recombinant cells. However, the isolated antibody is usually prepared by at least one purification step. A "primary antibody" is typically a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains (6) and two identical heavy chains (H). Each light chain is linked to a heavy chain via a covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy chain and light chain also has an intra-chain disulfide bridge of 149799.doc -33- 201106972. Each heavy chain has a variable domain (H) 在 at the - terminus. Each light chain has a variable domain at one end and a domain at the other end; 'aligns the domain with the first-frequent domain of the heavy bond' and the light chain variable domain is aligned with the heavy chain variable domain. The specific amino acid residue forms an interface between the light chain and the heavy chain variable domain. The "variable region" or "variable domain" refers to the amine-based Uf domain of the heavy or light chain of an antibody. The variable domain of the heavy chain can be called "". The variable domain of the light chain can be referred to as VL". These domains are typically the most variable part of an antibody and contain an antigen binding site. The term "variable" means that the sequences of certain portions of the variable domains vary widely among the various antibodies and that such portions are used for the ',' alpha 〇 and specificity of the particular antigen for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable domain. It is concentrated in three segments called the variable region (HVR) in the light chain variable domain and the heavy chain variable domain. The higher degree of conservation of the variable domain is referred to as the framework region (FR). The variable domains of the native heavy and light chains each comprise four regions, the 4 FR regions are predominantly in a beta sheet configuration, joined via three hvrs, thereby forming a loop connecting the beta sheet structures, and in some cases the loops Forming a portion of the p-sheet structure. The HVRs in each chain are tightly bound via the FR region and together with the HVR of the other chain contribute to the formation of the antigen binding site of the antibody (see Kabat k' Sequences of Proteins of Immunological Interest, 5th edition, National Institute of Health, Bethesda, MD (1991)). It is not directly related to the binding of antibodies to antigens, but exhibits various effector functions such as antibodies involved in antibody-dependent cellular toxicity. The "light chain" group of antibodies (immunoglobulins) from any vertebrate species 149799.doc -34- 201106972 The amino acid sequence in its constant domain can be classified into two distinct types (called κ and λ). one. Antibodies (immunoglobulins) can be classified into different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these immunoglobulins can be further divided into subclasses (isotypes)' such as IgGi, IgG2, IgG3, IgG4, IgAi And IgA2. The heavy-chain strange domains corresponding to different classes of immunoglobulins are called 〇α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described, for example, in Abbas et al., Ce//w/ar Mo/., 4th Edition (WB Saunders, Co. 2000). A portion of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides. The terms "full length antibody", "intact antibody" and "entire antibody" are used interchangeably herein. An antibody that is in a substantially intact form, rather than an antibody fragment as defined below. These terms especially refer to antibodies having a heavy chain comprising an Fc region. A "naked antibody" for the purposes herein is an antibody that does not bind to a cytotoxic moiety or a radiolabel. • An "antibody fragment" comprises a portion of an intact antibody, preferably comprising its antigen; a binding region. Examples of antibody fragments include Fab, Fab, F(ab,) 2 and Fv fragments; bifunctional antibodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain-digested antibodies produce two identical antigen-binding fragments called "Fab" fragments each having a single antigen-binding site; and a residual 149799.doc-35-201106972 "Fc" fragment whose name reflects its ability to crystallize readily . Pepsin treatment yields an F(ab')2 fragment that has two antigen binding sites and is still capable of cross-linking with the antigen. "F" is the smallest antibody fragment that contains the entire antigen binding site. In one embodiment, the 'dual state material consists of a tight, non-covalently bound one-heavy bond variable domain and one light chain variable domain dimer. In a single-stranded substance, one heavy chain variable domain and one light chain variable domain can be covalently linked via a flexible linker such that the light and heavy chains can be similar to a double stranded & The "dimerization" structure is combined. In this configuration, the three HVRs of each variable domain interact to set the antigen binding site on the surface of the dimer. In summary, the six HVRs confer antigen binding specificity to the antibody. Thus, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) is able to recognize and bind antigen but has a lower affinity than the entire binding site.

Fab片段含有重鏈可變域及輕鏈可變域且亦含有輕鏈之 恆定域及重鏈之第-怪定域（CH1)。Fab，片段因在重鍵⑽ 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）而與Fab片段不同。在本文中 才曰示其中恆疋域之半胱胺酸殘基帶有游離硫醇基之以匕，。 F(ab')2抗體片段最初係作為其間具有鉸鏈半胱胺酸之Fab， 片段對產生。亦已知抗體片段之其他化學偶合。 「單鏈Fv」或「scFv」抗體片段包含抗體之vh及vl 域，其中此等域存在於單一多肽鏈中。通常，scFv多肽進 一步包含VH與VL域之間之多肽連接子，其使scFv能夠形 149799.doc -36- 201106972 成抗原結合所需結構。欲回顧scFv，參見例如Pluckthiin， The Pharmacology of Monoclonal Antibodies，第]Λ3 卷， Rosenburg及Moore編，（Springer-Verlag, New York, 1994)， 第 269-315頁。 術語「雙功能抗體」係指具有兩個抗原結合位點之抗體 片段，該等片段在同一多肽鏈（VH-VL)中包含相連接之重 鏈可變域（VH)與輕鏈可變域（VL)。因使用連接子過短而無 法使同一鏈上兩個域之間配對，該等域被迫與另一條鏈之 互補域配對且產生兩個抗原結合位點。雙功能抗體可為二 價或雙特異性抗體。雙功能抗體更全面地描述於例如EP 404,097 ； WO 1993/01161 ； Hudson# A » Nat. Med. 9:129-134 (2003);及 Hollinger 等人，iVoc. iVa". 9〇: 6444-6448 (1993)中。三功能抗體及四功能抗體亦描述 於 Hudson等人，TVai. Med 9:129-134 (2003)中。 如本文所用之術語「單株抗體」係指自一群實質上同源 抗體獲得之抗體，亦即構成該群體之個別抗體除可微量存 在之可能突變（例如天然存在之突變）外均相同。因此，修 飾語「單株」表示不為個別抗體之混合物之抗體的特徵。 在某些實施例中，該單株抗體通常包括包含結合標靶之多 肽序列之抗體，其中藉由包括自複數個多肽序列選擇單個 標靶結合多肽序列之方法來獲得標靶結合多肽序列。舉例 而言，選擇方法可為自複數個純系（諸如融合瘤純系池、 噬菌體純系池或重組DNA純系池）選擇獨特純系。應暸 解，所選擇之標靶結合序列可進一步變異以例如提高對標 149799.doc •37· 201106972 靶之親和力、使標靶結合序列人類化、提高其在細胞培養 物中之產量、減少其活體内免疫原性、產生多特異性抗體 等，且包含變異之標靶結合序列的抗體亦為本發明之單株 抗體。與通常包括針對不同決定子（抗原決定基）之不同抗 體之多株抗體製劑相反，單株抗體製劑中之各單株抗體係 針對抗原上之單個決定子。單株抗體製劑除其特異性外， 有利之處亦在於其通常未受到其他免疫球蛋白污染。 修飾語「單株」表示自一群實質上同源抗體獲得之抗體 之特性，且不應理解為需要利用任何特定方法產生該抗 體。舉例而6，根據本發明使用之單株抗體可利用多種技 術製備’包括例如融合瘤方法（例如Kohler及Milstein, iVaiwre，256:495-97 (1975) ; Hongo等人，//少〜·而廳，14 (3): 253-260 (1995) ; Harlow等人，如价祕w d （Cold Spring Harbor Laboratory Press，第 2 版 1988) ; Hammerling等人’於：她⑽·以 r-Ce// 563-681 (Elsevier, N.Y·，1981)中）；重 組DNA方法（參見例如美國專利案第4,816,567號）；噬菌體 呈現技術（參見例如Clackson等人，TVaiwre，352: 624-628 (1991) ; Marks等人，J. Mo/. 5ζ·〇/· 222: 581-597 (1992); Sidhu等人，乂 Μσ/· 5Μ/· 338(2): 299-310 (2004); Lee等 人’乂 Mo/· 340(5): 1073-1093 (2004) ; Fellouse，The Fab fragment contains a heavy chain variable domain and a light chain variable domain and also contains a constant domain of the light chain and a first-order domain (CH1) of the heavy chain. Fab, a fragment differs from a 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 bond (10) domain. In this paper, the cysteine residues in the constant guanidine domain are shown to have a free thiol group. The F(ab')2 antibody fragment was originally produced as a Fab with a hinged cysteine in between. Other chemical couplings of antibody fragments are also known. A "single-chain Fv" or "scFv" antibody fragment comprises the vh and vl domains of an antibody, wherein such domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding to 149799.doc-36-201106972. For a review of scFv, see, for example, Pluckthiin, The Pharmacology of Monoclonal Antibodies, vol. 3, ed. Rosenburg and Moore, (Springer-Verlag, New York, 1994), pp. 269-315. The term "bifunctional antibody" refers to an antibody fragment having two antigen-binding sites comprising a linked heavy chain variable domain (VH) and a light chain variable domain in the same polypeptide chain (VH-VL). (VL). Because the linker is too short to pair between the two domains on the same strand, the domains are forced to pair with the complementary domain of the other strand and create two antigen-binding sites. The bifunctional antibody can be a bivalent or bispecific antibody. Bifunctional antibodies are more fully described, for example, in EP 404,097; WO 1993/01161; Hudson # A » Nat. Med. 9:129-134 (2003); and Hollinger et al., iVoc. iVa". 9〇: 6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al, TVai. Med 9: 129-134 (2003). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor mutations (e.g., naturally occurring mutations) that may be present in minor amounts. Therefore, the modifier "single plant" indicates the characteristics of an antibody that is not a mixture of individual antibodies. In certain embodiments, the monoclonal antibody typically comprises an antibody comprising a polypeptide sequence that binds to a target, wherein the target binding polypeptide sequence is obtained by a method comprising selecting a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method can be to select a unique pure line from a plurality of pure lines (such as a fusion tumor pure line pool, a phage pure line pool, or a recombinant DNA pure line pool). It will be appreciated that the selected target binding sequence can be further mutated, for example, to increase the affinity of the target 149799.doc • 37·201106972 target, humanize the target binding sequence, increase its yield in cell culture, and reduce its in vivo An antibody that is immunogenic, produces a multispecific antibody, and the like, and includes a target binding sequence of the mutation is also a monoclonal antibody of the present invention. In contrast to multi-drug antibody preparations which typically include different antibodies against different determinants (antigenic determinants), each individual plant anti-system in a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibody preparations are advantageous in that they are generally not contaminated by other immunoglobulins. The modifier "single plant" indicates the identity of an antibody obtained from a population of substantially homologous antibodies and is not to be construed as requiring the production of the antibody by any particular method. For example, 6, a monoclonal antibody used in accordance with the present invention can be prepared using a variety of techniques including, for example, fusion knob methods (e.g., Kohler and Milstein, iVaiwre, 256:495-97 (1975); Hongo et al., // less ~· Hall, 14 (3): 253-260 (1995); Harlow et al., nicknamed wd (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al.' Yu: she (10)·r-Ce// 563-681 (Elsevier, NY, 1981); recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567); phage display technology (see, e.g., Clackson et al, TVaiwre, 352: 624-628 (1991); Marks Et al., J. Mo/. 5ζ·〇/· 222: 581-597 (1992); Sidhu et al., 乂Μσ/· 5Μ/· 338(2): 299-310 (2004); Lee et al. Mo/· 340(5): 1073-1093 (2004) ; Fellouse,

Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004)； 及 Lee 等人 ’ J. Immunol. Methods 284(1-2): 119-132 (2004));及用於在動物中產生具有編碼人類免疫球蛋白之 149799.doc -38- 201106972 人類免疫球蛋白基因座或基因之一部分或全部之人類或人 類樣抗體的技術（參見例如WO 1998/24893 ; WO 1996/ 34096 ； WO 1996/33735 ; WO 1991/10741 ; Jakobovits 等 人，尸roc. #加/· Jai/· 5W. tASJ 90: 2551 (1993);Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al. 'J. Immunol. Methods 284(1-2): 119-132 (2004)); Techniques for producing human or human-like antibodies having a portion or all of a human immunoglobulin locus or gene encoding a human immunoglobulin of 149799.doc-38-201106972 in an animal (see, for example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., corpse roc. #加/· Jai/· 5W. tASJ 90: 2551 (1993);

Jakobovits# A J Nature 362: 255-258 (1993) ； Bruggemann 等人，Fear in Immunol. 7:33 (1993);美國專利案第 5,545,807 號、第 5,545,806 號、第 5,569,825 號、第 5,625,126 號、第 5,633,425 號及第 5,661，016號；Marks 等 k，Bio/Technology 10: 779-783 (1992) ; Lonberg 等人， Nature 3 68: 856-859 (1994) ； Morrison, Nature 3 68: 812-813 (1994) ; Fishwild等人，5/oiec/mo/· 14: 845-851 (1996) ; Neuberger, iVaiwre Bfoiec/mo/· 14: 826 (1996);及 Lonberg 及 Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)) 〇 本文之單株抗體特定包括「嵌合」抗體，其中重鏈及/ 或輕鏈之一部分與源自特定物種或屬於特定抗體種類或亞 類之抗體中的相應序列一致或同源，而該（該等）鏈之其餘 部分與源自另一物種或屬於另一抗體種類或亞類之抗體中 的相應序列一致或同源；以及該等抗體之片段，只要其展 現出所需生物學活性即可（參見例如美國專利案第 4,816,567號；及 Morrison 等人，Proc. A/W/. Jccd <Scz·· 81:6851-6855 (1984))。嵌合抗體包括 PRIMATIZED® 抗 體，其中抗體之抗原結合區源自藉由例如以相關抗原使獼 猴免疫而產生之抗體。 149799.doc -39- 201106972 「人類化」形式之非人類（例如鼠類）抗體為含有源自非 人類免疫球蛋白之最小序列的嵌合抗體。在一實施例中， 人類化抗體為人類免疫球蛋白（受者抗體），其中受者之 HVR的殘基已經具有所要特異性、親和力及/或容量之非 人類物種（供者抗體）（諸如小鼠、大鼠、兔或非人類靈長類 動物）之HVR的殘基置換。在一些情況下，人類免疫球蛋 白之FR殘基經相應非人類殘基置換。此外，人類化抗體可 包含未發現於受者抗體或供者抗體中之殘基。可進行此等 修飾以進一步改進抗體效能。通常，人類化抗體將包含實 質上全部之至少一個且通常兩個可變域，其中全部或實質 上全部南變環對應於非人類免疫球蛋白之高變環，且全部 或貫質上全部FR為人類免疫球蛋白序列之fr。人類化抗 體視情況亦將包含免疫球蛋白恆定區通常為人類免疫 球蛋白恆定區）之至少一部分。欲知詳情，參見J〇nes等 人，施321:522-525 (1986) ; Riechmann等人， 332.323-329 (1988);及 Presta，Cwrr· Ο/?· Βζ·ο/. 2:593-596 (1992)。亦參見例如 vaswani 及 Hamilton,Jakobovits # AJ Nature 362: 255-258 (1993); Bruggemann et al., Fear in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 And No. 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature 3 68: 856-859 (1994); Morrison, Nature 3 68: 812-813 (1994) Fishwild et al, 5/oiec/mo/· 14: 845-851 (1996); Neuberger, iVaiwre Bfoiec/mo/· 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65 -93 (1995)) The monoclonal antibodies specified herein include "chimeric" antibodies in which one of the heavy and/or light chains is identical to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass. Or homologous, and the remainder of the chain is identical or homologous to the corresponding sequence from another species or antibody belonging to another antibody class or subclass; and fragments of such antibodies, as long as they are revealed The desired biological activity is sufficient (see, for example, U.S. Patent No. 4,816,567; . Morrison et al, Proc A / W / Jccd < Scz ·· 81:. 6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies in which the antigen binding region of an antibody is derived from an antibody produced by, for example, immunizing a macaque with a related antigen. 149799.doc -39- 201106972 A "humanized" form of a non-human (eg, murine) antibody is a chimeric antibody containing minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody), wherein the residue of the recipient's HVR already has a non-human species (donor antibody) of the desired specificity, affinity and/or capacity (such as Residue replacement of HVR of mouse, rat, rabbit or non-human primate). In some cases, the FR residue of a human immunoglobulin 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 can be made to further improve antibody performance. Typically, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the southern variable loop corresponds to a hypervariable loop of a non-human immunoglobulin, and all or all of the FR For the human immunoglobulin sequence fr. The humanized anti-visual condition will also include at least a portion of the immunoglobulin constant region, typically the human immunoglobulin constant region. For more information, see J〇nes et al., 321:522-525 (1986); Riechmann et al., 332.323-329 (1988); and Presta, Cwrr·Ο/?· Βζ·ο/. 2:593- 596 (1992). See also vaswani and Hamilton, for example.

Allergy, Asthma & Immunol. 1:105-1 15 (1998) ； Harris, 价oc/zem. Soc. 似 23:1035-1038 (1995) ; Hurle及Allergy, Asthma & Immunol. 1:105-1 15 (1998) ; Harris, val/ocm. Soc. Similar 23:1035-1038 (1995) ; Hurle and

Gross，Cwrr. Aoiec/z. 5:428-433 (1994);及美國專利第 6,982,321 號及第 7,087,409號。 「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及/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。人類抗體之此定義尤其排除包 149799.doc •40- 201106972 含非人類抗原結合殘基之人類化抗體。可使用此項技術巾 已知之各種技術（包括噬菌體呈現文庫）產生人類抗體。 Hoogenboom 及 Winter, 乂 Mol. Biol., 227:381 (1991); Marks等人，/. Mo/· 222:581 (1991)。描述於以下文 獻中之方法亦可用於製備人類單株抗體：Cole等人， Monoclonal Antibodies and Cancer Therapy, Alan R. Liss > 第 77 頁（1985) ; Boerner 等人，《/· /wmwwo/.，147(1):86-95 (1991)中。亦參見van Dijk 及 van de Winkel，Cwrr. 5: 368-74 (2001)。人類抗體可藉由將抗原投 與轉殖基因動物來製備，該轉殖基因動物經修飾可回應抗 原攻毒而產生該等抗體、但其内源基因座已失能，例如經 免疫之異種移植小鼠（xenomice)(關於又£1^01^101；8£1^技 術，參見例如美國專利第6,075，181號及第6，15〇,584號）。 關於經由人類B細胞融合瘤技術產生之人類抗體，亦參見 例如 Li 等人，scz··仍乂，i〇3:3557_3562 (2006)° 術語「咼變區」、「HVR」或「HV」當用於本文中時係 指序列1¾變及/或形成結構上確定之環的抗體可變域區 域。通常’抗體包含六個HVR ;三個在VH中（HI、H2、 H3) ’且三個在VL中（LI、L2、L3)。在原生抗體中，H3及 L3顯示六個HVR之最高多樣性，且尤其咸信H3在賦予抗 體精細特異性方面起獨特作用。參見例如Xu等人， /廳謂13:37-45 (2000); Johnson及 Wu，於输如心 kGross, Cwrr. Aoiec/z. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409. A "human antibody" is an amino acid sequence having an amino acid sequence corresponding to an antibody produced by a human and/or an antibody produced by any of the techniques for producing a human antibody disclosed herein. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues in the package 149799.doc • 40- 201106972. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, 乂 Mol. Biol., 227:381 (1991); Marks et al., /. Mo/. 222:581 (1991). The methods described in the following literature can also be used to prepare human monoclonal antibodies: Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss > page 77 (1985); Boerner et al., /. /wmwwo/. , 147(1): 86-95 (1991). See also van Dijk and van de Winkel, Cwrr. 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, such as by immunized xenografts Mice (xenomice) (see also, for example, U.S. Patent Nos. 6,075,181 and 6,15, 584). For human antibodies produced by human B cell fusion tumor technology, see, for example, Li et al., scz··乂, i〇3:3557_3562 (2006)° The term “transformation zone”, “HVR” or “HV” As used herein, it is meant an antibody variable domain region that is sequenced and/or forms a structurally defined loop. Typically the 'antibody contains six HVRs; three in VH (HI, H2, H3)' and three in VL (LI, L2, L3). Among the native antibodies, H3 and L3 show the highest diversity of the six HVRs, and especially the H3 has a unique role in conferring the fine specificity of the antibody. See, for example, Xu et al., / Hall says 13:37-45 (2000); Johnson and Wu, lose in heart k

Molecular Biology 248:1-25 (Lo^ > Human Press, Totowa, 149799.doc -41- 201106972 NJ, 2003)。實情為，僅由重鏈組成之天然存在之駱駝科動 物抗體在不存在輕鏈的情況下亦具功能性及穩定性。參見 例如 Hamers-Casterman等人，TVaiwre 363:446-448 (1993); Sheriff等人，«SYrMci. 5/〇/· 3:733-736 (1996) 0 本文中使用並涵蓋許多HVR敍述。Kabat互補決定區 (CDR)係以序列可變性為基礎且最常使用（Kabat等人， Sequences of Proteins of Immunological Interest，第 5版， 公共衛生署（Public Health Service),國家衛生研究院 (National Institutes of Health), Bethesda, MD. (1991))。 Chothia改為指出結構環之位置（Chothia &LeskJ.Mo/· 价〇/· 196:901-917 (1987))。AbM HVR代表 Kabat HVR與 Chothia結構環之間的折衷處理，且由Oxford Molecular之 AbM抗體建模軟體使用。「接觸」HVR係基於對可用複合 物晶體結構之分析。此等HVR每一者之殘基註解如下。 環 Kabat AbM Chtrthia 接觸 LI L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 HI H31-H35B H26-H35B H26-H32 H30-H35B (Kabat 編號) HI H31-H35 H26-H35 H26-H32 H30-H35 (Chothia 編號） H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101 149799.doc 201106972 HVR可包含如下之「延長HVR」：VL中之24-36或24-34(L1)、46-56 或 50-56(L2)及 89-97 或 89-96(L3);及 VH 中 之 26-35(Η1)、50-65 或 49-65(H2)及 93-102、94-102 或 95-102(H3)。可變域殘基係根據Kabat等人（同上）關於該等定 義中之每一者所述進行編號。 '「構架」或「FR」殘基為除如本文所定義之HVR殘基以 外之可變域殘基。 八 術語「根據Kabat之可變域殘基編號」或「根據Kabati 〇 胺基酸位置編號」及其變化形式係指Kabat等人（同上）之用 於編譯抗體之重鏈可變域或輕鏈可變域之編號系統。使用 該編號系統，實際線性胺基酸序列可含有更少或其他胺基 酸（對應於可變域之FR或HVR之縮短，或***）。舉例而 言，重鏈可變域可包括位於H2殘基52後之單一胺基酸*** (殘基52a ’根據Kabat)及位於重鏈FR殘基82後之***殘基 (例如殘基82a、82b及82c等，根據Kabat)。可藉由在同源 Q 區比對抗體序列與「標準」Kabat編號序列來確定指定抗 體中殘基之Kabat編號。Molecular Biology 248: 1-25 (Lo^ > Human Press, Totowa, 149799.doc -41- 201106972 NJ, 2003). In fact, naturally occurring camelid antibodies consisting of only heavy chains are also functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al, TVaiwre 363:446-448 (1993); Sheriff et al, «SYrMci. 5/〇/· 3:733-736 (1996) 0 Many HVR narratives are used and encompassed herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institute of Health (National Institutes) Of Health), Bethesda, MD. (1991)). Chothia instead pointed out the location of the structural ring (Chothia & LeskJ.Mo/. Price / 196:901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on the analysis of the crystal structure of the available compounds. The residues of each of these HVRs are as follows. Ring Kabat AbM Chtrthia Contact LI L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 HI H31-H35B H26- H35B H26-H32 H30-H35B (Kabat number) HI H31-H35 H26-H35 H26-H32 H30-H35 (Chothia number) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96- H101 H93-H101 149799.doc 201106972 HVR can include the following "extended HVR": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 in VL (L3); and 26-35 (Η1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. Variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions. A 'framework' or "FR" residue is a variable domain residue other than the HVR residue as defined herein. The terms "variable domain residue number according to Kabat" or "based on Kabati amino acid position number" and variations thereof refer to the heavy chain variable or light chain of Kabat et al. (supra) for compiling antibodies. Variable number numbering system. Using this numbering system, the actual linear amino acid sequence may contain fewer or other amino acids (corresponding to shortening, or insertion of FR or HVR of the variable domain). For example, a heavy chain variable domain can include a single amino acid insertion after residue H2 (residue 52a' according to Kabat) and an insertion residue located after heavy chain FR residue 82 (eg, residue 82a, 82b and 82c, etc., according to Kabat). The Kabat numbering of residues in a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence in the homologous Q region.

Kabat編號系統通常在提及可變域中之殘基（大致為輕鏈 殘基1-107及重鏈殘基1-113)時使用（例如Kabat等人， Sequences of Immunological Interest.第 5版，公共禕ί 生署 國家衛生研究院，Bethesda，Md. (1991))。「£11編號系統」 或「EU索引」通常在提及免疫球蛋白重鍵怪定區中之殘 基時使用（例如Kabat等人（同上）報導之EU索引）。「根據 Kabat之EU索引」係指人類igGl EU抗體之殘基編號。除 149799.doc -43- 201106972 非本文中另有說明，否則提及抗體可變域中之殘基編號意 謂依據Kabat編號系統進行之殘基編號。除非本文中另有 說明’否則提及抗體恆定域中之殘基編號意謂依據EU編 號系統進行之殘基編號（參見例如美國臨時申請案第 60/640,323號關於EU編號之諸圖）。 「親和力成熟」抗體為其一或多個HVR中具有一或多個 變異的抗體，該等變異使得抗體對抗原之親和力提高（與 不具有彼等變異之親本抗體相比）。在一實施例中，親和 力成熟抗體對乾抗原具有奈莫耳（nanomolar)或甚至皮莫耳 (picomolar)親和力。可使用此項技術中已知之某些程序產 生親和力成熟抗體。舉例而言，Marks等人之Bio/The Kabat numbering system is generally used when referring to residues in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al, Sequences of Immunological Interest. 5th edition, Public Health Department National Institutes of Health, Bethesda, Md. (1991)). The "£11 numbering system" or "EU index" is usually used when referring to residues in the immunoglobulin heavy-bonding region (for example, the EU index reported by Kabat et al., supra). "EU index according to Kabat" refers to the residue number of the human igGl EU antibody. Except as otherwise stated herein, reference to residue numbers in the variable domains of an antibody means the numbering of residues based on the Kabat numbering system. Unless otherwise stated herein, otherwise reference to the residue numbering in the constant domain of the antibody means the numbering of the residues in accordance with the EU numbering system (see, for example, the U.S. Provisional Application Serial No. 60/640,323, the disclosure of which is incorporated herein by reference). "Affinity maturation" antibodies are antibodies that have one or more variations in one or more HVRs that increase the affinity of the antibody for the antigen (as compared to parental antibodies that do not have such variations). In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the dry antigen. Affinity matured antibodies can be produced using certain procedures known in the art. For example, Marks et al. Bio/

Technology 10:779-783 (1992)描述藉由 VH及 VL域改組進 行親和力成熟。HVR及/或構架殘基之隨機突變誘發描述 於例如Barbas等人 ’ Proc #加· t/以 91:3809- 3813 (1994) ; Schier 等人，Gene 169:147-155 (1995);Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutation induction of HVR and/or framework residues is described, for example, in Barbas et al. 'Proc #加·t/91:3809-3818 (1994); Schier et al, Gene 169:147-155 (1995);

Yelton 等人，乂 /wwwno/. 155:1994-2004 (1995) ； Jackson 等人 ’ 乂 /wmwno/· 154(7):3310-9 (1995);及 Hawkins 等 A » J. Mol. Biol. 226:889-896 (1992) t。 「阻斷」抗體或「拮抗劑」抗體為抑制或降低其所結合 之抗原之生物活性的抗體。某些阻斷抗體或拮抗劑抗體實 質上或完全抑制抗原之生物活性。 如本文中所用之「促效劑抗體」為部分或完全模擬相關 多肽之至少一種功能活性的抗體。 「生長抑制性」抗體為防止或減少表現抗體所結合之抗 149799.doc -44 - 201106972 原之細胞增瘦的抗體。舉例而言，抗體可防止或減少活體 外及/或活體内表現Axl之癌細胞的增瘦。 「誘導細胞凋亡」之抗體為誘導漸進式細胞死亡的抗 體，如藉由標準細胞凋亡分析法所測定，諸如磷脂結合蛋 白Vhnnexin V)結合、DNA斷裂、細胞皺縮、内質網擴 張、細胞斷裂、及/或膜囊（稱為細胞凋亡體）形成。 抗體「效應功能」係指由抗體之F c區（原生序列F c區或 〇 胺基酸序列變異體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（CDC) ; Fc受體結合；抗體依賴性細胞 介導之細胞毒性（ADCC);吞嗟作用；細胞表面受體（例如 B細胞受體）之下調；及B細胞活化。 本文中之術語「Fc區」用於定義免疫球蛋白重鏈之c端 區域’包括原生序列Fc區及變異型Fc區。雖然免疫球蛋白 重鏈之Fc區之邊界可變化’但人類IgG重鏈Fc區通常定義 Q 為自位置Cys226之胺基酸殘基或自pr〇23〇伸展至其叛基末 端。可移除Fc區之C端離胺酸（殘基447，根據EU編號系 統）’例如在產生或純化抗體期間或藉由對編碼抗體重鏈 • 之核酸進行重組工程改造來移除。因此，完整抗體之組成 : 可包含全部K447殘基皆移除之抗體群體、K447殘基未移 除之抗體群體’及具有存在Κ447殘基之抗體與不存在 Κ447殘基之抗體之混合物的抗體群體。 「功能性Fc區」具有原生序列Fc區之「效應功能」。例 示性「效應功能」包括Clq結合；CDC ; Fc受體結合； 149799.doc •45- 201106972 ADCC ;吞噬作用；細胞表面受體（例如b細胞受體；BCR) 之下調等。該等效應功能通常需要Fc區與結合域（例如抗 體可變域）組合且可使用例如本文定義中所揭示之各種分 析法來評估。 「原生序列Fc區」包含與自然界中發現之Fc區之胺基酸 序列一致之胺基酸序列。原生序列人類Fc區包括原生序列 人類IgG! Fc區（非A異型及A異型）、原生序列人類igG2 Fc 區、原生序列人類IgG3 Fc區，及原生序列人類IgG4 Fc區 以及其天然存在之變異體。 「變異型Fc區」包含因至少一個胺基酸修飾、較佳一或 多個胺基酸取代而與原生序列F c區之彼胺基酸序列不同的 胺基酸序列。較佳地，與原生序列卜區或親本多肽之以區 相比，變異Fc區具有至少一個胺基酸取代，例如在原生序 列Fc區中或親本多肽之Fc區中具有約一個至約十個胺基酸 取代，且較佳約一個至約五個胺基酸取代。本文中之變異 型Fc區較佳與原生序列Fc區及/或與親本多肽之Fc區具有 至少約80%之同源性，且最佳與其具有至少約9〇%之同源 性，更佳與其具有至少約9 5 %之同源性。 「Fc受體」或r FcR」描述結合抗體卜區之受體。在一 些貝施例中，FcR為原生人類fcr。在一些實施例中，FcR 為結合IgG抗體之FcRq受體）且包括FcyRI、f^rii及 FcyRIII亞類之受體，包括該等受體之對偶基因變異體及選 擇性拼接形式。FCYRII受體包括FcyRnA(「活化受體」）及 FcYRIIB(「抑制受體」），其胺基酸序列相似，主要區別在 149799.doc •46- 201106972 於其細胞質域。活化受體FcyRIIA在其細胞質域中含有基 於免疫受體絡胺酸之活化基元（ITAM)。抑制受體FcyRllB 在其細胞質域中含有基於免疫受體酪胺酸之抑制基元 (ITIM)(參見例如 DaSron, J㈣m. i?ev. 15:203-234 (1997))。FcR §平述於例如 Ravetch 及 Kinet，jnm/· i?ev. /wmw⑽/ 9:45 7-92 (1991)，Capel 專人，⑽心 4:25-34 (1994);及 de Haas 等人，J. cm Me<i. 126: 330-41 (1995)中。本文中術語「FcR」涵蓋其#Fcr，包括 〇 有待將來鑑別之FcR。 術語「Fc受體」或「FcR」亦包括新生兒受體FcRn，其 負責將母體IgG傳遞至胎兒中（Guyer等人，乂 /所則„0/. 117:587 (1976)及 Kim等人，《/· /wwwno/. 24:249 (1994))及 免疫球蛋白之穩定調節。量測FcRn結合之方法已知（參見 例如 Ghetie 及 Ward·, Immunol. Today 18(12):592-598 (1997) ; Ghetie等人，iViziMre 15(7):637-640 〇 (1997) ; Hinton 等人，《/. Bzo/. CAew. 279(8):6213-6216 (2004) ; WO 2004/92219 (Hinton 等人））° 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，或在投與具有變異型Fc區之多肽的靈長類動物 ； 中，分析人類FcRn高親和力結合多肽之活體内人類FcRn 結合及jk清半衰期。WO 2000/42072(Presta)描述與FcR之 結合增強或減弱的抗體變異體。亦參見例如Shields等人， J.价〇/· C7zem_ 9(2):6591-6604 (2001)。 「人類效應細胞」為表現一或多種FcR且執行效應功能 149799.doc •47- 201106972 之白血球。在某些實施例中，該等細胞至少表現FcyRIII且 執行ADCC效應功能。介導ADCC之人類白血球之實例包 括周邊血液單核細胞（PBMC)、自然殺手（NK)細胞、單核 細胞、細胞毒性T細胞及嗜中性白血球。可自原生來源（例 如jk液）分離效應細胞。 「抗體依賴性細胞介導之細胞毒性」或r ADCC」係指 一種細胞毒性形式，其中所分泌之1§結合於某些細胞毒性 細胞（例如NK細胞、嗜中性白血球及巨噬細胞）上所存在之 Fc受體（FcR)使得此等細胞毒性效應細胞能夠特異性結合 攜有抗原之標靶細胞且隨後用細胞毒素殺死該標靶細胞。 介導ADCC之初級細胞、NK細胞僅表現FcyRIII，而單核細 胞表現FcyRI、FcyRII及FcyRIII。FcR在造血細胞上之表現 概述於 Ravetch 及 Kinet, Rev. Immunol 9:457-92 (1991)之第464頁上的表3中。為評估相關分子的adcC活 性，可執行活體外ADCC分析法，諸如美國專利第 5,500,362號或第5,821,337號或美國專利第6,737,056號 (Presta)中所述之分析法。適用於該等分析法之效應細胞 包括PBMC及NK細胞。或者或另外，相關分子的adcC活 性可於活體内’例如在諸如Clynes等人，户见a 95:652-656 (1998)所揭示之動物模型中評估。 「補體依賴性細胞毒性」或「CDC」係指標靶細胞在補 體存在下溶解。經典補體路徑之活化係藉由補體系統之第 一組分（C 1 q)與結合其同源抗原之抗體（適當亞類之抗體）結 合來啟動。為評估補體活化，可進行CDC分析法，例如 I49799.doc •48- 201106972Yelton et al., 乂/wwwno/. 155:1994-2004 (1995); Jackson et al.' 乂/wmwno/· 154(7):3310-9 (1995); and Hawkins et al. A » J. Mol. Biol. 226: 889-896 (1992) t. A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking antibody or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. An "agonist antibody" as used herein is an antibody that partially or fully mimics at least one of the functional activities of a related polypeptide. The "growth inhibitory" antibody is an antibody which prevents or reduces the binding of the antibody to the original cell 149799.doc-44 - 201106972. For example, antibodies can prevent or reduce the leaning of cancer cells that exhibit Axl in vitro and/or in vivo. An antibody that induces apoptosis is an antibody that induces progressive cell death, as determined by standard apoptosis assays, such as phospholipid binding protein Vhnnexin V) binding, DNA fragmentation, cell shrinkage, endoplasmic reticulum expansion, Cell rupture, and/or formation of a membrane sac (called an apoptotic body). The antibody "effector function" refers to the biological activity caused by the Fc region of the antibody (the native sequence Fc region or the 〇 amino acid sequence variant Fc region), and which varies with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); swallowing; cell surface receptors (eg, B cell receptors) Bottom); and B cell activation. The term "Fc region" as used herein to define the c-terminal region of an immunoglobulin heavy chain 'includes a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region typically defines Q as an amino acid residue from position Cys226 or from pr〇23〇 to its tremor end. The C-terminal deaminase of the Fc region can be removed (residue 447, according to the EU numbering system)', for example, during recombinant production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, the composition of the intact antibody: an antibody population that includes all of the K447 residues removed, an antibody population that does not have a K447 residue removed, and an antibody that has a mixture of an antibody having a residue of Κ447 and an antibody lacking a residue of Κ447. group. The "functional Fc region" has the "effect function" of the native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; 149799.doc • 45-201106972 ADCC; phagocytosis; cell surface receptors (eg, b cell receptor; BCR) downregulation. Such effector functions typically require an Fc region to be combined with a binding domain (e. g., an antibody variable domain) and can be assessed using, for example, various assays disclosed in the definitions herein. The "primary sequence Fc region" contains an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The native sequence human Fc region includes the native sequence human IgG! Fc region (non-A isoform and A isoform), the native sequence human igG2 Fc region, the native sequence human IgG3 Fc region, and the native sequence human IgG4 Fc region and its naturally occurring variants . The "variant Fc region" comprises an amino acid sequence which differs from the amino acid sequence of the F c region of the native sequence by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the region of the native sequence region or the parent polypeptide, for example, from about one to about in the Fc region of the native sequence or in the Fc region of the parent polypeptide. The ten amino acids are substituted, and preferably from about one to about five amino acids. The variant Fc region herein preferably has at least about 80% homology to the native sequence Fc region and/or to the Fc region of the parent polypeptide, and preferably has at least about 9% homology to it, more Preferably, it has at least about 95% homology. "Fc receptor" or r FcR" describes a receptor that binds to an antibody region. In some of the examples, the FcR is a native human fcr. In some embodiments, the FcR is a FcRq receptor that binds to an IgG antibody) and includes receptors for the FcyRI, f^rii, and FcyRIII subclasses, including dual gene variants of such receptors, and alternatively spliced forms. FCYRII receptors include FcyRnA ("activating receptor") and FcYRIIB ("inhibiting receptor"), and their amino acid sequences are similar, mainly in 149799.doc • 46-201106972 in their cytoplasmic domain. The activating receptor FcyRIIA contains an immunoreceptor lysine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRllB contains an immunoreceptor tyrosine-based inhibitory element (ITIM) in its cytoplasmic domain (see, for example, DaSron, J(d) m. i?ev. 15:203-234 (1997)). FcR § is described, for example, in Ravetch and Kinet, jnm/·i?ev. /wmw(10)/ 9:45 7-92 (1991), Capel, (10) Heart 4:25-34 (1994); and de Haas et al. J. cm Me<i. 126: 330-41 (1995). The term "FcR" is used herein to encompass its #Fcr, including Fc FcR to be identified in the future. The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the delivery of maternal IgG to the fetus (Guyer et al., 乂/所 „0/. 117:587 (1976) and Kim et al. , // /wwwno/. 24:249 (1994)) and stable regulation of immunoglobulins. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward, Immunol. Today 18(12): 592-598 (1997); Ghetie et al., iViziMre 15(7): 637-640 〇 (1997); Hinton et al., /. Bzo/. CAew. 279(8): 6213-6216 (2004); WO 2004/92219 (Hinton et al.)) ° can be analyzed, for example, in a transgenic mouse that expresses human FcRn or in a transfected human fine: cell line, or in a primate that administers a polypeptide having a variant Fc region; Human FcRn high affinity binding polypeptides in vivo human FcRn binding and jk clear half-life. WO 2000/42072 (Presta) describes antibody variants that enhance or attenuate binding to FcR. See also, for example, Shields et al., J. C7zem_ 9(2): 6591-6604 (2001). "Human effector cells" are white blood cells that exhibit one or more FcRs and perform effector functions 149799.doc •47- 201106972 . In certain embodiments, the cells exhibit at least FcyRIII and perform an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from a native source, such as a jk fluid. "Antibody-dependent cell-mediated cytotoxicity" or r ADCC" refers to a cytotoxic form in which the secreted 1 § binds to certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages) The Fc receptor (FcR) present enables such cytotoxic effector cells to specifically bind to the target cell carrying the antigen and subsequently kill the target cell with a cytotoxin. Primary cells that mediate ADCC, NK cells only exhibit FcyRIII, while monocytes exhibit FcyRI, FcyRII, and FcyRIII. The performance of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Rev. Immunol 9:457-92 (1991). In order to evaluate the adcC activity of the relevant molecule, an in vitro ADCC assay can be performed, such as the one described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, or U.S. Patent No. 6,737,056 (Presta). Effector cells suitable for such assays include PBMC and NK cells. Alternatively or additionally, the adcC activity of the relevant molecule can be assessed in vivo, e.g., in an animal model such as that disclosed by Clynes et al., A 95:652-656 (1998). The "complement dependent cytotoxicity" or "CDC" indicator target cells are dissolved in the presence of complement. Activation of the classical complement pathway is initiated by binding the first component of the complement system (C 1 q) to an antibody that binds to its cognate antigen (an antibody of the appropriate subclass). To assess complement activation, a CDC assay can be performed, for example, I49799.doc •48- 201106972

Gazzano-Santoro 等人，·/· Immunol. Methods 202:163 (1996)中所述。具有變異Fc區胺基酸序列（具有變異型Fc區 之多肽）且C 1 q結合能力增加或減小之多肽變異體描述於例 如美國專利第6,194,551 B1號及WO 1999/51642中。亦參見 例如 Idusogie 等人，《/ 164: 4178-4184 (2000) 〇 Ο 〇 術語「包含F c區之抗體」係指包含F c區之抗體。可移除 Fc區之C端離胺酸（殘基447，根據EU編號系統），例如在抗 體純化期間或藉由對編碼抗體之核酸進行重組工程改造來 移除。因此’包含具有本發明之Fc區之抗體的組合物可包 含具有K447之抗體、所有K447均移除之抗體、或具有 K447殘基之抗體與不具有K447殘基之抗體的混合物。 「結合親和力」通常係指分子（例如抗體）之單一結合位 點與其結合搭配物（例如抗原）之間非共價相互作用力的總 和。除非另有說明，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）成員之間丨：丨相互作用之 固有結合親和力。分子X對其搭配物γ之親和力通常可由 解離承數（Kd)表不。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。低親和力抗體一般緩慢 :合抗原且傾向於容易解離，而高親和力抗體—般較快： :抗原且傾向於保持較長時間結合。此項技術中已知多種 測、’。口親和力之方法，其申任一種方法均可用於本發明 :二的。量測結合親和力之特定說明性及例示性實 述於下文中。 在貫施例中，本發明之「κ $「γ估乂么从Gazzano-Santoro et al., // Immunol. Methods 202: 163 (1996). Polypeptide variants having a variant Fc region amino acid sequence (polypeptide having a variant Fc region) and having an increased or decreased C1 q binding capacity are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999/51642. See also, for example, Idusogie et al., / 164: 4178-4184 (2000) 〇 〇 〇 The term "antibody comprising the F c region" refers to an antibody comprising a F c region. The C-terminal acyl acid of the Fc region can be removed (residue 447, according to the EU numbering system), for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, a composition comprising an antibody having an Fc region of the present invention may comprise an antibody having K447, an antibody in which all K447 is removed, or a mixture of an antibody having a K447 residue and an antibody having no K447 residue. "Binding affinity" generally refers to the sum of the non-covalent interaction forces between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects the 丨:丨 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of the molecule X for its conjugate γ is usually represented by the dissociation weight (Kd). Affinity can be measured by common methods known in the art, including the methods described herein; Low-affinity antibodies are generally slow: antigen-binding and tend to dissociate easily, while high-affinity antibodies are generally faster: antigens and tend to remain bound for a longer period of time. A variety of measurements are known in the art. A method of oral affinity, which can be used in the present invention: A specific illustrative and exemplary embodiment of measuring binding affinity is set forth below. In the embodiment, the "κ $" γ estimate of the present invention

Kd」或Kd值」係藉由放射 149799.doc •49- 201106972 性標記抗原結合性分析法（RIA)量測，該分析法利用相關 抗體之Fab形式及其抗原執行，如以下分析法所述。Fab對 抗原之溶液結合親和力如下量測：在連續滴定之未標記抗 原存在下，使Fab與最小濃度之（1251)標記之抗原平衡，接 著用塗佈抗Fab抗體之培養盤捕捉所結合抗原（參見例如 Chen等人，/. Mo/. 5b/·, 293:865-881(1999))。為建立分析 條件，用含5 pg/ml捕捉抗Fab抗體（Cappel Labs)之50 mM 碳酸鈉（pH 9.6)塗佈MICROTITER®多孔培養盤（Thermo Scientific)隔夜，且接著在室溫（約23°C)下用含2%(w/v)牛 血清白蛋白之PBS阻斷2至5小時。在非吸附培養盤（Nunc #269620)中，將100 pM或26 pM [1251]-抗原與相關Fab之連 續稀釋液混合（例如按照Presta等人，Cancer及以· 57:4593_ 4599 (1997)中之抗VEGF抗體Fab-12之評估）。接著培育相 關Fab隔夜；然而，可持續培育較長時間（例如約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性培 養盤中培育（例如歷時1小時）。隨後移除溶液且用含0.1 °/〇 TWEEN-20TMiPBS洗滌培養盤八次。當培養盤乾燥時， 每孔添加 150微升閃爍體（MICROSCINT-20tm ; Packard)， 且培養盤在TOPCOUNTtm γ計數器（Packard)上計數十分 鐘。得到小於或等於20%最大結合之各Fab濃度選用於競 爭性結合分析法。 根據另一實施例，藉由在25°C下，使用BIACORE®-2000 或 BIACORE®-3000(BIAcore，Inc·，Piscataway, NJ)，利用 以約10個反應單位（RU)固定之抗原CM5晶片，使用表面電 149799.doc -50- 201106972 漿子共振分析法來量測Kd或Kd值。簡言之，根據供應商說 明書，以尽乙基二曱基胺基丙基）_碳化二亞胺鹽酸 鹽（EDC)及羥基丁二醯亞胺（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 (卩114.8)稀釋抗原至5 4邑/1111(約0.2 41^)，再以5微升/分鐘之 流速注射，以獲得約10個反應單位（RU)之偶合蛋白。注射 抗原之後，注射1 Μ乙醇胺以阻斷未反應基團。為進行動 力學量測，在25°C下，以約25 μΐ/min之流速注射Fab於含 0.05% TWEEN-20tm界面活性劑之PBS(PBST)中之兩倍連 續稀釋液（0.78 nM至5〇0 nM)。使用簡單的一對一朗繆爾 結合模型（one-to-one Langmuir binding model)(BIACORE ® 評估軟體3.2版），藉由同時擬合結合及解離感應圖譜來計 算結合速率（kj及解離速率（k。^)。根據k^f/ku比率計算平 衡解離常數（Kd)。參見例如Chen等人，/. Mo/. 5z‘o/· 293:865-881 (1999)。若依據上述表面電漿共振分析法，結 合速率超過106 Μ·1 s_1，則可藉由使用螢光淬滅技術測定 結合速率，該技術係在遞增濃度之抗原（如分光計（諸如配 備停流模式之分光光度計（Aviv Instruments)或具有攪拌式 比色管的.8000系列SLM-AMINCO TM分光光度計 (ThermoSpectronic))所量測）存在下，在25°C下量測含於 PBS(pH 7.2)中之20 nM抗抗原抗體（Fab形式）之螢光發射強 度（激發=295 nm ;發射=340 nm ’ 16 nm帶通）的增大或減 小0 本發明之「結合速率」或「k〇n」亦可如上所述使用 149799.doc 51 201106972 biacorE®_2000 或 BIAC〇RE®_3〇〇〇 系統（BiAe〇re，Inc， Piscataway，NJ)來測定。 如本文中所用之術語「實質上類似」或「實質上相同」 表示兩個數值（例如一個與本發明抗體相關且另一個與參 考/比較杬體相關）之間具有足夠高之相似度，以致熟習此 項技術者會認為，在生物學特徵依據該等值（例如L值）度 量之情況下，兩個值之間之差異很小或無生物學及/或統 β十學顯著性。以參考/比較值計，該兩個值之間的差異為 例如小於約50%、小於約40%、小於約3〇%、小於約2〇% 及/或小於約10%。 如本文中所用之片語「實質上減小」或「實質上不同」 表示兩個數值（通常一個與分子相關且另一個與參考/比較 分子相關）之間具有足夠高之差異，以致熟習此項技術者 會認為，在生物學特徵依據該等值（例如Kd值）度量之之情 況下，該兩個值之間之差異具有統計學顯著性。以參考/ 比較分子之值計’該兩個值之間的差異為例如大於約 1 0%、大於約20%、大於約30%、大於約40%及/或大於約 50%。 「純化」意謂分子存在於樣品中之濃度為至少95%或至 少98%(以含有其之樣品重量計）。 「分離」核酸分子為一種核酸分子，其已與其通常所結 合之至少一種其他核酸分子（例如在其天然環境中）分離。 分離核酸分子進一步包括通常表現核酸分子之細胞中所含 之核酸分子，但該核酸分子存在於染色體外或存在於與其 149799.doc -52- 201106972 天然染色體位置不同之染色體位置。 如本文所使用之術語「載體」意指一種核酸分子，其能 夠轉運其所連接之另一核酸分子。一類載體為「質體」， 其指可接合其他DNA區段之環形雙股DNA。另一類載體為 病毋載體’其中可將其他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哺乳動物載 Q 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基因組中，且從而與宿主基因組 一起複製。此外，某些載體能夠導引其可操作地連接之基 -因之表現。該等載體在本文中稱為「重組性表現載體」或 簡稱「表現載體」。通常，適用於重組DNA技術中之表現 載體通常呈質體形式。在本說明書中，由於質體為最常用 之載體形式，故「質體」與「載體」可互換使用。 如本文中可互換使用之「聚核苷酸」或「核酸」係指任 〇 何長度之核苷酸聚合物，且包括DNA及RNA。核苷酸可為 脫氧核糖核苷酸 '核糖核苷酸、經修飾核苷酸或鹼基、 及/或其類似物，或可藉由DNA或RNA聚合酶或藉由合成 反應併入聚合物中之任何受質。聚核苷酸可包含經修飾之 核苷酸，諸如曱基化核苷酸及其類似物。若存在，則可在 裝配聚合物之前或之後對核苷酸結構進行修飾。核苷酸序 列可雜有非核普酸組分。聚核苷酸可包含合成之後所形成 之修飾，諸如與標記結合。其他類型之修飾包括：例如， 或夕個天然存在之核苷酸經類似物「帽」取代；核苷酸 H9799.doc •53· 201106972 間修飾，諸如具有不帶電荷之鍵聯（例如膦酸甲酯、磷酸 三酯、磷醢胺酸、胺基甲酸酯等）之彼等物及具有帶電荷 鍵聯（例如硫代磷酸酯、二硫代磷酸酯等）之彼等物、含有 側接部分（例如蛋白質（例如核酸酶、毒素、抗體、信號 肽、ply-L-離胺酸等）之彼等物、具有嵌入劑（例如吖啶 (acridine)、補骨脂素（psoralen)等）之彼等物、含有螯合劑 (例如金屬、放射性金屬、硼、氧化性金屬等）之彼等物、 含有烧化劑之彼等物、具有經修飾之鍵聯（例如α變旋異構 體核酸等）之彼等物，以及聚核苷酸之未修飾形式。此 外’通常存在於糖中之任何羥基均可經例如膦酸酯基、鱗 酸酯基置換’經標準保護基團保護或經活化以製備與其他 核苷酸之其他鍵聯；或可與固體或半固體支撐物結合。5, 及3'末端ΟΗ可經磷酸化或經】至2〇個碳原子之胺或有機封 端基團部分取代。其他羥基亦可衍生為標準保護基。聚核 苷酸亦可含有此項技術中通常已知之核糖或脫氧核糖之類 似物形式’包括例如2'-〇-甲基-核糖、2，_〇_烯丙基_核糖、 2·-氟•核糖或2’-迭氮基-核糖、碳環糖類似物、心變旋異構 糖、差向異構糖（绪如***糖（arabin〇se)、木糖或來蘇糖 (lyxose))、哌喃醣、呋喃醣、景天庚酮糖、無環類似物及 鹼性核苷類似物（諸如曱基核糖苷）。一或多個磷酸二酯鍵 聯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）磷酸酯基經P(0)s(「硫代酸酯基」）、p(s)s (「二硫代酸酯基」）、（〇)NR2(「醯胺酸酯基」）、p(〇)R、 P(0)0R’、CO或CH2(「曱縮醛」）置換的實施例，其中各尺 149799.doc -54- 201106972 或R'獨立地為Η或者視情況含有醚（_〇_)鍵聯之經取代或未 經取代之烷基（1-20個C)、芳基、烯基、環烷基、環烯基或 芳烷基。並非聚核苷酸中之所有鍵聯皆需相同。上述描述 適用於本文中所提及之所有聚核苷酸，包括RNA及DNA。 如本文中所使用之「募核苷酸」通常係指短聚核苷酸， ' 通常為單股、通常為合成的短聚核苷酸，其長度通常（但 不一定）小於約200個核苷酸。術語「募核苷酸」及「聚核 0 苷&」並不相互排斥。上文有關聚核苷酸之描述同樣且完 全適用於寡核苷酸。 除非另外指示，否則如本文中所用之術語「Αχ1」係指 來自任何脊椎動物來源（包括哺乳動物，諸如靈長類動物 (例如人類）及齧齒動物（例如小鼠及大鼠））之任何原生 AX1。該術語涵蓋「全長」、未經處理之Αχ1以及由在細胞 中處理所產生之任何形式之Axl ^該術語亦涵蓋天然存在 之Axl變異體，例如剪接變異體或對偶基因變異體。 Ο 除非另外指示，否則如本文中所用之術語「Axl配位 體」或「Gas6」係指來自任何脊椎動物來源（包括哺乳動 物，諸如靈長類動物（例如人類）及齧齒動物（例如小鼠及大 鼠））之任何原生Axl配位體。該術語涵蓋「全長」、未經處 理之Axl配位體以及由在細胞中處理所產生之任何形式之 Axl配位體。該術語亦涵蓋天然存在之變異型A"配位體， 例如剪接變異體或對偶基因變異體。 如本文中所用’「治療（treatment)」（及其變化形式，諸 如/「treat」或「treating」）係指試圖改變所治療個體或細 149799.doc -55- 201106972 胞之自然過程之臨床干預， 預且可出於預防之目的或在臨床 病理過程中執行。理想 療效果包括預防疾病發生或復 輕症狀、減少疾病之任何直接或間接病理性後果、 預防轉移、降低疾病進展速 逐手改善或減綾疾病狀態、及 症狀緩解或預後改良。在一此 二貫她例中，使用本發明之抗 緩疾病或病症之發展或減慢疾病或病症之進展。 「個體（individual/sub㈣」丨「患者」為脊椎動物。 在某些實施例中，脊椎動物為哺乳動物。哺乳動物包括 (但不限於）農畜（諸如牛）、運動型動物'窥物（諸如描、狗 及馬）、靈長類動物、小鼠及大鼠。在某些實施例中，哺 乳動物為人類。 術δ吾「醫樂調配物係指g由 切」你扣呈使活性成分之生物活性有效 之形式的製劑，且：a：不合料外淑分啡Λ u 、+ 3對杈與遠調配物之個體具有不可 接受之毒性的其他組分。該等調配物可為無菌的。 「無菌」調配物為無菌的或不含所有活微生物及其孢 子0 「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/分子之「治療有效量」可根據諸如以下 因素而變"固體之疾病狀態、年齡、性別及體重，以及物 質/分子在個體中誘發所要反應的能力。治療有效量涵蓋 使治療有利作用超過物質/分子之任何毒性或有害作用的 里。「預防有效I」係指有效達成所需預防結果所必需之 劑量及時間量。因為預防劑量係在疾病之前或在疾病早期 149799.doc -56- 201106972 用於個體，所以預防有效量通常（但不一定）會少於治療有 效量。 如本文中所用之術語「細胞毒性劑」係指抑制或阻止細 胞功能及/或引起細胞死亡或破壞的物質。該術語意欲包 括放射性同位素（例如At211、I131、1丨25、Y90、Rel86、The Kd" or Kd value" is measured by the 149799.doc •49-201106972 Sex Labeled Antigen Binding Assay (RIA), which is performed using the Fab form of the relevant antibody and its antigen, as described in the following assay . The solution binding affinity of the Fab to the antigen is measured by balancing the Fab with the minimally labeled (1251) labeled antigen in the presence of a continuously titrated unlabeled antigen, followed by capture of the bound antigen with a plate coated with an anti-Fab antibody ( See, for example, Chen et al., /. Mo/. 5b/., 293:865-881 (1999)). To establish analytical conditions, a MICROTITER® porous plate (Thermo Scientific) was coated overnight with 50 mM sodium carbonate (pH 9.6) containing 5 pg/ml anti-Fab antibody (Cappel Labs), and then at room temperature (approximately 23°) C) was blocked with 2% (w/v) bovine serum albumin in PBS for 2 to 5 hours. In a non-adsorbing plate (Nunc #269620), mix 100 μM or 26 pM [1251]-antigen with serial dilutions of the relevant Fab (eg, according to Presta et al., Cancer and 57:4593_4599 (1997) Evaluation of anti-VEGF antibody Fab-12). The relevant Fab is then incubated overnight; however, it can be incubated for a longer period of time (e.g., about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture culture tray for incubation at room temperature (e.g., for 1 hour). The solution was then removed and the plate was washed eight times with 0.1 °/〇 TWEEN-20TMiPBS. When the plates were dry, 150 microliters of scintillant (MICROSCINT-20tm; Packard) was added to each well, and the plates were counted on a TOPCOUNTtm gamma counter (Packard) for ten minutes. Each Fab concentration that yields a maximum binding of less than or equal to 20% is selected for the competitive binding assay. According to another embodiment, an antigen CM5 wafer immobilized in about 10 reaction units (RU) is utilized by using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25 °C. The surface electric 149799.doc -50- 201106972 slurry resonance analysis method was used to measure the Kd or Kd value. Briefly, carboxymethylated dextran was activated by ethyl dimethyl decylaminopropyl) carbodiimide hydrochloride (EDC) and hydroxybutaneimine (NHS) according to the supplier's instructions. Biosensor chip (CM5, BIACORE, Inc.). The antigen was diluted with 5 mM sodium acetate (卩114.8) to 5 4邑/1111 (about 0.241^) and injected at a flow rate of 5 μl/min to obtain about 10 reaction units (RU) of the coupled protein. After the injection of the antigen, 1 Μ ethanolamine was injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions (0.78 nM to 5) of Fab in PBS (PBST) containing 0.05% TWEEN-20 tm surfactant were injected at 25 ° C at a flow rate of approximately 25 μΐ/min. 〇0 nM). Using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software Version 3.2), the binding rate (kj and dissociation rate (k) is calculated by simultaneously fitting the binding and dissociation sensing spectra (k) ^). Calculate the equilibrium dissociation constant (Kd) according to the k^f/ku ratio. See, for example, Chen et al., /. Mo/. 5z'o/· 293:865-881 (1999). Resonance analysis, where the rate of binding exceeds 106 Μ·1 s_1, the rate of binding can be determined by using a fluorescence quenching technique, which is an increasing concentration of antigen (such as a spectrometer (such as a spectrophotometer equipped with a stop-flow mode ( 20 nM in PBS (pH 7.2) at 25 ° C in the presence of Aviv Instruments or a .8000 Series SLM-AMINCOTM Spectrophotometer (ThermoSpectronic) with agitated colorimetric tubes) The increase or decrease of the fluorescence emission intensity of the anti-antigen antibody (Fab form) (excitation = 295 nm; emission = 340 nm '16 nm band pass) 0 The "binding rate" or "k〇n" of the present invention may also Use 149799.doc 51 201106972 biacorE®_2000 or BIAC〇RE®_3〇 as described above The enthalpy system (BiAe〇re, Inc, Piscataway, NJ) is used for the determination. As used herein, the terms "substantially similar" or "substantially identical" mean two values (eg one associated with an antibody of the invention and the other There is a sufficiently high degree of similarity between the reference/comparisons of the carcass, so that those skilled in the art will recognize that the difference between the two values is determined by the biological characteristics based on the value (eg, L value). Little or no biological and/or statistical significance. The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 3%, less than about, based on the reference/comparative value. 2〇% and/or less than about 10%. As used herein, the phrase "substantially decreases" or "substantially different" means two values (usually one related to a molecule and the other related to a reference/comparative molecule) There is a sufficiently high difference between them so that those skilled in the art will recognize that the difference between the two values is statistically significant in the case where the biological characteristics are measured in terms of the values (e.g., Kd values). Reference / comparison molecule The difference between the two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%. "Purification" means that the molecule is present in the sample. The concentration is at least 95% or at least 98% (based on the weight of the sample containing it). The "isolated" nucleic acid molecule is a nucleic acid molecule that has been associated with at least one other nucleic acid molecule (eg, in its natural environment) ) Separation. The isolated nucleic acid molecule further comprises a nucleic acid molecule contained in a cell which typically expresses the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or is present at a chromosomal location different from the position of its natural chromosome 149799.doc-52-201106972. The term "vector," as used herein, refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is linked. One type of vector is a "plastid" which refers to a circular double strand of DNA that can engage other DNA segments. Another type of vector is the disease vector 'where other DNA segments can be ligated into the viral genome. • Certain vectors are capable of autonomous replication in host cells into which such vectors are introduced (e.g., bacterial vectors having bacterial origins of replication and free mammalian Q-hosts). Other vectors (e. g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell and thereby replicated along with the host genome. In addition, some carriers are capable of directing the basis of their operative connection. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors suitable for use in recombinant DNA techniques are typically in plastid form. In the present specification, since the plastid is the most commonly used carrier form, the "plastid" and the "carrier" are used interchangeably. As used herein, "polynucleotide" or "nucleic acid" refers to any length of nucleotide polymer and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide ribonucleotide, a modified nucleotide or base, and/or an analog thereof, or may be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Any of the qualities. The polynucleotide may comprise modified nucleotides such as thiolated nucleotides and analogs thereof. If present, the nucleotide structure can be modified before or after assembly of the polymer. The nucleotide sequence may be heterozygous with a non-nucleotide component. The polynucleotide may comprise modifications formed after synthesis, such as binding to a label. Other types of modifications include, for example, or the substitution of a naturally occurring nucleotide by the analog "cap"; modifications between nucleotides H9799.doc •53·201106972, such as having an uncharged linkage (eg, phosphonic acid) And a substance having a charge linkage (for example, phosphorothioate, phosphorodithioate, etc.), and a side thereof, such as a methyl ester, a phosphoric acid triester, a phosphonium acid, a urethane or the like. a moiety (eg, a protein (eg, nuclease, toxin, antibody, signal peptide, ply-L-lysine, etc.), having an intercalating agent (eg, acridin, psoralen, etc.) And other substances thereof, such as a chelating agent (for example, a metal, a radioactive metal, boron, an oxidizing metal, etc.), a substance containing a burning agent, and a modified linkage (for example, α-rotation isomer) The nucleic acid, etc., and the unmodified form of the polynucleotide. Further, any hydroxyl group normally present in the sugar may be protected by a standard protecting group, for example, by a phosphonate group, a carboxylic acid ester group. Activated to make other linkages with other nucleotides Or may be combined with a solid or semi-solid support. The 5, and 3' terminal oxime may be phosphorylated or partially substituted with an amine or an organic capping group of 2 carbon atoms. Other hydroxyl groups may also be derived from standard protection. Polynucleotides may also contain analogs of ribose or deoxyribose analogs generally known in the art, including, for example, 2'-quinone-methyl-ribose, 2,_〇-allyl-ribose, 2·. -Fluorine ribose or 2'-azido-ribose, carbocyclic analogue, cardiac spirulina, epimerose (such as arabinose, xylose or sucrose) Lyxose)), pentamose, furanose, sedoheptulose, acyclic analogs, and basic nucleoside analogs (such as thiol ribosides). One or more phosphodiester linkages can be alternatively linked Substituent substitution. These alternative linking groups include, but are not limited to, phosphate groups via P(0)s ("thioester"), p(s)s ("dithioester" And (〇) NR2 ("valerate"), p(〇)R, P(0)0R', CO or CH2 ("acetal"), wherein each ruler 149799.doc -54- 201106972 or R'Independence a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl group containing an ether (_〇_) linkage as appropriate Not all linkages in a polynucleotide need to be identical. The above description applies to all polynucleotides mentioned herein, including RNA and DNA. As used herein, "raised nucleotides" are usually Refers to a short polynucleotide, 'usually a single strand, usually a synthetic short polynucleotide, usually (but not necessarily) less than about 200 nucleotides in length. The terms "raised nucleotides" and "nuclear nucleus" 0 Glycosides &" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. The term "Αχ1" as used herein, unless otherwise indicated, refers to any native source from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats). AX1. The term encompasses "full length", untreated sputum 1 and any form of Axl produced by treatment in a cell. The term also encompasses naturally occurring Axl variants, such as splice variants or dual gene variants. The term "Axl ligand" or "Gas6" as used herein, unless otherwise indicated, refers to any vertebrate source (including mammals, such as primates (eg, humans) and rodents (eg, mice). And any native Axl ligand of rat)). The term encompasses "full length", untreated Axl ligands, and any form of Axl ligand produced by treatment in a cell. The term also encompasses naturally occurring variant A" ligands, such as splice variants or dual gene variants. As used herein, 'treatment' (and its variants, such as /"treat" or "treating" refers to a clinical intervention that attempts to alter the natural course of the individual being treated or the cell 149799.doc -55- 201106972 It can be performed for the purpose of prevention or in the course of clinical pathology. Ideal treatments include preventing or slowing down the symptoms of the disease, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the progression of the disease, improving or reducing the disease state, and improving the symptoms or prognosis. In one such second example, the use of the anti-stable disease or condition of the present invention or the progression of the disease or condition is slowed down. "Individual (sub)" or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, agricultural animals (such as cattle), sports animals 'spys ( Such as tracing, dogs and horses, primates, mice and rats. In some embodiments, the mammal is a human. Δ My "medical music formula refers to g by cutting" you buckle to make the activity A formulation in the form of a biologically active form of the ingredient, and: a: an unincorporated external ingredient, a compound having an unacceptable toxicity to the individual of the far-adjusted formulation. The formulation may be sterile. "Aseptic" formulation is sterile or free of all living microorganisms and spores. "Effective amount" means the amount of time and time necessary to effectively achieve the desired therapeutic or prophylactic result. The therapeutically effective amount can vary depending on factors such as the disease state of the solid, age, sex and weight, and the ability of the substance/molecule to induce a desired response in the individual. The therapeutically effective amount encompasses a therapeutically superior effect. Any toxic or detrimental effect of a substance/molecule. "Prophylactically effective I" means the dose and amount of time necessary to achieve the desired preventative effect. Because the preventive dose is before the disease or in the early stages of the disease 149799.doc -56- 201106972 is used in an individual, so a prophylactically effective amount will usually (but not necessarily) be less than a therapeutically effective amount. As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. The term is intended to include radioisotopes (eg, At211, I131, 1丨25, Y90, Rel86,

Re188、Sm153、Bi212、P32、Pb⑴及“之放射性同位素）、 化學治療劑（例如曱胺嗓吟（methotrexate)、阿黴素 〇 (adriamicin)、長春花生物鹼（Vinca alkaloid)(長春新鹼 (vincristine)、長春鹼（vinblastine)、依託泊苷（etop〇side))、 小紅莓（doxorubicin)、美法侖（melphalan)、絲裂黴素 C(mitomycin C)、苯丁 酸氮芥（chl〇rambucii)、道諾黴素 (daunorubicin)或其他嵌入劑、酵素及其片段（諸如核酸分 解酶）、抗生素及毒素（諸如小分子毒素，或細菌、真菌、 植物或動物來源之酶促活性毒素，包括其片段及/或變異 體）’以及下文揭示之各種抗腫瘤劑或抗癌劑。其他細胞 ◎ 毒性劑在下文中描述。殺腫瘤劑可引起腫瘤細胞毀壞。 「毒素」為能夠對細胞生長或增殖具有有害影響的任何 物質。 「化學治療劑」為可用於治療癌症之化合物。化學治療 ; 劑之實例包括烷基化劑’諸如塞替派（thi〇tepa)及環磷醯胺 (cyclosphosphamide ; CYTOXAN®);烷基磺酸酯，諸如白 消安（busulfan)、英丙舒凡（improsuifan)及娘泊舒凡 (piposulfan);氮丙〇定（aziridine)，諸如苯〇坐多巴 (benzodopa)、卡波酿i(carboquone)、米特多巴（meturedopa) 149799.doc -57- 201106972 及尤利多巴（uredopa);乙烯亞胺（ethylenimine)及曱基三聚 氰fe’包括，、甲密胺（altretamine)、曲他胺 (triethylenemelamine) '三伸乙基填醯胺、三伸乙基硫代填 醯胺及三羥甲基三聚氰胺；乙醯精（尤其布 拉他辛（bullatacin)及布拉他辛酮（bullatacin〇ne)) ; δ 9 四 氫***酚（屈***酚（dr〇nabin〇i)，MARINOL®) ; β-拉帕酮 (beta-lapachone);拉帕醇（iapach〇〇 ;秋水仙鹼 (colchicine);樺木酸（betulinic扣⑷；喜樹鹼 (camptothecin)(包括合成類似物拓朴替康（t〇p〇tecan ; HYCAMTIN®) ' CPT-ll(伊立替康（irin〇teCan ; CAMPTOSAR®))、乙醯基喜樹鹼、東莨菪素（sc〇p〇iectin) 及9-胺基喜樹驗）；苔蘚抑素（bry〇statin);卡利斯塔叮 (eallystatin) ; CC_l〇65(包括其合成類似物阿多來新 (adozelesin) 卡折來新（carzelesin)及比折來新 (bizelesin)) ’ 足葉草毒素（p〇d〇phyll〇toxin);足葉草酸 (podophyllinic acid);替尼泊苷（teniposide；);念珠藻環肽 (cryptophycin)(尤其念珠藻環肽1及念珠藻環肽8);海兔毒 素（dolastatin);多卡米辛（duocarmycin)(包括合成類似物 KW-2189 及 CB1-TM1);艾權素（eleutherobin);盤克斯塔叮 (pancratistatin);沙考的汀（sarcodictyin);海綿抑素 (spongistatin);氮芥（nitrogen mustard)，諸如苯丁 酸氮芥 (chlorambucil)、萘氮芥（chlornaphazine)、氯璘醯胺 (chlorophosphamide)、雌莫司汀（estramustine)、異環填醯 胺（ifosfamide)、雙氯乙基甲胺（mechlorethamine)、雙氯乙 149799.doc -58- 201106972 基曱胺氧化物鹽酸鹽、美法俞、新恩比興（novembichin)、 膽固醇對苯乙酸氮芬（phenesterine)、潑尼莫司汀 (prednimustine)、曲洛填胺（trofosfamide)、尿 α密咬芥 (uracil mustard);亞硝基脲，諸如卡莫司汀（carmustine)、 氯脲黴素（chlorozotocin)、福莫司汀（fotemustine)、洛莫司 、;丁（lomustine)、尼莫司 ί丁（nimustine)及雷莫司丁 (ranimnustine);抗生素，諸如稀二炔抗生素（例如刺孢黴 素（calicheamicin)，尤其刺孢黴素γΐΐ及刺孢黴素ωΐΐ (參見 Nicolaou^ A > Angew. Chem Inti. Ed. Engl., 33: 183-186 (1994)) ; CDP323(—種口服a-4整合素抑制劑）；達米 辛（dynemicin)，包括達米辛A;艾斯帕米辛（esperamicin); 以及新制癌菌素（neocarzinostatin)發色團及相關色蛋白烯 二炔抗生素發色團）、阿克拉黴素（aclacinomysin)、放線菌 素（actinomycin)、安麯黴素（authramycin)、偶氮絲胺酸 (azaserine)、博來徽素（bleomycin)、放線菌素C(cactinomycin)、 卡拉比辛（carabicin)、洋紅黴素（carminomycin)、嗜癌菌素 (carzinophilin)、色黴素（chromomycin)、放線菌素 D(dactinomycin)、道諾黴素、地托比星（detorubicin)、6-重氮基-5-側氡基-L-正白胺酸、小紅莓（包括 ADRIAMYCIN®、N-嗎啉基-小紅莓、氰基-(N-嗎啉基）-小 紅莓、2-(N-吡咯啉基）_小紅莓、鹽酸小紅莓脂質體注射液 (DOXIL®)、脂質體小紅葛 TLC D-99(MYOCET®)、聚乙二 醇化脂質體小紅莓（CAELYX®)及脫氧小紅莓 (deoxydoxorubicin))、表柔比星（epirubicin)、依索比星 149799.doc •59- 201106972 (esorubicin)、伊達比星（idarubicin)、麻西羅黴素 (marcellomycin)、絲裂黴素（諸如絲裂黴素C)、黴S分酸 (mycophenolic acid)、諾拉黴素（nogalamycin)、橄禮1 黴素 (olivomycin)、培洛黴素（peplomycin)、泊非黴素 (porfiromycin)、°票吟黴素、三鐵阿黴素（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)、淨司他丁（zinostatin)、左柔比星（zorubicin); 抗代謝物，諸如曱胺嗓吟、吉西他濱（gemcitabine ; GEMZAR®)、喃氟啶（tegafur ; UFTORAL®)、卡培他濱 (capecitabine ; XELODA®)、埃坡黴素（epothilone)及 5-氣 尿°密咬（5-FU);葉酸類似物，諸如迪諾特寧（denopterin)、 甲胺嗓呤、蝶羅0令（pteropterin)、三曱曲沙（trimetrexate); 嗓呤類似物，諸如氟達拉濱（fludarabine)、6-疏基嘌呤、 硫11米°票吟（thiamiprine)、硫鳥嘌吟；°密。定類似物，諸如安 西他濱（ancitabine)、阿紮胞苦（azacitidine)、6-氮雜尿誓、 卡莫氟（carmofur)、阿糖胞苷（cytarabine)、雙脫氧尿苷、 脫氧氟尿苷、依諾他濱（enocitabine)、氮尿苷 (floxuridine);雄激素，諸如卡普睾酮（calusterone)、屈他 雄酮丙酸醋（dromostanolone propionate)、環疏雄醇 (epitiostanol)、美雄烧（mepitiostane)、睾内醋（testolactone); 抗腎上腺，諸如胺魯米特（aminoglutethimide)、米托坦 (mitotane)、曲洛司坦（trilostane);葉酸補充劑，諸如夫羅 林酸（frolinic acid);乙醢葡搭醋（aceglatone);搭磷醯胺糖 149799.doc -60- 201106972Re188, Sm153, Bi212, P32, Pb(1) and "radioisotopes", chemotherapeutic agents (eg, methotrexate, adriamicin, vinca alkaloid (vinca alkaloid) (vincristine) Vincristine), vinblastine, etop〇side, doxorubicin, melphalan, mitomycin C, chlorambucil (chl) 〇rambucii), daunorubicin or other intercalating agents, enzymes and fragments thereof (such as nucleases), antibiotics and toxins (such as small molecule toxins, or enzymatically active toxins of bacterial, fungal, plant or animal origin) , including fragments and/or variants thereof' and various anti-tumor or anti-cancer agents disclosed below. Other cells ◎ Toxic agents are described below. Tumor-killing agents can cause destruction of tumor cells. "Toxins" are capable of cell growth. Or proliferate any substance that has a detrimental effect. "Chemotherapeutic agent" is a compound that can be used to treat cancer. Chemotherapy; Examples of agents include alkylating agents such as thiotepa (thi 〇tepa) and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsuifan and piposulfan; aziridine Aziridine), such as benzodopa, carboquone, meturedopa 149799.doc -57- 201106972 and uredopa; ethylenimine and Mercapto melamine fe' includes, altretamine, triethylenemelamine 'tri-ethyl acetamide, tri-ethyl thioacetamide and trimethylol melamine; Fine (especially bullatacin and bullatacin〇ne); δ 9 tetrahydrocannabinol (dr〇nabin〇i, MARINOl®); β-Lapbachone (beta-lapachone); Lappaol (iapach〇〇; colchicine; betulinic acid (betulinic deduction (4); camptothecin (including synthetic analogue topotecan (t〇p〇tecan; HYCAMTIN®) 'CPT-ll (irin〇teCan; CAMPTOSAR®), acetaminos camptothecin, sc〇p〇iectin And 9-Amino-HiStreet); Bryo〇statin; eallystatin; CC_l〇65 (including its synthetic analogue adozeesin) And bizelesin) 'p〇d〇phyll〇toxin; podophyllinic acid; teniposide; cryptophycin In particular, Candida cyclic peptide 1 and Nostoccal cyclic peptide 8); dolastatin; doocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; Pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, chlornaphazine, chloramphenicol Chlorophosphamide, estramustine, ifosfamide, mechlorethamine, dichloroethane 149799.doc -58- 201106972 hydrazide oxide hydrochloride , US law Yu, new engembichin, cholesterol phenesterine, prednisolone Prednimustine, trofosfamide, uracil mustard; nitrosourea, such as carmustine, chlorozotocin, floros Fentium (fotemustine), lomustine, lomustine, nimustine, and ramiminustine; antibiotics, such as diacetylene antibiotics (such as calicheamicin), especially Chrysosporin γ ΐΐ and calicheamicin ω ΐΐ (see Nicolaou ^ A > Angew. Chem Inti. Ed. Engl., 33: 183-186 (1994)); CDP323 (--oral a-4 integrin inhibitor) ); dynemicin, including Damisin A; esperamicin; and neocarzinostatin chromophore and related chromoprotein diacetylene antibiotic chromophore), Accra Aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabincin ), erythromycin (carminomycin), carzinophilin, color Chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-side thiol-L-positive leucine, cranberry (including ADRIAMYCIN®, N-morpholinyl-cranberry, cyano-(N-morpholinyl)-cranberry, 2-(N-pyrroyl)-cranberry, cranberry hydrochloride liposome injection (DOXIL®), liposome S. erythraea TLC D-99 (MYOCET®), PEGylated lipohydrate cranberry (CAELYX®) and deoxydoxorubicin (deoxydoxorubicin), epirubicin, Isoubicin 149799.doc •59- 201106972 (esorubicin), idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic mycophenolic Acid), nogalamycin, olivomycin, peplomycin, porfiromycin, penicillin, quelamycin , rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin , zorubicin; antimetabolites such as amidoxime, gemcitabine (GEMZAR®), fluflurane (tegafur; UFTORAL®), capecitabine (XELODA®), epo Epothilone and 5-gasuria (5-FU); folic acid analogues such as denoptin, methotrexate, pteropterin, triterpene Trimetrexate; 嗓呤 analogs, such as fludarabine, 6-mercaptopurine, sulfur 11 m ° thiamiprine, thioguanine; Analogs such as ancitabine, azacitidine, 6-azapine, carmofur, cytarabine, dideoxyuridine, deoxyfluoride Glycosides, enocitabine, floxuridine; androgens, such as caluterone, dromostanolone propionate, epitiostanol, meixiong (mepitiostane), testosterone (testolactone); anti-adrenal, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid ); ace gar vinegar (aceglatone); phosphatamide 149799.doc -60- 201106972

苷；胺基乙醯丙酸；恩尿嘴。定（eniluracil);安0丫 π定 (amsacrine);倍思塔布（bestrabucil);比生群（bisantrene); 艾達曲克（edatraxate);得弗伐胺（defofamine);秋水仙胺 (demecolcine);地'1 丫酿（diaziquone);艾弗鳥胺酸 (elfornithine);依利醋敍（elliptinium acetate);埃坡黴素； 依託格魯（etoglucid);硝酸鎵；羥基脲；香菇多糖 (lentinan)；羅尼達寧（lonidainine)；類美登素 (maytansinoid)，諸如美登素（maytansine)及胺沙托辛 (ansamitocin);米托胍月宗（mitoguazone);米托蒽酿 (mitoxantrone)；莫比達摩（mopidanmol);硝’拉維林 (nitraerine);喷司他丁（pentostatin);凡那明（phenamet); 0比柔比星（pirarubicin);洛索蒽醒（losoxantrone) ; 2-乙基 醢肼；丙卡巴肼（procarbazine) ; PSK®多醣複合物（JHS Natural Products, Eugene, OR);雷佐生（razoxane);根瘤 菌素（rhizoxin);西佐喃（sizofiran);鍺螺胺（spirogermanium); 細交鏈抱菌酮酸（tenuazonic acid);三亞胺酿；2,2',2'-三氯 三乙胺；單端孢黴烯族毒素（trichothecene)(尤其T-2毒素、 弗納庫林A(verracurin A)、桿抱菌素A(roridin A)及蛇形菌 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);二溴甘露醇（mitobronitol);二 漠·衛矛醇（mitolactol) ; 〇辰泊漠烧（pipobroman);曱托辛 (gacytosine);***糖苷（arabinoside ;「Ara-C」）；塞替 派；紫杉烧類（taxoid)，例如太平洋紫杉醇（paclitaxel ; 149799.doc -61 - 201106972 TAXOL®)、白蛋白工程改造之太平洋紫杉醇之奈米顆粒調 配物（ABRAXANETM)及多西他赛（TAXOTERE®);苯丁酸 氮芥（chloranbucil) ; 6-硫代鳥嘌呤；疏嗓α令；曱胺嗓呤； 舶藥劑，諸如順始（cisplatin)、奥沙利舶（oxaliplatin)(例如 ELOXATIN®)及卡I白（carboplatin);防止微管蛋白聚合形 成微管的長春花（vincas)，包括長春鹼（VELBAN®)、長春 新鹼（ONCOVIN®)、長春地辛（ELDISINE®、FILDESIN®) 及長春瑞賓（vinorelbine ; NAVELBINE®);依託泊苷（VP-16);異環磷醯胺；米托蒽醌；曱醯四氫葉酸（leucovorin); 諾凡特龍（novantrone);依達曲沙（edatrexate);道諾黴 素；胺基蝶呤；伊班膦酸鹽（ibandronate);拓撲異構酶抑 制劑RFS 2000 ;二氟曱基鳥胺酸（DMFO);類視黃素，諸 如視黃酸，包括貝瑟羅汀（bexarotene ; TARGRETIN®); 雙膦酸鹽，諸如氯屈膦酸鹽（clodronate)(例如BONEFOS® 或 OSTAC®)、依替膦酸鹽（etidronate ; DIDROCAL®)、 NE-58095、°坐來膦酸（zoledronic acid)/嗤來膦酸鹽 (zoledronate ; ZOMETA®)、阿侖膦酸鹽（alendronate ; FOSAMAX®)、帕米膦酸鹽（pamidronate ; AREDIA®)、替 魯膦酸鹽（tiludronate ； SKELID®)或利塞膦酸鹽 (risedronate ; ACTONEL®);曲沙他濱（troxacitabine)(l，3-二氧戊環核苷胞嘧啶類似物）；反義寡核苷酸，尤其抑制 與異常細胞增殖有關之信號傳導路徑中之基因之表現的反 義寡核苷酸，諸如PKC-(x、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 149799.doc •62· 201106972 苗，例如ALLOVECTIN®疫苗、LEUVECTIN®疫苗及 VAXID®疫苗；拓撲異構酶1抑制劑（例如LURTOTECAN®); rmRH(例如 ABARELIX®) ; BAY439006(索拉非尼 (sorafenib) ; Bayer) ; SU-11248(舒尼替尼（sunitinib)， SUTENT®，Pfizer);哌立福新（perifosine)、COX-2抑制劑 (例如塞來昔布（celecoxib)或依託昔布（etoricoxib))、蛋白 酶體抑制劑（例如PS341);硼替佐米（bortezomib ; VELCADE®) ; CCI-779 ;替吡法尼（tipifarnib ; R1 1577); 索拉非尼（orafenib)、ABT510 ; Bcl-2抑制劑，諸如奥利默 森納（oblimersen sodium ; GENASENSE®);匹克生璦 (pixantrone) ; EGFR抑制劑（參見以下定義）；酷胺酸激酶 抑制劑（參見以下定義）；絲胺酸-蘇胺酸激酶抑制劑，諸如 雷帕黴素（rapamycin)(西羅莫司（sirolimus)，RAPAMUNE®); 法尼基轉移酶（farnesyltransferase)抑制劑，諸如洛那法尼 (lonafarnib ; SCH 6636，SARASARTM);及上述任一者之 醫藥學上可接受之鹽、酸或衍生物；以及上述兩者或兩者 以上之組合，諸如CHOP，其為環磷醯胺、小紅莓、長春 新鹼及潑尼龍之組合療法之縮寫；及FOLFOX，其為奥沙 利鉑（ELOXATINtm)與5-FU及曱醯四氫葉酸之組合治療方 案的縮寫。 如本文中所定義之化學治療劑包括用以調節、減少、阻 斷或抑制可促進癌生長之激素之作用的「抗激素劑」或 「内分泌治療劑」。其可為激素本身，包括（但不限於）：具 有混合促效/拮抗概況之抗***，包括他莫昔芬 149799.doc -63- 201106972 (NOLVADEX®)、4-經基他莫昔芬、托瑞米芬（toremifene ; FARESTON®)、艾多昔芬（idoxifene)、曲洛昔芬 (droloxifene)、雷洛昔芬（rai〇xifene ; EVISTA®)、曲沃昔 芬（trioxifene)、雷洛昔芬（keoxifene)及選擇性***受體 調節劑（SERM)，諸如SERM3 ;不具有促效性質之純抗雌 激素，諸如氟維司群（fulvestrant ; FASLODEX®)及 EM800(該等藥劑可阻斷***受體（ER)二聚化、抑制 DNA結合、增加ER週轉且/或抑制ER含量）；芳香酶抑制 劑，包括類固醇芳香酶抑制劑（諸如福美司坦（formestane) 及依西美坦（exemestane ; AROMASIN®))及非類固醇芳香 酶抑制劑（諸如阿那曲唑（anastrazole ; ARIMIDEX®)、來 曲。坐（letrozole ; FEMARA®)及胺魯米特），以及其他芳香 酶抑制劑，包括伏氣0坐（vorozole ; RIVISOR®)、乙酸曱地 孕酮（megestrol acetate ; MEGASE®)、法屈0坐（fadrozole) 及4(5)-咪唑；黃體激素釋放激素促效劑，包括亮丙立德 (leuprolide ; LUPRON® 及 ELIGARD®)、戈舍瑞林 (goserelin)、布舍瑞林（buserelin)及曲特瑞林（tripterelin); 性類固醇，包括孕激素（諸如乙酸甲地孕酮及乙酸曱羥孕 酮）、***（諸如己浠雌紛（diethylstilbestrol)及普雷馬林 (premarin))及雄激素/類視黃素（諸如H經曱基睾_ (fluoxymesterone)、所有反式視黃酸及芬維A胺 (fenretinide));奥那司酮（onapristone);抗孕酮；***受 體下調劑（ERD);抗雄激素’諸如氟他胺（flutamide)、尼 魯胺（nilutamide)及比卡魯胺（bicalutamide);及上述任一者 149799.doc -64- 201106972 之醫藥學上可接受之鹽、酸或衍生物；以及上述兩者或兩 者以上之組合。 §用於本文中時，「生長抑制劑」係指活體外或活體内 , 抑制細胞（諸如表現Axl之細胞）生長之化合物或組合物。因 此，生長抑制劑可為大大降低s期細胞（諸如表現Axl之細 胞）百分率的藥劑。生長抑制劑之實例包括阻斷細胞週期 進程（除S期外之階段）之藥劑，諸如誘導G1停滯及Μ期停 〇 滯之藥劑。經典Μ期阻斷劑包括長春花（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）。阻滞 G1之彼等藥劑亦導致s期停滯，例如DNA烷化劑，諸如他 莫西芬、潑尼松（prednisone)、氮烯唑胺、氮芥、順鉑、甲 胺喋呤、5-氟尿啶及ara_c。更多資訊可見於Mendeis〇hn及 Israel 編，The Molecular Basis of Cancer·,第 1 章， Murakami等人之題為「Cell cycle regulati〇n，〇nc〇genes, 〇 and antineoplastic drugs」（WB Saunders， 1995)，例如第13頁。紫杉烷（太平洋紫杉醇及多西他赛 (docetaxel))均為源自紫杉樹之抗癌藥。源自歐洲紫杉之多 西他賽（TAXOTERE®，Rh〇ne-poulenc Rorer)為太平洋紫杉 醇（TAXOL®，BriSt〇l-Myers Squibb)之半合成類似物。紫杉 醇及多西他賽促進微管蛋白二聚體組裝成微管且藉由防止 解聚而使微管穩定，從而抑制細胞有絲***。 III.組合物及方法 本發明之特徵在於以組合療法使用Axl拮抗劑及VEGF拮 149799.doc -65- 201106972 抗劑治療諸如腫瘤之病理病狀。本發明亦提供抗Axl抗體 及其使用方法。Glycosides; amino acetopropionic acid; Eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colchicine ;1' brewing (diaziquone); efornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan ); lonidainine; maytansinoid, such as maytansine and ansamitocin; mitoguazone; mitoxantrone ; mopidanmol; nitraerine; pentostatin; phenamet; 0 pirarubicin; losoxantrone; -ethyl hydrazine; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; Syrrogermanium; tenuazonic acid; triammine; 2,2',2'-trichlorotriethylamine; Trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine toxin); urathan ); vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; Pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, such as paclitaxel; 149799.doc-61 - 201106972 TAXOL®), albumin engineered Pacific paclitaxel nanoparticle formulation (ABRAXANETM) and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; dredging α 曱; amidoxime; Pharmacy agents, such as cisplatin, oxaliplatin (such as ELOXATIN®) and carboplatin; preventing the polymerization of tubulin to form microtubules of periwinkle ( Vincas), including vinblastine (VELBAN®), vincristine (ONCOVIN®), long Essin (ELDISINE®, FILDESIN®) and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; Noventrone; edatrexate; daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorodecyl ornithine (DMFO); retinoids such as retinoic acid, including bexarotene; TARGRETIN®; bisphosphonates such as clodronate (eg BONEFOS® or OSTAC®), Etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (alendronate; FOSAMAX®), pa Methyl phosphonate (pamidronate; AREDIA®), tiludronate (SKELID®) or risedronate ( ACTONEL®); troxacitabine (l,3-dioxol) a cyclic nucleoside cytosine analog; an antisense oligonucleotide, particularly a gene in a signal transduction pathway involved in abnormal cell proliferation Expression of antisense oligonucleotides, such as PKC-(x, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE® vaccine and gene therapy epidemic 149799.doc • 62·201106972 For example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (eg LURTOTECAN®); rmRH (eg ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (eg celecoxib or etoricoxib), proteasome inhibitors (eg PS341); bortezomib; VELCADE®; CCI-779; Tipifarnib (R1 1577); Orafenib, ABT510; Bcl-2 inhibitors, such as Olimo Exlimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); valine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as Rapamycin (sirolimus, RAPAMUNE®) a farnesyltransferase inhibitor, such as lonafarnib (SCH 6636, SARASARTM); and a pharmaceutically acceptable salt, acid or derivative of any of the above; a combination of two or more, such as CHOP, which is an abbreviation for combination therapy with cyclophosphamide, cranberry, vincristine, and pour nylon; and FOLFOX, which is oxaliplatin (ELOXATINtm) and 5-FU and strontium Abbreviation for a combination treatment regimen of 醯tetrahydrofolate. A chemotherapeutic agent as defined herein includes an "antihormonal agent" or an "endocrine therapeutic agent" for regulating, reducing, blocking or inhibiting the action of a hormone which promotes cancer growth. It may be the hormone itself, including but not limited to: an antiestrogens with a mixed agonist/antagonistic profile, including tamoxifen 149799.doc -63-201106972 (NOLVADEX®), 4-amino tamoxifen , toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, thunder Keoxifene and selective estrogen receptor modulator (SERM), such as SERM3; pure antiestrogens without agonistic properties, such as fulvestrant (FASLODEX®) and EM800 (these agents) It can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover and / or inhibit ER content); aromatase inhibitors, including steroid aromatase inhibitors (such as formestane and Cimemet (exemestane; AROMASIN®) and non-steroidal aromatase inhibitors (such as anastrozole (ARIMIDEX®), lysine. (letrozole; FEMARA® and amine), and other aromatase Inhibitors, including volts 0 sitting (vorozole; RIVI SOR®), megestrol acetate (MEGASE®), fadrozole and 4(5)-imidazole; progesterone-releasing hormone agonists, including leuprolide; LUPRON® And ELIGARD®), goserelin, buserelin and tripterelin; sex steroids, including progestogens (such as megestrol acetate and hydroxyprogesterone acetate), Estrogens (such as diethylstilbestrol and premarin) and androgen/retinoids (such as H fluoxymesterone, all trans retinoic acid and fenretin A) Fenretinide; onapristone; antiprogesterone; estrogen receptor downregulator (ERD); antiandrogen such as flutamide, nilutamide and bikaru Bicalutamide; and a pharmaceutically acceptable salt, acid or derivative of any of the above 149799. doc-64-201106972; and a combination of two or more thereof. As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells, such as cells expressing Axl, in vitro or in vivo. Thus, the growth inhibitor can be an agent that greatly reduces the percentage of s phase cells, such as cells expressing Axl. Examples of growth inhibitors include agents that block cell cycle progression (stages other than S phase), such as agents that induce G1 arrest and stagnation. Classical sputum blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase inhibitors (such as doxorubicin, epirubicin, daunorubicin, and backing Bovine and bleomycin). Agents that block G1 also cause s-term arrest, such as DNA alkylating agents such as tamoxifen, prednisone, carbazolamide, nitrogen mustard, cisplatin, methotrexate, 5- Fluoridine and ara_c. More information can be found in Mendeis 〇hn and Israel, The Molecular Basis of Cancer, Chapter 1, Murakami et al., entitled "Cell cycle regulati〇n, 〇nc〇genes, 〇and antineoplastic drugs" (WB Saunders, 1995), for example on page 13. Taxanes (pacific paclitaxel and docetaxel) are anticancer drugs derived from yew trees. From the European yew, TAXOTERE®, Rh〇ne-poulenc Rorer is a semi-synthetic analogue of Pacific Taxol (TAXOL®, BriSt〇l-Myers Squibb). Taxol and docetaxel promote the assembly of tubulin dimers into microtubules and stabilize microtubules by preventing depolymerization, thereby inhibiting cell mitosis. III. Compositions and Methods The present invention is characterized by the use of an Axl antagonist and a VEGF antagonist 149799.doc-65-201106972 anti-allergic agent for the treatment of pathological conditions such as tumors. The invention also provides anti-Axl antibodies and methods of use thereof.

Axl拮抗劑 適用於本發明方法之Αχί拮抗劑包括特異性結合Αχί之多 肽、抗Axl抗體、Axl小分子、特異性結合Αχί之受體分子 及衍生物’及融合蛋白。Axl拮抗劑亦包括Axl多肽之拮抗 性變異體、siRNA、針對Axl及Axl配位體之RNA適體及肽 體。適用於本發明方法之Axl拮抗劑亦包括抗Axl配位體抗 體、抗Axl配位體多肽、特異性結合Αχί配位體之Axl受體 分子及衍生物。此等每一者之實例描述於下文中。在一些 實施例中，Axl拮抗劑不為華法林。 適用於本發明方法之抗Axl抗體包括以足夠親和力及特 異性結合Axl且可降低或抑制Axl活性之任何抗體。 抗Axl抗體在此項技術中已熟知且進一步描述於本文 中。參見例如 WO 2009/062690 ; WO 2009/06396 ;共同擁 有之同在申請中之美國專利申請案61/356,508(2010年6月 18曰申請）；5,468,634。在一實施例中，抗Axl抗體為單株 抗體。在一實施例中，抗Axl抗體為抗體片段，例如Fab、 Fab'-SH、Fv、scFv 或（Fab')2 片段。在一實施例中，抗 Αχ1 抗體為欲合、人類化或人類抗體。在一實施例中，抗Αχι 抗體經純化。在某些實施例中，組合物為用於治療癌症之 醫藥調配物。 由融合瘤產生之例示性單株抗體提供於本文中且描述於 實例8中。該等抗體稱為3G9、8B5、12A11及4F8。在一態 149799.doc •66· 201106972 樣中，提供與3G9、8B5、12A11及4F8競爭結合Axl之單株 抗體。亦提供所結合之抗原決定基與3G9、8B5、12A11及 4F8所結合之抗原決定基相同的單株抗體。 在一些實施例中，抗Axl抗體包含含有序列EVQLVESG GGLVQPGGSLRLSCAASGFSLSGSWIHWVRQAPGKGLEW VGWINPYRGYAYYADSVKGRFTISADTSKNTAYLQMNSLR AEDTAVYYCAREYSGWGGSSVGYAMDYWGQGTLV(SEQ ID NO:)的重鏈可變區，及含有序列DIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFG QGTKVEIKR(SEQIDNO)的輕鏈可變區。 抗Axl配位體Gas 6之抗體亦可用於本發明之方法中。 特異性結合Axl配位體（例如Gas6)之Axl分子或其片段可 用於本發明之方法中，例如結合且螯合Axl配位體蛋白 質、藉此阻止其信號傳導的Axl分子或其片段。Axl分子或 其Axl配位體結合片段較佳為可溶形式。在一些實施例 中，受體之可溶形式藉由結合Axl配位體而對Axl蛋白質之 生物活性施加抑制作用，藉此防止其結合標靶細胞表面上 存在之其天然受體。亦包括Axl融合蛋白，其實例描述於 下文中。亦參見Nagata，J Biol Chem 271:30022 (1996); 2005/0185471 ; McClosky等人，J Biol Chem 272: 23285 (1997) ; Fridell等人，J Biol Chem 273:7123 (1998)。 可溶性Axl蛋白質或嵌合Axl蛋白質包括不經由跨膜域而 固著至細胞表面之Axl蛋白質。因而，Axl之可溶形式（包 149799.doc •67- 201106972 括嵌合受體蛋白）儘管能夠結合Axl配位體且使Axl配位體 失活’但不包含跨膜域且因此通常不與表現分子的細胞之 細胞膜結合。 本發明之方法中可使用特異性結合Axl且阻斷或降低Axl 之活化，藉此防止其信號傳導的Axl配位體分子或其片 段。Axl Antagonists The antagonists suitable for use in the methods of the invention include polypeptides that specifically bind to Αχ, peptides that are resistant to Axl antibodies, small molecules of Axl, receptor molecules and derivatives that specifically bind to ’, and fusion proteins. Axl antagonists also include antagonistic variants of Axl polypeptides, siRNAs, RNA aptamers and peptides directed against Axl and Axl ligands. Axl antagonists suitable for use in the methods of the invention also include anti-Axl ligand antibodies, anti-Axl ligand polypeptides, Axl receptor molecules and derivatives that specifically bind to the Αχί ligand. Examples of each of these are described below. In some embodiments, the Axl antagonist is not warfarin. Anti-Axl antibodies suitable for use in the methods of the invention include any antibody which binds Axl with sufficient affinity and specificity and which reduces or inhibits Axl activity. Anti-Axl antibodies are well known in the art and are further described herein. See, e.g., WO 2009/062690; WO 2009/06396; commonly owned U.S. Patent Application Serial No. 61/356, 508, filed June 18, 2010; 5,468,634. In one embodiment, the anti-Axl antibody is a monoclonal antibody. In one embodiment, the anti-Axl antibody is an antibody fragment, such as a Fab, Fab'-SH, Fv, scFv or (Fab')2 fragment. In one embodiment, the anti-Αχ1 antibody is a human, human or human antibody. In one embodiment, the anti- Αχι antibody is purified. In certain embodiments, the composition is a pharmaceutical formulation for treating cancer. Exemplary monoclonal antibodies produced by fusion tumors are provided herein and are described in Example 8. These antibodies are referred to as 3G9, 8B5, 12A11 and 4F8. In a single 149799.doc •66·201106972 sample, monoclonal antibodies that compete with 3G9, 8B5, 12A11, and 4F8 for binding to Axl are provided. Monoclonal antibodies having the same epitope as the antigenic determinant to which 3G9, 8B5, 12A11 and 4F8 are bound are also provided. In some embodiments, the anti-Axl antibody comprises a heavy chain variable region sequence containing EVQLVESG GGLVQPGGSLRLSCAASGFSLSGSWIHWVRQAPGKGLEW VGWINPYRGYAYYADSVKGRFTISADTSKNTAYLQMNSLR AEDTAVYYCAREYSGWGGSSVGYAMDYWGQGTLV (SEQ ID NO :), the sequence and containing DIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFG QGTKVEIKR (SEQIDNO) light chain variable region. Antibodies against the Axl ligand Gas 6 can also be used in the methods of the invention. Axl molecules or fragments thereof that specifically bind to an Axl ligand (e.g., Gas6) can be used in the methods of the invention, e.g., Axl molecules or fragments thereof that bind to and sequester the Axl ligand protein, thereby preventing its signaling. The Axl molecule or its Axl ligand binding fragment is preferably in a soluble form. In some embodiments, the soluble form of the receptor exerts an inhibitory effect on the biological activity of the Axl protein by binding to the Axl ligand, thereby preventing its binding to its native receptor present on the surface of the target cell. Also included are Axl fusion proteins, examples of which are described below. See also Nagata, J Biol Chem 271:30022 (1996); 2005/0185471; McClosky et al, J Biol Chem 272: 23285 (1997); Fridell et al, J Biol Chem 273:7123 (1998). The soluble Axl protein or chimeric Axl protein includes Axl protein that is not fixed to the cell surface via the transmembrane domain. Thus, the soluble form of Axl (package 149799.doc •67-201106972 includes chimeric receptor proteins), although capable of binding to Axl ligands and inactivating Axl ligands, does not contain transmembrane domains and therefore is generally not associated with Cell membrane binding of cells expressing molecules. Axl ligand molecules or fragments thereof that specifically bind to Axl and block or reduce activation of Axl, thereby preventing its signaling, can be used in the methods of the present invention.

Axl siRNA描述且舉例說明於本文中且亦描述於例如w〇 2009/005813 中。 適體為核酸分子，其形成的三級結構特異性結合諸如 Axl配位體多肽之標把分子。此項技術中已熟知適體之產 生及治療用途。參見例如美國專利案第5,475,096號。Axl 配位體適體為聚乙二醇化修飾之寡核普酸，其採用使得其 能夠與細胞外Axl配位體結合之三維構形。關於適體之其 他資afl可見於美國專利申請公開案第20060148748號中。 肽體為連接至編碼免疫球蛋白分子之片段或部分之胺基 酸序列的肽序列。多肽可源自藉由任何特異性結合方法所 選擇之隨機化序列，該方法包括（但不限於）噬菌體呈現技 術。在一較佳實施例中，可將所選多肽連接至編碼免疫球 蛋白之Fc部分的胺基酸序列。特異性結合且拮抗Αχι配位 體或Axl之肽體亦適用於本發明之方法中。Axl siRNA is described and exemplified herein and is also described, for example, in WO 2009/005813. An aptamer is a nucleic acid molecule that forms a tertiary structure that specifically binds to a target molecule such as an Axl ligand polypeptide. The production and therapeutic use of aptamers is well known in the art. See, for example, U.S. Patent No. 5,475,096. The Axl ligand aptamer is a PEGylated modified oligonucleotide that adopts a three-dimensional configuration that enables it to bind to an extracellular Axl ligand. Other patents relating to aptamers can be found in U.S. Patent Application Publication No. 20060148748. A peptibody is a peptide sequence that is linked to an amino acid sequence encoding a fragment or portion of an immunoglobulin molecule. The polypeptide may be derived from a randomized sequence selected by any specific binding method, including, but not limited to, phage display technology. In a preferred embodiment, the selected polypeptide can be linked to an amino acid sequence encoding the Fc portion of the immunoglobulin. Peptibodies that specifically bind to and antagonize the 配ι ligand or Axl are also suitable for use in the methods of the invention.

Axl拮抗劑包括小分子，諸如R 428(Rigei ; 丨等 人，Cancer Research 70, 1544 (2〇1〇))及 pF〇234i〇66。在 一些實施例中，小分子Axl拮抗劑不為華法林。 VEGF拮抗劑 149799.doc •68- 201106972 VEGF拮抗劑係指能夠結合VEGF、降低VEGF表現量或 中和、阻斷、抑制、中止、降低或干擾VEGF生物學活性 (包括VEGF與一或多種VEGF受體結合及VEGF介導之血管 生成及内皮細胞存活或增殖）的分子。適用於本發明方法 中之VEGF拮抗劑包括特異性結合VEGF之多肽、抗VEGF 抗體及其抗原結合片段、特異性結合VEGF藉此隔絕其與 一或多種受體結合之受體分子及衍生物、融合蛋白（例如 VEGF-Trap(Regeneron))及 VEGF12i-白樹素（gelonin) (Peregrine)。VEGF拮抗劑亦包括VEGF多肽之拮抗性變異 體、針對VEGF之RNA適體及肽體。下文描述此等每一者 的實例。 適用於本發明方法中之抗VEGF抗體包括以足夠親和力 及特異性結合VEGF且可降低或抑制VEGF生物活性之任何 抗體或其抗原結合片段。抗VEGF抗體通常不結合諸如 VEGF-B或VEGF-C之其他VEGF同源物，亦不結合諸如 P1GF、PDGF或bFGF之其他生長因子。該等抗VEGF抗體 之實例包括（但不限於）本文中在「定義」之下所提供之抗 體。 兩種經最佳表徵之VEGF受體為VEGFR1 (亦稱為Fit-1)及 VEGFR2(關於鼠類同系物，亦稱為KDR及FLK-1)。各受體 對各VEGF家族成員之特異性可變化，但VEGF-A與Fit-1及 KDR均結合。全長Flt-1受體包括具有七個Ig域之細胞外 域、跨膜域及具有酪胺酸激酶活性之細胞内域。細胞外域 涉及結合VEGF且細胞内域涉及信號轉導。 149799.doc -69- 201106972 特異性結合VEGF之VEGF受體分子或其片段可在本發明 之方法中用於結合且螯合VEGF蛋白質，藉此防止其信號 傳導。在某些實施例中，VEGF受體分子或其VEGF結合片 段為可溶形式，諸如sFlt-1。受體之可溶形式藉由結合 VEGF來對VEGF蛋白質之生物活性發揮抑制作用，藉此阻 止VEGF與標靶細胞表面上存在之其天然受體結合。亦包 括VEGF受體融合蛋白，其實例描述於下文中。 嵌合VEGF受體蛋白為具有源自至少兩種不同蛋白質之 胺基酸序列的受體分子，其中至少一種蛋白質為能夠結合 VEGF且抑制VEGF之生物活性的VEGF受體蛋白（例如flt_l 或KDR受體）。在某些實施例中，本發明之嵌合VEGF受體 蛋白由源自僅兩種不同VEGF受體分子之胺基酸序列組 成；然而，包含一個、兩個、三個、四個、五個、六個或 所有七個來自fit-1及/或KDR受體之細胞外配位體結合區之 Ig樣結構域的胺基酸序列可連接至其他不相關蛋白質之胺 基酸序列，例如免疫球蛋白序列。可與Ig樣結構域組合之 其他胺基酸序列對於一般技術者顯而易知。嵌合VEGF受 體蛋白之實例包括可溶性Flt-1/Fc、KDR/Fc或FLt-1/KDR/Fc(亦稱為VEGF Trap)(參見例如PCT申請公開案第 WO 97/44453)。 本發明之可溶性VEGF受體蛋白或嵌合VEGF受體蛋白包 括不經由跨膜域固定至細胞表面之VEGF受體蛋白。因 此，VEGF受體之可溶形式（包括嵌合受體蛋白）儘管能夠結 合VEGF且使VEGF失活，但不包含跨膜域且因此通常不與 149799.doc -70- 201106972 表現分子的細胞之細胞膜結合。 適體為核酸分子’其形成的三級結構特異性結合諸如 VEGF多肽之標靶分子。此項技術中已熟知適體之產生及 治療用途。參見例如美國專利案第5,475,〇96號。VEGF適 體為聚乙二醇化修飾之券核苷酸，其採用使得其能夠與細 ’胞外VEGF結合之三維構形。靶向VEGF以治療年齡相關之 黃斑變性的治療有效適體之一實例為派加替尼 〇 (Pegaptanib ; Macugen™, 0SI)。關於適體之其他資訊可見 於美國專利申請公開案第20060148748號中。 肽體為連接至編碼免疫球蛋白分子之片段或部分之胺基 酸序列的肽序列。多肽可源自藉由任何特異性結合方法所 選擇之隨機化序列，該方法包括（但不限於）噬菌體呈現技 術。在一較佳實施例中，可將所選多肽連接至編碼免疫球 蛋白之Fc部分的胺基酸序列。特異性結合vegf且拮抗 VEGF之肽體亦適用於本發明之方法中。 〇 机後 1.抗體片段 本發明涵蓋抗體片段。抗體片段可藉由傳統方法產生， -諸如酶促消化’或藉由重組技術產生。在某些情形下，使 :用抗體片段優於使用整個抗體。較小尺寸之片段允許迅速 清除且更容易進入實體腫瘤中。欲回顧某些抗體片段，參 M, Hudson^ A > (2003) Nat. Med. 9:129-134 〇 已開發用於產生抗體片段的多種技術。傳統上，經由完 整抗體之蛋白水解消化獲得該等片段（參見例如M〇rim〇t〇 149799.doc -71 201106972 專尺 ’ Journal of Biochemical and Biophysical Methods 24:107-117 (1992);及 Brennan 等人，229:81 (1 985))。然而’該等片段現可由重組宿主細胞直接產生。 Fab、Fv及ScFv抗體片段皆可由大腸桿菌表現且分泌，從 而容易產生大量此等片段。可自上文所討論之抗體噬菌體 文庫中分離抗體片段。或者，可自大腸桿菌直接回收Fabi_ SH片段且以化學方式偶合而形成p(ab，）2片段（Carter等人， 扪1〇:163_167 (1992))。根據另一方法， F(ab’）2片段可自重組宿主細胞培養物直接分離。美國專利 第5,869,046號中描述包含救助受體結合抗原決定基殘基、 活體内半衰期延長的Fab及F(ab，）2片段。產生抗體片段之 其他技術對於熟習此項技術者顯而易知。在某些實施例 中，抗體為單鏈Fv片段（scFv)。參見w〇 93/16185、美國 專利第5,571，894號及第5,587,458號。Fv及scFv為完整結合 位點不含恆定區的唯一物質；因此，其可適於在活體内使 用期間減少非特異性結合。可構築scFv融合蛋白以使效應 蛋白在ScFv之胺基或羧基末端融合。參見办 B0rrebaeck編（同上）。抗體片段亦可為例如 「線性抗體」，例如美國專利第5,641，87〇號中所述。該等 線性抗體可為單特異性或雙特異性抗體。 2.人類化抗體 本發明涵盍人類化抗體。此項技術中已知將非人類抗體 人類化之多種方法。舉例而言，人類化抗體中可引入一或 多個來自非人類來源之胺基酸殘基。此等非人類胺基酸殘 149799.doc -72- 201106972 基通常稱為「輸入」殘基，其通常取自「輸入」可變域。 人類化基本上可依循Winter及同事之方法（jones等人， (1986) 321:522-525 ; Riechmann 等人，（1988) itowre 332:323-327 ; Verhoeyen等人，（1988) 加e 239:Axl antagonists include small molecules such as R 428 (Rigei; 丨 et al, Cancer Research 70, 1544 (2〇1〇)) and pF〇234i〇66. In some embodiments, the small molecule Axl antagonist is not warfarin. VEGF antagonist 149799.doc •68- 201106972 VEGF antagonist refers to the ability to bind VEGF, reduce VEGF expression or neutralize, block, inhibit, halt, reduce or interfere with VEGF biological activity (including VEGF and one or more VEGF receptors) Molecules of body binding and VEGF-mediated angiogenesis and endothelial cell survival or proliferation. VEGF antagonists suitable for use in the methods of the invention include polypeptides that specifically bind to VEGF, anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives that specifically bind to VEGF to thereby bind to one or more receptors, Fusion proteins (eg VEGF-Trap (Regeneron)) and VEGF12i-gelonin (Peregrine). VEGF antagonists also include antagonistic variants of VEGF polypeptides, RNA aptamers against VEGF, and peptibodies. Examples of each of these are described below. Anti-VEGF antibodies suitable for use in the methods of the invention include any antibody or antigen-binding fragment thereof which binds VEGF with sufficient affinity and specificity and which reduces or inhibits the biological activity of VEGF. Anti-VEGF antibodies typically do not bind to other VEGF homologs such as VEGF-B or VEGF-C, nor to other growth factors such as P1GF, PDGF or bFGF. Examples of such anti-VEGF antibodies include, but are not limited to, the antibodies provided herein under "Definitions". The two best characterized VEGF receptors are VEGFR1 (also known as Fit-1) and VEGFR2 (for murine homologs, also known as KDR and FLK-1). The specificity of each receptor for each VEGF family member can vary, but VEGF-A binds to both Fit-1 and KDR. The full-length Flt-1 receptor includes an extracellular domain having seven Ig domains, a transmembrane domain, and an intracellular domain having tyrosine kinase activity. The extracellular domain is involved in binding to VEGF and the intracellular domain is involved in signal transduction. 149799.doc -69- 201106972 A VEGF receptor molecule or fragment thereof that specifically binds VEGF can be used in the methods of the invention to bind and sequester VEGF protein, thereby preventing its signaling. In certain embodiments, the VEGF receptor molecule or VEGF binding fragment thereof is in a soluble form, such as sFlt-1. The soluble form of the receptor inhibits the biological activity of the VEGF protein by binding to VEGF, thereby preventing VEGF from binding to its native receptor present on the surface of the target cell. Also included are VEGF receptor fusion proteins, examples of which are described below. A chimeric VEGF receptor protein is a receptor molecule having an amino acid sequence derived from at least two different proteins, wherein at least one of the proteins is a VEGF receptor protein capable of binding to VEGF and inhibiting the biological activity of VEGF (eg, flt_l or KDR body). In certain embodiments, a chimeric VEGF receptor protein of the invention consists of an amino acid sequence derived from only two different VEGF receptor molecules; however, one, two, three, four, five are included , six or all of the amino acid sequences from the Ig-like domain of the extracellular ligand binding region of the fit-1 and/or KDR receptor can be linked to amino acid sequences of other unrelated proteins, such as immunization Globulin sequence. Other amino acid sequences that can be combined with Ig-like domains are readily apparent to those of ordinary skill in the art. Examples of chimeric VEGF receptor proteins include soluble Flt-1/Fc, KDR/Fc or FLt-1/KDR/Fc (also known as VEGF Trap) (see, e.g., PCT Application Publication No. WO 97/44453). The soluble VEGF receptor protein or chimeric VEGF receptor protein of the invention comprises a VEGF receptor protein that is not immobilized to the cell surface via a transmembrane domain. Thus, soluble forms of the VEGF receptor (including chimeric receptor proteins), while capable of binding to VEGF and inactivating VEGF, do not comprise a transmembrane domain and therefore typically do not interact with cells of the 149799.doc-70-201106972 expression molecule Cell membrane binding. The aptamer is a nucleic acid molecule' the tertiary structure it forms specifically binds to a target molecule such as a VEGF polypeptide. The production and therapeutic use of aptamers are well known in the art. See, for example, U.S. Patent No. 5,475, No. 96. The VEGF aptamer is a PEGylated modified conjugated nucleotide that employs a three-dimensional configuration that enables it to bind to the fine 'extracellular VEGF. An example of a therapeutically effective aptamer that targets VEGF to treat age-related macular degeneration is pecotinib (MacugenTM, 0SI). Further information on aptamers can be found in U.S. Patent Application Publication No. 20060148748. A peptibody is a peptide sequence that is linked to an amino acid sequence encoding a fragment or portion of an immunoglobulin molecule. The polypeptide may be derived from a randomized sequence selected by any specific binding method, including, but not limited to, phage display technology. In a preferred embodiment, the selected polypeptide can be linked to an amino acid sequence encoding the Fc portion of the immunoglobulin. Peptibodies that specifically bind to vegf and antagonize VEGF are also suitable for use in the methods of the invention. 〇 机 1. Antibody Fragments The present invention encompasses antibody fragments. Antibody fragments can be produced by conventional methods, such as enzymatic digestion' or by recombinant techniques. In some cases, it is preferred to use antibody fragments over the entire antibody. Fragments of smaller size allow for rapid clearance and easier access to solid tumors. To review certain antibody fragments, see M, Hudson^ A > (2003) Nat. Med. 9: 129-134 多种 A variety of techniques have been developed for the production of antibody fragments. Traditionally, such fragments have been obtained by proteolytic digestion of intact antibodies (see, for example, M〇rim〇t〇149799.doc-71 201106972 Specialized 'Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al. Man, 229:81 (1 985)). However, these fragments are now directly produced by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed and secreted by E. coli, so that a large number of such fragments are easily produced. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, the Fabi_SH fragment can be directly recovered from E. coli and chemically coupled to form a p(ab,)2 fragment (Carter et al., 扪1〇: 163_167 (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 rescue receptor binding epitope residue, an in vivo half-life extension are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments are readily apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and scFv are the only substances in which the complete binding site does not contain a constant region; therefore, it can be adapted to reduce non-specific binding during in vivo use. The scFv fusion protein can be constructed such that the effector protein is fused at the amino or carboxy terminus of the ScFv. See the B0rrebaeck series (ibid.). The antibody fragment can also be, for example, a "linear antibody" as described in U.S. Patent No. 5,641,87. The linear antibodies can be monospecific or bispecific antibodies. 2. Humanized antibodies The present invention encompasses humanized antibodies. A variety of methods for humanizing non-human antibodies are known in the art. For example, one or more amino acid residues from a non-human source can be introduced into the humanized antibody. These non-human amino acid residues 149799.doc -72- 201106972 are commonly referred to as "input" residues, which are typically taken from the "input" variable domain. Humanization can basically follow the methods of Winter and colleagues (Jones et al. (1986) 321:522-525; Riechmann et al. (1988) itowre 332:323-327; Verhoeyen et al. (1988) plus e 239:

1534-1536)藉由南變區序列取代人類抗體之相應序列來進 行。因此，該等「人類化」抗體為嵌合抗體（美國專利第 4,816,567號），其中實質上小於完整人類可變域已經非人 類物種之相應序列取代。實際上，人類化抗體通常為人類 抗體，其中一些高變區殘基及可能之一些FR殘基已經齧齒 動物抗體之類似位點之殘基取代。 選擇用於產生人類化抗體之人類可變域（輕鏈與重鏈）對 於減少抗原性極其重要。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篩檢齧齒動物抗體之 可變域序列。隨後將與齧齒動物之該序列最接近之人類序 列視作人類化抗體之人類構架。參見例如以邮等人， (1993) J. 151:2296 ; Ch〇thia等人，ο%?)1534-1536) by substituting the sequence of the human variable for the sequence of the human variable. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less than the corresponding sequence of a non-human species has been replaced by an intact human variable domain. In fact, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues have been replaced by residues at 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. The variable domain sequence of the rodent antibody is screened against a complete library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to this sequence of rodents is then considered a human framework for humanized antibodies. See, for example, E. et al., (1993) J. 151:2296; Ch〇thia et al., ο%?)

Mo/.此〇/. 196:901。另一方法係使用源自含有特定亞群之 輕鏈或重鏈之所有人類抗體之共同序列的特定構架。若干 架。參見例如Carter等 USA, 89:4285 ； Presta^ 不同的人類化抗體可使用相同構 k，（\992) Proc. Natl. Acad. Sci. 人，（1993) J. /w卿⑽/·，151:2623。 通常更需要使抗體在保持對抗原之高親和力及其他良好 生物特性下經人類化。為達成此目的，根據—種方法:藉 由使用親本序列及人類化序列之三維模型分析親本序列^ 149799.doc -73- 201106972 、u生人類化產物的過程來製備人類化抗體。三维免 疫球蛋白模型普遍可得且為熟習此項技術者熟知。可利用 電腦程式說明及呈現所選擇之候選免疫球蛋白序列之可能 二維構形結構。檢查此等呈現可分析殘基在候選免疫球蛋 白序列之功能中的可能作用，亦即，分析影響候選免疫球 蛋白結合其抗原之能力的殘基。藉此，可自接受者及輸入 序列選擇FR殘基且加以組合以便獲得所需抗體特徵，諸如 對標乾抗狀親和力增加。通f，冑變區殘基直接且最實 質性地涉及影響抗原結合。 3.人類抗體 可藉由將選自人類源噬菌體呈現文庫之Fv純系可變域序 列與如上所述之已知人難定域序列組合來構築本發明之 人類抗體。或者’可利㈣合瘤方法製造本發明之人類單 株抗體。用於產生人類單株抗體之人類骨髓瘤及小氣_人 類雜交骨髓瘤（heteromyei〇ma)細胞株已描述於例如K〇zb〇r 乂 /讲卿似/·，133: 3001 (1984) ; Br〇deur等人’Mo/. This 〇/. 196:901. Another method uses a specific framework derived from a common sequence of all human antibodies that contain a light or heavy chain of a particular subpopulation. Several shelves. See, for example, Carter et al., USA, 89: 4285; Presta^ Different humanized antibodies can use the same conformation k, (\992) Proc. Natl. Acad. Sci. Human, (1993) J. /w (10)/·, 151 :2623. It is often more desirable to subject the antibody to humanization while maintaining high affinity for the antigen and other good biological properties. To achieve this, a humanized antibody is prepared according to a method by analyzing a parental sequence of a parental sequence and a humanized sequence using a three-dimensional model of the parental sequence and the humanized sequence. Three-dimensional immunoglobulin models are commonly available and are well known to those skilled in the art. A computer program can be used to illustrate and present the possible two-dimensional configuration of the selected candidate immunoglobulin sequence. Examination of these presentations may analyze the possible role of the residues in the function of the candidate immunoglobulin sequence, i.e., to analyze residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thereby, FR residues can be selected from the recipient and the input sequence and combined to obtain the desired antibody characteristics, such as increased affinity for the stem. Passing f, the mutated region residues are directly and most substantively involved in affecting antigen binding. 3. Human antibody The human antibody of the present invention can be constructed by combining an Fv pure line variable domain sequence selected from a human-derived phage display library with a known human difficult-to-local sequence as described above. Alternatively, the human monoclonal antibody of the present invention can be produced by the 'four' tumor-binding method. Human myeloma and small gas-human hybrid myeloma (heteromyei〇ma) cell lines for the production of human monoclonal antibodies have been described, for example, in K〇zb〇r 乂/讲卿似/·, 133: 3001 (1984); Br 〇deur et al'

Antibody Production Techniques and Applications ,箄 s\-63 頁（Marcel Dekker，Inc.，New York，1987);及 Boernei^ 人，《/. /mmw«o/·, 147: 86 (1991)中。 現可產生轉殖基因動物（例如小鼠），其免疫後能夠在缺 乏内源免疫球蛋白產生之情況下產生全譜系之人類抗體。 舉例而言，已描述敌合及生殖系突變體小鼠中抗體重鏈連 接£ (JH)基因的同種接合子缺失會完全抑制内源抗體產 生。將人類生殖系免疫球蛋白基因陣列轉移至該等生殖系 149799.doc -74- 201106972 突變體小鼠體内將會在抗原攻擊後引起人類抗體之產生。 參見例如 Jakobovits等人 ’ Proc. Jcac/. Scz· C/a, 90: 2551 (1993) ; Jakobovits等人，iVaiwre, 362: 255 (1993);Antibody Production Techniques and Applications, 箄 s\-63 (Marcel Dekker, Inc., New York, 1987); and Boernei^, "/. /mmw«o/., 147: 86 (1991). It is now possible to produce a transgenic animal (e.g., a mouse) which, upon immunization, is capable of producing a full lineage of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain ligation (JH) gene in a hostile and germline mutant mouse completely inhibits endogenous antibody production. Transfer of human germline immunoglobulin gene arrays to these germ lines 149799.doc -74- 201106972 Mutant mice will cause human antibody production after antigen challenge. See, for example, Jakobovits et al., Proc. Jcac/. Scz. C/a, 90: 2551 (1993); Jakobovits et al., iVaiwre, 362: 255 (1993);

Bruggermann等人，/mmwwo/·, 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（其亦稱「抗原決定基印記 Q 法」），藉由如上所述之噬菌體呈現技術所獲得之非人類 抗體片段之重鏈或輕鏈可變區可以人類V域基因之譜系置 換’形成非人類鍵/人類鍵scFv或Fab喪合體之群。以抗原 進行選擇可分離非人類鏈/人類鏈嵌合scFv或Fab，其中人 類鏈可恢復在移除初級噬菌體呈現純系中之相應非人類鏈 時所損壞的抗原結合位點，亦即抗原決定基決定（印記）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（參見1993年4月1日公開之PCT w〇 ◎ 93/06213)。與傳統上藉由CDR移植使非人類抗體人類化不 同，此技術提供不具有非人類來源之1711或（：〇尺殘基的完全 人類抗體。 4·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的 單株抗體。在某些實施例中，雙特異性抗體為人類或人類 化抗體。纟某些實施例中，結合特異性之一係針對—而 另-者係針對其他任何抗原4某些實施射，雙特異性 抗體可結合AXl之兩個不同抗原決定基。雙特異性抗體亦 149799.doc •75- 201106972 可用於將細胞毒性劑定位於表現Axl之細胞。此等抗體具 有結合Axl之臂及結合細胞毒性劑之臂，該細胞毒性劑諸 如皂草素（saporin)、抗干擾素-α、長春花生物鹼、蓖麻毒 素Α鏈、甲胺喋呤或放射性同位素半抗原。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）。 此項技術中已知製造雙特異性抗體之方法。傳統上，雙 特異性抗體之重組產生法係基於兩對免疫球蛋白重鏈_輕 鏈之共同表現，其中兩個重鏈具有不同特異性（Milstein及 Cuello, 305: 537 (1983))。由於免疫球蛋白重鏈及 輕鍵之隨機分配’此專融合瘤（四源雜交瘤）產生10種不同 抗體分子之潛在混合物，其中僅一種具有正確雙特異性結 構。通常藉由親和層析步驟進行之正確分子之純化法相當 繁瑣’且產物產率較低。類似程序揭示於i 993年5月i 3日 公開之 WO 93/08829及 Traunecker 等人，£从50 /·，10: 3655 (1991)中。 根據不同方法，將具所需結合特異性（抗體_抗原結合位 點）之抗體可變域與免疫球蛋白恆定域序列融合。例如與 免疫球蛋白重鏈恆定域（包含敍鏈、CH2及CH3區之至少一 部分）融合。在某些實施例中，含有輕鏈結合所需位點的 第一重鏈悝定區（CH1)存在於至少一個融合部分。將編碼 免疫球蛋白重鏈融合體及（若需要）免疫球蛋白輕鏈2DNA ***個別表現載體中，且共轉染至適當宿主生物體中。從 而在使用不相等比率之三個多肽鏈以建構得到最佳產量的 149799.doc -76· 201106972 實施例中，為調整這三個多肽片段之相互比例提供極大靈 活性。然而，當相等比率之至少兩個多狀鍵的表現造成高 產量或當比率並非特別重要時，可將兩個或全部三個多肽 鍵之編碼序列***一個表現載體中。 在此方法之一實施例中，雙特異性抗體係由位於一臂中 ' 之雜交免疫球蛋白重鏈（具有第一結合特異性）與位於另一 臂中之雜交免疫球蛋白重鏈_輕鏈對（提供第二結合特異性） 〇 組成。已發現此不對稱結構有利於將所需雙特異性化合物 與非所要之免疫球蛋白鏈組合分離，因此免疫球蛋白輕鍵 存在於雙特異性分子之僅一半中可提供一種簡便之分離方 式。此方法揭示於wo 94/04690中。關於產生雙特異性抗 體之其他細節，參見例如Suresh等人，抓如心以 121:210 (1986)。 根據另一方法，可工程改造一對抗體分子間之界面以使 自重組細胞培養物回收雜二聚體之百分比最大化。該界面 O g 3抗體恆定域之Ch3域之至少一部分。以此方法，使第 k體分子界面之一或多條小胺基酸側鏈經較大側鏈（例 如酪胺酸或色胺酸）置換。藉由較小胺基酸側鏈（例如丙胺 酸或蘇胺酸）置換較大胺基酸側鏈可在第二抗體分子之界 產生尺寸與較大側鏈相同或類似之互補「空腔」。由 此提供增加雜二聚體（而非其他非所要最終產物，諸如同 二聚體）之產量的機制。 雙特異性抗體包括交聯或「異結合」抗體。舉例而言， 異結合之抗體之一可與抗生物素蛋0(avidin)偶合，另一 149799.doc •77- 201106972 者與生物素偶合。已提出該等抗體例如可使免疫系統細胞 靶向不需要細胞（美國專利案第4,676,980號）且可用於治療 HIV 感染（WO 91/00360、WO 92/00373 及 EP 03089)。可使 用任何適宜的交聯方法製造異結合抗體。適當交聯劑及多 種交聯技術在此項技術中已熟知且揭示於美國專利案第 4,676,980號中。 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。Brennan 等人，229: 81 (1985)描述一種程序，其中完整抗 體經蛋白質裂解而產生F(ab')2片段。此等片段在二硫醇錯 合劑亞砷酸鈉存在下還原以穩定鄰近二硫醇且防止分子間 二硫鍵形成。接著所產生之Fab’片段轉化為硫代硝基苯甲 酸酯（TNB)衍生物。接著藉由髄基乙胺還原將Faiy-TNB衍 生物之一再轉化為Fab'-硫醇，且與等莫耳量之另一種Fab1-TNB衍生物混合以形成雙特異性抗體。所產生之雙特異性 抗體可用作選擇性固定酵素之藥劑。 最新進展已可方便地自大腸桿菌直接回收Fab’-SH片 段，該等片段可經化學偶合而形成雙特異性抗體。 Shalaby等人，MW.，175: 217-225 (1992)描述完全 人類化雙特異性抗體F(ab’）2分子之製備。各Fab'片段分別 由大腸桿菌分泌且進行活體外定向化學偶合以形成雙特異 性抗體。由此形成之雙特異性抗體能夠結合過度表現 HER2受體之細胞及正常人類T細胞，且引發人類細胞毒性 淋巴細胞針對人類***腫瘤標靶之溶解活性。 149799.doc •78· 201106972 亦已描述直接在重組細胞培養物中形成且分離雙特異性 抗體片段之多種技術。舉例而言，已使用白胺酸拉鏈產生 雙特異性k體。Kostelny等人，乂 /所則仙厂，148(5):1547_ 15 53 (1992)。藉由基因融合將來自F〇s及Jun蛋白之白胺酸 拉鏈肽與兩種不同抗體之Fab·部分連接。抗體均二聚體在 鉸鏈區還原而形成單體，且接著再氧化而形成抗體雜二聚 體。此方法亦可用於產生抗體均二聚體。H〇llinger等人’ 尸roc. Α^αί/· Jcai/· 5W· 90:6444-6448 (1993)所述之「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) ’該連接子過短而無法使同一鏈上之兩 個結構域之間配對。因此，一個片段之VH及VL域被迫與 另一個片段之互補VL及VH域配對，由此形成兩個抗原結 合位點。另一種藉由使用單鏈Fv(sFv)二聚體形成雙特異性 抗體片段之朿略亦已報導。參見Gruber等人，J. ， 152:5368 (1994)° 涵蓋兩價以上之抗體。舉例而言，可製備三特異性抗 體。Tutt等人，/· /mwMwo/· 147: 60 (1991)。 5.多價抗體 多價抗體可比二價抗體更快地被表現抗體所結合之抗原 的細胞内化（及/或異化）。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）’多價抗體可容易藉由重組表現編碼抗體多 肽鏈之核酸來製得。多價抗體可包含二聚域及三個或三個 149799.doc •79· 201106972 以上抗原結合位點。在某些實施例中，二聚域包含Fc區或 鉸鏈區（或由Fc區或鉸鏈區組成）。在此情形中，抗體將包 含Fc區及三個或三個以上位於Fc區之胺基末端之抗原結合 位點。在某些實施例中’多價抗體包含三個至約八個抗原 結合位點（或由三個至約八個抗原結合位點組成）。在一該 實施例中，多價抗體包含四個抗原結合位點（或由四個抗 原結合位點組成）。多價抗體包含至少一個多肽鍵（例如兩 個多狀鍵），其中多狀鍵包含兩個或兩個以上的可變域。 舉例而言’多肽鏈可包含νϋ^(Χ1)η-νϋ2-(Χ2)η-Ρο，其中 VD1為第一可變域，VD2為第二可變域，Fc為一條多肽鏈 中之Fc區’ XI及X2表示胺基酸或多肽，且η為〇或1。舉例 而言’多肽鏈可包含：VH-CH1-柔性連接子-VH-CHl-Fc區 鏈或VH-CH1-VH-CH1-Fc區鏈。本文中之多價抗體可另外 包含至少兩個（例如四個）輕鏈可變域多肽。本文中之多價 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域0 6·單域抗體 在一些實施例中，本發明之抗體為單域抗體。單域抗體 為包含抗體之重鏈可變域之全部或一部分或者輕鏈可變域 之全部或一部分的單一多肽鏈。在某些實施例中，單域抗 體為人類單域抗體（Domantis，Inc·, Waltham，MA;參見例 如美國專利案第6,248,516 B1號）。在一實施例中，單域抗 體係由抗體重鏈可變域之全部或一部分組成。 149799.doc -80 · 201106972 7.抗體變異鱧 在一些貫施例中’本發明涵蓋本文所述之抗體的胺基酸 序列修飾。舉例而言，可能需要改良抗體之結合親和力 及/或其他生物學特性。可藉由將適當變異引入編碼抗體 之核苦酸序列中或藉由肽合成來製備抗體之胺基酸序列變 異體。該等修飾包括例如抗體胺基酸序列内之殘基缺失 及/或***及/或取代。缺失、***及取代可任意組合以獲 ❹ 得最終構築體’其限制條件為最終構築體具有所需特徵。 可在形成序列時將胺基酸變異引入標的抗體胺基酸序列 中。 種適用於鑑別抗體之作為較佳突變誘發位置之某些殘 基或區域的方法稱為「丙胺酸掃描突變誘發」，如Bruggermann et al., /mmwwo/., 7: 33 (1993). '· Gene shuffling can also be used to obtain human antibodies 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 "antigenic epitope imprinting Q method"), the heavy or light chain variable region of the non-human antibody fragment obtained by the phage display technology as described above can be replaced by the human V domain gene lineage 'Form a group of non-human bonds/human bond scFvs or Fabs. The non-human chain/human chain chimeric scFv or Fab can be isolated by antigen selection, wherein the human chain can restore the antigen binding site, ie, the epitope, which is damaged when the primary phage exhibits the corresponding non-human chain in the pure line. Decide (imprint) the choice of human chain collocation. When this process is repeated to replace the remaining non-human strands, human antibodies are obtained (see PCT w〇 ◎ 93/06213 published on April 1, 1993). Unlike the traditional humanization of non-human antibodies by CDR grafting, this technique provides a fully human antibody that does not have a non-human source of 1711 or (: a scorpion residue. 4. A bispecific antibody bispecific antibody is at least Two different antigens have binding specificity of monoclonal antibodies. In certain embodiments, the bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is directed to - and the other It is specific for any other antigen 4, and the bispecific antibody can bind to two different epitopes of AXl. The bispecific antibody is also 149799.doc •75- 201106972 can be used to localize cytotoxic agents to cells expressing Axl These antibodies have an arm that binds to Axl and an arm that binds to a cytotoxic agent such as saporin, anti-interferon-α, vinca alkaloid, ricin toxin chain, methotrexate Or a 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. It is known in the art to make bispecific antibodies. Traditionally, the recombinant production of bispecific antibodies is based on the common performance of two pairs of immunoglobulin heavy chain-light chains, two of which have different specificities (Milstein and Cuello, 305: 537 (1983) Due to the random distribution of immunoglobulin heavy and light bonds 'this fusion tumor (four-source hybridoma) produces a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Usually by affinity layer The purification of the correct molecule by the step of the analysis is quite cumbersome and the product yield is low. A similar procedure is disclosed in WO 93/08829 and Traunecker et al., published on May 3, 993, £50/·, 10: In 3655 (1991), an antibody variable domain having the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant domain sequence according to different methods, for example, with an immunoglobulin heavy chain constant domain (including Fusion, at least a portion of the CH2 and CH3 regions are fused. In certain embodiments, a first heavy chain definitive region (CH1) comprising a site required for light chain binding is present in at least one fused portion. The protein heavy chain fusion and, if desired, the immunoglobulin light chain 2 DNA are inserted into individual expression vectors and co-transfected into a suitable host organism, thereby providing optimal yields using three polypeptide chains of unequal ratios. 149799.doc -76· 201106972 In the examples, great flexibility is provided to adjust the mutual ratio of the three polypeptide fragments. However, when the performance of at least two polymorphic bonds of equal ratios results in high yield or when the ratio is not particularly important In this embodiment, the coding sequence of two or all three polypeptide bonds can be inserted into a representation vector. In one embodiment of the method, the bispecific antibody system consists of a hybrid immunoglobulin heavy chain located in one arm (having The first binding specificity is composed of a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) located in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from the undesired immunoglobulin chain combination, and thus the presence of immunoglobulin light bonds in only half of the bispecific molecule provides a convenient separation. This method is disclosed in wo 94/04690. For additional details on the production of bispecific antibodies, see, for example, Suresh et al., Scratch, 121:210 (1986). According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of recovered heterodimers from the recombinant cell culture. At least a portion of the Ch3 domain of the constant domain of the O g 3 antibody. In this way, one or more of the small amino acid side chains of the k-th molecular interface are replaced by a larger side chain (e.g., tyrosine or tryptophan). Substitution of the larger amino acid side chain by a smaller amino acid side chain (eg, alanine or threonine) can create a complementary "cavity" of the same or similar size to the larger side chain at the boundary of the second antibody molecule. . This provides a mechanism to increase the yield of heterodimers (rather than other undesirable end products, such as homodimers). Bispecific antibodies include cross-linked or "iso-binding" antibodies. For example, one of the hetero-binding antibodies can be coupled to avidin and the other 149799.doc •77-201106972 can be coupled to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and to treat HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Heteroconjugate antibodies can be made using any suitable crosslinking method. Suitable cross-linking agents and a variety of cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980. Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al., 229: 81 (1985) describe a procedure in which intact antibodies are cleaved by protein to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol-missing agent sodium arsenite to stabilize the adjacent dithiol and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Faay-TNB derivatives is then reconverted to Fab'-thiol by mercaptoethylamine reduction and mixed with another molar amount of another Fab1-TNB derivative to form a bispecific antibody. The bispecific antibody produced can be used as an agent for selectively immobilizing an enzyme. Recent advances have made it possible to conveniently recover Fab'-SH fragments directly from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, MW., 175: 217-225 (1992) describe the preparation of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment was secreted by E. coli and subjected to in vitro directed chemical coupling to form a bispecific antibody. The bispecific antibody thus formed is capable of binding to cells overexpressing the HER2 receptor and normal human T cells, and elicits the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. 149799.doc •78· 201106972 Various techniques for forming and isolating bispecific antibody fragments directly in recombinant cell culture have also been described. For example, leucine zippers have been used to generate bispecific k bodies. Kostelny et al., 乂 /所则仙厂, 148(5): 1547_ 15 53 (1992). The leucine zipper peptide from F〇s and Jun proteins was ligated to the Fab·portion of two different antibodies by gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer, which is then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. H〇llinger et al.'s corpse roc. Α^αί/· Jcai/· 5W· 90:6444-6448 (1993) "Bifunctional antibody" technology 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. The linker is too short to pair between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for the formation of bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J., 152:5368 (1994)° for antibodies of more than two valencies. For example, a trispecific antibody can be prepared. Tutt et al., /. /mwMwo/. 147: 60 (1991). 5. Multivalent antibodies Multivalent antibodies can be internalized (and/or catabolized) by the antigens to which the antibodies bind, faster than bivalent antibodies. The antibody of the present invention may be a multivalent antibody having three or more antigen-binding sites (except for an antibody of the IgM class) (for example, a tetravalent antibody). A multivalent antibody can easily express a nucleic acid encoding an antibody polypeptide chain by recombinant expression. To make it. The multivalent antibody may comprise a dimeric domain and three or three antigen binding sites above 149799.doc •79·201106972. In certain embodiments, the dimeric domain comprises an Fc region or a hinge region (or consists of an Fc region or a hinge region). In this case, the antibody will comprise an Fc region and three or more antigen binding sites located at the amino terminus of the Fc region. In certain embodiments, a multivalent antibody comprises three to about eight antigen binding sites (or consists of three to about eight antigen binding sites). In one such embodiment, the multivalent antibody comprises four antigen binding sites (or consists of four antigen binding sites). A multivalent antibody comprises at least one polypeptide bond (e.g., two polymorphic bonds), wherein the polymorphic bond comprises two or more variable domains. For example, a polypeptide chain may comprise νϋ^(Χ1)η-νϋ2-(Χ2)η-Ρο, wherein VD1 is a first variable domain, VD2 is a second variable domain, and Fc is an Fc region in a polypeptide chain. 'XI and X2 represent an amino acid or polypeptide, and η is 〇 or 1. For example, a polypeptide chain can comprise: a VH-CH1-flexible linker-VH-CH1-Fc region chain or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may additionally comprise at least two (e.g., four) light chain variable domain polypeptides. Multivalent antibodies herein may, 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 optionally comprise a CL domain 106. Single domain antibody. In some embodiments, the antibody of the invention is a single domain antibody. A single domain antibody is a single polypeptide chain comprising all or a portion of a heavy chain variable domain of an antibody or all or a portion of a light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). In one embodiment, the single domain antibody system consists of all or a portion of the antibody heavy chain variable domain. 149799.doc -80 · 201106972 7. Antibody variants 鳢 In some embodiments, the invention encompasses amino acid sequence modifications of the antibodies described herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. The amino acid sequence variant of the antibody can be prepared by introducing appropriate mutations into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Deletions, insertions, and substitutions can be arbitrarily combined to obtain the final construct', with the proviso that the final construct has the desired characteristics. Amino acid variations can be introduced into the target antibody amino acid sequence upon sequence formation. A method suitable for identifying certain residues or regions of an antibody as a preferred mutation-inducing position is called "alanine scanning mutation induction", such as

Cunningham 及 Wells (1989) 244:1081-1085 所描 述。其中，鑑別一個殘基或一組標靶殘基（例如帶電荷殘 基，諸如arg、asp、his、lys及glu)且用中性或帶負電荷之 〇 胺基酸（例如丙胺酸或聚丙胺酸）置換以影響胺基酸與抗原 之相互作用。接者，藉由在取代位點或針對取代位點引入 另外或其他變異體來改進對取代顯示功能敏感性之彼等胺 基酸位置。因此，雖然引入胺基酸序列變異之位點係預先 確定’但突變之性質本身無需預先確定。舉例而言，為分 析特定位點之突變效能，在靶密碼子或區域執行ala掃描或 隨機突變誘發且針對所需活性篩檢所表現之免疫球蛋白。 胺基酸序列***物包括長度範圍為一個殘基至含有一百 個或一百以上個殘基之多肽的胺基末端及/或羧基未端融 149799.doc -81 - 201106972 合體’以及具有單個或多個胺基酸殘基之序列内***物。 末端***物之貫例包括具有N末端甲硫胺醯基殘基之抗 體。抗體分子之其他***變異體包括抗體之N末端或C: 端與酵素（例如，肖於ADEPT而言）或延長抗體之血清半衰 期之多狀的融合體。 在某些實施例中，本發明之抗體經變異可增大或減小抗 體糖基化之程度。多肽之糖基化通常為]^連接型或〇連接 型。N連接型係指碳水化合物部分連接至天冬醯胺殘基之 側鏈。二肽序列天冬醯胺_Χ_絲胺酸及天冬醯胺蘇胺酸 (其中X為除脯胺酸以外之任何胺基酸）為碳水化合物部分 與天冬醯胺側鏈之酶促連接的識別序列。因此，多肽中存 在此等三肽序列之任一者可產生潛在糖基化位點。〇連接 型糖基化係指糖N-乙醯基半乳糖胺、半乳糖或木糖之一連 接至輕基胺基酸’最常見為絲胺酸或蘇胺酸，但亦可使用 5 -經基脯胺酸或5 -經基離胺酸。 抗體糖基化位點之添加或缺失宜藉由改變胺基酸序列以 使得產生或移除一或多個上述三肽序列來實現（針對N_連 接型糖基化位點）。亦可藉由一或多個絲胺酸或蘇胺酸殘 基之添加、缺失或取代來使原始抗體序列產生變異（針對〇 連接型糖基化位點）。Cunningham and Wells (1989) 244:1081-1085. Wherein, identifying a residue or a set of target residues (eg, charged residues such as arg, asp, his, lys, and glu) and using a neutral or negatively charged amidinoic acid (eg, alanine or poly Alanine) substitutions affect the interaction of the amino acid with the antigen. In turn, the position of the amino acid that is sensitive to the substitution display function is improved by introducing additional or other variants at the substitution site or for the substitution site. Therefore, although the site in which the amino acid sequence variation is introduced is determined in advance, the nature of the mutation itself does not need to be predetermined. For example, to analyze the mutational potency at a particular site, an ala scan or random mutation is performed at the target codon or region and the immunoglobulin exhibited by the desired activity screening is performed. Amino acid sequence inserts include an amine-based end and/or a carboxy-terminal end of a polypeptide ranging from one residue to a polypeptide containing one hundred or more residues, 149799.doc -81 - 201106972, and a single Or an intrasequence insert of a plurality of amino acid residues. Examples of terminal inserts include antibodies having N-terminal methionine residues. Other insertion variants of the antibody molecule include fusions of the N-terminus or C: terminus of the antibody to the enzyme (e.g., in the case of ADEPT) or to prolong the serum half-life of the antibody. In certain embodiments, the antibodies of the invention are mutated to increase or decrease the extent of antibody glycosylation. The glycosylation of a polypeptide is usually a ligated or hydrazone type. The N-linked type refers to a side chain in which the carbohydrate moiety is attached to the asparagine residue. The dipeptide sequence aspartame Χ Χ 丝 丝 丝 丝 及 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝 丝The identification sequence of the connection. Thus, any of these tripeptide sequences in the polypeptide can produce a potential glycosylation site. 〇 linked glycosylation refers to the attachment of one of the sugars N-ethyl galactosamine, galactose or xylose to a light amino acid, most commonly serine or threonine, but may also be used 5 - By phosphinic acid or 5-amino-amino acid. The addition or deletion of the antibody glycosylation site is preferably accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences are produced or removed (for N-linked glycosylation sites). The original antibody sequence can also be mutated (for 〇-linked glycosylation sites) by addition, deletion or substitution of one or more serine or threonine residues.

在抗體包含F c區之情況下’可改變與其連接之碳水化合 物。哺乳動物細胞產生之原生抗體通常包含分支鏈、雙觸 角（biantennary)募醣，其通常藉由N鍵聯連接至FC區之CH2 域之 Asn297。參見例如 Wright 等人，（1997) TIBTECH 149799.doc -82- 201106972 15:26-32。寡醣可包括各種碳水化合物，例如甘露糖、N-乙醯基葡糖胺（GlcNAc)、半乳糖及唾液酸，以及連接至雙 觸角寡醣結構之「莖幹」中之GlcNAc之海藻糖。在一些 實施例中，可對本發明抗體中之寡醣進行修飾以便產生具 有某些改良性質之抗體變異體。In the case where the antibody contains the F c region, the carbohydrate compound to which it is attached can be changed. Native antibodies produced by mammalian cells typically comprise a branched chain, biantennary glycoside, which is typically linked by N bonds to Asn297 in the CH2 domain of the FC region. See, for example, Wright et al. (1997) TIBTECH 149799.doc -82-201106972 15:26-32. The oligosaccharide may include various carbohydrates such as mannose, N-ethyl glucosamine (GlcNAc), galactose and sialic acid, and trehalose of GlcNAc linked to the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants having certain improved properties.

舉例而言，提供具有碳水化合物結構之抗體變異體，該 碳水化合物結構缺乏（直接或間接）連接至Fc區之海藻糖。 該等變異體可具有改良之ADCC功能。參見例如美國專利 公開案第 US 2003/0157108 號（Presta, L_) ; US 2004/ 0093621(Kyowa Hakko Kogyo Co., Ltd)。與「去海藻糖基 化」或「缺海藻糖」抗體有關之公開案的實例包括：US 2003/0157108 ； WO 2000/61739 ； WO 2001/29246 ； USFor example, an antibody variant having a carbohydrate structure that lacks (directly or indirectly) trehalose attached to the Fc region is provided. Such variants may have improved ADCC function. See, for example, U.S. Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "trehalose-based" or "trehalose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US

2003/0115614 ； US 2002/0164328 ； US 2004/0093621 ； US2003/0115614 ; US 2002/0164328 ; US 2004/0093621 ; US

2004/0132140 ； US 2004/0110704 ； US 2004/0110282 ; US2004/0132140 ; US 2004/0110704 ; US 2004/0110282 ; US

2004/0109865 ； WO 2003/085119 ； WO 2003/084570 ； WO 2005/035586 ； WO 2005/035778 ； WO 2005/053742 ； WO 2002/031140 ; Okazaki等人，乂 Mo/.价〇/· 336:1239-1249 (2004) ; Yamane-Ohnuki 等人，87: 614 (2004)。產生去海藻糖基化抗體之細胞株之實例包括缺乏 蛋白質海藻糖基化之Lecl3 CHO細胞（Ripka等人，Jrc/z. 价249:533-545 (1986);美國專利申請案第 US 2003/0157108 Α1 號，Presta，L ;及 WO 2004/056312 Al，Adams等人，尤其實例11)，及基因剔除細胞株，諸如 α-1，6-海藻糖基轉移酶基因、尸、基因剔除CHO細胞（參 149799.doc -83- 201106972 見例如 Yamane-Ohnuki 等人，Biotech. Bi〇enS- 87:614 (2004) ; Kanda，Y.等人，价扣/m〇/.价⑽狀.，94(4):680-688 (2006);及 WO 2003/085107)。 另外提供寡醣被切成兩分之抗體變異體，例如其中藉由 GlcNAc將連接至抗體之Fc區之雙觸角募醣切成兩分。該 等抗體變異體可具有減少之海藻糖化及/或改良之ADCC功 能。該等抗體變異體之實例描述於例如wo 2003/ 01 1878(Jean-Mairet 等人）、美國專利案第 6,602,684 號 (Umana等人）及 US 2005/0123546(Umana等人）中。亦提供 在連接至Fc區之募醣中具有至少一個半乳糠殘基之抗體變 異體。該等抗體變異體可具有改良之CDC功能。該等抗體 變異體描述於例如WO 1997/30087(Patel等人）、WO 1998/58964(Raju, S.)及 WO 1999/22764(Raju，S_)中。 在某些實施例中，抗體變異體包含具有一或多個進一步 改良ADCC之胺基酸取代（例如Fc區之位置298、333及/或 334(Eu殘基編號）處之取代）的1^區。該等取代可與上述任 何變異組合出現。 在某些實施例中’本發明涵蓋具有一些（但非全部）效應 功能之抗體變異體’從而使其成為許多應用之理想候選藥 物，其中抗體之活體内半衰期為重要的，但某些效應功能 (諸如補體及ADCC)為不必要或有害的。在某些實施例 中，可量測抗體之Fc活性以確保僅維持所要特性。可進行 活體外及/或活體内細胞毒性分析法以確認CDC及/或ADCC 活性之降低/耗竭。舉例而言，可進行Fc受體（FcR)結合分 149799.doc -84· 201106972 析法以確保抗體缺乏FcYR結合能力（從而可能缺乏ADCC活 性），但保留FcRn結合能力。介導ADCC之初級細胞、NK 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, Annu. Rev. Immunol. 9:457-92 (1991)之第 464頁之表 3 中。 評估相關分子之ADCC活性之活體外分析法之非限制性實 例描述於美國專利案第5,500,362號（參見例如1^113〖1'〇111， I.，KProc. Nat'l Acad. Sci. USA 83:7059-7063 (1986))A Hellstrom, A » Proc. ΝαίΊ Acad. Sci. USA 82:1499-1502 (1985) ; 5,821,337(參見 Bruggemann，M.等人，J.五x/?. Mei 166:1351-1361 (1987))中。或者，可使用非放射性分 析方法（參見例如流式細胞測量術之ACTITM非放射性細胞 毒性分析法（CellTechnology，Inc. Mountain View，CA);及 CytoTox 96®非放射性細胞毒性分析法（Promega, Madison， WI))。適用於該等分析法之效應細胞包括周圍血液單核細 胞（PBMC)及自然殺手（NK)細胞。或者或另外，可評估相 關分子在活體内，例如在動物模型（諸如揭示於Clynes等 人，尸roc. Scz·· 95:652-656 (1998)中之動物 模型）中之ADCC活性。亦可進行Clq結合分析法以確認抗 體無法與Clq結合且因此缺乏CDC活性。為了評估補體活 化，可執行CDC分析法（參見例如Gazzano-Santoro等人，J. Immunol. Methods 202:163 (1996) ； Cragg, M.S.等人， 5/ood 101:1045-1052 (2003);及 Cragg，M_S.及]\4丄 Glennie，5/ooof 103:2738-2743 (2004))。亦可使用此項技術 149799.doc •85- 201106972 中已知之方法測定FcRn結合及活體内清除/半衰期（參見例 如 Petkova, S.B.等人，7«〆/. Immunol. 18(12):1759-1769 (2006))。 提供具有一或多個胺基酸取代之其他抗體變異體。用於 取代性突變誘發之相關位點包括高變區，但亦涵蓋FR變 異。保守型取代顯示於表1中「較佳取代」標題下。稱為 「例示性取代」之更多實質性變異提供於表1中，或如下 文參考胺基酸類別進一步描述。可將胺基酸取代引入相關 抗體中且篩檢產物，例如針對所要活性（諸如改良之抗原 結合、降低之免疫原性、改良之ADCC或CDC等）篩檢產 物。 表1 原始殘基 例示性取代 較佳取代 Ala (A) Val ; Leu ; lie Val Atr(R) Lys ; Gin ; Asn Lys Asn (N) Gin ; His ; Asp，Lys ; Arg Gin Asp (D) Glu ； Asn Glu Cys (C) Ser ; Ala Ser Gln(〇) Asn ; Glu Asn Glu (E) Asp ; Gin Asp Gly (G) Ala Ala His (H) Asn ； Gin ； Lys ； Arg Arg lie (I) Leu ; Val ; Met ; Ala ; Phe ;正白胺酸 Leu Leu (L) 正白胺酸；He ; Val ; Met ; Ala ; Phe lie Lys (K) Arg ; Gin ; Asn Arg Met (M) Leu ; Phe ; lie Leu -86- 149799.doc 201106972 原始殘基 例示性取代 較佳取代 Phe(F) Trp ； Leu ； Val ； lie ； Ala ； Tyr Tyr Axl(P) Ala Ala Ser (S) Thr Thr Thr(T) Val ； Ser Ser Trp(W) Tyr ； Phe Tyr Tyr (Y) Trp ； Phe ； Thr ； Ser Phe Val (V) lie ; Leu ; Met ; Phe ; Ala ;正白胺酸 Leu 可藉由選擇影響以下方面之取代來實現抗體生物學性質 之改變：（a)取代區域中多肽主鏈之結構，例如呈片或螺旋 構形；（b)靶點處分子之電荷或疏水性；或（c)側鏈之大 小。胺基酸可根據其側鏈性質之相似性分類（A. L. Lehninger, Biochemistry，第二版，第 73-75 頁，Worth Publishers, New York (1975)) · (1) 非極性：Ala (A)、Val (V)、Leu (L)、lie (I)、 Axl(P)、Phe (F)、Trp (W)、Met (M) (2) 不帶電荷極性：Gly (G)、Ser (S)、Thr (T)、Cys 〇 (C)、Tyr (Y)、Asn (N)、Gin (Q) (3) 酸性：Asp (D)、Glu (E) (4) 鹼性：Lys (K)、Arg (R)、His(H) 或者，天然存在之殘基可基於共同側鏈性質分類： (1)疏水性：正白胺酸、Met、Ala、Val、Leu、lie ; (2) 中性親水性：Cys、Ser、Thr、Asn、Gin ; (3) 酸性：Asp、Glu ; (4) 驗性：His、Lys、Arg ; (5) 影響鏈取向之殘基：Gly、Pro ; 149799.doc -87- 201106972 (6)方族：Trp、Tyr、Phe 〇 非保守取代將引起此等類別之一之成員交換成另—類成 員。亦可將該等經取代之殘基引入保守性取代位點中或引 入其餘（非保守性）位點中。 一種類型之取代變異體涉及取代親本抗體（例如人類化 抗體或人類抗體）之一或多個高變區殘基。通常，所得選 用於進一步開發之變異體相對於產生其之親本抗體具有經 修改（例如改良）的生物特性。例示性取代變異體為親和力 成熟抗體，其宜使用基於噬菌體呈現之親和力成熟技術產 生。簡而言之，使數個高變區位點（例如6_7個位點）突變， 以在各位點處產生所有可能之胺基酸取代。由此產生之抗 體以與封裝於各粒子内之噬菌體鞘蛋白(例如M i 3之基因 ΙΠ產物）之至少一部分融合的形式由絲狀噬菌體粒子呈 現。接著篩檢噬菌體呈現之變異體之生物活性(例如结合 親和力）。為鑑利於修飾之候選高變區位點，可執行掃 描誘變(例如丙胺酸掃描)以鑑別對抗原結合有顯著作用的 咼變區殘基。或者或另外，可有益的暑八 ，皿妁疋分析抗原-抗體複 合物之晶體結構以鑑別抗體與抗原之間的接觸點。根據此 ^支術中已知之技術，包括本文詳述之技術，該等接觸殘 土及相鄰殘基為用於取代之候選殘基。一旦產生該等變異 體’即使用此項技術中已知 、 笪姑tv ^ 中描述之彼 #技術）師檢該組變里體 且… 擇在一或多個相關分析 法中'、有優良性質的變異體用於進一步開發。 編碼抗體之胺基酸序列變 #幻龙異體之核酸分子係由此項技術 149799.doc -88 - 201106972 已知之多種方法製備。該等方法包括（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 由對早先製備之抗體變異體或非變異型抗體進行寡核普酸 ‘（或疋點）之突變誘發、PCR突變誘發及卡匿式突變誘 發來製備。 可能需要將一或多個胺基酸修飾引入本發明抗體之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 0 胺基酸位置（包括鉸鏈半胱胺酸位置）含有胺基酸修飾（例如 取代）之人類Fc區序列（例如人類igGl、lgG2、IgG3或IgG4 Fc 區）。 根據本說明書及此項技術之教示，可預期在有些實施例 中’本發明之抗體與野生型相應抗體相比可包含一或多個 變異，例如Fc區中之一或多個變異。然而，此等抗體保留 與其野生型對應物實質上相同之治療效用所需特徵。舉例 而s ’咸信Fc區中產生的某些變異會改變（亦即改良或減 〇 弱 )C 1 q結合及/或補體依賴性細胞毒性（CDC)，例如w〇 99/5 1642中所述。關於Fc區變異體之其他實例，亦參見WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al., 乂Mo/.价/· 336:1239- 1249 (2004); Yamane-Ohnuki et al., 87: 614 (2004). Examples of cell lines that produce de-fucosylated antibodies include Lecl3 CHO cells lacking protein fucosylation (Ripka et al, Jrc/z. 249: 533-545 (1986); US Patent Application No. US 2003/ 0157108 Α1, Presta, L; and WO 2004/056312 Al, Adams et al, especially Example 11), and gene knockout cell lines, such as α-1,6-trehalyltransferase gene, cadaver, gene knockout CHO cells (See 149799.doc -83-201106972 See, for example, Yamane-Ohnuki et al, Biotech. Bi〇enS-87:614 (2004); Kanda, Y. et al., price deduction / m〇 /. Price (10) shape., 94 (4): 680-688 (2006); and WO 2003/085107). Further, an antibody variant in which the oligosaccharide is cut into two is provided, for example, in which the biantennary glycoside linked to the Fc region of the antibody is cut into two by GlcNAc. Such antibody variants may have reduced enzymatic saccharification and/or improved ADCC functionality. Examples of such antibody variants are described, for example, in WO 2003/01 1878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.) and US 2005/0123546 (Umana et al.). Antibody variants having at least one galactone residue in the glycoprotein linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.), WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S_). In certain embodiments, the antibody variant comprises 1^ having one or more amino acid substitutions that further improve ADCC (eg, substitutions at positions 298, 333, and/or 334 (Eu residue numbering) of the Fc region) Area. These substitutions can occur in combination with any of the above variations. In certain embodiments 'the invention encompasses antibody variants having some, but not all, effector functions' making them ideal candidates for many applications where the in vivo half-life of the antibody is important, but some effector functions (such as complement and ADCC) is unnecessary or harmful. In certain embodiments, the Fc activity of an antibody can be measured to ensure that only the desired properties 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, Fc receptor (FcR) binding can be performed 149799.doc -84·201106972 to ensure that the antibody lacks FcYR binding ability (and thus may lack ADCC activity), but retains FcRn binding ability. Primary cells that mediate ADCC, NK cells only exhibit FcyRIII, while monocytes exhibit FcyRI, FcyRII, and FcyRIII. The FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of related molecules are described in U.S. Patent No. 5,500,362 (see, for example, 1^113 〖1'〇111, I., KProc. Nat'l Acad. Sci. USA 83 :7059-7063 (1986)) A Hellstrom, A » Proc. ΝαίΊ Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J.5x/?. Mei 166: 1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the relevant molecule can be assessed in vivo, e.g., in an animal model such as the animal model disclosed in Clynes et al., corpse roc. Scz 95: 652-656 (1998). A Clq binding assay can also be performed to confirm that the antibody is unable to bind to Clq and thus lacks CDC activity. To assess complement activation, a CDC assay can be performed (see, eg, Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996); Cragg, MS et al, 5/ood 101: 1045-1052 (2003); Cragg, M_S. and] \4丄Glennie, 5/ooof 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life can also be determined using methods known in the art 149799.doc • 85-201106972 (see, for example, Petkova, SB et al, 7 «〆/. Immunol. 18(12): 1759-1769 (2006)). Other antibody variants with one or more amino acid substitutions are provided. Relevant sites for substitution mutation induction include hypervariable regions, but FR variants are also covered. Conservative substitutions are shown under the heading "Preferred substitutions" in Table 1. Further substantial variations known as "exemplary substitutions" are provided in Table 1, or as further described below with reference to the amino acid class. Amino acid substitutions can be introduced into the relevant antibody and the product screened, e.g., screened for the desired activity (e.g., improved antigen binding, reduced immunogenicity, modified ADCC or CDC, etc.). Table 1 Exemplary substitutions of the original residues preferred substitutions Ala (A) Val ; Leu ; lie Val Atr ( R ) Lys ; Gin ; Asn Lys Asn ( N ) Gin ; His ; Asp , Lys ; Arg Gin Asp (D) Glu Asn Glu Cys (C) Ser ; Ala Ser Gln (〇) Asn ; Glu Asn Glu (E) Asp ; Gin Asp Gly (G) Ala Ala His (H) Asn ; Gin ; Lys ; Arg Arg lie (I) Leu ; Val ; Met ; Ala ; Phe ; orthanoic acid Leu Leu ( L ) orthanoic acid ; He ; Val ; Met ; Ala ; Phe lie Lys ( K ) Arg ; Gin ; Asn Arg Met (M) Leu ; Lie Leu -86- 149799.doc 201106972 Illustrative substitution of the original residue preferred substitution of Phe(F) Trp ; Leu ; Val ; lie ; Ala ; Tyr Tyr Axl (P) Ala Ala Ser (S) Thr Thr Thr (T Ser Ser Trp(W) Tyr ; Phe Tyr Tyr (Y) Trp ; Phe ; Thr ; Ser Phe Val (V) lie ; Leu ; Met ; Phe ; Ala ; Substitution to achieve a change in the biological properties of the antibody: (a) the structure of the polypeptide backbone in the substitution region, eg, in a sheet or helical configuration; (b) the charge or hydrophobicity of the molecule at the target; or (c) side The size of the chain. Amino acids can be classified according to their similarity in side chain properties (AL Lehninger, Biochemistry, Second Edition, pp. 73-75, Worth Publishers, New York (1975)). (1) Non-polar: Ala (A), Val (V), Leu (L), lie (I), Axl (P), Phe (F), Trp (W), Met (M) (2) Uncharged polarity: Gly (G), Ser (S ), Thr (T), Cys 〇 (C), Tyr (Y), Asn (N), Gin (Q) (3) Acidity: Asp (D), Glu (E) (4) Alkaline: Lys (K , Arg (R), His (H) or, naturally occurring residues can be classified based on the properties of the common side chain: (1) Hydrophobicity: n-leucine, Met, Ala, Val, Leu, lie; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin; (3) Acidity: Asp, Glu; (4) Detectability: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; 149799.doc -87- 201106972 (6) Fang: Trp, Tyr, Phe 〇 Non-conservative substitution will cause members of one of these categories to be exchanged for another class member. The substituted residues can also be introduced into a conservative substitution site or introduced into the remaining (non-conservative) sites. One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variants selected for further development have modified (e.g., improved) biological properties relative to the parent antibody from which they are produced. Exemplary substitution variants are affinity matured antibodies which are preferably produced using affinity matured techniques based on phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each point. The antibody thus produced is expressed by filamentous phage particles in a form fused to at least a portion of a phage sheath protein (e.g., a gene of the gene of Mi 3 ) encapsulated in each particle. The biological activity (e.g., binding affinity) of the variant presented by the phage is then screened. To facilitate the modification of candidate hypervariable region sites, scanning mutagenesis (e.g., alanine scanning) can be performed to identify the mutated region residues that have a significant effect on antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and the antigen. According to the techniques known in the art, including the techniques detailed herein, the contact residues and adjacent residues are candidate residues for substitution. Once the variants are produced, use the technique described in this technique, described in the t t tv ^ ) ) 师 师 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 且 择 择 择 择 择 择 t t t t Variants of nature are used for further development. The amino acid sequence encoding the antibody is prepared by various methods known in the art 149799.doc-88 - 201106972. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or by oligonucleotides of previously prepared antibody variants or non-variant antibodies (or 疋 point) mutation induction, PCR mutation induction and cardinal mutation induction to prepare. It may be desirable to introduce one or more amino acid modifications into the Fc region of an antibody of the invention, thereby producing an Fc region variant. An Fc region variant may comprise a human Fc region sequence containing an amino acid modification (eg, a substitution) at one or more of the 0 amino acid positions (including the hinge cysteine position) (eg, human igG1, lgG2, IgG3, or IgG4 Fc) Area). In accordance with the present specification and teachings of the art, it is contemplated that in some embodiments the antibody of the invention may comprise one or more variations, such as one or more variations in the Fc region, as compared to a wild-type corresponding antibody. However, such antibodies retain substantially the same therapeutic utility characteristics as their wild-type counterparts. For example, some of the mutations produced in the Fc region of singularity will change (ie, improve or decrease) C 1 q binding and/or complement dependent cytotoxicity (CDC), such as in w〇99/5 1642 Said. See also other examples of Fc region variants.

Duncan 及 Winter，iVaiwre 322:738-40 (1988);美國專利案 •第5,648,260號；美國專利案第5,624,821號；及w〇 94/29351。WO 00/42072(Presta)及 w〇 2004/056312 (Lowman)描述與FcR結合增強或減弱的抗體變異體。此等 專利公開案之内容以引用之方式明確併入本文中。亦參見Duncan and Winter, iVaiwre 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and w〇 94/29351. WO 00/42072 (Presta) and w〇 2004/056312 (Lowman) describe antibody variants that increase or decrease binding to FcR. The contents of these patent publications are expressly incorporated herein by reference. See also

Shields等人，J. 5沁/· C/2em. 9(2): 6591-6604 (2001)。US 2005/0014934Al(Hinton等人）中描述半衰期延長且與新生 149799.doc -89- 201106972 兒Fc受體（FcRn)之結合增強的抗體，新生兒以受體負責將 母體IgG轉移至胎兒中（Guyer等人，J. /讀㈣ι17:587 (1976)及 Kim等人，//«膽⑽/· 24:249 (1994))。此等抗體 包含其中具有一或多個增強Fc區與FcRn結合之取代的Fc 區。美國專利第6，194,551B1號、W099/5 1642中描述Fc區 胺基酸序列已變異且Clq結合能力增大或減小之多肽變異 體。該等專利公開案之内容以引用的方式明確併入本文 中。亦參見Idusogie等人 ’ J. ⑽/ 164: 4178-4184 (2000) 〇 在另一態樣中，本發明提供在包含Fc區之Fc多肽之界面 中包含修飾的抗體，其中該等修飾有助於且/或促進雜二 聚化。該等修飾包含將突起引入第一 Fc多肽中以及將空腔 引入第一 Fc多肽中’其中該突起可定位於該空腔中以便促 進第一 Fc多肽與第二Fc多肽之複合。產生具有該等修飾之 抗體的方法已知於此項技術中，例如美國專利第5,73丨，丨68 號所述。 在另一態樣中，可能需要產生經半胱胺酸工程改造之抗 體，例如「thioMAb」，其中抗體之一或多個殘基經半胱胺 酸殘基取代。在特定實施例中，經取代殘基存在於抗體之 可達位點。藉由半胱胺酸取代彼等殘基，從而將反應性硫 醇基定位於抗體之可達位點且可用於使抗體與其他部分 (諸如樂物部分或連接子-藥物部分）結合’如本文中進一步 描述。在某些實施例中，任一或多個以下殘基可經半胱胺 酸取代：輕鏈之V205(Kabat編號）；重鏈之AU8(Elm 149799.doc -90· 201106972 號）；及重鏈Fc區之S400(EU編號）。 8.抗艎衍生物 本發明之抗體可進-步修飾成含有此項技術中已知且易 於獲得之其他非蛋白部分。適於抗體衍生化之部分較佳為 水溶性聚合物。水溶性聚合物之非限制性實例包括（但不 ㈣）聚乙二醇（PEG)、乙二醇/丙二醇共聚物、叛甲基纖維 素、葡聚糖、聚乙烯醇、聚乙烯吡咯啶酮、聚丨，％二氧戊 0 壞 '聚_1，3,6-三噁烷、乙烯/順丁烯二酸酐共聚物、聚胺基 酸（均聚物或無規共聚物）及葡聚糖或聚（N_乙烯吡咯啶嗣） 聚乙二醇、丙二醇均聚物、聚氧化丙烯/氧化乙烯共聚 物、聚氧乙烯多元醇（例如甘油）、聚乙烯醇及其混合物。 聚乙二醇丙醛因其於水中之穩定性而可具有製造優勢。聚 合物可具有任何分子量，且可分支或不分支。連接至抗體 之聚合物之數目可變化，且若連接一個以上聚合物，則其 可為相同或不同分子。通常’用於衍生化之聚合物之數目 〇 及/或類型可基於以下考慮來確定：包括（但不限於）待改良 之抗體特定性質或功能，抗體衍生物是否將用於限定條件 下之療法等。 -在另一實施例中，提供抗體與非蛋白部分之結合物，該 • 等結合物可藉由曝露於輻射來選擇性加熱。在一實施例 中’非蛋白部分為碳奈米管（Kam等人，尸roc· jVai/ C/U 1〇2: 11600-11605 (2005))。此輻射可具有任一波 長’且包括（但不限於）不損傷一般細胞、但能將非蛋白部 分加熱至可殺死抗體—非蛋白部分鄰近之細胞之溫度的波 149799.doc -91 · 201106972 長。 B.某些製造抗體之方法 1.某些基於融合瘤之方法 本發明之單株抗體可使用融合瘤方法製備，融合瘤方法 首先描述於Kohler等人，Waiwre, 256:495 (1975)中’且進 一步描述於關於人類-人類融合瘤的以下文獻中：例如 Hongo等人，14 (3): 253-260 (1995) ; Harlow 等人，Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press，第 2版 1988) ; Hammerling等人， ^ ： Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y·，1981);及 Ni，Χία«如/ 26(4): 265-268 (2006)。其他方法包括例如美國專利第7,189,826 號中所述之關於自融合瘤細胞株產生單株人類天然IgM抗 體之方法。人類融合瘤技術（三源雜交瘤技術）描述於 Vollmers 及 Brandlein, Histology and Histopathology, 20(3):927-937 (2005)及 Vollmers及 Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005)中。 對於各種其他融合瘤技術，參見例如US 2006/258841 ; US 2006/183887(完全人類抗體）；US 2006/059575 ; US 2005/287149 ; US 2005/100546 ; US 2005/026229 ;及美國 專利第7,078,492號及第7,153,507號。使用融合瘤方法產生 單株抗體之例示性方案描述如下。在一實施例中，使小鼠 或其他適當宿主動物（諸如倉鼠）免疫以誘使淋巴細胞產生 149799.doc -92- 201106972 或能夠產生特異性結合用於免疫之蛋白質之抗體。藉由多 次皮下（SC)或腹膜内（ip)注射包含Axl或其片段之多肽及佐 劑（諸如單磷醯基脂質A(MPL)/海藻糖二柯諾黴菌酸醋 (trehalose dicrynomycolate ； TDM)(Ribi Immunochem. Research，Inc.，Hamilton，MT))來使動物產生抗體。可使用 此項技術中熟知之方法（諸如重組方法）製備包含Αχί或其片 段之多肽，其中一些方法在本文中另外描述。分析經免疫 動物之血清中的抗Axl抗體’且視情況投與免疫強化劑。 自產生抗Axl抗體之動物分離淋巴細胞。或者，可於活體 外免疫淋巴細胞。 隨後使用適合融合劑（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。參見例如G〇ding, Monocloncil Antibodies. Principles and Practice ,第 59-103 頁（Academic Press，1986)。可使用有效融合、有助於所選 抗體產生細胞穩定高量產生抗體且對培養基（諸如HAT培養 基）敏感的骨髓瘤細胞。例示性骨髓瘤細胞包括（但不限於） 鼠類骨髓瘤細胞株’諸如可自沙克生物研究所細胞分配中 心（Salk Institute Cell Distribution Center)(San Diego, California USA)獲得之MOPC-21及MPC-11小鼠腫瘤所產生 的彼等細胞及可自美國菌種保存中心（American TypeShields et al., J. 5沁/· C/2em. 9(2): 6591-6604 (2001). An antibody with increased half-life and enhanced binding to the Fc receptor (FcRn) of the newborn 149799.doc-89-201106972 is described in US 2005/0014934 Al (Hinton et al.), and the neonatal receptor is responsible for the transfer of maternal IgG to the fetus ( Guyer et al., J. / ed. (iv) ι 17:587 (1976) and Kim et al., //«Bold (10)/· 24:249 (1994)). Such antibodies comprise an Fc region having one or more substitutions that enhance the binding of the Fc region to FcRn. Polypeptide variants in which the Fc region amino acid sequence has been mutated and the Clq binding capacity is increased or decreased are described in U.S. Patent Nos. 6,194,551 B1 and WO99/5 1642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. 'J. (10) / 164: 4178-4184 (2000) In another aspect, the invention provides a modified antibody comprising an interface at an Fc polypeptide comprising an Fc region, wherein the modification aids And/or promote heterodimerization. The modifications comprise introducing a protuberance into the first Fc polypeptide and introducing a cavity into the first Fc polypeptide ' wherein the protuberance can be localized in the cavity to facilitate recombination of the first Fc polypeptide with the second Fc polypeptide. Methods of producing antibodies having such modifications are known in the art, for example, as described in U.S. Patent No. 5,73, 丨68. In another aspect, it may be desirable to produce a cysteine engineered antibody, such as "thioMAb", wherein one or more residues of the antibody are substituted with a cysteine residue. In a particular embodiment, the substituted residue is present at a reachable site of the antibody. Substituting cysteine for their residues allows the reactive thiol group to be localized to the accessible site of the antibody and can be used to bind the antibody to other moieties such as the moiety or linker-drug moiety. Further described in this article. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; AU8 of the heavy chain (Elm 149799. doc-90·201106972); S400 (EU number) of the chain Fc region. 8. Anti-Indole Derivatives The antibodies of the invention can be further modified to contain other non-protein portions known in the art and readily available. The moiety suitable for antibody derivatization is preferably a water soluble polymer. Non-limiting examples of water soluble polymers include (but not (iv)) polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, methyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone , polyfluorene, % dioxol 0 bad 'poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and Sugar or poly(N_vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylene polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has manufacturing advantages due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. Generally, the number and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be modified, and whether the antibody derivative will be used in a defined condition. Wait. - In another embodiment, a combination of an antibody and a non-protein moiety is provided, which can be selectively heated by exposure to radiation. In one embodiment the 'non-protein portion is a carbon nanotube (Kam et al., corpse roc. jVai/C/U 1 〇 2: 11600-11605 (2005)). The radiation can have any wavelength 'and includes, but is not limited to, a wave that does not damage the general cells, but can heat the non-protein portion to a temperature that kills the antibody - a cell adjacent to the non-protein portion 149799.doc -91 · 201106972 long. B. Certain Methods of Making Antibodies 1. Certain Fusion Tumor-Based Methods The monoclonal antibodies of the present invention can be prepared using the fusion tumor method, first described in Kohler et al., Waiwre, 256:495 (1975). And further described in the following literature on human-human fusion tumors: for example, Hongo et al, 14 (3): 253-260 (1995); Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al, ^: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981); and Ni, Χία «eg / 26(4): 265-268 (2006). Other methods include, for example, a method for producing a single human native IgM antibody from a fusion tumor cell strain as described in U.S. Patent No. 7,189,826. Human fusion tumor technology (three-source hybridoma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3) :185-91 (2005). For various other fusion tumor technologies, see, for example, US 2006/258841; US 2006/183887 (complete human antibodies); US 2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and US Patent No. 7,078,492 And No. 7,153,507. An exemplary protocol for the production of monoclonal antibodies using the fusion tumor method is described below. In one embodiment, a mouse or other appropriate host animal, such as a hamster, is immunized to induce lymphocyte production 149799.doc-92-201106972 or to produce antibodies that specifically bind to the protein used for immunization. By subcutaneous (SC) or intraperitoneal (ip) injection of a polypeptide comprising Axl or a fragment thereof and an adjuvant such as monophosphoryl lipid A (MPL) / trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT)) to produce antibodies to animals. Polypeptides comprising Αχ or a fragment thereof can be prepared using methods well known in the art, such as recombinant methods, some of which are additionally described herein. The anti-Axl antibody in the serum of the immunized animal is analyzed and an immunopotentiator is administered as appropriate. Lymphocytes were isolated from animals producing anti-Axl antibodies. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form fusion tumor cells. See, for example, G〇ding, Monocloncil Antibodies. Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells that are effective for fusion, contribute to the stable production of antibodies by selected antibody producing cells, and are sensitive to a medium such as a HAT medium can be used. Exemplary myeloma cells include, but are not limited to, murine myeloma cell lines such as MOPC-21 and MPC available from the Salk Institute Cell Distribution Center (San Diego, California USA). -11 mouse tumors produced by their cells and available from the American Type Culture Center (American Type

Culture Collection, Rockville, Maryland USA)獲得之 SP-2 或X63-Ag8-653細胞。亦已描述用於產生人類單株抗體的 人類骨趙瘤及小鼠-人類雜交骨髓瘤細胞株（Kozbor，《/. /wimMno/·，133:3001 (1984) ; Brodeur 等人，Mo«oc/o«a/ 149799.doc -93- 201106972Culture Collection, Rockville, Maryland USA) obtained SP-2 or X63-Ag8-653 cells. Human bone tumors and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have also been described (Kozbor, "/. /wimMno/., 133:3001 (1984); Brodeur et al., Mo«oc /o«a/ 149799.doc -93- 201106972

Antibody Production Techniques and Applications，第 51-63 頁（Marcel Dekker，Inc.，New York, 1987))。 由此製備之融合瘤細胞接種且生長於適當培養基中，例 如含有一或多種抑制未融合之親代骨髓瘤細胞之生長或存 活之物質的培養基。舉例而言，若親代骨髓瘤細胞缺乏次 黃嘌呤鳥嘌呤磷酸核糖轉移酶（HGPRT或HPRT)，則融合瘤 培養基通常將包括次黃嘌呤、胺基喋呤及胸苷（HAT培養 基）’該等物質會阻止缺乏HGPRT之細胞之生長。無血清 之融合瘤細胞培養方法較佳用於減少使用動物源血清（諸 如胎牛血清），例如Even等人，h S/oiec/mo/ogj；, 24(3), 105-108 (2006)中所述。 作為提高融合瘤細胞培養物之生產力之手段的募肽描述 於 Franek, Trends in Monoclonal Antibody Research, 111-122 (2005)中。特定言之，標準培養基富含某些胺基酸（丙 胺酸、絲胺酸、天冬酿胺、捕胺酸）或蛋白質水解產物部 分’且由三個至六個胺基酸殘基構成之合成寡肽可顯著抑 制細胞凋亡。該等肽以毫莫耳濃度或高於毫莫耳濃度之濃 度存在。 可對培養融合瘤細胞之培養基分析結合Αχί之單株抗體 之產生。融合瘤細胞所產生之單株抗體的結合特異性可藉 由免疫沈澱法或藉由活體外結合分析法（諸如放射免疫分 析法（RIA)或酶聯免疫吸附分析法（ELISA))測定。單株抗 體之結合親和力可藉由例如史卡查分析（Scatchard analysis)來測定。參見例如Munson等人，j⑽/ 149799.doc -94- 201106972 107:220 (1980)。 銓別可產生具有所要特異性、親和力及/或活性之抗體 的融合瘤細胞之後，可藉由限制性稀釋程序次選殖純系且 . #由標準方法培養。參見例如Goding，同上。適用於此目 的之培養基包括例如D-ME1V[或RPMI-1640培養基。另外， 融合瘤細胞可在動物體活體内内培養為腹水腫瘤。次純系 所刀/必之單株抗體宜藉由習知免疫球蛋白純化程序（諸如 〇 蛋白A_瓊脂糖、羥磷灰石層析、凝膠電泳、透析或親和性 層析）自培養基、腹水或血清中分離。一種自融合瘤細胞 分離蛋白質之程序描述於us 2〇〇5/176122及美國專利第 6,919,436號中。該方法包括在結合過程中使用最少量之鹽 (諸如易溶鹽）及亦較佳在溶離過程中使用少量有機溶劑。 2·某些文庫篩檢方法 可藉由使用組合文庫篩檢具有所需活性之抗體來獲得本 發明之抗體。舉例而言’此項技術中已知多種產生噬菌體 ◎ 呈現文庫及篩檢該等文庫中具有所要結合特徵之抗體的方 法。該等方法一般描述於H〇〇genb〇〇m等人之〜 M〇/㈣—心/㈣ 178:1_37(〇,Brien等人編，Human : T〇towa，NJ，2001)中。舉例而言，如^等人，】m〇i _· Bi〇1. (2〇04)，340(5):1073_93中所述，一種產生相關抗體之 方法係使用噬菌體抗體文庫。 大體上，藉由筛檢含有噬菌體的噬菌體文庫來選擇合成 抗體純系，該噬菌體呈現與噬菌體鞘蛋白融合之抗體可變 區（Fv)之各種片段。藉由針對所需抗原進行親和層析來淘 149799.doc •95· 201106972 % 3玄專电囷體文庫。表現能夠與所需抗原結合之FV片段之 純系吸附至抗原且因此與文庫中非結合純系分離。接著將 結合純系自抗原溶離，且可藉由另外幾輪抗原吸附/溶離 進一步富集。本發明之任何抗體可如下獲得：設計適合抗 原篩檢程序以選擇相關嗤菌體純系，接著使用相關噬菌體 純系之Fv序列及Kabat等人，Sewe〇/ Pr0如·如〇/ /mmw⑽/«ierew，第 5版，NIH Publication 91-3242, Bethesda MD (1991)，第1-3卷中所述之適合恆定區（Fc)序 列來構築全長抗體純系。 在某些實施例中，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，可變區各來自輕鏈（VL)及 重鏈（VH)且均存在三個高變環（HVR)或互補決定區 (CDR)。可變域可作為單鏈Fv(scpv)片段（其中vh與VL經 由柔性短肽共價鍵聯）或作為Fab片段（其中VH與VL各與恒 定域融合且非共價相互作用）功能性地呈現於噬菌體上， 如Winter等人，則⑽厂，12: 433-455(1994)中所 述如本文所使用’ scFv編碼嗟菌體純系及Fab編碼嗔菌 體純系統稱為「Fv噬菌體純系」或「Fv純系」。 VH與VL基因譜系可藉由聚合酶鏈反應（pcR)分別選殖， 且在嗟菌體文庫令隨機重組，接著可搜尋抗原結合純系， 如 Winter專人，J⑽I〗： 433-455(1994)中所 述。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，可選殖可提供針對廣泛範圍之 非自體抗原以及自體抗原之單一人類抗體來源而無需任何 149799.doc -96- 201106972 免疫作用的天然譜系，如Griffiths等人，五ΜδΟ 乂 12: 725· 734 (1993)所述。最後，天然文庫亦可如下合成獲得：自 幹細胞選殖未經重排之V—基因片段並使用含有隨機序列之 t PCR引子編碼CDR3高變區及完成活體外重排，如Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The thus prepared fusion tumor cells are seeded and grown in a suitable medium, such as a medium containing one or more substances which inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the fusion tumor medium will typically include hypoxanthine, aminopurine and thymidine (HAT medium). Such substances will prevent the growth of cells lacking HGPRT. The serum-free fusion tumor cell culture method is preferably used to reduce the use of animal-derived serum (such as fetal bovine serum), for example, Even et al., h S/oiec/mo/ogj;, 24(3), 105-108 (2006) Said in the middle. Peptides as a means of increasing the productivity of fusion tumor cell cultures are described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). In particular, the standard medium is enriched with certain amino acids (alanine, serine, aspartic acid, amino acid) or protein hydrolysate fractions and consists of three to six amino acid residues. Synthetic oligopeptides can significantly inhibit apoptosis. The peptides are present at a concentration of millimolar or higher than the concentration of millimolar. The medium in which the fusion tumor cells are cultured can be analyzed for the production of a monoclonal antibody that binds to Αχί. The binding specificity of the monoclonal antibodies produced by the fusion tumor cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis. See, for example, Munson et al, j(10)/149799.doc-94-201106972 107:220 (1980). After screening for fusion tumor cells that produce antibodies with the desired specificity, affinity, and/or activity, the lines can be sub-selected by a limiting dilution procedure and cultured by standard methods. See, for example, Goding, ibid. The medium suitable for this purpose includes, for example, D-ME1V [or RPMI-1640 medium. In addition, the fusion tumor cells can be cultured as an ascites tumor in vivo in an animal body. The sub-pure monoclonal antibody must be prepared from the culture medium by a conventional immunoglobulin purification procedure (such as prion A_Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography). Separated from ascites or serum. A procedure for isolating proteins from a fusion cell is described in US Pat. No. 5,176, 436 and U.S. Patent No. 6,919,436. The method involves the use of a minimum amount of salt (such as a readily soluble salt) during the bonding process and preferably a small amount of organic solvent during the dissolution process. 2. Certain Library Screening Methods The antibodies of the present invention can be obtained by screening a combinatorial library for antibodies having the desired activity. For example, a variety of methods are known in the art for producing phage ◎ presenting libraries and screening for antibodies having the desired binding characteristics in the libraries. Such methods are generally described in H〇〇genb〇〇m et al. ~ M〇/(iv)-heart/(iv) 178:1_37 (〇, by Brien et al., Human: T〇towa, NJ, 2001). For example, as described in ^ et al., m〇i _· Bi〇1. (2〇04), 340(5): 1073_93, a method for producing a related antibody uses a phage antibody library. In general, a synthetic antibody-pure line is selected by screening a phage-containing phage library which exhibits various fragments of an antibody variable region (Fv) fused to a phage sheath protein. The 149799.doc •95·201106972 % 3 玄 囷 囷 囷 。 library was performed by affinity chromatography on the desired antigen. The pure line of the FV fragment that is capable of binding to the desired antigen is adsorbed to the antigen and is therefore isolated from the unbound pure line in the library. The bound pure phase is then eluted from the antigen and further enriched by additional rounds of antigen adsorption/dissolution. Any of the antibodies of the present invention can be obtained by designing an antigen screening program to select a relevant strain of the bacterium, followed by the Fv sequence of the relevant phage-free line and Kabat et al., Sewe 〇 / Pr0 such as 〇 / / mmw (10) / «ierew , 5th edition, NIH Publication 91-3242, Bethesda MD (1991), suitable for the constant region (Fc) sequence described in Volumes 1-3 to construct a full length antibody pure line. In certain embodiments, the antigen binding domain of an antibody is formed by two variable (V) regions having about 110 amino acids, each of which is derived from a light chain (VL) and a heavy chain (VH) and both There are three hypervariable loops (HVRs) or complementarity determining regions (CDRs). The variable domain can be functionally as a single-chain Fv (scpv) fragment (wherein vh and VL are covalently linked via a flexible short peptide) or as a Fab fragment (wherein VH and VL are each fused to a constant domain and non-covalently interact) functionally Presented on phage, as described in Winter et al., (10), 12: 433-455 (1994), as used herein, 'scFv encoding sputum pure line and Fab coding sputum pure system called "Fv phage pure line" Or "Fv pure system". The VH and VL gene lineages can be separately cloned by polymerase chain reaction (pcR), and random recombination can be performed in the sputum library, followed by searching for antigen-binding pure lines, such as Winter Special, J(10)I: 433-455 (1994) Said. Libraries from immunized sources provide high affinity antibodies against the immunogen without the need to construct fusion tumors. Alternatively, the alternative can provide a single human antibody source for a wide range of non-autoantigens and autoantigens without the need for any natural lineage of 149799.doc-96-201106972 immunity, such as Griffiths et al., ΜδΜ 乂12: 725·734 (1993). Finally, the natural library can also be obtained by culturing an unrearranged V-gene fragment from a stem cell and encoding the CDR3 hypervariable region using a t PCR primer containing a random sequence and performing an in vitro rearrangement, such as

Hoogenboom及 Winter，Μί?/· 5ζ·ο/·，227: 381-388 (1992)所 述。 在某些實施例中，使用絲狀噬菌體呈現與次要鞘蛋白 Q ρΠΙ融合的抗體片段。抗體片段可作為單鏈Fv片段呈現， 其中VH域與VL域藉由柔性多肽間隔基連接於同一多肽鏈 上，例如Marks等人 ’ X m〇/·出〇/·, 581-597(1991)中所述； 或作為Fab片段呈現，其中一鏈與？111融合而另一鏈分泌進 入細菌性宿主細胞周質内，在周質内藉由置換一些野生型 鞘蛋白組裝呈現於噬菌體表面上的Fab-鞘蛋白結構，例如 Hoogenboom等人，, 19: 4133 4137 中所述。 〇 通常，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。若需要偏重於抗Αχί純系的文庫，則個 體以Axl免疫以產生抗體反應’且回收脾細胞及/或循環b 細胞及其他周圍血液淋巴細胞（PBL)用於文庫建構。在較 佳實施例中’偏重於抗Axl純系之人類抗體基因片段文庫 可糟由在攜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）之轉殖基因小鼠中產生抗Αχ丨抗體 反應以使得Axl免疫引起Β細胞產生抗Axl之人類抗體來獲 得。下文描述產生人類抗體之轉殖基因小鼠的產生。 149799.doc -97- 201106972 針對抗Axl反應性細胞群之其他富集可藉由使用適當筛 檢程序分離表現Axl特異性膜結合抗體之B細胞來獲得，例 如使用Axl親和層析法分離細胞或使細胞吸附至螢光染料 標記之Axl、再進行流式活化細胞分選（FACS)。 或者’使用未免疫供體之脾細胞及/或B細胞或其他pBL 可更好地展現可能抗體譜系，且亦容許使用任何動物（人 類或非人類）物種（其中Axl不具抗原性）建構抗體文庫。對 於合併活體外抗體基因建構之文庫而言，自個體採集幹細 胞以提供編碼未重排抗體基因區段之核酸。相關免疫細胞 可自多種動物物種獲得，諸如人類、小鼠、大鼠、兔類、 狼、犬、貓、豬、牛、馬及鳥類物種等。 自相關細胞中回收編碼抗體可變基因區段（包括VH及VL 區段）之核酸並擴增。在重排VH及VL基因文庫之狀況中， 所需DNA可如下獲得：自淋巴細胞中分離基因組dnA或 mRNA ’接著利用與重排VH及VL基因之5,端及3,端匹配之 引子進行聚合酶鏈反應（PCR)，如Orlandi等人，Proc. iVai/. 5Ά (USA), 86: 3833-3837 (1989)中所述，從而 獲得用於表現之各種V基因譜系。V基因可自cDNA及基因 組DNA擴增’其中反向引子位於編碼成熟v域之外顯子之 5'端且前向引子定位於J區段内部，如〇riandi等人，（1989) 及 Ward等人，iVaiMre, 341: 544-546 (1989)中所述。然而， 自cDNA擴增時，反向引子亦可定位於前導外顯子中，如 Jones等人，9: 88-89 (1991)中所述，且前向引 子可位於值定區内部，如Sastry等人，尸roc. iVai/. vicac/. 149799.doc •98- 201106972Hoogenboom and Winter, Μί?/· 5ζ·ο/·, 227: 381-388 (1992). In certain embodiments, a filamentous phage is used to present an antibody fragment fused to a minor sheath protein Q ρΠΙ. The antibody fragment can be presented as a single-chain Fv fragment, wherein the VH domain and the VL domain are joined to the same polypeptide chain by a flexible polypeptide spacer, such as Marks et al. 'X m〇/·出〇/·, 581-597 (1991) Said; or presented as a Fab fragment, one of which is linked with? 111 is fused while the other chain is secreted into the periplasm of the bacterial host cell, and the Fab-sheath protein structure presented on the surface of the phage is assembled in the periplasm by replacing some wild-type sheath proteins, for example, Hoogenboom et al., 19: 4133 Said in 4137. 〇 Generally, nucleic acids encoding antibody gene fragments are obtained freely from immune cells harvested by humans or animals. If a library of anti-Αχ pure lines is desired, the individual is immunized with Axl to generate an antibody response' and spleen cells and/or circulating b cells and other peripheral blood lymphocytes (PBL) are recovered for library construction. In a preferred embodiment, a library of human antibody gene fragments that are biased towards anti-Axl pure lines can be poorly derived from transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system). An anti-sputum antibody response is produced to allow Axl immunity to cause sputum cells to produce anti-Axl human antibodies. The production of transgenic mice producing human antibodies is described below. 149799.doc -97- 201106972 Additional enrichment against anti-Axl reactive cell populations can be obtained by isolating B cells expressing Axl-specific membrane-bound antibodies using appropriate screening procedures, for example using Axl affinity chromatography to isolate cells or The cells were adsorbed to the fluorescent dye-labeled Axl and subjected to flow-activated cell sorting (FACS). Or 'using spleen cells and/or B cells or other pBLs that are not immune donors to better represent possible antibody lineages, and also allow the construction of antibody libraries using any animal (human or non-human) species (where Axl is not antigenic) . For libraries incorporating in vivo antibody gene construction, stem cells are harvested from the individual to provide nucleic acids encoding the unrearranged antibody gene segments. Related immune cells are available from a variety of animal species, such as humans, mice, rats, rabbits, wolves, dogs, cats, pigs, cattle, horses, and bird species. Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the relevant cells and amplified. In the case of rearrangement of the VH and VL gene libraries, the desired DNA can be obtained by isolating the genomic dnA or mRNA from the lymphocytes and then using the primers that match the 5, 3, and 3 ends of the rearranged VH and VL genes. Polymerase chain reaction (PCR), as described in Orlandi et al, Proc. iVai/. 5Ά (USA), 86: 3833-3837 (1989), thereby obtaining various V gene lineages for expression. The V gene can be amplified from cDNA and genomic DNA 'where the reverse primer is located at the 5' end of the exon of the mature v domain and the forward primer is located inside the J segment, eg 〇riandi et al. (1989) and Ward Et al., iVaiMre, 341: 544-546 (1989). However, in the case of cDNA amplification, the reverse primer can also be located in the leader exon, as described in Jones et al., 9: 88-89 (1991), and the forward primer can be located inside the value region, such as Sastry et al., corpse roc. iVai/. vicac/. 149799.doc •98- 201106972

Scr人86: 5728-5732 (1989)中所述。為最大化互補 性’可合併引子中的簡併性，如〇rlandi等人（丨989)或 Sastry等人（1989)中所述。在某些實施例中，文庫多樣性 可藉由使用靶向各V基因家族的pcR引子最大化，以擴增 存在於免疫細胞核酸樣品中之所有可利用的VH與VL排 列，例如Marks等人，/· M〇/_ 厂，222: 581-597 (1991)之 方法中所述或如Orum等人，及以，21: 4491-4498 (1993)之方法中所述。將擴增之dna選殖入表現載體 内時，可將稀少限制性位點引入PCR引子内之一端作為標 記，如Orlandi等人（1989)中所述，或利用標記引子進一步 進行 PCR擴增，如 Clackson 等人，TVaiwre, 352: 624-628 (1991)中所述。 合成性重排之V基因Ί普系可活體外源自v基因區段。人 類VH基因區段中的大部分已選殖且定序（報導於T〇niHns〇n 專人 ’·/. Λ/ο/· 5/〇厂，227: 776-798 (1992)中），且已繪'出圖 譜（報導於 Matsuda 等人，iVaiwre Gewei·, 3: 88-94 (1993) 中）；可使用此等選殖區段（包括HI與H2環之所有主要構 形）以及編碼各種序列及長度之H3環的PCR引子產生各種 VH基因譜系，如Hoogenboom 及 Winter，丄 Mo/. 227. 381-388 (1992)中所述。亦可獲得所有序列多樣性皆集中 於長H3環之單一長度上的VH譜系，如Barbas等人，Pr〇c iWzi/. ylcaci. 5Ά 89: 4457-4461 (1992)中所述。人類Scr. 86: 5728-5732 (1989). The degeneracy in the primers can be combined to maximize complementarity as described in 〇rlandi et al. (丨 989) or Sastry et al. (1989). In certain embodiments, library diversity can be maximized by using pcR primers that target each V gene family to amplify all available VH and VL sequences present in the immune cell nucleic acid sample, such as Marks et al. , / M 〇 / _ factory, 222: 581-597 (1991) as described in the method or as described in the method of Orum et al., and 21: 4491-4498 (1993). When the amplified dna is cloned into the expression vector, a rare restriction site can be introduced into one end of the PCR primer as a marker, as described in Orlandi et al. (1989), or further PCR amplification using a labeled primer. As described in Clackson et al., TVaiwre, 352: 624-628 (1991). Synthetic rearranged V gene sputum can be derived in vitro from the v gene segment. Most of the human VH gene segments have been cloned and sequenced (reported in T〇niHns〇n Specialist '·/. Λ/ο/· 5/〇, 227: 776-798 (1992)), and A map has been drawn (reported in Matsuda et al., iVaiwre Gewei., 3: 88-94 (1993)); these selection sections (including all major configurations of the HI and H2 rings) and various codes can be used. The PCR primers of the sequence and length of the H3 loop produce various VH gene lineages as described in Hoogenboom and Winter, 丄Mo/. 227. 381-388 (1992). It is also possible to obtain a VH lineage in which all sequence diversity is concentrated on a single length of the long H3 loop, as described in Barbas et al., Pr〇c iWzi/. ylcaci. 5Ά 89: 4457-4461 (1992). Humanity

Vk及νλ區段已選殖且定序（報導於Williams及Winter, £阶· ·/·/所顧⑽/·，23: 1456-1461 (1993)中）且可用於形成合成性 149799.doc -99- 201106972 輕鏈譜系。基於一系列之VH與VL摺疊及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之後，生殖系V基因區段可根據11〇〇#心〇〇111 及 Winter，i M〇/•如 〇/., 227: 381-388 (1992)之方法活體外 重排。 可藉由以多種方式將VH與VL基因譜系組合在一起來建 構抗體片段譜系。各譜系可建立於不同載體中，且載體可 活體外重組’例如Hogrefe等人，128: 119-126 (1993)中所述，或藉由組合感染（例如Waterh〇use等人， We/7^·，21: 2265_2266 (1993)中所述之比⑼系統） 活體内重組。活體内重組方法係利用F ab片段之雙鏈性質 來克服大腸桿菌轉型效率對文庫大小所施加之限制。天然 VH及VL譜系係分別選殖，一者選殖入噬菌粒中且另一者 選殖入噬菌體載體中。接著藉由噬菌體感染含有噬菌粒之 細菌來組合兩個文庫，以使得各細胞含有不同組合且文庫 大小僅受所存在細胞數量（約1〇12個純系）限制。兩載體均 含有活體内重組信號，以使得¥11與VL基因在單一複製子 上重組且共封裝於噬菌體病毒粒子中。此等大型文庫提供 大量具有良好親和力（Kd·1為約10_8 M)之多種抗體。 或者，該等譜系可依序選殖入同一載體，例如Barbas等 人，Proc.心"· w 仍乂，88: 7978_7982 (1991)中所 述，或藉由PCR組裝在一起且接著選殖，例如（^丨“匕⑽等 人，352: 624-628 (1991)中所述。亦可使用 PCR組 裝將VH及VL DNA與編碼柔性肽間隔基之DNA連接在一起 149799.doc 201106972 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在淋巴細胞内組合VH與VL基因且 接著選殖已連接基因之譜系，如Embleton等人，WMC/ 及 20:3831-37 (1992)中所述0 由天然文庫所產生之抗體（天然或合成）可具有中度親和 力（約106至1〇7 Μ-1之Kd-1)，但親和力成熟亦可藉由建構及 再選擇第二文庫來活體外模擬，如Winter等人（1994)(同 0 上）所述。舉例而言，可使用Hawkins等人，J. Mo/.別〇厂， 226: 889-896 (1992)之方法或 Gram等人，Pro。Jcad. 5W t/M，89: 3576-3 580 (1992)之方法中之易出錯聚合酶 (報導於 Leung等人 ’ rec/migwe, 1: 11-15 (1989)中）活體外 隨機引入突變。此外’親和力成熟可如下進行：在所選個 別Fv純系中使一或多個CDR隨機突變，例如使用具有全長 為相關CDR之隨機序列的引子進行PCR，且篩檢較高親和 力純系。WO 96〇7754(1"6年3月I4日公開）描述一種誘導 〇 免疫球蛋白輕鏈之互補決定區發生突變以建立輕鏈基因文 庫的方法。另一種有效方法為將藉由嗤菌體呈現所選擇的 VH域或VL域與獲自未免疫供體之天然存在之v域變異體 譜系重組且以數輪鏈改組篩檢較高親和力，如Marks等 .人，1〇: 779_783 (1992)中所述。此技術能夠 產生親和力為約1〇-9 Μ或10_9 μ以下之抗體及抗體片段。 可藉由此項技術中已知之各種技術完成文庫篩檢。舉例 而吕，可使用Axl塗佈吸附板之各孔，於固定至吸附板之 宿主細胞上表現或用於細胞分選，或與生物素結合以供塗 149799.doc -101- 201106972 有抗生物蛋白鏈菌素之珠粒捕捉，或用於淘選噬菌體呈現 文庫之任何其他方法中。 在適於使嗤菌體顆粒之至少一部分與吸附劑結合之條件 下’使嗟菌體文庫樣品與經固定Αχί接觸。通常選擇可模 擬生理條件的條件，包括pH值、離子強度、溫度及其類似 條件。洗蘇結合至固相之噬菌體且接著藉由酸溶離，例如 Barbas等人 ’ 勤".deal W 88: 7978-7982 (199 U中所述’或藉由驗溶離’例如Marks等人，J. Mo厂 方⑴/·’ 222·· 581-597 (1991)中所述’或藉由Axl抗原競爭， 例如以與Clackson等人，iVa/wre, 352:624-628 (1991)之抗 原競爭方法類似之程序進行Αχί抗原競爭。可在單輪選擇 中备集20-1，〇〇〇倍之噬菌體。此外，所富集之噬菌體可在 細菌培養物中生長並經歷其他多輪選擇。 選擇效率視許多因素而定’包括洗滌過程中之解離動力 學及單一噬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 紐時間洗滌、多價噬菌體呈現及抗原於固相中之高塗佈密 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。可使用長時間 洗滌及單價噬菌體呈現（如Bass等人’ 8: 309-314 (1990)及臂〇 92/〇969〇中所述）及低抗原塗佈密度（如 等人，別oiec/mo/_, 10: 779_783 (1992)中所述）來促進對具 有慢速解離動力學（及良好結合親和力）之抗體之選擇。 可在對Axl親和力不同之噬菌體抗體之間選擇，即使親 149799.doc -102· 201106972 和力稍微不同。然而，所選抗體之隨機突變（例如，如在 一些親和力成熟技術中所執行）可能產生許多突變體，大 多數結合抗原，且有一些具有較高親和力。在限制Αχΐ ' 下，可淘汰稀少的高親和力噬菌體。為了保留所有較高親 和力突變體，可將噬菌體與過量的經生物素標記之一 起培育，但經生物素標記之Axl之莫耳濃度比Αχ1之目標莫 耳親和力常數低。高親和力結合噬菌體接著可被塗有抗生 ◎ 物蛋白鏈菌素之順磁珠粒捕捉。該「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 大夏過I噬菌體分離。亦可操控用於洗滌結合至固相之噬 菌體的條件以基於解離動力學進行區分。 可基於活性選擇抗Axl純系。在某些實施例中，本發明 提供結合天然表現Axl之活細胞之抗Αχ1抗體。在一實施例 中，本發明提供阻斷Axl配位體與Axl之間結合但不阻斷 〇 Axl配位體與第二蛋白之間結合的抗Αχ1抗體。對應於該等 抗Axl抗體的Fv純系可如下選擇：（1)如上所述自噬菌體文 庫中分離抗Axl純系，及視需要擴增分離的噬菌體純系群 • 體（藉由在適當細菌宿主中培育該群體）；（2)針對分別需要 •‘ 阻斷活性及非阻斷活性來選擇Axl及第二蛋白；（3)將抗Αχ1 噬菌體純系吸附至固著Axl ; (4)使用過量的第二蛋白溶離 任何非所要純系’非所要純系識別的Αχ1結合決定子與第 二蛋白之結合決定子重疊或共有；及（5)溶離步驟（4)完成 後仍吸附的純系。具有所要阻斷/非阻斷特性之純系視需 149799.doc •103- 201106972 要可進一步藉由將本文中所述之選擇程序重複一或多次來 富集。 使用習知程序（例如使用設計成自融合瘤或噬菌體Dna 模板特異性擴增相關重鏈及輕鏈編碼區的寡核苷酸引子） 易於分離及定序編碼本發明之融合瘤源單株抗體或噬菌體 呈現Fv純系的DNA。DNA —經分離，便可置於表現載體 内’接著轉染至宿主細胞（諸如大腸桿菌細胞、猿c〇s細 胞、中國倉鼠卵巢（CHO)細胞或骨髓瘤細胞）中（否則宿主 細胞不產生免疫球蛋白），以於重組宿主細胞中獲得所需 單株抗體之合成。關於在細菌中重組表現編碼抗體之DNA 的評論文章包括Skerra等人，C«rr. Op⑻·0„〜廳⑽厂，5: 256 (1993)及 Pluckthun, /ievs，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序列可依據Kabat等 人（同上）獲得）組合以形成編碼全長或部分長度重鏈及/或 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。源自一種動物（諸如 人類）物種之可變域DNA且接著與另—動物物種之恆定區 DNA融合以形成「雜交」全長重鏈及/或輕鏈之編碼序列 的Fv純系包括於如本文所使用之「嵌合」及「融合」抗體 定義中。在某些實施例中，使來源於人類可變〇?^入之以純 系與人類恆定區DNA融合以形成全長或部分長度人類重鏈 及/或輕鏈之編碼序列。 149799.doc * 104- 201106972 編碼本發明之源自雜交瘤之抗Axl抗體的DNA亦可經修 飾’例如用人類重鏈恆定域及輕鏈恆定域編碼序列取代源 自融合瘤純系的同源鼠序列（例如Morrison等人，Pr〇c 81: 6851-6855 (1984)之方法）。編碼 源自融合瘤或Fv純系之抗體或片段的dna可藉由共價連接 至免疫球蛋白編碼序列、非免疫球蛋白多肽之全部或部分 編碼序列來進一步修飾。以此方式可製備具有本發明之源 Q 自FV純系或融合瘤純系之抗體之結合特異性的「嵌合」或 「融合」抗體。 3·載體、宿主細胞及重組方法 亦可使用重組方法產生抗體。為重組產生抗八“抗體， 將編碼該抗體之核酸分離且***可複製載體中以便進一步 選殖（擴增DNA)或用於表現。可使用習知程序（例如使用能 夠特異性結合編碼抗體重鏈及輕鏈之基因之寡核苷酸探 針）谷易離及疋序編碼抗體之DNA。可利用多種載體。 〇 載體組分一般包括（但不限於）以下一或多者：信號序列、 複製起點、一或多個標記基因、強化子元件、啟動子及轉 錄終止序列。 a)信號序列組分 . 本發明之抗體不僅可直接重組產生，而且可以與異源多 肽之融合多肽形式產生，該異源多肽較佳為信號序列或在 成熟蛋白質或多肽之N末端處具有特定裂解位點之其他多 肽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。對於不識別及處理 149799.doc -105- 201106972 原生抗體信號序列之原核宿主細胞而言，將該信號序列取 代為原核彳s號序列’例如選自驗性填酸酶、青黴素酶、 !PP或熱穩定性腸毒素II前導序列之群的原核信號序列。對 於酵母分泌’原生彳s號序列可取代為例如酵母轉化酶前導 序列、α因子如導序列（包括酵母菌及克魯 維酵母（尤/吵veromyeOa因子前導序列）或酸性磷酸酶前導 序列、白色念珠菌（C. albicans)葡糖澱粉酶前導序列或w〇 90/13646中所述之信號。在哺乳動物細胞表現中，可使用 哺乳動物信號序列以及病毒分泌性前導序列（例如單純疱 疹gD信號）。 b)複製起點 表現載體與選殖載體皆含有使載體能夠在一或多種所選 佰主細胞中複製的核酸序列。通常，在選殖载體中，此序 列為使載體能夠獨立於宿主染色體DNA複製的序列，且包 括複製起點或自主複製序列。已熟知多種細菌、酵母及病 毒之該等序列。質體ρΒΜ22之複製起點適於大多數革蘭 氏陰性（Gram-negative)細菌，2μ質體起點適於酵母，且各 種病毒起點（SV40、多形瘤、腺病毒、vsv或Βρν)適用於 哺乳動物細胞巾之選耗體。通常，哺乳動物表現載體不 需要複製起點組分（使用SV40起點的原因通常僅在於其含 有早期啟動子）。 ^ 3 c)選擇基因組分 ，亦稱為可選標記物。 針對抗生素或其他毒素 表現及選殖載體可含有選擇基因 典型選擇基因編碼的蛋白質（a)賦予 149799.doc -106- 201106972 (例如安比西林（ampicillin)、新黴素（ne〇mycin)、曱胺喋呤 或四環素（tetracycline))的抗性，（b)補充營養缺陷型不^ 或⑷供應不可自複雜培養基中得到之關鍵營養，例如編碼 桿狀菌（5此////)之D-丙胺酸消旋酶的基因。 ' 選擇方案之一實例係利用藥物阻滯宿主細胞之生長。經 異源基因成功轉型之彼等細胞產生賦予抗藥性之蛋白質且 ^此在選擇療程中存活。該顯性選擇之實例係使用藥物新 0 黴素、黴酚酸及濕黴素（hygromycin)。 適用於哺乳動物細胞之可選擇標記之另一實例為能夠鑑 別有能力接納編碼抗體之核酸之細胞的彼等標記，諸如 DHFR、麵醯胺酸合成酶（GS)、胸苷激酶、金屬硫蛋白工及 金屬硫蛋白11(較佳為靈長類動物金屬硫蛋白基因广腺苷 脫胺酶、鳥胺酸脫羧酶等。 舉例而言，藉由在含有甲胺喋呤（Mtx)(DHFR之競爭性 拮抗劑）之培養基中培養轉化子來鑑別經DHFR基因轉型之 〇 細胞。在此等條件下，卿R基因與任何其他共轉型核酸 一起擴增。可使用缺乏内源性DHFR活性之中國倉鼠卵巢 (CH0)細胞株（例如 ATCC CRL-9096)。 或者，藉由在含有L- f硫胺酸續醯亞胺（Msx)(GS之抑制 劑）之培養基中培養轉化子來鑑別經Gs基因轉型之細胞。 在此等條件下，GS基因與任何其他共轉型核酸一起擴增。 GS選擇/擴增系統可與上述DHFR選擇/擴增系統組合使 用。 或者，可藉由在含有針對可選擇標記之選擇劑（諸如胺 149799.doc •107- 201106972 基醣普抗生素，例如卡那黴素（kanamycin)、新黴素或 G4 1 8)之培養基中培養細胞來選擇經編碼相關抗體之DNA 序列、野生型DHFR基因及另一可選擇標記（諸如胺基醣普 31-磷酸轉移酶（ΑΡΗ))轉型或共轉型之宿主細胞（尤其含有 内源性DHFR之野生型宿主）。參見美國專利案第4,965,199 號。 適用於酵母中之選擇基因為存在於酵母質體YRp7中之 冲1 基因（Stinchcomb等人，施iwre, 282:39 (1979))。冲1 基 因為不能在色胺酸中生長的突變型酵母菌株（例如ATCc編 號44076或PEP4-1)提供選擇標記。jones，仏奸价八85:12 (1977)。酵母宿主細胞基因組中存在之丨損傷接著為偵 測在色胺酸不存在下由生長所引起之轉型提供有效環境。 類似地，/^2缺陷型酵母菌株（八丁(^ 20,622或38,626)由攜 有基因之已知質體補充。 此外’來源於1.6 μηι環狀質體PKD1之載體可用於轉型克 魯維酵母。或者，已報導乳酸克魯維酵母（尺之表 現系統’其用於大規模生產重組小牛凝乳酶。Van den Berg，出〇/7^以0/0幻;，8:ι35 (1990)。亦已揭示供工業克魯 維酵母菌株分泌成熟重組人類血清白蛋白的穩定多複本表 現載體。Fleer等人，仏〇/7^/2«〇/〇容少，9:968-975 (1991)。 d)啟動子組分 表現及選殖載體通常含有可被宿主生物體識別且可操作 地連接至編碼抗體之核酸之啟動子。適用於原核宿主之啟 動子包括；?/2〇A啟動子、β-内醯胺酶及乳糖啟動子系統、鹼 149799.doc •108· 201106972 性磷酸酶啟動子、色胺酸（trp)啟動子系統、及融合啟動子 (諸如tac啟動子）。然而’其他已知之細菌啟動子亦適用。 用於細菌系統中之啟動子亦含有可操作地連接至編碼抗體 之DNA之夏因-達爾加諾（shine-Dalgarno; S.D.)序列。 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3〇個鹼基處之富AT 區。發現於多種基因之轉錄起點上游7〇至8〇個鹼基處的另 0 一序列為CNC AΑτ區，其中N可為任何核苷酸。大部分真 核基因的3’端為AATAAA序列，該序列可為添加p〇丨y八尾 至編碼序列之3,端的信號。所有此等序列均適合***真核 表現載體中。 適用於酵母宿主之啟動子序列之實例包括3 _填酸甘油酸 激酶或其他醣解酶（諸如烯醇酶、甘油醛_3_磷酸脫氫酶、 己醣激酶、丙酮酸脫羧酶、磷酸果糖激酶、葡萄糖_6_磷酸 異構酶、3-磷酸甘油酸歧化酶、丙酮酸激酶、磷酸丙醣異 〇 構酶、磷酸葡糖異構酶及葡萄糖激酶）之啟動子， 作為具有轉錄受生長條件控制之額外優勢之可誘導啟動 子的其他酵母啟動子為以下之啟動子區：乙醇脫氫酶2、 • 異細胞色素C、酸性磷酸酶、與氮代謝相關之降解酶、金 .屬硫蛋白、甘油醛_3_磷酸脫氫酶及負責麥芽糖及半乳糖利 用之酵素。EP 73,657中進一步描述適用於酵母表現之載體 及啟動子。酵母增強子亦可有利地與酵母啟動子一起使 用。 哺乳動物宿主細胞中經由載體進行的抗體轉錄可藉由例 149799.doc -109- 201106972 如以下啟動子控制：獲自病毒（諸如多形瘤病毒、雞痘病 毒、腺病毒（諸如腺病毒2)、牛乳頭狀瘤病毒、鳥肉瘤病 毒、細胞巨大病毒、反轉錄病毒、B型肝炎病毒、猿猴病 毒40(SV40))之基因組的啟動子；或異源哺乳動物啟動子 (例如肌動蛋白啟動子或免疫球蛋白啟動子）；熱休克啟動 子’其限制條件為該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宜作為亦含有SV4〇病毒 複製起點之SV40限制片段獲得。人類細胞巨大病毒之立即 早期啟動子宜作為Hindlll E限制片段獲得。美國專利第 4,419，446號中揭示使用牛乳頭狀瘤病毒作為載體在哺乳動 物宿主中表現DNA之系統。美國專利第4,6〇1，978號中描述 此系統之改型。關於人類β干擾素cDNA在小鼠細胞中在單 純疮疹病毒之胸苷激酶啟動子控制下之表現，亦參見The Vk and νλ segments have been cloned and sequenced (reported in Williams and Winter, £8····/ (10)/·, 23: 1456-1461 (1993)) and can be used to form synthetic 149799.doc -99- 201106972 Light chain pedigree. Based on a series of VH and VL folds and the synthesis of L3 and H3 lengths, the V gene lineage will encode antibodies with substantial structural diversity. After amplifying the DNA encoding the v gene, the germline V gene segment can be in vitro weighted according to the method of 11〇〇#心〇〇111 and Winter, i M〇/•如〇/., 227: 381-388 (1992). row. The antibody fragment lineage can be constructed by combining the VH and VL gene lineages in a variety of ways. Each lineage can be established in a different vector and the vector can be recombined in vitro as described in, for example, Hogrefe et al, 128: 119-126 (1993), or by a combination infection (eg, Waterh〇use et al, We/7^) ·, 21: 2265_2266 (1993) than the ratio (9) system) in vivo recombination. In vivo recombination methods utilize the double-stranded nature of the Fab fragment to overcome the limitations imposed by the E. coli transformation efficiency on library size. The native VH and VL lineages are separately selected, one is selected into the phagemid and the other is selected into the phage vector. The two libraries are then combined by phage infection of bacteria containing phagemids such that each cell contains a different combination and the library size is limited only by the number of cells present (approximately 1 12 pure lines). Both vectors contain an in vivo recombination signal such that the ¥11 and VL genes are recombined on a single replicon and co-encapsulated in phage virions. These large libraries provide a large number of antibodies with good affinity (Kd·1 is about 10-8 M). Alternatively, the lineages can be sequenced into the same vector, for example as described by Barbas et al., Proc. Heart " w 乂 乂, 88: 7978_7982 (1991), or assembled by PCR and subsequently colonized For example, (^), 匕 (10) et al, 352: 624-628 (1991). PCR assembly can also be used to link VH and VL DNA to DNA encoding a flexible peptide spacer 149799.doc 201106972 to form Single-chain Fv (scFv) lineage. In another technique, "intracellular PCR assembly" is used to combine VH and VL genes in lymphocytes by PCR and then to select the lineage of linked genes, such as Embleton et al., WMC/ And the antibody (natural or synthetic) produced by the natural library described in 20:3831-37 (1992) may have moderate affinity (Kd-1 of about 106 to 1〇7 Μ-1), but affinity matured. In vitro simulations can be performed by constructing and reselecting a second library, as described by Winter et al. (1994) (same as 0). For example, Hawkins et al., J. Mo/. : Method of 889-896 (1992) or error-prone aggregation in the method of Gram et al., Pro. Jcad. 5W t/M, 89: 3576-3 580 (1992) Enzymes (reported in Leung et al. ' rec/migwe, 1: 11-15 (1989)) were randomly introduced in vitro. In addition, 'affinity maturation can be performed as follows: one or more CDRs are randomized in selected individual Fv lines Mutations, for example, using primers with a full-length sequence of relevant CDRs for PCR, and screening for higher affinity pure lines. WO 96〇7754 (1" published March 3, 4) describes an induced immunoglobulin light chain Mutation of the complementarity determining region to establish a light chain gene library. Another effective method is to present the selected VH domain or VL domain by the sputum cell and the naturally occurring v domain variant lineage obtained from the unimmunized donor. Recombination and screening for higher affinity by several rounds of chain shunting, as described in Marks et al., 1 〇: 779_783 (1992). This technique produces antibodies and antibodies with affinities of about 1〇-9 Μ or 10_9 μ. Fragments. Library screening can be accomplished by various techniques known in the art. For example, each well of an adsorption plate can be coated with Axl, expressed on a host cell immobilized to the adsorption plate, or used for cell sorting, Or combined with biotin For coating 149799.doc -101- 201106972 with anti-bio-streptomycin beads capture, or for any other method for panning phage display libraries. Suitable for at least a portion of sputum bacterial particles and adsorbents Under the conditions of the combination, the sample of the bacillus library was contacted with the fixed Αχ. Conditions that mimic physiological conditions are generally selected, including pH, ionic strength, temperature, and the like. The sputum is bound to the solid phase phage and then lysed by acid, for example as described by Barbas et al., 'Qin'. Deal W 88: 7978-7982 (in the case of 199 U, or by dissolution), eg, Marks et al., J Mo Factory (1)/·' 222·· 581-597 (1991) 'or compete by Axl antigen, for example to compete with the antigen of Clackson et al, iVa/wre, 352:624-628 (1991) The method is similar to the procedure for 抗原ί antigen competition. 20-1, 〇〇〇 phage can be prepared in a single round of selection. In addition, the enriched phage can grow in bacterial culture and undergo other rounds of selection. Efficiency depends on many factors 'including the dissociation kinetics during washing and whether multiple antibody fragments on a single phage can simultaneously bind to the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinity) can be used by using neon time Washing, multivalent phage display and high coating density of the antigen in the solid phase are preserved. High density not only stabilizes the phage via multivalent interaction, but also facilitates recombination of the dissociated phage. unit price Phage display (as described in Bass et al. 8: 309-314 (1990) and Brake 92/〇969〇) and low antigen coating density (eg, et al, oiec/mo/_, 10: 779_783 ( (1992) to promote the selection of antibodies with slow dissociation kinetics (and good binding affinity). Can be selected between phage antibodies with different Axl affinity, even pro 149799.doc -102· 201106972 and force Slightly different. However, random mutations in selected antibodies (eg, as performed in some affinity maturation techniques) may produce many mutants, most of which bind antigen and some have higher affinity. Eliminate rare high-affinity phage. In order to retain all higher affinity mutants, phage can be incubated with excess biotinylated, but the biotin-labeled Axl molar concentration is lower than the target molar affinity constant of Αχ1. The high affinity binding phage can then be captured by paramagnetic beads coated with anti-biotin phage. This "balance capture" enables sensitivity based on the binding affinity of the antibody. Sensitively selecting antibodies that allow for the isolation of a mutant line with only twice the high affinity from the phage that has a lower affinity. The conditions for washing the phage bound to the solid phase can also be manipulated based on the dissociation kinetics. The anti-Axl pure line may be selected based on the activity. In certain embodiments, the invention provides an anti-Αχ1 antibody that binds to a living cell that naturally expresses Axl. In one embodiment, the invention provides for blocking the Axl ligand with An anti-Αχ1 antibody that binds between Axl but does not block binding between the 〇Axl ligand and the second protein. The Fv pure line corresponding to the anti-Axl antibodies can be selected as follows: (1) isolating the anti-Axl pure line from the phage library as described above, and amplifying the isolated phage pure line group as needed (by culturing in a suitable bacterial host) (2) Axl and a second protein are selected for each of the required 'blocking activity and non-blocking activity; (3) the anti-Αχ1 phage pure line is adsorbed to the immobilized Axl; (4) the second is used in excess The protein dissolves any undesired homologues of the Αχ1 binding determinant recognized by the non-purified line and the binding determinant of the second protein overlaps or shares; and (5) the pure line that remains adsorbed after the dissolution step (4) is completed. Pure line with desired blocking/non-blocking properties 149799.doc • 103- 201106972 To be further enriched by repeating the selection procedure described herein one or more times. Easy to isolate and sequence encoding the fusion tumor-derived monoclonal antibodies of the present invention using conventional procedures (eg, using oligonucleotide primers designed to specifically amplify relevant heavy and light chain coding regions from a fusion tumor or phage DNA template) Or the phage exhibits Fv pure lineage DNA. DNA - after isolation, can be placed in a performance vector' and then transfected into host cells (such as E. coli cells, 猿c〇s cells, Chinese hamster ovary (CHO) cells or myeloma cells) (otherwise host cells do not produce Immunoglobulin) to obtain the synthesis of the desired monoclonal antibody in a recombinant host cell. Commentary on the recombinant expression of DNA encoding antibodies in bacteria includes Skerra et al., C«rr. Op(8)·0„~ Hall (10), 5: 256 (1993) and Pluckthun, /ievs, 130: 151 (1992). A DNA encoding a 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 according to Kabat et al., supra) to form a full length or partial length of the code. Pure lines of chains and/or light chains. It will be appreciated that any isotype constant region can be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be obtained from any human or animal species. An Fv-derived line derived from a variable domain DNA of an animal (such as a human) species and then fused to a constant region DNA of another animal species to form a "hybrid" full-length heavy and/or light chain coding sequence is included as herein Use the "chimeric" and "fusion" antibody definitions. In certain embodiments, a human-derived constant region DNA is fused to human-derived to form a coding sequence for a full-length or partial-length human heavy and/or light chain. 149799.doc * 104- 201106972 The DNA encoding the hybridoma-resistant anti-Axl antibody of the present invention may also be modified, for example, by replacing the homologous mouse derived from the fusion tumor pure line with the human heavy chain constant domain and the light chain constant domain coding sequence. Sequence (e.g., Morrison et al., Pr. 81: 6851-6855 (1984)). A dna encoding an antibody or fragment derived from a fusion tumor or an Fv pure line can be further modified by covalent attachment to an immunoglobulin coding sequence, all or part of a coding sequence of a non-immunoglobulin polypeptide. In this manner, a "chimeric" or "fusion" antibody having the binding specificity of the source Q of the present invention from an FV pure or fusion-tumor antibody can be prepared. 3. Vectors, Host Cells, and Recombinant Methods Antibodies can also be produced using recombinant methods. For the production of an anti-eight" antibody, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further selection (amplification of the DNA) or for expression. A conventional procedure can be used (eg, using a specific antibody capable of specific binding) Oligonucleotide probes for the genes of the strands and light chains) DNAs encoding the antibodies and the sequences may be utilized. A variety of vectors may be utilized. The carrier components generally include, but are not limited to, one or more of the following: signal sequences, The origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. a) Signal sequence components. The antibodies of the present invention can be produced not only directly by recombinant but also by fusion polypeptides of heterologous polypeptides, Preferably, the heterologous polypeptide is a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence is preferably recognized and processed by the host cell (ie, by signal peptidase) Signal sequence for cleavage). For prokaryotic host cells that do not recognize and handle the 149799.doc-105-201106972 native antibody signal sequence, the signal is The column is substituted with a prokaryotic signal sequence of the prokaryotic 彳 s sequence 'for example, a population selected from the group consisting of a confirmatory hydratase, a penicillinase, a !PP or a thermostable enterotoxin II leader sequence. For the yeast secretion, the 'native 彳 s sequence can be substituted. For example, yeast invertase leader sequence, alpha factor such as leader sequence (including yeast and Kluyveromyces (extra/veromyeOa factor leader sequence) or acid phosphatase leader sequence, C. albicans glucoamylase The leader sequence or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leader sequences (eg, herpes simplex gD signal) can be used. b) Replication origin expression vector and selection The vectors all contain a nucleic acid sequence which enables the vector to replicate in one or more selected sputum master cells. Typically, in a selection vector, the sequence is a sequence which enables the vector to replicate independently of the host chromosomal DNA and includes an origin of replication or Autonomously replicating sequences. These sequences of bacteria, yeast and viruses are well known. The origin of replication of plastid ρΒΜ22 is suitable for most Gram-negative (Gram-negative) bacteria, 2μ plastid origin suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, vsv or Βρν) are suitable for mammalian cell towel selection. Usually, mammalian expression vector There is no need to copy the starting component (the reason for using the SV40 origin is usually that it contains an early promoter). ^ 3 c) Selecting a genetic component, also known as a selectable marker. For antibiotics or other toxins and selection vectors may contain Selecting the protein encoded by the gene's typical selection gene (a) confers resistance to 149799.doc-106-201106972 (eg, ampicillin, ne〇mycin, amidoxime or tetracycline), (b) supplementing auxotrophs or (4) supplying essential nutrients that are not available from complex media, such as the gene encoding D-alanine racemase from Bacillus (5////). One example of a selection protocol is the use of drugs to block the growth of host cells. The cells that have been successfully transformed by the heterologous gene produce a protein that confers resistance and this survives during the elective course of treatment. An example of this dominant selection is the use of the drugs neomycin, mycophenolic acid and hygromycin. Another example of a selectable marker suitable for use in mammalian cells is one that is capable of identifying cells capable of receiving a nucleic acid encoding the antibody, such as DHFR, fascia synthase (GS), thymidine kinase, metallothionein. And metallothionein 11 (preferably primate metallothionein gene adenosine deaminase, ornithine decarboxylase, etc., for example, by containing methotrexate (Mtx) (DHFR Transformants are cultured in a culture medium to identify DHFR-transformed sputum cells. Under these conditions, the cleavage R gene is amplified together with any other co-transformed nucleic acid. A Chinese lacking endogenous DHFR activity can be used. Hamster ovary (CH0) cell line (eg, ATCC CRL-9096). Alternatively, Gs can be identified by culturing transformants in a medium containing L-f thioglycolate (Msx) (inhibitor of GS) Gene-transformed cells. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acid. The GS selection/amplification system can be used in combination with the DHFR selection/amplification system described above. Selection mark Selecting a medium (such as an amine 149799.doc • 107-201106972 basal antibiotic, such as kanamycin, neomycin or G4 18) to culture the cells to select the DNA sequence encoding the relevant antibody, wild a host cell of a type DHFR gene and another selectable marker (such as aminoglycoside 31-phosphate transferase (ΑΡΗ)) for transformation or co-transformation (especially a wild-type host containing endogenous DHFR). See U.S. Patent No. 4,965,199 The selection gene for yeast is the rush 1 gene present in the yeast plastid YRp7 (Stinchcomb et al., iwre, 282:39 (1979)). The rush 1 gene is a mutation that cannot grow in tryptophan. A yeast strain (eg, ATCc No. 44076 or PEP4-1) provides a selectable marker.jones, smuggling price 85:12 (1977). The sputum damage present in the yeast host cell genome is then detected in the absence of tryptophan The transformation caused by growth provides an effective environment. Similarly, the /^2 deficient yeast strain (Eight (^ 20, 622 or 38, 626) is supplemented by a known plastid carrying a gene. In addition, 'derived from 1.6 μηι cyclic plastid PKD1 Carry It can be used to transform Kluyveromyces. Or, Kluyveromyces cerevisiae has been reported (the performance system of the ruler's for large-scale production of recombinant calf chymosin. Van den Berg, 〇/7^ with 0/0 illusion ;, 8:ι35 (1990). A stable multi-replica expression vector for the secretion of mature recombinant human serum albumin by industrial Kluyveromyces strains has also been revealed. Fleer et al., 仏〇/7^/2«〇/〇容, 9:968-975 (1991). d) Promoter Components The expression and selection vectors typically contain a promoter that is recognized by the host organism and operably linked to the nucleic acid encoding the antibody. Promoters suitable for use in prokaryotic hosts include: ?/2〇A promoter, β-endosaminolase and lactose promoter system, base 149799.doc •108· 201106972 phosphatase promoter, tryptophan (trp) promoter Subsystems, and fusion promoters (such as the tac promoter). However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems also contain a shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody. Promoter sequences for use in eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 3 bases upstream of the transcription start site. Another sequence found at 7 to 8 bases upstream of the transcription start of multiple genes is the CNC AΑτ region, where N can be any nucleotide. The 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal that adds p〇丨y octagonal to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors. Examples of promoter sequences suitable for use in a yeast host include 3 - glycerol acid kinase or other glycolytic enzymes (such as enolase, glyceraldehyde_3_phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, fructose phosphate) Promoter of kinase, glucose-6-phosphate isomerase, 3-phosphoglycerate dismutase, pyruvate kinase, triose phosphate, phosphoglucose isomerase, and glucokinase Additional yeast promoters with an additional advantage of conditional control are the following promoter regions: alcohol dehydrogenase 2, • isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, gold. Protein, glyceraldehyde _3_phosphate dehydrogenase and enzyme responsible for the utilization of maltose and galactose. Vectors and promoters suitable for yeast expression are further described in EP 73,657. Yeast enhancers can also be advantageously used with yeast promoters. Antibody transcription via a vector in a mammalian host cell can be controlled by the following promoter 149799.doc-109-201106972: from a virus (such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2) Promoter of the genome of bovine papilloma virus, avian sarcoma virus, giant cell virus, retrovirus, hepatitis B virus, simian virus 40 (SV40); or a heterologous mammalian promoter (eg actin promoter) A sub- or immunoglobulin promoter; a heat shock promoter' is limited in that the promoter is compatible with the host cell system. The early and late promoters of the SV40 virus are preferably obtained as SV40 restriction fragments that also contain the SV4 prion replication origin. The immediate promoter of the human cell giant virus should be obtained as a restriction fragment of Hindlll E. 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. A modification of this system is described in U.S. Patent No. 4,6,1,978. For the performance of human beta interferon cDNA in mouse cells under the control of the thymidine kinase promoter of the pure sore virus, see also

Reyes 等人，TVaiwre 297:598-601 (1982)。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。 C)強化子元件組分 尚等真核細胞轉錄編碼本發明抗體之dNA通常藉由將強 化子序列***載體中來增強。現已知多種源自哺乳動物基 因（球蛋白、彈性蛋白酶、白蛋白、心胎蛋白及胰島素）的 強化子序列。然而，通常使用真核細胞病毒之強化子。實 例包括複製起點（bp 100-270)後側之SV4〇強化子、細胞巨 大病毒早期啟動子強化子、複製起點後側之多形瘤病毒強 化子及腺病毒強化子。關於用於活化真核啟動子之增強元 149799.doc -110- 201106972 件’亦可參見Yanjv, 297:17-18 (1982)。強化子可煎 接至載體中位於抗體編碼序列之5,或3,位置，但較佳位於 啟動子之5’位點。 f) 轉錄終止組分Reyes et al., TVaiwre 297: 598-601 (1982). Alternatively, the long terminal repeat of Rous Sarcoma Virus can be used as a promoter. C) Enhancer element components The dNA of the eukaryotic cell transcription encoding the antibody of the invention is typically enhanced by insertion of the enhancer sequence into the vector. A variety of enhancer sequences derived from mammalian genes (globin, elastase, albumin, heart protein, and insulin) are known. However, enhancers of eukaryotic cell viruses are commonly used. Examples include the SV4〇 enhancer on the back of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer on the posterior side of the replication origin, and the adenovirus enhancer. Regarding the enhancement element used to activate the eukaryotic promoter 149799.doc -110- 201106972 piece 'see also Yanjv, 297: 17-18 (1982). The enhancer can be decimated into the vector at position 5, or 3, of the antibody coding sequence, but is preferably located at the 5' position of the promoter. f) transcription termination component

L 在真核宿主細胞（酵母、真菌、昆蟲、植物、動物 '人 類或來自其他多細胞生物體之有核細胞）中使用之表現載 體亦含有終止轉錄及穩定mRNA所需的序列。該等序列通 q 常可自真核細胞或病毒DNA或cDNA之5'且有時3'非轉譯區 獲得。此等區域含有在編碼抗體之mRNA之非轉譯部分中 以聚腺苷酸化片段形式轉錄的核苷酸區段。一種適用之轉 錄終止組分為牛生長激素多聚腺苷酸化區。參見WO 94/11 026及其中所揭示之表現載體。 g) 選擇及轉型宿主細胞 適用於在本文中之載體中選殖或表現DNA之宿主細胞為 上述原核細胞、酵母或高等真核細胞。適於此目的之原核 〇 細胞包括真細菌’諸如革蘭氏陰性或革蘭氏陽性生物體， 例如腸内囷科（Enterobacteriaceae)，諸如埃希氏菌屬 (仏⑷（例如大腸桿菌）、腸内菌屬（五加、 •歐文氏菌屬CErm’m’a)、克雷伯氏菌屬、變形桿 .菌屬（iVo/ewi)、沙門氏菌屬（化/w〇„e//a)(例如鼠傷寒沙門 氏菌//α ί少则”·謂））、沙雷氏菌屬（心"加⑷（例 如黏/沙雷氏菌（《Serra/ia marcescaws))及志贺氏菌屬 (以如/⑷以及芽孢桿菌屬⑺⑽·叫（諸如枯草芽孢桿菌 μ⑽出）及地衣芽孢桿菌例如1989年4月 149799.doc -111 - 201106972 12日公開之DD 266,710中揭示之地衣芽孢桿菌41p))、假單 胞菌屬諸如綠膿假單胞菌（p "細㈣》 及鏈黴菌屬（汾repiomKw)。儘管諸如大腸桿菌B、大腸桿 菌 X1776(ATCC 31，537)及大腸桿菌 W3110(ATCC 27,325)之 其他菌株亦適合，但一較佳大腸桿菌選殖宿主為大腸桿菌 294(ATCC 3 1，446)。此等實例具說明性而非限制性。 可在細議中產生全長抗體、抗體融合蛋白及抗體片段， 尤其當不需要糖基化及Fc效應功能時，諸如當治療性抗體 與自身顯示有效破壞腫瘤細胞之細胞毒性劑（例如毒素）結 合時。全長抗體在循環中具有較長半衰期。大腸桿菌之生 產更快且更具成本效率。關於抗體片段及多肽在細菌中之 表現，參見例如 U.S. 5,648,237(Carter 等人）、u.s. 5,789,199(Joly# A) ' U.S. 5,840,523(Simmons # A) > 述轉譯起始區（丁IR)及用於優化表現及分泌之信號序列。 亦參 lChar\ton，Methods in Molecular Biology，第248卷 (B.K.C. Lo編，Humana press, Totowa，NJ，2〇〇3)，第 245_ 254頁，其描述大腸桿菌中抗體片段之表現。表現之後， 抗體可自可溶部分中之大腸桿菌細胞糊狀物分離且可視同 型而疋經由例如蛋白A或G管柱加以純化。最終純化可類 似於純化例如CH〇細胞中所表現之抗體的方法進行。 除原核生物外，諸如絲狀真菌或酵母之真核細胞微生物 為適用於編碼抗體之載體的選殖或表現宿主。在低等真核 細胞佰主微生物中最常使用釀酒酵母（Saccharomyces CereV】-Siae)或普通麵包酵母（baker’s yeast)。然而，大量其 149799.doc •112· 201106972L The expression vector used in eukaryotic host cells (yeast, fungi, insect, plant, animal 'human or nucleated cells from other multicellular organisms) also contains sequences required for termination of transcription and stabilization of mRNA. Such sequences are often obtained from 5' and sometimes 3' non-translated regions of eukaryotic cells or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One suitable transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11 026 and the expression vectors disclosed therein. g) Selection and transformation of host cells Host cells suitable for the selection or expression of DNA in the vectors herein are prokaryotic cells, yeast or higher eukaryotic cells as described above. Prokaryotic sputum cells suitable for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (仏(4) (eg E. coli), intestines) Endophyte (five plus, Erwinia cerevisiae CErm'm'a), Klebsiella, deformed rod, genus (iVo/ewi), Salmonella (chemical / w〇„e//a) (eg, Salmonella typhimurium / / α ί 则 ) ·)), Serratia (heart " plus (4) (such as Serra / ia marcescaws) and Shigella (Bacillus licheniformis 41p as disclosed in DD 266,710, as disclosed in DD 266, 710, as described in /(4) and Bacillus (7) (10), (such as Bacillus subtilis μ (10)) and Bacillus licheniformis, for example, April 149 799. doc -111 - 201106972 12) Pseudomonas such as Pseudomonas aeruginosa (p "fine (4)" and Streptomyces (汾repiomKw). Despite such as E. coli B, E. coli X1776 (ATCC 31, 537) and Escherichia coli W3110 ( Other strains of ATCC 27,325) are also suitable, but a preferred E. coli selection host is E. coli 294 (AT). CC 3 1,446). These examples are illustrative and not limiting. Full length antibodies, antibody fusion proteins and antibody fragments can be produced in detail, especially when glycosylation and Fc effect functions are not required, such as when Sexual antibodies bind to cytotoxic agents (such as toxins) that are themselves effective in destroying tumor cells. Full-length antibodies have a longer half-life in circulation. E. coli production is faster and more cost effective. About antibody fragments and peptides in bacteria For performance, see, for example, US 5,648,237 (Carter et al.), us 5,789,199 (Joly # A) 'US 5,840,523 (Simmons # A) > translation initiation region (DIR) and signal sequences for optimizing expression and secretion. Also refer to lChar\ton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana press, Totowa, NJ, 2〇〇3), pp. 245-254, which describes the performance of antibody fragments in E. coli. The antibody can be isolated from the E. coli cell paste in the soluble fraction and can be purified by, for example, protein A or G column. The final purification can be similar to purification such as CH〇. The method of expressing the antibody in the cell is carried out. In addition to the prokaryote, a eukaryotic cell microorganism such as a filamentous fungus or yeast is a colonization or expression host suitable for use in a vector encoding the antibody. Saccharomyces cerevisiae (Saccharomyces CereV)-Siae or baker's yeast is most commonly used. However, a large number of its 149799.doc •112· 201106972

他屬、種及菌株通常可利用並適用於本文，諸如裂瘦酵母 (Schizosaccharomyces尸〇所办e);克魯維酵母宿主，諸如乳 酸克魯維酵母、脆壁克魯維酵母（尺./ragz7b)(ATCC 12,424)、保加利亞克魯維酵母（尤 16,045)、威克克魯維酵母（ii：_w/cA：eram//)(ATCC 24,178)、 瓦提克魯維酵母（尺.而/i/〇(ATCC 56,500)、果蠅克魯維酵 母菌（AT.i/rc^op/n'/arwwXATCC 3 6,906)、耐熱克魯維酵母菌 ([ 及馬克斯克魯維酵母（足，所;解 脂耶氏酵母（}arro\Wa)(EP 402,226);甲醇酵母（P/c/^a paW〇rh)(EP 183,070);假絲酵母半知菌木黴 {Trichoderma reesia)(E? 244,234);粗厚神經胞子菌 (iVewroipora crassa);許旺酵母屬（Sc/iwawm’ow少ces)，諸如 西方許旺酵母（《Sc/zwawm’o/wyce·? occWwia/z··?);及纖維狀真 菌’諸如脈孢菌屬（iVewroaora)、青黴菌屬（户_以7//_)、 彎^員徽（Tb/ypoc/a山'Mm)及曲黴菌宿主，諸如溝 〇 巢麯黴及黑麵黴（Am’ger)。關於討論酵母及絲 狀真菌用於製備治療性蛋白質之用途的評述’參見例如His genus, species, and strains are generally available and suitable for use herein, such as Schizosaccharomyces (e.), Kluyveromyces hosts, such as Kluyveromyces lactis, Kluyveromyces cerevisiae. Ragz7b) (ATCC 12,424), Kluyveromyces cerevisiae (especially 16,045), Kluyveromyces cerevisiae (ii:_w/cA:eram//) (ATCC 24,178), Kluyveromyces cerevisiae (foot. i/〇 (ATCC 56,500), Kluyveromyces cerevisiae (AT.i/rc^op/n'/arwwXATCC 3 6,906), Kluyveromyces cerevisiae ([ and Markus rotissus Yarrowia lipolytica (}arro\Wa) (EP 402, 226); methanol yeast (P/c/^a paW〇rh) (EP 183,070); Candida subsp. trichoderma {Trichoderma reesia) (E? 244,234); iVewroipora crassa; Schwannella (Sc/iwawm'ow less ces), such as the western Schwann yeast ("Sc/zwawm'o/wyce·? occWwia/z··?) And fibrous fungi such as iVewroaora, Penicillium (household 7//_), bender emblem (Tb/ypoc/a mountain 'Mm) and Aspergillus host, such as gully Aspergillus niger and Ampere (Am'ger). Review of yeast and filamentous fungi discussed for the preparation of therapeutic protein & apos see, e.g.

Gerngross，ΛΓα/· ^oiec/z. 22:1409-1414 (2004)。 可選擇糖基化路徑已「人類化」的某些真菌及酵母菌 株從而產生具有部分或完全人類糖基化模式之抗體。參 見例如Li等人，#加·价24..210-215 (2006)(描述甲醇 酵母中糖基化路徑之人類化）；及Gerngross等人（同上）。 適用於表現糖基化抗體之宿主細胞亦可源自多細胞生物 體（無脊椎動物及脊椎動物）。無脊椎動物細胞之實例包括 149799.doc -113- 201106972 植物及昆蟲細胞。已鑑別多種桿狀病毒株及變異體以及來 自諸如以下宿主的相應許可性昆蟲宿主細胞厂^地黏蟲 (Spodoptera frugiperda)(毛A) '埃及伊故以以以狀訂沖) (蚊子）、白紋伊蚊（Ua ⑽）（蚊子）、黑腹果繩 me/w叹似如）（果蠅）及家蠶（5〇_只_^·)。用 於轉染之多種病毒株公開可得’例如苜卷丫紋夜蛾 〇4wiograp/7a ca/(/bmka)NPV 之 L-1 變異體及家蠶 Npv 之Gerngross, ΛΓα/· ^oiec/z. 22:1409-1414 (2004). Certain fungal and yeast strains whose glycosylation pathway has been "humanized" can be selected to produce antibodies with partial or complete human glycosylation patterns. See, for example, Li et al., #加价24.210-215 (2006) (describes the humanization of the glycosylation pathway in methanol yeast); and Gerngross et al. (supra). Host cells suitable for use in the expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include 149799.doc-113-201106972 plant and insect cells. A variety of baculovirus strains and variants have been identified as well as corresponding licensed insect host cell plants (Spodoptera frugiperda) (Mao A) from the following host, 'Egyptian I used to make a shape (mosquito), Aedes albopictus (Ua (10)) (mosquito), black bellied fruit me/w sigh like) (Drosophila) and silkworm (5〇_only _^·). A variety of strains for transfection are publicly available, such as the L-1 variant of the 4wiograp/7a ca/(/bmka)NPV and the silkworm Npv.

Bm-5病毒株，且該等病毒可根據本發明用作本文中之病 毒，尤其用於轉染草地黏蟲細胞。 棉花、玉米、馬鈴薯、大豆、矮牵牛、番茄、浮萍（浮 萍科、紫苜蓿（截形苜蓿⑶/β))及菸 草之植物細胞培養物亦可用作宿主。參見例如美國專利第 5,959，177 5虎、弟 6,040,498 號、第 6,420,548 號、第 7，125,978號及第6,417,429號（描述在轉殖基因植物中產生 抗體之 PLANTIBODIEStm 技術）。 脊椎動物細胞可用作宿主’且在培養物（組織培養物）中 增殖脊椎動物細胞已成為常規程序。適用哺乳動物宿主細 胞株之實例為經SV40轉型之猴腎CV1細胞株（COS-7， ATCC CRL 1651);人類胚腎細胞株（293細胞或經次選殖以 便在懸浮培養物中生長之293細胞，Graham等人，*/. Gen Wro/· 36:59 (1977);幼倉鼠腎細胞（BHK，ATCC CCL 10);小鼠足細胞（TM4，Mather,价〇/.及叩⑺汰23:243-251 (1980));猴腎細胞（CV1 ATCC CCL 70);非洲綠猴腎細胞 (VERO-76，ATCC CRL-1587);人類子宮頸癌細胞（HELA ’ 149799.doc -114· 201106972 ATCC CCL 2);犬腎細胞（MDCK，ATCC CCL 34);水牛鼠 肝細胞（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，ATCC CCL51) ; TRI 細胞（Mather 等 A » Annals N.Y. Acad. Sci. 383:44-68 (1982)) ; MRC 5多田 胞；FS4細胞；及人類肝腫瘤細胞株（Hep G2)。其他適用 哺乳動物宿主細胞株包括中國倉鼠卵巢（CHO)細胞，包括 DHFR_ CHO 細胞（Urlaub等人，Proc. iVai/. Jcai 5cz·· t/SJ 77:4216 (1980));及骨髓瘤細胞株，諸如NSO及Sp2/0。欲 回顧適用於產生抗體之某些哺乳動物宿主細胞株，參見例 如 Yazaki 反 Methods in Molecular Biology，第 248 卷 (B.K.C. Lo編 ’ Humana Press, Totowa, NJ，2003)，第 255-268 頁。 宿主細胞用產生抗體之上述表現或選殖載體轉型，且在 適當時經修改的習知營養培養基中培養以便誘導啟動子、 Q 選擇轉化子或擴增編碼所需序列之基因。 h)培養宿主細胞 用於產生本發明抗體之宿主細胞可於多種培養基中培 -養。市購培養基（諸如Ham氏FlO(Sigma)、最低必需培養基 • (MEM)(Sigma)、RPMI-1640(Sigma)及杜貝科氏修飾之伊格 氏培養基（Dulbecco’s Modified Eagle's Medium ; DMEM)(Sigma))適用於培養宿主細胞。此外，Ham等人， Mei/z. £«ζ· 58:44 (1979) ; Barnes 等人， 102:255 (1980);美國專利第 4,767,704 號、第 4,657,866 149799.doc • 115- 201106972 號、第 4,927,762號、第 4,560,655號或第 5,122,469號；WO 90/03430，WO 87/00195 ;或美國專利參考案3〇,985中所 述之任何培養基可用作宿主細胞之培養基。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、躬、鎂及填 酸鹽）、緩衝液（諸如HEPES)、核苷酸（諸如腺苷及胸苦）、 抗生素（諸如GENTAMYCIN™藥物）、微量元素（定義為無 機化合物，通常以在微莫耳濃度範圍内之最終濃度存在） 及葡萄糖或等效能源。亦可包括熟習此項技術者已知之適 當濃度的任何其他必需補充劑。培養條件（諸如溫度、pH 值及其類似條件）為前述選擇用於表現之宿主細胞所用的 條件，且對一般技術者顯而易知。 i)抗體純化 使用重組技術時，抗體可在細胞内、周質間隙中產生， 或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，藉由例如離心或超濾移除微粒碎片（宿主細胞或 已溶解片段）。Carter 等人，价1〇:163_167 (1992)描述將分泌至大腸桿菌之周質間隙中之抗體分離的 程序。簡言之，在乙酸鈉（pH 3.5)、EDTA及苯基甲基磺醯 氟（PMSF)存在下，經約30分鐘將細胞糊狀物解凍。細胞碎 片可利用離心移除。在抗體分泌至培養基中之情形下，通 帛首先使用市售蛋白濃縮過濾器（例如Amic〇n或Miiiip0re Pellicon超濾裝置）濃縮此等表現系統之上清液。任何前述 步驟中可包括諸如PMSF之蛋白酶抑制劑以抑制蛋白水 149799.doc •116· 201106972 解’且可包括抗生素以防止外來污染物生長。 由細胞製備之抗體組合物可使用例如羥磷灰石層析、疏 水相互作用層析、凝膠電泳、透析及親和性層析來純化， 親和性層析為通常較佳之純化步驟之一。蛋白人作為親和 性配位體之適用性視存在於抗體中之任何免疫球蛋白&域 的種類及同型而定。蛋白A可用於純化基於人類γ1、丫2或 γ4重鏈的抗體（Lindmark等人，/· /所所⑽从似62m 0 (1983))。蛋白G推薦用於所有小鼠同型及人類Y3(Guss等 人，五5:15671575 (1986))。雖然親和性配位體所連 接之基質最常為瓊脂糖，但可利用其他基質。與用瓊脂糖 可達成之流速及處理時間相比，機械穩定性基質（諸如可 控孔隙玻璃或聚（苯乙烯二乙稀基）苯）允許更快之流速及更 短之處理時間。當抗體包含Ch3域時，可使用BakeAond ΑΒΧ™樹脂α τ歸％ phi出psburg，Νί)純化。視欲回收 ^抗體而定’亦可利用純化蛋白質之其他技術，諸如離子 〇 交換柱分離法、乙醇沈澱法、逆相HPLC、二氧化矽層 析、肝素SEPHAR0SETM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 酸銨沈澱。 繼任何初步純化步驟之後，可使用阳值為約2 5_4 5之間 的溶離緩衝液使包含所關注之抗體及污染物之混合物進行 低阳疏水性相互作用層析，較佳在低鹽濃度（例如約0-0.25 Μ鹽）下進行。 通常’用於製備供研究、測試及臨床使用之抗體之各種 149799.doc •117- 201106972 方法在此項技術中已充分確立，與上述方法一致且/或被 熟習此項技術者視為適用於所關注之特定抗體。 c.免疫結合物 本發明亦提供包含與一或多種細胞毒性劑結合之抗體的 免疫結合物（可互換地稱為「抗體_藥物結合物」或 「ADC」），細胞毒性劑諸如化學治療劑、藥物、生長抑 制劑、毒素（例如蛋白質毒素，細菌、真菌、植物或動物 來源之酶促活性毒素，或其片段）或放射性同位素（亦即放 射性結合物）。 免疫結合物已在癌症治療中用於細胞毒性劑（亦即殺死 細胞或抑制細胞生長或增殖之藥物）之局部傳遞（Lambert， J_ (2005) 灯 5:543-549 ; Wu等 人，（2005) TVaiwre 少 23(9):1 137-1 146 ; Payne， G. (2003) i 3:207-212 ; Syrigos 及 Epenetos (1999) Anticancer Research 19:605-614 ; Niculescu-Duvaz 及 Springer (1997) Ji/v. De/ίν. 26:151-172 ;美國專 利第4,975，278號）。免疫結合物允許藥物部分乾向傳遞至 腫瘤且在其中進行細胞内積聚，其中未結合藥物之全身投 與會對正常細胞以及試圖消除之腫瘤細胞造成不可接受之 毒性程度（Baldwin等人，(1986年3月15日）第603-05 頁；Thorpe (1985)「Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review，」於 M〇f2〇c/〇«<3/Bm-5 strains, and such viruses can be used according to the invention as viruses herein, especially for transfecting grass worm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Apricot, Aster (Cut (3)/β)) and tobacco can also be used as hosts. See, for example, U.S. Patent No. 5,959,177, 5, vol. 6, 040, 498, 6, 420, 548, 7,125, 978, and 6, 417, 429 (the PLANTIBODIEStm technique for the production of antibodies in transgenic plants). It is a routine procedure for vertebrate cells to be used as a host' and to proliferate vertebrate cells in cultures (tissue cultures). Examples of suitable mammalian host cell strains are 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 for growth in suspension culture) Cells, Graham et al, */. Gen Wro/· 36:59 (1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse podocytes (TM4, Mather, valence/. and 叩 (7) :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 '149799.doc -114· 201106972 ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065) Mouse breast tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al. A » Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 multi-field; FS4 cells; and human liver tumor cell lines (Hep G2). Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR_CHO cells (Urlaub et al., Proc. iVai/. Jcai 5). Cz·· t/SJ 77:4216 (1980)); and myeloma cell lines, such as NSO and Sp2/0. To review certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki Anti Methods in Molecular Biology , vol. 248 (BKC Lo, 'Humana Press, Totowa, NJ, 2003), pp. 255-268. Host cells are transformed with the above-described expression or selection vectors that produce antibodies, and modified nutrient media, as appropriate The medium is cultured to induce a promoter, a Q-selective transformant, or a gene encoding a desired sequence. h) Culture of Host Cells Host cells for producing the antibodies of the present invention can be cultured in a variety of media. Commercially available media (such as Ham's FlO (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) )) Suitable for culturing host cells. In addition, Ham et al., Mei/z. £«ζ·58:44 (1979); Barnes et al., 102:255 (1980); US Patent Nos. 4,767,704, 4,657,866 149799.doc • 115-201106972, Any medium described in U.S. Patent Nos. 4,927,762, 4,560, 655, or 5,122, 469; WO 90/03430, WO 87/00195; or U.S. Patent No. 3,985, may be used as a medium for host cells. 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, barium, magnesium and sulphate), buffers (such as HEPES). , nucleotides (such as adenosine and chest pain), antibiotics (such as GENTAMYCINTM drugs), trace elements (defined as inorganic compounds, usually present in the final concentration in the range of micromolar concentrations) and glucose or equivalent energy. It may also include any other necessary supplements of the appropriate concentration known to those skilled in the art. The culture conditions (such as temperature, pH, and the like) are the conditions for the aforementioned selection of host cells for expression, and are apparent to those of ordinary skill. i) Antibody Purification When recombinant techniques are used, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, as a first step, microparticle fragments (host cells or solubilized fragments) are removed by, for example, centrifugation or ultrafiltration. Carter et al., Price: 163_167 (1992) describe procedures for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonamide fluoride (PMSF) over about 30 minutes. Cell debris can be removed by centrifugation. In the case where the antibody is secreted into the medium, the supernatant of these performance systems is first concentrated using a commercially available protein concentration filter (e.g., Amic〇n or Miiiip0re Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit protein water 149799.doc • 116· 201106972 solution and may include antibiotics to prevent the growth of foreign contaminants. The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography. Affinity chromatography is one of the generally preferred purification steps. The suitability of a protein human as an affinity ligand depends on the type and isotype of any immunoglobulin & domain present in the antibody. Protein A can be used to purify antibodies based on the human γ1, 丫2 or γ4 heavy chain (Lindmark et al., /10/10) from 62m 0 (1983). Protein G is recommended for all mouse isotypes and human Y3 (Guss et al., 5:15671575 (1986)). While the matrix to which the affinity ligand is attached is most often agarose, other matrices may be utilized. Mechanically stable matrices (such as controlled pore glass or poly(styrenediethyl)benzene) allow for faster flow rates and shorter processing times than can be achieved with agarose achievable flow rates and processing times. When the antibody comprises a Ch3 domain, it can be purified using BakeAond(R)TM resin alpha τ, % phi, psburg, 。ί). Other techniques for purifying proteins, such as ion exchange column separation, ethanol precipitation, reverse phase HPLC, ceria chromatography, heparin SEPHAR0SETM chromatography, anion or cation exchange resins (also known as recovery of antibodies) For example, polyaspartic acid column chromatography, chromatofocusing, sds_page and ammonium sulfate precipitation. Following any preliminary purification step, a mixture comprising the antibody of interest and a contaminant can be subjected to a low-yang hydrophobic interaction chromatography, preferably at a low salt concentration, using a dissolution buffer having a positive value between about 25 and 45. For example, about 0-0.25 Μ salt). Often 'various' for the preparation of antibodies for research, testing and clinical use. 149799.doc • 117-201106972 Methods are well established in the art and are consistent with the above methods and/or are known to those skilled in the art. Specific antibodies of interest. c. Immunoconjugates The invention also provides immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising antibodies that bind to one or more cytotoxic agents, cytotoxic agents such as chemotherapeutic agents , drugs, growth inhibitors, toxins (eg protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioisotopes (ie, radioconjugates). Immunoconjugates have been used in the treatment of cancer for the local delivery of cytotoxic agents (ie, drugs that kill cells or inhibit cell growth or proliferation) (Lambert, J_ (2005) Lamp 5: 543-549; Wu et al., ( 2005) TVaiwre 23 (9): 1 137-1 146; Payne, G. (2003) i 3: 207-212; Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; Niculescu-Duvaz and Springer (1997) Ji/v. De/ίν. 26:151-172; U.S. Patent No. 4,975,278). The immunoconjugate allows for partial delivery of the drug to the tumor and intracellular accumulation therein, wherein systemic administration of unbound drug causes unacceptable levels of toxicity to normal cells and tumor cells that are attempted to be eliminated (Baldwin et al., 1986 March 15th, pp. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," at M〇f2〇c/〇«<3/

AntibodiQS '84: Biological And Clinical Applications (A. Pinchera等人編）第475-5 06頁）。已報導多株抗體與單株抗 149799.doc -118- 201106972 體均適用於此專录略中（Rowland等人，（1986) Cczwcer iwmwwo/. /mmMwoi/zer. 21:1 83-87)。該等方法中使用的藥物 包括道諾黴素、阿黴素、曱胺喋呤及長春地辛（Rowland等 人，（1986)同上）。用於抗體-毒素結合物之毒素包括諸如 白喉毒素之細菌毒素；諸如萬麻毒蛋白之植物毒素；小分 子毒素，諸如格爾德黴素（geldanamycin)(Mandler等人， (2000) J. Cancer 92(19):1573-1581 ; Mandler等 人 ’ (2000) S/oorg'am.c c& Chem. Letters 10:1025- 1028 ; Mandler等人，（2002) C/zew. 13:786- 791)，類美登素（EP 1391213 ; Liu等人，（1996) iVoc.AntibodiQS '84: Biological And Clinical Applications (A. Pinchera et al., pp. 475-5 06). It has been reported that multiple antibodies and monoclonal antibodies 149799.doc-118-201106972 are suitable for use in this catalogue (Rowland et al., (1986) Cczwcer iwmwwo/. /mmMwoi/zer. 21:1 83-87). The drugs used in these methods include daunorubicin, doxorubicin, amidoxime and vindesine (Rowland et al., (1986) supra). Toxins for antibody-toxin conjugates include bacterial toxins such as diphtheria toxin; phytotoxins such as marigold; small molecule toxins such as geldanamycin (Mandler et al., (2000) J. Cancer 92(19): 1573-1581; Mandler et al.' (2000) S/oorg'am.c c& Chem. Letters 10:1025-1028; Mandler et al., (2002) C/zew. 13:786-791 ), class maytansin (EP 1391213; Liu et al., (1996) iVoc.

Jcod 5W. i/iSvi 93:8618_8623)及加里刹徽素（calicheamicin) (Lode等人，（1998) Cancer Λα. 58:2928 ; Hinman等人， (1993) Cancer Λα. 53:3336-3342)。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來發揮其細 胞毒性作用。有些細胞毒性藥物當與大抗體或蛋白質受體 配位體結合時易失活或活性降低。 ZEVALIN®(替伊莫單抗替烏克坦（ibritumomab tiuxetan)， Biogen/Idec)為抗體-放射性同位素結合物，其由藉由硫脲 連接子-螯合劑相結合之抗CD20抗原（發現於正常及惡性B 淋巴細胞之表面上）之鼠類IgGlK單株抗體與114η或9GY放射 性同位素組成（Wiseman 等人（2000)五wr. «/owr. 27 (7):766-77; Wisemanf A(2002) Blood 99 (12) :4336-42; Witzig等人（2002) J. Owco/· 20 (10) :2453-63; Witzig 等人（2002) J. C/M. 0«co/. 20 (15) :3262-69)。儘管 149799.doc -119- 201106972 ZEVALIN具有抗B細胞非霍奇金氏淋巴瘤（NHL)的活性， 但投藥在大部分患者中引起嚴重及長期性血細胞減少症。 MYLOTARG™(吉妥珠單抗奥唑米星（gemtuzumab ozogamicin)，Wyeth Pharmaceuticals)(— 種由相連接之 huCD33抗體與刺孢黴素組成的抗體藥物結合物）於2000年 獲准用於注射治療急性骨髓性白血病（Drwg·? 〇/ ί/ze Fwiwre (2000) 25 (7):686 ;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）。康圖單抗美坦 辛（Cantuzumab mert an sine) (Immunogen, Inc.)(—種由經由 二硫化物連接子SPP相連接之huC242抗體與類美登素藥物 部分DM 1構成的抗體-藥物結合物）正進入治療表現CanAg 之癌症的II期試驗，諸如結腸癌、胰臟癌、胃癌及其他癌 症。MLN-2704(Millennium Pharm., BZL Biologies, Immunogen Inc.)(—種由相連接之抗***特異性膜抗原 (PSMA)單株抗體與類美登素藥物部分DM1構成的抗體-藥 物結合物）正開發用於***腫瘤之潛在性治療。奥利斯 坦叮（auristatin)肽、奥利斯坦叮E(AE)及單曱基奥利斯坦 叮（monomethylauristatin ; MMAE)(海兔毒素之合成類似 物）與後合單株抗體cBR96(對癌科Lewis Y具有特異性）及 cACIO(對惡性血液病CD30具有特異性）結合（D〇r〇nina等人 (2003) Nature Biotechnology 21 (7):778-784)且正在治療性 開發中。 在某些實施例中’免疫結合物包含抗體及化學治療劑或 149799.doc •120- 201106972 其他毒素。本文中（例如上文）描述了適用於產生免疫結合 物之化學治療劑。可使用的酶促活性毒素及其片段包括： 白喉A鏈、白喉毒素之非結合活性片段、外毒素A鏈（來自 綠膿假單胞菌）、篦麻毒素A鏈、相思豆毒素A鏈、蒴蓮根 毒蛋白A鏈、α-帚麴菌素（alpha-sarcin)、油掏（Aleurites fordii)蛋白質、康乃馨蛋白質、美洲商陸（Phyt〇laca americana)蛋白質（PAPI、PAPII 及 PAP-S)、苦瓜（momordicaJcod 5W. i/iSvi 93:8618_8623) and calicheamicin (Lode et al., (1998) Cancer Λα. 58:2928; Hinman et al., (1993) Cancer Λα. 53:3336-3342). Toxins can exert their cytotoxic effects by a mechanism including tubulin binding, DNA binding or topoisomerase inhibition. Some cytotoxic drugs are susceptible to inactivation or decreased activity when combined with large antibody or protein receptor ligands. ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate consisting of an anti-CD20 antigen bound by a thiourea linker-chelator (found in normal And murine IgGlK monoclonal antibodies on the surface of malignant B lymphocytes and 114η or 9GY radioisotope composition (Wiseman et al. (2000) V. wr. «/owr. 27 (7): 766-77; Wisemanf A (2002 ) Blood 99 (12): 4336-42; Witzig et al. (2002) J. Owco/. 20 (10): 2453-63; Witzig et al. (2002) J. C/M. 0 «co/. 20 ( 15) : 3262-69). Although 149799.doc -119- 201106972 ZEVALIN has anti-B cell non-Hodgkin's lymphoma (NHL) activity, administration causes severe and long-term cytopenia in most patients. MYLOTARGTM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate consisting of a linked huCD33 antibody and calicheamicin, was approved for injection in 2000. Myeloid leukemia (Drwg·? 〇/ ί/ze Fwiwre (2000) 25 (7): 686; U.S. Patent Nos. 4,970,198, 5,079,033, 5,558,089, 5,560,040, 5,693,762, 5,739,116, No. 5767285, No. 5773001). Cantuzumab mert an sine (Immunogen, Inc.) (an antibody-drug combination consisting of a huC242 antibody linked via a disulfide linker SPP and a maytansinoid drug moiety DM 1 Phase II trials for the treatment of cancers that exhibit CanAg, such as colon cancer, pancreatic cancer, stomach cancer, and other cancers. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.) (an antibody-drug conjugate consisting of a linked anti-prostate specific membrane antigen (PSMA) monoclonal antibody and a maytansinoid drug moiety DM1) Potential treatment for prostate tumors is being developed. Auristatin peptide, Orlistin E (AE) and monomethylauristatin (MMAE) (synthetic analog of dolastatin) and post-monoclonal antibody cBR96 (for cancer) Lewis Y has specificity and cACIO (specific for hemorrhagic disease CD30) binding (D〇r〇nina et al. (2003) Nature Biotechnology 21 (7): 778-784) and is in therapeutic development. In certain embodiments, the immunoconjugate comprises an antibody and a chemotherapeutic agent or 149799.doc • 120- 201106972 other toxins. Chemotherapeutic agents suitable for use in the production of immunoconjugates are described herein (e.g., above). The enzymatically active toxins and fragments thereof which can be used include: diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin toxin A chain, Alfalfa root protein A chain, alpha-sarcin, Aleurites fordii protein, carnation protein, Phyt〇laca americana protein (PAPI, PAPII and PAP-S), Bitter gourd (momordica

charantia)抑制劑、麻瘋樹毒蛋白（curcin)、巴豆毒蛋白 (crotin)、肥皂草（sapaonarja 〇fficinaiis)抑制劑、白樹素 (gelonin)、有絲***素（mitogeiHn)、侷限麴菌素 (restrictocin)、紛徽素、伊諾黴素（en〇mycin)及單端抱黴烯 族毒素（tricothecene)。參見例如1993年1〇月28曰公開之 WO 93/21232。多種放射性核素可用於產生放射性結合之 抗體。實例包括212Bi、1311、mIn、川丫及⑻以。抗體與細 胞毒性劑之結合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N_ 丁二醯亞胺 醋（SPDP)、5胺基硫雜環戊帥τ)、_㈣之雙官能街 生物（諸如二亞胺代己二酸二甲醋鹽酸鹽）、活性醋（諸如辛 二酸二丁二醯亞胺醋）、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- ° 签—胺）、雙重氮鹽衍生物 (Γ如雙（對重氮鹽苯甲酿基）·乙二胺）、二異氛酸醋（諸如甲 本2,6_二異氮酸醋）及雙活性氟化合物（諸如1>5_二氟_24二 石肖基苯）。舉例而言，昆麻毒素免疫毒素可如抑伽等人， Science，238:1098 (1987)中所述力 149799.doc -121. 201106972 異硫氰基苯曱基-3-曱基二伸乙三胺五乙酸（ΜΧ-DTPA)為一 種用於使放射性核苷酸與抗體結合之例示性螯合劑。參見 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、單端孢黴稀族毒素 及CC1065以及此等毒素之具有毒素活性之衍生物）之彡士合 物0 1·美登素與類美登素 在一些實施例中，免疫結合物包含抗體（全長或片段）與 一或多種類美登素分子之結合物。 類美登素為藉由抑制微管蛋白聚合而發揮作用之有絲分 裂抑制劑。美登素最先係自東非灌木齒葉美登木 (1\^71611此561^13)分離（美國專利第3,896，111號）。隨後發 現某些微生物亦產生類美登素，諸如美登醇及C-3美登醇 酯（美國專利第4,15 1,042號）。合成的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。 類美登素藥物部分為抗體藥物結合物中引人注目之藥物 部分，此歸因於其：（i)藉由醱酵或化學修飾、醱酵產物衍 149799.doc -122- 201106972 金i製備相對各易；⑻易用適於經由非二硫化物連接子 、抗體結合之官能基衍生化；（m)在血衆中穩定；及（iv) 對多種腫瘤細胞株有效。 _ +#|胃美登素之免疫結合物、其製造方法及其治療用途 、揭不於例如美國專利第5,208,020號、第5,416,064號及歐洲 專利EP 〇 425 235 B1中，其揭示内容以引用的方式明確併 入本文中。Liu等人，Proc Natl Acad Sci USA 93:8618_ ο 8623 (1996)描述的免疫結合物包含相連接之類美登素(命 名為DM1)與針對人類結腸直腸癌之單株抗體c242。已發 現該結合物對所培養之結腸癌細胞具有高細胞毒性且在活 體内腫瘤生長分析法中顯示抗腫瘤活性。Chari等人， Cancer Research 52:127 131 (1992)描述免疫結合物其中 類美登素經由二硫化物連接子與結合至人類結腸癌細胞株 上之抗原的鼠類抗體八7結合’或與結合HER_2/neu致癌基 因之另種乳類單株抗體TA_ 1結合。已活體外測試τα. 1 _ 〇 類美登素結合物對人類乳癌細胞株SK-BR-3之細胞毒性， δ亥細胞株每個細胞表現3χ1〇5個HER_2表面抗原。藥物結合 物達成的細胞毒性度與游離類美登素藥物類似，細胞毒性Charantia) inhibitor, curcin, crotin, sapaonarja 〇fficinaiis inhibitor, gelonin, mitogen (mitogeiHn), restrictocin (restrictocin), Enzyme, en〇mycin and trichothecene. See, for example, WO 93/21232, published January 28, 1993. A variety of radionuclides are available for the production of radiolabeled antibodies. Examples include 212Bi, 1311, mIn, Chuanxiong, and (8). Combinations of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propionic acid N-butylidene imidate (SPDP). , 5-aminothiolane τ), _ (four) bifunctional street organisms (such as diimine adipate dimethyl vinegar hydrochloride), active vinegar (such as dibutyl succinate) , aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzylidene) hexyl-amine), double nitrogen derivatives (such as bis (p-diazonium) Benzoyl) ethylenediamine), diacetoic acid vinegar (such as a 2,6-diisoxamic acid vinegar) and a double active fluorine compound (such as 1>5_difluoro-24-distone). For example, a quintoxin immunotoxin can be as described in gamma et al., Science, 238: 1098 (1987). 149799.doc -121. 201106972 isothiocyanobenzoyl-3-mercaptodiethyl Triamine pentaacetic acid (ΜΧ-DTPA) is an exemplary chelating agent for binding radioactive nucleotides to antibodies. See WO 94/11026. Also included herein are antibodies and one or more small molecule toxins (such as echinomycin, maytansinoids, dolastatin, auristatin, trichothecene, and CC1065, and derivatives of these toxins)彡 彡 0 1 1 美 美 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 与 。 。 。 Maytansin is a mitotic inhibitor that acts by inhibiting tubulin polymerization. Maydensin was first isolated from the East African shrub tooth leaf Meidenmu (1\^71611 561^13) (US Patent No. 3,896,111). It was subsequently discovered that certain microorganisms also produced maytansinoids such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,15,042). 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 , 4, 309, 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 and In No. 4,371,533. The maytansinoid moiety is a striking drug moiety in an antibody drug conjugate, which is attributed to: (i) by fermentation or chemical modification, fermentation product derivative 149799.doc -122- 201106972 gold i preparation (8) Ease of use is suitable for derivatization via a non-disulfide linker, antibody-bound functional group; (m) stable in blood groups; and (iv) effective against a variety of tumor cell lines. _ +#|Immunoconjugates of sedatives, methods for their manufacture, and therapeutic uses thereof, as disclosed in, for example, U.S. Patent No. 5,208,020, U.S. Patent No. 5,416,064, The manner is explicitly incorporated herein. The immunoconjugate described by Liu et al., Proc Natl Acad Sci USA 93:8618_ ο 8623 (1996) comprises a conjugated maytansinoid (named DM1) and a monoclonal antibody c242 against human colorectal cancer. This conjugate has been found to be highly cytotoxic to colon cancer cells cultured and to exhibit antitumor activity in an in vivo tumor growth assay. Chari et al, Cancer Research 52: 127 131 (1992) describe immunoconjugates in which the maytansinoid binds to or binds to a murine antibody VIII that binds to an antigen on a human colon cancer cell line via a disulfide linker. Another species of HER_2/neu oncogene binds to the monoclonal antibody TA_1. The cytotoxicity of the τα. 1 _ 〇 meimeisin conjugate to the human breast cancer cell line SK-BR-3 has been tested in vitro, and the δ hai cell strain exhibits 3 χ 1 〇 5 HER 2 surface antigen per cell. The cytotoxicity achieved by the drug conjugate is similar to that of the free maytansinoid drug, cytotoxicity

I •度可藉由增加每個抗體分子之類美登素分子之數目來增 加Α7_類美登素結合物在小鼠體内顯示低全身性細胞毒 性。 抗體-類美登素結合物係藉由將抗體與類美登素分子化 學連接來製備而不顯著降低抗體或類美登素分子之生物學 活性》參見例如美國專利第5,2〇8,〇2〇號（其揭示内容以引 149799.doc •123- 201106972 用方式明確地併入本文中）。平均每個抗體分子結合3_4個 類美登素分子已顯示出增強標靶細胞之細胞毒性之功效而 對抗體功能或溶解性無不利影響，即使一個分子毒素/抗 體預計亦會增強細胞毒性而超過使用裸抗體。類美登素已 為此項技術中所熟知，且可藉由已知技術合成或自天然來 源分離。適當之類美登素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。較佳之類美 登素為美登醇及在美登素醇分子之芳環中或其他位置處經 修飾之美登醇類似物（諸如各種美登醇酯）。 此項技術中已知之用於製備抗體_類美登素結合物的多 種連接基團包括例如美國專利第5,2θ8,θ2θ號或歐洲專利〇 425 235 B1 ’ Chari 等人，Cancer Research 52:127. 131(1992)及美國專利申請案第1〇/96〇6〇2號（2〇〇4年1〇月8 曰申請）中所揭示的連接基團，其揭示内容以引用方式明 確併入本文中。可如2004年1〇月8曰申請之美國專利申請 案第10/960,602號中所揭示，製備包含連接組分SMCC之抗 體-類美登素結合物。如以上鑑別之專利中所揭示連接 基團包括二硫基、硫醚基、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，較佳為二 硫基及硫醚基。本文中描述及舉例說明其他連接基團。 杬體與類美登素之結合物可使用多種雙官能蛋白偶合劑 製得，該等蛋白偶合劑諸如3_(2_吡啶基二硫基）丙酸义丁 二醯亞胺酯（SPDP)、4-(Ν-順丁烯二醯亞胺基甲基）環己烷_ 1-甲酸丁二醯亞胺酯（SMCC)、亞胺基硫雜環戊烷（ΙΤ)、醯 149799.doc -124*. 201106972 亞胺酯之雙官能衍生物（諸如二亞胺代己二酸二甲酯鹽酸 鹽）、活性酯（諸如辛二酸二丁二醯亞胺酯）、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（諸如曱苯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）。特別較佳之偶合劑包 括提供二硫鍵之3-(2-吡啶基二硫基）丙酸N-丁二醯亞胺酯 (SPDP)(Carlsson等人，如173:723-737 (1978))及 4-(2-吡啶基硫基）戊酸N-丁二醯亞胺酯（SPP)。 視連接類型而定，連接子可連接於類美登素分子之各種 位置。舉例而言’可使用習知偶合技術、藉由與羥基反應 形成酯鍵。反應可發生於具有羥基之C-3位置、經經甲基 修飾之C-14位置、經羥基修飾之c-1 5位置及具有羥基之c_ 20位置處。在一較佳實施例中，鍵聯形成於美登醇或美登 醇類似物之C-3位置處。 2·奥利斯坦叮及海兔毒素 在一些實施例中’免疫結合物包含抗體與海兔毒素或海 兔毒素肽類似物及衍生物奥利斯坦叮之結合物（美國專利 第5635483號、第578〇588號）。海兔毒素及奥利斯坦叮已 顯示可干擾微管動力學、GTP水解及細胞核及細胞*** (Woyke等人 ’（2001) Antimicrob. Agents and Chemother. 45(12):358〇-3584)且具有抗癌（US 5663149)及抗真菌活性 (Pettit等人 ’（1998) Antimicrob· Agents Chemother. 42. 2961 -965)。海兔毒素或奥利斯坦叮藥物部分可經由肽藥物 149799.doc -125- 201106972 部分之N(胺基）末端或C(羧基）末端與抗體連接（WO 02/088172)。 例示性奥利斯坦叮實施例包括「Monomethyl valine Compounds Capable of Conjugation to Ligands」（2004年 11 月5日申請之美國專利第10/983,340號，其全部揭示内容以 引用方式明確併入本文中）中所揭示的N末端連接之單曱基 奧利斯坦叮藥物部分DE及DF。 基於肽之藥物部分通常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。該等肽鍵可根據例如 肽化學領域中熟知之液相合成方法（參見E. Schrader及K. Liibke，「The Peptides」，第 1 卷，第 76-136 頁，1965, Academic Press)製備。可根據以下文獻中之方法製備奥利 斯坦叮/海兔毒素藥物部分：US 5635483 ; US 5780588 ; Pettit等人，（1989) J. Am. Chem. Soc. 111:5463-5465 ; Pettit 等人（1998) Anti-Cancer Drug Design 13:243-277; Pettit, G.R.等人，Synthesis, 1996，719-725 ;及 Pettit 等 人，（1996) J. Chem. Soc· Perkin Trans. 1 5:859-863。亦參 見 Doronina (2003) Nat Biotechnol 21 (7):778-784 ； 「Monomethylvaline Compounds Capable of Conjugation to Ligands」，2004年11月5日申請之美國專利第10/983,340 號，其全文以引用方式併入本文中（揭示例如連接子及製 備與連接子結合之單甲基纈胺酸化合物（諸如MMAE及 MMAF)的方法）。 3.刺孢擻素 149799.doc •126- 201106972 在其他實施例中，免疫結合物包含抗體與一或多個刺孢 黴素分子之結合物。刺胞黴素家族之抗生素能夠在亞皮莫 耳濃度下產生雙股DNA斷裂。有關刺胞黴素家族結合物之 製備，參見美國專利第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)。可使用之刺孢黴素之結構 類似物包括（但不限於）γΐΙ、α2Ι、《31、N-乙醯基-γιι、 PSAG及 0Il(Hinman等人，Cancer Research 53:3336-3342 (1993) ; Lode等人 ’ Cancer Research 58:2925-2928 (1998); 及頒予American Cyanamid之上述美國專利）。可與抗體結 合之另一種抗腫瘤藥物為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。因此， 細胞經由抗體介導之内化作用攝取此等藥劑可大大增強其 細胞毒性作用。 4.其他細胞毒性劑 可與抗體結合之其他抗瘤劑包括BCNU、鏈脲佐菌素 (streptozoicin)、長春新鹼及5-氟尿嘧啶（該家族之藥劑統 稱為LL-E33288複合物，如美國專利5,〇53,394、5,770,710 中所述）以及艾斯帕米辛（eSperamicin)(美國專利 5,877,296) ° 可使用的if；促活性毒素及其片段包括白喉A鍵、白喉毒 素之非結合活性片段、外毒素A鏈（獲自綠膿假單胞菌）、 篦麻毒素A鏈、相思豆毒素a鍵、蒴蓮根毒素a鏈、α_帚麴 149799.doc -127· 201106972 菌素、油桐蛋白、康乃馨蛋白、美洲商陸蛋白(顧、 PAPII及PAp_S)、苦瓜抑制劑、麻瘋樹毒蛋白、巴豆毒蛋 白、肥皂草抑制劑、白樹素、有絲***素、偈限麵菌素、 酚黴素、伊諾黴素及單端孢黴烯族毒素。參見例如1993年 10月28日所公開之WO 93/21232。 本發明進一步涵蓋抗體與具有核分解活性之化合物（例 如核糖核酸酶或DNA内切酶，諸如去氧核糖核酸酶； DNase)之間所形成的免疫結合物。 為選擇性破壞腫瘤，抗體可包含高度放射性原子。可使 用多種放射性同位素產生放射性結合抗體。實例包括 At、I13 丨、I丨25、γ9。、Re186、Rei88、％153、出212、卩32、I degree can be increased by increasing the number of maytansine molecules per antibody molecule. The Α7_maytansin conjugate exhibits low systemic cytotoxicity in mice. An antibody-maytansin-binding system is prepared by chemically linking an antibody to a maytansinoid molecule without significantly reducing the biological activity of the antibody or maytansinoid molecule. See, for example, U.S. Patent No. 5,2,8, 〇 2 nickname (the disclosure of which is incorporated herein by reference in its entirety by reference 149799.doc. On average, each antibody molecule binds to 3_4 maytansinoid molecules and has been shown to enhance the cytotoxicity of target cells without adversely affecting antibody function or solubility, even though a molecular toxin/antibody is expected to enhance cytotoxicity beyond Use naked antibodies. Maytansine is well known in the art and can be synthesized by known techniques or isolated from natural sources. Appropriate, such as, for example, US Patent No. 5,2,8, 2, 2, and other patents and non-patent publications mentioned above. Preferably, medenoxine is maytansinol and a maytansinoid analog (such as various maytansinol) modified in the aromatic ring of the maytansinol molecule or elsewhere. A variety of linking groups known in the art for preparing antibody-maytansin conjugates include, for example, U.S. Patent No. 5, 2 theta 8, θ 2θ or European Patent 〇 425 235 B1 ' Chari et al., Cancer Research 52: 127 The linking group disclosed in 131 (1992) and U.S. Patent Application Serial No. 1/96/6, 2, filed on Jan. 4, 2011, the disclosure of which is expressly incorporated by reference. In this article. An antibody-maytansinoid conjugate comprising the linking component SMCC can be prepared as disclosed in U.S. Patent Application Serial No. 10/960,602, the entire disclosure of which is incorporated herein by reference. The linking group as disclosed in the above identified patent includes a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group or an esterase labile group. Preferred are disulfide groups and thioether groups. Other linking groups are described and exemplified herein. The combination of steroids and maytansinoids can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propanoic acid succinimide (SPDP), 4-(Ν-m-butylene iminomethyl)cyclohexane _ 1-carboxylic acid butyl sulfoxide (SMCC), iminothiolane (ΙΤ), 醯 149799.doc - 124*. 201106972 Bifunctional derivatives of imidates (such as diimine dimethyl adipate hydrochloride), active esters (such as dibutyl succinate), aldehydes (such as pentane) Dialdehyde), azide-based compound (such as bis(p-azidobenzylidene) hexamethylenediamine), double nitrogen salt derivative (such as bis(p-diazonium benzylidene)-ethylenediamine) , a 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-butanediimide (SPDP) which provides a disulfide bond (Carlsson et al., 173: 723-737 (1978)). And 4-(2-pyridylthio)pentanoic acid N-butanediimide (SPP). Depending on the type of linkage, the linker can be attached to various locations of the maytansinoid molecule. 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 methyl-modified C-14 position, a hydroxyl group-modified c-1 5 position, and a c_20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of the maytansinol or maytansinoid analog. 2. Orlistan and Sea Rabbit Toxins In some embodiments, the immunoconjugate comprises a combination of an antibody with a dolastatin or a dolastatin peptide analogue and a derivative, Orlistan (U.S. Patent No. 5,563, 482, 578〇588). The rabbit toxin and orlistan oxime have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 358〇-3584) and have Anticancer (US 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42. 2961-965). The dolastatin or orlistin drug moiety can be linked to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug 149799.doc-125-201106972 (WO 02/088172). Exemplary Olivier 叮 embodiments include "Monomethyl valine Compounds Capable of Conjugation to Ligands" (U.S. Patent No. 10/983,340, filed on Nov. 5, 2004, the entire disclosure of The disclosed N-terminally linked monothiol orlistan drug moiety DE and DF. Peptide-based drug moieties can generally be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schrader and K. Liibke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). The Olivier/Hypotoxin drug fraction can be prepared according to the methods in the following literature: US 5635483; US 5780588; Pettit et al., (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al. 1998) Anti-Cancer Drug Design 13: 243-277; Pettit, GR et al., Synthesis, 1996, 719-725; and Pettit et al., (1996) J. Chem. Soc· Perkin Trans. 1 5: 859-863 . See also Doronina (2003) Nat Biotechnol 21 (7): 778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Patent No. 10/983,340, filed on Nov. 5, 2004, which is incorporated by reference in its entirety Herein, methods for e.g., linkers and preparation of monomethylproline compounds (such as MMAE and MMAF) in combination with a linker are disclosed. 3. Leptosin 149799.doc • 126- 201106972 In other embodiments, the immunoconjugate comprises a combination of an antibody and one or more echinomycin molecules. Antibiotics from the echinomycin family are capable of producing double strand DNA breaks at sub-picol concentrations. For the preparation of echinomycin family conjugates, see U.S. Patent Nos. 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. To American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, γ ΐΙ, α 2 Ι, 31, N-ethinyl-γιι, PSAG, and 01 (Hinman et al, Cancer Research 53: 3336-3342 (1993) Lode et al. 'Cancer Research 58: 2925-2928 (1998); and the aforementioned US patent to American Cyanamid). Another anti-tumor drug that can be combined with an antibody is QFA, which is an antifolate. Both echinomycin and QFA have intracellular sites of action and are not susceptible to crossing the plasma membrane. Thus, uptake of such agents via antibody-mediated internalization greatly enhances their cytotoxic effects. 4. Other cytotoxic agents Other anti-neoplastic agents that can bind to antibodies include BCNU, streptozoicin, vincristine, and 5-fluorouracil (the family of drugs are collectively referred to as LL-E33288 complex, such as the US patent) 5, 〇53,394, 5,770,710) and eSperamicin (U.S. Patent 5,877,296) ° can be used; pro-active toxins and fragments thereof include diphtheria A bonds, non-binding active fragments of diphtheria toxin, Toxin A chain (obtained from Pseudomonas aeruginosa), ricin A chain, abrin toxin a bond, lotus root toxin a chain, α_帚麴149799.doc -127· 201106972 bacteriocin, tung oil protein, Carnation protein, Pokeweed protein (Gu, PAPII and PAp_S), Momordica melon, Jatropha, crotonin, saponin, leucovorin, mitogen, quercetin, phenolic acid, y Noromycin and trichothecenes. See, for example, WO 93/21232, published October 28, 1993. The invention further encompasses immunoconjugates formed between an antibody and a compound having nuclear cleavage activity, such as a ribonuclease or endonuclease, such as a deoxyribonuclease; DNase. To selectively destroy a tumor, the antibody can comprise highly radioactive atoms. Radioactively bound antibodies can be produced using a variety of radioisotopes. Examples include At, I13 丨, I 丨 25, γ9. , Re186, Rei88, %153, out 212, 卩32,

Pb212及Lu之放射性同位素。當使用結合物進行偵測時，其 可包含用於閃爍攝影研究之放射性原子，例如Tc"m或 I123 ;或用於核磁共振（NMR)成像（亦稱為磁共振成像， mri)之自旋標記’諸如峨_丨23及硪_丨3〗、銦-丨丨丨、氟_丨9、 碳-13、氮-1 5、氧-17、釓、猛或鐵。 可以已知方式將放射性標記或其他標記併入結合物中。 舉例而言，肽可生物合成，或可藉由化學胺基酸合成、使 用適當胺基酸前驅物（包括例如替代氫的氟_丨9)來合成。諸 如tc或I 、Rel86、Re188及In111之標記可經由肽中之半 胱胺酸殘基連接。釔-90可經由離胺酸殘基連接。可使用 IODOGEN 方法（Fraker 等人，（1978) Biochem. Biophys· Res. Commun· 80: 49_57)併入碘 _123。「Monoclonal Antibodies in Immunoscintigraphy」（Chatal, CRC Press 149799.doc -128· 201106972 1989)詳細描述了其他方法。 抗體與細胞毒性劑之結合物可使用多種雙官能蛋白偶合 劑製得，該等蛋白偶合劑為諸如3-(2-吡啶基二硫基）丙酸 N-丁二醯亞胺酯（SPDP)、4-(N-順丁烯二醯亞胺基甲基）環 己烷-1-甲酸丁二醯亞胺酯（SMCC)、亞胺基硫雜環戊烷 (IT)、醯亞胺酯之雙官能衍生物（諸如二亞胺代己二酸二曱 酯鹽酸鹽）、活性酯（諸如辛二酸二丁二醯亞胺酯）、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（諸如曱苯2,6-二異氰酸酯）及雙活性 氟化合物（諸如1,5 -二氟-2，4-二石肖基苯）。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-異硫氰基苯甲基-3-曱基二伸 乙三胺五乙酸（MX-DTPA)為一種用於使放射性核苷酸與抗 體結合之例示性螯合劑。參見W094/11026。連接子可為 有利於細胞毒性藥物釋放於細胞中之「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、二曱基連接子或含二硫鍵連接子 (Chari等人，52:127-131 (1992);美國專 利第 5,208,020號）。 該等化合物明確涵蓋（但不限於）與以下交聯試劑所製備 之 ADC : BMPS、EMCS、GMBS、HBVS、LC-SMCC、 MBS、ΜΡΒΗ、SBAP、SIA、SIAB、SMCC、SMPB、 SMPH、sulfo-EMCS、sulfo-GMBS、sulfo-KMUS、sulfo- 149799.doc -129- 201106972 MBS、sulfo-SIAB、sulfo-SMCC 及 sulfo-SMPB 及 SVSB((4- 乙烯基砜）苯甲酸丁二醯亞胺酯），該等交聯試劑可市構（例 如構自 Pierce Biotechnology, Inc., Rockford, IL., U.S.A)。 參見 2003-2004 Applications Handbook and Catalog第 467- 498 頁。 5.製備抗體藥物結合物 在抗體藥物結合物（ADC)中，抗體（Ab)經由連接子（l)與 一或多個藥物部分（D)(例如每個抗體約1至約2〇個藥物部 分）結合。式I之ADC可藉由若干途徑，使用熟習此項技術 者已知之有機化學反應、條件及試劑來製備，包括：（”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，接著與藥物部分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形成D_L，接著與 抗體之親核基團反應。本文描述用於製備ADC之其他方 法。Radioisotopes of Pb212 and Lu. When conjugates are used for detection, they may contain radioactive atoms for scintigraphy studies, such as Tc"m or I123; or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) spins. The marks 'such as 峨_丨23 and 硪_丨3〗, indium-bismuth, fluorine_丨9, carbon-13, nitrogen-1 5, oxygen-17, hydrazine, violent or iron. Radioactive labels or other labels can be incorporated into the conjugate in a known manner. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using a suitable amino acid precursor (including, for example, fluorine_丨9 instead of hydrogen). Labels such as tc or I, Rel86, Re188 and In111 can be linked via a cysteine residue in the peptide.钇-90 can be linked via an amine acid residue. Iodine _123 can be incorporated using the IODOGEN method (Fraker et al., (1978) Biochem. Biophys. Res. Commun. 80: 49_57). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 149799. doc-128. 201106972 1989). Combinations of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein couplers such as 3-(2-pyridyldithio)propionic acid N-butyl succinimide (SPDP). , 4-(N-m-butylene iminomethyl)cyclohexane-1-carboxylic acid butyl succinimide (SMCC), imidothiolane (IT), yttrium imidate Bifunctional derivatives (such as diimidodidecyl adipate hydrochloride), active esters (such as dibutyl succinate), aldehydes (such as glutaraldehyde), diazide Base compounds (such as bis(p-azidobenzylidene) hexamethylenediamine), double nitrogen salt derivatives (such as bis(p-diazonium benzylidene)-ethylenediamine), diisocyanates (such as toluene) 2,6-diisocyanate) and a double active fluorine compound (such as 1,5-difluoro-2,4-dichthyl benzene). For example, a ricin toxin immunotoxin can be prepared as described in Vitetta et al., 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-mercaptodiethylammonium triamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radioactive nucleotides to antibodies. See W094/11026. The linker can be a "cleavable linker" that facilitates the release of cytotoxic drugs into the cell. For example, an acid labile linker, a peptidase-sensitive linker, a photolabile linker, a dimercapto-linker or a disulfide-containing linker can be used (Chari et al., 52: 127-131 ( 1992); U.S. Patent No. 5,208,020). These compounds expressly cover, but are not limited to, ADCs prepared with the following crosslinking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, ΜΡΒΗ, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo- EMCS, sulfo-GMBS, sulfo-KMUS, sulfo- 149799.doc -129- 201106972 MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB ((4-vinyl sulfone) benzoic acid butyl imidate The cross-linking reagents are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA). See pages 467-498 of the 2003-2004 Applications Handbook and Catalog. 5. Preparation of an antibody drug conjugate In an antibody drug conjugate (ADC), the antibody (Ab) is linked to one or more drug moieties (D) via a linker (1) (eg, about 1 to about 2 drugs per antibody) Partial) combined. The ADC of Formula I can be prepared by a number of routes using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: ("reacting the nucleophilic group of the antibody with a bivalent linker reagent via covalent The bond forms Ab-L, which is then reacted with drug moiety D; and (2) reacts the nucleophilic group of the drug moiety with a divalent linker reagent to form D_L via a covalent bond, followed by reaction with the nucleophilic group of the antibody. Other methods for preparing ADCs are described herein.

Ab-(L-D)p j 連接子可由一或多種連接子組分構成。例示性連接子組 分包括6-順丁烯二醯亞胺基己醯基（「Mc」）、順丁烯二醯 亞胺基丙醯基（「MP」）、纈胺酸-瓜胺酸（「val-cit」）、丙 胺酸.***酸（「ala-phe」）、對胺基苯曱氧基羰基 (「PAB」）、4-(2-吡啶基硫基）戊酸N_ 丁二醯亞胺酯 (「SPP」）、4_(N_順丁烯二醯亞胺基甲基）環己烷小甲酸 N-丁 —醯亞胺酯（「SMCC」）及（4_碘-乙醯基）胺基苯甲酸 N-丁二醯亞胺酯（「SIAB」）。其他連接子組分已為此項技 149799.doc -130- 201106972 術中所知且有些描述於本文中。亦參見 「Monomethylvaline Compounds Capable of Conjugation to Ligands」，2004年11月5曰申請之美國專利第1〇/983,wo 號’其全部内谷以引用方式併入本文中。 在一些實施例中，連接子可包含胺基酸殘基。例示性胺 基酸連接子組分包括二肽、三肽、四肽或五肽。例示性二 肽包括纈胺酸-瓜胺酸（vc或val-cit)、丙胺酸-***酸（af Q 或ala-Phe)。例示性三肽包括甘胺酸-纈胺酸-瓜胺酸（gly-Val_cit)及甘te酸-甘胺酸-甘胺酸（gly_gly_gly)。包含胺基 酸連接子組分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 對胺基酸連接子組分進行設計且優化以對特定酵素（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 酶促裂解具有選擇性。 抗體上之親核基團包括（但不限於）：⑴N末端胺基；（Η) 〇 側鍵胺基’例如離胺酸；（in)側鏈硫醇基，例如半胱胺 酸；及（iv)抗體經糖基化之糖羥基或胺基。胺基、硫醇基 及羥基呈親核性且能夠與連接子部分及連接子試劑上之親 •電子基團（包括：（i)活性酯，諸如NHS酯、HOBt酯、鹵甲 :酸醋及酸鹵化物；（ii)烷基及苯甲基函化物，諸如齒乙醯 胺；（iii)搭、酮、羧基及順丁烯二醯亞胺基;)反應形成共價 鍵。某些抗體具有可還原之鏈間二硫鍵，亦即半胱胺酸 橋抗體可猎由诸如DTT(二硫蘇糖酵）之還原劑處理而具 有反應性以便與連接子試劑結合。因此，每個半胱胺酸橋 149799.doc -131· 201106972 在理論上將形成兩個反應性硫醇親核體。其他親核基團可 經由離胺酸與2-亞胺基硫雜環戊烷（特勞特試劑（心扣以 reagent))反應使胺轉化為硫醇而引入抗體中。反應性硫醇 基可藉由引入-個、兩個、三個、四個或四個以上半脱胺 酸殘基而引入抗體（或其片段）中（例如製備包含一或多個非 原生半胱胺酸胺基酸殘基之突變體抗體）。 亦可藉由修飾抗體以引入可與連接子試劑或藥物上之親 核性取代基反應的親電子性部分來製備抗體藥物結合物。 糖基化抗體中之糖可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越或嗣基。所得 亞胺希夫鹼（Schiff base)基團可形成穩定鍵聯，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 基化抗體中之碳水化合物部分與半乳糖氧化酶或偏過峨酸 納之反應可於蛋白質中產生可與藥物上之適當基團反應的 幾基（酸及 _基團）（Hermans〇n，Bi〇c〇njugate τ_η 咖十 在另貫把例中，含有N末端絲胺酸或蘇胺酸殘基之蛋白 質可與偏過碘酸鈉反應’從而產生醛而非第一胺基酸 (Geoghegan 及 Stroh，（1992) Bi〇c〇njugate 3:138_ 146’· US 5362852)。«可與藥物部分或連接子親核體反 應。 類似地，藥物部分上之親核基團包括（但不限於）：胺、 硫醇、經基、醯肼 '海、肼、硫半卡（thi〇semi⑽以贿）、 肼Μ酸鹽及芳基醯肼基團，該等基團能夠與連接子部分及 連接子試劑上之親電子基團（包括：⑴活性醋，諸如麵 149799.doc • J32- 201106972 酯、H〇m酯、鹵甲酸酯及酸鹵化物；（ii)烷基及苯甲基鹵 化物，諸如鹵乙醯胺；（iii)醛、酮、羧基及順丁烯二醯亞 胺基）反應形成共價鍵。 或者，包含抗體及細胞毒性劑之融合蛋白可藉由例如重 組技術或肽合成法製造。DNA之長度可包含編碼結合物之 兩個部分的各別區域，該等區域可彼此相鄰或由編碼不破 壞結合物所需特性之連接子肽之區域分隔。 Q 在另一實施例中，抗體可與用於預靶向腫瘤的「受體」 (諸如抗生物蛋白鏈菌素）結合，其中將抗體_受體結合物投 與患者，隨後使用廓清劑將未結合之結合物自循環系統中 移除，且接著投與與細胞毒性劑（例如放射性核苷酸）結合 的「配位體」（例如抗生物素蛋白）。 本發明之抗Axl抗體可藉由此項技術中已知之各種分析 法表徵其物理/化學性質及/或生物活性。 a) 活性分析法 〇 在一態樣中，提供用於鑑別具有生物活性之抗Axl抗體 之分析法。生物活性可包括例如抑制配位體與Αχί結合、 抑制Axl之磷酸化或下調Αχί表現。亦提供在活體内及/或 活體外具有該生物活性之抗體。 在某些實施例中，測試本發明抗體抑制配位體與Αχ1結 合、抑制Axl磷酸化或下調Αχί表現之能力。 b) 結合分析法及其他分析法 在一態樣中’測試本發明抗體之抗原結合活性，例如藉 由諸如ELISA、西方墨點法等已知方法測試。在另一態樣 149799.doc -133· 201106972 中，可使用競爭分析法鑑別與3G9、8B5、12A11或4F8競 爭結合Axl之單株抗體。在某些實施例中，該競爭抗體結 合的抗原決定基與3G9、8B5、12A11或4F8所結合之抗原 決定基（例如線性或構形抗原決定基）相同。例示性競爭分 析法包括（但不限於）常規分析法，諸如Harlow及Lane (1988) vl 第 14 章（ColdThe Ab-(L-D)p j linker can be composed of one or more linker components. Exemplary linker components include 6-maleimidoiminohexyl ("Mc"), maleimide-propyl propyl ("MP"), lysine- citrulline ("val-cit"), alanine, phenylalanine ("ala-phe"), p-aminophenyloxycarbonyl ("PAB"), 4-(2-pyridylthio)pentanoic acid N-butyl Terpene imidate ("SPP"), 4_(N-m-butylene iminomethyl)cyclohexane N-butyl sulfonate ("SMCC") and (4_iodine-B N-butyl succinimide ("SIAB"). Other linker components are known in the art and are described in the text 149799.doc-130-201106972. See also "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Patent No. 1/983, filed on Nov. 5, 2004, the entire disclosure of which is incorporated herein by reference. 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 valine-citrulline (vc or val-cit), alanine-phenylalanine (af Q or ala-Phe). Exemplary tripeptides include glycine-proline-glycine (gly-Val_cit) and glyco-glycine-glycine (gly_gly_gly). Amino acid residues comprising an amino acid 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 to be selective for enzymatic cleavage of specific enzymes, such as tumor-associated proteases, cathepsins B, c and D, or plasmin. Nucleophilic groups on the antibody include, but are not limited to, (1) an N-terminal amine group; (Η) a fluorene-terminated amine group such as an amide acid; (in) a side chain thiol group, such as cysteine; Iv) a glycosyl or amine group to which the antibody is glycosylated. The amine group, thiol group and hydroxyl group are nucleophilic and capable of interacting with the linker moiety and the pro-electron group on the linker reagent (including: (i) an active ester such as NHS ester, HOBt ester, halo: acid vinegar And an acid halide; (ii) an alkyl group and a benzyl group, such as chitosan; (iii) a ketone, a ketone, a carboxyl group, and a maleimide group; Some antibodies have a reducible interchain disulfide bond, i.e., a cysteine bridge antibody can be hunted by a reducing agent such as DTT (dithiothreitol) to be reactive for binding to a linker reagent. Therefore, each cysteine bridge 149799.doc -131· 201106972 will theoretically form two reactive thiol nucleophiles. Other nucleophilic groups can be introduced into the antibody via the reaction of an amine acid with a 2-iminothiolane (a reagent) to convert the amine to a thiol. A reactive thiol group can be introduced into an antibody (or a fragment thereof) by introducing one, two, three, four or more semi-deaminating acid residues (eg, preparing one or more non-native half) A mutant antibody to a cysteine amino acid residue). Antibody drug conjugates can also be prepared by modifying the antibody to introduce an electrophilic moiety that is reactive with the linker reagent or the nucleophilic substituent on the drug. The sugar in the glycosylated antibody can be oxidized, for example, by a periodate oxidizing agent to form an oxime group which can react with the linker reagent or the amine group of the drug moiety. The resulting imine Schiff base group can form a stable linkage or can be reduced by, for example, a borohydride reagent to form a stable amine linkage. In one embodiment, the reaction of the carbohydrate moiety of the glycosylated antibody with galactose oxidase or sodium perrhenate can produce a number of bases in the protein that are reactive with suitable groups on the drug (acids and groups) (Hermans〇n, Bi〇c〇njugate τ_η) In another example, a protein containing an N-terminal serine or a threonine residue can react with sodium metaperiodate to produce an aldehyde instead of The first amino acid (Geoghegan and Stroh, (1992) Bi〇c〇njugate 3: 138_ 146' US 5362852). «Can react with a drug moiety or a linker nucleophile. Similarly, the nucleophile on the drug moiety Groups include, but are not limited to, amines, thiols, thiols, 醯肼'sea, hydrazine, thiocabine (thi〇semi (10) for bribes), citrates and aryl sulfonium groups, such groups The group is capable of interacting with the linker moiety and the electrophilic group on the linker reagent (including: (1) active vinegar, such as face 149799.doc • J32-201106972 ester, H〇m ester, haloformate and acid halide; (ii An alkyl group and a benzyl halide such as a hydrazine; (iii) an aldehyde, a ketone, a carboxyl group and a maleic acid The imino group reaction forms a covalent bond. Alternatively, the fusion protein comprising the antibody and the cytotoxic agent can be produced by, for example, recombinant techniques or peptide synthesis. The length of the DNA can comprise separate regions encoding the two portions of the conjugate, The regions may be adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate. Q In another embodiment, the antibody may be associated with a "receptor" for pretargeting the tumor (such as an antibody) Bio-Streptavidin) in which an antibody-receptor conjugate is administered to a patient, followed by removal of the unbound conjugate from the circulatory system using an analysing agent, and subsequent administration to a cytotoxic agent (eg, a radionuclide) Acid-bound "ligand" (e.g., avidin). The anti-Axl antibodies of the invention can be characterized for their physical/chemical properties and/or biological activity by various assays known in the art. Analytical methods provide an assay for identifying biologically active anti-Axl antibodies in one aspect. Biological activity can include, for example, inhibiting ligand binding to Αχ, inhibiting phosphorylation of Axl or downregulating expression. Antibodies having such biological activity in vivo and/or in vitro are also provided. In certain embodiments, the antibodies of the invention are tested for their ability to inhibit ligand binding to Αχ1, inhibit Axl phosphorylation, or downregulate 表现. b) Binding assays and other assays The antigen binding activity of the antibodies of the invention is tested in one aspect, for example by known methods such as ELISA, Western blotting, and the like. In another aspect, 149799.doc-133.201106972, competition assays can be used to identify monoclonal antibodies that compete with 3G9, 8B5, 12A11, or 4F8 for binding to Axl. In certain embodiments, the competitor antibody-binding epitope is identical to an epitope (e.g., a linear or conformational epitope) to which 3G9, 8B5, 12A11 or 4F8 binds. Exemplary competitive analysis methods include, but are not limited to, conventional analytical methods such as Harlow and Lane (1988) vl Chapter 14 (Cold

Spring Harbor Laboratory, Cold Spring Harbor, NY)中提供 之常規分析法。對抗體所結合之抗原決定基定位之詳細例 示性方法提供於 Methods in Molecular Biology，第 66 卷 (Humana Press, Totowa，NJ)中之 Morris (1996)「Epitope Mapping Protocols」中。兩種抗體若一者將另一者之結合 阻斷50%或50%以上，則稱該兩種抗體結合於相同抗原決 定基。 在例示性競爭分析法中，在包含結合Axl之第一經標記 抗體（例如3G9、8B5、12A11或4F8)及經測試能夠與第一抗 體競爭結合Axl之第二未標記抗體的溶液中培育固定Axl。 第二抗體可存在於融合瘤上清液中。作為對照，在包含第 一經標記抗體、但不包含第二未標記抗體的溶液中培育固 定之Axl。在允許第一抗體與Axl結合之條件下培育之後， 移除過量的未結合抗體，且量測與固定之Axl結合之標記 之量。若相對於對照樣品，測試樣品中與固定之Axl結合 之標記之量大幅減少，則指示第二抗體與第一抗體競爭結 合 Axl。 在一態樣中，本發明之抗體可藉由一系列分析法進一步 149799.doc -134- 201106972 表徵’該等分析法包括（但不限於）N末端定序、胺基酸分 析、非變性尺寸排阻高壓液相層析（HpLC)、質譜分析、離 子交換層析及木瓜蛋白酶消化。 應瞭解，可使用本發明之免疫結合物替代抗Axl抗體或 除抗Axl抗體之外亦可使用本發明之免疫結合物來執行任 何上述分析法。 方法 〇 〇 組合療法 本發明之特徵在於Axl拮抗劑與vegf拮抗劑之組合使 用°該組合之有利效應包括（但不限於）治療劑組合所產生 之樂物動力學或樂效學共同作用。本發明尤其適用於治療 各階段之各類型癌症。 術語癌症包含多種辦+、戍、产..x 夕種增生病症，包括（但不限於）癌前期生 二且二=瘤及惡性腫瘤。良性腫瘤保持定位於初始位點 潤、侵襲或轉移至遠端位點之能力。惡性腫 ==壞其周圍的其他組織。其亦能夠脫離初始位 體的其他部分（轉移）。原發性 擴政至身 型分類；轉移性腫瘤係依計 據其發生之組織類 膻層係依據癌細胞所來源之 類。惡性腫瘤細胞隨日㈣變得更 、㈤“ 細胞。癌細胞外形之此變二且似手更不像正常 為充分分化(低級)、中度：:腫:錢’且癌細胞描述 級)。充分分化細胞之外形 化或未分化(高 正常細胞。未分化細胞為…/類似於其所來源之 胞為已變件如此異常以致無法判定細 149799.doc -135· 201106972 胞起源的細胞。 癌症分期系統描述在解剖學上癌症已擴散之程度且試圖 將具有類似預後及治療之患者歸為同一階段組。可進行數 種測試以有助於對癌症分期’包括活組織檢查及某些成像 測試，諸如胸部X射線、***X射線攝像（mamm〇gram)、 骨骼掃描、CT掃描及MRI掃描。亦使用血液測試及臨床評 估來評估患者總體健康且偵測癌症是否已擴散至某些器 官。 為對癌症分期，美國癌症聯合委員會（American Joint Committee on Cancer)首先使用TNM分類系統將癌症（尤其 實體腫瘤）按字母歸類。癌症以字母τ(腫瘤尺寸）、N(可觸 知結點）及/或Μ(轉移）指明。T1、Τ2、丁3及了4描述原發性 病變之尺寸增加；NG、N1、Ν2、⑽指示涉及進行性發展 之結點；且Μ0及]νπ反映不存在或存在遠端轉移。 在第二種分期方法（亦稱為總體分期或羅馬數字分期） 中將癌症刀成0至1 V期（合併原發性病變之尺寸以及結節 擴散及遠端轉移；、 . L, . ^ ^ 将砂之存在）。在此系統令，將病例分為由羅 馬數字I至IV表示之四個階段，或歸類為「復發性」。對於 某些癌症，0期稱為「疮从 々「π. 冉馬原位」或Tis」，諸如對於乳癌而 °私為乳腺官原位癌或小葉原位癌。高級腺瘤亦可歸類Conventional assays available in Spring Harbor Laboratory, Cold Spring Harbor, NY). A detailed exemplary method for epitope localization of antibodies is provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ). If two antibodies block the binding of the other by 50% or more, the two antibodies are said to bind to the same antigenic determinant. In an exemplary competitive assay, incubation is carried out in a solution comprising a first labeled antibody that binds Axl (eg, 3G9, 8B5, 12A11, or 4F8) and a second unlabeled antibody that is tested to compete with the first antibody for binding to Axl. Axl. The second antibody can be present in the supernatant of the fusion tumor. As a control, the immobilized Axl was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing the binding of the first antibody to Axl, excess unbound antibody is removed and the amount of label bound to the immobilized Axl is measured. If the amount of label bound to the immobilized Axl in the test sample is substantially reduced relative to the control sample, then the second antibody is indicated to compete with the first antibody for binding to Axl. In one aspect, the antibodies of the invention can be characterized by a series of assays further 149799.doc-134-201106972 'These assays include, but are not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size Exclusion high pressure liquid chromatography (HpLC), mass spectrometry, ion exchange chromatography and papain digestion. It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-Axl antibodies. Methods 〇 组合 Combination Therapy The present invention is characterized by the combination of an Axl antagonist and a vegf antagonist. Advantageous effects of the combination include, but are not limited to, a combination of therapeutic kinetics or philosophical effects produced by a combination of therapeutic agents. The invention is particularly useful for treating various types of cancer at various stages. The term cancer encompasses a variety of conditions, including, but not limited to, precancerous and secondary tumors and malignant tumors. Benign tumors remain capable of localizing, invading, or transferring to the distal site at the initial site. Malignant swelling == Bad other tissues around it. It can also be separated from other parts of the initial position (transfer). The primary expansion is to the classification of the body; the metastatic tumor is based on the tissue-derived layer that occurs according to the source of the cancer cells. Malignant tumor cells become more (4) "Cells. The shape of cancer cells changes and the hands are less like normal to fully differentiate (lower), moderate: swollen: money' and cancer cell description level). Fully differentiated cells are differentiated or undifferentiated (high normal cells. Undifferentiated cells are.../similar to the cells from which they are derived are so abnormal that it is impossible to determine the cells of the cell originating from 149799.doc-135·201106972. The staging system describes the extent to which anatomically cancer has spread and attempts to group patients with similar prognosis and treatment into the same phase group. Several tests can be performed to help stage the cancer' including biopsy and some imaging tests. Such as chest X-ray, mammography, bone scan, CT scan and MRI scan. Blood tests and clinical evaluations are also used to assess the overall health of the patient and to detect whether the cancer has spread to certain organs. For cancer staging, the American Joint Committee on Cancer first uses the TNM classification system to classify cancers (especially solid tumors) by letter. Cancer is indicated by the letters τ (tumor size), N (tactile nodes) and/or sputum (metastasis). T1, Τ2, butyl 3 and 4 describe the increase in size of the primary lesion; NG, N1, Ν2 (10) Indications of nodes involved in progressive development; and Μ0 and ]νπ reflect the absence or presence of distant metastases. In the second staging method (also known as global staging or Roman numeral staging), the cancer knife is 0 to 1 V. Period (combined with the size of the primary lesion and the nodular spread and distal metastasis; , . L, . ^ ^ the presence of sand). In this system, the case is divided into four stages represented by Roman numerals I to IV. Or classified as "recurrent." For some cancers, stage 0 is called "sac from 々" π. 冉 horse in situ" or Tis", such as for breast cancer, private for breast cancer in situ or lobular Carcinoma. Advanced adenomas can also be classified

1通# I期癌症為通常可治癒之小局部癌症，而IV 期通常表不無法進行手術或轉移性癌症。⑽及出期癌症 通常局部發展及/或顯示涉及局部淋巴結。通常，較高級 數字指示較曹士 # + 疾病’包括較大腫瘤尺寸及/或癌症擴散 149799.doc •136· 201106972 至附近淋巴結及/或鄰近原發性腫瘤之器官。此等階段定 義明確，但各類型癌症的定義不同且為熟習此項技術者所 知。 許多癌症登記處（諸如美國國家癌症研究所之監視、流 行病學及最終結果計劃（NCI，s Surveillance，Epidemiology， and End Resuhs pr〇gram，SEER))係使用總結分期。此系 統用於所有類型之癌症。其將癌症病例分成五個主要類 為別： 、 〇 原位為早期癌症，其僅存在於其開始之細胞層中。 局部化為偈限於開始器官’而無擴散證據之癌症。 區域性為已擴散超出初始（原發性）位點至附近淋巴結或 益官及組織之癌症。 遠端為已自原發性位點擴散至遠端器官或遠端淋巴結之 癌症。 未知用於描述無足夠資訊指示階段之病例。 〇 此外常見的是’在原發性腫瘤已移除之後數月或數年， 癌症復發。在所有可見腫瘤已根除之後復發之癌症稱為復 發性疾病。在原發性腫瘤區域内復發之疾病為局部復發， ’ 且以轉移形式復發的疾病稱為遠端復發。 腫瘤可為貫體腫瘤或非實體腫瘤或軟組織腫瘤。軟組織 腫瘤之實例包括白血病（例如慢性骨髓性白血病、急性骨 髓性白金病、成年急性淋巴母細胞白血病、急性骨髓性白 病成熟B細胞急性淋巴母細胞白血病、慢性淋巴球性 白血病、前淋巴球性白血病或毛細胞白血病）或淋巴瘤（例 149799.doc •137- 201106972 如非霍奇金氏淋巴瘤、皮膚τ細胞淋巴瘤或霍 實體腫瘤包括除血液、骨髓或淋巴系統以外的身體组織之 任何癌症。龍腫射進―步分為上皮細胞起源之實體腫 瘤及非上皮細胞起源之實體腫瘤。上皮細胞實體腫瘤之實 例包括胃腸道、結腸、***、***、肺、腎、肝、姨、 即巢、頭及頸部、口腔、胃、十二指腸、小腸、大腸、肛 門、膽囊、唇、鼻咽、皮膚、子宮、雄性生殖器、泌尿器 官、膀胱及皮膚之腫瘤。非上皮起源之實體腫瘤包括肉 瘤、腦腫瘤及骨腫瘤。 除上述治療方案以外，可對患者進行外科手術移除癌細 胞及/或放射療法。 在某些實施例中，對本文中之患者進㈣斷測試，例如 在治療之前及/或治療期間及/或治療之後進行診斷測試。 通常，若進行診斷測試，則樣品可獲自需要治療之患者。 若個體患有癌症，則樣品可為腫瘤樣品或其他生物樣品， 諸如生物體液，包括（但不限於）血液、尿、唾液、腹水、 或諸如血清及血漿之衍生物，及其類似物。 在些貫知例中’個體癌症表現（在一些實施例中，過 度表現）Axl及/或VEGF ^測定Αχ1及VEGF表現之方法在此 項技術中已知且本文中描述某些方法。 在一態樣中’本發明提供治療癌症、抑制不良細胞增 殖、抑制癌症轉移及活體内或活體外誘導腫瘤細胞之細胞 ;周亡的方法’該方法包含在允許本發明之抗體與Axl結合 之條件下使細胞暴露於抗體。在某些實施例中，細胞為骨 149799.doc -138- 201106972 髓性白血病細胞、肺癌細胞、胃癌細胞、乳癌細胞、前列 腺癌細胞、腎細胞癌細胞及神經膠母細胞瘤細胞。在—實 施例中，本發明之抗體可用於抑制Axl之活性，該方法包 含使Axl暴露於本發明之抗體以便抑制Αχί之活性。 在一態樣中，本發明提供治療癌症之方法，其包含向個 體投與有效量之本發明抗體。在某些實施例中，治療癌症 之方法包含向個體投與有效量之醫藥調配物，其包含本發 Q 明之抗體及視情況選用之至少一種其他治療劑（諸如下文 提供之治療劑）。 本發明之抗體在治療中可單獨使用或與其他組合物組合 使用。舉例而言’本發明之抗體可與至少一種其他治療劑 及/或佐劑共投與。在某些實施例中，其他治療劑為抗 VEGF抗體。 上述該等組合療法涵蓋組合投藥（其中兩種或兩種以上 治療劑包括在同一或各別調配物中），及分別投藥，在此 〇 情況下本發明抗體之投與可在投與其他治療劑及/或佐劑 之前、同時及/或之後進行。本發明之抗體亦可與放射療 法組合使用。 在一實施例中，將本發明之抗體用於結合個體中之Axl 的方法中’該個體罹患與Αχί表現及/或活性增加相關之病 症，該方法包含將抗體投與個體以結合個體中之Αχ1。在 一實施例中’ Axl為人類Axl且個體為人類個體。 診斷方法及偵測方法 在一態樣中’本發明之抗體適用於偵測生物樣品中Axl 149799.doc •139· 201106972 之存在。如本文中所用之術語「俄測」涵蓋定量或定性偵 測。在某些實施例中’生物樣品包含細胞或組織諸如腫 瘤組織。 在一態樣中，本發明提供偵測生物樣品中Αχ1之存在的 方法。在某些實施例中，該方法包含在允許抗Αχ1抗體與 Axl結合之條件下使生物樣品與抗Αχ1抗體接觸，及偵測抗 Axl抗體與Axl之間是否形成複合物。 在一態樣中，本發明提供診斷與Αχ1表現增加相關之病 症的方法。在某些實施例中，該方法包含：使測試細胞與 抗Axl抗體接觸；藉由偵測抗Αχ1抗體與Αχ1之結合來測定 測試細胞中Axl之表現量（定量或定性及對測試細胞之 Axl表現1與對照細胞（例如與測試細胞來源於相同組織之 正常細胞’或Axl表現量與此正常細胞相當之細胞）之Axl 表現量加以比較’其中測試細胞之AX丨表現量高於或低於 對照細胞皆指示存在與Αχί表現增加相關之病症。在某些 實施例中’測試細胞獲自懷疑患有與Αχί表現增加相關之 病症之個體。在某些實施例中’病症為細胞增殖性病症， 諸如癌症或腫瘤。 可使用本發明之抗體診斷之例示性病症包括骨髓性白血 病、肺癌（例如非小細胞肺癌（NSCLc))、胃癌、乳癌、前 列腺癌、腎細胞癌、胰臟癌及神經膠母細胞瘤。 可使用某些其他方法偵測抗體與Αχί之結合。該等方法 包括（但不限於）此項技術中熟知之抗原結合分析法，諸如 西方墨點法、放射免疫分析法、ELISA(酶聯免疫吸附分析 149799.doc •140- 201106972 法）、「夾心」免疫分析法、免疫沈殺分析法、螢光免疫分 析法、蛋白A免疫分析法及免疫組織化學（IHC)。 在某些實施例中，抗體經標記。標記包括（但不限於）直 接谓測之標記或部分（諸如蝥光、發色團、電子密度、化 學發光及放射性標記），以及間接偵測之部分，例如經由 ' 酶促反應或分子間相互作用間接偵測之部分，諸如酵素或 配位體。例示性標記包括（但不限於）放射性同位素32p、 Q 14(：、1251、4及1311 ;螢光團，諸如稀土螯合物或螢光素 (fluorescein)及其衍生物；若丹明（rh〇damine)及其衍生 物；丹醯基（dansyl);傘酮（umbelliferone);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），螢火蟲發光素（111(^61>丨11);2,3-二氫酜嗓二_;辣根 過氧化酶（HRP);鹼性磷酸酶；半乳糖苷酶；葡糖澱粉 酶，〉谷菌酶，醣氧化酶，例如葡萄糖氧化酶、半乳糖氧化 酶及葡萄糖-6-磷酸脫氫酶；雜環氧化酶，諸如尿酸酶及黃 〇 嘌呤氧化酶，以及使用過氧化氫氧化染料前驅物之酵素 (諸如HRP、乳酸過氧化酶或微過氧化酶）、生物素/抗生物 素蛋白、自旋標記、噬菌體標記、穩定游離基團及其類似 物。 在某些實施例中，將抗體固定於不溶性基質上。固定可 能引起抗Axl抗體與溶液中保持游離之任何Αχΐ分離。固定 s知如下元成.在分析程序之前，降低抗Αχΐ抗體溶解 生，吸附至水不溶性基質或表面（Bennkh等人，U S 3,720,760)，或共價偶合（例如使用戊二醛交聯”或在抗 149799.doc -141 - 2011069721 pass # Stage I cancer is a small, localized cancer that is usually curable, while stage IV usually does not allow surgery or metastatic cancer. (10) and out-of-stage cancer usually develop locally and/or show local lymph nodes. Typically, higher-level numbers indicate that the Cao's # + disease' includes larger tumor size and/or cancer spread 149799.doc • 136· 201106972 to nearby lymph nodes and/or organs adjacent to the primary tumor. These stages are defined explicitly, but the definitions of each type of cancer are different and are known to those skilled in the art. Many cancer registries (such as the National Cancer Institute's Surveillance, Epidemiology, and End Resuhs pr〇gram (SEER)) use summary staging. This system is used for all types of cancer. It divides cancer cases into five main categories: 、 In situ is an early cancer that exists only in the cell layer at which it begins. Localization is a cancer that is limited to starting organs and without evidence of proliferation. Regionality is cancer that has spread beyond the initial (primary) site to nearby lymph nodes or pros and cons. The distal end is a cancer that has spread from the primary site to the distal or distal lymph nodes. Unknown to describe cases that do not have enough information to indicate the stage. 〇 It is also common to see 'recurrence of cancer months or years after the primary tumor has been removed. A cancer that recurs after all visible tumors have been eradicated is called a recurrent disease. A disease that recurs in a primary tumor area is a local recurrence, and a disease that recurs in a metastatic form is called a distal recurrence. The tumor can be a pericy tumor or a non-solid tumor or a soft tissue tumor. Examples of soft tissue tumors include leukemia (eg, chronic myelogenous leukemia, acute myelodynic leukemia, adult acute lymphoblastic leukemia, acute myeloid white matter mature B cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, anterior lymphocyticity) Leukemia or hairy cell leukemia) or lymphoma (eg 149799.doc • 137- 201106972 such as non-Hodgkin's lymphoma, cutaneous tau cell lymphoma or hulk solid tumor including body tissues other than blood, bone marrow or lymphatic system Any cancer. The dragon is swollen into a solid tumor of epithelial origin and a solid tumor of non-epithelial origin. Examples of epithelial solid tumors include the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, sputum, That is, tumors of the nest, head and neck, mouth, stomach, duodenum, small intestine, large intestine, anus, gallbladder, lip, nasopharynx, skin, uterus, male genitalia, urinary organs, bladder and skin. Solid tumors of non-epithelial origin include Sarcoma, brain tumors and bone tumors. In addition to the above treatment options, patients can be treated Surgical removal of cancer cells and/or radiation therapy. In certain embodiments, a diagnostic test is performed on a patient herein, for example, prior to and/or during treatment and/or after treatment. For a diagnostic test, the sample can be obtained from a patient in need of treatment. If the individual has cancer, the sample can be a tumor sample or other biological sample, such as a biological fluid, including but not limited to blood, urine, saliva, ascites, or Derivatives such as serum and plasma, and the like. In some examples, 'individual cancer performance (in some embodiments, overexpression) Axl and/or VEGF^ methods for determining Αχ1 and VEGF expression in this technique Certain methods are known and described herein. In one aspect, the invention provides a method of treating cancer, inhibiting cell proliferation, inhibiting cancer metastasis, and inducing tumor cells in vivo or in vitro; The invention comprises exposing the cells to an antibody under conditions which permit binding of the antibody of the invention to Axl. In certain embodiments, the cells are bone 149799.doc-138-201106972 Leukemia cells, lung cancer cells, gastric cancer cells, breast cancer cells, prostate cancer cells, renal cell cancer cells, and glioblastoma cells. In the examples, the antibody of the present invention can be used to inhibit the activity of Axl, and the method comprises Axl is exposed to an antibody of the invention to inhibit the activity of Αχ. In one aspect, the invention provides a method of treating cancer comprising administering to an individual an effective amount of an antibody of the invention. In certain embodiments, treating cancer The method comprises administering to the individual an effective amount of a pharmaceutical formulation comprising an antibody of the present invention and optionally at least one other therapeutic agent (such as a therapeutic agent provided below). The antibody of the present invention may be used alone in therapy or Used in combination with other compositions. For example, an antibody of the invention can be co-administered with at least one other therapeutic agent and/or adjuvant. In certain embodiments, the additional therapeutic agent is an anti-VEGF antibody. The above combination therapies include combination administration (in which two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case administration of the antibody of the present invention may be administered to other treatments. The agent and/or adjuvant are administered before, simultaneously and/or after. The antibody of the present invention can also be used in combination with radiation therapy. In one embodiment, the use of an antibody of the invention in a method of binding to Axl in an individual afflicts the subject with a condition associated with increased performance and/or activity, the method comprising administering an antibody to the individual to bind to the individual Αχ1. In one embodiment 'Axl is human Axl and the individual is a human individual. Diagnostic Methods and Detection Methods In one aspect, the antibodies of the invention are suitable for detecting the presence of Axl 149799.doc • 139·201106972 in a biological sample. The term "Russian" as used herein encompasses quantitative or qualitative detection. In certain embodiments the biological sample comprises cells or tissues such as tumor tissue. In one aspect, the invention provides a method of detecting the presence of Αχ1 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with an anti-Αχ1 antibody under conditions that permit binding of the anti-Αχ1 antibody to Axl, and detecting whether a complex is formed between the anti-Axl antibody and Axl. In one aspect, the invention provides a method of diagnosing a condition associated with increased performance of Αχ1. In certain embodiments, the method comprises: contacting the test cell with an anti-Axl antibody; determining the amount of Axl expression in the test cell by detecting binding of the anti-Αχ1 antibody to Αχ1 (quantitative or qualitative and Axl to the test cell) Performance 1 is compared to the Axl performance of control cells (eg, normal cells from which the test cells are derived from the same tissue) or Axl expression is equivalent to this normal cell. 'The AX丨 performance of the test cells is higher or lower. Control cells all indicate the presence of a condition associated with increased expression of Αχ. In certain embodiments, the test cells are obtained from an individual suspected of having a condition associated with increased performance of the sputum. In certain embodiments, the condition is a cell proliferative disorder. Such as cancer or tumors. Exemplary conditions that can be diagnosed using the antibodies of the invention include myeloid leukemia, lung cancer (eg, non-small cell lung cancer (NSCLc)), gastric cancer, breast cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, and nerves. Glioblastoma. Some other methods can be used to detect the binding of antibodies to Αχί. These methods include, but are not limited to, this technique. Well-known antigen binding assays, such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay 149799.doc • 140-201106972 method), "sandwich" immunoassay, immunostaining assay, firefly Photoimmunoassay, Protein A Immunoassay, and Immunohistochemistry (IHC). In certain embodiments, the antibody is labeled. The label includes, but is not limited to, a direct pre-measured label or portion (such as calendering, hair color) Groups, electron density, chemiluminescence, and radioactive labeling, as well as indirect detection, such as parts that are indirectly detected by 'enzymatic or intermolecular interactions, such as enzymes or ligands. Exemplary markers include (but not Limited to) radioisotopes 32p, Q 14 (:, 1251, 4 and 1311; fluorophores such as rare earth chelates or fluorescein and its derivatives; rhodamine and its derivatives ; dansyl; umbelliferone; luciferase, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), firefly luciferin (111 (^61>丨11) ) ; 2,3-dihydroindole II; horseradish peroxidase (HRP); alkaline phosphatase; galactosidase; glucoamylase, glutamic enzyme, sugar oxidase, such as glucose oxidase, Galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes using hydrogen peroxide dye precursors such as HRP, lactate peroxidase or micro Peroxidase), biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like. In certain embodiments, the antibody is immobilized on an insoluble matrix. Immobilization may cause anti-Axl antibodies Separate from any hydrazine remaining free in the solution. The immobilization is known as follows. Before the analysis procedure, the anti-spasm antibody is reduced, adsorbed to a water-insoluble matrix or surface (Bennkh et al., US 3,720, 760), or covalently coupled (for example, using glutaraldehyde cross-linking) or Anti-149799.doc -141 - 201106972

Axl抗體與Axl之間形成複合物之後，降低抗Axl抗體溶解 性，例如藉由免疫沈澱法降低抗Axl抗體溶解性。 應瞭解，可使用本發明之免疫結合物代替抗Axl抗體或 除抗Axl抗體之外亦使用本發明之免疫結合物來執行任何 上述診斷或偵測實施例。 本發明亦提供測定對Axl拮抗劑治療之反應的方法，包 含偵測圖6中所示之任一基因之表現（多肽，核酸）。在一些 實施例中，用Axl拮抗劑（諸如抗Axl抗體）治療後， MYCN、HLX、GAS7、HDAC9、E2F1、CXCR4、PMCH及 ANG-2中一或多者之表現增加。在一些實施例中，用Axl 拮抗劑（諸如抗Axl抗體）治療後，IFI44L、GJA4、Axl、 IFIT1、SCG5、CYTL1、DPP4 及 DKK3 中一或多者之表現 降低。在一些實施例中，增加及/或降低之表現為尚未經 Axl拮抗劑治療之個體（或經Axl拮抗劑治療之前的個體）中 的相對表現。 化學治療劑 本發明之組合療法可進一步包含一或多種化學治療劑。 組合投藥包括使用各別調配物或單一醫藥調配物進行共投 藥或同時投藥，及依任一次序之連續投藥，其中較佳存在 兩種（或所有）活性劑同時發揮其生物活性的時間段。 化學治療劑（若投與）通常以其已知劑量投與，或由於藥 物之組合作用或可歸因於投與抗代謝物化學治療劑之消極 副作用而視情況降低。可根據製造商之說明書或如熟練從 業者根據經驗所確定來使用該等化學治療劑之製劑及給藥 149799.doc -142- 201106972 時程。 以上揭示可組合之各種化學治療劑。— „ 甘—些實施例中， 待組合之化學治療劑選自由以下組成 X <群.紫杉醇（包括 多西他赛及太平洋紫杉醇）、長春花（諸如 ^ 長春知負或長春 化鹼）、始化合物（諸如卡銘或順始）、芳香酶抑制劑（諸士 來曲峻、阿那曲錢依西美坦）、抗***（例如氟維司群 或他莫昔芬）、依託泊苷、嗟替派、 a 壤磷醯胺、甲胺喋After the complex is formed between the Axl antibody and Axl, the solubility of the anti-Axl antibody is lowered, for example, by immunoprecipitation to reduce the solubility of the anti-Axl antibody. It will be appreciated that any of the above diagnostic or detection embodiments can be performed using the immunoconjugates of the invention in place of or in addition to anti-Axl antibodies. The invention also provides a method of determining the response to treatment with an Axl antagonist comprising detecting the expression (polypeptide, nucleic acid) of any of the genes shown in Figure 6. In some embodiments, the performance of one or more of MYCN, HLX, GAS7, HDAC9, E2F1, CXCR4, PMCH, and ANG-2 is increased following treatment with an Axl antagonist (such as an anti-Axl antibody). In some embodiments, one or more of IFI44L, GJA4, Axl, IFIT1, SCG5, CYTL1, DPP4, and DKK3 are reduced in performance following treatment with an Axl antagonist (such as an anti-Axl antibody). In some embodiments, the increase and/or decrease manifests as a relative performance in an individual who has not been treated with an Axl antagonist (or an individual prior to treatment with an Axl antagonist). Chemotherapeutic Agents The combination therapies of the invention may further comprise one or more chemotherapeutic agents. Combination administration includes co-administration or simultaneous administration using separate formulations or a single pharmaceutical formulation, and continuous administration in either order, wherein preferably there is a period of time during which both (or all) of the active agents simultaneously exert their biological activity. The chemotherapeutic agent (if administered) is usually administered at a known dose, or is reduced as appropriate due to the combined action of the drug or the negative side effects attributable to administration of the antimetabolite chemotherapeutic agent. Formulations and administration of such chemotherapeutic agents can be carried out according to the manufacturer's instructions or as determined by the skilled practitioner based on experience. 149799.doc -142- 201106972 Time course. The various chemotherapeutic agents that can be combined are disclosed above. — 甘—In some embodiments, the chemotherapeutic agent to be combined is selected from the group consisting of X < group. paclitaxel (including docetaxel and paclitaxel), periwinkle (such as ^Changchunzhi or vinca citrate), Compounds (such as Carmine or Shun), aromatase inhibitors (Zhu Shilai Qujun, Anasicidacetam), antiestrogens (such as fulvestrant or tamoxifen), etoposide嗟 派 、, a lycopene, methotrexate

呤、脂質阿黴素、聚乙二醇化脂質阿黴素、卡西他賓、士、 西他濱、COX-2抑制劑（例如賽利克西）或蛋白酶體抑制二 (例如PS342)。在-些實施例中，化學治療劑為紫杉醇及 翻化合物（諸如卡鉑）。 調配物、劑量及投藥 本發明所用治療劑將以符合優良醫藥實 給予且投與。本文中所考慮之因素包括所治療=病 症、所治療之特定健、個別患者之臨床錄、病症之病 因、藥劑傳遞位點、投藥方法、投藥時程、待組合藥劑的 藥物-藥物相互作用及醫學從業者已知之其他因素。 治療性調配物係使用此項技術中已知之標準方法，藉由 混合具有所需純度之活性成分與可選的生理學上可接受之 載劑、賦形劑或穩定劑（Remingt〇n，s pharmaceuticai Sciences (第 20 版），A. Gennar〇 編，2〇〇〇, Uppinc〇tt，Indole, lipid doxorubicin, pegylated lipid doxorubicin, citacitabine, g., citridin, a COX-2 inhibitor (e.g., cylixix) or proteasome inhibitor II (e.g., PS342). In some embodiments, the chemotherapeutic agent is paclitaxel and a compound (such as carboplatin). Formulations, Dosage, and Administration The therapeutic agents used in the present invention will be administered and administered in accordance with good pharmaceuticals. Factors considered herein include the treatment = condition, the particular health being treated, the clinical record of the individual patient, the etiology of the disorder, the site of administration of the drug, the method of administration, the time course of administration, the drug-drug interaction of the agent to be combined, and Other factors known to medical practitioners. Therapeutic formulations are prepared by mixing active ingredients of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers using standard methods known in the art (Remingt〇n, s Pharmaceuticai Sciences (20th Edition), A. Gennar, 2, Uppinc〇tt,

Williams & Wilkins，Phi][adelphia，pA)來製備。可接受之載 dJ包括生理食鹽水或緩衝劑，諸如填酸鹽、檸檬酸鹽及其 他有機酸；抗氧化劑，包括抗壞血酸；低分子量（小於約 149799.doc -143- 201106972 ίο個殘基）多肽；蛋白質，諸如血清白蛋白、明膠或免疫 球蛋白；親水聚合物，諸如聚乙烯吡咯啶g同；胺基酸，諸 如甘胺酸、麩醯胺酸、天冬醯胺酸、精胺酸或離胺酸；單 醣、二醣及其他碳水化合物，包括葡萄糖、甘露糖或糊 精；螯合劑，諸如EDTA;糖醇，諸如甘露糖醇或山梨糖 醇；成鹽相對離子，諸如鈉；及/或非離子界面活性劑， 諸如 TWEENtm、plur〇nicstm或 pEG。 調配物視情況但較佳含有醫藥學上可接受之鹽，較佳為 氣化鈉且較佳處於約生理學濃度下。本發明之調配物可視 If况含有醫藥學上可接受之防腐劑。在一些實施例中，防 腐劑濃度範圍通常為H2 ()%(v/v)。適當防腐劑包括醫 樂技術中已知者。笨甲醇、苯盼、間甲紛、對經基苯甲酸 ”曰及對經基苯甲酸丙醋為較佳防腐劑。本發明之調配物 了視情況包括濃度為〇 〇〇5至〇 〇2%之醫藥學上 面活性劑。 饮又·^介 ’d疋適應症需要時，本文中之調配物 以上活性化合物，較佳A且亡有 佳為具有不會對彼此產生不利影響 互補活性的活性化合物。 “、y響 之量組合存在。 以分子宜以有效達成預期目 活性成分亦可截留於 所製備之微谬囊(例如=由凝聚技罐^ 及聚(甲基丙烯酸甲酷):膠囊：甲基纖維气或：膠微夥: 統(例如脂質體、白、, 、截留於膠狀藥物傳遞: 米囊劑)或截留於***洛由 心液“顆粒及; 中。該等技術揭示於Remingt()n 149799.doc -144 - 201106972Williams & Wilkins, Phi] [adelphia, pA) to prepare. Acceptable dJ includes physiological saline or buffers such as sulphates, citrates and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 149799.doc - 143 - 201106972 ίο residues) polypeptides a protein such as serum albumin, gelatin or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidine g; an amino acid such as glycine, glutamic acid, aspartic acid, arginine or a lysine; a monosaccharide, a disaccharide, and other carbohydrates, including glucose, mannose or dextrin; a chelating agent such as EDTA; a sugar alcohol such as mannitol or sorbitol; a salt-forming relative ion such as sodium; / or a nonionic surfactant such as TWEENtm, plur〇nicstm or pEG. The formulation will optionally contain a pharmaceutically acceptable salt, preferably sodium vaporification and preferably at about physiological concentrations. The formulations of the present invention may contain a pharmaceutically acceptable preservative, as the case may be. In some embodiments, the preservative concentration typically ranges from H2 ()% (v/v). Suitable preservatives include those known in the art of medicine. Stupid methanol, benzene, dimethyl ketone, p-benzoic acid hydrazine and p-propyl benzoic acid propyl vinegar are preferred preservatives. The formulations of the present invention include concentrations of 〇〇〇5 to 视2 as appropriate. % of the above active agents of medicine. Drinking ··介介'd indications, when needed, the above active compounds, preferably A and good to have an activity that does not adversely affect each other's complementary activities Compound. ", y ring volume combination exists. The active ingredient may be effectively retained in the molecule to be retained in the prepared microcapsule (for example, = by a coagulation tank and poly (methacrylic acid): capsule: methyl fiber or gel: System (eg liposome, white,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 201106972

Pharmaceutical Sciences(同上）中 〇 可製備持續釋放製劑。持續釋放製劑之適合實例包括含 有抗體之半透性固體疏水性聚合物基質，該等基質呈成型 物品之形式，例如薄膜或微膠囊。持續釋放基質之實例包 括聚酯、水凝膠（例如聚（曱基丙烯酸2_羥基乙酯）或聚（乙 '烯醇））、聚乳酸交酯（美國專利第3,773,919號）、L_麩胺酸 與γ乙基-L-麩胺酸酯之共聚物、不可降解性乙烯_乙酸乙烯 Q 酯、可降解性乳酸·乙醇酸共聚物（諸如LUpR〇N DEPOT，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-D-(-)-3-羥基丁酸。雖然諸如乙烯_乙酸 乙烯醋及乳酸-乙醇酸之聚合物釋放分子能夠逾1〇〇天，但 某些水凝勝釋放蛋白質的時段更短。當囊封抗體長時間保 留於體内時，由於在3rC下暴露於水分，其可變性或聚 集，導致生物活性損失及可能之免疫原性變化。視所涉及 之機制而定，可設計合理策略以達成穩定化。舉例而言， 〇 #發現聚集機制為經由硫基-二硫化物互換形成分子間°s_s 鍵，則可藉由修飾氫硫基殘基、由酸性溶液;東乾、控a制水 分含量、使用適當添加劑及開發特定聚合物基質組合物來 本發明之治療劑係根據已知方法投與人類患者，諸如快 速靜脈内投與或在一段時間連續輸注、肌肉内、、 腦脊髓内、皮下1節内、滑膜内、鞘内、經口、局部赤 吸入路徑。在VEGF括抗劑之情況下，若廣泛 ： 性與―结抗作用相_，則尤其需要局部投藥。治 149799.doc -145- 201106972 應用亦可使用離體策略。離體策略涉及以編碼Axl及/或 VEGF拮抗劑之聚核苷酸轉染或轉導自個體獲得之細胞。 接著，使轉染或轉導細胞返回至個體。該等細胞可為多種 類型中之任一種，包括（但不限於）造血細胞（例如骨腾細 胞、巨噬細胞、單核細胞、樹突狀細胞、T細胞或b細 胞）、纖維母細胞、上皮細胞、内皮細胞、角質細胞或肌 細胞。 舉例而言’若Axl及/或VEGF拮抗劑為抗體，則藉由包 括非經腸、皮下、腹膜内、肺内及鼻内之任何適合方式投 與該抗體，且若需要局部免疫抑制治療，則藉由病灶内方 式投與。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，抗體宜藉由脈衝輸注來投與，尤其 遞減劑量之抗體。給藥較佳藉由注射進行，最佳靜脈内或 皮下注射，此部分地視投藥為短期或長期而定。 在另一實例中，當病症或腫瘤位置允許時，可局部投與Pharmaceutical Sciences (supra) 〇 A sustained release formulation can be prepared. Suitable examples of sustained release formulations include semipermeable solid hydrophobic polymeric matrices containing antibodies in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(ethylene 'enol)), polylactide (U.S. Patent No. 3,773,919), L-Bran Copolymer of aminic acid with γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate Q, degradable lactic acid·glycolic acid copolymer (such as LUpR〇N DEPOT, copolymer of lactic acid-glycolic acid And injectable microspheres composed of leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than one day, some water gels have a shorter period of time to release proteins. When the encapsulated antibody is retained in the body for a long period of time, its variability or aggregation due to exposure to moisture at 3rC results in loss of biological activity and possible immunogenic changes. Depending on the mechanism involved, a reasonable strategy can be devised to achieve stabilization. For example, 〇# found that the aggregation mechanism is to form an intermolecular °s_s bond via a thio-disulfide interchange, which can be modified by modifying the thiol residue, from an acidic solution; Appropriate Additives and Development of Specific Polymer Matrix Compositions The therapeutic agents of the present invention are administered to human patients according to known methods, such as rapid intravenous administration or continuous infusion over a period of time, intramuscular, intracerebrospinal, subcutaneous Internal, intrasynovial, intrathecal, oral, and local red inhalation paths. In the case of a VEGF antagonist, if it is broad: sex and nociceptive phase, it is particularly necessary to administer the drug locally. Treatment 149799.doc -145- 201106972 Applications can also use an ex vivo strategy. An ex vivo strategy involves transfecting or transducing cells obtained from an individual with a polynucleotide encoding an Axl and/or VEGF antagonist. The transfected or transduced cells are then returned to the individual. The cells can be of any of a variety of types including, but not limited to, hematopoietic cells (eg, osteoblasts, macrophages, monocytes, dendritic cells, T cells or b cells), fibroblasts, Epithelial cells, endothelial cells, keratinocytes or muscle cells. For example, if the Axl and/or VEGF antagonist is an antibody, the antibody is administered by any suitable means including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and if local immunosuppressive therapy is desired, Then it is administered by means of the lesion. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, antibodies are preferably administered by pulse infusion, particularly by decreasing the dose of antibody. The administration is preferably carried out by injection, preferably intravenously or subcutaneously, depending in part on the short-term or long-term administration. In another example, local administration may be performed when a condition or tumor location permits

Axl及/或VEGF结抗劑化合物，例如藉由直接注射局=投 與，且可定期重複注射。Axl及/或VEGF拮抗劑亦可全身 性傳遞至個體或直接傳遞至腫瘤細胞，例如在外科切除腫 瘤之後傳遞至腫瘤或腫瘤床，以防止或減少局部復發或轉 移。 在抑制劑為抗體的情況下，所投抗體較佳為裸抗體。然 而，所投抑制劑可與細胞毒性劑結合。戶斤結合之抑制劑 及/或與其結合之抗原較佳經細胞内化，從而增加免疫结 合物殺死其所結合之標心胞之治療功效。在-較佳實施 149799.doc •146. 201106972 例中，細胞毒性劑乾向或干擾癌細胞中之核酸。該等細胞 毒性劑之實例包括類美登素、加里刹徵素、核糖核酸酶及 DNA核酸内切酶。 本文中所列之任一治療劑之適當劑量為目前使用之劑量 且可由治療醫師適當降低或提高。 治療劑組合通常在限定時間段（通常數分鐘、數小時、 數天或數週，視所選組合而定）投與。組合療法意欲包含 〇 依序投與此等治療劑（亦即，其中在不同時間投與各治療 劑）以及貫質上同時投與此等治療劑或至少兩種治療劑。 治療劑可由相同途徑或不同途徑投與。舉例而言，可藉 2靜脈内注射投與組合中之¥咖及/或Axl拮抗劑，而組 合中之蛋白質激酶抑制劑可經口投與。或者，例如，視特 定：療劑而定，可經口投與兩種治療劑或可藉由靜脈内注 射扠/、兩種/σ療劑。視特定藥劑而定，亦可改變治療劑之 投與次序。 〇 本案涵蓋藉由基因療法來投與VEGF及/或AxW抗劑。 關於使用基因療法產生細胞内抗體，參見例如^㈣年3月 14 曰公開之WO96/07321。 ‘應瞭解，可使用本發明之免疫結合物代替抗體或除抗體 之外亦使用本發明之免疫結合物來執行任何上述治療方 法。 D·製品 在本發明之另—態樣中，提供-種含有適用於治療、預 防及/或δ乡斷上述病症之物質的製品。該製品包含容器及 149799.doc -147- 201106972 位㈣容器上或與該容器相關之標鐵或藥品說明書。適入 之容器包括例如瓶子、小瓶、注射器等。容器^諸2 璃或塑膠之多種材料形成。 谷益可由诸如玻璃或塑膠之多 :材 各器容納單獨組合物或該組合物與有好 t預防及/或診斷病狀之另—種組合物之組合，且可二 2 Ί接取例如容器可為具有可由皮下注射針刺穿之 =靜脈内溶液袋或小瓶)。組合物中至少-種活性劑 為本發明之抗體卷务 合物用於治療所:=、、：。標記或藥品說明書指示組 物Μ一 卜，製品包含⑷其巾含有組合 ♦益’其中該組合物包含本發明之抗體或免疫結 I ，及⑻其中含有組合物之第二容器，丨中該組合物包 3^種細胞毒性劑或其他治療劑。本發明之此實施例中 的製-可it #包含藥品說明書，該藥品說明書指示組合 物可用於治療特定病狀。或者或另外，製品可進一步包含 3有醫樂學上可接受之缓衝液（諸如注射用之抑菌水 (B㈣)、磷酸鹽緩衝生理食鹽水、林格氏溶液⑻_ S〇1Utl〇n)及右旋糖溶液）的第二（或第三）容器。其可進一步 就商業及使用者觀點而言所需之其他物質，包括其他 緩衝劑、稀釋劑、過據器、針及注射器。 ’、 實例 乂下為本發明之方法及組合物之實例。應瞭解，鑒於上 文提供之一般說明，可實施各種其他實施例。 A.物質及方法 抗體。抗體獲自以下供應商··針對人類Axl之小鼠單株 149799.doc -148- 201106972 抗體（mAb)(純系 6C8，Abnova, Taiwan) ’填酸化 Akt(Ser 473)小鼠mAb及Akt多株抗體（Cell Signaling)，針對人類 Gas6之小鼠mAb(R&D System)。用於磷酸化Axl之ELISA套 組購自 R&D System。 細胞株及培養物。人類癌瘤細胞株來自ATCC。初級人 類臍靜脈内皮細胞（HUVEC)及肺動脈平滑肌細胞（PASMC) 來自Cambrex(Walkersville，MD)且根據供應商建議培養。 siRNA轉染。Axl及螢光素酶對照物（GL2)之siRNA購自 Dharmacon。基於先前公開之序列（Holland, 2005)之其他 Axl siRNA(Axl2及Axl4)係内部合成。siRNA轉染時，使用 脂染胺（lipofectamine)RNAiMax(Invitrogen)以 50 pmol siRNA轉染3 xlO5個細胞/孔（6孔培養盤）HUVEC，且轉染後 24至96小時藉由FACS定量所阻斷之Axl基因表現。 產生可誘導性Αχ丨shRNA細胞株。shRNA載體（pHUSH-GW)包含shRNA表現穿梭質體及反轉錄病毒載體骨架，該 反轉錄病毒載體骨架含有TetR-IRES-Puro卡匣以使得 shRNA表現能夠被四環素調節。使用脂染胺2000試劑 (Invitrogen)轉染293GP2細胞。轉染後48小時，收集傳染 性上清液，過濾且接著與所靶向之癌症細胞株一起培育24 小時。將對嘌呤黴素處理具有抗性之細胞合併以產生表現 AxlshRNA之穩定細胞株。 共培養。添加HUVEC前一天將PASMC(2.4xl05個細胞/ 孔）接種於塗有膠原蛋白之12孔培養盤中。用50 Pm〇l siRNA轉染早期繼代HUVEC(p<5)，且在轉染後24小時， 149799.doc -149- 201106972 以胰蛋白酶處理細胞且將4χ 104個細胞置放於單層PASMC 上。共培養細胞3天且藉由結合FITC之抗CD31抗體染色來 表徵。在ImageXpressMICR()成像系統下觀察且拍攝血管。 藉由MetaXpress軟體分析總管長度。 微陣列。用Axl或對照物siRNA轉染HUVEC。轉染後72 小時，使用RNeasy Plus小型套組（Qiagen)提取RNA。在 Affymetrix GeneChip Human Genome U133 Plus 2.0陣列 上、根據製造商方案執行HUVEC之mRNA表現分析。資料 分析及探針設定如本文中所描述。 產生抗Axl單株抗體。Axl細胞外域在CHO細胞中表現且 自CHO細胞純化。使用經純化AxlECD使小鼠免疫。針對 抑制Baf3 - Axl細胞之Gas6依賴性增殖之能力篩檢融合瘤上 清液。所選抗體進行次選殖及純化。 異種移植實驗。所有研究皆根據「實驗動物之護理及使 用準貝ij (Guidance for the Care and Use of Laboratory Animals)」（NIH)進行且由機構性動物護理及使用委員會 (Institutional Animal Care and Used Committee ; IACUC)批 准。本文中描述詳細方案。 正位轉移模型。依序用AxlshRNA及dsRedLuc感染乳癌 細胞株MDA-MB-231以產生可表現Dox可誘導性AxlshRNA 與螢光素酶報導基因的穩定細胞株。將1 〇5個細胞植入 SCID小鼠之乳腺脂肪塊中。植入後次日’將小鼠分為Dox 處理組及未處理組（每組n=5)。當原發性腫瘤尺寸達到 1000 mm3時，移除腫瘤。移除原發性腫瘤後5週’移除肺 149799.doc 150· 201106972 用於生物發光成像。 生物發光成像》將200 μι含25 mg/mL D-螢光素 (Invitrogen，Carlsbad，CA)之PBS腹膜内注入小鼠且在成像 期間使用異氟烷（Henry Schein，Sparks，NV)經由鼻錐麻醉 小既。使用固疋至不透光成像室之冷卻式加強電荷輕合裝 置攝影機（Stanford Photonics, Palo Alto, CA)獲得生物發光 影像。影像獲取時間通常小於5分鐘，且藉由將參考影像 Q 與生物發光資料影像共寄存來處理。離體成像時，獲取 肺，在PBS中沖洗且浸泡於1〇〇 μ丨25 mg/mL D_螢光素納鹽 中。 實例1. Axl頻繁過度表現於癌細胞株中 使用微陣列分析法篩檢一大批代表多種腫瘤類型之癌細 胞株用於Axl mRNA表現。Axl展現自僅可偵測之量至極高 量之廣泛表現’甚至在相同組織類型中（圖1A)。Αχί過度 表現於多種癌細胞株中，其中多形性神經膠母細胞瘤 Q (GBM)及胰臟癌中頻率最高。使用免疫墨點法測定所選癌 細胞株中Axl蛋白含量。Axl高度表現於所有經測試之癌細 胞株中’包括GBM、***癌、胰臟癌、結腸癌、NSCLC 及乳癌（SH-SY5Y(—種已知不表現Axl之神經母細胞瘤細胞 株）用作陰性對照）（圖1B)。此外，配位體Gas6共同表現於 此等癌細胞株中（圖1B)。為測定Axl是否經組成性活化， 藉由ELISA量測磷酸化Axl之含量。所有經測試之癌細胞 株均具有可偵測之磷酸化Axl含量（圖1C)。用外源添加之 Gas6處理細胞可顯著誘導A172、Calul及H1299細胞中之 149799.doc -151 - 201106972 進一步Axl磷酸化，但不能誘導其他細胞株中之進一步Axl 磷酸化。此等結果表明Axl在腫瘤細胞株中在不同程度上 被組成性活化（可能經由内源Gas6之自分泌作用）。 實例2.阻斷癌細胞株中之Axl基因表現可降低細胞存活 率且減弱其遷移 為檢查Axl表現在腫瘤生長、遷移及轉移中之功能性意 義，建立其中Axl表現可藉由多西環素（doxycycline)可誘導 性AxlshRNA阻斷的穩定細胞株。首先測試六種Axl siRNA 序列抑制人類臍靜脈内皮細胞（HUVEC)中Axl表現之能 力。所有六種Axl特異性siRNA皆使Axl蛋白含量降低超過 90%，如轉染後48小時FACS所量測（圖10A及圖10B)，而對 照物siRNA對Axl蛋白質無影響。接著，使用以一種Axl siRNA(Axl-4)為基礎之短髮夾RNA(shRNA)產生可誘導性 阻斷Axl基因表現之穩定細胞株。此等細胞株作為細胞池 產生以防止純系變異。 用Dox處理穩定表現AxlshRNA之癌細胞株72小時使得 Axl蛋白質表現幾乎被完全抑制（圖2A)。阻斷Axl基因表現 伴隨下游信號傳導事件之抑制。儘管Gas6在未經Dox處理 之親本H1299及H1299AxlshRNA細胞中誘導Akt磷酸化， 但Gas6誘導之Akt磷酸化在阻斷Axl基因表現之細胞中完全 中止，與經可溶性受體AxlFc處理之細胞類似（圖2B)。 為測試阻斷Axl基因表現對活體外癌細胞之作用，執行 細胞增殖及遷移分析法。Dox誘導阻斷Axl基因表現可降低 A549(50%)及 MDA-MB-23 1(330/。）之細胞存活率（圖 2C)。儘 149799.doc -152· 201106972 管在Gas6不存在下阻斷HI299細胞中之Axl基因表現不影響 細胞存活率，但其使Gas6介導之培養於低血清中之細胞之 存活率停止增加（圖2C)。阻斷Axl基因表現可顯著減弱 MDA-MB-231(40%)及 A549(48%)細胞（圖 2D)之遷移。此等 結果表明，視細胞情形而定，Gas6-Axl信號傳導在不同程 度上促進癌細胞生長及遷移。 實例3.阻斷Axl基因表現可降低異種移植模型中之腫 瘤生長 為評估活體内癌細胞生長是否需要Axl表現，測試阻斷 Axl基因表現對小鼠中之NSCLC A549及H1299及乳癌MDA-MB-23 1異種移植物之生長的影響。將穩定表現AxlshRNA 之細胞分別皮下植入裸小鼠（A549及H1299)或SCID小鼠 (MDA-MB-231)中。對於A549及H1299模型，植入後將小 鼠隨機分為處理組及對照組且分別投與含多西環素（Dox) 或蔗糖之飲用水。與未處理小鼠相比，Dox誘導之Axl沉默 引起A549腫瘤生長能力之顯著減弱（圖3A)。Dox誘導之阻 斷H1299細胞中之Axl基因表現引起裸小鼠中腫瘤生長之中 度（25%)抑制（圖3B)。對於MDA-MB-231模型，讓異種移植 腫瘤生長至200 mm3之平均尺寸；接著將小鼠隨機分為未 處理組或Dox處理組。阻斷Axl基因表現顯著降低所建立之 MDA-MB-231腫瘤之生長（33%抑制）（圖3C)。在所建立之 A549腫瘤模型中獲得類似結果（資料未圖示）。研究結束 時，藉由西方墨點法分析腫瘤溶解產物來檢驗Axl基因表 現之有效阻斷（圖3)。此等結果表明ΑχΗ言號傳導有助於 149799.doc -153- 201106972 A549、出299及河0八-]^8-231腫瘤異種移植物之活體内生 長。 實例4.阻斷Αχ丨基因表現可抑制MDA-MB-231乳癌細胞 轉移至肺 已證明Axl促進活體外癌細胞之遷移及侵襲性（圖2d，The Axl and/or VEGF antagonist compound is administered, for example, by direct injection, and the injection can be repeated periodically. The Axl and/or VEGF antagonist can also be delivered systemically to the individual or directly to the tumor cells, e.g., after surgical removal of the tumor to the tumor or tumor bed to prevent or reduce local recurrence or metastasis. In the case where the inhibitor is an antibody, the antibody to be administered is preferably a naked antibody. However, the inhibitors administered can be combined with cytotoxic agents. The inhibitor of the binding and/or the antigen bound thereto is preferably intracellularly internalized, thereby increasing the therapeutic efficacy of the immunological complex to kill the labeled cardiac cell to which it binds. In the preferred embodiment 149799.doc • 146. 201106972, the cytotoxic agent interferes with or interferes with nucleic acids in cancer cells. Examples of such cytotoxic agents include maytansinoids, calicheamicin, ribonucleases and DNA endonucleases. Suitable dosages for any of the therapeutic agents listed herein are those currently in use and may be suitably reduced or increased by the treating physician. The combination of therapeutic agents is typically administered for a defined period of time (typically minutes, hours, days or weeks, depending on the combination selected). Combination therapies are intended to comprise 〇 sequential administration of such therapeutic agents (i.e., wherein each therapeutic agent is administered at different times) and concurrent administration of such therapeutic agents or at least two therapeutic agents. The therapeutic agent can be administered by the same route or by different routes. For example, a combination of a ¥ca and/or an Axl antagonist can be administered by intravenous injection, and the combined protein kinase inhibitor can be administered orally. Alternatively, for example, depending on the particular therapeutic agent, the two therapeutic agents can be administered orally or by intravenous injection of the fork/two sputum therapeutic agents. Depending on the particular agent, the order of administration of the therapeutic agent can also be changed. 〇 This case covers the administration of VEGF and/or AxW antagonists by gene therapy. For the production of intracellular antibodies using gene therapy, see, for example, WO 96/07321, published March 14, 2004. "It will be appreciated that any of the above therapeutic methods can be performed using the immunoconjugates of the invention in place of or in addition to antibodies. D. Articles In another aspect of the invention, there is provided an article comprising a substance suitable for use in the treatment, prevention, and/or delta-distance of the above conditions. The article comprises a container and a standard iron or pharmaceutical product on or associated with the container of 149799.doc - 147 - 201106972 (4). Suitable containers include, for example, bottles, vials, syringes and the like. The container is formed of a variety of materials such as glass or plastic.谷益 can be used in a variety of materials such as glass or plastic: a separate composition of the material or a combination of the composition with another composition that has a good preventive and/or diagnosing condition, and can be taken, for example, from a container. It may be an intravenous solution bag or vial that can be pierced by a hypodermic needle. At least one active agent in the composition is used in the treatment of the antibody roll composition of the present invention: =, , :. The label or the drug product instructions indicate that the composition comprises (4) the towel contains a combination of ♦ 'the composition comprising the antibody or immunological I of the invention, and (8) the second container containing the composition, the combination Pack 3 cytotoxic agents or other therapeutic agents. The present invention in this embodiment of the invention contains a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the preparation may further comprise 3 medicinally acceptable buffers (such as bacteriostatic water for injection (B(iv)), phosphate buffered saline, Ringer's solution (8) _S〇1Utl〇n), and The second (or third) container of the dextrose solution. It may further be desirable for commercial and user reasons, including other buffers, diluents, filters, needles and syringes. Examples are examples of the methods and compositions of the present invention. It will be appreciated that various other embodiments may be implemented in light of the general description provided above. A. Substances and methods Antibodies. The antibody was obtained from the following suppliers: mouse monoclonal against human Axl 149799.doc -148- 201106972 antibody (mAb) (pure line 6C8, Abnova, Taiwan) 'Accumulated Akt (Ser 473) mouse mAb and Akt multiple strain Antibody (Cell Signaling), a mouse mAb (R&D System) directed against human Gas6. The ELISA kit for phosphorylating Axl was purchased from R&D System. Cell lines and cultures. Human cancer cell lines are from ATCC. Primary human umbilical vein endothelial cells (HUVEC) and pulmonary artery smooth muscle cells (PASMC) were obtained from Cambrex (Walkersville, MD) and cultured according to the supplier's recommendations. siRNA transfection. siRNA for Axl and luciferase control (GL2) was purchased from Dharmacon. Other Axl siRNAs (Axl2 and Axl4) based on previously disclosed sequences (Holland, 2005) are internal synthesis. When siRNA was transfected, HUVEC was transfected with 3 pO5 cells/well (6-well plate) with 50 pmol siRNA using lipofectamine RNAiMax (Invitrogen), and blocked by FACS 24 to 96 hours after transfection. Broken Axl gene expression. An inducible Αχ丨shRNA cell line was produced. The shRNA vector (pHUSH-GW) comprises shRNA expressing a shuttle plastid and a retroviral vector backbone containing a TetR-IRES-Puro cassette to enable shRNA expression to be regulated by tetracycline. 293GP2 cells were transfected with lipofectamine 2000 reagent (Invitrogen). Forty-eight hours after transfection, infectious supernatants were collected, filtered and then incubated with the targeted cancer cell line for 24 hours. Cells resistant to puromycin treatment were pooled to produce stable cell lines expressing Axl shRNA. Co-culture. PASMC (2.4 x 105 cells/well) was inoculated into a collagen-coated 12-well culture dish the day before the addition of HUVEC. Early passage HUVECs were transfected with 50 Pm〇l siRNA (p<5), and 24 hours after transfection, 149799.doc -149- 201106972 cells were trypsinized and 4χ104 cells were placed in monolayer PASMC on. Cells were co-cultured for 3 days and characterized by staining with FITC-conjugated anti-CD31 antibody. Blood vessels were observed and photographed under the ImageXpressMICR() imaging system. The length of the manifold is analyzed by the MetaXpress software. Microarray. HUVECs were transfected with Axl or control siRNA. 72 hours after transfection, RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). mRNA performance analysis of HUVECs was performed on an Affymetrix GeneChip Human Genome U133 Plus 2.0 array according to the manufacturer's protocol. Data analysis and probe settings are as described herein. An anti-Axl monoclonal antibody was produced. The Axl extracellular domain is expressed in CHO cells and purified from CHO cells. Mice were immunized with purified AxlECD. The fusion tumor supernatant was screened for the ability to inhibit the Gas6-dependent proliferation of Baf3 - Axl cells. Selected antibodies are subjected to secondary selection and purification. Xenograft experiments. All studies were conducted in accordance with the "Guidance for the Care and Use of Laboratory Animals" (NIH) and approved by the Institutional Animal Care and Used Committee (IACUC). . Detailed solutions are described herein. A positive transfer model. The breast cancer cell line MDA-MB-231 was sequentially infected with AxlshRNA and dsRedLuc to produce a stable cell line expressing Dox-inducible Axl shRNA and luciferase reporter gene. One 〇5 cells were implanted into the mammary fat mass of SCID mice. Mice were divided into Dox treated and untreated groups (n=5 per group) the day after implantation. Tumors were removed when the primary tumor size reached 1000 mm3. Removal of the lungs 5 weeks after removal of the primary tumor 149799.doc 150· 201106972 For bioluminescence imaging. Bioluminescence imaging: 200 μM PBS containing 25 mg/mL D-luciferin (Invitrogen, Carlsbad, CA) was intraperitoneally injected into mice and isoflurane (Henry Schein, Sparks, NV) was used via the nose cone during imaging. The anesthesia is small. Bioluminescence images were obtained using a cooled enhanced charge light fixture camera (Stanford Photonics, Palo Alto, CA) that was fixed to an opaque imaging chamber. The image acquisition time is usually less than 5 minutes and is handled by co-registering the reference image Q with the bioluminescent image. For in vitro imaging, the lungs were harvested, rinsed in PBS and soaked in 1 μ μ 25 mg/mL D_luciferin sodium salt. Example 1. Axl is frequently overexpressed in cancer cell lines A large number of cancer cell lines representing various tumor types were screened for Axl mRNA expression using microarray analysis. Axl exhibits a wide range of performance from a detectable amount to a very high amount' even in the same tissue type (Fig. 1A). Αχί Excess is expressed in a variety of cancer cell lines, with the highest frequency of pleomorphic glioblastoma Q (GBM) and pancreatic cancer. The Axl protein content in selected cancer cell lines was determined using an immunostick method. Axl is highly expressed in all tested cancer cell lines including GBM, prostate cancer, pancreatic cancer, colon cancer, NSCLC and breast cancer (SH-SY5Y (a neuroblastoma cell strain known not to exhibit Axl) As a negative control) (Fig. 1B). Further, the ligand Gas6 was collectively expressed in these cancer cell lines (Fig. 1B). To determine if Axl was constitutively activated, the amount of phosphorylated Axl was measured by ELISA. All tested cancer cell lines had detectable phosphorylated Axl levels (Fig. 1C). Treatment of cells with exogenously added Gas6 significantly induced further Axl phosphorylation in A172, Calul and H1299 cells, but did not induce further Axl phosphorylation in other cell lines. These results indicate that Axl is constitutively activated to varying degrees in tumor cell lines (possibly via autocrine effect of endogenous Gas6). Example 2. Blocking the expression of Axl gene in cancer cell lines can reduce cell viability and attenuate its migration. To test the functional significance of Axl expression in tumor growth, migration and metastasis, establish Axl expression by doxycycline (doxycycline) a stable cell line that is inducible by Axl shRNA blockade. The ability of six Axl siRNA sequences to inhibit Axl expression in human umbilical vein endothelial cells (HUVEC) was first tested. All six Axl-specific siRNAs reduced the Axl protein content by more than 90%, as measured by FACS 48 hours after transfection (Figures 10A and 10B), whereas the control siRNA had no effect on Axl protein. Next, a short hairpin RNA (shRNA) based on an Axl siRNA (Axl-4) was used to generate a stable cell strain which inducibly blocked the expression of the Axl gene. These cell lines are produced as cell pools to prevent pure lineage variation. Treatment of cancer cells stably expressing Axl shRNA with Dox for 72 hours resulted in almost complete inhibition of Axl protein expression (Fig. 2A). Blocking Axl gene expression is accompanied by inhibition of downstream signaling events. Although Gas6 induces Akt phosphorylation in the Dox-treated parental H1299 and H1299Axl shRNA cells, Gas6-induced Akt phosphorylation is completely halted in cells that block Axl gene expression, similar to cells treated with soluble receptor AxlFc ( Figure 2B). To test the effect of blocking the expression of Axl gene on cancer cells in vitro, cell proliferation and migration assays were performed. Dox-induced blockade of Axl gene expression reduced cell viability of A549 (50%) and MDA-MB-23 1 (330/.) (Fig. 2C). 149799.doc -152· 201106972 Blocking the Axl gene expression in HI299 cells in the absence of Gas6 does not affect cell viability, but it stops the increase in the survival rate of Gas6-mediated cells cultured in low serum (Fig. 2C). Blocking Axl gene expression significantly attenuated migration of MDA-MB-231 (40%) and A549 (48%) cells (Fig. 2D). These results indicate that Gas6-Axl signaling promotes cancer cell growth and migration to varying degrees depending on the cell condition. Example 3. Blocking Axl gene expression can reduce tumor growth in xenograft models To assess whether Axl expression is required for cancer cell growth in vivo, the test blocks Axl gene expression in NSCLC A549 and H1299 and breast cancer MDA-MB- in mice. 23 1 Effects of xenograft growth. Cells stably expressing AxlshRNA were subcutaneously implanted into nude mice (A549 and H1299) or SCID mice (MDA-MB-231), respectively. For the A549 and H1299 models, the mice were randomly divided into a treatment group and a control group after implantation, and were administered with drinking water containing doxycycline (Dox) or sucrose, respectively. Dox-induced Axl silencing caused a significant decrease in A549 tumor growth ability compared to untreated mice (Fig. 3A). Dox-induced inhibition of Axl gene expression in H1299 cells caused moderate (25%) inhibition of tumor growth in nude mice (Fig. 3B). For the MDA-MB-231 model, xenograft tumors were grown to an average size of 200 mm3; mice were then randomized into untreated or Dox treated groups. Blocking the Axl gene showed a significant decrease in the growth of the established MDA-MB-231 tumor (33% inhibition) (Fig. 3C). Similar results were obtained in the established A549 tumor model (data not shown). At the end of the study, tumor lysates were analyzed by Western blotting to examine the effective blockade of Axl gene expression (Figure 3). These results indicate that rumor conduction contributes to the in vivo growth of 149799.doc-153-201106972 A549, 299 and WT-8-]^8-231 tumor xenografts. Example 4. Blocking Αχ丨 gene expression inhibits MDA-MB-231 breast cancer cells Metastasis to the lung Axl has been shown to promote migration and invasiveness of in vitro cancer cells (Fig. 2d,

Zhang等人，2008 ; Tai等人，2008);然而，仍需確定Axl 在癌症轉移中之作用。利用正位模型及藉由生物發光成像 監視腫瘤轉移來解決此問題。產生表現D〇x可誘導性 AxlshRNA與螢光素酶報導基因之穩定mDA_mb_23丨細胞 株。如活體外螢光素酶分析法所測定，此細胞株顯示高生 物發光度（資料未圖示）。 圖4A概述針對此項研究之實驗設計。首先提問Αχΐ表現 對乳癌細胞擴散至遠端器官位點是否有影響。細胞植入後 次曰開始多西環素處理。植人後2週，良好建立原發性腫 瘤。當原發性腫瘤達到1000 mm3之尺寸時將其移除，且移 除原發腫瘤後k視肺轉移5週。移除原發性腫瘤後5週， 在對照組5隻小鼠中的3隻小鼠肺中偵測到大規模轉移性病 灶而、、呈D〇x處理之動物（n=5)中無肺轉移跡象（圖4B，第t 、、且）肺中腫瘤病灶之存在係藉由狀£染& (圖4c)及藉由可 確定性地標記人類腫瘤細胞的c_Met(已知表現於mda_mb_ 231、.、田胞中之蛋白質）IHC(未圖示）確認。此等結果表明'Μ 促進乳癌細胞轉移至肺。接著提問—旦癌細胞定殖於肺中 後轉移ί生生長是否需要Αχ1表現。為解決此問題，讓原 發性腫瘤尺寸達到1〇〇〇 mm3，移除原發性腫瘤接著開始 149799.doc -154- 201106972 用Dox處理。移除原發性腫瘤後監視小鼠5週。經對照物處 理之小鼠與經Dox處理之小鼠均具有轉移性病灶（每組5隻 小鼠中的3隻小鼠）（圖4B，第2組）。此等結果表明一旦癌細 胞自身在肺中建立後，Axl對轉移性病灶生長並非關鍵。 實例5.阻斷HUVEC中之Axl基因表現可減少内皮小管 形成 先前研究已表明，Axl不僅過度表現於腫瘤中，而且過 度表現於周圍血·管細胞中（Hutterer等人）。為進一步評估 Axl在調節血管生成中之潛在作用，測試阻斷Axl基因表現 對内皮小管形成之影響。FACS分析證明Axl表現於HUVEC 表面上（圖5A)。轉染後48小時，在經Axl特異性siRNA轉染 之HUVEC中，Axl蛋白含量下調超過90%，而對照物螢光 素酶siRNA(GL2)無影響（圖5A)。阻斷HUVEC中之Axl基因 表現對細胞生長無顯著影響（圖1 0C)。 接著使用初級HUVEC/PASMC(肺動脈平滑肌細胞）共培 養分支小管形成分析法評估Axl在血管生成中之重要性。 與經對照物siRNA轉染之細胞相比，經Axl siRNA轉染之 HUVEC中依據小管長度定量之管形成減少約40%(圖5B)。 此等結果表明Axl涉及内皮小管形態形成之調節。 實例6. Axl調節血管生成因子Dickkopf同系物3(DKK3) 及促血·管生成素-2(Ang-2)之表現 為瞭解Axl調節血管生成所涉及的機制，對經Axl或對照 物siRNA轉染之HUVEC執行基因分析。圖6A列舉阻斷Axl 基因表現後HUVEC中上調或下調幅度最大的八種基因。 149799.doc -155- 201106972 其中兩種基因Ang-2及DKK3為已知的血管生成調節劑。藉 由對DKK3及Ang-2執行定量RT-PCR來確認微陣列結果。 此外，如ELISA所量測，阻斷Axl基因表現後，HUVEC溶 解產物及培養基中DKK3及Ang-2蛋白質之含量亦變化（圖 11)。 為瞭解Axl與DKK3或Ang-2之間的關係，測試阻斷DKK3 及Ang-2 siRNA基因表現對Axl表現之影響。阻斷DKK3及 Ang-2基因表現均不影響Axl蛋白含量（圖11)。此外，阻斷 DKK3基因表現對Ang-2無影響，阻斷Ang-2基因表現亦不 影響DKK3表現（圖11)。接著測試DKK3及Ang-2 siRNA對 内皮小管形成之作用。藉由RNAi阻斷DKK3基因表現可使 小管形成減少至與阻斷Axl基因表現類似之程度，而單獨 Ang-2 siRNA對小管形成無顯著作用（圖6C)。此等結果表 明DKO及Ang-2為Αχ1之下游標靶且可藉由不同信號傳導 路徑調節其表現。 實例7.阻斷Αχ丨基因表現與抗VEGF具有相加效應 因為VEGF為調節内皮細胞功能之主要因子，所以在j 管形成分析法中測試阻斷Axl基因表現與抗vegf單株抗體 -起之作用。用單獨抗而處理細胞弓丨起劑量依賴性小 管形成減少（未圖示），阻斷Axl基因表現與抗vegf一起可 增強對小管㈣之抑制（圖7)，阻斷咖3基因表現亦具有 類似結果（圖7)。此等結果表明，下調Αχ1表現與抗v咖具 有抑制小管形成的相加效應、。 實例8.產生阻斷Αχ丨功能之單株抗體 149799.doc -156- 201106972 使用RNAi之驗證資料表明Axl涉及腫瘤生長、轉移以及 血管生成之調節。因此著手開發針對Axl之單株抗體。開 發的基於細胞之分析法係直接測試Axl mAb阻斷Axl/Gas6 介導之細胞生長之能力。產生過度表現Axl之Baf3前B細胞 的穩定細胞株（圖12A)。Baf3細胞中Axl之表現引起Gas6依 賴性生長（圖12B)，其可由AxlFc以劑量依賴性方式抑制（圖 12C)。用人類AxlECD使小鼠免疫，且使用此分析法篩檢 一組融合瘤上清液。鑑別四種抗體阻斷Gas6誘導之 Baf3Axl細胞生長（圖12D)。經純化之抗體對Baf3Axl細胞 生長顯示劑量依賴性抑制，最強阻斷劑12A11展現約100 ng/ml之 IC5〇(圖 8A) 〇 為瞭解此等mAb抑制Axl之機制，進一步表徵此等抗 體。在固相ELISA中，Axl mAb 3G9及8B5阻斷配位體與受 體結合，而12A11及4F8對配位體結合無影響（圖8B1)。藉 由使用FACS測定Axl mAb阻斷Gas6與A549細胞之細胞表 面Axl結合之能力來確認此結果（圖8B2)。Axl mAb 12A11、8B5及3G9下調A549細胞中之受體表現，而4F8無 影響（圖8C)。4F8為西方墨點法偵測Axl之唯一抗體，表明 其識別線性抗原決定基（資料未圖示）。此外，12A11及3G9 抑制H1299細胞中經Gas6誘導之Axl磷酸化（圖8C)。 此等單株抗體不與鼠類Axl交叉反應（圖13 A)，其亦不與 相關受體Tyro3及Mer交叉反應（圖13B)。在交叉阻斷分析 法中，Axl mAb彼此不競爭（資料未圖示），表明其結合不 同抗原決定基。為定位mAb之結合抗原決定基，活體外轉 149799.doc -157- 201106972 錄及轉譯Axl ECD之不同部分（圖8D)且接著用作ELISA中 之抗原。3G9及8B5之抗原決定基似乎位於第一 Ig域内，與 其阻斷配位體與受體結合之能力一致，而12A11及4F8之抗 原決定基定位於第一纖維結合蛋白域中（圖8 D)。 實例9. Axl mAb抑制A549 NSCLC異種移植腫瘤生長 為評估mAb抑制Axl是否影響活體内腫瘤細胞生長，將 A549細胞皮下植入裸小鼠中。當腫瘤尺寸達到1 〇〇 mm3時 (圖9A中第0天），將動物隨機分組且用30 mg/kg Axl mAb或 對照抗體處理，每週2次。與對照物相比，Axl mAb顯著減 弱A549腫瘤生長，給藥後第40天抑制約40%(圖9A)。為研 究Axl mAb抑制腫瘤生長之機制，執行藥效學研究。投與 抗體後〇、24、48及72小時後切除腫瘤且產生腫瘤溶解產 物。西方墨點法分析表明，給藥後24小時Axl mAb下調腫 瘤中Axl表現，此下調持續72小時（圖9B)。對腫瘤樣品之 Ki67及卡斯蛋白酶3染色表明，給藥後72小時，Axl mAb 12A11引起Ki67染色減少20%(圖9C)及卡斯蛋白酶3染色增 加約50%(未圖示）。此等結果表明Axl mAb藉由下調受體表 現來減弱A549異種移植物生長，從而又引起腫瘤細胞之細 胞凋亡增強及細胞增殖減少。此等結果與阻斷A549細胞中 之Axl基因表現一致，證明Axl在調節此NSCLC細胞株之腫 瘤生長中起重要作用。 討論 此研究中已研究Axl在腫瘤形成（包括腫瘤生長、轉移以 及血管生成）中之多種作用。已使用可誘導性shRNA抑制 149799.doc -158- 201106972 人類癌細胞株中之Axl表現且證明Axl表現促進NSCLC及乳 癌模型中之腫瘤生長。分別使用Axl顯性負性突變體或 shRNA之先前研究表明，Axl表現促進神經膠母細胞瘤 (Vajkoczy等人，2006)及乳癌（Holland等人，2005)異種移 植模型中之腫瘤生長。然而，此等研究均在對照細胞株與 ' 其中在植入小鼠前Axl或其信號傳導經下調之細胞株之間 進行腫瘤生長速率比較。儘管此方法適用，但其無法評估 Axl抑制對腫瘤生長之直接影響。本發明方法係利用 〇Zhang et al, 2008; Tai et al, 2008); however, the role of Axl in cancer metastasis still needs to be determined. This problem is solved by using a positive model and monitoring tumor metastasis by bioluminescence imaging. A stable mDA_mb_23丨 cell line expressing D〇x inducible AxlshRNA and luciferase reporter gene was generated. This cell line showed high bioluminescence as measured by in vitro luciferase assay (data not shown). Figure 4A summarizes the experimental design for this study. First, ask questions about whether there is an effect on the spread of breast cancer cells to distant organ sites. After the cells were implanted, doxycycline treatment was initiated. Two weeks after implantation, a primary tumor was well established. When the primary tumor reached a size of 1000 mm3, it was removed and the primary tumor was removed and the lung metastasized for 5 weeks. Five weeks after the removal of the primary tumor, large-scale metastatic lesions were detected in the lungs of 3 of the 5 mice in the control group, and none of the animals treated with D〇x (n=5) Signs of lung metastasis (Fig. 4B, t, and) The presence of tumor lesions in the lungs is characterized by staining & (Fig. 4c) and c_Met (which is known to be expressed in mda_mb_) by definitively labeling human tumor cells. 231., protein in the cell, IHC (not shown) confirmed. These results indicate that 'Μ promotes the transfer of breast cancer cells to the lungs. Then ask questions - if the cancer cells colonize the lungs, it is necessary to transfer ί1 growth. To address this issue, the primary tumor size was 1〇〇〇 mm3, and the primary tumor was removed and then started. 149799.doc -154- 201106972 Treated with Dox. Mice were monitored for 5 weeks after removal of the primary tumor. Both the control treated mice and the Dox treated mice had metastatic lesions (3 out of 5 mice per group) (Fig. 4B, Group 2). These results indicate that Axl is not critical for the growth of metastatic lesions once the cancer cells themselves are established in the lung. Example 5. Blocking of Axl gene expression in HUVEC reduces endothelial tubule formation Previous studies have shown that Axl is not only overexpressed in tumors but also in peripheral blood and tubular cells (Hutterer et al.). To further assess the potential role of Axl in regulating angiogenesis, the effect of blocking Axl gene expression on endothelial tubule formation was tested. FACS analysis demonstrated that Axl was expressed on the surface of HUVEC (Fig. 5A). At 48 hours post-transfection, Axl protein content was down-regulated by more than 90% in HUVEC transfected with Axl-specific siRNA, whereas control luciferase siRNA (GL2) had no effect (Fig. 5A). Blocking the Axl gene expression in HUVEC had no significant effect on cell growth (Fig. 10C). The primary HUVEC/PASMC (Pulmonary Artery Smooth Muscle Cells) co-culture branch tube formation assay was then used to assess the importance of Axl in angiogenesis. Tube formation quantified by tube length was reduced by about 40% in HUVEC transfected with Axl siRNA compared to control siRNA transfected cells (Fig. 5B). These results indicate that Axl is involved in the regulation of endothelial tubular morphogenesis. Example 6. Axl regulates the expression of angiogenic factor Dickkopf homolog 3 (DKK3) and hematopoietic-tubulin-2 (Ang-2) to understand the mechanism involved in Axl regulation of angiogenesis, and transduction of Axl or control siRNA The HUVEC was subjected to genetic analysis. Figure 6A shows eight genes that up-regulate or down-regulate HUVECs after blocking Axl gene expression. 149799.doc -155- 201106972 Two of the genes Ang-2 and DKK3 are known angiogenesis regulators. The microarray results were confirmed by performing quantitative RT-PCR on DKK3 and Ang-2. In addition, as measured by ELISA, the content of DKK3 and Ang-2 protein in HUVEC-dissolved product and culture medium also changed after blocking the expression of Axl gene (Fig. 11). To understand the relationship between Axl and DKK3 or Ang-2, the effect of blocking the expression of DKK3 and Ang-2 siRNA genes on Axl expression was tested. Blocking DKK3 and Ang-2 gene expression did not affect Axl protein content (Figure 11). In addition, blocking DKK3 gene expression had no effect on Ang-2, and blocking Ang-2 gene expression did not affect DKK3 expression (Fig. 11). The effects of DKK3 and Ang-2 siRNA on endothelial tubule formation were then tested. Blocking DKK3 gene expression by RNAi reduced tubule formation to a similar extent as blocking Axl gene expression, whereas Ang-2 siRNA alone had no significant effect on tubule formation (Fig. 6C). These results indicate that DKO and Ang-2 are downstream targets of Αχ1 and their performance can be modulated by different signaling pathways. Example 7. Blocking Αχ丨 gene expression has an additive effect on anti-VEGF Since VEGF is a major factor regulating endothelial cell function, it is tested in the j-tube formation assay to block the expression of Axl gene and anti-vegf monoclonal antibody. effect. Treatment of cells with anti-inhibition dose-dependent decrease in tubule formation (not shown), blocking Axl gene expression together with anti-vegf enhances inhibition of tubules (IV) (Fig. 7) Similar results (Figure 7). These results indicate that the down-regulation of Αχ1 performance and the anti-v coffee have an additive effect of inhibiting tubule formation. Example 8. Generation of monoclonal antibodies that block sputum function 149799.doc -156- 201106972 Validation data using RNAi indicate that Axl is involved in tumor growth, metastasis, and regulation of angiogenesis. Therefore, the development of monoclonal antibodies against Axl was initiated. The cell-based assay developed directly tested the ability of Axl mAb to block Axl/Gas6-mediated cell growth. A stable cell line of Baf3 pre-B cells overexpressing Axl was generated (Fig. 12A). The expression of Axl in Baf3 cells caused Gas6 dependent growth (Fig. 12B), which was inhibited by AxlFc in a dose-dependent manner (Fig. 12C). Mice were immunized with human AxlECD and a set of fusion tumor supernatants were screened using this assay. Four antibodies were identified to block Gas6-induced Baf3Axl cell growth (Fig. 12D). The purified antibody showed a dose-dependent inhibition of Baf3Axl cell growth, and the strongest blocker 12A11 exhibited an IC5 约 of about 100 ng/ml (Fig. 8A). To understand the mechanism by which these mAbs inhibit Axl, these antibodies were further characterized. In the solid phase ELISA, Axl mAb 3G9 and 8B5 blocked the ligand binding to the receptor, while 12A11 and 4F8 had no effect on ligand binding (Fig. 8B1). This result was confirmed by measuring the ability of Axl mAb to block the binding of Gas6 to A549 cells on the cell surface by using FACS (Fig. 8B2). Axl mAbs 12A11, 8B5 and 3G9 down-regulated receptor expression in A549 cells, while 4F8 had no effect (Fig. 8C). 4F8 is the only antibody that detects Axl by Western blotting, indicating that it recognizes linear epitopes (data not shown). In addition, 12A11 and 3G9 inhibited Gas6-induced Axl phosphorylation in H1299 cells (Fig. 8C). These monoclonal antibodies did not cross-react with murine Axl (Fig. 13A), nor did they cross-react with the related receptors Tyro3 and Mer (Fig. 13B). In the cross-blocking assay, Axl mAbs did not compete with each other (data not shown), indicating that they bind to different epitopes. To localize the binding epitope of the mAb, a different portion of the Axl ECD (Figure 8D) was recorded and translated in vitro (Figure 8D) and used as an antigen in the ELISA. The epitopes of 3G9 and 8B5 appear to be located in the first Ig domain, consistent with their ability to block ligand-receptor binding, while the epitopes of 12A11 and 4F8 are localized in the first fibronectin domain (Fig. 8D) . Example 9. Axl mAb inhibits A549 NSCLC xenograft tumor growth To assess whether mAb inhibition of Axl affects tumor cell growth in vivo, A549 cells were subcutaneously implanted into nude mice. When the tumor size reached 1 〇〇 mm3 (Day 0 in Figure 9A), animals were randomized and treated with 30 mg/kg Axl mAb or control antibody twice a week. Axl mAb significantly attenuated A549 tumor growth compared to the control, inhibiting about 40% on day 40 after administration (Fig. 9A). To study the mechanism by which Axl mAb inhibits tumor growth, pharmacodynamic studies were performed. After administration of the antibody, the tumor was excised after 24, 48, and 72 hours, and a tumor lysate was produced. Western blot analysis showed that Axl mAb down-regulated Axl expression in tumors 24 hours after dosing, which was down-regulated for 72 hours (Fig. 9B). Ki67 and caspase 3 staining of tumor samples showed that Axl mAb 12A11 caused a 20% reduction in Ki67 staining (Fig. 9C) and an increase in caspase 3 staining of about 50% (not shown) 72 hours after administration. These results indicate that Axl mAb attenuates A549 xenograft growth by down-regulating receptor expression, which in turn leads to enhanced apoptosis and decreased cell proliferation of tumor cells. These results are consistent with the blockade of the Axl gene in A549 cells, demonstrating that Axl plays an important role in regulating tumor growth in this NSCLC cell line. Discussion The role of Axl in tumor formation, including tumor growth, metastasis, and angiogenesis, has been studied in this study. Inducible shRNA inhibition has been used 149799.doc -158- 201106972 Axl expression in human cancer cell lines and demonstrates that Axl expression promotes tumor growth in NSCLC and breast cancer models. Previous studies using Axl dominant negative mutants or shRNA, respectively, have shown that Axl expression promotes tumor growth in xenografts (Vajkoczy et al., 2006) and breast cancer (Holland et al., 2005) xenograft models. However, these studies compared tumor growth rates between control cell lines and 'cells where Axl or its signal transduction was down-regulated before implantation in mice. Although this method is applicable, it cannot assess the direct effect of Axl inhibition on tumor growth. The method of the invention utilizes

AxlshRNA之可誘導表現且在Axl表現存在或不存在下量測 相同細胞株之生長。本發明結果表明Axl表現對腫瘤生長 之影響視所用細胞模型而定。儘管阻斷A549 NSCLC中之 Axl基因表現幾乎完全抑制腫瘤生長，但阻斷Axl基因表現 對H1299 NSCLC及MDA-MB-231之影響為中度的（約30%生 長抑制）。A549細胞對阻斷Axl基因表現之敏感度較高可能 歸因於此等細胞中存在增加之Axl基因複本數目（未公開結 〇 果）。 轉移在所有癌症死亡中佔約90% ;且在涉及癌形成的所 有過程中，局部侵襲及轉移形成在臨床上最相關。最新實 ' 驗進展已鑑別多種分子路徑及細胞機制可解釋轉移之多階 ·. 段過程。此等階段包括腫瘤侵襲、腫瘤細胞經由血液或淋 巴系統擴散、定殖於遠端器官中及轉移之過度生長 (Christofori，2006)。Axl表現與多種癌症中之侵襲及轉移 相關，包括乳癌（Meris等人，2002 ; Zhang等人，2008)、 肺癌（Shieh等人，2005)及胃癌（Sainaghi等人，2005)以及 149799.doc -159- 201106972 神經膠母細胞瘤（Hutterer等人，2008)。已活體外證明Αχ1 涉及促進癌細胞遷移及侵襲（Zhang等人，2008 ; Tai等人， 2008 ; Vajkoczy等人，2006)。在乳癌模型中，Αχ1之異位 表現足以向弱侵襲性MCF7細胞賦予高侵襲性表型。相 反’藉由阻斷shRNA基因表現或抗Axl抗體抑制Αχί信號傳 導可減少高侵襲性乳癌細胞之遷移及侵襲^儘管此等研究 確定活體外Axl促進細胞遷移及侵襲之作用，但尚未說明 Axl表現是否引起轉移。此研究中已使用正位乳癌模型研 究Axl在轉移中之功能性意義。本發明結果表明mdA-MB-23 1細胞需要Axl表現以在肺中建立轉移性病灶，因為 shRNA阻斷Axl基因表現可完全中止來自原發性腫瘤之細 胞定殖於肺中之能力。然而，一旦轉移性病灶建立於肺 中，Axl似乎對轉移之過度生長不具有顯著影響。此結果 表明Axl與MDA-MB-23 1癌細胞轉移之早期有關，且提供 Axl與轉移直接有關的第一活體内證據。Inducible expression of AxlshRNA and measurement of growth of the same cell line in the presence or absence of Axl expression. The results of the present invention indicate that the effect of Axl expression on tumor growth depends on the cell model used. Although blocking the Axl gene expression in A549 NSCLC almost completely inhibited tumor growth, the effect of blocking Axl gene expression on H1299 NSCLC and MDA-MB-231 was moderate (about 30% growth inhibition). The higher sensitivity of A549 cells to block the expression of the Axl gene may be due to the increased number of Axl gene copies in these cells (unpublished results). Metastasis accounts for approximately 90% of all cancer deaths; and local invasion and metastasis formation are most clinically relevant in all processes involving cancer formation. Recent developments have identified multiple molecular pathways and cellular mechanisms that explain the multi-step process of migration. These stages include tumor invasion, spread of tumor cells via the blood or lymphatic system, colonization in distant organs, and excessive growth of metastasis (Christofori, 2006). Axl performance is associated with invasion and metastasis in a variety of cancers, including breast cancer (Meris et al, 2002; Zhang et al, 2008), lung cancer (Shieh et al, 2005) and gastric cancer (Sainaghi et al, 2005) and 149799.doc - 159- 201106972 Glioblastoma (Hutterer et al., 2008). It has been demonstrated in vitro that Αχ1 is involved in promoting cancer cell migration and invasion (Zhang et al, 2008; Tai et al, 2008; Vajkoczy et al, 2006). In the breast cancer model, the ectopic appearance of Αχ1 is sufficient to confer a highly invasive phenotype to weakly invasive MCF7 cells. Conversely, 'inhibition of shRNA gene expression or anti-Axl antibody inhibition of Αχί signaling can reduce migration and invasion of highly invasive breast cancer cells. Although these studies have determined that Axl promotes cell migration and invasion in vitro, it has not yet demonstrated Axl expression. Whether it causes a transfer. The functional breast cancer model has been used in this study to study the functional significance of Axl in metastasis. The results of the present invention indicate that mdA-MB-23 1 cells require Axl expression to establish a metastatic lesion in the lung because shRNA blocking the Axl gene expression completely abolishes the ability of the cells from the primary tumor to colonize the lung. However, once metastatic lesions are established in the lungs, Axl appears to have no significant effect on excessive growth of metastases. This result indicates that Axl is involved in the early stage of MDA-MB-23 1 cancer metastasis and provides the first in vivo evidence that Axl is directly involved in metastasis.

Gas6/Axl信號傳導因調節血管平滑肌細胞及内皮細胞之 存活及遷移而在血管生物學中起重要作用（Melaragn〇等 人，1999)。Gas6/Axl在損傷動脈中之表現受到上調且 Gas6經由PI3K/Akt之活化誘導A類清除性受體，引起泡沫 細胞形成，此為動脈粥樣硬化之重要步驟（Ca〇等人， 2001)。活化Axl信號傳導可介導内皮細胞之層流剪切應力 之抗細胞凋亡效應’可能經由其與ανβ3整合素複合物之結 合介導此效應（D’Arcangelo等人，2006)。Αχι與Gas6共同 表現於神經膠質瘤中與腫瘤相關之血管細胞中（Hutterer等 149799.doc -160- 201106972 人，2008)。在原生人類乳癌樣品中，發現腫瘤受質細胞 中之強Axl染色（未公開觀測結果）。因此，Axl不僅可在腫 瘤細胞中影響腫瘤形成，而且調節腫瘤受質功能，諸如血· 管生成。此研究中，執行實驗以瞭解涉及Axl調節内皮細 胞功能之機制。本發明資料表明内皮細胞增殖不需要Axl ' 表現，因為RNAi阻斷Axl基因表現對此等細胞之生長具有 極小影響。然而，抑制Axl表現可減少内皮小管形成。 _ 為瞭解有關機制，分析mRNA表現以鑑別可受Axl表現調 ❹ 節之基因。阻斷Axl基因表現引起DKK3之下調及Ang-2之 上調，DKK3及Ang-2已知為涉及血管生成的兩種因子。 DKK3為Wnt信號傳導之Dickkopf分泌性調節劑家族的一員 (Krupnik等人，1999)，且強表現於小鼠及雞之正發育心臟 及血管中（Monaghan等人，1999)。最新研究證明DKK3蛋 白質表現於高度血管化贅瘤之血管中，包括GBM、高級非 霍奇金氏淋巴瘤、黑色素瘤及結腸直腸癌（Monaghan等 Q 人，1999)。B16F10細胞中鼠類DKK3之穩定過度表現可顯 著增加C57/BL6黑色素瘤模型中之微血管密度。此外，對 初級内皮群落形成細胞之活體外研究表明，siRNA阻斷 - DKK3基因表現並不顯著影響細胞增殖，而是減少基質膠 -. (matrigel)中之小管形成（Untergasser等人，2008)。此等結 果與阻斷Axl或DKK3基因表現後在HUVEC中之發現類 似。阻斷HUVEC中之Axl基因表現引起Ang-2之上調。促 血管生成素信號傳導系統在調節血管生成、血管恆定及血 管退化中起重要作用（Yancopoulos等人，2000)。此信號傳 149799.doc -161 - 201106972 導系統係由促血管生成素1及促血管生成素2及其受體Tie2 組成（Dumonut等人，1994)。Ang-1為經由pi3K/Akt信號傳 導路徑（Peters等人，2004)促進EC存活及内皮完整（§111^等 人，1998)之促效劑。另一方面，Ang_2(Mais〇npierre等 人，1997)為抑制Ang-Ι之作用以促進血管失去穩定的信號 傳導拮抗劑。在VEGF存在下，Ang-2提供重要的血管生成 刺激物（Yancopoulos等人，2000)。然而，當vEgf信號傳 導丈抑制或缺乏時，Ang-2對血管之促失穩作用引起細胞 死亡及毛細血管退化（Yanc〇p〇ul〇s等人，2〇〇〇)。本發明結 果證明，阻斷Axl基因表現與抗VGEF具有抑制内皮小管形 成的相加效應。當Axl表現受抑制時，可經由Ang_2之上調 來介導此相加效應。總而言之，本發明結果提出藉由Αχΐ 調節内皮細胞功能的以下模型。Ec中Αχ1之表現引起促血 管生成因子DKK3之上調及Ang_2之下調，從而又促進八叫_ 1/Tie2之信號傳導且引起血管生成增加。Gas6/Axl signaling plays an important role in vascular biology by regulating the survival and migration of vascular smooth muscle cells and endothelial cells (Melaragn et al., 1999). Gas6/Axl is up-regulated in injured arteries and Gas6 induces class A scavenging receptors via activation of PI3K/Akt, causing foam cell formation, an important step in atherosclerosis (Ca〇 et al., 2001). The anti-apoptotic effect of activated Axl signaling, which mediates laminar shear stress of endothelial cells, may mediate this effect via its association with the ανβ3 integrin complex (D’Arcangelo et al., 2006). Αχι and Gas6 are expressed together in tumor-associated vascular cells in gliomas (Hutterer et al. 149799. doc-160-201106972, 2008). In the native human breast cancer samples, strong Axl staining in tumor-derived cells was found (unpublished observations). Therefore, Axl not only affects tumor formation in tumor cells, but also regulates tumor function, such as blood-tube production. In this study, experiments were performed to understand the mechanisms involved in Axl regulation of endothelial cell function. The present invention indicates that Axl' expression is not required for endothelial cell proliferation because RNAi blockade of Axl gene expression has minimal effect on the growth of such cells. However, inhibition of Axl expression reduces endothelial tubule formation. _ To understand the mechanism, analyze mRNA performance to identify genes that can be regulated by Axl. Blocking Axl gene expression caused up-regulation of DKK3 and Upregulation of Ang-2, which are known to be involved in two factors of angiogenesis. DKK3 is a member of the Dickkopf secretory regulator family of Wnt signaling (Krupnik et al., 1999) and is strongly expressed in the developing heart and blood vessels of mice and chickens (Monaghan et al., 1999). Recent studies have shown that DKK3 protein is expressed in the blood vessels of highly vascularized tumors, including GBM, advanced non-Hodgkin's lymphoma, melanoma, and colorectal cancer (Monaghan et al., Q, 1999). Stable overexpression of murine DKK3 in B16F10 cells significantly increased microvessel density in the C57/BL6 melanoma model. In addition, in vitro studies of primary endothelial colony forming cells showed that siRNA blockade-DKK3 gene performance did not significantly affect cell proliferation, but reduced tubule formation in Matrigel (Untergasser et al., 2008). These results are similar to those found in HUVEC after blocking the expression of the Axl or DKK3 genes. Blocking the Axl gene expression in HUVEC caused up-regulation of Ang-2. The angiopoietin signaling system plays an important role in regulating angiogenesis, vascular constants, and vascular degeneration (Yancopoulos et al., 2000). This signal transmission 149799.doc -161 - 201106972 is composed of angiopoietin 1 and angiopoietin 2 and its receptor Tie2 (Dumonut et al., 1994). Ang-1 is an agonist that promotes EC survival and endothelial integrity via a pi3K/Akt signaling pathway (Peters et al., 2004) (§ 111^ et al., 1998). On the other hand, Ang_2 (Mais〇npierre et al., 1997) is a signal transduction antagonist that inhibits the action of Ang-Ι to promote the instability of blood vessels. Ang-2 provides important angiogenic stimuli in the presence of VEGF (Yancopoulos et al., 2000). However, when vEgf signaling is inhibited or deficient, Ang-2 promotes cell dysfunction and capillary degeneration (Yanc〇p〇ul〇s et al., 2〇〇〇). The results of the present invention demonstrate that blocking the expression of the Axl gene and anti-VGEF have an additive effect of inhibiting endothelial tubule formation. When Axl expression is inhibited, this additive effect can be mediated via an upregulation of Ang_2. In summary, the results of the present invention propose the following model for regulating endothelial cell function by Αχΐ. The expression of Αχ1 in Ec caused up-regulation of the pro-angiogenic factor DKK3 and down-regulation of Ang_2, which in turn promoted signaling of octopus _ 1/Tie2 and caused an increase in angiogenesis.

Axl在腫瘤形成中所起的多種作用使得其成為受關注之 癌症治療標靶。抗Axl抗體為可用於研究Αχ1在腫瘤形成中 之作用的特定試劑且提供抑制患者中Αχ1依賴性信號傳導 之治療可能。拮抗性mAb可由多種機制實現其功能：（1)阻 斷配位體與受體結合，（2)促進受體降解，阻斷受體二 聚，或（4)介導ADCC。因為許多腫瘤可展現配位體非依賴 性活化Axl，所以重要的是開發且比較經由不同作用模式 起作用之mAb且基於功效選擇主導mAb。此研究中已表徵 一組針對Axl之融合瘤抗體。已針對阻斷Αχ1生物功能之單 149799.doc •162- 201106972 株抗體開發出基於細胞之篩檢。本文中所表徵之mAb包括 兩種阻斷配位體與受體結合之mAb(3G9及8B5)，以及一種 不干擾配位體結合但抑制受體磷酸化之mAb(12All)。所 有3種mAb皆誘導受體表現之下調。最重要的是，此等 mAb減弱A549 NSCLC腫瘤生長。此等結果不僅證實Axl在 促進腫瘤生長中之作用（如shRNA研究所表明），而且表明 單株抗體可提供治療過度表現Axl之癌症的有效策略。因 為此等mAb不與鼠類Axl交叉反應，所以無法評估Axl對於 活體内腫瘤血管生成之重要性及其對腫瘤基質之影響。目 前已開發出交叉反應性mAb以研究活體内Axl之此等潛在 功能。 總而言之，本發明已驗證Axl在促進腫瘤生長中之作用 且提供乳癌細胞轉移至肺需要Axl表現之第一證據。本發 明已證明Axl經由促血管生成素及DKK3信號傳導系統起作 用來增強内皮小管形成。本發明資料表明針對Axl之治療 性抗體不僅可在腫瘤本身中、而且可在周圍基質中阻斷 Axl功能。Axl抑制與抗VEGF之相加效應表明阻斷Axl功能 可增強抗血管生成療法。 實例10 物質及方法 抗體及細胞株。抗體獲自以下供應商：針對人類Axl之 小鼠單株抗體（mAb)(Abnova, Taiwan)，填酸化Akt小鼠 mAb及Akt多株抗體（Cell Signaling)。用於石粦酸化Axl之小 鼠重組Gas6及ELISA套組購自R&D System。人類癌瘤細胞 149799.doc -163 - 201106972 株獲自ATCC且在補充有10% FBS之PRMI1640培養基中培 養。融合瘤抗人類Axl單株抗體12All&3GW^、*Genentech 提供（參見Li(2009))。 產生噬菌體抗Αχ丨單株抗體。為產生抗體，淘選使用人 類噬菌體抗體文庫，其在所選互補決定區（HI、Η2、Η3) 中具有模擬人類IgG譜系之天然多樣性的合成多樣性。Fab 片段二價呈現於Ml 3噬菌體顆粒表面上。針對人類及鼠類 Axl ECD多輪淘選噬菌體抗體文庫。與人類Axl ECD-His及 鼠類Axl ECD-Fc融合蛋白結合之噬菌體抗體係藉由ELISA 及DNA定序鑑別，且重組抗體純系以表現全長IgG(Liang 等人，2007)。個別純系短暫表現於哺乳動物細胞中且以 蛋白A管柱純化（Carter，1992)。 篩檢嗔菌體純系抑制Baf3-Axl細胞之Gas6依賴性增殖之 能力。選擇在抑制Baf3 Axl細胞增殖中展現最高效能的兩 個純系用於親和力成熟。 對於親和力成熟，在Ml 3噬菌體表面上呈現單價Fab之 嗜菌質體（Liang等人，2007)充當用於接枝噬菌體Ab之輕 鏈（VL)可變域及重鏈（VH)可變域之文庫模板。親和力成熟 係採用軟隨機化策略，如（Liang等人，2007)所述，且使用 高產量單點競爭性噬菌體ELISA快速篩檢高親和力純系， 如（Sidhu等人，2004)所述。 量測抗Axl抗體之親和力。測定抗Axl抗體之結合親和力 時，使用由BIAcore™-3000儀器量測表面電漿子共振 (SRP)。為量測抗Axl抗體與人類Axl ECD-His蛋白之間的 149799.doc -164- 201106972 親和力，藉由可達成約250個反應單位（RU)之塗有小鼠抗 人類IgG的CM5生物感測晶片捕捉抗Axl人類IgG。動力學 量測時，在25°C下以30 μΐ/min之流速注射人類Axl ECD-The multiple roles of Axl in tumor formation make it a target for cancer treatment of interest. Anti-Axl antibodies are specific agents that can be used to study the role of Αχ1 in tumor formation and provide a therapeutic potential to inhibit Αχ1-dependent signaling in a patient. Antagonistic mAbs can function by a variety of mechanisms: (1) blocking ligand binding to the receptor, (2) promoting receptor degradation, blocking receptor dimerization, or (4) mediating ADCC. Since many tumors can exhibit ligand-independent activation of Axl, it is important to develop and compare mAbs that function via different modes of action and to dominate the mAb based on efficacy. A set of fusion tumor antibodies against Axl has been characterized in this study. Cell-based screening has been developed for antibodies that block the biological function of Αχ1, 149799.doc • 162-201106972. The mAbs characterized herein include two mAbs (3G9 and 8B5) that block ligand binding to the receptor, and a mAb (12All) that does not interfere with ligand binding but inhibits receptor phosphorylation. All three mAbs induced receptor downregulation. Most importantly, these mAbs attenuated A549 NSCLC tumor growth. These results not only confirm the role of Axl in promoting tumor growth (as indicated by the shRNA study), but also suggest that monoclonal antibodies provide an effective strategy for treating cancers that overexpress Axl. Since mAb does not cross-react with murine Axl, the importance of Axl for tumor angiogenesis in vivo and its effect on tumor stroma cannot be assessed. Cross-reactive mAbs have been developed to study these potential functions of Axl in vivo. In summary, the present invention has verified the role of Axl in promoting tumor growth and provides the first evidence that Axl expression is required for breast cancer cells to metastasize to the lung. The present invention has demonstrated that Axl acts to enhance endothelial tubule formation via angiopoietin and the DKK3 signaling system. The present invention demonstrates that therapeutic antibodies directed against Axl block Axl function not only in the tumor itself, but also in the surrounding matrix. The additive effect of Axl inhibition and anti-VEGF suggests that blocking Axl function enhances anti-angiogenic therapy. Example 10 Substance and method Antibodies and cell lines. Antibodies were obtained from the following mouse monoclonal antibodies (mAb) against human Axl (Abnova, Taiwan), acidified Akt mouse mAb and Akt polyclonal antibody (Cell Signaling). The mouse Recombinant Gas6 and ELISA kits for the dendrite Axl were purchased from R&D System. Human carcinoma cells 149799.doc -163 - 201106972 strains were obtained from ATCC and cultured in PRMI 1640 medium supplemented with 10% FBS. The fusion tumor anti-human Axl monoclonal antibody 12All & 3GW^, *Genentech provides (see Li (2009)). A phage anti-sputum monoclonal antibody is produced. To generate antibodies, a human phage antibody library was used for panning, which has synthetic diversity mimicking the natural diversity of the human IgG lineage in the selected complementarity determining regions (HI, Η2, Η3). The Fab fragment is bivalently presented on the surface of the Ml 3 phage particle. A phage antibody library was panned against human and murine Axl ECD. Phage resistance systems that bind to human Axl ECD-His and murine Axl ECD-Fc fusion proteins were identified by ELISA and DNA sequencing, and recombinant antibodies were pure to express full length IgG (Liang et al., 2007). Individual pure lines are transiently expressed in mammalian cells and purified by Protein A column (Carter, 1992). Screening of the sputum strains inhibited the Gas6-dependent proliferation of Baf3-Ax1 cells. Two pure lines that exhibited the highest potency in inhibiting Baf3 Axl cell proliferation were selected for affinity maturation. For affinity maturation, plaques that exhibit a monovalent Fab on the surface of Ml 3 phage (Liang et al., 2007) serve as a light chain (VL) variable domain and a heavy chain (VH) variable domain for grafting phage Ab Library template. Affinity maturation was performed using a soft randomization strategy as described (Liang et al., 2007) and rapid screening of high affinity lines using high yield single point competitive phage ELISA as described (Sidhu et al., 2004). The affinity of the anti-Axl antibody was measured. Surface plasmon resonance (SRP) was measured by a BIAcoreTM-3000 instrument when determining the binding affinity of the anti-Axl antibody. To measure 149799.doc -164-201106972 affinity between anti-Axl antibody and human Axl ECD-His protein, CM5 biosensing coated with mouse anti-human IgG by approximately 250 reaction units (RU) The wafer captures anti-Axl human IgG. For kinetic measurements, human Axl ECD- was injected at a flow rate of 30 μΐ/min at 25 °C.

His於PBT緩衝液（含有0.05% Tween 20之PBS)中之兩倍連 續稀釋液（440 nM-28 nM)。使用簡單的一對一朗繆爾結合 ' 模型（BIAcore評估軟體3.2版）計算結合速率及解離速 率（k〇ff)。平衡解離常數（尤D)依據hff/tn比率計算。為量測 a 抗Axl抗體對鼠類Axl ECD-Fc融合蛋白之親和力，藉由可 〇 達成約150個反應單位（RU)之塗有小鼠抗人類IgG的CM5生 物感測晶片捕捉鼠類Axl ECD人類IgG融合蛋白。動力學量 測時，在25°C下以30 ml/min之流速注射抗Axl Fab片段於 PBST緩衝液（含有0.05% Tween 20之PBS)中之兩倍連續稀 釋液（200 nM-12 nM)。 細胞增殖分析法。細胞以5000個細胞/孔接種於96孔培 養盤中且用多種濃度之Axl mAb處理72小時。使用 Q CellTiter-Glo發光細胞存活率分析法（Promega)、根據製造 商說明書量測細胞增殖。 ELISA及螢光活化細胞分選（FACS)» ELISA分析法執行 •如下：用 0.5% BSA、PBS、0.05% Tween 20(PBST)阻斷塗 . 有山羊抗人類IgG Fc之培養盤。對於交叉反應分析法，在 室溫下將所塗培養盤與人類Axl.Fc、小鼠Axl.Fc或人類 Mer.Fc、Tyro-3 .Fc —起培育1小時，在PBST中洗蘇4次， 與抗Axl mAb及結合HRP之抗小鼠Ig—起培育。對於結合 分析法，在室溫下將所塗培養盤與人類Axl.Fc—起培育1 149799.doc -165 - 201106972 小時，在PBST中洗滌4次，在室溫下與rmGas 6及抗Axl mAb—起培育1小時。培養盤在PBST中洗滌4次，與結合生 物素之抗mGas6及抗生物蛋白鏈菌素-HRP—起培育。根據 製造商說明書、使用R&D ELISA套組量測所分泌之Ang-2 及DKK3。使用標準技術藉由FACS測定細胞表面上之Axl 表現。簡言之，收集細胞，在冰上用抗Axl mAb( 12 All ’ 10 ug/ml)染色30分鐘，在PBS中洗滌2次且接著用結合PE 之第二抗體染色。為測定抗體對Gas6與細胞表面上之Axl 結合的阻斷作用，收集細胞，用抗Axl mAb染色30分鐘且 在冰上與rmGas6 —起培育3 0分鐘。在PB S中洗蘇2次且用 結合生物素之抗Gas6及結合PE之抗生物蛋白鏈菌素染色。 樣品在BDFACScalibur流式細胞儀上分析。 異種移植實驗。所有研究皆根據「實驗動物之護理及使 用準則」（NIH)進行且由機構性動物護理及使用委員會 (IACUC)批准。將基質膠中總共5χ106(Α549)或107個細胞 (MDA-MB-231)分別皮下植入裸小鼠（A549)或SCID小鼠 (MDA-MB-231)之右侧腹中。當平均腫瘤尺寸達到100mm3 時，將小鼠隨機分為不同處理組（每組n= 10)。經由腹膜内 注射（IP)以10-3 0 mg/kg投與抗Axl或對照IgGl抗體，以1-2 mg/kg投與抗VEGF，每週2次。每天經口管飼100 mg/kg埃 羅替尼。處理開始時，分別地，每天皮下投與6.25 mg/kg 太平洋紫杉醇歷時5天，及皮下投與100 mg/kg單一劑量之 卡鉑。使用雙因子變異數分析（Anova two-way)執行統計分 析以比較不同處理組之腫瘤生長。 149799.doc -166 - 201106972 對於藥效學（PD)研究，用抗體處理小鼠0、24、72及168 小時。在各時間點切除腫瘤，處理以用於免疫組織化學染 色及影像分析，且用於產生細胞溶解產物以用於西方墨點 分析。 對於轉移研究，經由尾靜脈注射將經螢光素酶報導基因 穩定轉染之5xl05個MDA-MB-231(Li等人，2009)植入SCID 小鼠中。藉由生物發光偵測法監視腫瘤細胞向各器官之轉 移，如（Li等人，2009)所述。 免疫組織化學。將異種移植腫瘤樣品固定於10%中性緩 衝福馬林（formalin)中，處理，包埋於石蝶中且以4 μηι切 片。薄切片接著用針對Ki67、***卡斯蛋白酶3及MECA32 之一次抗體處理，接著用結合生物素之二次抗體及DAB色 素原處理。 自Cureline Inc.獲得原發性人類乳癌組織微陣列，包括 乳腺管及轉移性腺癌。使用先前描述之抗Axl單株抗體（Li 等人，2009)執行Axl IHC，且使用CD68染色巨噬細胞。對 於雙重 Axl/CD68 IHC，首先使用 Vector ABC Elite HRP試 劑及DAB受質以2 pg/ml執行Axl染色。亦使用ABC Elite-HRP試劑、但改用Vector SG色素原（藍色/灰色）以0.5 pg/ml 依序執行CD68染色。在兩種複合物之間執行第二標靶抗 原修復步驟以溶離第一複合物，從而避免兩種標記物之交 叉反應性。 血管密度量測及資料分析。腫瘤樣品用MECA32(—種泛 内皮細胞標記物）染色。藉由Ariol SL-50自動化載片掃描 149799.doc -167- 201106972 平台（Genetix Ltd.; Hampshire, UK)獲得100倍最終放大率 影像。腫瘤特定區域作為個別8位元影像輸出以供 Metamorph 套裝軟體（MDS Analytical Technologies, Ontario, Canada)分析。使用藍色正規化演算法、經由蘇木 精對比染色分離棕色DAB特異性染色，如（Brey等人， 2003)所述。分段演算法鑑別血管且基於尺寸及形狀移除 雜訊。細胞係基於蘇木精染色之尺寸、形狀及密度鑑別為 腫瘤或非腫瘤。非腫瘤區域係依據非腫瘤細胞密度與腫瘤 細胞密度之對比加以鑑別。完成分析後，人工再檢視影像 以移除錯誤鑑別為血管或腫瘤區域之假影。記錄個別血管 以及各影像中腫瘤及非腫瘤區域的面積量測。使用JMP 8.0 軟體（SAS Institute,Inc·, North Carolina, USA)分析基於 影像分析之原始數值。執行學生t檢驗以比較各對平均 值，其中ρ<〇·〇5。 分離腫瘤相關巨噬細胞（ΤΑΜ)及偵測所分泌之細胞激 素。切開腫瘤，斬為小塊及在含2.5% FBS、0.2單位/毫升 Liberase Blendzymes II及 5 單位/毫升 DNasel之 RPMI1640培 養基（Roche)中培育。使用MACS解離器（Miltenyi Biotec)解 離腫瘤細胞且在室溫下保持20分鐘。添加EDTA(最終濃度 0.002%)以終止反應。製備單細胞懸浮液且使用RBC溶解 緩衝液（eBioscience)移除紅血球。以107個細胞/毫升將細 胞再懸浮於含有1% FBS之PBS中且與20 pg/ml FcRII、 FcRIII及FcRIV —起培育20分鐘。添加抗F4/80-PE (eBioscience)及抗 CDllc-APC(BD Pharmingen)(0.2 pg/106 149799.doc -168- 201106972 個細胞）且在冰上培育30分鐘。藉由FACSAria(BD Biosciences)分選F4/80及CDllc陽性細胞。將總共2χ105個 F4/80及CD 11c陽性細胞接種於96孔培養盤中且培養隔夜。 收集培養基且使用Bio-Plex小鼠細胞激素分析法（Bio-Rad) 、 根據製 造商說 明書測 定細胞 激素及 趨化因 子之含 量。 抗原決定基定位 藉由標準分子生物學技術製備入\1-2(?11反111^\1(1-134Aa)/HuIgGlFc)、Axl-3(PRK HuAxl(l-221Aa)/HuIgGlFc)、 Axl-4(PRK HuAxl(l-324Aa)/HuIgGlFc)及 Axl-5(PRK HuAxl (l-435Aa)/HuIgGlFc)質體構築體。藉由直接定序及/或限 制消化確認所有質體。使用Promega L2080 TNT SP6快速 轉錄/轉譯系統套組活體外轉錄及轉譯編碼Axl細胞外域之 多個部分（aal-134、aal-221、aal-324、aal-435)的質體且 用作ELISA中之抗原。用於此等實驗之Axl部分包括： 人類Axl(包含Axl之Igl)之胺基酸1-134 : MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPF VGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILE LADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQC LVFLGHQTFVSQPGYVG (SEQ ID NO:18)，及 人類Axl(包含Axl纖維結合蛋白域）之胺基酸221-324 : ITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTL QAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHP HTPYHIRVACTSSQGPSSWTHWL (SEQ ID NO:19)。 149799.doc -169- 201106972 抗體競爭實驗 為測定噬菌體抗Axl mAb YW3 27.6或YW3 27.42是否與融 合瘤抗Axl mAb 12A11及3G9競爭，A549細胞用抗Axl嗤菌 體抗體（各為50 ug/ml)及融合瘤抗Axl抗體（各為10 ug/ml) 共染色30分鐘。細胞在PBS中洗滌2次且用抗小鼠Ig-PE染 色。如上針對FACS分析所述，樣品在BDFACScalibur流式 細胞儀（BD Biosciences)上分析。 結果 產生阻斷Αχ丨功能之噬菌體源單株抗體。為鑑別與鼠類His was in two-fold serial dilutions (440 nM-28 nM) in PBT buffer (PBS containing 0.05% Tween 20). The binding rate and dissociation rate (k〇ff) were calculated using a simple one-to-one Langmuir combination model (BIAcore Evaluation Software Version 3.2). The equilibrium dissociation constant (especially D) is calculated from the hff/tn ratio. To measure the affinity of a anti-Axl antibody to the murine Axl ECD-Fc fusion protein, rodent Axl was captured by a CM5 biosensing wafer coated with mouse anti-human IgG to achieve approximately 150 reaction units (RU) ECD human IgG fusion protein. For kinetic measurements, two-fold serial dilutions (200 nM-12 nM) of anti-Axl Fab fragments in PBST buffer (PBS containing 0.05% Tween 20) were injected at a flow rate of 30 ml/min at 25 °C. . Cell proliferation assay. Cells were seeded at 5,000 cells/well in 96-well culture dishes and treated with various concentrations of Axl mAb for 72 hours. Cell proliferation was measured using the Q CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. ELISA and fluorescence activated cell sorting (FACS) » ELISA assay performed • As follows: Blocking with 0.5% BSA, PBS, 0.05% Tween 20 (PBST). Plate with goat anti-human IgG Fc. For cross-reactivity analysis, the plate was incubated with human Axl.Fc, mouse Axl.Fc or human Mer.Fc, Tyro-3.Fc for 1 hour at room temperature, and washed 4 times in PBST. , incubated with anti-Axl mAb and anti-mouse Ig binding to HRP. For binding assays, the plate was incubated with human Axl.Fc at room temperature for 1 149799.doc -165 - 201106972 hours, washed 4 times in PBST, at room temperature with rmGas 6 and anti-Axl mAb - incubated for 1 hour. The culture plates were washed 4 times in PBST and incubated with biotin-conjugated anti-mGas6 and anti-bio-streptavidin-HRP. The secreted Ang-2 and DKK3 were measured using the R&D ELISA kit according to the manufacturer's instructions. Axl expression on the cell surface was determined by FACS using standard techniques. Briefly, cells were harvested, stained with anti-Axl mAb (12 All '10 ug/ml) for 30 minutes on ice, washed twice in PBS and then stained with a secondary antibody bound to PE. To determine the blocking effect of antibodies on the binding of Gas6 to Axl on the cell surface, cells were harvested, stained with anti-Axl mAb for 30 minutes and incubated with rmGas6 for 30 minutes on ice. The cells were washed twice in PB S and stained with biotin-resistant anti-Gas6 and PE-resistant streptavidin. Samples were analyzed on a BDFACScalibur flow cytometer. Xenograft experiments. All studies were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH) and approved by the Institutional Animal Care and Use Committee (IACUC). A total of 5χ106 (Α549) or 107 cells (MDA-MB-231) in Matrigel were subcutaneously implanted into the right abdomen of nude mice (A549) or SCID mice (MDA-MB-231), respectively. When the average tumor size reached 100 mm3, mice were randomized into different treatment groups (n=10 per group). Anti-Axl or control IgGl antibodies were administered via intraperitoneal injection (IP) at 10-3 0 mg/kg, and anti-VEGF was administered at 1-2 mg/kg twice a week. Oral gavage of 100 mg/kg erlotinib daily. At the beginning of the treatment, 6.25 mg/kg of paclitaxel was administered subcutaneously for 5 days, and a single dose of 100 mg/kg of carboplatin was administered subcutaneously. Statistical analysis was performed using a two-way variance analysis (Anova two-way) to compare tumor growth in different treatment groups. 149799.doc -166 - 201106972 For pharmacodynamic (PD) studies, mice were treated with antibodies for 0, 24, 72 and 168 hours. Tumors were excised at various time points, processed for immunohistochemical staining and image analysis, and used to generate cell lysates for Western blot analysis. For metastasis studies, 5xl05 MDA-MB-231 (Li et al., 2009) stably transfected with the luciferase reporter gene was implanted into SCID mice via tail vein injection. The transfer of tumor cells to various organs is monitored by bioluminescence detection as described (Li et al., 2009). immunochemistry. Xenograft tumor samples were fixed in 10% neutral buffered formalin, processed, embedded in stone butterflies and cut at 4 μηι. The thin sections were then treated with primary antibodies against Ki67, split caspase 3 and MECA32, followed by secondary antibodies bound to biotin and DAB chromogen. Primary human breast cancer tissue microarrays, including breast ducts and metastatic adenocarcinoma, were obtained from Cureline Inc. Axl IHC was performed using the previously described anti-Axl monoclonal antibody (Li et al, 2009) and macrophages were stained with CD68. For the dual Axl/CD68 IHC, Axl staining was first performed at 2 pg/ml using Vector ABC Elite HRP reagent and DAB substrate. CD68 staining was also performed sequentially at 0.5 pg/ml using ABC Elite-HRP reagent but using Vector SG chromogen (blue/grey). A second target antigen repair step is performed between the two complexes to dissolve the first complex, thereby avoiding cross-reactivity of the two labels. Vascular density measurement and data analysis. Tumor samples were stained with MECA32 (a pan-endothelial cell marker). The 100-fold final magnification image was obtained by the Ariol SL-50 automated slide scan 149799.doc -167- 201106972 platform (Genetix Ltd.; Hampshire, UK). Specific areas of the tumor were exported as individual 8-bit images for analysis by the Metamorph suite of software (MDS Analytical Technologies, Ontario, Canada). Brown DAB-specific staining was isolated via hematoxylin contrast staining using a blue normalization algorithm as described (Brey et al., 2003). The segmentation algorithm identifies blood vessels and removes noise based on size and shape. Cell lines were identified as tumors or non-tumors based on the size, shape and density of hematoxylin staining. Non-tumor regions were identified based on a comparison of non-tumor cell density to tumor cell density. After the analysis is completed, the image is manually reviewed to remove artifacts that are incorrectly identified as blood vessels or tumor areas. Area measurements of individual blood vessels and tumor and non-tumor areas in each image were recorded. Raw values based on image analysis were analyzed using JMP 8.0 software (SAS Institute, Inc., North Carolina, USA). A Student's t test is performed to compare pairs of averages, where ρ<〇·〇5. Tumor-associated macrophages (ΤΑΜ) are isolated and secreted cytokines are detected. Tumors were dissected and minced into small pieces and incubated in RPMI 1640 medium (Roche) containing 2.5% FBS, 0.2 units/ml Liberase Blendzymes II and 5 units/ml DNasel. Tumor cells were dissociated using a MACS dissociator (Miltenyi Biotec) and kept at room temperature for 20 minutes. EDTA (final concentration 0.002%) was added to terminate the reaction. Single cell suspensions were prepared and red blood cells were removed using RBC lysis buffer (eBioscience). The cells were resuspended at 107 cells/ml in PBS containing 1% FBS and incubated with 20 pg/ml FcRII, FcRIII and FcRIV for 20 minutes. Anti-F4/80-PE (eBioscience) and anti-CDllc-APC (BD Pharmingen) (0.2 pg/106 149799.doc -168-201106972 cells) were added and incubated on ice for 30 minutes. F4/80 and CDllc positive cells were sorted by FACSAria (BD Biosciences). A total of 2χ105 F4/80 and CD 11c positive cells were seeded in 96-well plates and cultured overnight. The medium was collected and the content of cytokines and chemoattractants was determined according to the manufacturer's instructions using the Bio-Plex Mouse Cytokine Assay (Bio-Rad). The epitope localization was prepared by standard molecular biology techniques into \1-2 (?11 anti111^1(1-134Aa)/HuIgGlFc), Axl-3 (PRK HuAxl(l-221Aa)/HuIgGlFc), Axl -4 (PRK HuAxl (l-324Aa)/HuIgGlFc) and Axl-5 (PRK HuAxl (l-435Aa)/HuIgGlFc) plastid constructs. All plastids were confirmed by direct sequencing and/or restriction digestion. The Promega L2080 TNT SP6 rapid transcription/translation system was used to in vitro transcription and translation of plastids encoding multiple parts of the Axl extracellular domain (aal-134, aal-221, aal-324, aal-435) and used as ELISA. Antigen. The Axl portion used in these experiments includes: Human Axl (Igl containing Axl) amino acid 1-134: MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPF VGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILE LADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQC LVFLGHQTFVSQPGYVG (SEQ ID NO: 18), and human Axl (containing Axl fibronectin domain) Amino acid 221-224: ITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTL QAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHP HTPYHIRVACTSSQGPSSWTHWL (SEQ ID NO: 19). 149799.doc -169- 201106972 Antibody competition assay to determine whether phage anti-Axl mAb YW3 27.6 or YW3 27.42 competes with fusion tumor anti-Axl mAb 12A11 and 3G9, and A549 cells use anti-Axl sputum antibody (50 ug/ml each) The fusion tumor anti-Axl antibodies (10 ug/ml each) were co-stained for 30 minutes. The cells were washed twice in PBS and stained with anti-mouse Ig-PE. Samples were analyzed on a BDFACScalibur flow cytometer (BD Biosciences) as described above for FACS analysis. As a result, a phage-derived monoclonal antibody that blocks sputum function was produced. For identification and rodents

Axl及人類Axl交叉反應之抗體，使用在所選互補決定區中 具有模擬人類IgG之天然多樣性之合成多樣性的噬菌體呈 現抗體文庫（Lee等人，2004)。藉由ELISA及DNA定序鑑別 與人類Axl ECD及鼠類Axl ECD均結合之噬菌體抗體，且 重組抗體純系以表現全長IgG(Liang等人，2007)。接著針 對抑制Baf3Axl細胞之Gas6依賴性生長之能力篩檢一組全 長IgG(Li等人，2009)，對純系YW327.6之一進行親和力成 熟及純化。 親和力成熟之Axl mAb YW327.6S2以高親和力結合人類 Axl與鼠類Axl，Kd分別為約1 nM及545 pM(圖14A)。特定 而言，Ka 為 1.7xl05，kd 為 1_7><104 且 KD 為 9.9><10_10。此抗 體亦結合獼猴Axl，但其不與相關受體Tyro3及Mer交叉反 應（圖14B)。如無細胞ELISA與細胞表面FACS所證明， YW327.6S2以劑量依賴性方式阻斷配位體Gas6與Axl結合 (圖 14C)。 149799.doc -170- 201106972 親和力成熟之Axl MAb YW327.6S11、YW327.42S8 及 YW327.42S31 亦已表徵。YW327.6S11、YW327.42S8 及 YW327.42S3 1以南親和力結合人類Axl與鼠類Axl。舉例而 言，對抗體結合人類Axl之biacore分析產生以下結果：Axl and human Axl cross-reactive antibodies use a phage display antibody library with synthetic diversity mimicking the natural diversity of human IgG in selected complementarity determining regions (Lee et al., 2004). Phage antibodies bound to both human Axl ECD and murine Axl ECD were identified by ELISA and DNA sequencing, and the recombinant antibodies were pure to express full length IgG (Liang et al., 2007). A set of full-length IgG (Li et al., 2009) was then screened for inhibition of the Gas6-dependent growth of Baf3Axl cells, and one of the pure lines of YW327.6 was affinity matured and purified. The affinity matured Axl mAb YW327.6S2 binds human Axl and murine Axl with high affinity, Kd of about 1 nM and 545 pM, respectively (Fig. 14A). Specifically, Ka is 1.7xl05, kd is 1_7><104 and KD is 9.9><10_10. This antibody also binds to macaque Axl, but it does not cross-react with the related receptors Tyro3 and Mer (Fig. 14B). As demonstrated by cell-free ELISA and cell surface FACS, YW327.6S2 blocked ligand Gas6 binding to Axl in a dose-dependent manner (Fig. 14C). 149799.doc -170- 201106972 Affinity matured Axl MAb YW327.6S11, YW327.42S8 and YW327.42S31 have also been characterized. YW327.6S11, YW327.42S8 and YW327.42S3 1 combined with human Axl and murine Axl with affinity. For example, the biacore analysis of antibodies in combination with human Axl produced the following results:

Hu AxlHu Axl

Ka kd KD YW327.6S11 1.7χ105 1.7x104 1.3x10! YW327.42S8 5.2χ104 1.3χ104 2.5x10! YW327.42S31 6.3 χΙΟ4 1.5χ104 2.4x10'Ka kd KD YW327.6S11 1.7χ105 1.7x104 1.3x10! YW327.42S8 5.2χ104 1.3χ104 2.5x10! YW327.42S31 6.3 χΙΟ4 1.5χ104 2.4x10'

此等抗體不與Tyro3及Mer交叉反應。YW327.6S2(及親本 抗體YW 327.6)阻斷配位體Gas6與Axl結合，而 YW327.42S8 及 YW327.42S31(及親本 Mab YW327.42)不阻 斷配位體Gas6與Axl結合。These antibodies do not cross-react with Tyro3 and Mer. YW327.6S2 (and the parent antibody YW 327.6) blocked the binding of the ligand Gas6 to Axl, while YW327.42S8 and YW327.42S31 (and the parent Mab YW327.42) did not block the binding of the ligand Gas6 to Axl.

YW327.6S2包含含有序列EVQLVESGGGLVQPGGSLRLSCA ASGFSLSGSWIHWVRQAPGKGLEWVGWINPYRGYAYYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYSG WGGSSVGYAMDYWGQGTLV(SEQ ID ΝΟ:1)的重鍵可變 區，及含有序列 DIQMTQSPSSLSASVGDRVTITCRAS QDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKR (SEQIDN0:2)的輕鏈可變區。YW327.6S2 contain sequences comprising EVQLVESGGGLVQPGGSLRLSCA ASGFSLSGSWIHWVRQAPGKGLEWVGWINPYRGYAYYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYSG WGGSSVGYAMDYWGQGTLV (SEQ ID ΝΟ: 1) the variable region of the heavy key, and contains the sequence DIQMTQSPSSLSASVGDRVTITCRAS QDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKR (SEQIDN0: 2) light chain variable region.

Axl抗原決定基分析 使用ELISA分析結合人類Axl細胞外域之多個部分的Axl 抗體。獲得結果。 149799.doc -171 - 201106972 YW327.6 YW327.42Axl epitope assay Axl antibodies that bind to multiple portions of the human Axl extracellular domain were analyzed using ELISA. Get the results. 149799.doc -171 - 201106972 YW327.6 YW327.42

Axl2 (aal-134) + -Axl2 (aal-134) + -

Axl-3 (aal-221) + -Axl-3 (aal-221) + -

Axl-4 (aal-324) + + YW3 27.6結合包含人類Axl之胺基酸1-134的Axl-Fc融合 物。相反，YW327.42結合包含人類Axl之胺基酸1-324之 Axl-Fc融合物，但不結合包含胺基酸1-134及1-221之Axl Fc融合物。因此得出結論：YW327.6結合由人類Axl之胺 基酸1-134組成之多肽且YW327.42結合需要人類Axl胺基酸 222-234。胺基酸1-134含有Axl Igl域且胺基酸222-234含有 Axl纖維結合蛋白域。 抗Axl融合瘤抗體之競爭實驗 測定抗體YW3 26.6及YW3 27.42是否可與抗Axl融合瘤抗 體12A11及3G9(Li, (2009))競爭結合人類Axl。在對人類 Axl之抗體競爭結合實驗中，抗體YW327.6不與融合瘤抗 體12A11或3G9中任一者競爭結合人類Axl，證明此等抗體 不識別相同抗原決定基。抗體YW327.42與融合瘤抗體 12A11競爭結合人類Axl，但不與融合瘤抗體3G9競爭結合 人類Axl。 YW327.6S2下調Axl表現，抑制其活化、信號傳導及 Gas6依賴性Baf3Ax丨細胞增殖 為測試YW327.6S2是否影響Axl生物功能，首先評估其 對Axl表現及信號傳導之影響。用YW327.6S2處理NSCLC 細胞株A549引起細胞表面上Axl表現之快速下調（圖14D， 149799.doc -172- 201106972Axl-4 (aal-324) + + YW3 27.6 binds to the Axl-Fc fusion comprising amino acid 1-134 of human Axl. In contrast, YW327.42 binds to the Axl-Fc fusion comprising amino acids 1-324 of human Axl, but does not bind to the Axl Fc fusion comprising amino acids 1-134 and 1-221. Therefore, it was concluded that YW327.6 binds to a polypeptide consisting of human Axl amino acid 1-134 and YW327.42 binds to human Axl amino acid 222-234. The amino acid 1-134 contains the Axl Igl domain and the amino acid 222-234 contains the Axl fibronectin domain. Competition experiments against anti-Axl fusion tumor antibodies Whether antibodies YW3 26.6 and YW3 27.42 compete with anti-Axl fusion tumor antibodies 12A11 and 3G9 (Li, (2009)) for binding to human Axl. In the antibody-binding assay for human Axl, antibody YW327.6 did not compete with any of the fusion tumor antibodies 12A11 or 3G9 for binding to human Axl, demonstrating that these antibodies do not recognize the same epitope. Antibody YW327.42 competes with fusion tumor antibody 12A11 for binding to human Axl but does not compete with fusion tumor antibody 3G9 for binding to human Axl. YW327.6S2 down-regulates Axl expression, inhibits its activation, signal transduction, and Gas6-dependent Baf3Ax丨 cell proliferation. To test whether YW327.6S2 affects Axl biological function, first evaluate its effect on Axl expression and signaling. Treatment of NSCLC cell line A549 with YW327.6S2 caused a rapid down-regulation of Axl expression on the cell surface (Fig. 14D, 149799.doc -172- 201106972)

上圖）且此下調持續24小時（圖14D，下圖）。用Gas6處理 H1299 NSCLC細胞可誘導Axl磷酸化，當細胞用 YW327.6S2預培育時，Axl磷酸化受抑制（圖14E，上圖）。 因此，用YW327.6S2預培育H1299細胞可阻斷Gas6誘導之 下游信號傳導分子Akt磷酸化（圖14E，下圖）。藉由 YW3 27.6S2下調Axl表現且使其信號傳導失活可有效抑制 Baf3Axl細胞之Gas6依賴性生長，其中IC50為340 ng/ml(圖 14F)。 YW327.6S2降低A549異種移植物生長且增強抗VEGF之 作用。在前述研究中，已顯示藉由RNAi抑制Axl或用抗人 類Axl融合瘤單株抗體處理可顯著減弱A549 NSCLC腫瘤生 長（Li等人，2009)。因此首先測試YW327.6S2對此模型中 之Μ瘤生長之影響。10 mg/kg單獨YW327_6S2，每週兩次 給藥方案顯著降低A549腫瘤生長（圖15A)，且此抑制效果 與抗人類Axl融合瘤抗體相當（圖15C)。Above) and this downgrade lasts for 24 hours (Fig. 14D, bottom). Treatment of H1299 NSCLC cells with Gas6 induced Axl phosphorylation, and Axl phosphorylation was inhibited when cells were pre-incubated with YW327.6S2 (Fig. 14E, top panel). Thus, pre-incubation of H1299 cells with YW327.6S2 blocked the phosphorylation of Gaskt-induced downstream signaling molecules Akt (Fig. 14E, lower panel). The down-regulation of Axl expression by YW3 27.6S2 and its signal transduction inactivation effectively inhibited Gas6-dependent growth of Baf3Ax1 cells with an IC50 of 340 ng/ml (Fig. 14F). YW327.6S2 reduces the growth of A549 xenografts and enhances the anti-VEGF effect. In the foregoing studies, inhibition of Axl by RNAi or treatment with anti-human Axl fusion tumor monoclonal antibodies has been shown to significantly attenuate A549 NSCLC tumor growth (Li et al, 2009). Therefore, the effect of YW327.6S2 on the growth of the tumor in this model was first tested. 10 mg/kg YW327_6S2 alone, twice weekly dosing regimen significantly reduced A549 tumor growth (Fig. 15A), and this inhibitory effect was comparable to that of the anti-human Axl fusion tumor antibody (Fig. 15C).

Axl表現於内皮細胞上且增強VEGF誘導之内皮小管形成 (Li等人，2009 ; Holland等人，2005);已測試抗 Axl mAb 是否可增強抗VEGF之抗腫瘤生長性質（Liang等人， 2006)。單獨抗VEGF抗體及單獨YW327.6S2對A549腫瘤生 長具有類似作用（圖15 A)。與任一種單獨抗體相比，兩種 抗體之組合可增強對腫瘤生長之抑制，其中單一藥劑抑制 3 0%，相比之下，組合處理抑制60%(圖15 A)。 此研究中之動物每週給藥2次歷時60天且隨後在第85天 檢查踵瘤生長之延緩（當腫瘤尺寸超過800 mm3時將動物自 149799.doc •173- 201106972 研究移除；無動物因中毒而移除）。單一藥劑相比， 丫\¥3 27.682與抗乂£0卩組合可顯著延緩腫瘤生長（圖15八）。 自最後一次給藥至實驗結束（第85天）所耗時間期間，組合 處理組中不存在腫瘤再生長，此組中之所有動物在實驗結 束時存活，如卡普蘭-麥爾（Kaplan-Meier)曲線所示（圖 15B)。 抗人類Axl融合瘤單株抗體12A11 (其不與鼠類Axl交叉反 應）顯著減弱A549異種移植腫瘤生長（Li等人，2009)，其亦 增強抗VEGF之作用，如圖15C所示。此為所期望的，因為 12A11直接抑制腫瘤細胞生長且抗VEGF影響腫瘤血管結 構。 YW327.6S2下調受體表現且誘導A549腫瘤細胞之細胞凋 亡。為開始瞭解介導YW327.6S2對減少腫瘤生長之作用的 機制，執行藥效學研究。用YW327.6S2處理含有A549腫瘤 之小鼠且在給藥後0、24、72及168小時切除腫瘤。西方墨 點法分析腫瘤溶解產物顯示，投與抗體後24小時，Axl表 現下調且持續168小時（圖15D)，表明YW327.6S2之抗腫瘤 生長效應部分地由Axl表現之下調介導。 為確定YW327.6S2是否對腫瘤細胞增殖及細胞凋亡具有 直接影響，切除經對照物或YW327.6S2處理兩週之A549異 種移植腫瘤且執行***卡斯蛋白酶3(CC3)及Ki67 IHC。與 經對照物處理之腫瘤相比，經YW327.6S2處理之腫瘤展現 增加之CC3(圖15E)，表明YW327.6S2誘導腫瘤細胞之細胞 凋亡。經對照物處理之腫瘤與經YW327.6S2處理之腫瘤之 149799.doc 174· 201106972 間不存在Ki67陽性細胞核之顯著差異，表明YW327.6S2不 直接影響腫瘤細胞增殖。 為研究YW327.6S2是否影響腫瘤相關血管結構，用單獨 YW327.6S2或用YW327.6S2與抗VEGF之組合處理含有 A549腫瘤之小鼠。切除腫瘤且用MECA32(—種泛内皮標 記物）染色以檢查瘤内血管密度。與對照物相比，單獨 YW327.6S2不顯著降低血管密度，但YW327.6S2與抗 VEGF之組合引起腫瘤相關血管密度之顯著降低（圖15F) 〇 相反，單獨12A11或其與抗VEGF之組合不顯著影響瘤内血 管密度。 YW327.6S2增強埃羅替尼及化學療法之作用。為測試 YW327.6S2是否可增加NSCLC之標準護理之治療指數，執 行YW327.6S2與EGFR小分子抑制劑（SMI)埃羅替尼及化學 療法之組合處理。 A549含有野生型EGFR且對埃羅替尼僅中度敏感（Yauch 等人，2005);因此研究抗Axl mAb是否可使此等細胞對 EGFR SMI敏感。YW327.6S2及埃羅替尼當作為單一藥劑 投與時引起腫瘤生長降低30%，但組合投與時降低腫瘤生 長速率超過50%(圖16A)，表明抗Axl mAb增強埃羅替尼之 抗腫瘤生長作用。 接著研究抗Axl mAb是否能夠增加NSCLC之標準化學療 法之治療指數。含有A549異種移植物之小鼠用一輪化學療 法處理，此輪化學療法係由在處理開始時（第0天，圖16B) 投與太平洋紫杉醇（6.25毫克/公斤/天，5天）及卡鉑（100 149799.doc -175- 201106972 mg/kg，單一劑量）組成。單獨化學療法與單獨投與 YW327.6S2對腫瘤生長具有類似作用，且兩者之組合可增 強對腫瘤生長之抑制（圖16B)。 在MDA-MB-231乳癌異種移植模型中，YW327.6S2降低 血管密度且抑制腫瘤相關巨噬細胞分泌發炎性細胞激素。 因為shRNA阻斷Axl基因表現對MDA-MB-231異種移植腫 瘤生長僅具有中度作用（Li等人，2009)，所以提問 YW327.6S2在此模型中是否有效。在此模型中，單獨 YW327.6S2能夠降低腫瘤生長（25°/。）且與用作單一藥劑之 抗VEGF具有類似效果（圖17A)。組合療法引起腫瘤生長降 低5 0%，表明YW3 27.6S2增強抗VEGF之作用（圖17A)。相 反，在此模型中，不與鼠類Axl交叉反應之抗Axl融合瘤抗 體12All(Li等人，2009)作為單一藥劑對腫瘤生長不具有 顯著作用，抗VEGF亦無影響（圖1 7B)。西方墨點分析顯示 YW327.6S2與12A11者均下調腫瘤中之Axl表現（圖17C及圖 17D)。此等結果表明YW327.6S2之抗腫瘤生長效應可藉由 調節腫瘤基質功能來介導。 為進一步研究YW327.6S2會如何調節腫瘤基質功能，用 單獨YW3 27.6S2或其與抗VEGF之組合處理含有MDA-MB-23 1之小鼠。在投與抗體後之多個時間點，切除腫瘤且用 MECA32染色以檢查瘤内血管密度。與對照物相比， YW327.0S2與抗VEGF均顯著降低血管密度（圖17E)。且兩 種抗體之組合進一步降低腫瘤相關灰管密度。此等結果表 明YW3 27.6S2藉由改變血管功能來部分地降低MDA-MB- 149799.doc -176- 201106972 23 1腫瘤生長。 在原發性人類乳癌樣品中，發現Axl蛋白質強表現於浸 潤巨噬細胞中（圖17F)且因此提問Axl mAb YW327.6S2是否 會影響腫瘤相關巨噬細胞（ΤΑΜ)功能。用YW327.6S2、 12Α11或對照抗體處理MDA-MB-231異種移植腫瘤1週，且 藉由分選F4/80陽性細胞來分離ΤΑΜ。細胞在不含血清之 培養基中培養隔夜，且收集上清液並針對各種細胞激素及 趨化因子之存在進行分析法。與經對照抗體處理之ΤΑΜ相 比，來自經YW327.6S2及12Α11處理之腫瘤的ΤΑΜ產生的 發炎性細胞激素及趨化因子量低得多（圖17G)。與此等抗 體下調腫瘤細胞上之Axl表現（圖17C及圖17D)相反，用任 一種Axl-mAb處理均不影響ΤΑΜ上之Axl表現量（資料未圖 示）。此等結果表明Axl mAb很可能以間接方式（可能藉由 阻斷腫瘤與基質細胞之間的聯絡）調節ΤΑΜ分泌發炎性細 胞激素/趨化因子。 YW327.6S2降低MDA-MB-231乳癌細胞轉移至骨骼。前 述研究中已顯示，shRNA阻斷Axl基因表現可抑制正位模 型中MDA-MB-23 1乳癌細胞轉移至肺（Li等人，2009)，因 此測試YW327.6S2是否影響此等細胞之轉移。經由尾靜脈 將穩定表現螢光素酶報導基因之MDA-MB-23 1細胞注入 SCID小鼠中。注射後四週，在對照抗體處理組中的所有5 隻動物之顱面區域、脛骨及股骨偵測到強螢光信號。在對 照組中，利用生物發光所偵測之位點在各動物中為5、5、 4、3及1個，總共18個（圖18A)。在經YW327.6S2處理之小 149799.doc -177- 201106972 鼠中’利用生物發光所偵測之位點顯著減少，各動物中為 0、1、2、3及1個位點且整個組為總共7個（圖1 8A)。藉由 組織分析檢驗骨骼中轉移性病灶之存在（圖1 8B)。此等結 果表明YW327.6S2能夠降低MDA-MB-231乳癌細胞轉移至 遠端器官。 討論 已開發且表徵展現交叉物種反應性且阻斷Αχί在腫瘤形 成中之多種功能的人類抗Axl單株抗體（YW327.6S2)。除作 為第一個所報導之針對Axl之完全人類化阻斷抗體外， YW3 27 ·6 S2不僅充當分析Axl活化/信號傳導在癌症發展及 進程之多方面中之影響的有力工具，而且代表治療多種癌 症之可能治療劑。 本發明結果表明YW327.6S2藉由下調Axl表現及抑制配 位體Gas6與受體結合從而引起Αχί及其下游信號傳導之失 活來阻斷Axl功能（圖14)。因為許多癌症表現組成性活化Axl is expressed on endothelial cells and enhances VEGF-induced endothelial tube formation (Li et al, 2009; Holland et al, 2005); whether anti-Axl mAb has been tested to enhance anti-tumor growth resistance of anti-VEGF (Liang et al, 2006) . The anti-VEGF antibody alone and YW327.6S2 alone had a similar effect on A549 tumor growth (Fig. 15 A). The combination of the two antibodies enhanced inhibition of tumor growth compared to either of the individual antibodies, with a single agent inhibiting 30% compared to 60% inhibition of the combination treatment (Figure 15 A). Animals in this study were administered twice a week for 60 days and then on day 85 to check for delays in tumor growth (when tumor size exceeded 800 mm3, animals were removed from study 149799.doc • 173-201106972; no animal Removed due to poisoning). Compared with the single agent, the combination of 丫\¥3 27.682 and anti-乂0卩 significantly delayed tumor growth (Fig. 15). There was no tumor regrowth in the combination treatment group from the last administration to the end of the experiment (Day 85), and all animals in this group survived at the end of the experiment, such as Kaplan-Meier The curve is shown (Figure 15B). Anti-human Axl fusion tumor monoclonal antibody 12A11 (which does not cross-react with murine Axl) significantly attenuated A549 xenograft tumor growth (Li et al, 2009), which also potentiated the anti-VEGF effect, as shown in Figure 15C. This is desirable because 12A11 directly inhibits tumor cell growth and anti-VEGF affects tumor vascular structures. YW327.6S2 downregulates receptor expression and induces cell apoptosis in A549 tumor cells. To begin understanding the mechanisms that mediate the effects of YW327.6S2 on reducing tumor growth, pharmacodynamic studies were performed. Mice containing A549 tumors were treated with YW327.6S2 and tumors were excised at 0, 24, 72 and 168 hours after dosing. Western blot analysis of tumor lysates showed that Axl expression was down-regulated and continued for 168 hours 24 hours after administration of the antibody (Fig. 15D), indicating that the anti-tumor growth effect of YW327.6S2 was partially mediated by a down-regulation of Axl expression. To determine whether YW327.6S2 had a direct effect on tumor cell proliferation and apoptosis, A549 xenograft tumors treated with control or YW327.6S2 for two weeks were excised and split caspase 3 (CC3) and Ki67 IHC were performed. Tumors treated with YW327.6S2 exhibited increased CC3 compared to control treated tumors (Fig. 15E), indicating that YW327.6S2 induced apoptosis in tumor cells. There was no significant difference in Ki67-positive nuclei between the control-treated tumor and the YW327.6S2-treated tumor 149799.doc 174·201106972, indicating that YW327.6S2 does not directly affect tumor cell proliferation. To investigate whether YW327.6S2 affects tumor-associated vascular structures, mice bearing A549 tumors were treated with YW327.6S2 alone or in combination with YW327.6S2 and anti-VEGF. Tumors were excised and stained with MECA32 (a pan-endothelial marker) to examine intratumoral vessel density. Compared to the control, YW327.6S2 alone did not significantly reduce vascular density, but the combination of YW327.6S2 and anti-VEGF caused a significant decrease in tumor-associated vascular density (Fig. 15F). Conversely, 12A11 alone or in combination with anti-VEGF was not Significantly affect intratumoral vascular density. YW327.6S2 enhances the effects of erlotinib and chemotherapy. To test whether YW327.6S2 can increase the therapeutic index of NSCLC's standard care, perform a combination of YW327.6S2 with EGFR small molecule inhibitor (SMI) erlotinib and chemotherapy. A549 contains wild-type EGFR and is only moderately sensitive to erlotinib (Yauch et al, 2005); therefore, whether anti-Axl mAbs can make these cells sensitive to EGFR SMI is investigated. YW327.6S2 and erlotinib caused a 30% reduction in tumor growth when administered as a single agent, but decreased tumor growth rate by more than 50% when administered in combination (Figure 16A), indicating that anti-Axl mAb enhances erlotinib resistance Tumor growth. It was then investigated whether anti-Axl mAb could increase the therapeutic index of standard chemotherapy for NSCLC. Mice containing A549 xenografts were treated with one round of chemotherapy, which was administered with paclitaxel (6.25 mg/kg/day, 5 days) and carboplatin at the start of treatment (Day 0, Figure 16B). (100 149799.doc -175- 201106972 mg / kg, single dose) composition. Chemotherapy alone and YW327.6S2 alone have similar effects on tumor growth, and the combination of the two enhances inhibition of tumor growth (Fig. 16B). In the MDA-MB-231 breast cancer xenograft model, YW327.6S2 reduced vascular density and inhibited the secretion of inflammatory cytokines by tumor-associated macrophages. Since shRNA blocking Axl gene expression has only a modest effect on MDA-MB-231 xenograft tumor growth (Li et al., 2009), it is questioned whether YW327.6S2 is effective in this model. In this model, YW327.6S2 alone was able to reduce tumor growth (25°/.) and had a similar effect to anti-VEGF used as a single agent (Fig. 17A). Combination therapy caused tumor growth to decrease by 50%, indicating that YW3 27.6S2 potentiated the anti-VEGF effect (Fig. 17A). In contrast, in this model, the anti-Axl fusion tumor antibody 12All (Li et al., 2009) that does not cross-react with murine Axl has no significant effect on tumor growth as a single agent, and anti-VEGF has no effect (Fig. 17B). Western blot analysis showed that both YW327.6S2 and 12A11 down-regulated Axl expression in tumors (Fig. 17C and Fig. 17D). These results indicate that the anti-tumor growth effect of YW327.6S2 can be mediated by regulating tumor matrix function. To further investigate how YW327.6S2 regulates tumor stroma function, mice containing MDA-MB-23 1 were treated with YW3 27.6S2 alone or in combination with anti-VEGF. At various time points after antibody administration, tumors were excised and stained with MECA32 to examine intratumoral vessel density. Both YW327.0S2 and anti-VEGF significantly reduced vascular density compared to the control (Fig. 17E). And the combination of the two antibodies further reduces tumor associated gray tube density. These results indicate that YW3 27.6S2 partially reduced MDA-MB-149799.doc-176-201106972 23 1 tumor growth by altering vascular function. In primary human breast cancer samples, Axl protein was found to be strongly expressed in infiltrating macrophages (Fig. 17F) and therefore asked whether Axl mAb YW327.6S2 would affect tumor-associated macrophage (ΤΑΜ) function. MDA-MB-231 xenograft tumors were treated with YW327.6S2, 12Α11 or control antibody for 1 week, and sputum was isolated by sorting F4/80 positive cells. The cells were cultured overnight in serum-free medium, and the supernatant was collected and assayed for the presence of various cytokines and chemokines. The amount of inflammatory cytokines and chemokines produced by sputum from YW327.6S2 and 12Α11 treated tumors was much lower compared to the control antibody treated sputum (Fig. 17G). In contrast to these antibodies down-regulating Axl expression on tumor cells (Fig. 17C and Fig. 17D), treatment with either Axl-mAb did not affect Axl expression on the sputum (data not shown). These results indicate that the Axl mAb is likely to regulate sputum secretion of inflammatory cytokines/chemokines in an indirect manner (possibly by blocking the communication between the tumor and stromal cells). YW327.6S2 reduces the transfer of MDA-MB-231 breast cancer cells to the bone. It has been shown in the foregoing studies that shRNA blocking Axl gene expression inhibits the metastasis of MDA-MB-23 1 breast cancer cells to the lung in the orthotopic model (Li et al., 2009), thus testing whether YW327.6S2 affects the metastasis of these cells. MDA-MB-23 1 cells stably expressing the luciferase reporter gene were injected into SCID mice via the tail vein. Four weeks after the injection, strong fluorescent signals were detected in the craniofacial region, the tibia and the femur of all five animals in the control antibody-treated group. In the control group, the sites detected by bioluminescence were 5, 5, 4, 3, and 1 in each animal, for a total of 18 (Fig. 18A). In the small 149799.doc -177-201106972 treated by YW327.6S2, the sites detected by bioluminescence were significantly reduced, 0, 1, 2, 3 and 1 loci in each animal and the whole group was A total of 7 (Figure 18A). The presence of metastatic lesions in the bone was examined by tissue analysis (Fig. 1 8B). These results indicate that YW327.6S2 is capable of reducing the metastasis of MDA-MB-231 breast cancer cells to distant organs. Discussion Human anti-Axl monoclonal antibodies (YW327.6S2) have been developed and characterized to exhibit cross-species reactivity and block multiple functions of Αχί in tumor formation. In addition to being the first fully humanized blocking antibody against Axl, YW3 27 ·6 S2 not only serves as a powerful tool for analyzing the effects of Axl activation/signaling in many aspects of cancer development and progression, but also represents treatment Possible therapeutic agents for a variety of cancers. The results of the present invention indicate that YW327.6S2 blocks Axl function by down-regulating Axl expression and inhibiting the binding of the ligand Gas6 to the receptor, thereby causing the inactivation of Αχ and its downstream signaling (Fig. 14). Because many cancers are constitutively activated

Axl且不再對外源Gas6有反應，所以¥貿327 682下調癌細 胞中之Axl表現的能力代表其抑制作用之重要機制（Li等 人，2009)。 在A549 NSCLC模型中，YW327.6S2當作為單一藥劑投 與時顯著減弱腫瘤生長（圖15A)。此抑制效果與抗Αχ1融合 瘤抗體在此模型中之抑制效果相當（Li等人（2〇〇9)，圖 i5C)。YW327.6S2快速下調異種移植物中之Αχΐ表現（圖 1 5D)，且誘導腫瘤細胞之細胞凋亡（圖丨5E)，此可能為介 導其腫瘤生長抑制作用的機制之一。Αχ1藉由調節〇〇：3及 149799.doc -178- 201106972 促血管生成素/Tie2路徑調節内皮細胞功能的先前發現（Li 等人，2009)提高抗Axl mAb可增強抗VEGF降低腫瘤生長 之作用的可能性。本發明結果（圖1 5 A及圖1 5F)與此假設一 致，亦即YW327.6S2藉由增強抗VEGF降低瘤内血管密度 之作用來影響腫瘤血管結構。實際上，在A549模型中， YW327.6S2與抗VEGF之共投與引起腫瘤停滯，此停滯在 停止處理後維持至少4週（圖15B)。融合瘤抗體12A11亦增 強抗VEGF在此模型中之抗腫瘤作用（圖15C)。然而，與 YW327.6S2不同，12A11對腫瘤血管結構不具有直接影 響；而是其直接抑制腫瘤細胞增殖及誘導細胞凋亡（Li等 人，2009)。當兩種藥劑一起使用時，12A11對腫瘤生長之 作用及抗VEGF對腫瘤血管結構之作用引起增加之效果。 諸如埃羅替尼之EGFR小分子抑制劑可有效治療具有 EGFR突變或擴增之NSCLC腫瘤（Lynch等人，2004 ; Paez 等人，2004 ; Eberhard 等人，2005 ; Giaccone 等人， 2005 ; Tsao等人，2005)。亦已知用Her2/EGFR小分子抑制 劑拉帕替尼（Lapatinib)或抗Her2抗體赫赛汀（Herceptin)處 理乳癌細胞可誘導Axl表現且因此導致癌細胞對此等療法 產生抗性（Liu等人，2009)。最近發現在已獲得針對埃羅替 尼之抗性的HCC827 NSCLC(具有EGFR突變及擴增之細胞 株）細胞中可誘導Axl表現且阻斷抗性細胞中之Axl基因表 現可恢復該等細胞對埃羅替尼之敏感性，表明Axl可在 NSCLC對埃羅替尼之抗性中起作用。因為A549細胞含有 野生型EGFR且對活體外EGFR抑制僅具中度敏感性（Yauch 149799.doc -179- 201106972 等人，2005)，所以提問抗Axl mAb是否會使此等細胞對埃 羅替尼敏感。本發明結果顯示YW327.6S2增強埃羅替尼降 低腫瘤生長之作用（圖1 6A)，表明抗Axl mAb可增強EGFR 抑制劑在單獨EGFR抑制難治性腫瘤中的作用（可能藉由直 接降低腫瘤細胞中之Axl表現）。 全身化學療法在NSCLC之治療範例中占最大比重。對於 復發性或晚期疾病，經由卡鉑/太平洋紫杉醇組成之化學 療法治療之患者的反應率為15%且中值存活期為10.3個月 (Sandley等人，2006)。本發明在A549 NSCLC模型中之結 果顯示YW327.6S2能夠增強卡鉑/太平洋紫杉醇之抗腫瘤功 效（圖16B)，表明阻斷Axl功能可提高此疾病之化學療法之 治療指數。本發明結果與最近證明Axl小分子抑制劑與順 鉑協同抑制4T1乳癌正位模型中之肝微轉移之報導（Holland 等人，2010)—致。 MDA-MB-231乳癌模型中，單獨YW327.6S2能夠顯著減 弱腫瘤生長（圖17A)。不與鼠類Axl交叉反應且因此僅會影 響腫瘤細胞之抗Axl融合瘤抗體對此模型中之腫瘤生長不 具有顯著作用（圖17B)。此等結果表明YW327.6S2可能經由 其對腫瘤基質之作用來發揮其抗腫瘤效應。本發明結果表 明YW3 27.6S2降低瘤内血管密度（圖17E)且增強抗VEGF之 作用。YW327.6S2經由其對腫瘤血管結構之作用可影響腫 瘤生長。 已長期瞭解癌症之發展與炎症之間的相關性（Balkwi 11及 Mantovani, 2001 ; Coussens及 Werb,2002)。與感染及刺激 149799.doc -180- 201106972 相關之慢性炎症可能產生培養基因組病變及腫瘤起始之環 境。愈來愈多的證據表明ΤΑΜ在包括促進血管生成及基質 再成型的腫瘤進展中具有因果作用（Balkwill等人，2005 ; Pollard, 2004)。導致此作用之信號被認為是發炎性細胞激 素，包括腫瘤壞死因子-a(TNF-a)、介白素6(IL-6)及過多 的趨化因子。此等細胞激素及趨化因子不僅募集刺激腫瘤 進展之免疫細胞至特定位點，而且顯示其受體表現於腫瘤 細胞上，從而其可增強腫瘤生長及遷移（Haghnegahdar等 人，2000)。在原發性人類乳癌中，Axl蛋白高量表現於腫 瘤相關巨噬細胞（ΤΑΜ)上（圖17F)。本文證明抗Axl mAb可 經由間接機制調節此等細胞之功能。本發明資料顯示，用 YW327.6S2或12A11(其不與鼠類Axl交叉反應且因此對 ΤΑΜ不應具有直接作用）處理MDA-MB-231可抑制ΤΑΜ分 泌發炎性細胞激素及趨化因子（圖17G)。因為Axl-mAb似乎 不是對ΤΑΜ上之Axl表現具有顯著作用，而是下調腫瘤細 胞上之受體表現，所以抗Axl-mAb可藉由阻斷腫瘤與基質 細胞之間的聯絡來調節ΤΑΜ分泌細胞激素/趨化因子。此 等結果與最新報導（腫瘤細胞可藉由教導浸潤性白血球誘 導Gas6之表現來促進自身生長（Loges等人，2010);且Axl 之小分子抑制劑降低4T1***腫瘤細胞中之GM-CSF表現 (Holland等人，2010))—致。 先前研究已確定Axl在促進腫瘤細胞遷移、侵襲及轉移 中之作用（Zhang等人，2008 ; Li等人，2009 ; Tai等人， 2008 ; Vajkoczy等人，2006 ; Gjerdrum等人，2010)。本文 149799.doc -181 - 201106972 顯示YW327.6S2能夠減少MDA-MB-231乳癌細胞轉移至 骨。此等結果與先前資料（藉由RNAi使乳癌細胞中之Axl沉 默可抑制正位模型中之乳癌細胞轉移至肺（Li等人， 2009 ; Gjerdrum等人，2010))—致，表明此抗Axl抗體可不 僅在治療性原發腫瘤上、而且在治療轉移性疾病上具有治 療潛能。 總而言之，已開發阻斷Axl功能之人類單株抗體。此抗 Axl mAb經由多種機制發揮其抗腫瘤作用，該等機制包括 誘導腫瘤細胞之細胞凋亡、調節血管生成及調節腫瘤相關 免疫細胞功能。此外，此抗Axl mAb增強抗VEGF、EGFR SMI以及化學療法之抗腫瘤功效，因此可代表臨床配置中 之新穎治療方法，其中此等療法為標準護理。 參考文獻Axl no longer responds to exogenous Gas6, so the ability of K. 327 682 to down-regulate Axl expression in cancer cells represents an important mechanism of its inhibition (Li et al., 2009). In the A549 NSCLC model, YW327.6S2 significantly attenuated tumor growth when administered as a single agent (Figure 15A). This inhibitory effect is comparable to that of the anti-Αχ1 fusion tumor antibody in this model (Li et al. (2〇〇9), Figure i5C). YW327.6S2 rapidly down-regulated the sputum expression in xenografts (Fig. 15D) and induced apoptosis of tumor cells (Fig. 5E), which may be one of the mechanisms that mediate its tumor growth inhibition. Αχ1 by regulating 〇〇:3 and 149799.doc -178- 201106972 Angiopoietin/Tie2 pathway regulates endothelial cell function (Li et al., 2009) Enhancing anti-Axl mAb enhances anti-VEGF and reduces tumor growth The possibility. The results of the present invention (Fig. 15A and Fig. 15F) are consistent with this hypothesis, that is, YW327.6S2 affects tumor vascular structure by enhancing the effect of anti-VEGF on reducing intratumoral vascular density. In fact, in the A549 model, co-administration of YW327.6S2 with anti-VEGF caused tumor stagnation, which was maintained for at least 4 weeks after discontinuation of treatment (Fig. 15B). The fusion tumor antibody 12A11 also potentiated the anti-tumor effect of anti-VEGF in this model (Fig. 15C). However, unlike YW327.6S2, 12A11 has no direct effect on tumor vascular structure; rather, it directly inhibits tumor cell proliferation and induces apoptosis (Li et al., 2009). When both agents are used together, the effect of 12A11 on tumor growth and the effect of anti-VEGF on tumor vascular structure results in an increased effect. EGFR small molecule inhibitors such as erlotinib are effective in treating NSCLC tumors with EGFR mutations or amplification (Lynch et al, 2004; Paez et al, 2004; Eberhard et al, 2005; Giaccone et al, 2005; Tsao et al. People, 2005). It is also known that treatment of breast cancer cells with the Her2/EGFR small molecule inhibitor Lapatinib or the anti-Her2 antibody Herceptin induces Axl expression and thus causes cancer cells to develop resistance to these therapies (Liu et al. People, 2009). It has recently been found that Axl expression can be induced in cells of HCC827 NSCLC (cell line with EGFR mutation and expansion) that have acquired resistance to erlotinib and that Axl gene expression in resistant cells can be restored to restore these cell pairs. The sensitivity of erlotinib indicates that Axl plays a role in the resistance of NSCLC to erlotinib. Because A549 cells contain wild-type EGFR and are only moderately sensitive to EGFR inhibition in vitro (Yauch 149799.doc -179-201106972 et al., 2005), ask whether anti-Axl mAb will make these cells erlotinib sensitive. The results of the present invention show that YW327.6S2 enhances the effect of erlotinib on reducing tumor growth (Fig. 16A), indicating that anti-Axl mAb enhances the role of EGFR inhibitors in inhibiting refractory tumors by EGFR alone (possibly by directly reducing tumor cells) In the Axl performance). Systemic chemotherapy is the largest proportion of NSCLC treatments. For patients with relapsed or advanced disease, patients treated with chemotherapy with carboplatin/paclitaxel had a response rate of 15% and a median survival of 10.3 months (Sandley et al., 2006). The results of the present invention in the A549 NSCLC model show that YW327.6S2 enhances the anti-tumor efficacy of carboplatin/paclitaxel (Fig. 16B), indicating that blocking Axl function increases the therapeutic index of chemotherapy for this disease. The results of the present invention are consistent with the recent demonstration that Axl small molecule inhibitors and cisplatin synergistically inhibit liver micrometastases in the 4T1 breast cancer orthotopic model (Holland et al., 2010). In the MDA-MB-231 breast cancer model, YW327.6S2 alone significantly reduced tumor growth (Fig. 17A). Anti-Axl fusion tumor antibodies that do not cross-react with murine Axl and therefore only affect tumor cells have no significant effect on tumor growth in this model (Fig. 17B). These results indicate that YW327.6S2 may exert its anti-tumor effect via its action on tumor stroma. The results of the present invention show that YW3 27.6S2 reduces intratumoral vascular density (Fig. 17E) and enhances the anti-VEGF effect. YW327.6S2 affects tumor growth via its effect on tumor vascular structures. A long-standing correlation between cancer development and inflammation has been known (Balkwi 11 and Mantovani, 2001; Coussens and Werb, 2002). Chronic inflammation associated with infection and irritation 149799.doc -180- 201106972 may result in the development of genomic lesions and the initial onset of the tumor. Increasing evidence suggests that sputum has a causal role in tumor progression including angiogenesis and matrix remodeling (Balkwill et al., 2005; Pollard, 2004). The signal that causes this effect is considered to be inflammatory cytokines, including tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and excessive chemokines. These cytokines and chemokines not only recruit immune cells that stimulate tumor progression to specific sites, but also show that their receptors are expressed on tumor cells, thereby enhancing tumor growth and migration (Haghnegahdar et al., 2000). In primary human breast cancer, Axl protein is highly expressed on tumor-associated macrophages (ΤΑΜ) (Fig. 17F). It is demonstrated herein that anti-Axl mAbs can modulate the function of such cells via indirect mechanisms. The present invention shows that treatment of MDA-MB-231 with YW327.6S2 or 12A11 (which does not cross-react with murine Axl and therefore should not have a direct effect on sputum) inhibits sputum secretion of inflammatory cytokines and chemokines (Fig. 17G). Because Axl-mAb does not appear to have a significant effect on Axl expression on sputum, but downregulates receptor expression on tumor cells, anti-Axl-mAb can regulate sputum secreting cells by blocking the connection between tumor and stromal cells. Hormone / chemokine. These results and the latest reports (tumor cells can promote self-growth by teaching invasive leukocyte-induced Gas6 expression (Loges et al., 2010); and small molecule inhibitors of Axl reduce GM-CSF expression in 4T1 breast tumor cells (Holland et al., 2010)). Previous studies have determined the role of Axl in promoting tumor cell migration, invasion, and metastasis (Zhang et al, 2008; Li et al, 2009; Tai et al, 2008; Vajkoczy et al, 2006; Gjerdrum et al, 2010). This article 149799.doc -181 - 201106972 shows that YW327.6S2 can reduce the metastasis of MDA-MB-231 breast cancer cells to bone. These results are consistent with previous data (silencing of Axl in breast cancer cells by RNAi inhibits the metastasis of breast cancer cells in the orthotopic model to the lungs (Li et al., 2009; Gjerdrum et al., 2010)), indicating that this anti-Axl Antibodies can have therapeutic potential not only in therapeutic primary tumors, but also in the treatment of metastatic disease. In summary, human monoclonal antibodies that block Axl function have been developed. This anti-Axl mAb exerts its anti-tumor effects via a variety of mechanisms including induction of apoptosis of tumor cells, regulation of angiogenesis, and regulation of tumor-associated immune cell functions. In addition, this anti-Axl mAb enhances the anti-tumor efficacy of anti-VEGF, EGFR SMI, and chemotherapy, and thus represents a novel therapeutic approach in clinical settings where such therapies are standard care. references

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Zhang YX, Knyazev PG, Cheburkin YV, Sharma K, Knyazev YP, Orfi L 等人，（2008). AXL is a potential target for therapeutic intervention in breast cancer progression. Cancer Res 68:1905-15 。 如下所述，融合瘤細胞株3G9.19.7依據布達佩斯條約 (Budapest Treaty)條款寄存於美國菌種保存中心（ATCC), 10801 University Blvd., Manassas, VA 20110-2209, USA ： 物種 物質 寄存編號 存曰期 小鼠 3G9.19.7 _ _ 本文中之寄存係依據國際認可用於專利程序目的之微生 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。此舉確保自寄存 之曰起保持寄存物之存活培養物3 〇年。寄存物可依據布達 佩斯條約之條款纟至ATCC獲得，且服從Genentech, Inc。與 ATCC之間的協議，從而確保一旦相關美國專利頒佈或一 旦任何美國或國外專利申請案向公眾公開（以先到者為 準）’公眾即可永久及無限制獲得寄存物之培養物的子 代’且確保美國專利與商標委員（U.S. Commissioner of Patents and Trademarks)可有權根據 35 U.S.C. 122 及依照其 之委員細則（包括37 C.F.R. 1.14，其特定參考886 OG 638) 149799.doc •192- 201106972 確定獲得子代者。 雖然上文已出於清楚理解之目的藉助於說明及實例詳細 地描述本發明，但該等描述及實例不應解釋為限制本發明 之範疇。本文中所引用之所有專利及科學文獻之揭示内容 以全文引用的方式明確地併入本文中。 ' 【圖式簡單說明】 圖1顯示癌細胞株中之Axl表現。圖塊A :癌細胞株中之 Axl mRNA含量：在代表244個樣品（針對各腫瘤類型指定 樣品編號）的 Affymetrix GeneChip Human Genome U133 Plus 2.0陣列上編輯若干微陣列實驗所得的癌細胞株中Axl mRNA含量。選擇可代表Axl之表現的探針集 202686_s_at。圖塊B :對所選癌細胞株中Axl及Gas6之西 方墨點分析。U87及A172 :神經膠質瘤；DU145 :*** 癌；Pan0327 :胰臟癌；RKO :結腸癌；MDA-MB-231 : 乳癌；Calul、A549、H1299 及 HCC366 : NSCLC ； Q SHSY5Y :神經母細胞瘤；圖塊C :癌細胞株中之構酸化 Axl。使細胞在不含血清之培養基中饑餓隔夜且用 Gas6(200 ng/ml)處理30分鐘。藉由ELISA量測磷酸化Axl ; 圖2顯示阻斷癌細胞株中之Axl基因表現可降低細胞存活 率且減弱遷移。圖塊A :阻斷癌細胞株中之可誘導性Axl基 因表現（西方墨點分析）。藉由多西環素（doxycycline)處理 細胞72小時來誘導AxlshRNA表現。圖塊B :阻斷Axl基因 表現可中止下游信號傳導。使細胞血清饑餓24小時，用 100 ng/ml AxlFc預培育2小時且用Gas6處理30分鐘。針對 149799.doc -193 - 201106972 磷酸化Akt及Akt執行西方墨點分析；圖塊c :阻斷Αχ1基因 表現對細胞存活率之影響。細胞於丨〇%血清（左圖）中或於 具有或不具有Gas6之1%血清（H1299，右圖）中培養。藉由 Dox處理細胞72小時來誘導AxlshRNA，且使用CellTiter Glo分析法量測細胞存活率（n=8)。圖塊〇 :阻斷Αχί基因表 現可減弱細胞遷移。如所述執行遷移分析法。誤差長條圖 表示標準差（n=8); 圖3顯示阻斷Αχ丨基因表現可降低異種移植模型中之腫瘤 生長。對於各圖塊而言’左側曲線圖顯示腫瘤生長曲線且 右圖顯示對腫瘤溶解產物中Αχί表現之西方墨點分析。圖 塊 A : A549AxlshRNA。圖塊 B : H1299AxlshRNA。圖塊 C : MDA-MB-231 AxlshRNA。該等曲線圖代表三個獨立實 驗。誤差長條圖表示平均值標準誤差（每個實驗中，每組 n=10)； 圖4顯示阻斷Axl基因表現可抑制MDA-MB-23 1乳癌細胞 轉移至肺部。圖塊A :實驗設計之示意性說明。圖塊b : 原發性腫瘤移除後5週肺部之生物發光成像。圖塊c :原發 性腫瘤移除後5週肺部之蘇木素及曙紅染色（H&E staining)。圓圈表示腫瘤塊。此圖代表三個獨立實驗（每個 實驗中，每組n=5); 圖5顯示阻斷HUVEC中之Axl基因表現會減少内皮小管 形成。圖塊A :用對照物（GL2)或Axl siRNA轉染後96小時 對HUVEC細胞表面上之Axl表現的FACS分析。圖塊B : Axl siRNAs減少内皮管形成。左圖：在imageXpressMICRO成 149799.doc -194- 201106972 像系統下觀察且拍攝小管。右圖：藉由使用MetaXpress軟 體量測小管長度來定量總小管形成； 圖6顯示Axl基因表現被阻斷之HUVEC之基因表現概 況。圖塊A :阻斷Axl基因表現後、上調及下調幅度最大之 基因之清單。用對照物或Axl siRNA轉染HUVEC且在轉染 後72小時製備RNA。微陣列及資料分析描述於材料及方法 中。相較於對照物之基因表現變化倍數如圖所示。清單中 基因之定義：MYCN : N-Myc ; HLX : H2.0樣間源盒； GAS7 :生長抑制特異性7 ; HDAC9 :組蛋白脫乙醯基酶 9 ; E2F1 : E2F轉錄因子1 ; CXCR4 :趨化因子（C-X-C基 元）受體4 ; PMCH :促黑色素聚集激素；ANG-2 :促血管 生成素2(angiopoietin 2) ; IFI44L :干擾素誘導蛋白44樣； GJA4 :間隙接合蛋白，a4 ; IFIT1 :具有三角形四肽重複1 之干擾素誘導蛋白；SCG5 :分泌粒蛋白V(secretogranin V)，CYTL1 :細胞激素樣1 ; DPP4 :二肽基肽酶4 ; DKK3 : Dickkopf 3。圖塊B :阻斷Axl基因表現後對DKK3 及Ang-2 mRNA含量之定量RT-PCR。圖塊C:阻斷DKK3或 Ang-2 siRNA基因表現對内皮小管形成之影響。左圖：在 ImageXpressMICRO成像系統下觀察且拍攝小管（左圖）。藉 由使用MetaXpress軟體量測小管長度來定量總小管形成（右 圖）； 圖7顯示阻斷Axl基因表現與抗VEGF具有相加效應。在 抗VEGF存在或不存在的情況下用對照物、Axl或DKK3 siRNA轉染HUVEC細胞且與PASMC—起共培養。共培養後 149799.doc -195- 201106972 3天藉由結合FITC之抗CD31抗體染色來分析小管形成。上 圖：在ImageXpressMICRO成像系統下觀察且拍攝小管。 下圖：藉由使用MetaXpress軟體量測小管長度來定量總小 管形成； 圖8顯示Axl mAb之表徵。圖塊A : Axl mAb抑制Baf3 Axl 細胞生長。Baf3Axl細胞在含有200 ng/ml Gas6之培養基中 生長且用指定濃度之Axl mAb處理48小時。藉由CellTiter Glo分析法量測細胞存活率。圖塊B : mAb 3G9及8B5阻斷 Gas6與 Axl結合（B1 : ELISA且 B2 : FACS)。圖塊 C : mAb下 調A549中之Axl表現（西方墨點分析）及H1299細胞中經Gas6 誘導之Axl磷酸化（ELISA)。圖塊D :抗原決定基定位。活 體外轉錄及轉譯Axl ECD之各部分且用作ELISA中之抗 原； 圖9顯示Axl mAb抑制A549異種移植腫瘤生長。圖塊A : 腫瘤生長曲線。當平均腫瘤尺寸達到1 00 mm3時開始腹膜 内投與單株抗體，30 mg/kg，每週兩次。第一劑（第〇天）及 最後一劑（第40天）mAb投與用箭頭指示。該曲線圖代表三 個獨立實驗。誤差長條圖表示平均值標準誤差（每個實驗 中，每組n=10)。圖塊B : Axl mAb下調Axl表現。以30 mg/kg之mAb處理小鼠且在指定時間點切除腫瘤。腫瘤之 細胞溶解產物用於西方墨點法分析Axl表現。圖塊C :投與 Axl mAbl2All後72小時腫瘤樣品之Ki67染色； 圖10顯示藉由siRNA阻斷HUVEC中之Axl基因表現。用 螢光素對照物（GL2)siRNA或用Axl特異性（m-4、Axl2及 149799.doc •196· 201106972Zhang YX, Knyazev PG, Cheburkin YV, Sharma K, Knyazev YP, Orfi L et al, (2008). AXL is a potential target for therapeutic intervention in breast cancer progression. Cancer Res 68: 1905-15. As described below, the fusion cell line 3G9.19.7 is deposited with the American Type Culture Collection (ATCC) under the terms of the Budapest Treaty, 10801 University Blvd., Manassas, VA 20110-2209, USA: Species Material Registration Number Mouse 3G9.19.7 _ _ This document is based on the Budapest Treaty and the Regulations of the International Convention for the Protection of Microorganisms for the Purpose of the Invention. Thereunder) (the Budapest Treaty) is carried out. This ensures that the surviving culture of the deposit is kept for 3 years from the time of storage. Deposits may be obtained from the ATCC under the terms of the Treaty of Budapest and are subject to Genentech, Inc. Agreement with the ATCC to ensure that once the relevant US patent is issued or once any US or foreign patent application is made available to the public (whichever comes first), the public can obtain a permanent and unrestricted access to the culture of the deposit. The child's and the US Commissioner of Patents and Trademarks are entitled to be under 35 USC 122 and in accordance with its committee rules (including 37 CFR 1.14, its specific reference 886 OG 638) 149799.doc •192- 201106972 Determined to get the offspring. The present invention has been described above in detail by way of illustration and example, and the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the Axl expression in cancer cell lines. Panel A: Axl mRNA content in cancer cell lines: Axl mRNA was edited in several cancer cell lines obtained from several microarray experiments on an Affymetrix GeneChip Human Genome U133 Plus 2.0 array representing 244 samples (specified sample numbers for each tumor type) content. Select probe set 202686_s_at that represents the performance of Axl. Panel B: Western blot analysis of Axl and Gas6 in selected cancer cell lines. U87 and A172: glioma; DU145: prostate cancer; Pan0327: pancreatic cancer; RKO: colon cancer; MDA-MB-231: breast cancer; Calul, A549, H1299 and HCC366: NSCLC; Q SHSY5Y: neuroblastoma; Panel C: Acidified Axl in cancer cell lines. The cells were starved overnight in serum-free medium and treated with Gas6 (200 ng/ml) for 30 minutes. Phosphorylation of Axl was measured by ELISA; Figure 2 shows that blocking the expression of the Axl gene in cancer cell lines reduced cell viability and attenuated migration. Panel A: Blocking the inducible Axl gene expression in cancer cell lines (Western blot analysis). Axl shRNA expression was induced by treating cells with doxycycline for 72 hours. Block B: Blocking Axl gene performance can halt downstream signaling. The cells were serum starved for 24 hours, pre-incubated with 100 ng/ml AxlFc for 2 hours and treated with Gas6 for 30 minutes. Western blot analysis was performed on 149799.doc -193 - 201106972 phosphorylated Akt and Akt; block c: the effect of blocking Αχ1 gene expression on cell viability. The cells were cultured in sputum serum (left panel) or in 1% serum with or without Gas6 (H1299, right panel). Axl shRNA was induced by Dox treatment of cells for 72 hours, and cell viability was measured using the CellTiter Glo assay (n=8). Block 〇: Blocking Αχί gene expression can attenuate cell migration. The migration analysis method was performed as described. The error bar graph represents the standard deviation (n=8); Figure 3 shows that blocking gene expression can reduce tumor growth in xenograft models. For each tile, the left graph shows the tumor growth curve and the right panel shows the Western blot analysis for the tumor lysate. Panel A: A549AxlshRNA. Figure B: H1299AxlshRNA. Panel C: MDA-MB-231 AxlshRNA. These graphs represent three independent experiments. The error bar graph represents the mean standard error (n=10 per group in each experiment); Figure 4 shows that blocking Axl gene expression inhibits MDA-MB-23 1 breast cancer cell metastasis to the lungs. Block A: Schematic description of the experimental design. Figure b: Bioluminescence imaging of the lungs 5 weeks after primary tumor removal. Panel c: Hematoxylin and eosin staining of the lungs 5 weeks after primary tumor removal. Circles indicate tumor masses. This figure represents three independent experiments (n=5 per group in each experiment); Figure 5 shows that blocking the expression of the Axl gene in HUVEC reduces endothelial tubule formation. Panel A: FACS analysis of Axl expression on the surface of HUVEC cells 96 hours after transfection with control (GL2) or Axl siRNA. Panel B: Axl siRNAs reduce endothelial tube formation. Left: In imageXpressMICRO into 149799.doc -194- 201106972 Observe and take a small tube under the system. Right panel: Total tubule formation was quantified by measuring the tubule length using MetaXpress software; Figure 6 shows the gene expression profile of HUVEC with Axl gene expression blocked. Block A: A list of genes that block, up- and down-regulate the Axl gene expression. HUVECs were transfected with control or Axl siRNA and RNA was prepared 72 hours after transfection. Microarrays and data analysis are described in materials and methods. The fold change in gene performance compared to the control is shown in the figure. The definition of the gene in the list: MYCN: N-Myc; HLX: H2.0 sample source box; GAS7: growth inhibition specificity 7; HDAC9: histone deacetylase 9; E2F1: E2F transcription factor 1; CXCR4: Chemokine (CXC motif) receptor 4; PMCH: melanin-aggregating hormone; ANG-2: angiopoietin 2; IFI44L: interferon-inducible protein 44; GJA4: gap junction protein, a4; IFIT1: an interferon-inducible protein having a triangular tetrapeptide repeat 1; SCG5: secretegranin V, CYTL1: cytokine-like 1; DPP4: dipeptidyl peptidase 4; DKK3: Dickkopf 3. Panel B: Quantitative RT-PCR of DKK3 and Ang-2 mRNA content after blocking the expression of the Axl gene. Panel C: Blocking the effect of DKK3 or Ang-2 siRNA gene expression on endothelial tubule formation. Left: Observe and take a small tube (left) under the ImageXpressMICRO imaging system. Total tubule formation was quantified by measuring the tubule length using the MetaXpress software (right panel); Figure 7 shows that the blockade of Axl gene expression has an additive effect on anti-VEGF. HUVEC cells were transfected with control, Axl or DKK3 siRNA in the presence or absence of anti-VEGF and co-cultured with PASMC. After co-culture 149799.doc -195- 201106972 Three-day analysis of tubule formation was performed by staining with FITC-conjugated anti-CD31 antibody. Above: Observe and take a small tube under the ImageXpressMICRO imaging system. Bottom panel: Total tubule formation was quantified by measuring the tubule length using the MetaXpress software; Figure 8 shows the characterization of the Axl mAb. Panel A: Axl mAb inhibits the growth of Baf3 Axl cells. Baf3Axl cells were grown in medium containing 200 ng/ml Gas6 and treated with the indicated concentrations of Axl mAb for 48 hours. Cell viability was measured by the CellTiter Glo assay. Panel B: mAb 3G9 and 8B5 block Gas6 binding to Axl (B1: ELISA and B2: FACS). Panel C: mAb down-regulates Axl expression in A549 (Western blot analysis) and Gas6-induced Axl phosphorylation (ELISA) in H1299 cells. Panel D: epitope locating. Each part of Axl ECD was transcribed and translated in vitro and used as an antigen in ELISA; Figure 9 shows that Axl mAb inhibits A549 xenograft tumor growth. Block A: Tumor growth curve. When the average tumor size reached 100 mm3, intraperitoneal administration of monoclonal antibody, 30 mg/kg, was started twice a week. The first dose (day ) day) and the last dose (day 40) of mAb administration are indicated by arrows. The graph represents three independent experiments. The error bar graph represents the mean standard error (n=10 per group for each experiment). Block B: Axl mAb down-regulates Axl performance. Mice were treated with 30 mg/kg mAb and tumors were excised at the indicated time points. The cell lysate of the tumor was used for Western blot analysis of Axl expression. Panel C: Ki67 staining of tumor samples 72 hours after administration of Axl mAbl2All; Figure 10 shows blocking of Axl gene expression in HUVEC by siRNA. Use luciferin control (GL2) siRNA or specificity with Axl (m-4, Axl2 and 149799.doc •196· 201106972)

Axl4)siRNA轉染HUVEC。藉由FACS分析轉染後48小時細 胞表面上之Axl表現。圖塊A : FACS分析之Axl表現概況。 條形圖表示FACS之定量。圖塊B : siRNA序列（SEQ ID NO:3-16)。圖塊C:阻斷Axl基因表現對HUVEC生長之影 響。轉染後72小時藉由CellTiter Glo分析法測定細胞存活 率； 圖11顯示藉由RNAi阻斷DKK3或Ang2基因表現不會影響 HUVEC中之Axl蛋白質含量。用對照物（GL2)、Axl、Dkk3 或Ang-2 siRNA轉染HUVEC。轉染後48小時，藉由FACS分 析Axl蛋白質含量且藉由ELIS A測定培養基中DKK3及Ang-2之蛋白質含量。以相較於對照物之基因表現變化倍數表 不， 圖12顯示用於篩檢Axl單株抗體（mAb)之以細胞為基礎之 分析法。圖塊A : Axl穩定表現於Baf3細胞中（西方墨點 法）。圖塊B : Baf3Axl細胞之Gas6依賴性生長。圖塊C : AxlFc抑制Baf3Axl細胞之Gas6依賴性生長。圖塊D :篩檢 阻斷Gas6依賴性Baf3Axl生長之Axl mAb。Baf3Axl細胞在 200 ng/ml Gas6(C及D)存在下、用指定劑量之AxlFc(C)或 融合瘤上清液（D)培養72小時。藉由CellTiter Glo測定細胞 存活率。誤差長條圖表示標準差（n=8); 圖13顯示Axl mAb之交又反應性。圖塊a : Axl mAb不與 鼠類Axl交叉反應。圖塊B : Axl mAb不與Mer及Tyro-3交叉 反應。使用人類AxlFc、鼠類AxlFc、及人類MerFc及Tyro-3Fc作為供抗體結合之抗原執行ELISA ; 149799.doc -197· 201106972 圖 14 : Axl mAb YW327。6S2之表徵。Α·使用 BIAcore量 測YW327.6S2之親和力。使用一對一朗繆爾（Langmuir)結 合模型計算結合速率及解離速率（kQff)。平衡解離常數 (Kd)係由kon/koff比率推算。B. YW327.6S2之交叉反應性。 YW327.6S2與鼠類Axl及獼猴Axl而不與Tyro3或Mer交叉反 應。用抗人類IgG Fc塗佈培養盤，接著與人類Axl、Mer、 Tyro3 Fes、小鼠AxlFcs或獼猴AxlFcs —起培育。洗務後， 添加同型對照抗體或YW327.6S2，接著添加結合HRP之抗 人類IgG.C· YW327.6S2阻斷Gas6與Axl結合。上圖： ELISA。用抗人類IgG Fc塗佈培養盤且與人類Axl-Fc—起 培育。洗滌後，在抗體存在或不存在下添加Gas6。藉由結 合生物素之抗Gas6抗體及抗生物蛋白鏈菌素-HRP結合物 偵測Gas6之結合。下圖：FACS。收集HUVEC且用 YW327.6S2或對照抗體處理，接著在冰上與Gas6—起培 育30分鐘。藉由結合生物素之抗Gas6抗體及抗生物蛋白鏈 菌素-PE結合物偵測Gas6與細胞表面之結合。D. YW327.6S2 下調 Axl表現。A549細胞與 1 pg/ml YW327.6S2 一起培育指定時間，且藉由FACS測定細胞表面Axl表現（上 圖）並藉由西方墨點法分析總蛋白質表現（下圖）。E. YW327.6S2抑制Gas6誘導之Axl磷酸化及信號傳導。H1299 細胞在不含血清之培養基中培養隔夜，用YW327.6S2預培 育4小時並用Gas6處理30分鐘。藉由ELISA量測磷酸化 Axl(上圖）且藉由西方墨點法分析磷酸化Akt(下圖）。F. YW327.6S2抑制Baf3Axl細胞生長。Baf3Axl細胞在含有 149799.doc •198· 201106972 200 ng/ml Gas6之培養基中生長且用指定濃度之 YW327.6S2處理72小時。藉由CellTiter Glo分析法量測細 胞存活率； 圖15 : YW327.6S2減弱A549異種移植腫瘤生長且增強抗 VEGF之作用。A.腫瘤生長曲線。當平均腫瘤尺寸達到100 " mm3時開始（第0天）腹膜内投與mAb，10 mg/kg(YW327.6S2 及同型對照抗體）或1 mg/kg(抗VEGF)，每週兩次。誤差長 條圖表示平均值之標準誤差（每個實驗中，每組n=10)。 〇 p = 0.0003(YW327.6S2相較於對照物），p=10'n(YW327.6S2 相較於組合療法）。B.各處理組之卡普蘭-麥爾曲線。當其 腫瘤尺寸達到800 mm3時，即自研究中移除該小鼠，且對 各組中剩餘動物（剩餘百分比）作圖。C. 12A11增強抗VEGF 之作用。當平均禮瘤尺寸達到100 mm3時開始（第0天）分別 以30 mg/kg及1 mg/kg腹膜内投與12A11及抗VEGF，每週 兩次。誤差長條圖表示平均值標準誤差（每個實驗中，每 ◎ 組 n=10)。ρ=0.006(12Α11相較於對照物）；ρ=0·0001(12Α11 相較於組合療法）。D. YW327.6S2下調Axl表現。用10 mg/kg之YW327.6S2處理小鼠且在指定時間點切除腫瘤。 藉由西方墨點法針對Axl表現分析腫瘤之細胞溶解產物。 E. YW327.6S2誘導細胞凋亡。切除經對照物或YW327.6S2 處理2週之腫瘤且執行CC3 IHC以量測細胞凋亡。F. YW327.6S2增強抗VEGF在降低瘤内血管密度中之作用。 給藥後0小時及72小時切除如上述D中所處理之小鼠之腫瘤 且藉由MECA32免疫組織化學染色來顯現腫瘤血管結構且 149799.doc •199· 201106972 藉由景/像刀析疋量（以平方微米表示）。對各對樣品執行學 生t檢驗（對於對照物相較於組合療法，卩<〇〇5); 圖16 : YW327 6S2增強A549異種移植模型中埃羅替尼及 化學療法之抗腫瘤作用。A. YW327 6S2增強埃羅替尼之作 用。抗體投與與圖2中相同。以1〇〇毫克/公斤/天經口管飼 投與埃羅替尼。每組n=1〇 D p=h7xl〇-9(YW327 6s2相較於 對照物），15=2.3\1〇-1。(丫貿327.682相較於組合療法）。；6 YW327.6S2增強化學療法。抗體投與與圖2中相同。治療 開始時（第0天）分別皮下投與6·25毫克/公斤/天之太平洋紫 杉醇5天及1〇〇 mg/kg單一劑量之卡鉑。每組η=ι〇。 p=3xl(T5(YW327.6S2相較於對照物），ρ；=10-9(化學療法相 較於對照物）’ ρ=1 〇_5(組合療法相較於單獨化學療法）； 圖I7 : YW327_6S2藉由調節腫瘤受質功能來減弱MDA-MB-231異種移植腫瘤生長。a及B. YW327.6S2而非12A11 降低MDA-MB-231腫瘤生長且增強抗VEGF之作用。平均 腫瘤尺寸達到100 mm3時開始（第〇天），以20 mg/kg (YW327.6S2 及同型對照抗體）、30 mg/kg(12All)及 2 mg/kg(抗VEGF)腹膜内投與mAb，每週兩次。誤差長條圖 表示平均值標準誤差（每個實驗中，每組n=l〇)。P=8.5x10·6 (YW327.6S2相較於對照物），p=2.8xl(T8(YW327.6S2相較 於組合療法），ρ=〇·〇5(12Α11相較於對照物），p=〇.i45(抗 VEGF相較於組合療法）。C及D. YW327.6S2下調Axl表現。 用20 mg/kg之mAb處理含有MDA_MB231異種移植腫瘤（平 均尺寸500 mm3)之小鼠且在指定時間點切除腫瘤。腫瘤之 149799.doc •200- 201106972 細胞溶解產物用於西方墨點法分析Axl表現。E. YW3 27.6S2降低腫瘤相關血管結構之密度。0小時及給藥 後1週切除如上述C中所處理之小鼠之腫瘤且藉由MECA32 免疫組織化學染色來顯現腫瘤血管結構且藉由影像分析定 量（以平方微米表示）。對各對樣品執行學生t檢驗（對於 〜 YW327.6S2相較於對照物、抗VEGF相較於對照物，及抗 VEGF相較於組合療法，p<0.05)。F. Axl高度表現於原發性 ^ 人類乳癌之浸潤性巨噬細胞中。使用免疫組織化學檢查79 ❹ 種原發性腫瘤。21%此等腫瘤在浸潤性巨噬細胞中表現高 含量Axl。藉由抗CD68染色來鑑別巨噬細胞，且藉由抗 Axl/CD68雙重IHC測定巨噬細胞上Axl之表現。使用一系 列切片。G. YW327.6S2抑制腫瘤相關巨噬細胞發炎性分泌 細胞激素/趨化因子。用20 mg/kg之對照抗體、YW327.6S2 或12A11處理含有MDA-MB231異種移植腫瘤（平均尺寸500 mm3)之小鼠且在處理1週後切除腫瘤。藉由分選F4/80陽性 Q 細胞來分離腫瘤相關巨嗟細胞且培養隔夜。使用Bio-Plex 小鼠細胞激素分析套組量測分泌入培養基中之細胞激素及 趨化因子； '· 圖18 : YW327.6S2減少MDA-MB-231乳癌細胞向骨骼之 ·. 轉移。A.尾靜脈注射腫瘤細胞後4週進行的生物發光成 像。將 200μί 含 25 mg/mL D-螢光素（Invitrogen)之 PBS 腹膜 内（i.p)注入小鼠中且在成像期間使用異氟烷經由鼻錐麻醉 小鼠。用具有增強式電荷耦合器件攝影機之光子成像器 (Biospace Lab，Paris, France)獲得生物發光影像。B.脛骨 149799.doc -201 - 201106972 切片之Η及E染色。實驗結束時收集組織且固定於4%曱醛 中，切片且用H&E染色。上圖中之圓圈顯示侵襲骨骼之腫 瘤細胞。下圖詳細顯示骨髓中之贅生性細胞；及 圖 19 :例示性人類 Axl序列 RefSeq NM—001699(SEQ ID NO:17)。 149799.doc 202- 201106972 序列表 <110>美商建南德克公司 <120>組合療法 <130> P4347R1 <140> 099124592 <141> 2010/07/26 <150> US 61/228,915 <151> 2009-07-27 <150> US 61/356,498 <151> 2010-06-19 <160> 19 <210> 1 <211> 121 <212> PRT <2]3>人造序列 <220>Axl4) siRNA was transfected into HUVEC. The Axl expression on the surface of the cells 48 hours after transfection was analyzed by FACS. Block A: Axl performance profile for FACS analysis. The bar graph represents the quantification of FACS. Panel B: siRNA sequences (SEQ ID NOS: 3-16). Panel C: Blocking the effect of Axl gene expression on HUVEC growth. Cell viability was determined by CellTiter Glo assay 72 hours after transfection; Figure 11 shows that blocking DKK3 or Ang2 gene expression by RNAi does not affect Axl protein content in HUVEC. HUVECs were transfected with control (GL2), Axl, Dkk3 or Ang-2 siRNA. 48 hours after transfection, the Axl protein content was analyzed by FACS and the protein content of DKK3 and Ang-2 in the medium was determined by ELIS A. Figure 2 shows a cell-based assay for screening Axl monoclonal antibodies (mAbs), as shown by the fold change in gene performance compared to the control. Panel A: Axl is stably expressed in Baf3 cells (Western dot method). Panel B: Gas6-dependent growth of Baf3Axl cells. Panel C: AxlFc inhibits Gas6-dependent growth of Baf3Axl cells. Panel D: Screening Axl mAbs that block the growth of Gas6-dependent Baf3Axl. Baf3Axl cells were cultured for 72 hours in the presence of 200 ng/ml Gas6 (C and D) with the indicated dose of AxlFc (C) or fusion tumor supernatant (D). Cell viability was determined by CellTiter Glo. The error bar graph represents the standard deviation (n=8); Figure 13 shows the recombination of the Axl mAb. Block a: Axl mAb does not cross-react with murine Axl. Block B: Axl mAb does not cross-react with Mer and Tyro-3. ELISA was performed using human AxlFc, murine AxlFc, and human MerFc and Tyro-3Fc as antigens for antibody binding; 149799.doc -197· 201106972 Figure 14: Characterization of Axl mAb YW327.6S2. Α·Use BIAcore to measure the affinity of YW327.6S2. The binding rate and dissociation rate (kQff) were calculated using a one-to-one Langmuir combination model. The equilibrium dissociation constant (Kd) is derived from the kon/koff ratio. B. Cross-reactivity of YW327.6S2. YW327.6S2 cross-reacts with murine Axl and macaque Axl without Tyro3 or Mer. Plates were plated with anti-human IgG Fc followed by incubation with human Axl, Mer, Tyro3 Fes, mouse AxlFcs or macaque AxlFcs. After washing, an isotype control antibody or YW327.6S2 was added, followed by the addition of HRP-conjugated anti-human IgG.C·YW327.6S2 to block the binding of Gas6 to Axl. Above: ELISA. Plates were plated with anti-human IgG Fc and incubated with human Axl-Fc. After washing, Gas6 is added in the presence or absence of antibodies. The binding of Gas6 was detected by binding of biotin-resistant Gas6 antibody and anti-bio-streptomycin-HRP conjugate. Below: FACS. HUVECs were harvested and treated with YW327.6S2 or control antibody, followed by incubation with Gas6 for 30 minutes on ice. The binding of Gas6 to the cell surface was detected by binding biotin-resistant Gas6 antibody and anti-bio-streptavidin-PE conjugate. D. YW327.6S2 downgrades Axl performance. A549 cells were incubated with 1 pg/ml YW327.6S2 for the indicated time, and cell surface Axl expression was determined by FACS (top panel) and total protein performance was analyzed by Western blot (bottom panel). E. YW327.6S2 inhibits Gas6-induced Axl phosphorylation and signal transduction. H1299 cells were cultured overnight in serum-free medium, pre-incubated with YW327.6S2 for 4 hours and treated with Gas6 for 30 minutes. Phosphorylated Axl (top panel) was measured by ELISA and phosphorylated Akt was analyzed by Western blot method (bottom panel). F. YW327.6S2 inhibits the growth of Baf3Axl cells. Baf3Axl cells were grown in medium containing 149799.doc • 198·201106972 200 ng/ml Gas6 and treated with the indicated concentrations of YW327.6S2 for 72 hours. Cell viability was measured by the CellTiter Glo assay; Figure 15: YW327.6S2 attenuates A549 xenograft tumor growth and enhances anti-VEGF action. A. Tumor growth curve. MAb, 10 mg/kg (YW327.6S2 and isotype control antibody) or 1 mg/kg (anti-VEGF) were administered intraperitoneally (on day 0) when the mean tumor size reached 100 " mm3, twice a week. The error bar graph represents the standard error of the mean (n=10 per group in each experiment). 〇 p = 0.0003 (YW327.6S2 compared to the control), p=10'n (YW327.6S2 compared to combination therapy). B. Kaplan-Mal curve for each treatment group. When the tumor size reached 800 mm3, the mice were removed from the study and plotted against the remaining animals (percentage remaining) in each group. C. 12A11 enhances the anti-VEGF effect. When the average tumor size reached 100 mm3 (day 0), 12A11 and anti-VEGF were administered intraperitoneally at 30 mg/kg and 1 mg/kg, twice a week. The error bar graph represents the mean standard error (n=10 per group for each experiment). ρ = 0.006 (12Α11 compared to the control); ρ=0·0001 (12Α11 compared to combination therapy). D. YW327.6S2 downgrades Axl performance. Mice were treated with 10 mg/kg of YW327.6S2 and tumors were excised at the indicated time points. Cell lysates of tumors were analyzed for Axl expression by Western blotting. E. YW327.6S2 induces apoptosis. Tumors treated with control or YW327.6S2 for 2 weeks were excised and CC3 IHC was performed to measure apoptosis. F. YW327.6S2 enhances the role of anti-VEGF in reducing intratumoral vascular density. Tumors of mice treated as described in D above were excised at 0 hours and 72 hours after administration and tumor vascular structures were visualized by MECA32 immunohistochemical staining and 149799.doc •199· 201106972 by spectroscopy (in square micron). Student's t-test was performed on each pair of samples (for controls compared to combination therapy, 卩 < 〇〇 5); Figure 16: YW327 6S2 enhances the anti-tumor effect of erlotinib and chemotherapy in the A549 xenograft model. A. YW327 6S2 enhances the effect of erlotinib. Antibody administration was the same as in Figure 2. The erlotinib was administered by oral gavage at 1 mg/kg/day. Each group n = 1 〇 D p = h7xl -9 (YW327 6s2 compared to the control), 15 = 2.3 \ 1 〇 -1. (丫 327 327.682 compared to combination therapy). ; 6 YW327.6S2 enhanced chemotherapy. Antibody administration was the same as in Figure 2. At the beginning of treatment (Day 0), 6.25 mg/kg/day of paclitaxel was administered subcutaneously for 5 days and a single dose of carboplatin at 1 mg/kg. Each group η=ι〇. p=3xl (T5 (YW327.6S2 compared to control), ρ;=10-9 (chemotherapy compared to control)' ρ=1 〇_5 (combination therapy compared to chemotherapy alone); I7: YW327_6S2 attenuates MDA-MB-231 xenograft tumor growth by regulating tumor receptor function. a and B. YW327.6S2 but not 12A11 reduces MDA-MB-231 tumor growth and enhances anti-VEGF effect. Mean tumor size Start at 100 mm3 (day day), intraperitoneal administration of mAb at 20 mg/kg (YW327.6S2 and isotype control antibody), 30 mg/kg (12All) and 2 mg/kg (anti-VEGF), weekly Twice. The error bar graph indicates the mean standard error (n=l〇 for each group in each experiment). P=8.5x10·6 (YW327.6S2 compared to the control), p=2.8xl (T8( YW327.6S2 compared to combination therapy), ρ=〇·〇5 (12Α11 compared to control), p=〇.i45 (anti-VEGF compared to combination therapy) C and D. YW327.6S2 down-regulated Axl performance Mice treated with MDA_MB231 xenograft tumors (mean size 500 mm3) were treated with 20 mg/kg mAb and tumors were excised at the indicated time points. Tumor 149799.doc •200- 201106972 Cell lysate for Western ink The Axl expression was analyzed by E. YW3 27.6S2 to reduce the density of tumor-associated vascular structures. Tumors of mice treated as described in C above were excised at 0 hours and 1 week after administration and tumor blood vessels were visualized by MECA32 immunohistochemical staining. Structure and quantified by image analysis (in square micron). Student's t-test was performed on each pair of samples (for ~YW327.6S2 compared to control, anti-VEGF compared to control, and anti-VEGF compared to combination therapy) , p < 0.05). F. Axl is highly expressed in invasive macrophages of primary breast cancer. Immunohistochemical examination of 79 primary tumors. 21% of these tumors in infiltrating macrophages High content of Axl was expressed. Macrophages were identified by anti-CD68 staining, and Axl expression on macrophages was determined by anti-Axl/CD68 dual IHC. A series of sections were used. G. YW327.6S2 inhibited tumor-associated macrophages Cellular inflammatory secretion of cytokines/chemokines. Mice with MDA-MB231 xenograft tumors (mean size 500 mm3) were treated with 20 mg/kg of control antibody, YW327.6S2 or 12A11 and tumors were resected 1 week after treatment. .borrow F4/80-positive Q cells were sorted to isolate tumor-associated giant sputum cells and cultured overnight. The cytokines and chemokines secreted into the culture medium were measured using the Bio-Plex Mouse Cytokine Analysis Kit; '· Figure 18: YW327 .6S2 reduces the migration of MDA-MB-231 breast cancer cells to the bones. A. Bioluminescence imaging performed 4 weeks after the tail vein injection of tumor cells. 200 μί of PBS containing 25 mg/mL D-luciferin (Invitrogen) was intraperitoneally (i.p) injected into mice and the mice were anesthetized via a nose cone using isoflurane during imaging. Bioluminescence images were obtained using a photon imager (Biospace Lab, Paris, France) with an enhanced charge coupled device camera. B. tibia 149799.doc -201 - 201106972 Sliced sputum and E staining. At the end of the experiment, tissues were harvested and fixed in 4% furfural, sectioned and stained with H&E. The circle in the above image shows the tumor cells that invade the bone. The following figure shows in detail the neoplastic cells in the bone marrow; and Figure 19: Exemplary human Axl sequence RefSeq NM-001699 (SEQ ID NO: 17). 149799.doc 202- 201106972 Sequence Listing <110>US-based Nandek Corporation<120> Combination Therapy<130> P4347R1 <140> 099124592 <141> 2010/07/26 <150> US 61 /228,915 <151> 2009-07-27 <150> US 61/356,498 <151> 2010-06-19 <160> 19 <210> 1 <211> 121 <212> PRT <2]3>artificialsequence<220>

<223>合成序列 <400> 1<223>Synthesis sequence <400> 1

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

Gly Ser Trp lie His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 35 40 45Gly Ser Trp lie His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 35 40 45

Glu Trp Va] Gly Trp lie Asn Pro Tyr Arg Gly Tyr Ala Tyr Tyr 50 55 60Glu Trp Va] Gly Trp lie Asn Pro Tyr Arg Gly Tyr Ala Tyr Tyr 50 55 60

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

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

Thr Ala Val Tyr Tyr Cys Ala Arg Glu Tyr Ser Gly Trp Gly Gly 95 100 105Thr Ala Val Tyr Tyr Cys Ala Arg Glu Tyr Ser Gly Trp Gly Gly 95 100 105

Ser Ser Val Gly Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu 110 115 120Ser Ser Val Gly Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu 110 115 120

Val <210> 2 <21]> 108 <212> PRT <213>人造序列 <220> <223>合成序列 <400> 2Val <210> 2 <21]> 108 <212> PRT <213> artificial sequence <220><223> synthetic sequence <400>

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

Thr Ala Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 35 40 45Thr Ala Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 35 40 45

Leu Leu lie Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Leu Leu lie Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 149799.doc 201106972 65 70 75Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 149799.doc 201106972 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

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210> 3 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 3 cguacgcgga auacuucgat t 21 <210> 4 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 4 ucgaaguauu ccgcguacgt t 21 <210> 5 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 5 gaaagaagga gacccgttat t 21 <210> 6 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400〉 6 taacgggtct ccttctttct t 21 <210〉 7 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 7 ccaagaagat ctacaatggt t 21 <210> 8 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 8 2- 149799.doc 201106972 ccattgtaga tcttcttggt t 21 <210> 9 <2U> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 9 ggaactgcat gctgaatgat t zi <210> 10 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 10 f tcattcagca tgcagttcct t 21Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220><223> Synthetic sequence <400> 3 cguacgcgga auacuucgat t 21 <210> 4 <211> 21 <212> RNA <213> artificial sequence <220><223> synthetic sequence <400> 4 ucgaaguauu ccgcguacgt t 21 <210> 5 <211> 21 <212> RNA <213> artificial sequence <220><223> synthetic sequence <400> 5 gaaagaagga gacccgttat t 21 <210> 6 <211> 21 <212> RNA <213> artificial sequence <220><223> synthetic sequence <400> 6 taacgggtct ccttctttct t 21 <210> 7 <211> 21 <212> RNA <213> Artificial sequence <220><223> synthetic sequence <400> 7 ccaagaagat ctacaatggt t 21 <210> 8 <211> 21 <212> RNA <213> artificial sequence <220><223> Synthetic sequence <400> 8 2- 149799.doc 201106972 ccattgtaga tcttcttggt t 21 <210> 9 <2 U > 21 <212> RNA <213> artificial sequence <220><223> synthetic sequence <400> 9 ggaactgcat gctgaatgat t zi <210> 10 <211> 21 <212> RNA < 213 > artificial sequence <220><223> synthetic sequence <400> 10 f tcattcagca tgcagttcct t 21

<210> 11 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 11 ... gaaggagacc cgttatggat t ii <210> 12 <211> 21 <212> RNA <2}3>人造序列 <220> <223>合成序列 <400> 12 tccataacgg gtctccttct t n <210〉 13 <211> 21 <212> RNA <213〉人造序列 <220> <223>合成序列 <400> 13 t t gacatcctct ttctcctgct t ll <210> 14 <2\\> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 14 , gcaggagaaa gaggatgtct t /1 <210> 15 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 149799.doc 201106972 <400> 15 gatttggaga acacactgat t 21 <210> 16 <211> 21 <212> RNA <213>人造序列 <220> <223>合成序列 <400> 16 tcagtgtgtt ctccaaatct t 21 <210> 17 <211> 875 <212〉 PRT <213>智人 <400> 17<210> 11 <211> 21 <212> RNA <213> artificial sequence <220><223> synthetic sequence <400> 11 ... gaaggagacc cgttatggat t ii <210> 12 < 21 <212> RNA <2}3> artificial sequence <220><223> synthetic sequence <400> 12 tccataacgg gtctccttct tn <210> 13 <211> 21 <212> RNA <;213> artificial sequence <220><223> synthetic sequence <400> 13 tt gacatcctct ttctcctgct t ll <210> 14 <2\\> 21 <212> RNA <213> artificial sequence<213>;220><223>Synthesis sequence <400> 14 , gcaggagaaa gaggatgtct t /1 <210> 15 <211> 21 <212> RNA <213> artificial sequence <220><223> Sequence 149799.doc 201106972 <400> 15 gatttggaga acacactgat t 21 <210> 16 <211> 21 <212> RNA <213> artificial sequence <220><223> synthetic sequence <400> Tcagtgtgtt ctccaaatct t 21 <210> 17 <211> 875 <212> PRT <213> Homo sapiens <400>

Met Ala Trp Arg Cys Pro Arg Met Gly Arg Val Pro Leu Ala Trp 15 10 15Met Ala Trp Arg Cys Pro Arg Met Gly Arg Val Pro Leu Ala Trp 15 10 15

Cys Leu Ala Leu Cys Gly Trp Ala Cys Met Ala Pro Arg Gly Thr 20 25 30Cys Leu Ala Leu Cys Gly Trp Ala Cys Met Ala Pro Arg Gly Thr 20 25 30

Gin Ala Glu Glu Ser Pro Phe Val Gly Asn Pro Gly Asn lie Thr 35 40 45Gin Ala Glu Glu Ser Pro Phe Val Gly Asn Pro Gly Asn lie Thr 35 40 45

Gly Ala Arg Gly Leu Thr Gly Thr Leu Arg Cys Gin Leu Gin Val 50 55 60Gly Ala Arg Gly Leu Thr Gly Thr Leu Arg Cys Gin Leu Gin Val 50 55 60

Gin Gly Glu Pro Pro Glu Val His Trp Leu Arg Asp Gly Gin lie 65 70 75Gin Gly Glu Pro Pro Glu Val His Trp Leu Arg Asp Gly Gin lie 65 70 75

Leu Glu Leu Ala Asp Ser Thr Gin Thr Gin Val Pro Leu Gly Glu 80 85 90Leu Glu Leu Ala Asp Ser Thr Gin Thr Gin Val Pro Leu Gly Glu 80 85 90

Asp Glu Gin Asp Asp Trp lie Trp Ser Gin Leu Arg lie Thr Ser 95 100 305Asp Glu Gin Asp Asp Trp lie Trp Ser Gin Leu Arg lie Thr Ser 95 100 305

Leu Gin Leu Ser Asp Thr Gly Gin Tyr Gin Cys Leu Val Phe Leu 110 115 120Leu Gin Leu Ser Asp Thr Gly Gin Tyr Gin Cys Leu Val Phe Leu 110 115 120

Gly His Gin Thr Phe Val Ser Gin Pro Gly Tyr Val Gly Leu Glu 125 130 135Gly His Gin Thr Phe Val Ser Gin Pro Gly Tyr Val Gly Leu Glu 125 130 135

Gly Leu Pro Tyr Phe Leu Gla Glu Pro Glu Asp Arg Thr Val Ala 140 145 150Gly Leu Pro Tyr Phe Leu Gla Glu Pro Glu Asp Arg Thr Val Ala 140 145 150

Ala Asn Thr Pro Phe Asn Leu Ser Cys Gin Ala Gin Gly Pro Pro 155 160 165Ala Asn Thr Pro Phe Asn Leu Ser Cys Gin Ala Gin Gly Pro Pro 155 160 165

Glu Pro Val Asp Leu Leu Trp Leu Gin Asp Ala Val Pro Leu Ala 170 175 180Glu Pro Val Asp Leu Leu Trp Leu Gin Asp Ala Val Pro Leu Ala 170 175 180

Thr Ala Pro Gly His Gly Pro Gin Arg Ser Leu His Val Pro Gly 185 190 195Thr Ala Pro Gly His Gly Pro Gin Arg Ser Leu His Val Pro Gly 185 190 195

Leu Asn Lys Thr Ser Ser Phe Ser Cys Glu Aia His Asn Ala Lys 200 205 210Leu Asn Lys Thr Ser Ser Phe Ser Cys Glu Aia His Asn Ala Lys 200 205 210

Gly Trp Ser Arg Thr Ala Thr lie Thr Val Leu Pro Gin Gin Pro 215 220 225Gly Trp Ser Arg Thr Ala Thr lie Thr Val Leu Pro Gin Gin Pro 215 220 225

Arg Asn Leu His Leu Val Ser Arg Gin Pro Thr Glu Leu Glu Val 230 235 240Arg Asn Leu His Leu Val Ser Arg Gin Pro Thr Glu Leu Glu Val 230 235 240

Ala Trp Thr Pro Gly Leu Ser Gly lie Tyr Pro Leu Thr His Cys 245 250 255Ala Trp Thr Pro Gly Leu Ser Gly lie Tyr Pro Leu Thr His Cys 245 250 255

Thr Leu Gin Ala Val Leu Ser Asp Asp Gly Met Gly lie Gin Ala 260 265 270 -4- 149799.doc 201106972Thr Leu Gin Ala Val Leu Ser Asp Asp Gly Met Gly lie Gin Ala 260 265 270 -4- 149799.doc 201106972

Gly Glu Pro Asp Pro Pro Glu Glu Pro Leu Thr Ser Gin Ala Ser 275 280 285Gly Glu Pro Asp Pro Pro Glu Glu Pro Leu Thr Ser Gin Ala Ser 275 280 285

Val Pro Pro His Gin Leu Arg Leu Gly Ser Leu His Pro His Thr 290 295 300Val Pro Pro His Gin Leu Arg Leu Gly Ser Leu His Pro His Thr 290 295 300

Pro Tyr His lie Arg Val Ala Cys Thr Ser Ser Gin Gly Pro Ser 305 310 315Pro Tyr His lie Arg Val Ala Cys Thr Ser Ser Gin Gly Pro Ser 305 310 315

Ser Trp Thr His Trp Leu Pro Val Glu Thr Pro Glu Gly Val Pro 320 325 330Ser Trp Thr His Trp Leu Pro Val Glu Thr Pro Glu Gly Val Pro 320 325 330

Leu Gly Pro Pro Glu Asn lie Ser Ala Thr Arg Asn Gly Ser Gin 335 340 345Leu Gly Pro Pro Glu Asn lie Ser Ala Thr Arg Asn Gly Ser Gin 335 340 345

Ala Phe Val His Trp Gin Glu Pro Arg Ala Pro Leu Gin Gly Thr 350 355 360Ala Phe Val His Trp Gin Glu Pro Arg Ala Pro Leu Gin Gly Thr 350 355 360

Leu Leu Gly Tyr Arg Leu Ala Tyr Gin Gly Gin Asp Thr Pro Glu 365 370 375Leu Leu Gly Tyr Arg Leu Ala Tyr Gin Gly Gin Asp Thr Pro Glu 365 370 375

Val Leu Met Asp lie Gly Leu Arg Gin Glu Val Thr Leu Glu Leu 380 385 390Val Leu Met Asp lie Gly Leu Arg Gin Glu Val Thr Leu Glu Leu 380 385 390

Gin Gly Asp Gly Ser Val Ser Asn Leu Thr Val Cys Val Met TyrGin Gly Asp Gly Ser Val Ser Asn Leu Thr Val Cys Val Met Tyr

395 400 405395 400 405

Thr Met Gly Asp Gly Pro Txp Ser Leu Pro Val Pro Leu Glu Ala 410 415 420Thr Met Gly Asp Gly Pro Txp Ser Leu Pro Val Pro Leu Glu Ala 410 415 420

Trp Arg Pro Val Lys Glu Pro Ser Thr Pro Ala Phe Ser Trp Pro 425 430 435Trp Arg Pro Val Lys Glu Pro Ser Thr Pro Ala Phe Ser Trp Pro 425 430 435

Trp Trp Tyr Val Leu Leu Gly Ala Trp Ala Ala Ala Cys Val Leu 440 445 450 lie Leu Ala Leu Phe Leu Val His Arg Arg Lys Lys Glu Thr Arg 455 460 465Trp Trp Tyr Val Leu Leu Gly Ala Trp Ala Ala Ala Cys Val Leu 440 445 450 lie Leu Ala Leu Phe Leu Val His Arg Arg Lys Lys Glu Thr Arg 455 460 465

Tyr Gly Glu Val Phe Glu Pro Thr Val Glu Arg Gly Glu Leu Trp 470 475 4S0Tyr Gly Glu Val Phe Glu Pro Thr Val Glu Arg Gly Glu Leu Trp 470 475 4S0

Arg Tyr Arg Val Arg Lys Ser Tyr Ser Arg Arg Thr Thr Glu Ala 485 490 495Arg Tyr Arg Val Arg Lys Ser Tyr Ser Arg Arg Thr Thr Glu Ala 485 490 495

Thr Leu Asn Ser Leu Gly ile Ser Glu Glu Leu Lys Glu Lys Leu 500 505 510Thr Leu Asn Ser Leu Gly ile Ser Glu Glu Leu Lys Glu Lys Leu 500 505 510

Arg Asp Val Met Val Asp ATg His Lys Val Ala Leu Gly Lys Thr 515 520 525Arg Asp Val Met Val Asp ATg His Lys Val Ala Leu Gly Lys Thr 515 520 525

Leu Gly Glu Gly Glu Phe Gly Ala Val Met Glu Gly Gin Leu Asn 530 535 540Leu Gly Glu Gly Glu Phe Gly Ala Val Met Glu Gly Gin Leu Asn 530 535 540

Gin Asp Asp Ser Ile Leu Lys Val Ala Val Lys Thr Met Lys Ile 545 550 555Gin Asp Asp Ser Ile Leu Lys Val Ala Val Lys Thr Met Lys Ile 545 550 555

Ala Ile Cys Thr Arg Ser Glu Leu Glu Asp Phe Leu Ser Glu Ala 560 565 570Ala Ile Cys Thr Arg Ser Glu Leu Glu Asp Phe Leu Ser Glu Ala 560 565 570

Val Cys Met Lys Glu Phe Asp His Pro Asn Val Met Arg Leu Ile 575 580 585Val Cys Met Lys Glu Phe Asp His Pro Asn Val Met Arg Leu Ile 575 580 585

Gly Val Cys Phe Gin Gly Ser Glu Arg Glu Ser Phe Pro Ala Pro 590 595 600Gly Val Cys Phe Gin Gly Ser Glu Arg Glu Ser Phe Pro Ala Pro 590 595 600

Trp Ile Leu Pro Phe Met Lys His Gly Asp Leu His Ser Phe Leu 605 610 615Trp Ile Leu Pro Phe Met Lys His Gly Asp Leu His Ser Phe Leu 605 610 615

Leu Tyr Ser Arg Leu Gly Asp Gin Pro Trp Leu Pro Thr Gin Met 620 625 630Leu Tyr Ser Arg Leu Gly Asp Gin Pro Trp Leu Pro Thr Gin Met 620 625 630

Leu Val Lys Phe Met Ala Asp Ile Ala Ser Gly Met Glu Tyr Leu 635 640 645 149799.doc 201106972Leu Val Lys Phe Met Ala Asp Ile Ala Ser Gly Met Glu Tyr Leu 635 640 645 149799.doc 201106972

Ser Thr Lys Arg Phe lie His Arg Asp Leu Ala Ala Arg Asn Cys 650 655 660Ser Thr Lys Arg Phe lie His Arg Asp Leu Ala Ala Arg Asn Cys 650 655 660

Met Leu Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly Leu 665 670 675Met Leu Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly Leu 665 670 675

Ser Lys Lys lie Tyr Asn Gly Asp Tyr Tyr Arg Gin Gly Arg lie 680 685 690Ser Lys Lys lie Tyr Asn Gly Asp Tyr Tyr Arg Gin Gly Arg lie 680 685 690

Ala Lys Met Pro Val Lys Trp lie Ala lie Glu Ser Leu Ala Asp 695 700 705Ala Lys Met Pro Val Lys Trp lie Ala lie Glu Ser Leu Ala Asp 695 700 705

Arg Trp Thr Ser Lys Ser Asp Val Trp Ser Phe Gly Val Thr Met 710 715 720Arg Trp Thr Ser Lys Ser Asp Val Trp Ser Phe Gly Val Thr Met 710 715 720

Trp Glu lie Ala Thr Arg Gly Gin Thr Pro Tyr Pro Gly Val Glu 725 730 735Trp Glu lie Ala Thr Arg Gly Gin Thr Pro Tyr Pro Gly Val Glu 725 730 735

Asn Ser Glu lie Tyr Asp Tyr Leu Arg Gin Gly Asn Arg Leu Lys 740 745 750Asn Ser Glu lie Tyr Asp Tyr Leu Arg Gin Gly Asn Arg Leu Lys 740 745 750

Gin Pro Ala Asp Cys Leu.Asp Gly Leu Tyr Ala Leu Met Ser Arg 755 760 765Gin Pro Ala Asp Cys Leu.Asp Gly Leu Tyr Ala Leu Met Ser Arg 755 760 765

Cys Trp Glu Leu Asn Pro Gin Asp Arg Pro Ser Phe Thr Glu Leu 770 775 780Cys Trp Glu Leu Asn Pro Gin Asp Arg Pro Ser Phe Thr Glu Leu 770 775 780

Arg Glu Asp Leu Glu Asn Thr Leu Lys Ala Leu Pro Pro Ala Gin 785 790 795Arg Glu Asp Leu Glu Asn Thr Leu Lys Ala Leu Pro Pro Ala Gin 785 790 795

Glu Pro Asp Glu lie Leu Tyr Val Asn Met Asp Glu Gly Gly Gly 800 805 810Glu Pro Asp Glu lie Leu Tyr Val Asn Met Asp Glu Gly Gly Gly 800 805 810

Tyr Pro Glu Pro Pro Gly Ala Ala Gly Gly Ala Asp Pro Pro Thr 815 820 825Tyr Pro Glu Pro Pro Gly Ala Ala Gly Gly Ala Asp Pro Pro Thr 815 820 825

Gin Pro Asp Pro Lys Asp Ser Cys Ser Cys Leu Thr Ala Ala Glu 830 835 840Gin Pro Asp Pro Lys Asp Ser Cys Ser Cys Leu Thr Ala Ala Glu 830 835 840

Val His Pro Ala Gly Arg Tyr Val Leu Cys Pro Ser Thr Thr Pro 845 850 855Val His Pro Ala Gly Arg Tyr Val Leu Cys Pro Ser Thr Thr Pro 845 850 855

Ser Pro Ala Gin Pro Ala Asp Arg Gly Ser Pro Ala Ala Pro Gly 860 865 870Ser Pro Ala Gin Pro Ala Asp Arg Gly Ser Pro Ala Ala Pro Gly 860 865 870

Gin Glu Asp Gly Ala 875 <210> 18 <211> 134Gin Glu Asp Gly Ala 875 <210> 18 <211> 134

<212> PRT <213>智人 <400> 18<212> PRT <213> Homo sapiens <400> 18

Met Ala Trp Arg Cys Pro Arg Met Gly Arg Val Pro Leu Ala Trp 15 10 15Met Ala Trp Arg Cys Pro Arg Met Gly Arg Val Pro Leu Ala Trp 15 10 15

Cys Leu Ala Leu Cys Gly Trp Ala Cys Met Ala Pro Arg Gly Thr 20 25 30Cys Leu Ala Leu Cys Gly Trp Ala Cys Met Ala Pro Arg Gly Thr 20 25 30

Gin Ala Glu Glu Ser Pro Phe Val Gly Asn Pro Gly Asn He Thr 35 40 45Gin Ala Glu Glu Ser Pro Phe Val Gly Asn Pro Gly Asn He Thr 35 40 45

Gly Ala Arg Gly Leu Thr Gly Thr Leu Arg Cys Gin Leu Gin Val 50 55 60Gly Ala Arg Gly Leu Thr Gly Thr Leu Arg Cys Gin Leu Gin Val 50 55 60

Gin Gly Glu Pro Pro Glu Val His Trp Leu Arg Asp Gly Gin lie 65 70 75Gin Gly Glu Pro Pro Glu Val His Trp Leu Arg Asp Gly Gin lie 65 70 75

Leu Glu Leu Ala Asp Ser Thr Gin Thr Gin Val Pro Leu Gly Glu 80 85 90Leu Glu Leu Ala Asp Ser Thr Gin Thr Gin Val Pro Leu Gly Glu 80 85 90

Asp Glu Gin Asp Asp Trp lie Val Val Ser Gin Leu Arg lie Thr 95 100 105 -6- 149799.doc 201106972Asp Glu Gin Asp Asp Trp lie Val Val Ser Gin Leu Arg lie Thr 95 100 105 -6- 149799.doc 201106972

Ser Leu Gin Leu Ser Asp Thr Gly Gin Tyr Gin Cys Leu Val Phe 110 115 120Ser Leu Gin Leu Ser Asp Thr Gly Gin Tyr Gin Cys Leu Val Phe 110 115 120

Leu Gly His Gin Thr Phe Val Ser Gin Pro Gly Tyr Val Gly 125 130 <210> 19 <212> 104 <212> PRT <213>智人 <400> 19 lie Thr Val Leu Pro Gin Gin Pro Arg Asn Leu His Leu Val Ser 1 5 10 15Leu Gly His Gin Thr Phe Val Ser Gin Pro Gly Tyr Val Gly 125 130 <210> 19 <212> 104 <212> PRT <213> Homo sapiens <400> 19 lie Thr Val Leu Pro Gin Gin Pro Arg Asn Leu His Leu Val Ser 1 5 10 15

Arg Gin Pro Thr Glu Leu Glu Val Ala Trp Thr Pro Gly Leu Ser 20 25 30Arg Gin Pro Thr Glu Leu Glu Val Ala Trp Thr Pro Gly Leu Ser 20 25 30

Gly lie Tyr Pro Leu Thr His Cys Thr Leu Gin Ala Val Leu Ser 35 40 45Gly lie Tyr Pro Leu Thr His Cys Thr Leu Gin Ala Val Leu Ser 35 40 45

Asp Asp Gl.y Met Gly lie Gin Ala Gly Glu Pro Asp Pro Pro Glu 50 55 60Asp Asp Gl.y Met Gly lie Gin Ala Gly Glu Pro Asp Pro Pro Glu 50 55 60

Glu Pro Leu Thr Ser Gin Ala Ser Val Pro Pro His Gin Leu ArgGlu Pro Leu Thr Ser Gin Ala Ser Val Pro Pro His Gin Leu Arg

Leu Gly Ser Leu His Pro His Thr Pro Tyr His He Arg Val Ala 80 85 90Leu Gly Ser Leu His Pro His Thr Pro Tyr His He Arg Val Ala 80 85 90

Cys Thr Ser Ser Gin Gly Pro Ser Ser Trp Thr His Trp Leu 95 100Cys Thr Ser Ser Gin Gly Pro Ser Ser Trp Thr His Trp Leu 95 100

149799.doc149799.doc

Claims (1)

201106972 七、申請專利範圍： 1. 一種治療個體癌症之方法，其包括向該個體投與治療有 效量之Axl拮抗劑及VEGF拮抗劑。 2. —種抑制個體癌症轉移之方法，其包括向該個體投與治 ' 療有效量之Axl拮抗劑及VEGF拮抗劑。 3. 一種抑制個體血管生成之方法，其包括向該個體投與治 療有效量之Axl拮抗劑及VEGF拮抗劑。 4. 一種抑制個體細胞增殖之方法，其包括向該個體投與治 療有效量之Axl拮抗劑及VEGF拮抗劑。 5. 如請求項1、2、3或4之方法，其中該癌症為乳癌、結腸 直腸癌、直腸癌、非小細胞肺癌、非霍奇金氏淋巴瘤 (non-Hodgkins lymphoma ； NHL)、腎細胞癌、前歹丨]腺 癌、肝癌（諸如肝細胞癌）、胰臟癌、軟組織肉瘤、卡波 西氏肉瘤（kaposi’s sarcoma)、類癌瘤、頭頸癌、黑色素 瘤、卵巢癌、胃癌、間皮瘤及多發性骨聽瘤。 q 6.如請求項1、2、3或4之方法，其中該Axl拮抗劑為抗Axl 抗體。 7.如請求項6之方法，其中該抗Axl抗體包括含有序列 EVQLVESGGGLVQPGGSLRLSCAASGFSLSGSWIHWVRQ APGKGLEWVGWINPYRGYAYYADSVKGRFTISADTSKN TAYLQMNSLRAEDTAVYYCAREYSGWGGSSVGYAMDY WGQGTLV(SEQ ID ΝΟ:1)的重鏈可變區，及含有序列 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQK PGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP 149799.doc 201106972 EDFATYYCQQSYTTPPTFGQGTKVEIKR(SEQ ID NO:2) 的輕鍵可變區。 8. 如請求項6之方法，其中該抗Axl抗體為抗體3G9或抗體 3G9之人類化形式。 9. 如請求項1、2、3或4之方法，其中該VEGF拮抗劑為抗 VEGF抗體。 10_如請求項9之方法，其中該抗VEGF抗體為貝伐珠單抗 (bevacizumab)。 149799.doc201106972 VII. Patent Application Range: 1. A method for treating cancer in an individual comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. 2. A method of inhibiting cancer metastasis in an individual comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. 3. A method of inhibiting angiogenesis in a subject comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. 4. A method of inhibiting proliferation of an individual cell comprising administering to the individual a therapeutically effective amount of an Axl antagonist and a VEGF antagonist. 5. The method of claim 1, 2, 3 or 4, wherein the cancer is breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), kidney Cell carcinoma, anterior sacral adenocarcinoma, liver cancer (such as hepatocellular carcinoma), pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid tumor, head and neck cancer, melanoma, ovarian cancer, stomach cancer, Mesothelioma and multiple bone tumors. The method of claim 1, 2, 3 or 4, wherein the Axl antagonist is an anti-Axl antibody. 7. The method of item 6 of the request, wherein the anti-Axl antibody comprises a sequence comprising EVQLVESGGGLVQPGGSLRLSCAASGFSLSGSWIHWVRQ APGKGLEWVGWINPYRGYAYYADSVKGRFTISADTSKN TAYLQMNSLRAEDTAVYYCAREYSGWGGSSVGYAMDY WGQGTLV (SEQ ID ΝΟ: 1) a heavy chain variable region and contains sequences DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQK PGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP 149799.doc 201106972 EDFATYYCQQSYTTPPTFGQGTKVEIKR (SEQ ID NO: 2) Light key variable area. 8. The method of claim 6, wherein the anti-Axl antibody is a humanized form of antibody 3G9 or antibody 3G9. 9. The method of claim 1, 2, 3 or 4 wherein the VEGF antagonist is an anti-VEGF antibody. The method of claim 9, wherein the anti-VEGF antibody is bevacizumab. 149799.doc
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