TW200813092A

TW200813092A - Anti-TAT226 antibodies and immunoconjugates

Info

Publication number: TW200813092A
Application number: TW96109168A
Authority: TW
Inventors: Wei-Ching Liang; Chie Sakanaka; Yan Wu
Original assignee: Genentech Inc
Priority date: 2006-03-17
Filing date: 2007-03-16
Publication date: 2008-03-16
Also published as: TWI406871B

Abstract

Anti-TAT226 antibodies and immunoconjugates thereof are provided. Methods of using anti-TAT226 antibodies and immunoconjugates thereof are provided.

Description

200813092 九、發明說明：【發明所屬之技術領域】本發明係關於抗TAT226抗體及其免疫接合物。本發明進一步係關於使用抗TAT226抗體及其免疫接合物之方法。【先前技術】已證明結合至表現於癌細胞表面之多狀之抗體為有效抗癌療法。此等抗體係經由各種機制起作用’該等機制包括例如活化抗體依賴性細胞介導之細胞毒性（ADCC);由具有補體依賴性細胞毒性（CDC)之抗體誘導；增強細胞激素釋放及誘導細胞凋亡。參見例如Cardarelli等人（2002) Cancer Immunol· J^imunother· 51:15-24 〇舉例而言’ HERCEPTIN® 及 RITUXAN®(二者均來自 Genentech Inc·， South San Francisco，California)分別為已成功地用以治療乳癌及非霍奇金氏淋巴瘤（non_Hodgkin’s lymphoma)之抗體。HERCEPTIN®為選擇性地結合至人類表皮生長因子受體2(HER2)原癌基因之細胞外結構域的重組DNA衍生之人化單株抗體。在25-30%之原發性乳癌中觀測到HER2蛋白過度表現。RITUXAN⑧為對抗在正常及惡性B淋巴細胞表面上發現之CD20抗原的基因改造嵌合鼠/人類單株抗體。該兩種抗體均於CHO細胞中重組產生。HERCEPTIN®似乎 (至少部分）藉由抑制血管生成來起作用（Izumi等人（2002) TVaiwre 416:279-28 0)且RITUXAN®似乎（至少部分）藉由誘導細胞〉周亡來起作用（Cardarelli等人（2002) Cancer Immunol. Immunother. 51:15-24) 〇 119007.doc 200813092 免疫接合物或π抗體-藥物接合物π適用於在癌症治療中局部傳遞細胞毒性劑。參見例如Syrigos等人（1999) Anticancer 19:605-614 ; Niculescu-Duvaz 等人 (1997) 乂办.Drwg 26:151-172 ;美國專利第 4,975,278號。免疫接合物使得可將藥物部分靶向傳遞至腫瘤，而全身投予未接合之細胞毒性劑可對正常細胞以及待消除之腫瘤細胞產生不可接受程度之毒性。參見Baldwin 等人（1986 年 3 月 15 曰）第 603-05 頁；Thorpe (1985) Monoclonal Antibodies f84: Biological and Clinical Applications (A. Pinchera 等人編）中之’’Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review，’’第 475-506頁。已研發且將繼續研發靶向細胞表面多肽之免疫接合物以用於治療癌症。關於論述參見例如Hamann等人 (2005)幻φβ" 77^r· (2005) 15:1087-1103 〇顯然，持續需要靶向細胞表面多肽之藥劑以用於診斷及/ 或治療目的。本文所述之本發明滿足此需要且提供其他益處。本文引用之所有文獻，包括專利申請案及公開案之全文係以引用的方式併入本文中。【發明内容】本發明提供抗TAT226抗體及其使用方法。在一態樣中，提供結合至TAT226之抗體，其中該抗體包含至少一個、兩個、三個、四個、五個或六個選自以下之 HVR ·· 119007.doc 200813092 (1) 包含SEQ ID NO:4之胺基酸序列之HVR-Hl ; (2) 包含SEQ ID NO:5之胺基酸序列之HVR-H2 ; (3) 包含與SEQ ID ΝΟ:11之一致序列相符之胺基酸序列的 HVR-H3 ; (4) 包含SEQ ID NO:12之胺基酸序列之HVR-L1 ; (5) 包含SEQ ID NO:13之胺基酸序列之HVR-L2 ;及 (6) 包含與SEQ ID NO: 19之一致序列相符之胺基酸序列的 HVR-L3。200813092 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to an anti-TAT226 antibody and an immunoconjugate thereof. The invention further relates to methods of using anti-TAT226 antibodies and immunoconjugates thereof. [Prior Art] It has been demonstrated that an antibody that binds to a polymorphism expressed on the surface of a cancer cell is an effective anticancer therapy. These anti-systems function via a variety of mechanisms including, for example, activation of antibody-dependent cell-mediated cytotoxicity (ADCC); induction by antibodies with complement-dependent cytotoxicity (CDC); enhancement of cytokine release and induction of cells Apoptosis. See, for example, Cardarelli et al. (2002) Cancer Immunol·J^imunother· 51:15-24 〇 For example, 'HERCEPTIN® and RITUXAN® (both from Genentech Inc., South San Francisco, California) have been successfully An antibody used to treat breast cancer and non-Hodgkin's lymphoma. HERCEPTIN® is a recombinant human-derived monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. Overexpression of HER2 protein was observed in 25-30% of primary breast cancers. RITUXAN8 is a genetically engineered chimeric murine/human monoclonal antibody against CD20 antigen found on the surface of normal and malignant B lymphocytes. Both antibodies were recombinantly produced in CHO cells. HERCEPTIN® appears to act (at least in part) by inhibiting angiogenesis (Izumi et al. (2002) TVaiwre 416:279-28 0) and RITUXAN® appears to (at least in part) function by inducing cell deaths (Cardarelli) Et al. (2002) Cancer Immunol. Immunother. 51:15-24) 〇119007.doc 200813092 Immunoconjugate or π-antibody-drug conjugate π is suitable for the local delivery of cytotoxic agents in the treatment of cancer. See, for example, Syrigos et al. (1999) Anticancer 19: 605-614; Niculescu-Duvaz et al. (1997) 乂. Drwg 26: 151-172; U.S. Patent No. 4,975,278. The immunoconjugate allows targeted delivery of the drug moiety to the tumor, whereas systemic administration of the unconjugated cytotoxic agent produces an unacceptable level of toxicity to normal cells and tumor cells to be eliminated. See Baldwin et al. (March 15, 1986), pp. 603-05; Thorpe (1985) Monoclonal Antibodies f84: Biological and Clinical Applications (A. Pinchera et al.), ''Antibody Carriers Of Cytotoxic Agents In Cancer Therapy : A Review, ''pp. 475-506. Immunoconjugates targeting cell surface polypeptides have been developed and will continue to be developed for the treatment of cancer. For a discussion, see, for example, Hamann et al. (2005) Fantasy φβ " 77^r· (2005) 15:1087-1103 显然 Clearly, there is a continuing need for agents that target cell surface polypeptides for diagnostic and/or therapeutic purposes. The invention described herein satisfies this need and provides other benefits. All documents cited herein, including the entire contents of the patent application and publications, are hereby incorporated by reference. SUMMARY OF THE INVENTION The present invention provides an anti-TAT226 antibody and methods of use thereof. In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises at least one, two, three, four, five or six HVRs selected from the group consisting of: 119007.doc 200813092 (1) comprising SEQ ID NO: HVR-H1 of the amino acid sequence of 4; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (3) amino group comprising the sequence identical to SEQ ID NO: 11. HVR-H3 of the acid sequence; (4) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (6) inclusion HVR-L3 of the amino acid sequence corresponding to the sequence identical to SEQ ID NO: 19.

f ' 在另一態樣中，結合至TAT226之抗體包含（a)包含與SEQ ID ΝΟ:11之一致序列相符之胺基酸序列的HVR-H3及（b)至少一個、兩個、三個、四個或五個選自以下之HVR : (1) 包含SEQ ID NO:4之胺基酸序列之HVR-H1 ; (2) 包含SEQ ID NO:5之胺基酸序列之HVR-H2 ; (3) 包含8£(^10>!0:12之胺基酸序列之11¥11氺1; (5) 包含SEQ ID NO:13之胺基酸序列之HVR-L2 ;及 (6) 包含與SEQ ID NO: 19之一致序列相符之胺基酸序列 I 的 HVR-L3。在一實施例中，抗體包含具有與SEQ ID NO: 19之一致序列相符之胺基酸序列的HVR-L3。在一實施例中，抗體另外包含具有SEQ ID NO:4之胺基酸序列之HVR-H1及具有 SEQ ID NO:5之胺基酸序列之HVR-H2。在一實施例中，抗體另外包含具有SEQ ID N0:12之胺基酸序列之HVR-L1及具有8£卩10>^0:13之胺基酸序列之11¥11-1^2。在一實施例中，抗體包含具有選自SEQ ID ΝΟ:6·10之胺 119007.doc 200813092 基酸序列之HVR-H3。在一實施例中，抗體另外包含具有選自SEQ ID NChl4-18之胺基酸序列之HVR-L3。在一實施例中，HVR-H3包含SEQ ID NO:9之胺基酸序列，且HVR-L3包含SEQ ID NO: 17之胺基酸序列。在一實施例中， HVR-H3包含SEQ ID NChlO之胺基酸序列，且HVR-L3包含 SEQ ID NO:18之胺基酸序列。在一實施例中，抗體另外包含具有SEQ ID NO:4之胺基酸序列之HVR-H1及具有SEQ ID NO:5之胺基酸序列之HVR-H2。在一實施例中，抗體另外包含具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有 SEQ ID NChl3之胺基酸序列之HVR-L2。在一態樣中，提供結合至TAT226之抗體，其中該抗體包含至少一個、兩個、三個、四個、五個或六個選自以下之 HVR ： (1) 包含SEQ ID ΝΟ:1之胺基酸序列之HVR-H1 ; (2) 包含SEQ ID NO:2之胺基酸序列之HVR-H2 ; (3) 包含SEQ ID NO:3之胺基酸序列之HVR-H3 ; (4) 包含8£卩10>^0:12之胺基酸序列之11¥11氺1; (5) 包含8£卩1〇]^0:13之胺基酸序列之11¥11-1^2;及 (6) 包含SEQIDNO:14之胺基酸序列之HVR-L3。f ' In another aspect, the antibody that binds to TAT226 comprises (a) HVR-H3 comprising at least one amino acid sequence consistent with the sequence of SEQ ID NO: 11 and (b) at least one, two, three , four or five HVRs selected from the group consisting of: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (3) HVR-L2 comprising the amino acid sequence of 8 £(^10>!0:12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (6) inclusion HVR-L3 of the amino acid sequence I in accordance with the sequence SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-L3 having an amino acid sequence consistent with the sequence of SEQ ID NO: 19. In one embodiment, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4 and HVR-H2 having the amino acid sequence of SEQ ID NO: 5. In one embodiment, the antibody further comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and 11 ¥ 11-1^2 having an amino acid sequence of 8 卩 10 > ^ 0: 13. In one embodiment, the antibody comprises an antibody Amine from SEQ ID 6:6·10 119007.doc 200813092 base acid sequence HVR-H3. In one embodiment, the antibody further comprises HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NChl 4-18. In one embodiment, HVR-H3 comprises the amino acid of SEQ ID NO: a sequence, and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, HVR-H3 comprises the amino acid sequence of SEQ ID NChlO and HVR-L3 comprises the amino group of SEQ ID NO: The acid sequence. In one embodiment, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4 and HVR-H2 having the amino acid sequence of SEQ ID NO: 5. In one embodiment, The antibody further comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NChl3. In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises At least one, two, three, four, five or six HVRs selected from the group consisting of: (1) HVR-H1 comprising the amino acid sequence of SEQ ID: 1; (2) comprising SEQ ID NO: HVR-H2 of the amino acid sequence of 2; (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (4) 11 of the amino acid sequence comprising 8 卩 10 >¥11氺1; (5) Contains 8£卩1〇] 11:11-1^2 of the amino acid sequence of ^0:13; and (6) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

在另一態樣中，結合至TAT226之抗體包含（a)包含SEQ ID NO:3之胺基酸序列之HVR-H3及（b)至少一個、兩個、三個、四個或五個選自以下之HVR : (1) 包含SEQ ID ΝΟ:1之胺基酸序列之HVR-H1 ; (2) 包含SEQ ID NO:2之胺基酸序列之HVR-H2 ; 119007.doc -9- 200813092 (3) 包含SEQ ID NO:12之胺基酸序列之HVR-Ll ; (4) 包含8£卩10 1^0:13之胺基酸序列之^1¥11丄2;及 (5) 包含8丑(5 10>^0:14之胺基酸序列之11¥11丄3。在一實施例中，抗體包含具有SEQ ID NO: 14之胺基酸序列之HVR-L3。在一實施例中，抗體另外包含具有SEQ ID ΝΟ:1之胺基酸序列之HVR-H1及具有SEQ ID NO:2之胺基酸序列之HVR-H2。在一實施例中，抗體另外包含具有 SEQ IDN0:12之胺基酸序列之HVR-L1及具有SEQID >10:13之胺基酸序列之11¥11丄2。在某些實施例中，以上任何抗體另外包含至少一個選自由VH子群III 一致框架及VL子群I 一致框架之框架。In another aspect, the antibody that binds to TAT226 comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and (b) at least one, two, three, four or five selected From the following HVRs: (1) HVR-H1 comprising the amino acid sequence of SEQ ID ΝΟ: 1; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; 119007.doc -9- 200813092 (3) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (4) ^1¥11丄2 comprising an amino acid sequence of 8 卩10 1^0:13; and (5) inclusion 8 ugly (5 10>^0:14 amino acid sequence 11 ¥11丄3. In one embodiment, the antibody comprises HVR-L3 having the amino acid sequence of SEQ ID NO: 14. In an embodiment In addition, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 1 and HVR-H2 having the amino acid sequence of SEQ ID NO: 2. In one embodiment, the antibody further comprises SEQ ID NO: HVR-L1 of the amino acid sequence of 12 and 11 ¥11丄2 having the amino acid sequence of SEQ ID > 10: 13. In certain embodiments, any of the above antibodies additionally comprises at least one selected from the group consisting of VH subgroup III Consistent framework and framework of the VL subgroup I consistent framework.

在一態樣中，提供結合至TAT226之抗體，其中該抗體包含與選自SEQ ID ΝΟ··21-25之胺基酸序列具有至少90%序列一致性的重鏈可變結構域。在一實施例中，該抗體另外包含與選自SEQ ID ΝΟ:26-31之胺基酸序列具有至少90% 序列一致性的輕鏈可變結構域。在一實施例中，該抗體包含與SEQ ID ΝΟ:24之胺基酸序列具有至少90%序列一致性之重鏈可變結構域。在一實施例中，該抗體另外包含與 SEQ ID ΝΟ:29之胺基酸序列具有至少90%序列一致性之輕鏈可變結構域。在一實施例中，重鏈可變結構域包含SEQ ID ΝΟ:24之胺基酸序列，且輕鏈可變結構域包含SEQ ID NO:29之胺基酸序列。在一實施例中，該抗體包含與SEQ ID NO:25之胺基酸序列具有至少90%序列一致性之重鏈可變結構域。在一實施例中，該抗體另外包含與SEQ ID 119007.doc -10- 200813092 NO:30之胺基酸序列具有至少90%序列一致性之輕鏈可變結構域。在一實施例中，重鏈可變結構域包含SEQ ID NO:25之胺基酸序列，且輕鏈可變結構域包含SEQ ID NO:30之胺基酸序列。在一態樣中，提供結合至TAT226之抗體，其中該抗體包含與SEQ ID NO:20之胺基酸序列具有至少90%序列一致性之重鏈可變結構域。在一實施例中，該抗體另外包含與 SEQ ID NO:26之胺基酸序列具有至少90%序列一致性之輕鏈可變結構域。在一實施例中，重鏈可變結構域包含SEQ ID ΝΟ··20之胺基酸序列，且輕鏈可變結構域包含SEQ ID NO:26之胺基酸序列。在某些實施例中，提供編碼以上任何抗體之聚核苷酸。在一實施例中，提供包含聚核苷酸之載體。在一實施例中，提供包含載體之宿主細胞。在一實施例中，宿主細胞為真核的。在一實施例中，宿主細胞為CHO細胞。在一實施例中，提供一種製造抗TAT226抗體之方法，其中該方法包含在適合表現編碼抗體之聚核苷酸之條件下培養宿主細胞且分離該抗體。在一態樣中，提供結合至表現於細胞表面之TAT226之抗體。在一實施例中，該抗體結合至來自SEQ ID ΝΟ··75之胺基酸21-115之ΤΑΤ226區内的抗原決定基。在一實施例中，該細胞為癌細胞。在一實施例中，該癌細胞為印巢癌細胞、腦腫瘤細胞或威爾姆氏腫瘤細胞（Wilm’s tumor cell)。在某些實施例中，以上任何抗體均為單株抗體。在一實 119007.doc • 11- 200813092 施例中，該抗體為選自Fab、Fab’-SH、Fv、scFv或（Fab，）2 片段之抗體片段。在一實施例中，該抗體為人化的。在一實施例中，該抗體為人類的。在一實施例中，該抗體結合至與選自 YW0.32、YW0.49、YWO.49.B7、YWO.49.C9、 YWO.49.H2及YWO.49.H6之抗體相同之抗原決定基。在一態樣中，提供一種偵測生物樣本中TAT226存在之方法，該方法包含在容許抗體結合至TAT226之條件下使該生物樣本與以上任何抗體接觸且偵測在抗體與TAT226之間是否形成複合物。在一實施例中，該生物樣本包含卵巢腫瘤細胞、腦腫瘤細胞或威爾姆氏腫瘤細胞。在一態樣中，提供一種診斷與TAT226表現之增加相關聯之細胞增殖性病症的方法，該方法包含使測試細胞與以上任何抗體接觸；藉由偵測抗體與TAT226之結合確定 TAT226之表現程度；且比較測試細胞對TAT226之表現程度與對照細胞對TAT226之表現程度，其中與對照細胞相比，測試細胞對TAT226之較高表現程度指示存在與 TAT226之表現增加相關聯之細胞增殖性病症。在一實施例中，測試細胞為來自懷疑患有細胞增殖性病症之患者的細胞。在一實施例中，細胞增殖性病症係選自卵巢癌及威爾姆氏腫瘤。在一實施例中，該方法包含確定TAT226在測試細胞表面上之表現程度且比較TAT226在測試細胞表面上之表現程度與TAT226在對照細胞表面上之表現程度。本發明進一步提供免疫接合物及其使用方法。在一態樣中，免疫接合物包含以上任何共價連接至細胞 119007.doc -12- 200813092 毒性劑之抗TAT226抗體。在一實施例中，細胞毒性劑係選自毒素、化療劑、抗生素、放射性同位素及核分解酶。在一態樣中，提供具有式Ab_(L_D)p之免疫接合物，其中： (a) Ab為以上任何抗TAT226抗體， (b) L為連接子； (c) D為式DE或DF之藥物In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises a heavy chain variable domain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 26-31. In one embodiment, the antibody comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:29. In one embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 24 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29. In one embodiment, the antibody comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID 119007.doc-10-200813092 NO:30. In one embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 25 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:30. In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 26. In one embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID -20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 26. In certain embodiments, a polynucleotide encoding any of the above antibodies is provided. In one embodiment, a vector comprising a polynucleotide is provided. In one embodiment, a host cell comprising a vector is provided. In one embodiment, the host cell is eukaryotic. In one embodiment, the host cell is a CHO cell. In one embodiment, a method of making an anti-TAT226 antibody is provided, wherein the method comprises culturing a host cell under conditions suitable to represent a polynucleotide encoding the antibody and isolating the antibody. In one aspect, an antibody that binds to TAT226 that is expressed on the cell surface is provided. In one embodiment, the antibody binds to an epitope in the ΤΑΤ226 region of amino acid 21-115 from SEQ ID ΝΟ.75. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is a nested cancer cell, a brain tumor cell, or a Wilm's tumor cell. In certain embodiments, any of the above antibodies are monoclonal antibodies. In a method of 119007.doc • 11-200813092, the antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv or (Fab,) 2 fragments. In one embodiment, the antibody is humanized. In one embodiment, the antibody is human. In one embodiment, the antibody binds to the same antigen as the antibody selected from the group consisting of YW0.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6. base. In one aspect, a method of detecting the presence of TAT226 in a biological sample comprising contacting the biological sample with any of the above antibodies and detecting formation between the antibody and TAT226 is provided under conditions permitting binding of the antibody to TAT226 Complex. In one embodiment, the biological sample comprises ovarian tumor cells, brain tumor cells, or Wilm's tumor cells. In one aspect, a method of diagnosing a cell proliferative disorder associated with an increase in TAT226 expression is provided, the method comprising contacting a test cell with any of the above antibodies; determining the degree of performance of the TAT226 by detecting binding of the antibody to TAT226 And comparing the degree of performance of the test cells to TAT226 to the extent of performance of the control cells to TAT226, wherein the higher degree of performance of the test cells to TAT226 compared to the control cells indicates the presence of a cell proliferative disorder associated with increased performance of TAT226. In one embodiment, the test cells are cells from a patient suspected of having a cell proliferative disorder. In one embodiment, the cell proliferative disorder is selected from the group consisting of ovarian cancer and Wilford's tumor. In one embodiment, the method comprises determining the extent of expression of TAT226 on the surface of the test cells and comparing the extent of expression of TAT226 on the surface of the test cells to the extent of expression of TAT226 on the surface of the control cells. The invention further provides immunoconjugates and methods of use thereof. In one aspect, the immunoconjugate comprises any of the above anti-TAT226 antibodies covalently linked to a cell 119007.doc -12-200813092 toxic agent. In one embodiment, the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope, and a nucleolytic enzyme. In one aspect, an immunoconjugate having the formula Ab_(L_D)p is provided, wherein: (a) Ab is any of the above anti-TAT226 antibodies, (b) L is a linker; (c) D is a formula DE or DF drug

R9 〇R9 〇

1111

.R R10 Df 且其中R及R各自為甲基’ R3及R4各自為異丙基， R7為第二丁基，各R8係獨立地選自CH3、〇-CH3、 OH及 H; R9為 H; R10 為芳基；Z為 _0_ 或 ; R1 為 Η、CVCs 烧基或-(CH2)2-0-(CH2)2-CU(ch2)2-0 CH3 ;且 R18 為-C(R8)2-C(R8)2-芳基；且 (d) p在約1至8之範圍内。在一實施例中，抗體（Ab)包含1)包含與SEQ IE) n〇:114 一致序列相符之胺基酸序列的HVR-H3及2)至少一個、请個、三個、四個或五個選自以下之HVR : ⑴包含SEQ ID NO:4之胺基酸序列之HVR-H1 ; 119007.doc -13- 200813092 (ii)包含SEQ ID NO:5之胺基酸序列之HVR-H2 ; (出）包含8丑(）1〇]^0:12之胺基酸序列之11¥11-1^1; (iv) 包含SEQ ID NO:13之胺基酸序列之HVR-L2 ;及 (v) 包含與SEQ ID ΝΟ··19之一致序列相符之胺基酸序列之 HVR-L3。在一實施例中，抗體包含具有與SEQ ID NO: 19之一致序列相符之胺基酸序列之HVR-L3。在一實施例中，抗體包含具有SEQ ID NO:9之胺基酸序列之HVR-H3及具有SEQ ID NO: 17之胺基酸序列之HVR-L3。在一實施例中，抗體包含具有SEQ ID NO:10之胺基酸序列之HVR-H3及具有 8£()10 1^0:18之胺基酸序列之11¥11丄3。在一實施例中，抗體另外包含具有SEQ ID ΝΟ··4之胺基酸序列之HVR-H1 ; 具有SEQ ID ΝΟ:5之胺基酸序列之HVR-H2;具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之HVR-L2。在一實施例中，抗體包含與選自 SEQ ID NO:21-25之胺基酸序列具有至少90%序列一致性之重鏈可變區及與選自SEQ ID NO:26-31之胺基酸序列具有至少90%序列一致性之輕鏈可變區。在一實施例中，抗體包含與SEQ ID NO:24之胺基酸序列具有至少90%序列一致性之重鏈可變區及與SEQ ID NO:29之胺基酸序列具有至少90%序列一致性之輕鏈可變區。在一實施例中，抗體包含具有SEQ ID NO:24之胺基酸序列之重鏈可變區及具有 SEQ ID NO:29之胺基酸序列之輕鏈可變區。在一實施例中，抗體包含與SEQ ID NO:25之胺基酸序列具有至少90% 119007.doc -14· 200813092 序列一致性之重鏈可變區及與SEQ ID NO:30之胺基酸序列具有至少90%序列一致性之輕鏈可變區。在一實施例中，抗體包含具有SEQ ID ΝΟ:25之胺基酸序列之重鏈可變區及具有SEQIDNO:30之胺基酸序列之輕鏈可變區。另外提供關於以上任何免疫接合物之以下實施例。在一實施例中，免疫接合物具有活體外或活體内細胞殺死活性。在一實施例中，連接子係經由抗體上之硫醇基連接至抗體。在一實施例中，連接子可藉由蛋白酶裂解。在一實施例中’連接子包含val_cit二肽。在一實施例中，連接子包含對胺基苄基單元。在一實施例中，將對胺基苄基單元置於藥物與連接子中之蛋白酶裂解位點之間。在一實施例中’對胺基苄基單元為對胺基苄氧羰基（PAB)。在一實施例中’連接子包含6-馬來醯亞胺己醯基。在一實施例中，將6-馬來醯亞胺己醯基置於抗體與連接子中之蛋白酶裂解位點之間。以上實施例可單獨或以彼此之任何組合形式發生。在一實施例中，藥物係選自MMAE及MMAF。在一實施例中’免疫接合物具有下式. R R10 Df and wherein R and R are each methyl ' R 3 and R 4 are each isopropyl, R 7 is a second butyl group, and each R 8 is independently selected from the group consisting of CH 3 , 〇-CH 3 , OH and H; R 9 is H R10 is aryl; Z is _0_ or; R1 is Η, CVCs alkyl or -(CH2)2-0-(CH2)2-CU(ch2)2-0 CH3; and R18 is -C(R8) 2-C(R8)2-aryl; and (d)p is in the range of from about 1 to 8. In one embodiment, the antibody (Ab) comprises 1) HVR-H3 comprising an amino acid sequence consistent with the sequence of SEQ IE) n〇: 114 and 2) at least one, three, four or five An HVR selected from the group consisting of: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; 119007.doc-13-200813092 (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (out) 11 ¥ 11-1^1 comprising 8 amino acid sequences of 丑 ) ) ^ ^ ^ ; ; ; ; ; ; ; ; ; ; ; ; ; iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv iv v) HVR-L3 comprising an amino acid sequence conforming to the sequence identical to SEQ ID ΝΟ·19. In one embodiment, the antibody comprises HVR-L3 having an amino acid sequence consistent with the sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 9 and HVR-L3 having the amino acid sequence of SEQ ID NO: 17. In one embodiment, the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 10 and 11 ¥11丄3 having an amino acid sequence of 8 £() 10 1^0:18. In one embodiment, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID ΝΟ; 4; HVR-H2 having the amino acid sequence of SEQ ID NO: 5; amine having SEQ ID NO: HVR-L1 of the acid sequence and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. In one embodiment, the antibody comprises a heavy chain variable region having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-25 and an amine group selected from the group consisting of SEQ ID NOs: 26-31 The acid sequence has a light chain variable region with at least 90% sequence identity. In one embodiment, the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 and at least 90% identical to the amino acid sequence of SEQ ID NO: 29. Sexual light chain variable region. In one embodiment, the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 29. In one embodiment, the antibody comprises a heavy chain variable region having at least 90% 119007.doc -14.200813092 sequence identity to the amino acid sequence of SEQ ID NO: 25 and an amino acid of SEQ ID NO: A sequence has a light chain variable region with at least 90% sequence identity. In one embodiment, the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30. Additional examples of any of the above immunoconjugates are provided. In one embodiment, the immunoconjugate has an in vitro or in vivo cell killing activity. In one embodiment, the linker is linked to the antibody via a thiol group on the antibody. In one embodiment, the linker can be cleaved by a protease. In one embodiment the ' linker comprises a val_cit dipeptide. In one embodiment, the linker comprises a p-aminobenzyl unit. In one embodiment, the aminobenzyl unit is placed between the protease and the protease cleavage site in the linker. In one embodiment the 'p-aminobenzyl unit is p-aminobenzyloxycarbonyl (PAB). In one embodiment the ' linker comprises a 6-maleimide hexyl group. In one embodiment, a 6-maleimide hexyl group is placed between the antibody and the protease cleavage site in the linker. The above embodiments may be produced singly or in any combination with each other. In one embodiment, the drug is selected from the group consisting of MMAE and MMAF. In one embodiment, the immunoconjugate has the following formula

其中Ab為以上任何抗TAT226抗體，S為硫原子且p介於2至 5之範圍内。在一實施例中，免疫接合物具有下式 119007.doc -15- 200813092Wherein Ab is any of the above anti-TAT226 antibodies, S is a sulfur atom and p is in the range of 2 to 5. In one embodiment, the immunoconjugate has the formula 119007.doc -15- 200813092

其中Ab為以上任何抗TAT226抗體’ s為硫原子且p介於2至 5之範圍内。在一態樣中，提供一種包含以上任何免疫接合物及醫藥學上可接受之載劑的醫藥組合物。在一態樣中，提供一種治療細胞增殖性病症之方法，其中該方法包含向個體投予醫藥組合物。在一實施例中，細胞增殖性病症係選自卵巢、癌、子宮癌、腦腫瘤及威爾姆氏腫瘤。在一實施例中，細胞增殖性病症係與TAT226在細胞表面上之表現增加相關聯。在一態樣中，提供一種抑制細胞增殖之方法，其中該方法包含在容許免疫接合物結合至TAT226之條件下將細胞暴露於以上任何免疫接合物。在一實施例中，細胞為腫瘤細胞。在一實施例中，腫瘤細胞為卵巢腫瘤細胞、子宮腫瘤細胞、腦腫瘤細胞或威爾姆氏腫瘤細胞。在一實施例中，細胞為異種移植物。在一實施例中，暴露發生於活體外。在一實施例中，暴露發生於活體内。【實施方式】提供結合至TAT226之經分離抗體。另外提供包含抗 TAT226抗體之免疫接合物。例如，本發明之抗體及免疫接合物適用於診斷或治療與TAT226之變化表現（例如增加之表現）相關聯之病症。在某些實施例中，本發明之抗體或免疫接合物適用於診斷或治療細胞增殖性病症，諸如腫瘤 119007.doc •16- 200813092 或癌症。在某些實施例中，本發明《抗體或免疫接合物適用於偵測TAT226，例如於細胞表面上表現之TAT226。提供編碼抗TAT226抗體之聚核苷酸。提供包含編碼抗 TAT226抗體之聚核苷酸之載體且提供包含此等載體之宿主細胞。亦提供包含本發明之聚核苷酸、抗1八丁226抗體或免接合物中之任一或多者之組合物，包括醫藥調配物。 I.通用技術本文所述或參考之技術及程序一般為熟習此項技術者所熟知且通常係使用習知方法來採用，諸如在以下文獻中所述之廣泛使用之方法· Sambrook等人，]Molecular Cloning: A Laboratory Manual 第 3 版（2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.；Wherein Ab is any of the above anti-TAT226 antibodies 's are sulfur atoms and p is in the range of 2 to 5. In one aspect, a pharmaceutical composition comprising any of the above immunoconjugates and a pharmaceutically acceptable carrier is provided. In one aspect, a method of treating a cell proliferative disorder, wherein the method comprises administering to a subject a pharmaceutical composition. In one embodiment, the cell proliferative disorder is selected from the group consisting of ovarian, cancer, uterine cancer, brain tumor, and Wilm's tumor. In one embodiment, the cell proliferative disorder is associated with increased expression of TAT226 on the cell surface. In one aspect, a method of inhibiting cell proliferation is provided, wherein the method comprises exposing the cell to any of the above immunoconjugates under conditions that permit binding of the immunoconjugate to TAT226. In one embodiment, the cells are tumor cells. In one embodiment, the tumor cells are ovarian tumor cells, uterine tumor cells, brain tumor cells, or Wilm's tumor cells. In one embodiment, the cells are xenografts. In one embodiment, the exposure occurs outside the body. In one embodiment, the exposure occurs in a living body. [Embodiment] An isolated antibody that binds to TAT226 is provided. An immunoconjugate comprising an anti-TAT226 antibody is also provided. For example, the antibodies and immunoconjugates of the invention are useful for the diagnosis or treatment of conditions associated with altered behavior of TAT226 (e.g., increased performance). In certain embodiments, the antibodies or immunoconjugates of the invention are useful for the diagnosis or treatment of cell proliferative disorders, such as tumors 119007.doc • 16-200813092 or cancer. In certain embodiments, the antibodies or immunoconjugates of the invention are useful for detecting TAT226, such as TAT226, which is expressed on the cell surface. A polynucleotide encoding an anti-TAT226 antibody is provided. A vector comprising a polynucleotide encoding an anti-TAT226 antibody is provided and a host cell comprising such a vector is provided. Compositions comprising any one or more of the polynucleotides of the invention, anti-I-octabuter 226 antibodies or exfoliants, including pharmaceutical formulations, are also provided. I. General Techniques The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are commonly employed using conventional methods, such as those described in the following documents. [Sambrook et al.] Molecular Cloning: A Laboratory Manual Third Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;

Current Protocols in Molecular Biology (F· M· Ausubel等人編，（2003)) ; the series Methods in Enzymology (Academic Press, Inc.): Per 2: A Practical Approach (M. J. MacPherson，B_ D. Hames及 G. R. Taylor編（1995))，Harlow 及 Lane 編（1988) Antibodies，A Laboratory Manual 及 Animal Cell Culture (R. I. Freshney 編（1987)); Oligonucleotide Synthesis (M. J. Gait編，1984) ； Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis 編，1998) Academic Press ; Animal Cell Culture (R. I. Freshney)編，(1987); Introduction to Cell and Tissue Culture (J· Ρ· Mather及 Ρ· E. Roberts, 1998) Plenum Press ； Cell and Tissue Culture: 119007.doc -17- 200813092Current Protocols in Molecular Biology (F. M. Ausubel et al., (2003)); the series Methods in Enzymology (Academic Press, Inc.): Per 2: A Practical Approach (MJ MacPherson, B_D. Hames and GR Taylor) (1995)), Harlow and Lane (1988) Antibodies, A Laboratory Manual and Animal Cell Culture (RI Freshney, ed. (1987); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JE Cellis, 1998) Academic Press; Edited by Animal Cell Culture (RI Freshney), (1987); Introduction to Cell and Tissue Culture (J· Ρ· Mather and Ρ E. Roberts, 1998) Plenum Press ; Cell and Tissue Culture: 119007.doc -17- 200813092

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Monoclonal Antibodies: A Practical Approach (P.Monoclonal Antibodies: A Practical Approach (P.

Shepherd及 C. Dean編，Oxford University Press, 2000)； Using Antibodies: A Laboratory Manual (E. Harlow及0· Lane (Cold Spring Harbor Laboratory Press, 1999) ; The Antibodies (M. Zanetti 及 J. D. Capra 編，Harwood Academic Publishers，1995);及 Cancer: Principles and Practice of Oncology (V. T. DeVita等人編，J· B. Lippincott Company，1993)。 II.定義及縮寫 Α·定義 ’’經分離"抗體為經識別且自其天然環境之組份分離及/或回收之抗體。其天然環境之污染物組份為干擾抗體研究、診斷或治療用途之物質，且可包括酶、激素及其他蛋白質 119007.doc -18- 200813092 或非蛋白質溶質。在一些實施例中，將抗體（丨）純化至如例如勞裏法（Lowry method)所測定之大於95重量％之抗體且在一些實施例中，大於99重量％ ; (2)純化至藉由使用例如旋杯式定序儀足以獲得至少15個殘基之^^端或内部胺基酸序列之転度，或（3)藉由在還原或非還原條件下使用例如庫馬斯冗藍（Coomassie blue)或銀染色之SDS-PAGE純化至均質。經分離抗體包括重組細胞中原位之抗體，此係因為抗體天…；環i兄之至少一種組份將不存在。然而，經分離抗體通常將藉由至少一個純化步驟來製備。 ’’經分離’’核酸分子為自通常在例如其天然環境中與其相關聯之至少一種其他核酸分子分離之核酸分子。經分離核酉文刀子另外包括通常表現核酸分子之細胞中所含之核酸分子，但該核酸分子係存在於染色體外或存在於不同於其天然染色體位置之染色體位置。經純化’’意謂分子以在含有其之樣本中至少95重量％或至少98重量％之濃度存在於該樣本中。如本文所用之術語”大體上相似"或"大體上相同"表示兩個數值（例如，一者與本發明之抗體相關聯且另一者與參考/比較抗體相關聯）之間足夠高之相似度，以使得熟習此項技術者認為兩個值之間之差異在由該等值（例如尺(1值）所量測之生物特徵情形中幾乎無或無生物及/或統計學顯著性。該兩個值之間之差異作為參考/比較值之函數，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%。 119007.doc -19- 200813092 如本文所用之短語”大體上降低”或”大體上不同”表示兩個數值（一般為一者與分子相關聯且另一者與參考/比較分子相關聯）之間足夠高之差異度，以使得熟習此項技術者認為兩個值之間之差異在由該等值（例如Kd值）所量測之生物特徵情形中具有統計學顯著性。該兩個值之間之差異作為參考/比較分子之值的函數，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%。如本文所用之術語f’載體"意欲係指能夠轉運其所連接至之另一核酸的核酸分子。一種類型之載體為”質體"，其係指可接合額外DNA區段之環狀雙鏈DNA。另一種類型之載體為噬菌體載體。另一種類型之載體為病毒載體，其中額外DNA區段可接合至病毒染色體組中。某些載體能夠在引入其之宿主細胞（例如具有細菌複製起點之細菌載體及游離型哺乳動物載體）中自主複製。可將其他載體（例如非游離型哺乳動物載體）在引入宿主細胞中時整合於宿主細胞之染色體組中，且藉此與宿主染色體組一起複製。此外，某些載體能夠指引其可操作性地連接之基因的表現。此等載體在本文中稱作”重組表現載體”或簡言之稱作”表現載體’•。一般而言，用於重組DNA技術中之表現載體通常為質體形式。在本說明書中，由於質體為最常用之載體形式，因此”質體”與”載體"可交替使用。如本文可交替使用之，，聚核苷酸，，或"核酸”係指任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、經修飾之核苷酸或鹼及/或其類 119007.doc -20- 200813092 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，諸如甲基化核苷酸及其類似物。若存在，則可在組裝聚合物之前或之後對核苷酸結構進行修飾。核苷酸序列可藉由非核芽酸組份中斷。聚核苷酸可包含在合成後進行之修飾，諸如與一標記結合。其他類型之修飾例如包括”帽子”；以類似物取代天然存在之一或多種核苷酸；核苷酸間修飾，諸如彼等具有不帶電鍵聯（例如膦酸曱酯、磷酸三_、鱗醯胺酸酯、胺基甲酸酯等）及具有帶電鍵聯（例如硫代磷酸 S旨、二硫代磷酸酯等）者；彼等含有附屬部分者，諸如蛋白（例如核酸if、毒素、抗體、信號肽、離胺酸等），彼等具有嵌入劑者（例如吖啶、補骨脂素等）；彼等含有螯合劑者（例如金屬、放射性金屬、硼、氧化性金屬等）；彼等含有烷化劑者·，彼等具有經修飾鍵聯者（例如α 變旋異構核酸等）以及未經修飾形式之聚核苷酸。此外，可將通常存在於糖中之任何羥基（例如）經膦酸酯基、磷酸酉曰基置換；由標準保護基保護或經活化以製備額外核苷酸之額外鍵聯，或可結合至固體或半固體支撐物。5，及3,端〇Η可經磷酸化或經胺或具有！至2〇個碳原子之有機封端基團部分取代。其他羥基亦可衍生為標準保護基。聚核苷酸亦可含有一般為此項技術中已知之類似形式之核糖或去氧核糖’包括例如2，-〇-甲基_ ; 2，_〇_烯丙基2，_氣-或2，_疊氮基-核糖；碳環基糖類似物；α_變旋異構糖；差向異構糖，諸如***糖、木糖或來蘇糖；哌喃糖；呋喃糖；景 H9007.doc -21 - 200813092 天庚酮糖（sedoheptulose);非環式類似物及諸如甲基核糖苷基之鹼性核苷類似物。一或多個磷酸二酯鍵可經替代性鍵聯基團置換。該等替代性鍵聯基團包括（但不限於）其中磷酸酯由p(o)s(”硫代酯”）、p(s)s(”二硫代酯”）、（〇)NR2 (’·醯胺酯，，）、P(〇)R、P(0)0R’、CO 或 CH2C，甲縮醛，，）置換之實施例，其中各R或R’獨立地為Η或視情況含有醚（_〇_)鍵之經取代或未經取代烷基（1-20個C)、芳基、烯基、環烧基、環烯基或芳烧基（araldyl)。聚核苷酸中之所有鍵無需均相同。先前描述適用於本文提及之所有聚核苷酸，包括 RNA及 DNA。如本文所用之，，寡核苷酸”一般係指長度一般（但非必需）小於約2 0 0個核皆酸之短、一般為單鏈、一般為合成之聚核苷酸。術語”寡核苷酸”及”聚核苷酸，，並不相互排斥。以上關於聚核苷酸之描述可等同地且完全適用於寡核苦酸。關於參考多肽序列之’’胺基酸序列一致性百分比（％)"係定義為在對準序列且（若需要）引入間隙以達成最大序列一致性百分比之後，候選序列中與參考多肽序列中之胺基酸殘基相同之胺基酸殘基的百分比，且不認為任何保守性取代為序列一致性之部分。為測定胺基酸序列一致性百分比目的之對準可經此項技術中之各種方式來達成，例如使用可公開獲得之電腦軟體，諸如BLAST、BLAST-2、ALIGN 或Megalign(DNASTAR)軟體。彼等熟習此項技術者可確定用於對準序列之適當參數，包括需要用以達成對所比較序列全長之最大對準的任何演算法。然而，為本文之目的， 119007.doc -22- 200813092 使用序列比較電腦程式ALIGN-2產生胺基酸序列一致性％值。ALIGN-2序列比較電腦程式係由Genentech，Inc·創造，且源代碼已在U.S· Copyright Office，Washington D.C.， 20559中以使用者文件申請，其係於U.S. Copyright註冊號 TXU510087 下註冊。ALIGN-2 程式可自 Genentech，Inc·， South San Francisco，California公開獲得或可自源代碼編譯。ALIGN-2程式應經編譯以用於UNIX作業系統，較佳為數位UNIX V4.0D。所有序列比較參數係藉由ALIGN-2程式設定且不變化。在採用ALIGN-2用於胺基酸序列比較之情況下，指定胺基酸序列A與指定胺基酸序列B之胺基酸序列一致性％(可替代性地表述為與指定胺基酸序列B具有或包含特定胺基酸序列一致性％之指定胺基酸序列A)係如下計算：Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and 0. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, Harwood) Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., ed., J. B. Lippincott Company, 1993). II. Definitions and abbreviations Α · Definitions ''Separated" antibodies are identified and An antibody that is isolated and/or recovered from components of its natural environment. The contaminant component of its natural environment is a substance that interferes with the research, diagnosis, or therapeutic use of antibodies, and may include enzymes, hormones, and other proteins. 119007.doc -18- 200813092 or a non-protein solute. In some embodiments, the antibody (丨) is purified to greater than 95% by weight of the antibody as determined, for example, by the Lowry method and, in some embodiments, greater than 99% by weight; 2) purification to obtain a twist of at least 15 residues or an internal amino acid sequence by using, for example, a rotary cup sequencer, or (3) by reduction or non-reduction Purified to homogeneity under original conditions using, for example, Coomassie blue or silver stained SDS-PAGE. The isolated antibody includes antibodies in situ in recombinant cells, because of antibody days... at least one group of the ring brothers The fraction will not be present. However, the isolated antibody will typically be prepared by at least one purification step. The ''isolated'' nucleic acid molecule is a nucleic acid isolated from at least one other nucleic acid molecule that is normally associated with it, for example, in its natural environment. Molecules. The isolated nuclear knives additionally include nucleic acid molecules contained in cells that normally represent nucleic acid molecules, but the nucleic acid molecules are present extrachromosomally or at a chromosomal location different from their natural chromosomal location. The molecule is present in the sample at a concentration of at least 95% by weight or at least 98% by weight in the sample containing it. As used herein, the term "substantially similar" or "substantially identical" means two values ( For example, a sufficiently high similarity between one associated with an antibody of the invention and the other associated with a reference/comparative antibody) , So that those skilled in the art that this term consider the difference between two values of little or no biological and / or statistical significant and in the case of the equivalent biological characteristics (e.g. feet (value 1) as measured in the. The difference between the two values is a function of the reference/comparison value, e.g., less than about 50%, less than about 40%, less than about 3%, less than about 2%, and/or less than about 10%. 119007.doc -19- 200813092 The phrase "substantially lower" or "substantially different" as used herein denotes two values (generally one associated with a molecule and the other associated with a reference/comparative molecule). The difference is sufficiently high that the person skilled in the art believes that the difference between the two values is statistically significant in the biometric case measured by the equivalent (e.g., Kd value). The difference between the two values is a function of the value of the reference/comparative molecule, e.g., greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%. The term f'carrier" as used herein is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is "plastid", which refers to circular double-stranded DNA that can bind additional DNA segments. Another type of vector is a phage vector. Another type of vector is a viral vector in which additional DNA segments are present. Can be ligated into the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (eg, a bacterial vector with a bacterial origin of replication and a free mammalian vector). Other vectors (eg, non-episomal mammalian vectors) When introduced into a host cell, it is integrated into the genome of the host cell and thereby replicated with the host genome. In addition, certain vectors are capable of directing the expression of the operably linked genes. It is called a "reformed expression vector" or simply a "expression carrier". In general, expression vectors 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, the polynucleotide, or "nucleic acid" Nucleotide polymers of any length, and include DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or a base, and/or a class thereof, 119007.doc -20-200813092, or may be borrowed by DNA or RNA polymerase or Any substrate that is incorporated into the polymer by a synthetic reaction. The polynucleotide may comprise modified nucleotides such as methylated nucleotides and analogs thereof. If present, the nucleotide structure can be modified before or after assembly of the polymer. The nucleotide sequence can be interrupted by a non-nuclear acid component. The polynucleotide may comprise modifications that are made after synthesis, such as binding to a label. Other types of modifications include, for example, "hats"; substitution of one or more nucleotides naturally occurring with an analog; internucleotide modifications, such as those having an uncharged linkage (eg, phosphonium phosphonate, phosphoric acid tris, scales) "proline, urethane, etc." and those having a charge linkage (eg, thiophosphoric acid, dithiophosphate, etc.); those containing a subsidiary, such as a protein (eg, nucleic acid if, toxin, Antibodies, signal peptides, lysines, etc.), those with intercalating agents (such as acridine, psoralen, etc.); those containing chelating agents (such as metals, radioactive metals, boron, oxidizing metals, etc.); Those containing an alkylating agent, such as those having a modified linkage (for example, a α-rotational isomeric nucleic acid, etc.) and an unmodified form of the polynucleotide. In addition, any hydroxyl group typically present in the sugar can be replaced, for example, by a phosphonate group, a phosphonium phosphate group; an additional linkage that is protected or activated by a standard protecting group to make additional nucleotides, or can be Solid or semi-solid support. 5, and 3, the end can be phosphorylated or amine or have! Partially substituted with an organic capping group of 2 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. The polynucleotide may also contain ribose or deoxyribose sugars generally of a similar form known in the art including, for example, 2,-〇-methyl _; 2, 〇〇 ally ally 2, _ gas - or 2 , _ azido-ribose; carbocyclic sugar analog; α_orromeric isomeric sugar; epimeric sugar, such as arabinose, xylose or lyxose; palladium; furanose; H9007. Doc-21 - 200813092 sedoheptulose; acyclic analogs and basic nucleoside analogs such as methyl ribosyl groups. One or more phosphodiester linkages may be replaced by an alternative linkage group. Such alternative linking groups include, but are not limited to, wherein the phosphate is composed of p(o)s("thioester"), p(s)s("dithioester"), (〇)NR2 ( An example of a substitution of '· 醯酯 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , A substituted or unsubstituted alkyl group (1-20 C), an aryl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group or an araldyl group having an ether (_〇_) bond. All of the bonds in the polynucleotide need not be the same. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA. As used herein, an oligonucleotide "generally" refers to a short, generally single-stranded, generally synthetic polynucleotide having a length (typically, but not necessarily) less than about 2,000 nucleotides. Nucleotide "and" polynucleotides are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. The 'amino acid sequence identity percent (%)" for a reference polypeptide sequence is defined as the candidate sequence and the reference polypeptide sequence after the alignment sequence and, if necessary, the introduction of a gap to achieve a maximum sequence identity percentage. The percentage of amino acid residues of the same amino acid residue is not considered to be part of the sequence identity. Alignment of the purpose of determining the percent identity of the amino acid sequence can be achieved by various means in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for the alignment sequence, including any algorithms needed to achieve maximum alignment of the full length of the sequences being compared. For the purposes of this paper, however, 119007.doc -22- 200813092 uses the sequence comparison computer program ALIGN-2 to generate amino acid sequence identity % values. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been filed in U.S. Copyright Office, Washington D.C., 20559 as a user file, which is registered under U.S. Copyright registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California or can be compiled from source code. The ALIGN-2 program should be compiled for use in a UNIX operating system, preferably a digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and did not change. In the case where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence sequence of the amino acid sequence A and the designated amino acid sequence B is specified to be % identical (alternatively expressed as a specified amino acid sequence) The designated amino acid sequence A of B having or containing % identity of a particular amino acid sequence is calculated as follows:

l〇〇x 分數 X/Y 其中X為在A與B之程式對準中，藉由序列對準程式 ALIGN-2標記為相同匹配之胺基酸殘基的數目，且其中γ 為B中胺基酸殘基之總數。應瞭解，在胺基酸序列a之長度不等於胺基酸序列B之長度時，A與B之胺基酸序列一致性％將不等於B與A之胺基酸序列一致性％。除非另外特定描述，否則本文所用之所有胺基酸序列一致性％值係如前段所述使用ALIGN-2電腦程式獲得。除非另外指出，否則如本文所用之術語”TAT226"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）及齧齒類動物（例如小鼠及大鼠））之任何原生 119007.doc -23- 200813092 TAT226 〇術語涵蓋丨丨全長"未加工TAT226及由加工細胞所產生之任何形式ΤΑΤ226。術語亦涵蓋天然產生之ΤΑΤ226 變異體，例如剪接變異體或對偶基因變異體。”成熟形式” 之ΤΑΤ226為已經受加工之ΤΑΤ226形式，例如已經受Ν端 (例如信號序列）及/或C端裂解及/或由連接GPI固著劑修飾之ΤΑΤ226形式。在一實施例中，"成熟形式”之ΤΑΤ226係於細胞表面上表現。 π抗體n(Ab)及”免疫球蛋白n(Ig)為具有類似結構特徵之糖蛋白。儘管抗體對特異性抗原展現結合特異性，但免疫球蛋白包括一般缺少抗原特異性之抗體及其他抗體樣分子。後一種多肽例如由淋巴系統以低含量產生及由骨髓瘤以增加之含量產生。術語’’抗體”及”免疫球蛋白”最廣義上可互換使用且包括單株抗體（例如全長或完整單株抗體）、多株抗體、單價抗體、多價抗體、多特異性抗體（例如雙特異性抗體，只要其展現所需生物活性即可），且亦可包括某些抗體片段（如本文更詳細描述）。抗體可為嵌合抗體、人類抗體、人化抗體及/或親和力成熟之抗體。術語抗TAT226抗體"或π結合至TAT226之抗體"係指能夠以充分親和力結合ΤΑΤ226之抗體，以使得抗體適合用作靶向ΤΑΤ226之診斷及/或治療劑。較佳地，如例如藉由放射免疫檢定（RIA)所量測，抗ΤΑΤ226抗體與非相關、非 ΤΑΤ226蛋白之結合程度小於抗體與ΤΑΤ226結合之約1〇% 〇在某些實施例中，結合至ΤΑΤ226之抗體具有$1 μΜ、$100 119007.doc -24- 200813092 二M^lG nM、g nM或巧」nM之解離常數州。在某些實施例中，抗TAT226抗體結合至在來自不同物種二 TAT226中保守之TAT226抗原決定基。〜抗體之"可變區”或"可變結構域"係指抗體重鏈或輕鏈之胺基端結構域。重鏈可變結構域可稱作"VH"。輕鏈可變結構域可稱作"VL”。該等結構域—般為抗體之最可變部分: 含有抗原結合位點。術語”可變”係指抗體間可變結構域之特定部分之序列普遍不同且用於各特定抗體與其特定抗原之結合且特異性γ 然而，可變性在抗體之整個可變結構域中並非均勻分佈。其集中於均位於輕鏈及重鏈可變結構域中稱為互補判定區 (CDR)或尚變區（HVR)之三個區段中。可變結構域之較高度保守部分稱作框架區（FR)。原生重鏈及輕鏈之可變結構域各自包含大量採用β折疊構型、由三個CDR連接之四個 FR區，其形成環連接且在一些情況下形成ρ折疊結構之部分。將各鏈中之CDR藉由FR區緊鄰地固持在一起，且與其他鏈之CDR —起有助於形成抗體之抗原結合位點（參見 Kabat 專人 ’ Sequences of Proteins of ImmunologicalL〇〇x fraction X/Y where X is the number of amino acid residues labeled as the same match by the sequence alignment program ALIGN-2 in the alignment of A and B, and wherein γ is the amine in B The total number of base acid residues. It will be appreciated that when the length of the amino acid sequence a is not equal to the length of the amino acid sequence B, the % identity of the amino acid sequence of A and B will not be equal to the % identity of the amino acid sequence of B and A. Unless otherwise specifically stated, all amino acid sequence identity % values used herein were obtained using the ALIGN-2 computer program as described in the previous paragraph. The term "TAT226" 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). 119007.doc -23- 200813092 TAT226 〇 Terminology encompasses 丨丨 full length "unprocessed TAT226 and any form produced by processed cells ΤΑΤ226. The term also encompasses naturally occurring ΤΑΤ226 variants, such as splice variants or dual gene variants. The "mature form" ΤΑΤ 226 is in the form of a ΤΑΤ 226 that has been processed, for example, in the form of a ΤΑΤ 226 that has been cleaved by a scorpion (eg, a signal sequence) and/or a C-terminus and/or modified by a GPI-attachment. In one embodiment, The "mature form" is based on the cell surface. π antibody n (Ab) and "immunoglobulin n (Ig) are glycoproteins with similar structural features. Although antibodies exhibit binding specificity for specific antigens, immunoglobulins include antibodies and other antibodies that are generally lacking in antigen specificity. The latter polypeptide is produced, for example, by the lymphatic system at low levels and by increased levels of myeloma. The terms 'antibody' and 'immunoglobulin' are used interchangeably in the broadest sense and include monoclonal antibodies (eg full length or Complete monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (eg, bispecific antibodies, as long as they exhibit the desired biological activity), and may also include certain antibody fragments (eg, More detailed description). The antibody may be a chimeric antibody, a human antibody, a humanized antibody, and/or an affinity matured antibody. The term "anti-TAT226 antibody" or "antibody that binds to TAT226" refers to an antibody capable of binding ΤΑΤ226 with sufficient affinity to render the antibody suitable for use as a diagnostic and/or therapeutic agent for target 226. Preferably, the anti-ΤΑΤ226 antibody binds to an unrelated, non-ΤΑΤ226 protein less than about 1% by weight of the antibody and ΤΑΤ226, as measured, for example, by radioimmunoassay (RIA). In some embodiments, The antibody to 226 has a dissociation constant state of $1 μΜ, $100 119007.doc -24- 200813092 two M^lG nM, g nM or Q"nM. In certain embodiments, the anti-TAT226 antibody binds to a TAT226 epitope that is conserved in a different species from TAT226. ~ Antibody "variable region" or "variable domain" refers to the amino-terminal end domain of an antibody heavy or light chain. The heavy chain variable domain can be referred to as "VH". The variable domain can be called "VL". These domains are generally the most variable part of an antibody: contain an antigen binding site. The term "variable" refers to the fact that the sequences of specific portions of the variable domains between antibodies are generally different and are used for binding and specificity of each particular antibody to its particular antigen. However, the variability is not uniform throughout the entire variable domain of the antibody. distributed. It is concentrated in three segments, each of which is referred to as a complementarity determining region (CDR) or a variable region (HVR), in the light and heavy chain variable domains. The higher conserved portion of the variable domain is referred to as the framework region (FR). The variable domains of the native heavy and light chains each comprise a plurality of four FR regions joined by three CDRs in a beta sheet configuration which form a loop junction and in some cases form part of a p-fold structure. The CDRs in each chain are held together in close proximity by the FR region, and the CDRs of other chains contribute to the formation of the antigen binding site of the antibody (see Kabat Specialist' Sequences of Proteins of Immunological

Interest ’ 第五版，National Institute of Health，Bethesda， MD (1991))。恆定結構域不直接涉及在抗體與抗原之結合中’但展現各種效應功能，諸如抗體參與抗體依賴型細胞毒性。可將來自任何脊椎動物物種之抗體（免疫球蛋白）之”輕鏈” 基於其恆定結構域之胺基酸序列指定為稱為κ及λ之兩種完 119007.doc -25- 200813092 全不同類型中之一種。抗體（免疫球蛋白）可視其重鏈恆定結構域之胺基酸序列而指定為不同種類。存在五種主要種類之免疫球蛋白： IgA、IgD ' IgE、IgG及IgM，且可將其中若干進一步分為例如 IgG!、IgG2、IgG3、IgG4、IgAdIgA2之子類（同型）。將對應於不同免疫球蛋白種類之重鏈恆定結構域分別稱為 α、δ、ε、γ及μ。不同種類免疫球蛋白之次單位結構及三維構型係眾所熟知，且一般例如描述於Abbas等人Cellular ( and M〇L Immunology，第4版（2000)中。抗體可為藉由抗體與一或多種其他蛋白或肽之共價或非共價締合所形成之較大融合分子的部分。術語’’全長抗體"、"完整抗體”及，’全抗體"在本文中可互換使用，以係指其大體上完整形式之抗體，而非如下文所疋義之抗體片段。該等術語尤其係指具有含有F c區之重鏈之抗體。 π抗體片段’’僅包含完整抗體之一部分，其中該部分保持通常與彼部分存在於完整抗體中時相關之至少一種，且盡可能為大部分或所有功能。在一實施例中，抗體片段包含完整抗體之抗原結合位點，且因此保持結合抗原之能力。在另一實施例中，抗體片段（例如包含Fc區之抗體片段）保持通常與Fc區存在於完整抗體中時相關之至少一種生物功能’該等生物功能諸如FcRn結合，抗體半衰期調變、 ADCC功能及補體結合。在一實施例中，抗體片段為具有與完整抗體大體上類似之活體内半衰期之單價抗體。舉例 119007.doc -26- 200813092Interest's fifth edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of the antibody to the antigen 'but exhibit various effector functions, such as antibodies involved in antibody-dependent cytotoxicity. The "light chain" of antibodies (immunoglobulins) from any vertebrate species can be designated as the amino acid sequence based on its constant domain as the two types called κ and λ. 119007.doc -25- 200813092 One of them. The antibody (immunoglobulin) can be assigned to a different species depending on the amino acid sequence of its heavy chain constant domain. There are five major classes of immunoglobulins: IgA, IgD 'IgE, IgG, and IgM, and several of them can be further divided into subclasses (isotypes) such as IgG!, IgG2, IgG3, IgG4, IgAdIgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformation of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al. Cellular (and M〇L Immunology, 4th edition (2000). Antibodies can be made with antibodies and Or a portion of a larger fusion molecule formed by covalent or non-covalent association of a plurality of other proteins or peptides. The terms ''full length antibody", "intact antibody" and, 'full antibody" are interchangeable herein. An antibody that is used in its substantially intact form, rather than an antibody fragment as defined below. These terms especially refer to an antibody having a heavy chain comprising a Fc region. The π antibody fragment '' contains only intact antibodies. a portion, wherein the portion retains at least one of the associations typically associated with the presence of the portion in the intact antibody, and is as much or all of the functionality as possible. In one embodiment, the antibody fragment comprises the antigen binding site of the intact antibody, and thus Maintaining the ability to bind antigen. In another embodiment, an antibody fragment (eg, an antibody fragment comprising an Fc region) remains normally associated with the presence of an Fc region in an intact antibody. One less biological function 'such biological functions as FcRn binding, antibody half-life modulation, ADCC function and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to the intact antibody. Example 119007 .doc -26- 200813092

接至Fc序列之抗原結合臂。Attached to the antigen binding arm of the Fc sequence.

易於結晶之能力的一個殘餘”Fc，，片段。受，及名稱反映其胃蛋白酶處理產生具有兩個抗原組合位點且仍可交聯抗原之F(ab，)2片段。 nFvn為含有完全抗原結合位點之最小抗體片段。在一實施例中，雙鏈Fv物種由緊密非共價締合之一個重鏈與一個輕鏈可變結構域之二聚體組成。在單鏈Fv(scFv)物種中，一個重鏈與一個輕鏈可變結構域可由彈性肽連接子共價連接，以使得輕鏈與重鏈可以與雙鏈Fv物種中類似之，，二聚，，結構締合。在此構型中，各可變結構域之三個CDR相互作用以定義VH-VL二聚體表面上之抗原結合位點。總而言之，/、個CDR賦予抗體以抗原結合特異性。然而，即使單一可變結構域（或僅包含三個對抗原具特異性之CDR之半數Fv)亦具有識別及結合抗原之能力，儘管其具有比全結合位點較低之親和力。A residual "Fc," fragment of the ability to crystallize, and the name reflects its pepsin treatment to produce a F(ab,)2 fragment that has two antigen-binding sites and is still cross-linkable. nFvn is a complete antigen The minimal antibody fragment of the binding site. In one embodiment, the double-stranded Fv species consists of a heavy chain of tight non-covalent associations and a dimer of one light chain variable domain. In single-chain Fv (scFv) In a species, one heavy chain and one light chain variable domain can be covalently linked by an elastin linker such that the light and heavy chains can be associated with a similar, dimeric, structural association with a double-stranded Fv species. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In summary, the CDRs confer antigen binding specificity to the antibody. However, even a single The variable domain (or half of the Fv comprising only three antigen-specific CDRs) also has the ability to recognize and bind antigen, although it has a lower affinity than the full binding site.

Fab片段含有重鏈及輕鏈可變結構域，且亦含有輕鏈之恆定結構域及重鏈之第一恆定結構域（CH1)。Fab，片段因為在包括來自抗體欽鍵區之一或多個半脱胺酸之重鍵CH1 結構域的羧基端添加數個殘基而不同於Fab片段。Fab，-SH 在本文中命名為Fab’，其中恆定結構域之半胱胺酸殘基帶有游離硫醇基。F(ab’）2抗體片段初始係以其間具有鉸鏈半胱胺酸之Fab’片段對之形式而產生。抗體片段之其他化合 119007.doc -27- 200813092 偶合亦為已知。 π單鏈Fv"或nscFv"抗體片段包含抗體之VH及VL結構域，其中該等結構域係存在於單一多肽鏈中。一般而言， scFv多肽在VH與VL結構域之間另外包含多肽連接子，其使得scFv可形成抗原結合所需之結構。關於scFV之論述參見 Pluckthun ，在 The Pharmacology of Monoclonal Antibodies，第 113 卷中，Rosenburg 及 Moore 編，Springer-Verlag，New York，第 269-315 頁（1994)。術語”雙功能抗體’’係指具有兩個抗原結合位點之小抗體片段，該等片段包含連接至相同多肽鏈中之輕鏈可變結構域（VL)之重鏈可變結構域（VH)(VH-VL)。藉由使用過短而不能使相同鏈上之兩個結構域之間成對的連接子，迫使該等結構域與另一鏈之互補結構域成對且產生兩個抗原結合位點。雙功能抗體可為二價的或雙特異性的。雙功能抗體係更全面描述於例如EP 404,097 ; WO 93/11161 ; Hudson 等人（2003) Mei 9:129-134;及 Hollinger 等人，Proc·The Fab fragment contains a heavy chain and a light chain variable domain and also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab, the fragment differs from the Fab fragment by the addition of several residues at the carboxy terminus including the heavy bond CH1 domain from one or more of the semi-deaminating acids of the antibody. Fab, -SH is designated herein as Fab', wherein the cysteine residue of the constant domain carries a free thiol group. The F(ab')2 antibody fragment is initially produced in the form of a Fab' fragment pair with hinged cysteine. Other combinations of antibody fragments 119007.doc -27- 200813092 Coupling is also known. A π single-chain Fv" or nscFv" antibody fragment comprises the VH and VL domains of an antibody, wherein the domains are present in a single polypeptide chain. In general, the scFv polypeptide additionally comprises a polypeptide linker between the VH and VL domains which allows the scFv to form the desired structure for antigen binding. For a discussion of scFV, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994). The term "bifunctional antibody" refers to a small antibody fragment having two antigen binding sites comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain. (VH-VL). By using a link that is too short to be paired between two domains on the same chain, forcing the domains to be paired with the complementary domains of the other chain and creating two Antigen binding sites. Bifunctional antibodies can be bivalent or bispecific. Bifunctional anti-systems are more fully described, for example, in EP 404,097; WO 93/11161; Hudson et al. (2003) Mei 9:129-134; Hollinger et al., Proc·

Natl. Acad· Sci. 90:6444-6448 (1993)中。三功能抗體及四功能抗體亦描述於Hudson等人（2003) Md· 9:129-134 中。如本文所用之術語，，單株抗體”係指由大體上均質之抗體群體（亦即除可微量存在之可能突變（例如天然產生之突變）以外’包含該群體之個別抗體係相同的）所獲得之抗體。因此’修飾語”單株，，表示不為離散抗體之混合物之抗體特徵。在某些實施例中，此單株抗體通常包括包含結合靶之 119007.doc -28 - 200813092 多肽序列之抗體，丨中該靶結合多肽序列係藉由包括自複數個夕狀序列選擇單一靶結合多肽序列之方法而獲得。舉例而^，選擇方法可為自複數個純系（諸如融合瘤純系、噬菌體純系或重組DNA純系之總和）選擇獨特純系。應瞭解，所選擇之靶結合序列可經進一步改變，例如以改良對靶之親和力；使靶結合序列人化；改良其在細胞培養物中之產生，降低其活體内免疫原性；產生多特異性抗體等，且包含經改變靶結合序列之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）之不同抗體的多株抗體製劑相反，單株抗體製劑之各單株抗體係針對抗原上之單一決定子。除其特異性之外，單株抗體製劑之優勢在於其通&不文其他免疫球蛋白之污染。修飾語”單株”表示由大體上均質之抗體群體所獲得之抗體的特徵，且不將其理解為需要藉由任何特定方法產生抗體。舉例而言，待根據本發明使用之單株抗體可藉由各種技術來製造，該等技術包括例如融合瘤法（例如Kohler等人 ’ TVaiwre，256: 495 (1975) ; Harlow等人，Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press，第 2 版 1988) ; Hammerling 等人，在 Monoclonal Antibodies and Τ-CeII Hybridomas 563-681 (Elsevier, N.Y·，1981)中）、重組DNA法（參見例如美國專利第 4,816,567號）、噬菌體呈現技術（參見例如(：13^^〇11等人， 352: 624-628 (1991) ; Marks等人，/· Mo/· 5b/· 222·· 581-597 (1992) ; Sidhu等人，J. Mo/. 5b/· 338(2): 119007.doc •29- 200813092 299-310 (2004) ; Lee等人，/· Mo/· 5b/· 340(5): 1073-1093 (2004) i Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004)；及 Lee 等人 ’ J. Immunol· Methods 284(1-2): 119-132(2004))及在具有編碼人類免疫球蛋白序列之部分或所有人類免疫球蛋白基因座或基因之動物體内產生人類或人類樣抗體之技術（參見例如WO 98/24893 ; WO 96/34096 ； WO 96/33735 ； WO 91/10741 ； Jakobovits 等人，尸roc· deal 90: 2551 (1993);Natl. Acad. Sci. 90:6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al. (2003) Md. 9: 129-134. As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies (ie, the same as the individual resistance systems comprising the population, except for possible mutations that may be present in trace amounts (eg, naturally occurring mutations)) The obtained antibody. Therefore, the 'modifier' is a single strain, indicating an antibody characteristic that is not a mixture of discrete antibodies. In certain embodiments, the monoclonal antibody typically comprises an antibody comprising a 119007.doc -28 - 200813092 polypeptide sequence that binds to a target, wherein the target binding polypeptide sequence selects a single target binding by including a plurality of equated sequences Obtained by the method of polypeptide sequence. For example, the selection method may be to select a unique pure line from a plurality of pure lines (such as the sum of the fusion tumor pure line, the phage pure line or the recombinant DNA pure line). It will be appreciated that the selected target binding sequence may be further altered, for example to improve affinity for the target; to humanize the target binding sequence; to improve its production in cell culture, to reduce its in vivo immunogenicity; to produce multispecific An antibody or the like, and an antibody comprising a target binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody against the individual antibody preparation is directed against a single determinant on the antigen. In addition to its specificity, the advantage of a monoclonal antibody preparation is that it is contaminated with other immunoglobulins. The modifier "single plant" refers to the characteristics of an antibody obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques including, for example, fusion knob methods (e.g., Kohler et al. 'TVaiwre, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al., Monoclonal Antibodies and Τ-CeII Hybridomas 563-681 (Elsevier, NY, 1981), recombinant DNA method (see for example, USA) Patent No. 4,816,567), phage display technology (see, for example, (: 13^^〇11 et al, 352: 624-628 (1991); Marks et al, /· Mo/· 5b/· 222·· 581-597 ( 1992); Sidhu et al., J. Mo/. 5b/· 338(2): 119007.doc •29- 200813092 299-310 (2004); Lee et al.,/· Mo/· 5b/· 340(5) : 1073-1093 (2004) i Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.' J. Immunol. Methods 284(1-2): 119- 132 (2004)) and the production of humans or humans in animals having part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences Antibody technology (see, e.g. WO 98/24893; WO 96/34096; WO 96/33735; WO 91/10741; Jakobovits et al., P roc · deal 90: 2551 (1993);

Jakobovits# A 5 Nature 362: 255-258 (1993) ； Bruggemann 等人，M /mmw似/· 7:33 (1993);美國專利第 5,545,807 m ;第 5,545,806 號；第 5,569,825 號；第 5,625,126 號；第 5,633,425 號；第 5,661，016 號；Marks 等尺，Bio· 10·· 779-783 (1992); Lonberg等人，Jakobovits # A 5 Nature 362: 255-258 (1993); Bruggemann et al., M /mmw like / 7:33 (1993); U.S. Patent No. 5,545,807 m; 5,545,806; 5,569,825; 5,625,126; 5, 633, 425; No. 5, 661, 016; Marks et al., Bio 10·· 779-783 (1992); Lonberg et al.

Nature 368: 856-859 (1994) ； Morrison, Nature 368: 812-8 1 3 (1994) ; Fishwild 等人，Λ^ίι/re 14:845- 851 (1996) ； Neuberger, Nature Biotechnol. 14: 826 (1996) 及 Lonberg 及 Huszar，Intern. Rev. Immunol. 13: 65-93 (1995)) 〇本文之單株抗體特別包括”嵌合”抗體，其中重鏈及/輕鏈之部分與衍生自特定物種或屬於特定抗體種類或子類之抗體中之對應序列相同或同源，而鍵之剩餘部分與衍生自另一物種或屬於另一抗體種類或子類之抗體中之對應序列相同或同源；以及此等抗體之片段，只要其展現所需生物活性即可（美國專利第4,81 6,567號；及Morrison等人，Pro 119007.doc -30- 200813092 t/以 81:6851-6855 (1984))。非人類（例如鼠）抗體之”人化”形式為含有衍生自非人類免疫球蛋白之最小序列之嵌合抗體。在一實施例中，人化抗體為其中受體高變區之殘基經具有所需特異性、親和力及/或容量之非人類物種（供體抗體諸如小鼠、大鼠、兔或非人類靈長類動物）之高變區之殘基置換的人類免疫球蛋白（受體抗體）。在一些情況下，人類免疫球蛋白之框架區（FR)殘基係由對應之非人類殘基置換。此外，人化抗體可包含在受體抗體或供體抗體中未發現之殘基。可進行該等修飾以進一步改進抗體效能。一般而言，人化抗體將包含大體上所有之至少一個且通常兩個可變結構域，其中所有或大體上所有高變環對應於非人類免疫球蛋白之彼等環’且所有或大體上所有FR為人類免疫球蛋白序列之彼等 FR。人化抗體視情況亦將包含免疫球蛋白（通常為人類免疫球蛋白）恆定區（Fc)之至少一部分。關於進一步詳述，參見 Jones 等人，iVaiwre 321:522-525 (1986) ; Riechmann 等 k ’ Nature 332:323-329 (1988);及 Presta，Cwrr. 0ρ· Arwci. 5b/· 2:593-596 (1992)。亦參見以下綜述文章及其中引用之參考文獻：Vaswani及Hamilton，d⑽.Nature 368: 856-859 (1994); Morrison, Nature 368: 812-8 1 3 (1994); Fishwild et al., Λ^ίι/re 14:845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)) The monoclonal antibodies herein include, in particular, "chimeric" antibodies in which portions of the heavy and/or light chains are derived from specific The corresponding sequence in a species or antibody belonging to a particular antibody class or subclass is identical or homologous, and the remainder of the bond is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass And fragments of such antibodies as long as they exhibit the desired biological activity (U.S. Patent No. 4,81,567; and Morrison et al, Pro 119007.doc -30-200813092 t/81:6851-6855 (1984) )). A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a non-human species (donor antibody such as mouse, rat, rabbit or non-human) in which the residue of the hypervariable region of the receptor has the desired specificity, affinity and/or capacity. Human immunoglobulin (receptor antibody) substituted with residues in the hypervariable region of primates. In some cases, the framework (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to their respective loops of non-human immunoglobulins' and all or substantially All FRs are their FRs of human immunoglobulin sequences. The humanized antibody will also optionally comprise at least a portion of the immunoglobulin (typically human immunoglobulin) constant region (Fc). For further details, see Jones et al, iVaiwre 321:522-525 (1986); Riechmann et al k ' Nature 332:323-329 (1988); and Presta, Cwrr. 0ρ·Arwci. 5b/· 2:593- 596 (1992). See also the following review article and references cited therein: Vaswani and Hamilton, d(10).

Asthma & Immunol. 1:105-115 (1998) ； Harris, Biochem. Soc· TVawaciiow 23:1035-1038 (1995) ; Hurle 及 Gross， Cwrr. 5:428-433 (1994)。 ”人類抗體”為具有與人類產生之抗體之胺基酸序列對應之胺基酸序列及/或已使用如本文所揭示之製造人類抗體 119007.doc -31 - 200813092 之任何技術製得之抗體。人類抗體之該定義特別排除包含非人類抗原結合殘基之人化抗體。當於本文中使用時，術語π高變區M、MHVR”或’’HV"係指序列高變及/或形成結構上限定之環之抗體可變結構域的區域。一般而言，抗體包含六個高變區；三個在VH(H1、 H2、H3)中且三個在VL(L1、L2、L3)中。在原生抗體中， H3及L3呈現最大多樣性之六個高變區，且尤其咸信H3具有賦予抗體優良特異性之獨特作用。Xu等人（2000) Immunity 13:37-45 ; Johnson Bl Wu (2003) ^ Methods in Molecular Biology 248:l-25(Lo編，Human Press，Totowa， NJ)中。實際上，僅由重鏈組成之天然產生之駱駝抗體 (camelid antibody)在不存在輕鏈下具有功能性且為穩定的。Hamers-Casterman 等人（1993) 363:446-448 ;Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. TVawaciiow 23: 1035-1038 (1995); Hurle and Gross, Cwrr. 5: 428-433 (1994). A "human antibody" is an amino acid sequence having an amino acid sequence corresponding to a human-derived antibody and/or an antibody which has been produced using any of the techniques for producing human antibodies 119007.doc-31 - 200813092 as disclosed herein. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. As used herein, the term π hypervariable region M, MHVR" or ''HV" refers to a region of a sequence variable and/or an antibody variable domain that forms a structurally defined loop. In general, the antibody comprises Six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among the native antibodies, H3 and L3 show the six hypervariable regions with the greatest diversity. And especially the H3 has a unique role in conferring excellent specificity to antibodies. Xu et al. (2000) Immunity 13: 37-45; Johnson Bl Wu (2003) ^ Methods in Molecular Biology 248: l-25 (Lo edit, Human In Press, Totowa, NJ). In fact, the naturally occurring camelid antibody consisting of only heavy chains is functional and stable in the absence of light chains. Hamers-Casterman et al. (1993) 363: 446-448;

Sheriff等人(1996) 5ζ·ο/· 3:733-736 〇本文中使用且涵蓋多種高變區之定義。Kabat互補判定區（CDR)係基於序列可變性且最常用（Kabat等人， Sequences of Proteins of Immunological Interest，第 5版， Public Health Service, National Institutes of Health, Bethesda，MD. (1991))。相反，Chothia係指結構化環之位置（Chothia及 Lesk J· Mo/·万沁/· 196:901-917 (1987))。AbM 高變區代表Kabat CDR與Chothia結構化環之間之折衷，且為Oxford Molecular’s AbM抗體模型化軟體所使用。"接觸" 高變區係基於對可用複合晶體結構之分析。來自該等高變區各自之殘基係如下所述。 119007.doc -32- 200813092 環 Kabat AbM Chothia 接觸 L1 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 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat編號） H1 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 高變區可包含如下之’’擴展高變區” ：VL中24-36或24_ 34(L1)、46-56 或 50-5 6(L2)及 89-97 或 89-96(L3)及 VH 中 26-35(H1)、50-65 或 49-65(H2)及 93-102、94-102或 95-102(H3)。可變結構域殘基係根據Kabat等人（同上）關於該等各自之定義進行編號。 π框架”或’’FR”殘基為不同於本文所定義之高變區殘基之彼等可變結構域殘基。術語”如Kabat中之可變結構域殘基編號”或’’如Kabat中之胺基酸位置編號’’及其變化形式係指在Kabat等人之 Sequences of Proteins of Immunological Interest，第 5版Sheriff et al. (1996) 5ζ·ο/· 3:733-736 〇 The definitions of various hypervariable regions are used throughout this document. 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 Institutes of Health, Bethesda, MD. (1991)). Conversely, Chothia refers to the location of the structural ring (Chothia and Lesk J. Mo/· Wan沁/· 196:901-917 (1987)). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structuring loop and is used by Oxford Molecular's AbM antibody modeling software. "Contact" Hypervariables are based on analysis of available composite crystal structures. The residues from each of the hypervariable regions are as follows. 119007.doc -32- 200813092 Ring Kabat AbM Chothia Contact L1 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 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat number) H1 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 The hypervariable zone may contain the following ''extended hypervariable zone'): 24-36 or 24_34 (L1), 46-56 or 50-5 6 (L2) in VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in 89-97 or 89-96 (L3) and VH. Variable domain Residues are numbered according to Kabat et al. (supra) for their respective definitions. π-framework or ''FR' residues are those variable domain residues other than the hypervariable region residues defined herein. The term "such as the variable domain residue number in Kabat" or "such as the amino acid position number in Kabat" and its variants refers to the Kabat et al. Sequences of Proteins of Immunological Interest, 5th Edition

Public Health Service, National Institutes of Health, Bethesda，MD (1991)中用於抗體編譯之重鏈可變結構域或輕鏈可變結構域之編號系統。使用此編號系統，實際為線性之胺基酸序列可含有對應於縮短或***可變結構域之FR 或HVR之較少數或額外胺基酸。舉例而言，重鏈可變結構域可包括在H2之殘基52後***之單一胺基酸（根據Kabat之 119007.doc -33- 200813092 殘基52a)及在重鍵FR殘基82後***之殘基（例如根據Kabat 之殘基82a、82b及82c等）。可藉由在抗體之序列同源區與π標準” Kabat編號序列對準來確定指定抗體之殘基Kabat 編號。 ’’親和力成熟”抗體為在其一或多個HVR中具有一或多處變化之抗體，與不具有彼等變化之親本抗體相比，該等變化致使改良抗體對抗原之親和力。在一實施例中，親和力成熟抗體對靶抗原具有奈莫耳或甚至皮莫耳之親和力。親和力成熟抗體係藉由此項技術中已知之程序而產生。Public Health Service, National Institutes of Health, Bethesda, MD (1991) Numbering system for antibody-compiled heavy chain variable domains or light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the FR or HVR that shortens or inserts the variable domain. For example, the heavy chain variable domain can include a single amino acid inserted after residue 52 of H2 (according to Kabat's 119007.doc-33-200813092 residue 52a) and inserted after the heavy bond FR residue 82 Residues (e.g., according to Kabat residues 82a, 82b, and 82c, etc.). The Kabat numbering of the residues of a given antibody can be determined by alignment of the sequence homology region of the antibody with the π-standard Kabat numbering sequence. The 'affinity matured' antibody has one or more changes in one or more of its HVRs. Such antibodies result in improved affinity of the antibody for the antigen as compared to parent antibodies that do not have such changes. In one embodiment, the affinity matured antibody has a affinity for the target antigen for naim or even picomoles. Affinity mature anti-systems are produced by procedures known in the art.

Marks 等人 10:779-783 (1992)描述藉由 VH 及VL結構域改組之親和力成熟。HVR及/或框架殘基之隨機突變係由以下文獻描述：Barbas等人/V% ί/α 91:3809-3813 (1994); Schier等人 Gene 169:147-155 (1995) ; Yelton 等人 J· /mmwo/. 155:1994-2004 (1995) ; Jackson等人，《/· 154(7):3310-9 (1995); 及 Hawkins等人 J. Mo/. 5/0/. 226:889-896 (1992)。 ”阻斷”抗體或，，拮抗，，抗體為抑制或降低其所結合抗原之生物活性之抗體。某些阻斷抗體或拮抗抗體大體上或完全抑制抗原之生物活性。如本文所用之，，促效抗體，，為模擬所關注多肽之至少一種功能活性之抗體。抗體”效應功能，，係指彼等可歸因於抗體Fc區（原生序列Fc 區或胺基酸序列變異Fc區）之生物活性，且因抗體同型而不同。抗體效應功能之實例包括：Clq結合及補體依賴性 119007.doc -34- 200813092 細胞毒性；Fc受體結合；抗體依賴性細胞介導之細胞毒性 (ADCC);吞噬作用；細胞表面受體（例如B細胞受體）下調及B細胞活化。 nFc受體”或” FcR”描述結合至抗體Fc區之受體。在一些實施例中，FcR為原生人類FcR。在一些實施例中，FcR為結合IgG抗體之受體（γ受體）且包括FcYRI、FcyRII及FcyRIII 子類之受體，包括該等受體之對偶基因變異體及替代性之剪接形式。FcyRII受體包括FcyRIIA(n活化受體”）及 FqRIIBC’抑制受體”），其具有主要在其細胞質域中不同之類似胺基酸序列。活化受體FcyRIIA在其細胞質域中含有基於免疫受體酪胺酸之活化基元（ITAM)。抑制受體 FcyRIIB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 DaSron，/mmwno/· 15:203-234 (1997)) o FcR/系於 Ravetch及 Kinet，dwm/· 9:457-92 (1991) ; Capel 等人，Immunomethods 4:25-34 (1994) ;及 de Haas 等人，J· Cm Mei 126:330-41 (1995) 中論述。包括彼等欲在未來識別者之其他FcR係以術語nFcRn涵蓋於本文中。術語nFc受體”或”FcR"亦包括負責將母體IgG轉移至胎兒 (Guyer 等人，J. /mmw⑽/. 1 17:587 (1976)及 Kim 等人，/· ImmunoL 24:249 (1994))且調節免疫球蛋白穩定性之新生兒受體，FcRn。量測與FcRn結合之方法為已知的（參見例如Ghetie 1997，Hinton 2004)。可於（例如）表現人類FcRn之轉殖基因小鼠或經轉染人類細胞株或投予Fc變異多肽之靈 119007.doc -35- 200813092 長類動物中檢定人類FcRn高親和力結合多肽與人類FcRn 之活體内結合及血清半衰期。 WO 00/42072(Presta)描述與FcRs具有改良或減低之結合的抗體變異體。該等專利公開案之内容係以特定引用的方式併入本文。亦參見Shields等人/· 5ζ·<9/· CAem· 9(2): 6591-6604 (2001)。 π人類效應細胞π為表現一或多種FcR且執行效應功能之白血球。在某些實施例中，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)、天然殺死（NK)細胞、單核細胞、細胞毒性T細胞及嗜中性白血球。效應細胞可自例如血液之原生來源分離。 π抗體依賴性細胞介導之細胞毒性"或’’ADCC”係指其中經分泌Ig結合至存在於特定細胞毒性細胞（例如天然殺死 (NK)細胞、嗜中性白血球及巨噬細胞）上之Fc受體（FcR)上而使得該等細胞毒性效應細胞特異性地結合至攜帶抗原之靶細胞且隨後以細胞毒性殺死靶細胞之細胞毒性形式。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表現係概述於 Ravetch 及 Kinet，如m / 所加 / 9:457-92 (1991) 第464頁之表3中。為評估所關注分子之ADCC活性，可進行諸如美國專利第5,500,362號或第5,821，337號或Presta美國專利第6,737,056號中所述之活體外ADCC檢定。適用於此等檢定之效應細胞包括周邊血液早核細胞（PBMC)及天 119007.doc -36- 200813092 然殺死（NK)細胞。或者，或另外，所關注分子2aDCC活性可於活體内評估，例如於諸如Clynes等人，户见 95:652-656 (1998)中揭示之動物模型中評估。 π補體依賴性細胞毒性”或”CDC”係指靶細胞在補體存在下之溶解。經典補體路徑之活化係藉由將補體系統之第一組份（Clq)結合至與其同源抗原結合之抗體（適當子類之抗體）來起始。為評估補體活化，可進行例如於Gazzan〇_ Santoro等人，J· 从以心心 2〇2:163 (i996)中所述之CDC檢定。具有變化之Fc區胺基酸序列及增加或減低之Clq結合能力之多肽變異體係描述於美國專利第6，194,551 B1號及w〇 99/51642中。彼等專利公開案之内容係以特定引用的方式併入本文中。亦參見Idus〇gie等人乂历㈣〇厂164: 4178_ 4184 (2000) 〇術語·’包含Fc區之多肽，，係指包含卜區之多肽，諸如抗體或免疫黏附素。可例如在多肽純化期間或藉由重組工程設计編碼多肽之核酸來移除Fc區之C端離胺酸（根據eu編號系統之殘基447)。因此，包含具有本發明之Fc區之多肽的組合物可包含具有K447之多肽；所有Κ447經移除之多肽或具有與不具有K447殘基之多肽混合物。為本文目的之，，受體人類框架”為包含衍生自人類免疫球蛋白框架或人類一致框架之VL或Vh框架之胺基酸序列的框架。”衍生自”人類免疫球蛋白框架或人類一致框架之受體人類框架可包含其相同胺基酸序列，或其可含有先前存 119007.doc -37- 200813092 在（pre-existing)之胺基酸序列改變。在一些實施例中，先前存在之胺基酸改變數目為10或更小、9或更小、8或更小、7或更小、6或更小、5或更小、4或更小、3或更小或2 或更小。當先前存在之胺基酸改變係存在於VH中時，彼等改變較佳僅發生於位置71H、73H及78H中之三個、兩個或一個位置；例如彼等位置之胺基酸殘基可為71A、73T 及/或78A。在一實施例中，VL受體人類框架之序列與VL 人類免疫球蛋白框架序列或人類一致框架序列相同。 "人類一致框架"為表示在人類免疫球蛋白VL或VH框架序列之選擇中最常發生之胺基酸殘基的框架。一般而言，人類免疫球蛋白VL或VH序列係選自可變結構域序列之子群。一般而言，序列之子群為如Kabat等人，Sequences of Proteins of Immunological Interest，第 5版Public Health Service，National Institutes of Health，Bethesda，MD (1991) 中之子群。在一實施例中，對於VL而言，子群為如同上文之Kabat等人中之子群κ I。在一實施例中，對於VH而言，子群為如同上文之Kabat等人中之子群III。 nVH子群III一致框架π包含由Kabat等人（同上文）中之可變重子群III中之胺基酸序列所獲得之一致序列。在一實施例中，VH子群III 一致框架胺基酸序列包含以下序列各自之至少一部分或所有：EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:50)-H1-WVRQAPGKGLEWV(SEQ ID NO:51)-H2-RFTISADTSKNTAYLQMNSLRAEDTAVYYC(SEQ ID NO:59)-H3-WGQGTLVTVSS(SEQ ID NO:35) 〇 119007.doc -38 - 200813092 "VL子群I一致框架”包含由Kabat等人（同上文）中之可變輕κ子群I中之胺基酸序列所獲得之一致序列。在一實施例中，VH子群I 一致框架胺基酸序列包含以下序列各自之至少一部分或所有：DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:60)-L1-WYQQKPGKAPKLLIY(SEQ ID NO:61)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:62)-L3-FGQGTKVEIK(SEQ ID NO:63)。 n結合親和力” 一般係指分子（例如抗體）之單一結合位點與其結合搭配物（例如抗原）之間非共價相互作用之總強度。除非另外指出，否則如本文所用之”結合親和力”係指反映結合對成員（例如抗體與抗原）之間1:1相互作用之固有結合親和力。分子X對其搭配物Y之親和力一般可藉由解離常數（Kd)來表示。親和力可藉由此項技術中已知之通用方法（包括本文所述之彼等方法）來量測。低親和力抗體一般與抗原緩慢結合且傾向於易於解離，而高親和力抗體一般與抗原較快速結合且傾向於更長久地保持結合。此項技術中已知各種量測結合親和力之方法，任何該等方法均可用於本發明之目的。特定說明性實施例係描述於下文中。在一實施例中，根據本發明2nKd”或”Kd值”係藉由以所關注之Fab型抗體及所述之其抗原藉由以下檢定進行之經放射性標記抗原結合檢定（RIA)來量測。在未標記抗原之連續滴定存在下，藉由使Fab與最小濃度之經（1251)-標記之抗原平衡，接著捕獲與經抗Fab抗體塗覆之板結合之抗原來量測Fab對抗原之溶液結合親和力（Chen等人，（1999) J. 119007.doc -39- 200813092Marks et al. 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutations in HVR and/or framework residues are described by Barbas et al/V% ί/α 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. /mmwo/. 155:1994-2004 (1995); Jackson et al.,/· 154(7):3310-9 (1995); and Hawkins et al. J. Mo/. 5/0/. 226: 889-896 (1992). "Blocking" an antibody or, antagonizing, an antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonizing antibodies substantially or completely inhibit the biological activity of the antigen. As used herein, an agonist antibody is an antibody that mimics at least one functional activity of a polypeptide of interest. Antibody "effector function" refers to the biological activity attributable to the Fc region of the antibody (either the native sequence Fc region or the amino acid sequence variant Fc region), and differs depending on the antibody isotype. Examples of antibody effector functions include: Clq Binding and complement dependence 119007.doc -34- 200813092 Cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg B cell receptors) and B Cell activation. The nFc receptor "or" FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is a receptor that binds to an IgG antibody (gamma receptor) and includes receptors for the FcYRI, FcyRII, and FcyRIII subclasses, including dual gene variants of such receptors, and alternative spliced forms. FcyRII receptors include FcyRIIA (n-activated receptors) and FqRIIBC' inhibitory receptors), which have similar amino acid sequences that differ primarily in their cytoplasmic domain. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See DaSron, /mmwno/. 15:203-234 (1997)) o FcR/ is in Ravetch and Kinet, dwm/. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994) And de Haas et al., J. Cm Mei 126: 330-41 (1995). Other FcR lines including those that are intended to be recognized in the future are covered by the term nFcRn. The term nFc receptor" or "FcR" also includes the transfer of maternal IgG to the fetus (Guyer et al, J. /mmw (10) /. 17 17:587 (1976) and Kim et al, /. ImmunoL 24:249 (1994) And a neonatal receptor that regulates the stability of immunoglobulins, FcRn. Methods for measuring binding to FcRn are known (see, for example, Ghetie 1997, Hinton 2004). Human FcRn high affinity binding polypeptide and human FcRn can be assayed in, for example, a transgenic mouse expressing human FcRn or a transfected human cell line or a Fc variant polypeptide 119007.doc-35-200813092 elongate In vivo binding and serum half-life. WO 00/42072 (Presta) describes antibody variants with improved or reduced binding to FcRs. The contents of these patent publications are incorporated herein by reference in their entirety. See also Shields et al. / 5ζ·<9/· CAem·9(2): 6591-6604 (2001). The π human effector cell π is a white blood cell that exhibits one or more FcRs and performs effector functions. In certain embodiments, the cell exhibits at least FcyRIII and performs an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural kill (NK) cells, mononuclear cells, cytotoxic T cells, and neutrophils. Effector cells can be isolated, for example, from the native source of blood. π antibody-dependent cell-mediated cytotoxicity" or ''ADCC') refers to the secretion of secreted Ig to specific cytotoxic cells (eg, natural killing (NK) cells, neutrophils, and macrophages). The Fc receptor (FcR) on the cytotoxic form such that the cytotoxic effector cells specifically bind to the target cell carrying the antigen and subsequently kill the target cell by cytotoxicity. Primary cells mediated by ADCC, NK cells Only FcyRIII is expressed, whereas monocytes exhibit FcyRI, FcyRII, and FcyRIII. The FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, as shown in Table 3 on page 464 of m/Adda/9:457-92 (1991). In order to evaluate the ADCC activity of the molecule of interest, an in vitro ADCC assay such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, or to the disclosure of U.S. Patent No. 6,737,056, which is incorporated herein by reference. Early nuclear cells (PBMC) and day 119007.doc -36- 200813092 kill (NK) cells. Alternatively, or in addition, the 2aDCC activity of the molecule of interest can be assessed in vivo, for example in Clynes, etc. , See households 95: 652-656 (1998) discloses an animal model was evaluated in the π complement dependent cytotoxicity "or" CDC "refers to the target cell lysis in the presence of complement. Activation of the classical complement pathway is initiated by binding the first component of the complement system (Clq) to an antibody (a suitable subclass of antibody) that binds to its cognate antigen. To assess complement activation, for example, the CDC assay described in Gazzan〇_ Santoro et al, J., in Hearts 2〇2:163 (i996) can be performed. Polypeptide variants having altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described in U.S. Patent Nos. 6,194,551 B1 and WO 99/51642. The contents of their patent publications are hereby incorporated by reference in their entirety. See also Idus〇gie et al. (4) 〇 164: 4178_ 4184 (2000) 〇 Terminology · A polypeptide comprising an Fc region, refers to a polypeptide comprising a region, such as an antibody or immunoadhesin. The C-terminal lysine of the Fc region (residue 447 according to the eu numbering system) can be removed, for example, during polypeptide purification or by recombinant engineering designing the nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region of the invention may comprise a polypeptide having K447; all polypeptides with a deletion of 447 or a mixture with a polypeptide having no K447 residue. For the purposes of this document, the acceptor human framework" is a framework comprising amino acid sequences derived from the human immunoglobulin framework or the VL or Vh framework of the human consensus framework." Derived from "human immunoglobulin framework or human consensus framework The acceptor human framework may comprise the same amino acid sequence, or it may contain a prior amino acid sequence change of 119007.doc -37-200813092. In some embodiments, a pre-existing amine The number of base acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. When a previously present amino acid change is present in VH, such changes preferably occur only at three, two or one of positions 71H, 73H and 78H; for example, amino acid residues at positions May be 71A, 73T and/or 78A. In one embodiment, the sequence of the VL receptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence. "Human consensus framework" The most selective selection of globulin VL or VH framework sequences The framework of the amino acid residues that occur. In general, the human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. In general, subgroups of sequences are, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Subversion 5 of Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, for VL, the subgroup is a subgroup of Kabat et al. In an embodiment, for VH, the subgroup is subgroup III as in Kabat et al. above. The nVH subgroup III consensus framework π comprises the variable baryon group by Kabat et al. (supra) A consensus sequence obtained from the amino acid sequence of III. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 50)-H1- WVRQAPGKGLEWV (SEQ ID NO: 51) - H2-RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO: 59) - H3-WGQGTLVTVSS (SEQ ID NO: 35) 〇 119007.doc -38 - 200813092 "VL Subgroup I Consistent Framework" encompassed by Kabat Wait for Variable) in the light of the above, the obtained κ consensus sequence of subgroup I of the amino acid sequence. In one embodiment, the VH subgroup I consensus framework amino acid sequence comprises at least a portion or all of each of: 03. SEQ ID NO: 62) - L3-FGQGTKVEIK (SEQ ID NO: 63). n binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, "binding affinity" as used herein Refers to the intrinsic binding affinity of a 1:1 interaction between a binding member (eg, an antibody and an antigen). The affinity of molecule X for its conjugate Y is generally represented by the dissociation constant (Kd). Affinity can be used by this General methods known in the art, including those described herein, are used to measure. Low-affinity antibodies generally bind slowly to antigens and tend to dissociate readily, while high-affinity antibodies generally bind more rapidly to antigens and tend to be longer The bonding is maintained. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative embodiments are described below. In one embodiment, 2nKd in accordance with the present invention "or" Kd value" by radioactivity of the Fab-type antibody of interest and the antigen thereof by the following assay Note antigen binding assay (RIA) to measure. Fab-to-antigen solution is measured by balancing the Fab with a minimal concentration of the (1251)-labeled antigen in the presence of a continuous titration of the unlabeled antigen followed by capture of the antigen bound to the anti-Fab antibody coated plate. Combining affinity (Chen et al., (1999) J. 119007.doc -39- 200813092

Mo/ 293:865-881)。為確立檢定條件，將微量滴定盤 (Dynex)以50 mM碳酸納（pH值為9_6)中之5 pg/ml所俘獲之抗Fab抗體（Cappel Labs)隔夜塗覆且隨後於室溫（約23。〇下以PBS中之2%(w/v)牛血清白蛋白阻斷歷時兩至五小時。在無吸附劑之盤（Nunc #269620)中，將 100 p]y[或 26 pM [1251] 抗原與所關注之Fab連續稀釋液混合（例如，與抗VEGF抗體，Fab-12之評估一致，Presta等人，（1997) C㈣ 57:4593-4 599)。接著將所關注之Fab培育隔夜；然而，培育可持續更長時期（例如約65小時）以確保達到平衡。其後，於室溫下將混合物轉移至捕獲盤中以供培育（例如一小時）。接著將溶液移除且將盤以PBS中之0.1%吐溫 20(Tween-20)洗滌八次。當盤乾燥時，每孔添加150 μΐ之閃爍體（MicroScint-20; Packard)且將盤於 Topcount γ計數器 (Packard)上計數10分鐘。選擇產生小於或等於20%最大結合濃度之各Fab以用於競爭性結合檢定中。根據另一實施例，藉由於25°C下使用BIAcoreTM-2000或 BIAcoreTM-3000(BIAcore，Inc·，Piscataway，NJ)，使用表面電漿共振檢定以約10個反應單位（RU)之固定抗原CM5晶片量測Kd或Kd值。簡言之，根據供應商說明以N-乙基-N*_ (3-二甲胺基丙基）-碳化二醯亞胺鹽酸鹽（EDC)及N-羥基琥珀醯亞胺（NHS)活化羧甲基化葡聚糖生物感應器晶片（CM5, BIAcore Inc·)。在以每分鐘5 μΐ之流動速率注射之前，將抗原以pH 4·8之10 mM乙酸納稀釋至5 pg/ml(約0.2 μΜ)以達成約10個反應單位（RU)之偶合蛋白。注射抗原後，注射 119007.doc -40- 200813092 1 Μ乙醇胺以阻斷未反應之基團。為動力學量測之目的，於25°C下以約25 μΐ/min之流動速率在含有0.05%吐溫20之 PBS(PBST)中注射Fab之兩倍連續稀釋液（0.78 nM至500 ηΜ)。使用簡單的一對一朗缪耳結合模型（Langmuir binding model)(BIAcore Evaluation Software 3.2版本），藉由同時擬合締合及解離生物感應曲線計算締合速率（kQn)及解離速率（kQff)。將平衡解離常數（Kd)以kwf/kcn之比率計算。例如參見 Chen，Y_ 等人（1999) J· Μο/ 价〇/· 293:865-881。若藉由上述表面電漿共振檢定之締合速率大於106 M·1 ，則可在如由諸如裝備有滯流之分光光度計（Aviv Instruments) 或具有攪拌光析管之8000系列SLM-Aminco分光光度計 (ThermoSpectronic)之分光計中所量測之增加濃度之抗原存在下，於25°C下藉由使用量測20 nM抗-抗原抗體（Fab形式）在pH 7.2之PBS中之螢光放射強度增加或降低之螢光淬滅技術（激發=295 nm;放射=340 nm，16 nm帶通）來測定締合速率。本發明之”締合速率’’或”k。/亦可如上文所述使用 BIAcoreTM-2000 或 BIAcoreTM-3000 系統（BIAcore， Inc·， Piscataway，NJ)測定。 ’’病症’’為受益於以本發明之物質/分子或方法治療之任何病況或疾病。其包括慢性或急性病症，包括彼等使哺乳動物易患所論及病症之病理學病況。本文中待治療之病症之非限制性實例包括癌病，諸如腫瘤，例如癌瘤（上皮腫瘤）及母細胞瘤（胚胎組織衍生之腫瘤），且在一些實施例中為 119007.doc -41 - 200813092 卵巢癌、子宮癌（包括子宮内膜癌）、腦腫瘤（例如星形細胞瘤及神經膠質瘤）及腎癌，包括腎胚細胞瘤（例如威爾姆氏腫瘤）。術語"細胞增殖性病症"及”增殖性病症"係指與—定程度之異常細胞增殖相關聯之病症。在—實施例中，細胞增疫性病症為癌症。 ^如本文所用之"腫瘤"係指所有贅生性細胞生長及增殖（不管為惡性或良性）及所有癌前期及癌細胞及組織。如本文所提及之術語"癌症"、"癌"、"細胞增瘦性病症"、"增殖性病症”及π腫瘤”不相互排除。術語，’癌症”及”癌”係指或描述通常特徵在於未經調節之、、、田胞生長/增殖之哺乳動物生理學病況。癌症之實例包括 (但不限於）癌瘤、淋巴瘤（例如霍奇金氏及非霍奇金氏淋巴瘤）、母細胞瘤、肉瘤及白血病。此等癌症之更特別實例包括鱗狀細胞癌、小細胞肺癌、非小細胞肺癌、肺腺癌、肺鱗狀細胞癌、腹膜癌、肝細胞癌、腸胃癌、胰腺癌、神、、&勝貝瘤 '子宮頸癌、卵巢癌、肝癌、膀胱癌、肝腫瘤、礼癌、結腸癌、結腸直腸癌、子宮内膜癌或子宮癌、唾液腺癌、腎癌、肝癌、***癌、陰門癌、甲狀腺癌、肝癌瘤、白血病及其他淋巴增殖性病症及各種類型之頭頸癌。如本文所用之”治療”係指試圖改變所治療個體或細胞之自然進程之臨床介入，且可為預防之目的或在臨床病理過 &期間進行。所需治療效應包括預防疾病之發生或復發；緩解症狀；減少疾病之任何直接或間接病理結果；預防癌 119〇〇7.doc -42- 200813092 Π預:低:病發展速率，·改善或減輕疾病病況且緩解或病或病症之;展~ =例中，本⑽ "個體，，為二二T或病症之發展。動物。哺乳些實施例中，脊椎動物為哺乳物、寵物（語但不限於）家畜（諸如牛）、競技類動在某些實施例中，哺乳動物為人類。]鼠及大鼠Mo/293:865-881). To establish assay conditions, a microtiter plate (Dynex) was coated overnight with anti-Fab antibody (Cappel Labs) captured at 5 pg/ml in 50 mM sodium carbonate (pH 9-6) and then at room temperature (approximately 23 The armpits were blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours. In the non-adsorbent plate (Nunc #269620), 100 p]y [or 26 pM [1251] The antigen is mixed with the serial dilution of the Fab of interest (eg, consistent with the evaluation of the anti-VEGF antibody, Fab-12, Presta et al, (1997) C (iv) 57: 4593-4 599). The Fab of interest is then incubated overnight. However, the incubation can last for a longer period of time (eg, about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture tray for incubation (eg, one hour) at room temperature. The solution is then removed and The plate was washed eight times with 0.1% Tween-20 in PBS. When the plate was dry, 150 μM scintillator (MicroScint-20; Packard) was added to each well and plated on a Topcount gamma counter (Packard). Count for 10 minutes. Select each Fab that produces a maximum binding concentration of less than or equal to 20% for use in a competitive binding assay. According to another embodiment, a fixed antigen CM5 of about 10 reaction units (RU) is determined using surface plasma resonance assay using BIAcoreTM-2000 or BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25 °C. The wafer measures Kd or Kd values. Briefly, according to the supplier's instructions, N-ethyl-N*_(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- Hydroxyaluminum succinimide (NHS) activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc.). The antigen was applied at 10 mM pH 4·8 before injection at a flow rate of 5 μM per minute. The sodium acetate was diluted to 5 pg/ml (about 0.2 μΜ) to achieve about 10 reaction units (RU) of the coupled protein. After the antigen was injected, 119007.doc -40-200813092 1 Μ ethanolamine was injected to block the unreacted groups. For the purpose of kinetic measurement, two-fold serial dilutions of Fab (0.78 nM to 500 ηΜ) were injected into PBS (PBST) containing 0.05% Tween 20 at a flow rate of about 25 μΐ/min at 25 °C. Using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) with simultaneous Bonding association and dissociation biosensor curve calculated association rate (kQn) and dissociation rates (kQff). The equilibrium dissociation constant (Kd) is calculated as the ratio of kwf/kcn. See, for example, Chen, Y_ et al. (1999) J. Μο/ Price 〇 / 293: 865-881. If the association rate by the above surface plasma resonance test is greater than 106 M·1 , it can be split by, for example, an 8000 series SLM-Aminco equipped with a stagnation spectrophotometer (Aviv Instruments) or a stirred phototube. Fluorescence emission in PBS at pH 7.2 using a 20 nM anti-antigen antibody (Fab format) at 25 ° C in the presence of an increased concentration of antigen measured in a spectrometer of ThermoSpectronic Fluorescence quenching techniques with increased or decreased intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) were used to determine the association rate. The "association rate" or "k" of the present invention. / can also be determined using BIAcoreTM-2000 or BIAcoreTM-3000 systems (BIAcore, Inc., Piscataway, NJ) as described above. The 'disease' is any condition or disease that benefits from treatment with the substance/molecule or method of the present invention. It includes chronic or acute conditions, including those pathological conditions in which the mammal is susceptible to the condition in question. Non-limiting examples of conditions to be treated herein include cancers, such as tumors, such as carcinomas (epithelial neoplasms) and blastomas (embryonic tissue-derived tumors), and in some embodiments, 119007.doc-41 - 200813092 Ovarian cancer, uterine cancer (including endometrial cancer), brain tumors (such as astrocytoma and glioma), and kidney cancer, including nephroblastoma (such as Wilm's tumor). The term "cell proliferative disorder" and "proliferative disorder" refers to a disorder associated with a defined degree of abnormal cell proliferation. In an embodiment, the cell-proliferative disorder is cancer. ^ As used herein "tumor" means all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues. As mentioned herein, the terms "cancer", "cancer""Cell augmentation disorders", "proliferative disorders" and π tumors are not mutually exclusive. The terms, 'cancer' and 'cancer' are generally characterized by unregulated, morphological growth. / Proliferating mammalian physiological condition. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, intestinal cancer, pancreatic cancer, deity, & Shengbei tumor 'cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, genital cancer , thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders and various types of head and neck cancer. "Treatment" as used herein refers to a clinical intervention that attempts to alter the natural course of the individual or cell being treated, and may be performed for prophylactic purposes or during clinical pathology. The desired therapeutic effects include prevention of the occurrence or recurrence of the disease; alleviation of symptoms; reduction of any direct or indirect pathological outcome of the disease; prevention of cancer 119 〇〇 7.doc -42- 200813092 Π pre: low: rate of disease progression, improvement or mitigation Disease condition and relief or disease or condition; exhibition ~ = in the case, the (10) & "individual," is the development of T2 or T. animal. In some embodiments, the vertebrate is a mammal, a pet (not limited to) a domestic animal (such as a cow), a competitive animal. In some embodiments, the mammal is a human. Rat and rat

曰1有二量:係指有效達成所需治療或預防結果所必需之劑 1及日f間量。防有效里係4日有效達成所需預防結果所必需之劑量及時間量。通常但並非必需，由於預防劑量係在疾病之前或早本發明之物質/分子之"治療有效量，，可根據諸如疾病病況、個體年齡、性別及體重以及物質/分子在個體内引發所需反應之能力之諸因素而不同。治療有效量亦涵蓋治療有益效應超過物質/分子之任何毒性或有害效應之量。"預期用於受檢者，因此預防有效量應小於治療有效量。如本文所用之術語"細胞毒性劑"係指抑制或阻礙細胞功能及/或引起細胞死亡或破壞之物質。術語意欲包括放射性同位素（例如 At211、I131、I125、Y90、Re186、Rel88、曰1 has two quantities: refers to the amount of agent 1 and day f necessary to effectively achieve the desired treatment or prevention results. The effective dose is the dose and time required to effectively achieve the desired preventive effect on the 4th. Usually, but not necessarily, since the prophylactic dose is a therapeutically effective amount of the substance/molecule of the present invention prior to or prior to the disease, it may be elicited in an individual according to, for example, the disease condition, the age, sex and weight of the individual, and the substance/molecule. The factors of the ability to react vary. A therapeutically effective amount also encompasses an amount that treats a beneficial effect over any toxic or detrimental effect of the substance/molecule. " is expected to be used in the subject, so the preventive effective amount should be less than the therapeutically effective amount. The term "cytotoxic agent" as used herein refers to a substance that inhibits or blocks cellular function and/or causes cell death or destruction. The term is intended to include radioisotopes (eg At211, I131, I125, Y90, Re186, Rel88,

Sm1Sm1

Bi212、P32、Pb212及Lu之放射性例如甲胺喋呤、阿黴素（adriamicin)、長春藤驗（長春新驗、長春驗、依託泊苷（etoposide))、多柔比星 (doxorubicin)、美法侖（melphalan)、絲裂黴素 c、苯丁酸氮芥、道諾黴素（daunorubicin);或其他嵌入劑、酶及其片 119007.doc -43- 200813092 段，諸如核分解酶、抗生素及毒素，諸如細菌、真菌、植物或動物來源之小分子毒素或酶活性毒素（包括其片段及/ 或變異體）及下文揭示之各種抗腫瘤劑或抗癌劑。其他細胞毒性劑描述於下文中。殺腫瘤劑引起腫瘤細胞之破壞。 ”毒素π為能夠對細胞生長或增殖具有有害效應之任何物質。 ’’化療劑π為適用於治療癌症之化合物。化療劑之實例包括烷基化劑，諸如塞替派（thiotepa)及CYTOXAN®環磷醯胺；石黃酸烧酯，諸如白消安（busulfan)、英丙舒凡 (improsulfan)及派泊舒凡（piposulfan);氮丙咬，諸如苯佐多帕（benzodopa)、卡波S昆（carboquone)、麥曲多帕 (meturedopa)及尤利多帕（uredopa);伸乙基亞胺及甲基三聚氰胺（methylamelamine)，包括六甲蜜胺、三伸乙基三聚氰胺、三伸乙基磷醯胺、三伸乙基硫代磷醯胺及三羥曱基三聚氰胺；乙醯精寧（acetogenin)(尤其為泡番荔枝辛 (bullatacin)及泡番篇枝辛酮（bullatacinone)) ; δ-9-四經基 ***紛（曲***紛（dronabinol), MARINOL®) ; β-拉帕 _ (β-lapachone);拉帕醇（lapachol);秋水仙驗（colchicine);樺木酸（betulinic acid);喜樹驗（camptothecin)(包括合成類似物拓朴替康（topotecan)(HYCAMTIN®)、CPT-11(伊立替康 (irinotecan)， CAMPTOSAR®)、乙醯基喜樹鹼 (acetylcamptothecin)、司考波萊辛（scopolectin)及 9-胺基喜樹驗）；苔薄蟲素（bryostatin);開利斯塔、;丁（callystatin); CC-1065(包括其阿多來新（adozelesin)、卡折來新 119007.doc •44- 200813092 (carzelesin)及比折來新（bizelesin)合成類似物）；鬼臼毒素 (podophyllotoxin)；鬼臼毒素酸（podophyllinic acid);替尼泊甙（teniposide);念珠藻環肽（cryptophycin)(尤其為念妹藻環肽1及念珠藻環肽8);海兔毒素（dolastatin);多卡黴素 (duocarmycin)(包括合成類似物，KW-2189及 CB1-TM1); 艾權素（eleutherobin);潘卡替斯汀（pancratistatin);沙科地辛（sarcodictyin);斯旁斯 >T (spongistatin);芥子氮，諸如苯丁酸氮芥、萘氮芥、膽磷醯胺、雌莫司汀 (estramustine)、異環碌醯胺、氮芬（mechlorethamine)、鹽酸氧化氮芥、美法侖、新恩比星（novembichin) '苯芥膽留醇（phenesterine)、潑尼莫司汀（prednimustine)、曲洛碟胺 (trofosfamide)、尿卩密唆芥子氣（uracil mustard);亞硝基脲，諸如卡莫司汀（carmustine)、氣脲黴素 (chlorozotocin)、福莫司、汀（fotemustine)、洛莫司汀 (lomustine)、尼莫司汀（nimustine)及萊尼莫司、;丁 (ranimnustine);抗生素，諸如烯二快抗生素（例如卡奇徽素（calicheamicin)，尤其為卡奇黴素γΐΐ及卡奇黴素coll(例如參見 jg ㈣ w, "·五d·五 33:183-186 (1994)); 地奈黴素（dynemicin)，包括地奈黴素A;艾司匹拉黴素 (esperamicin);以及新抑癌蛋白發色團及相關色素蛋白浠二炔抗生素發色團）、阿克萊諾米星（aclacinomysins)、放線菌素、奥萊米星（authramycin)、偶氮絲胺酸 (azaserine)、博萊黴素（bleomycins)、放線菌素C、卡拉比星（carabicin)、洋紅黴素（carminomycin)、嗜癌菌素 119007.doc -45- 200813092 (carzinophilin)、卡莫黴素（chromomycinis)、放線菌素 D、道諾黴素、地托比星（detorubicin)、6-重氮基-5-侧氧基-L-正白胺酸、ADRIAMYCIN®多柔比星（包括嗎啉基-多柔比星、氰基嗎啉基-多柔比星、2-吡咯基-多柔比星及去氧多柔比星）、表柔比星（epirubicin)、依索比星（esorubicin)、黃膽素（idarubicin)、麻西羅黴素（marcellomycin)、諸如絲裂黴素C之絲裂黴素、黴紛酸、諾加黴素（nogalamycin)、橄欖黴素（olivomycins)、培洛黴素（peplomycin)、波替諾黴素（p〇tf*iromycin)、嘌羅黴素（puromycin)、炔萊黴素 (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);抗代謝劑，諸如甲胺喋呤及5-氟尿嘧啶（5-FU);葉酸類似物，諸如迪諾特寧 (denopterin)、曱胺嗓呤、嗓羅呤（pteropterin)、三甲曲沙 (trimetrexate);嗓呤類似物，諸如氟達拉賓（fludarabine)、 6-Μ σ票呤（mercaptopurine)、硫口米嗓吟（thiamiprine)、硫鳥嗓呤（thioguanine);鳴°定類似物，諸如安西他濱 (ancitabine)、阿紮胞^(azacitidine)、6-氮尿苷（azauridine)、卡莫氟（carmofur)、阿糖胞苷（cytarabine)、雙去氧尿苷 (dideoxyuridine)、去氧氟尿普（doxifluridine)、依諾他濱 (enocitabine)、氮尿苦（floxuridine);雄激素，諸如卡普睾酮（calusterone)、丙酸屈他雄酮（dromostanolone propionate)、環硫雄醇（epitiostanol)、美雄烧 119007.doc -46- 200813092 (mepitiostane)、睾内赂（testolactone);抗腎上腺素，諸如胺魯米特（aminoglutethimide)、米托坦（mitotane)、曲洛司坦（trilostane);葉酸補充物，諸如酸葉酸（frolinic acid); 醋葡駿内醋（aceglatone);酸填醯胺糖苦（aldophosphamide glycoside);胺基乙醯丙酸；尹陸萊辛（eniluracil);安°丫唆 (amsacrine);貝曲布辛（bestrabucil);比生群（bisantrene); 伊達曲仙（edatraxate);德弗法明（defofamine);秋水仙胺 (demecolcine)；地吖 g 昆(diaziquone)；伊弗尼辛 (elfornithine);依利醋錄（elliptinium acetate);艾普塞隆 (epothilone);依託格魯（etoglucid);瑣酸鎵；經基尿素；蘑兹多糠（lentinan);洛尼達寧（lonidainine);麥坦西諾 (maytansinoids)，諸如美登素（maytansine)及安沙米托辛 (ansamitocins)；米托脈腙（mitoguazone)；米托蒽酉昆 (mitoxantrone)；莫 σ比丹莫（mopidanmol);尼曲伊寧 (nitraerine);喷司他丁（pentostatin);蛋胺氮芥 (phenamet); 比柔比星（pirarubicin); 洛索蒽酉昆 (losoxantrone) ； 2-乙醯肼；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);細交鏈孢菌酮酸（tenuazonic acid);三亞胺g昆（triaziquone) ; 2,2’，2"-三氣三乙胺；單端孢黴烯（trichothecene)(尤其為T-2毒素、維萊庫寧A (verracurin A)、桿孢菌素 A(roridin A)及安古定 (anguidine));烏拉坦（urethan);長春地辛（vindesine) 119007.doc -47- 200813092 (ELDISINE®、FILDESIN®);達卡巴嗪（dacarbazine);甘露莫司汀（mannomustine);二漠甘露醇（mitobronitol);二漠衛矛醇（mitolactol);旅泊漠烧（pipobroman);瓜西托辛 (gacytosine);***糖普（arabinoside)(nAra-Cn);塞替派 (thiotepa);紫杉醇類，例如TAXOL®紫杉醇（Bristol-Myers Squibb Oncology，Princeton，N.J·)、ABRAXANETM游離型十六醇聚氧乙烯醚、經白蛋白工程加工之紫杉醇奈米粒子調配物（American Pharmaceutical Partners, Schaumberg, Illinois)及 TAXOTERE® 多烯紫杉醇（Rh0ne-Poulenc Rorer， Antony，France);氯萊布辛（chloranbucil);吉西他濱 (gemcitabine)(GEMZAR®); 6-硫鳥嗓呤；疏σ票吟；甲胺蝶呤；翻類似物，諸如順始及卡銘；長春花鹼 (vinblastine)(VELBAN®);鉑；依託泊苷（VP-16);異環磷醢胺；米托蒽酿i (mitoxantrone);長春新驗（vincristine) (ONCOVIN®);奥賽力鉑（oxaliplatin)；氯扣沃新 (leucovovin);長春瑞濱（vinorelbine)(NAVELBINE®);諾消靈（novantrone);依達曲沙（edatrexate);道諾黴素；胺基蝶呤（aminopterin);伊班膦酸鹽（ibandronate);拓撲異構酶抑制劑RFS 2000 ;二氟曱基鳥胺酸（DMFO);諸如視黃酸之類視色素；卡西他濱（capecitabine)(XELODA®); 以上任何醫藥學上可接受之鹽、酸或衍生物；以及以上兩種或兩種以上之組合，諸如CHOP(環磷醯胺、多柔比星、長春新鹼與潑尼龍之組合療法之縮寫）及FOLFOX(以奥赛力鉑（ELOXATINtm)與5-FU及氣扣沃新組合之治療方案的 119007.doc -48- 200813092 縮寫）。該定義中亦包括用以調節、降低、阻斷或抑制可促進癌生長之激素效應且通常為全身或全體治療形式之抗激素劑。其自身可為激素。實例包括抗***及選擇性*** 受體調節劑（SERM)，包括例如他莫昔芬（tamoxifen)(包括 NOLVADEX® 他莫昔芬）、EVISTA® 雷諾昔酚（EVISTA® raloxifene)、屈洛昔芬（droloxifene)、4-經基他莫昔芬、曲沃昔芬（trioxifene)、科奥昔芬（keoxifene)、LY117018、奥那司酮（onapristone)及 FARESTON®托瑞米芬（FARESTON® toremifene);抗-孕酮；***受體下調劑（ERD);用以抑制或中止卵巢之藥劑，例如黃體生成激素釋放激素 (LHRH)促效劑，諸如LUPRON⑧及ELIGARD®乙酸亮丙立德（ELIGARD® leuprolide acetate)、乙酸戈舍瑞林 (goserelin acetate)、乙酸布舍瑞林（buserelin acetate)及曲特來寧（tripterelin);其他抗雄激素，諸如氟他胺 (flutamide)、尼魯胺（nilutamide)及比卡魯胺（bicalutamide); 及抑制調節腎上腺中***產生之酶芳香酶的芳香酶抑制劑，諸如4(5)-味吐、胺魯米特（aminoglutethimide)、 MEGASE®乙酸甲地孕酮、AROMASIN®依西美坦 (AROMASIN® exemestane)、福美斯坦（formestanie)、法屈 ^(fadrozole)、RIVISOR® 伏氣 ^(RIVISOR® vorozole)、 FEMARA® 雷曲嗤（FEMARA® letrozole)及 ARIMIDEX® 安美達鍵（ARIMIDEX® anastrozole)。此外，化療劑之此定義包括雙膦酸鹽，諸如氣屈膦酸鹽（clodronate)(例如 119007.doc -49- 200813092 BONEFOS® 或 OSTAC®)、DIDROCAL® 依替膦酸鹽 (DIDROCAL® etidronate)、NE-58095、ZOMETA®唑來膦酸/唑來膦酸鹽、FOSAMAX®阿侖膦酸鹽、AREDIA®帕米膦酸鹽、SKELID®替魯膦酸鹽或ACTONEL®利塞膦酸鹽 (risedronate);以及曲沙他濱（troxacitabine)(l，3-二氧戊環核苷胞嘧啶類似物）；反義寡核苷酸，尤其為彼等抑制異常細胞增殖中涉及之信號路徑中之基因表現的寡核苷酸，諸如PKC-α、Raf、H-Ras及表皮生長因子受體（EGF-R); 疫苗，諸如THERATOPE®疫苗及基因療法疫苗，例如 ALLOVECTIN® 疫苗、LEUVECTIN® 疫苗及 VAXID® 疫苗；LURTOTECAN⑧1型拓撲異構酶抑制劑；ABARELIX® rmRH ;拉帕替尼二甲苯橫酸鹽（lapatinib ditosylate)(亦稱為GW5 72016之ErbB-2及EGFR雙酪胺酸激酶小分子抑制劑）及以上任何醫藥學上可接受之鹽、酸或衍生物。本文使用之’’生長抑制劑”係指抑制活體外或活體内細胞 (諸如表現TAT226之細胞）生長之化合物或組合物。因此，生長抑制劑可為顯著降低S相細胞（諸如表現TAT226之細胞）百分比之生長抑制劑。生長抑制劑之實例包括阻斷細胞週期進程（除S相以外之位置）之藥劑，諸如誘導G1停滯及Μ相停滯之藥劑。經典Μ相阻斷劑包括長春蔓（vinca)(長春新驗及長春驗）、紫杉烧（taxane)及II型拓樸異構酶抑制劑，諸如多柔比星、表柔比星、道諾黴素、依託泊苷及博萊黴素。停滞G1之彼等藥劑亦溢流入S相停滞中，例如 DNA烧化劑，諸如他莫昔芬、強的松（prednisone)、達卡 119007.doc -50- 200813092 巴嗪、氮芥、順鉑、甲胺喋呤、5-氟尿嘧啶及阿糖胞苷 (ara-C)。其他資訊可見於Murakami等人之The Molecular Basis of Cancer，Mendelsohn 及 Israel編，第 1章，標題為 nCell cycle regulation, oncogenes, and antineoplastic drugs’’（WB Saunders: Philadelphia，1995)中，尤其為第 13 頁。备、杉烧（备、杉醇及多稀紫杉醇（docetaxel))均為衍生自紫杉樹之抗癌藥物。自歐洲紫杉衍生之多烯紫杉醇 (TAXOTERE®，Rhone-P〇ulenc R0rer)為紫杉醇（TAX〇L⑧，Radioactivity of Bi212, P32, Pb212 and Lu such as methotrexate, adriamicin, vinca test (vinchun new test, changchun test, etoposide), doxorubicin, beauty Melphalan, mitomycin c, chlorambucil, daunorubicin; or other intercalating agents, enzymes and fragments thereof 119007.doc -43- 200813092, such as nucleolytic enzymes, antibiotics And toxins, such as small molecule toxins or enzymatically active toxins (including fragments and/or variants thereof) of bacterial, fungal, plant or animal origin, and various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Tumor killing agents cause destruction of tumor cells. "Toxin π is any substance that has a detrimental effect on cell growth or proliferation. ''The chemotherapeutic agent π is a compound suitable for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® Cyclophosphamide; pyruvate, such as busulfan, improsulfan, and piposulfan; azepine, such as benzodopa, Kappa S Carboquone, meturedopa and uredopa; methyl imino and methylamelamine, including hexamethylene melamine, tri-ethyl melamine, tri-ethyl phosphine Amine, tri-ethyl thiophosphoniumamine and trishydroxyl melamine; acetogenin (especially bulbatacin and bulbatacinone); δ-9 - four-base marijuana (dronabinol, MARINOL®); β-lapach _ (β-lapachone); lapachol; colchicine; betulinic acid; Camptothecin (including synthetic analog topotecan (topot) Ecan) (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminopyrazine; Bryostatin; california, callystatin; CC-1065 (including its adozelesin, katsin 119007.doc •44-200813092 (carzelesin) and the fold Biz 新 (bizelesin) synthetic analogue); podophyllotoxin; podophyllinic acid; teniposide; cryptophycin (especially nymphalin 1) And Nostoccal cyclic peptide 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancastatin ( Pancratistatin); sarcodictyin; spongistatin; mustard nitrogen, such as chlorambucil, naphthyl mustard, cholestyramine, estramustine, heterocyclic Methionine, mechlorethamine, nitrous oxide mustard, melphalan, neombibichin, benzene mustard Phenosterine), prednimustine, trofosfamide, uracil mustard; nitrosourea, such as carmustine, chlorozotocin ), tomis, fotemustine, lomustine, nimustine, and lenimos, ranimnustine; antibiotics, such as enemetric fast antibiotics (such as the Kaqi emblem) Calicheamicin, especially calicheamicin γ 卡 and calicheamicin coll (see, for example, jg (iv) w, "·5d·5:33:183-186 (1994)); dynemicin, Including dinabin A; esperamicin; and new tumor suppressor protein chromophore and related pigment protein 浠 diacetylene antibiotic chromophore), aclacinomysins, actinomycetes Au, authramycin, azaserine, bleomycins, actinomycin C, caracalcin, carminomycin, carcinogen 119007.doc -45- 200813092 (carzinophilin), chromomycinis, release line D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-positemin, ADRIAMYCIN® doxorubicin (including morpholinyl-doxorubicin) Star, cyanomorpholino-doxorubicin, 2-pyrrolyl-doxorubicin and deoxydoxonol), epirubicin, esorubicin, phos (idarubicin), marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin ), potentinomycin (p〇tf*iromycin), puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin (streptozocin), tuberculin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil ( 5-FU); folic acid analogs, such as denopterin, amidoxime, pteropterin, trimetrexate; purine analogs, such as Fludarabine, 6-Μ σ 呤 mer mer (mercaptopurine), thiamiprine (thiomiprine), thioguanine (thioguanine); mingding analogs, such as ancitabine (ancitabine), Aza Azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enoxa Etocitabine, floxuridine; androgen, such as caluterone, dromostanolone propionate, epitiostanol, meixiong 119007.doc -46 - 200813092 (mepitiostane), testolactone; anti-adrenalin, such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as acid folic acid Acid); aceglatone; acid-filled aldophosphamide glycoside; alanine-propionic acid; eniluracil; amsacrine; betrixine (bestrabucil); bisantrene; Idaqu (edatraxate); defofamine; demecolcine; diziquone; elfornithine; elliptinium acetate; epothilone ; etoglucid; etoglucid; gallium glutamate; transbasic urea; lentinan; lonidainine; maytansinoids, such as maytansine and ansami Ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; egg amine nitrogen Mustard (phenamet); pirarubicin; losoxantrone; 2-acetamidine; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); Razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2 ',2"-three gas triethylamine; trichothecene (especially T-2 toxin, Ville Verracurin A, roridin A, and anguidine; urethan; vindesine 119007.doc -47- 200813092 (ELDISINE®, FILDESIN) ®);dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine ; arabinoside (nAra-Cn); thiotepa; paclitaxel, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ·), ABRAXANETM free cetyl polyoxyethylene ether , paclitaxel nanoparticle formulation processed by albumin engineering (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® docetaxel (Rh0ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; sulphate; methotrexate; analogs such as cis and carmine; vinblastine (VELBAN®); platinum; etoposide (VP-16); Isocyclophosphamide; Mitox Brewing i (mitoxa Ntrone);vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novotron (novantrone); Edsarexate; daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorodecylguanine acid (DMFO); a visual pigment such as leucovorin; capecitabine (XELODA®); any of the above pharmaceutically acceptable salts, acids or derivatives; and combinations of two or more of the above, such as CHOP (cyclophosphine)醯、、、、 119 119 及及及及及及及 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 119 200813092 Abbreviation). Also included in the definition are anti-hormonal agents that modulate, reduce, block or inhibit the hormonal effects that promote cancer growth, and are generally systemic or therapeutic forms of the whole. It can be a hormone itself. Examples include anti-estrogen and selective estrogen receptor modulators (SERMs) including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene (EVISTA® raloxifene), Droloxifene, 4-amino tamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene Toremifene); anti-progesterone; estrogen receptor down-regulator (ERD); an agent used to inhibit or stop the ovaries, such as luteinizing hormone releasing hormone (LHRH) agonists, such as LUPRON8 and ELIGARD® leuprolide (ELIGARD® leuprolide acetate), goserelin acetate, buserelin acetate and tripterelin; other antiandrogens, such as flutamide, niru Amine (nilutamide) and bicalutamide; and aromatase inhibitors that inhibit the regulation of estrogen-producing enzymes in the adrenal gland, such as 4(5)-sweet, aminoglutethimide, MEGASE® Megestrol acetate , AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® FERMA® letrozole and ARIMIDEX® ARIMIDEX® anastrozole. In addition, this definition of chemotherapeutic agent includes bisphosphonates such as clodronate (eg 119007.doc -49-200813092 BONEFOS® or OSTAC®), DIDROCAL® etidronate (DIDROCAL® etidronate) , NE-58095, ZOMETA® zoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate or ACTONOL® risedronate ( Risedronate); and troxacitabine (l,3-dioxolan nucleoside cytosine analog); antisense oligonucleotides, especially in the signal pathways involved in their inhibition of abnormal cell proliferation Gene-expressing oligonucleotides such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccines and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; LURTOTECAN81 topoisomerase inhibitor; ABARELIX® rmRH; lapatinib ditosylate (also known as GW5 72016 ErbB-2 and EGFR double tyrosine kinase small molecule inhibition) And any pharmaceutically acceptable salt Acid or derivative thereof. As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells in vitro or in vivo, such as cells expressing TAT226. Thus, growth inhibitors can significantly reduce S phase cells (such as cells expressing TAT226) Percentage of growth inhibitors. Examples of growth inhibitors include agents that block cell cycle progression (except for the S phase), such as agents that induce G1 arrest and stagnation. Classical phase blockers include vinca ( Vinca) (Changchun new test and Changchun test), taxane and type II topoisomerase inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide and Bole The antiseptic agents of G1 also overflow into the S phase arrest, such as DNA burning agents, such as tamoxifen, prednisone, Dhaka 119007.doc -50-200813092, prazin, nitrogen mustard, Cisplatin, methotrexate, 5-fluorouracil, and cytarabine (ara-C). Additional information can be found in Murakami et al., The Molecular Basis of Cancer, Mendelsohn and Israel, Chapter 1, titled nCell cycle regulation , oncogene s, and antineoplastic drugs'' (WB Saunders: Philadelphia, 1995), especially page 13. Preparation, cedar (prepared, cedarol and docetaxel) are derived from yew tree anti-cancer Drug. Taxol derived from European yew (TAXOTERE®, Rhone-P〇ulenc R0rer) is paclitaxel (TAX〇L8,

Bristol-Myers Squibb)之半合成類似物。紫杉醇及多烯紫杉醇促進由微管蛋白二聚體組裝微管且藉由防止解聚合穩定微管，其致使抑制細胞中之有絲***。術語”細胞内代謝物”係指由抗體-藥物接合物（ADC)之細胞内之代謝過程或反應所產生之化合物。代謝過程或反應可為酶促過程，諸如蛋白水解裂解ADC之肽連接子或水解諸如腙、酯或醯胺之官能基。細胞内代謝物包括（但不限於）在進入、擴散、吸收或轉運至細胞中之後經受細胞内裂解之抗體及游離藥物。術語π細胞内裂解π係指抗體藥物接合物（ADc)細胞内之代6射過私或反應，错此破壞共價連接，亦即藥物部分（D) 與抗體（Ab)之間之連接子，導致游離藥物自細胞内之抗體解離。因此ADC之裂解部分為細胞内代謝物。術語"生物有效度”係指投予患者之指定量藥物之全身有效度（亦即血液/血漿含量）。生物有效度為表示對藥物自投予劑型達到總循環之時間（速率）及總量（程度）之量測的絕 119007.doc -51- 200813092 對形式。、、、田胞骨性活性”係指抗體·藥物接合物或抗體_藥物接合物之細胞内代謝物之細胞殺死、細胞生長抑制或生長抑制效應。細胞毒性活性可表現為KM值，其為一半細胞存活時每單位體積之濃度（莫耳或質量）。烷基’’為含有常態、第二、第三或環狀碳原子之Ci_Cw 丈工。貫例為甲基（Me、-CH3)、乙基（Et、-(：Η2(：Η3)、1_丙基 (〜Pr、正丙基、-CH2CH2CH3)、2·丙基(i-pr、異丙基、-CH(CH3)2)、 1- 丁基（n-Bu、正丁基、-CH2cH2CH2CH3)、2-甲基 j 丙基 (i-Bu、異丁基、_Ch2cH(CH3)2)、2_ 丁基（s_Bu、第二丁基-CH(CH3)CH2CH3)、2-甲基-2·丙基（t-Bu、第三丁基、-C(CH3)3)、1-戊基（正戊基、_CH2CH2CH2CH2CH3)、 2_戊基(-CH(CH3)CH2CH2CH3)、3-戊基(-CH(CH2CH3)2)、2_ 甲基-2-丁基(-C(CH3)2CH2CH3)、3-甲基-2·丁基(-CH(CH3)CH(CH3)2)、 3-甲基-1_ 丁基（_CH2CH2CH(CH3)2)、2-甲基·1_ 丁基 CH2CH(CH3)CH2CH3)、1-己基(-CH2CH2CH2CH2CH2CH3)、 2- 己基（-CH(CH3)CH2CH2CH2CH3)、3-己基（-CH(CH2CH3) (CH2CH2CH3))、2-甲基-2-戊基（-C(CH3)2CH2CH2CH3)、3-甲基-2-戊基（-CH(CH3)CH(CH3)CH2CH3)、4-甲基-2_戊基 (CH(CH3)CH2CH(CH3)2)、3-甲基-3_戊基(-C(CH3)(CH2CHA^ 2-甲基-3-戊基（-CH(CH2CH3)CH(CH3)2)、2,3_ 二甲基-2-丁基(-C(CH3)2CH(CH3)2)、3,3-二甲基-2-丁基(-CH(CH3)C(CH3)3)。如本文所用之術語”<^-(：8烷基”係指具有1至8個碳原子之直鏈或支鏈、飽和或不飽和烴。代表性’’Ci-Cs烷基"包括 119007.doc -52- 200813092 (但不限於)-甲基、-乙基、-正丙基、-正丁基、-正戊基、_ 正己基、-正庚基、-正辛基、-正壬基及-正癸基；而支鍵 c^c：8烷基包括（但不限於異丙基、-第二丁基、-異丁基、-弟二丁基、-異戍基、2 -甲基丁基，不飽和Ci-Cg燒基包括（但不限於）_乙烯基、-烯丙基、-^丁烯基、_2_丁稀基、-異丁烯基、-1-戊稀基、-2-戊稀基、-3-曱基-1-丁烯基、-2-甲基-2_丁烯基、-2,3-二曱基-2-丁烯基、1-己基、 2-己基、3 -己基、-乙快基、-丙炔基、-1-丁炔基、-2 -丁快基、-1-戊炔基、-2-戊炔基、-3-甲基-1-丁炔基、甲基、乙基、丙基、異丙基、正丁基、異丁基、第二丁基、第三丁基、正戊基、異戊基、新戊基、正己基、異己基、2-甲基戊基、3-曱基戊基、2,2-二甲基丁基、2,3_二曱基丁基、 2,2-二甲基戊基、2,3_二曱基戊基、3,3-二甲基戊基、 2，3,4 -二甲基戊基、3 -甲基己基、2,2 -二甲基己基、2,4 -二甲基己基、2,5_二甲基己基、3,5·二甲基己基、2,4-二甲基戊基、2·曱基庚基、3-曱基庚基、正庚基、異庚基、正辛基及異辛基。Ci-Cs烧基可未經取代或經一或多個包括（但不限於）以下基團之基團取代：-CrCs烷基、_〇-((：!-C8烷基）、-芳基、-C(0)R，、-0C(0)R，、-C(0)0R，、-C(0)NH2、 C(0)NHR丨、_C(0)N(R丨)2-NHC(0)R，、-S03R，、-S(0)2R丨、 -S(0)R*、-〇H、_ 函素、-N3、-NH2、-NH(R’）、-N(R’）2及 _CN ;其中各R’係獨立地選自h、-q-Cs烷基及芳基。 n烯基”為具有至少一個不飽和（亦即碳-碳，印2雙鍵）位點之含有常態、第二、第三或環狀碳原子之匸2-〇18烴。實例 119007.doc •53- 200813092 包括（但不限於）：伸乙基或乙烯基(_CH=CH2)、烯丙基(_CH2CH= CH2)、環戊烯基（-c^7)及 5_ 己烯基（-Ch2CH2CH2CH2Ch= CH2) 〇 ”炔基”為具有至少一個不飽和（亦即碳_碳，sp參鍵）位點之含有常態、第二、第三或環狀碳原子之c2_ci8烴。實例包括（但不限於）：乙炔基（_C=CH)及炔丙基（_ch2C^CH)。伸烧基係指具有1 -18個碳原子且具有藉由自母體烧烴之相同或兩個不同碳原子移除兩個氫原子衍生之兩個單價基團中心的飽和、支鏈或直鏈或環狀烴基。典型伸烧基包括（但不限於）：亞甲基（-CH2-)、1，2-乙基（_CH2CH2-)、1，3-丙基（_CH2CH2CH2-)、1，4· 丁基（-CH2CH2CH2CH2-)及其類似基團。Semi-synthetic analog of Bristol-Myers Squibb. Paclitaxel and docetaxel promote assembly of microtubules by tubulin dimers and stabilize microtubules by preventing depolymerization, which results in inhibition of mitosis in cells. The term "intracellular metabolite" refers to a compound produced by a metabolic process or reaction within the cell of an antibody-drug conjugate (ADC). The metabolic process or reaction can be an enzymatic process, such as proteolytic cleavage of a peptide linker of an ADC or hydrolysis of a functional group such as a hydrazine, an ester or a guanamine. Intracellular metabolites include, but are not limited to, antibodies that undergo intracellular lysis following entry, diffusion, absorption, or transport into cells, and free drugs. The term π intracellular cleavage π refers to the intracellular generation of an antibody drug conjugate (ADc) that has been eclipsed or reacted, thereby disrupting the covalent linkage, ie, the linker between the drug moiety (D) and the antibody (Ab). , causing free drug to dissociate from the antibodies in the cell. Thus the cleavage portion of the ADC is an intracellular metabolite. The term "bioavailability" refers to the systemic effectiveness (i.e., blood/plasma content) of a given amount of a drug administered to a patient. The bioavailability is the time (rate) and total for the total time of administration of the drug to the dosage form. The amount (degree) of the measurement 119007.doc -51- 200813092 on the form.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, , cell growth inhibition or growth inhibition effects. The cytotoxic activity can be expressed as a KM value which is the concentration per unit volume (mol or mass) at which half of the cells survive. The alkyl group '' is a Ci_Cw group containing a normal, second, third or cyclic carbon atom. The examples are methyl (Me, -CH3), ethyl (Et, -(:Η2(:Η3), 1-propyl (~Pr, n-propyl, -CH2CH2CH3), 2·propyl (i-pr) , isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2cH2CH2CH3), 2-methyljpropyl (i-Bu, isobutyl, _Ch2cH(CH3) 2), 2_butyl (s_Bu, second butyl-CH(CH3)CH2CH3), 2-methyl-2.propyl (t-Bu, tert-butyl, -C(CH3)3), 1- Pentyl (n-pentyl, _CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3) 2CH2CH3), 3-methyl-2.butyl (-CH(CH3)CH(CH3)2), 3-methyl-1_butyl (_CH2CH2CH(CH3)2), 2-methyl·1_butyl CH2CH (CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl ( -C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (CH(CH3)CH2CH(CH3)2) 3-methyl-3-pentyl (-C(CH3)(CH2CHA^2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2- Butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(C) H3) C(CH3)3). The term "<^-(:8 alkyl" as used herein refers to a straight or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative' 'Ci-Cs alkyl" includes 119007.doc -52- 200813092 (but not limited to) -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, _n-hexyl, -positive Heptyl, -n-octyl, -n-decyl and -n-decyl; and the branch c^c:8 alkyl includes (but not limited to, isopropyl, -t-butyl, -isobutyl, -di Dibutyl, -isodecyl, 2-methylbutyl, unsaturated Ci-Cg alkyl including, but not limited to, _vinyl, -allyl, -butenyl, _2-butadienyl, -isobutenyl,-1-pentyl,-2-pentyl,-3-mercapto-1-butenyl,-2-methyl-2-butenyl,-2,3-didecyl 2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -ethylidene, -propynyl,-1-butynyl,-2-butanthyl,-1-pentynyl, 2-pentynyl,-3-methyl-1-butynyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, second butyl, tert-butyl, N-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl 2-methylpentyl, 3-mercaptopentyl, 2,2-dimethylbutyl, 2,3-didecylbutyl, 2,2-dimethylpentyl, 2,3-dioxin Pentyl, 3,3-dimethylpentyl, 2,3,4-dimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-Dimethylhexyl, 3,5·dimethylhexyl, 2,4-dimethylpentyl, 2·decylheptyl, 3-decylheptyl, n-heptyl, isoheptyl, N-octyl and isooctyl. The Ci-Cs alkyl group may be unsubstituted or substituted with one or more groups including, but not limited to, the following groups: -CrCs alkyl, _〇-((:!-C8 alkyl), -aryl , -C(0)R,, -0C(0)R,, -C(0)0R,, -C(0)NH2, C(0)NHR丨, _C(0)N(R丨)2- NHC(0)R,, -S03R,, -S(0)2R丨, -S(0)R*, -〇H, _ tex, -N3, -NH2, -NH(R'), -N (R') 2 and _CN; wherein each R' is independently selected from the group consisting of h, -q-Cs alkyl and aryl. n-alkenyl" has at least one unsaturation (ie, carbon-carbon, 2 pairs) a )2-〇18 hydrocarbon containing a normal, second, third or cyclic carbon atom. Examples 119007.doc •53- 200813092 Includes (but is not limited to): Ethyl or vinyl (_CH= CH2), allyl (_CH2CH=CH2), cyclopentenyl (-c^7) and 5-hexenyl (-Ch2CH2CH2CH2Ch=CH2) 〇"alkynyl" have at least one unsaturated (ie carbon-carbon) The sp2 bond contains a c2_ci8 hydrocarbon of a normal, second, third or cyclic carbon atom. Examples include, but are not limited to, ethynyl (_C=CH) and propargyl (_ch2C^CH). Stretching base means having from 1 to 18 carbon atoms a saturated, branched or straight chain or cyclic hydrocarbon radical having two monovalent radicals derived by the removal of two hydrogen atoms from the same or two different carbon atoms of the parent hydrocarbon. Typical extensions include (but not Limited to: methylene (-CH2-), 1,2-ethyl (_CH2CH2-), 1,3-propyl (_CH2CH2CH2-), 1,4·butyl (-CH2CH2CH2CH2-) and the like .

Ci-Ci。伸烧基π為式-(CHQmo-之直鏈飽和烴基。CrC^o 伸烷基之實例包括亞甲基、伸乙基、伸丙基、伸丁基、伸戊基、伸己基、伸庚基、伸辛基、伸壬基及伸癸基。 M伸浠基’’係指具有2 -1 8個破原子且具有藉由自母體稀烴之相同或兩個不同碳原子移除兩個氫原子衍生之兩個單價基團中心的不飽和、支鏈或直鏈或環狀烴基。典型伸烯基包括（但不限於）：1,2-伸乙基（-CH=CH-)。 π伸炔基”係指具有2-1 8個碳原子且具有藉由自母體炔烴之相同或兩個不同碳原子移除兩個氫原子衍生之兩個單價基團中心的不飽和、支鏈或直鏈或環狀烴基。典型伸炔基包括（但不限於）：乙炔基（-CeC-)、炔丙基（-CH2C^C-)及4-戊炔基（-CH2CH2CH2CeC-)。 119007.doc •54- 200813092 芳基”係指碳環芳族基。芳基之實例包括（但不限於）苯基、萘基及蒽基。碳環芳族基或雜環芳族基可未經取代或經一或多個包括（但不限於）以下基團之基團取代：_Ci_C8 烧基、烧基）、_芳基、_c(〇)R，、-〇C(0)R，、-C(0)0R，、-C(0)NH2、-C(0)NHR’、-C(0)N(R，)2-NHC(0)R，、-S(0)2R，、-S(0)R’、-〇H、-鹵素、_Ns、-NH2、一nh(r，）、_n(r，)2 及-CN ;其中各r’係獨立地選自H、_Ci-c8烷基及芳基。伸芳基”為如以下結構所示之具有兩個共價鍵且可為鄰位、間位或對位構型之芳基：Ci-Ci. The stretching group π is a linear-saturated hydrocarbon group of the formula -CHQmo-. Examples of the CrC^o alkylene group include a methylene group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and a hexylene group. M, octyl, thiol and hydrazine. M 浠 ' '' means having 2 to 18 broken atoms and having two or more different carbon atoms removed from the parent dilute hydrocarbon An unsaturated, branched or straight chain or cyclic hydrocarbon group centered on two monovalent groups derived from a hydrogen atom. Typical alkenyl groups include, but are not limited to, 1,2-extended ethyl (-CH=CH-). "π"alkynyl" means an unsaturated group having 2 to 8 carbon atoms and having two monovalent groups derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne Chain or linear or cyclic hydrocarbon groups. Typical alkynyl groups include, but are not limited to, ethynyl (-CeC-), propargyl (-CH2C^C-), and 4-pentynyl (-CH2CH2CH2CeC-). 119007.doc •54- 200813092 aryl” means a carbocyclic aromatic group. Examples of aryl group include, but are not limited to, phenyl, naphthyl and anthracenyl. Carbocyclic aromatic or heterocyclic aromatic groups may Substituted or Or a plurality of groups including, but not limited to, the following groups: _Ci_C8 alkyl, alkyl), aryl, _c(〇)R, , -〇C(0)R,, -C(0) 0R, , -C(0)NH2, -C(0)NHR', -C(0)N(R,)2-NHC(0)R,, -S(0)2R, , -S(0) R', -〇H, -halogen, _Ns, -NH2, -nh(r,), _n(r,)2 and -CN; wherein each r' is independently selected from H, _Ci-c8 alkyl and aryl base. The aryl group is an aryl group having two covalent bonds and which may be in the ortho, meta or para configuration as shown in the following structure:

其中笨基可未經取代或經至多四個包括（但不限於）以下基團之基團取代：-CKC8烷基、烷基）、_芳基、_c(〇)R， ^ -0C(0)Rf > -C(0)〇Rf > -C(0)NH2 - -C(0)NHRf - -C(0)N(Rf)2-NHC(0)R’、-S(0)2R’、-S(0)R’、-〇H、·_ 素、、NH2 1/ ' _NH(R )、-N(R’)2及-CN ;其中各R’係獨立地選自H、_Wherein the stupid group may be unsubstituted or substituted with up to four groups including, but not limited to, the following groups: -CKC8 alkyl, alkyl), _aryl, _c(〇)R, ^ -0C(0 ) Rf > -C(0)〇Rf > -C(0)NH2 - -C(0)NHRf - -C(0)N(Rf)2-NHC(0)R', -S(0) 2R', -S(0)R', -〇H, ·_, , NH2 1/ ' _NH(R ), -N(R') 2 and -CN; wherein each R' is independently selected from H , _

Ci-Cs烧基及芳基。 n芳烷基”係指其中鍵結至碳原子（通常為末端或sp3碳原子）之氫原子之一經芳基置換之非環狀烷基。典型芳烷基包括（但不限於）节基、2-苯基乙烷_丨_基、2_苯基乙烯基、奈基甲基、2-奈基乙烧_ι_基、2_萘基乙烯基、萘幷苄基、2-奈幷笨基乙烷-丨-基及其類似基團。芳烷基包含6 至20個碳原子，例如芳烷基之烷基部分（包括烷基、烯基 119007.doc -55- 200813092 或炔基）為1至6個碳原子且芳基部分為5至14個碳原子。 "雜芳烧基"係指其中鍵結至碳原子(通常為末端或:卩3碳原子）之氫原子之一經雜芳基置換之非環狀燒基。典型雜芳烷基包括（但不限於）2_苯幷咪唑基甲基、2•呋喃基乙基及其類似基團。雜芳烷基包含6至2〇個碳原子，例如雜芳烷基之烷基部分(包括烷基、烯基或炔基)為⑴個碳原子且雜芳基部分為5至14個碳原子及丨至3個選自n' 〇、卩及^ $ 之雜原子。雜芳烷基之雜芳基部分可為具有3至7個環成員 ' 之單環（2至6個碳原子）或具有7至10個環成員之雙環（4至9 個碳原子及1至3個選自N、〇、！>及8之雜原子），例如：雙環[4,5]、[5,5]、[5,6]或[6,6]系統。Ci-Cs alkyl and aryl. "Aralkyl" means an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (usually a terminal or sp3 carbon atom) is replaced by an aryl group. Typical aralkyl groups include, but are not limited to, a benzyl group, 2-phenylethane-丨-yl, 2-phenylvinyl, naphthylmethyl, 2-naphthylacetone_ι_yl, 2-naphthylvinyl, naphthoquinonebenzyl, 2-nene An alkyl ethane-fluorenyl group and the like. The aralkyl group contains 6 to 20 carbon atoms, such as an alkyl portion of an aralkyl group (including an alkyl group, an alkenyl group 119007.doc -55-200813092 or an alkynyl group). ) is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms. "Heteroaryl" refers to a hydrogen atom bonded to a carbon atom (usually a terminal or a 卩3 carbon atom). A non-cyclic alkyl group substituted by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2, furanylethyl and the like. Heteroaralkyl contains 6 to 2 carbon atoms, for example, an alkyl moiety (including an alkyl group, an alkenyl group or an alkynyl group) of a heteroarylalkyl group is (1) carbon atoms and a heteroaryl moiety is 5 to 14 carbon atoms and 丨 to 3 Selected from n' 〇, 卩 and a hetero atom of ^. The heteroaryl portion of the heteroaralkyl group may be a single ring (2 to 6 carbon atoms) having 3 to 7 ring members ' or a double ring having 7 to 10 ring members (4 to 9) One carbon atom and one to three hetero atoms selected from N, 〇, !> and 8, for example: bicyclo[4,5], [5,5], [5,6] or [6,6] system.

"經取代之烷基"、"經取代之芳基，，及"經取代之芳烷基，· 分別意謂其中一或多個氫原子各自獨立地經取代基置換之院基、芳基及芳烧基。典型取代基包括（但不限於）_χ、_R 、-〇·、-OR、-SR、_s.、-NR2、-NR3、=NR、-CX3、-CN 、_〇CN、-SCN、-N=C=0、_NCS、-NO、_N02、=N2、-N3 、NC(=〇)R、-C(=0)R、-C(=0)NR2、-S03-、-so3h、-s(=o)2r 、-os(=o)2〇r、_s(=o)2nr、-s(=o)r、_〇p(=〇)(or)2、 -P(=0)(0R)2、-P03-、_P03H2、-C(=0)R、-c(=0)x、-C(=S)R 、-C02R、_C02_、-C(=S)OR、-C(=0)SR、-C(=S)SR、-C(=〇)NR2 、-C(=S)NR2、-C(=NR)NR2，其中各X獨立地為鹵素：F、 Cl、Br或I ;且各R獨立地為-Η、C2_C18烷基、（：6-(：20芳基' C3-C14雜環、保護基或前藥部分。如上所述之伸烷基、伸烯基及伸炔基亦可類似地經取代。 119007.doc •56- 200813092 "雜芳基"及"雜環"係指其中—或多個環原子為雜原子（例如氮、氧及硫)之環系統。雜環基包含個碳原子及i 至3個選自N、0、PAS之雜原子。雜環可為具有⑴個環成貝之單裱（2至6個碳原子及丨至3個選自N、〇、卩及8之雜原子）或具有7至U)個環成員之雙環（4至9個碳原子及⑴個選自N、Ο、P及S之雜原子），例如：雙環[4,5]、[5,5]、 [5,6]或[6,6]系統。雜環係描述於Paquette，Leo A· ; ”Principles of M〇dern Heterocyclic Chemistry^ W. A. Benjamin, New York, 1968)，尤其是第 1、3、4、6、7及 9章；"The Chemistry of"Substituted alkyl", "substituted aryl, and "substituted aralkyl, respectively, meaning that one or more of the hydrogen atoms are independently replaced by a substituent , aryl and aryl groups. Typical substituents include, but are not limited to, χ, _R, -〇·, -OR, -SR, _s., -NR2, -NR3, =NR, -CX3, -CN, _〇CN, -SCN, -N =C=0, _NCS, -NO, _N02, =N2, -N3, NC(=〇)R, -C(=0)R, -C(=0)NR2, -S03-, -so3h, -s (=o)2r , -os(=o)2〇r, _s(=o)2nr, -s(=o)r, _〇p(=〇)(or)2, -P(=0)( 0R)2, -P03-, _P03H2, -C(=0)R, -c(=0)x, -C(=S)R, -C02R, _C02_, -C(=S)OR, -C( =0) SR, -C(=S)SR, -C(=〇)NR2, -C(=S)NR2, -C(=NR)NR2, wherein each X is independently halogen: F, Cl, Br Or I; and each R is independently -Η, C2_C18 alkyl, (:6-(:20 aryl 'C3-C14 heterocycle, protecting group or prodrug moiety. Alkyl, alkenyl group as described above) And an alkynyl group can be similarly substituted. 119007.doc •56-200813092 "heteroaryl" and "heterocycle" means that one or more ring atoms are heteroatoms (eg nitrogen, oxygen and a ring system of sulfur. The heterocyclic group contains one carbon atom and i to three heteroatoms selected from N, 0, and PAS. The heterocyclic ring may be a monocyclic (1) ring having 2 to 6 carbon atoms and丨 to 3 selected from N, 〇, 卩 and 8 a heteroatom) or a bicyclic ring having 7 to U) ring members (4 to 9 carbon atoms and (1) a hetero atom selected from N, Ο, P, and S), for example, bicyclo[4,5], [5, 5], [5,6] or [6,6] systems. Heterocycles are described in Paquette, Leo A· ; "Principles of M〇dern Heterocyclic Chemistry ^ WA Benjamin, New York, 1968), especially number 1. Chapters 3, 4, 6, 7 and 9; "The Chemistry of

Heterocyclic Compounds, A series of Monographs11 (John Wiley & Sons，New York，1950至今），尤其是第 13、14、 16、19及 28卷；及/· c/zew· 5W· (1960) 82:5566 中。雜環之實例以實例而非限制之方式包括吡啶基、二氫吡啶基、四氫吡啶基（哌啶基）、噻唑基、四氫噻吩基、經硫氧化之四氫°塞吩基、u密σ定基、吱σ南基、σ塞吩基、吼p各基、吡唑基、咪唑基、四唑基、苯幷呋喃基、噻萘基、吲哚基、吲哚啉基、喹啉基、異喹啉基、苯幷咪唑基、哌啶基、4-派咬酮基、吼洛咬基、2-吼略酮基、α比洛琳基、四氫呋喃基、雙-四氫呋喃基、四氫哌喃基、雙-四氫哌喃基、四氫喹啉基、四氫異喹啉基、十氫喹啉基、八氫異喹啉基、吖辛因基、三嗪基、6Η-1，2,5-噻二嗪基、2Η，6Η-1，5,2_二噻嗪基、噻吩基、噻嗯基、哌喃基、異苯幷呋喃基、咣烯基、咄基、啡噁噻基、2Η-吡咯基、異噻唑基、 119007.doc -57- 200813092 異鳴、哇基、啦嗪基、噠嗪基、吲嗪基、異吲哚基、3仏吲口朵基、1H-吲峻基、嘌呤基、4H_喹嗪基、呔嗪基、喑啶基、啥°若琳基、啥ϋ坐琳基、吟嘆琳基、嗓^定基、卡峻基、味嗤基、β_咔啉基、啡啶基、吖啶基、嘧啶基、啡啉基、啡嗓基、啡噻嗪基、呋吖基、啡噁嗪基、異咣基、咣基、咪唑啶基、咪唑啉基、吡唑啶基、吼唑啉基、哌嗪基、’ °朵琳基、異吲哚啉基、嗝啶基、嗎啉基、噁唑啶基、苯幷三嗤基、苯幷異噁唑基、羥基吲哚基、苯幷噁唑琳基及°引σ木滿一^綱基（isatinoyl)。以實例而非限制之方式，碳鍵結之雜環係於以下位置鍵結·吡啶之位置2'3、4、5或6;噠嗪之位置3、4、5或Heterocyclic Compounds, A series of Monographs 11 (John Wiley & Sons, New York, 1950-present), especially Volumes 13, 14, 16, 19 and 28; and /· c/zew· 5W· (1960) 82:5566 in. Examples of heterocycles include, by way of example and not limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidinyl), thiazolyl, tetrahydrothiophenyl, sulfur-oxidized tetrahydro-thiophene, u Σσ定基,吱σ南基, σ塞基基,吼p, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thionaphthyl, anthracenyl, porphyrin, quinoline , isoquinolyl, benzoimidazolyl, piperidinyl, 4-piperidone, indole, 2-indolyl, alpha-pirolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetra Hydroperyl, bis-tetrahydropyranyl, tetrahydroquinolyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, octyl octyl, triazinyl, 6 Η- 1,2,5-thiadiazinyl, 2Η,6Η-1,5,2-dithiazinyl, thienyl, thienyl, piperidyl, isobenzofuranyl, nonenyl, fluorenyl, Phenylthio, 2Η-pyrrolyl, isothiazolyl, 119007.doc -57- 200813092 oxime, wowyl, oxazinyl, pyridazinyl, pyridazinyl, isodecyl, 3 oxime , 1H-sulfonyl, fluorenyl, 4H_quinolinyl, pyridazinyl, Acridine, 啥°若琳基, 啥ϋ 琳基吟, 吟琳基嗓嗓吟吟、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、 Orolinyl, morphinyl, phenothiazine, furazinyl, phenoxazinyl, isodecyl, fluorenyl, imidazolidinyl, imidazolinyl, pyrazolyl, oxazolinyl, piperazinyl, ' °Dolinyl, isoindolyl, acridinyl, morpholinyl, oxazolidinyl, benzoquinone, benzoxazole, hydroxy hydrazino, benzoxazole ° σσ木满一^基基 (isatinoyl). By way of example and not limitation, the carbon-bonded heterocyclic ring is bonded to the position 2'3, 4, 5 or 6 of the pyridine; the position of the oxazine is 3, 4, 5 or

6，％咬之位置2、4、5或6 ;吡嗪之位置2、3、5或6 ;呋喃、四氫呋喃、硫呋喃、噻吩、吡咯或四氫吡咯之位置 2、3、4或5 ;噁唑、咪唑或噻唑之位置2、4或$ ;異噁嗤比嗤或異嗟嗤之位置3、4或5 ;氮丙唆之位置2或3 ; 。丫丁唆之位置2、3或4;噎琳之位置2、3、4、5、6、7或8 或異啥啉之位置……卜…或卜更通常^碳鍵結之雜環包括2·吼咬基、基、6 -°tb咬基、3-嚏嗪基基、2-嘧啶基、4-嘧啶基基、3 - σ比嘻基、5 - σ比嗪基基或5 - σ塞嗤基。丨-°比。定基、4- 口比π定基、5-。比唆 4-噠嗪基、5-噠嗪基、6-噠嗪 5密咬基、6·嘴咬基、2·吼嗪 6-吼嗪基、2-噻唑基、4-噻唑以實例而非限制之方結：氣丙咬、。丫丁咬、式’氮鍵結之雜環係於以下位置鍵比各、°比洛咬、2 _ π比洛淋、3 - 〇比υ各 119007.doc -58- 200813092 啉、咪唑、咪唑啶、2-咪唑啉、3_咪唑啉、吡唑、吡唑啉、2-吡唑啉、3-吡唑啉、哌啶、哌嗪、吲哚、吲哚啉、 1H-吲唑之位置1 ;異吲哚或異吲哚啉之位置2 ;嗎啉之位置4及咔唑或β-咔啉之位置9。更通常地，氮鍵結之雜環包括1-氮丙啶基、1-吖唉基（azetedyl)、吡咯基、丨_咪唑基、1-吼0坐基及1-旅咬基。6, 2 bit position 2, 4, 5 or 6; pyrazine position 2, 3, 5 or 6; furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole position 2, 3, 4 or 5; The position of the oxazole, imidazole or thiazole is 2, 4 or $; the position of the oxindole is 3, 4 or 5 than the position of hydrazine or hydrazine; the position of the acetophenone is 2 or 3; Location of 丫丁唆2, 3 or 4; position of 2、琳, 2, 3, 4, 5, 6, 7 or 8 or the position of isoporphyrin... Bu... or more usually carbon-bonded heterocycle 2. A bite base, a base, a 6-°tb bite group, a 3-pyridazinyl group, a 2-pyrimidinyl group, a 4-pyrimidinyl group, a 3 - σ thiol group, a 5 - σ-pyridyl group or a 5- σ塞嗤基.丨-° ratio. Base, 4-port ratio π-base, 5-. Specific examples of 4-pyridazinyl, 5-pyridazinyl, 6-pyridazine-5 cryptyl, 6-neck, 2-pyridazine 6-pyridazinyl, 2-thiazolyl, 4-thiazole Unrestricted square knot: a gas bite. The squid bite, the type of nitrogen-bonded heterocyclic ring is in the following positions: 各洛 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Position of pyridine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, hydrazine, porphyrin, 1H-carbazole 1; position of isoindole or isoindoline 2; position of morpholine 4 and position of carbazole or β-carboline 9. More typically, the nitrogen-bonded heterocyclic ring includes 1-aziridine, 1-azetyl, pyrrolyl, oxime-imidazolyl, 1-indolyl and 1-branched.

Cs-Cs雜ί哀”係指其中環碳原子之一至四者獨立地經選自由〇、S與Ν組成之群之雜原子置換的芳族或非芳族c3_C8 碳環。雜環之代表性實例包括（但不限於）苯幷呋喃基、苯幷噻吩、吲哚基、苯幷咄唑基、香豆素基、異喹啉基、吡咯基、噻吩基、呋喃基、噻唑基、咪唑基、吡唑基、***基、喹啉基、嘧啶基、吡啶基、吡啶酮基、吡嗪基、噠嗪基、異噻唑基、異噁唑基及四唑基。c3_C8雜環可未經取代或經至多七個包括（但不限於）以下基團之基團取代·· -CVCs烷基、-CKCrCs烷基）、-芳基、-c(0)R，、"Cs-Cs" means an aromatic or non-aromatic c3_C8 carbocyclic ring in which one to four of the ring carbon atoms are independently replaced by a hetero atom selected from the group consisting of ruthenium, S and ruthenium. Examples include, but are not limited to, benzofuranyl, benzoquinone, fluorenyl, benzoxazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thienyl, furyl, thiazolyl, imidazolyl , pyrazolyl, triazolyl, quinolyl, pyrimidinyl, pyridyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl. c3_C8 heterocyclic ring may not be Substituting or replacing up to seven groups including, but not limited to, the following groups: -CVCs alkyl, -CKCrCs alkyl), -aryl, -c(0)R,

-0C(0)R’、_C(0)〇R，、-C(0)NH2、-C(0)NHR，、-C(0)N(Rf)2 、-NHC(0)R’、-S(0)2R’、-S(0)R，、-OH、-函素、-N3、-NH2 、-NH(R’）、-N(R，)2及-CN ;其中各R，係獨立地選自h、-CV C8烷基及芳基。 "CrC8雜環基"係指其中雜環基之氫原子之一經一鍵置換的以上定義之C3-C8雜環基。C3-C8雜環基可未經取代或經至多六個包括（但不限於）以下基團之基團取代：-(^-(^烷基、-O-CCi-Cs烷基）、-芳基、-C(0)R，、-〇C(0)R，、-C(0)0R，、-C(0)NH2、_C(0)NHR，、_C(0)N(R，）2、_NHC(0)R·、 119007.doc -59- 200813092 -S(0)2R’、-S(0)R’、-OH、- i 素、-N3、-NH2、-NH(R，）、 -N(Rf)2及-CN ;其中各R*係獨立地選自h、-Ci-Cs烷基及芳基。 ”碳環n意謂具有3至7個碳原子之單環形式或7至12個碳原子之雙環形式之飽和或不飽和環。單環碳環具有3至6個環原子，更通常為5或6個環原子。雙環碳環具有例如以雙環[4,5]、[5,5]、[5,6]或[6,6]系統形式排列之7至12個環原子’或以雙環[5,6]或[6,6]系統排列之9或1〇個環原子。單環碳環之實例包括環丙基、環丁基、環戊基、丨_環戊·丨_烯基、1-環戊-2-烯基、1-環戊_3_烯基、環己基、卜環己_卜烯基、1-¾己-2-烯基、1-環己-3-烯基、環庚基及環辛基。 nC3-C8^環”為3_ m、或8-員飽和或不飽和非芳族碳環。代表性CyC：8碳環包括（但不限於）_環丙基、-環丁基、-環戊基、-環戊二烯基、_環己基、-環己烯基、-H 環己二烯基、-1，4_環己二烯基、_環庚基、·口-環庚二烯基、-1，3,5-環庚三稀基、_環辛基及環辛二稀基。c3_c8碳環基可未經取代或經一或多個包括(但不限於)以下基團之基團取代：-CVC8烧基、_〇_(Ci_C8燒基）、芳基、 ^ -〇C(0)R· ^ -C(0)〇R. . .C(〇)NH2，-C(〇)NHR. . -C(〇)N(R')2- NHC(〇)R，、-S(0)2R，、_s(〇)R，、·〇Η、齒素、_乂、顧2 、-NH(R·)、-N(RI)2及· CN ;其中各㈣獨立地選自h、_-0C(0)R', _C(0)〇R,, -C(0)NH2, -C(0)NHR, -C(0)N(Rf)2, -NHC(0)R', -S(0)2R', -S(0)R, -OH, -, -N3, -NH2, -NH(R'), -N(R,)2, and -CN; , are independently selected from the group consisting of h, -CV C8 alkyl and aryl. "CrC8 heterocyclic group" means a C3-C8 heterocyclic group as defined above in which one of the hydrogen atoms of the heterocyclic group is replaced by a bond. The C3-C8 heterocyclic group may be unsubstituted or substituted with up to six groups including, but not limited to, the following groups: -(^-(^alkyl, -O-CCi-Csalkyl), -aryl Base, -C(0)R,, -〇C(0)R,, -C(0)0R, , -C(0)NH2, _C(0)NHR,, _C(0)N(R,) 2, _NHC(0)R·, 119007.doc -59- 200813092 -S(0)2R', -S(0)R', -OH, -i, -N3, -NH2, -NH(R, And -N(Rf)2 and -CN; wherein each R* is independently selected from the group consisting of h, -Ci-Cs alkyl and aryl. "Carbocycle n means a monocyclic form having 3 to 7 carbon atoms. Or a saturated or unsaturated ring of a bicyclic form of 7 to 12 carbon atoms. The monocyclic carbocyclic ring has 3 to 6 ring atoms, more usually 5 or 6 ring atoms. The bicyclic carbon ring has, for example, a bicyclic ring [4, 5 ], [5, 5], [5, 6] or [6, 6] 7 to 12 ring atoms arranged in system form ' or 9 or 1 in a double ring [5, 6] or [6, 6] system环 a ring atom. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, oxime-cyclopentanyl-alkenyl, 1-cyclopent-2-enyl, 1-cyclopenta-3 -alkenyl, cyclohexyl, cyclohexyl-b-alkenyl, 1-3⁄4-hex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl and cyclooctyl The nC3-C8^ ring" is a 3-m, or 8-membered, saturated or unsaturated, non-aromatic carbocyclic ring. Representative CyC: 8 carbocycles include, but are not limited to, _cyclopropyl, cyclohexyl, cyclopentane , -cyclopentadienyl, _cyclohexyl, -cyclohexenyl, -H cyclohexadienyl, -1,4-cyclohexadienyl, _cycloheptyl, s-cycloheptadiene a group, a -1,3,5-cycloheptatriene, a cyclooctyl group, and a cyclooctyldiyl group. The c3_c8 carbocyclic group may be unsubstituted or include one or more of the following groups including, but not limited to, the following groups Group substitution: -CVC8 alkyl, _〇_(Ci_C8 alkyl), aryl, ^ -〇C(0)R· ^ -C(0)〇R. . .C(〇)NH2,-C( 〇)NHR. . -C(〇)N(R')2- NHC(〇)R,, -S(0)2R,,_s(〇)R,,·〇Η, 齿素,_乂,顾2, -NH(R·), -N(RI)2, and CN; wherein each (4) is independently selected from h, _

Ci_C8烧基及芳基。 "C^-C8碳環基丨’係指其中碳環美 r及衣暴之虱原子之一經一鍵置換的以上定義之(：3-(：8碳環基。、 119007.doc -60- 200813092 ’’連接子’’係指包含將抗體共價連接至藥物部分之共價鍵或原子鏈之化學部分。在各種實施例中，連接子包括二價基，諸如烷二基、芳二基、雜芳二基；諸如以下之部分：-(CR2)nO(CR2)n-、烷氧基之重複單元（例如聚伸乙氧基、PEG、聚亞甲氧基）及烷胺基（例如聚伸乙胺基、 JeffamineTM);及二酸酯及醯胺，包括琥珀酸酯、琥珀醯胺、二羥乙酸酯、丙二酸酯及己醯胺。術語’’對掌性”係指具有鏡像搭配物之非重疊特性之分子，而術語π非對掌性’’係指關於其鏡像搭配物重疊之分子。術語”立體異構體”係指具有相同化學組成，但原子或基團之空間排列不同之化合物。 ”非對映異構體”係指具有兩個或兩個以上對掌性中心且其分子彼此不為鏡像之立體異構體。非對映異構體具有不同物理特性，例如熔點、沸點、光譜特性及反應性。非對映異構體之混合物可於高解析分析程序（諸如電泳及層析）下分離。 ’’對映異構體”係指為彼此為不可重疊鏡像之化合物的兩種立體異構體。本文所用之立體化學定義及慣例一般係依據S. P. Parker 編之 McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company，New York ;及 Eliel，E.及 Wilen, S·， Stereochemistry of Organic Compounds (1994) John Wiley & Sons，Inc·，New York。許多有機化合物係以 119007.doc -61- 200813092 光活性形式存在，亦即其具有旋轉平面偏振光平面之能力。在描述光活性化合物中，字首D及l或R及s係用以表示分子關於其對掌性中心之絕對構型。字首4及丨或（+)及㈠係用以表示以化合物旋轉平面偏振光之標記，其中㈠或1 意謂化合物為左旋的。以（+)或d為字首之化合物為右旋的。對於指定化學結構而言，該等立體異構體除為彼此之鏡像以外為相同的。特定立體異構體亦可稱作對映異構體且此等異構體之混合物通常稱作對映異構體混合物。對映異構體之50:50混合物稱作外消旋混合物或外消旋體，其可在化學反應或過程中不存在立體選擇性或立體特異性時產生。術語’’外消旋混合物，，及，，外消旋體”係指無光活性之兩種對映異構體物種之等莫耳混合物。脫離基"係指可經另一種官能基取代之官能基。某些脫離基於此項技術中已熟知且實例包括（但不限於）豳化物（例如氯化物、>臭化物、峨化物）、甲石黃醯基（meSyl)、對甲苯磺醯基（tosyl)、三氟甲基磺醯基（三氟甲磺酸酯基（triflate)) 及三氟甲基磺酸酯基。 B·縮寫連接子組份： MC = 6-馬來醯亞胺己醯基 =纈胺酸·瓜胺酸（可被蛋白酶裂解之連接子中的例示性二肽）瓜胺酸=2-胺基-5-脲基戊酸 ?八8 =對胺基苄氧羰基（"自毁性（5^1£*1111111〇1&1^6)’’連接 119007.doc -62- 200813092 子組份之實例）Ci_C8 alkyl and aryl. "C^-C8 Carbocyclic 丨' refers to the above definition in which one of the ruthenium atoms of the carbocyclic ring and the clothing storm is replaced by a bond (: 3-(:8 carbocyclic group., 119007.doc -60) - 200813092 ''Linker'' refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, the linker comprises a divalent group, such as an alkanediyl group, a aryl group a heteroaryl group; such as the following: -(CR2)nO(CR2)n-, a repeating unit of an alkoxy group (eg, a poly(ethyleneoxy) group, a PEG group, a polymethylene group), and an alkylamino group ( For example, polyethylamine, JeffamineTM; and diesters and decylamines, including succinate, succinimide, glycolate, malonate, and hexamethyleneamine. Refers to a molecule that has a non-overlapping property of a mirror image, and the term π non-pivoted '' refers to a molecule that overlaps its mirror image. The term "stereoisomer" refers to an atom or group that has the same chemical composition. The space of the group is arranged in different compounds. "Diastereomer" means having two or more pairs of palms. And the molecules are not mirror images of each other. The diastereomers have different physical properties such as melting point, boiling point, spectral properties and reactivity. Mixtures of diastereomers can be used in high resolution analysis procedures ( Separation, such as electrophoresis and chromatography. ''Enantiomers' are two stereoisomers of compounds that are non-superimposable mirror images of each other. The stereochemical definitions and conventions used herein are generally based on SP Parker. McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many The organic compound is present in a photoactive form of 119007.doc -61 - 200813092, ie its ability to rotate a plane of polarized light. In describing photoactive compounds, the prefixes D and l or R and s are used to indicate molecular Its absolute configuration to the palm center. The prefixes 4 and 丨 or (+) and (1) are used to indicate the rotation of a plane-polarized light with a compound, where (a) or 1 means a compound The compound of the formula (+) or d is dextrorotatory. For a given chemical structure, the stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as Enantiomers and mixtures of such isomers are often referred to as enantiomeric mixtures. The 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which is available in chemistry. Produced when there is no stereoselectivity or stereospecificity in the reaction or process. The term ''racemic mixture, and, racemic' refers to an equimolar mixture of two enantiomeric species that are optically inactive. "Debonding group" means that it can be substituted with another functional group. Functional groups. Some detachments are well known in the art and include, but are not limited to, tellurides (e.g., chlorides, >smell, telluride), mesylate (meSyl), p-toluenesulfonyl ( Tosyl), trifluoromethylsulfonyl (triflate) and trifluoromethanesulfonate. B. Abbreviation linker component: MC = 6-maleimide Sulfhydryl=proline citrate (an exemplary dipeptide in a linker that can be cleaved by protease) citrulline = 2-amino-5-ureidovaleric acid VIII 8 = p-aminobenzyloxycarbonyl ("self-destructive (5^1£*1111111〇1&1^6)'' connection 119007.doc -62- 200813092 sub-component example)

Me-Val-Cit=N_甲基-纈胺酸-瓜胺酸（其中連接子肽鍵經改質以防止其被組織蛋白酶B裂解） MC(PEG)6-OH=馬來醯亞胺己醯基-聚乙二醇（可連接至抗體半胱胺酸）細胞毒性藥物： MMAE=單甲基 auristatin E(MW 718) MMAF=於藥物之C端具有***酸之auristatin E (MMAE)變異體（MW 731.5)Me-Val-Cit=N_methyl-proline-citrulline (where the linker peptide bond is modified to prevent it from being cleaved by cathepsin B) MC(PEG)6-OH=maleimide Mercapto-polyethylene glycol (can be linked to the antibody cysteine) Cytotoxic drugs: MMAE = monomethyl auristatin E (MW 718) MMAF = auristatin E (MMAE) variant with phenylalanine at the C-terminus of the drug (MW 731.5)

MMAF-DMAEA=於與C端***酸之醯胺鍵中具有 DMAEA(二甲胺基乙胺）之MMAF(MW 801.5) MMAF-TEG=具有經酯化為***酸之四乙二醇之 MMAF MMAF-NtBu=作為醯胺連接至MMAF之C端之N-第三丁基其他縮寫如下·· AE為auristatin E ; Boc為TV-(第三丁氧魏基）；cit為瓜胺酸；dap為多拉苯丙普寧（dolaproine) ; DCC 為1，3-二環己基碳化二醯亞胺；DCM為二氯曱烷；DEA為二乙胺；DEAD為偶氮二甲酸二乙酯；DEPC為氰酸磷醯基二乙酯；DIAD為偶氮二甲酸二異丙酯；DIEA為二異丙基乙胺；dil為多拉異白胺酸；DMA為二甲基乙醯胺； DMAP為4-二甲胺基吡啶；DME為乙二醇二甲醚（或1，2-二甲氧基乙烷）；DMF為愚7V-二甲基甲醯胺；DMSO為二甲亞颯；doe為多拉苯寧（dolaphenine) ; dov為二甲基纖胺 119007.doc -63- 200813092 酸；DTNB為5,5·-二硫雙（2-硝基苯甲酸）；DTPA為二伸乙三胺五乙酸；DTT為二硫蘇糠醇；EDCI為1-(3-二曱胺基丙基）-3-乙基碳化二醯亞胺鹽酸鹽；EEDQ為2-乙氧基-1-乙氧羰基-1，2-二氫喹啉；ES-MS為電喷質譜分析；EtOAc為乙酸乙酯；Fmoc為7V_(9-苐基甲氧羰基）；gly為甘胺酸； HATU為0-(7-氮雜苯幷***-1-基）_N，N，N丨，N，_四甲錁六氟磷酸鹽；HOBt為1-羥基苯幷***；HPLC為高壓液相層析； ile為異白胺酸；lys為離胺酸；MeCN(CH3CN)為乙腈； MeOH為甲醇；Mti*為4-大茴香基二苯基甲基（或4-甲氧基三苯曱基）；nor為去甲麻黃鹼；PBS為磷酸鹽緩衝生理食鹽水（pH 7.4) ; PEG為聚乙二醇；Ph為苯基；Pnp 為對硝基苯基；MC為6-馬來醯亞胺己醯基；phe為L-苯丙胺酸；PyBrop為溴基參吡咯啶基六氟磷酸鱗；SEC為尺寸排阻層析法；Su為琥珀醯亞胺；TFA為三氟乙酸；TLC為薄層層析法；UV為紫外線；且val為纈胺酸。 III·組合物及其製造方法提供結合至TAT226之抗體。提供包含抗TAT226抗體之免疫接合物。本發明之抗體及免疫接合物適用於例如診斷或治療與TAT226之變化表現（例如增加之表現）相關聯之病症。在某些實施例中，本發明之抗體或免疫接合物適用於診斷或治療細胞增殖性病症，諸如癌症。 A.抗TAT226抗體 TAT226("腫瘤相關抗原靶第226號，，）為經加工且於特定細胞類型（包括腫瘤細胞）表面上表現之蛋白。詳言之，先 119007.doc -64· 200813092MMAF-DMAEA=MMAF (MW 801.5) with DMAEA (dimethylaminoethylamine) in the guanamine bond of C-terminal phenylalanine MMAF-TEG=MMAF MMAF with tetraethylene glycol esterified to phenylalanine -NtBu=N-tert-butyl as the indoleamine attached to the C-terminus of MMAF Other abbreviations are as follows: AE is auristatin E; Boc is TV-(third butoxy-wei); cit is citrulline; dap is Dolaproine; DCC is 1,3-dicyclohexylcarbodiimide; DCM is dichlorodecane; DEA is diethylamine; DEAD is diethyl azodicarboxylate; DEPC is cyanide Acid diphosphonium diethyl ester; DIAD is diisopropyl azodicarboxylate; DIEA is diisopropylethylamine; dil is doramamic acid; DMA is dimethylacetamide; DMAP is 4- Dimethylaminopyridine; DME is ethylene glycol dimethyl ether (or 1,2-dimethoxyethane); DMF is fool 7V-dimethylformamide; DMSO is dimethyl hydrazine; doe is more Dolaphenine; dov is dimethyl fibrin 119007.doc -63- 200813092 acid; DTNB is 5,5·-dithiobis(2-nitrobenzoic acid); DTPA is diamethylenediamine Acetic acid; DTT is dithiothreitol; EDCI is 1-(3-dioxin) Propyl)-3-ethylcarbodiimide hydrochloride; EEDQ is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; ES-MS is analyzed by electrospray mass spectrometry; EtOAc is ethyl acetate; Fmoc is 7V-(9-fluorenylmethoxycarbonyl); gly is glycine; HATU is 0-(7-azabenzotriazol-1-yl)_N,N,N丨, N, _ tetramethyl hexafluorophosphate; HOBt is 1-hydroxybenzotriazole; HPLC is high pressure liquid chromatography; ile is isoleucine; lys is lysine; MeCN (CH3CN) is acetonitrile; MeOH Is methanol; Mti* is 4-anisyldiphenylmethyl (or 4-methoxytriphenylhydrazine); nor is norephedrine; PBS is phosphate buffered physiological saline (pH 7.4); PEG is polyethylene glycol; Ph is phenyl; Pnp is p-nitrophenyl; MC is 6-maleimine hexanyl; phe is L-phenylalanine; PyBrop is bromopyrrolidyl hexafluoro Phosphate scale; SEC is size exclusion chromatography; Su is amber quinone imine; TFA is trifluoroacetic acid; TLC is thin layer chromatography; UV is ultraviolet; and val is valine. III. Composition and method of producing the same An antibody that binds to TAT226 is provided. An immunoconjugate comprising an anti-TAT226 antibody is provided. The antibodies and immunoconjugates of the invention are useful, for example, in the diagnosis or treatment of a condition associated with a manifested change in TAT226 (e.g., increased performance). In certain embodiments, an antibody or immunoconjugate of the invention is useful for the diagnosis or treatment of a cell proliferative disorder, such as cancer. A. Anti-TAT226 Antibody TAT226 ("Tumor-associated antigen target No. 226,) is a protein that is processed and expressed on the surface of specific cell types, including tumor cells. In detail, first 119007.doc -64· 200813092

前已報導人類TAT226於特定類型之腫瘤（包括卵巢、子宮、子宮内膜、腎、肺、胰腺、腎上腺及肝細胞腫瘤）中過度表現。參見例如美國專利申請公開案US 2003/0148408 Al、US 2004/0229277 A1 及 US 2003/0100712 A1(將 TAT226 稱作 ’’PR09917”）；及美國專利第 6,710，170 B2 號 (SEQ ID N0315)。與TAT226相關之其他資料庫條目及揭示内容如下：NCBI寄存編號AY358628 —1(將人類TAT226稱作 ’’PSCA Hlog’，）； NCBI 寄存編號 AAQ88991.1 及 ”gi，，第 37182378 號（將人類 TAT226 稱作 ’，PSCA Hlog’，）； RIKEN cDNA 2700050C12 ； US 2003/0096961 A1(SEQ IDHuman TAT226 has previously been reported to be overexpressed in certain types of tumors, including ovaries, uterus, endometrium, kidney, lung, pancreas, adrenal gland, and hepatocyte tumors. See, for example, U.S. Patent Application Publication No. US 2003/0148408 Al, US 2004/0229277 A1 and US 2003/0100712 A1 (TAT226 is referred to as ''PR09917'); and U.S. Patent No. 6,710,170 B2 (SEQ ID N0315). Other database entries and disclosures related to TAT226 are as follows: NCBI registration number AY358628-1 (referred to as human 'TAT226' 'PSCA Hlog',); NCBI registration number AAQ88991.1 and "gi,, No. 37182378 (will be human TAT226 is called ', PSCA Hlog',); RIKEN cDNA 2700050C12; US 2003/0096961 A1 (SEQ ID

NO:16) ； US 2003/0129192 A1(SEQ ID NO:215) ； US 2003/0206918 Al(實例 5 ； SEQ ID NO:215) ； USNO: 16); US 2003/0129192 A1 (SEQ ID NO: 215); US 2003/0206918 Al (Example 5; SEQ ID NO: 215); US

2003/0232056 A1(SEQ ID NO:215) ； US 2004/0044179 A1(SEQ ID NO:16) ； US 2004/0044180 A1(SEQ ID NO:16); US 2005/0238649 Al(將人類 TAT226 稱作，’PSCA HlogM) ； WO 2003/025148(SEQ ID NO:292) ； WO 2003/105758(SEQ ID NO:14);及 EP 1347046(SEQ ID NO:2640)。全長TAT226於細胞内經受加工以產生在細胞表面上表現之成熟形式蛋白。舉例而言，如SEQ ID NO:75所示之全長人類TAT226含有來自胺基酸1-20或1-22之預測信號肽序列，其經預測將自蛋白裂解。預測胺基酸116-141之C端將自蛋白裂解且GPI部分於胺基酸115處連接至蛋白。預測來自SEQ ID NO:75之胺基酸21-115或23-115之成熟形式人類 119007.doc -65- 200813092 TAT220將經由GPI部分固著至細胞表面。猴及齧齒動物 TAT226(參見例如SEQ ID ΝΟ:76·78)與人類TAT226高度相似且因此咸信其將於等效胺基酸位置被裂解及修飾。參見圖1。所得來自胺基酸21-115或23-115之成熟形式之人類、猴及齧齒動物ΤΑΤ226(如圖1所示）係100%相同的。人類ΤΑΤ226之其他特徵包括已經經驗證實之胺基酸45處之預測]^糖基化位點及來自8£()1〇]^〇:75之胺基酸94-107 之預測Ly6/u-PAR結構域。人類ΤΑΤ226與***幹細胞抗原（PSCA)(—種經由GPI鍵聯表現於細胞表面上之***癌特異性腫瘤抗原）具有約32%之胺基酸同源性。參見Reiter 等人（1998) Pr%· 心W· 95:1735-1740。 PSCA在超過80%之***癌中過度表現。（同上）。如同 TAT226，其含有預測Ly6/u-PAR結構域，其涉及在諸如信號轉導及細胞黏著之細胞功能中。（同上）。在一態樣中，本發明提供結合至TAT226之抗體。在一些實施例中，提供結合至成熟形式TAT226之抗體。在一個此實施例中，成熟形式之TAT226具有來自SEQ ID NO:75之胺基酸21-115或23-115之胺基酸序列。在一些實施例中， TAT226之抗體結合至於細胞表面上表現之成熟形式 TAT226。在一些實施例中，結合至於細胞表面上表現之成熟形式TAT226的抗體抑制細胞生長。在一些實施例中，抗 TAT226抗體結合至於細胞表面上表現之成熟形式TAT226 且抑制細胞增殖。在某些實施例中，抗TAT226抗體結合至於細胞表面上表現之成熟形式TAT226且誘導細胞死亡。在 119007.doc -66- 200813092 一些實施例中，抗TAT226抗體結合至於癌細胞表面上表現之成熟形式TAT226。在一些實施例中，抗TAT226抗體結合至相對於相同組織來源之正常細胞於癌細胞表面上過度表現之成熟形式TAT226。在一態樣中，抗TAT226抗體為單株抗體。在一態樣中，抗TAT226抗體為抗體片段，例如Fab、Fab’-SH、Fv、scFv 或（Fab，）2片段。在一態樣中，抗TAT226抗體為嵌合、人化或人類抗體。在一態樣中’純化本文所述之任何抗TAT226 抗體。如實例B中所述，本文提供衍生自噬菌體庫之例示性單株抗體。用於篩檢庫之抗原為具有SEQ ID NO:75之胺基酸 1-115序列之多肽，對應於缺少為假定GPI連接位點C端之胺基酸的TAT226形式。將由庫篩檢產生之抗體命名為 YW0.32及YW0.49。YW0.49為親和力成熟的以產生 YWO.49.B7、YWO.49.C9、YWO.49.H2 及 YWO.49.H6。 YW0.32 、 YW0.49 、 YWO.49.B7 、 YWO.49.C9 、 YWO.49.H2及YWO.49.H6之重鏈及輕鏈可變結構域序列之對準分別展示於圖11及12中。在一態樣中，提供與 YW0.32、YW0.49、YWO.49.B7、 YWO.49.C9、YWO.49.H2 或 YWO.49.H6競爭結合至TAT226 之單株抗體。亦提供結合至與YWO.32、YW0.49、 YWO.49.B7、YWO.49.C9、YWO.49.H2 或 YWO.49.H6相同之抗原決定基之單株抗體。在本發明之一態樣中，提供編碼抗TAT226抗體之聚核苷 119007.doc -67- 200813092 酸。在某些實施例中，提供包含編碼抗TAT226抗體之聚核苷酸之載體。在某些實施例中，提供包含此等載體之宿主細胞。在本發明之另一態樣中，提供包含抗TAT226抗體或編碼抗TAT226抗體之聚核苷酸之組合物。在某些實施例中，本發明之組合物為用於治療諸如彼等於本文中列舉者之細胞增殖性病症之醫藥調配物。例示性抗TAT226抗體之詳述如下： 1、抗TAT226抗體之特定實施例在一態樣中，本發明提供包含選自以下之至少一個、兩個、三個、四個、五個或六個HVR之抗體：（a)包含SEQ ID NChl或SEQ ID NO:4之胺基酸序列之HVR-H1 ; (b)包含 SEQ ID NO:2 或 SEQ ID NO:5之胺基酸序列之 HVR-H2 ; (c) 包含選自8丑(5 10>^0:3及6-11之胺基酸序列之11¥11-113;((1) 包含SEQ ID NO:12之胺基酸序列之HVR-L1 ; (e)包含SEQ IDNO:13之胺基酸序列之HVR_L2;及（f)包含選自SEQID >^0:14-19之胺基酸序列之11¥11氺3。在一態樣中，本發明提供包含至少一個、至少兩個或所有三個選自以下之VH HVR序列之抗TAT226抗體：（a)包含 SEQ ID ΝΟ:1 或 SEQ ID NO:4之胺基酸序列之 HVR-H1 ; (b) 包含SEQ ID NO:2或SEQ ID NO:5之胺基酸序列之HVR-H2 ;及（c)包含選自SEQ ID N03及6·11之胺基酸序列之 HVR-H3。在一態樣中，本發明提供包含具有SEQ ID ΝΟ:1 或SEQ ID NO:4之胺基酸序列之HVR_H1的抗TAT226抗體。在一態樣中，本發明提供包含具有SEQ ID NO:2或 119007.doc -68 - 200813092 SEQ ID NO:5之胺基酸序列之HVR-H2的抗TAT226抗體。在一態樣中，本發明提供包含具有選自SEQ ID NO:3及6-11之胺基酸序列之HVR-H3的抗TAT226抗體。在一態樣中，本發明提供包含具有SEQ ID NO:9或10之胺基酸序列之HVR-H3的抗TAT226抗體。在一態樣中，本發明提供包含具有選自SEQ ID NO:3及 6-11之胺基酸序列之HVR-H3及具有選自SEQ ID NO: 1及 SEQ ID NO:4之胺基酸序列之HVR-H1的抗TAT226抗體。在一態樣中，本發明提供包含具有選自SEQ ID ΝΟ:6-11之胺基酸序列之HVR-H3及具有SEQ ID NO:4之HVR-H1的抗 TAT226抗體。在一態樣中，本發明提供包含具有SEQ ID NO:9 或 10 之 HVR-H3 及具有 SEQ ID NO:4 之 HVR-H1 的抗 TAT226抗體。在一態樣中，本發明提供包含具有SEQ ID NO:3 之 HVR-H3及具有 SEQ ID ΝΟ:1 之 HVR-H1 的抗 TAT226 抗體。在一態樣中，本發明提供包含具有選自SEQ ID NO:3及 6-11之胺基酸序列之HVR-H3及具有選自SEQ ID NO:2及 SEQ ID NO:5之胺基酸序列之HVR-H2的抗TAT226抗體。在一態樣中，本發明提供包含具有選自SEQ ID ΝΟ:6·11之胺基酸序列之HVR-H3及具有SEQ ID ΝΟ:5之HVR-H2的抗 ΤΑΤ226抗體。在一態樣中，本發明提供包含具有SEQ ID NO:9 或 10 之 HVR-H3 及具有 SEQ ID NO:5 之 HVR-H2 的抗 TAT226抗體。在一態樣中，本發明提供包含具有SEQ ID NO:3之 HVR-H3及具有 SEQ ID NO:2之 HVR-H2 的抗 TAT226 119007.doc -69- 200813092 抗體。在一態樣中，本發明提供包含具有SEQ ID ΝΟ·.1或SEQ ID NO:4之胺基酸序列之HVR-H1 ;具有SEQ ID NO:2或 SEQ ID NO:5之胺基酸序列之HVR-H2 ;及具有選自SEQ ID ΝΟ··3及6-11之胺基酸序列之HVR-H3的抗TAT226抗體。在一實施例中，HVR_H1包含SEQ ID ΝΟ:4之胺基酸序列； HVR-H2包含SEQ ID ΝΟ:5之胺基酸序列；且HVR-H3包含選自SEQ ID ΝΟ·_6-11之胺基酸序列。在一實施例中， ' HVR-H1包含SEQ ID ΝΟ:4之胺基酸序列；HVR-H2包含 SEQ ID ΝΟ:5之胺基酸序列；且HVR-H3包含SEQ ID ΝΟ:9 或10。在一實施例中，HVR-H1包含SEQ ID ΝΟ:1之胺基酸序列；HVR-H2包含SEQ ID ΝΟ:2之胺基酸序列；且HVR-Η3包含SEQIDNO:3之胺基酸序歹ij。在一態樣中，本發明提供包含至少一個、至少兩個或所有三個選自以下之VL HVR序列之抗TAT226抗體：（a)包含 SEQ ID NO:12之胺基酸序列之HVR-L1 ; (b)包含SEQ ID NO:13之胺基酸序列之HVR-H2 ;及（c)包含選自SEQ ID NO: 14-19之胺基酸序列之HVR-H3。在一態樣中，本發明提供包含具有選自SEQ ID NO:14-19之胺基酸序列之HVR-L3的抗TAT226抗體。在一態樣中，本發明提供包含具有 SEQ ID NO:17或18之胺基酸序列之HVR-L3的抗TAT226抗在一態樣中，本發明提供包含以下之抗TAT226抗體： (a)包含選自SEQ ID NO:3及6-11之胺基酸序列之11¥11-：»3及 119007.doc -70- 200813092 (13)包含選自8£()1〇]^0:14-19之胺基酸序列之11¥尺-1^3。在一態樣中，本發明提供包含以下之抗TAT226抗體：（a)包含SEQ ID NO:3之胺基酸序列之HVR-H3及（b)包含SEQ ID NO:14之胺基酸序列之HVR-L3。在一些實施例中， TAT226抗體另外包含（a)包含SEQ ID ΝΟ:1之HVR-H1及包含 SEQ ID NO:2之 HVR-H2。在一態樣中，本發明提供包含以下之抗TAT226抗體， (a)包含選自SEQ ID NCh6-ll之胺基酸序列之HVR_H3及（b) 包含選自SEQ ID NO: 14-19之胺基酸序列之HVR-L3。在一些實施例中，HVR-H3包含SEQ ID NO:9之胺基酸序列且 11¥尺-1^3包含8丑(5 1〇]^0:17之胺基酸序列。在一些實施例中，HVR-H3包含SEQ ID NO:10之胺基酸序列且HVR-L3包含SEQ ID NO: 18之胺基酸序列。在一些實施例中， TAT226抗體另外包含具有SEQ ID NO:4之HVR-H1及具有 SEQ ID NO:5之HVR-H2。在一態樣中，本發明提供包含以下之抗TAT226抗體： (a)包含SEQ ID ΝΟ:1或SEQ ID NO:4之胺基酸序列之HVR-Hl ; (b)包含SEQ ID NO:2或SEQ ID NO:5之胺基酸序列之 11乂11-112;((〇包含選自8丑(^10 1^0:3及6-11之胺基酸序列之 11乂11-113;((1)包含8丑(5 10>^0:12之胺基酸序列之11¥11-LI ; (e)包含SEQ ID NO:13之胺基酸序列之HVR-L2 ;及（f) 包含選自SEQ ID NO :14-19之胺基酸序列之HVR-L3。在一態樣中，本發明提供包含以下之抗TAT226抗體： (a)包含SEQ ID ΝΟ:1之胺基酸序列之HVR-H1 ; (b)包含 119007.doc -71 - 200813092 SEQ ID NO:2之胺基酸序列之HVR-H2 ; (c)包含SEQ ID NO:3之胺基酸序列之HVR-H3 ; (d)包含SEQ ID NO:12之胺基酸序列之HVR-L1 ; (e)包含SEQ ID NO:13之胺基酸序列之11¥11-]^2;及（〇包含8丑(5 10>^0:14之胺基酸序列之11¥11-L3 ° 在一態樣中，本發明提供包含以下之抗TAT226抗體： (a)包含SEQ ID NO:4之胺基酸序列之HVR-H1 ; (b)包含 SEQ ID NO:5之胺基酸序列之HVR-H2 ; (c)包含選自SEQ ID ΝΟ:6-11之胺基酸序列之HVR-H3 ; (d)包含SEQ ID NO:12之胺基酸序列之HVR-L1 ; (e)包含SEQ ID NO:13之胺基酸序列之HVR-L2 ;及（f)包含選自SEQ ID NO:14-19之胺基酸序列之HVR-L3。在一態樣中，本發明提供包含以下之抗TAT226抗體： (a)包含SEQ ID NO:4之胺基酸序列之HVR-H1 ; (b)包含 SEQ ID NO:5之胺基酸序列之HVR-H2 ; (c)包含SEQ ID NO:9之胺基酸序列之HVR-H3 ; (d)包含SEQ ID NO:12之胺基酸序列之HVR-L1 ; (e)包含SEQ ID NO:13之胺基酸序列之^^/11-乙2;及（]〇包含選自8£()1〇]^0:17之胺基酸序列之 HVR-L3。在一態樣中，本發明提供包含以下之抗TAT226抗體： (a)包含SEQ ID NO:4之胺基酸序列之HVR-H1 ; (b)包含 SEQ ID NO:5之胺基酸序列之HVR-H2 ; (c)包含SEQ ID NO:10之胺基酸序列之HVR-H3 ; (d)包含SEQ ID NO:12之胺基酸序列之HVR-L1 ; (e)包含SEQ ID NO:13之胺基酸序 119007.doc -72- 200813092 列之HVR-L2 ;及（f)包含SEQ ID NO:18之胺基酸序列之 HVR-L3。在一些實施例中，抗TAT226抗體為親和力成熟的以獲得所要之靶結合親和力。在一些實施例中，抗體之任一或多個胺基酸係於以下HVR位置（Kabat編號）經取代：H98、 H99、H100、H100B、L90、L92、L93、L96 及 L97。舉例而言，在一些實施例中，可以任何組合進行以下任一或多種取代：2003/0232056 A1 (SEQ ID NO: 215); US 2004/0044179 A1 (SEQ ID NO: 16); US 2004/0044180 A1 (SEQ ID NO: 16); US 2005/0238649 Al (referred to as human TAT226, 'PSCA HlogM); WO 2003/025148 (SEQ ID NO: 292); WO 2003/105758 (SEQ ID NO: 14); and EP 1347046 (SEQ ID NO: 2640). Full length TAT226 is processed in cells to produce mature form proteins that are expressed on the cell surface. For example, full length human TAT226 as set forth in SEQ ID NO: 75 contains a predicted signal peptide sequence from amino acid 1-20 or 1-22 which is predicted to cleave from the protein. It is predicted that the C-terminus of the amino acid 116-141 will be cleaved from the protein and the GPI moiety will be linked to the protein at the amino acid 115. The mature form of the amino acid 21-115 or 23-115 from SEQ ID NO: 75 is predicted to be human 119007.doc-65-200813092 TAT220 will be fixed to the cell surface via the GPI moiety. The monkey and rodent TAT226 (see, e.g., SEQ ID NO: 76.78) is highly similar to human TAT226 and is therefore believed to be cleaved and modified at the equivalent amino acid position. See Figure 1. The resulting mature human form of human, monkey and rodent 226 (shown in Figure 1) from amino acid 21-115 or 23-115 is 100% identical. Other features of human ΤΑΤ226 include the prediction of 45 amino acids that have been empirically proven] glycosylation sites and predicted Ly6/u- from 8£()1〇]^:75 amino acid 94-107 PAR domain. Human sputum 226 has about 32% amino acid homology with prostate stem cell antigen (PSCA), a prostate cancer-specific tumor antigen that appears on the cell surface via a GPI linkage. See Reiter et al. (1998) Pr%·Heart W. 95: 1735-1740. PSCA is overexpressed in more than 80% of prostate cancers. (ibid.). Like TAT226, it contains a predicted Ly6/u-PAR domain involved in cellular functions such as signal transduction and cell adhesion. (ibid.). In one aspect, the invention provides an antibody that binds to TAT226. In some embodiments, an antibody that binds to the mature form of TAT226 is provided. In one such embodiment, the mature form of TAT226 has the amino acid sequence from amino acid 21-115 or 23-115 of SEQ ID NO:75. In some embodiments, the antibody of TAT226 binds to the mature form of TAT226 expressed on the cell surface. In some embodiments, an antibody that binds to the mature form of TAT226 expressed on the surface of the cell inhibits cell growth. In some embodiments, the anti-TAT226 antibody binds to the mature form of TAT226 expressed on the cell surface and inhibits cell proliferation. In certain embodiments, the anti-TAT226 antibody binds to the mature form of TAT226 expressed on the cell surface and induces cell death. In some embodiments, 119007.doc-66-200813092, the anti-TAT226 antibody binds to the mature form of TAT226 expressed on the surface of cancer cells. In some embodiments, the anti-TAT226 antibody binds to a mature form of TAT226 that is overexpressed on the surface of cancer cells relative to normal cells of the same tissue source. In one aspect, the anti-TAT226 antibody is a monoclonal antibody. In one aspect, the anti-TAT226 antibody is an antibody fragment, such as a Fab, Fab'-SH, Fv, scFv or (Fab,) 2 fragment. In one aspect, the anti-TAT226 antibody is a chimeric, humanized or human antibody. Any of the anti-TAT226 antibodies described herein are purified in one aspect. As described in Example B, exemplary monoclonal antibodies derived from phage libraries are provided herein. The antigen used in the screening library is a polypeptide having the amino acid 1-115 sequence of SEQ ID NO: 75, corresponding to the TAT226 form lacking the amino acid which is the C-terminus of the putative GPI junction site. The antibodies produced by the library screening were named YW0.32 and YW0.49. YW0.49 is affinity matured to produce YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The alignment of the heavy and light chain variable domain sequences of YW0.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 are shown in Figure 11, respectively. And 12 in. In one aspect, a monoclonal antibody that competes with YW0.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6 for binding to TAT226 is provided. A monoclonal antibody that binds to the same epitope as YWO.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6 is also provided. In one aspect of the invention, a polynucleoside 119007.doc-67-200813092 acid encoding an anti-TAT226 antibody is provided. In certain embodiments, a vector comprising a polynucleotide encoding an anti-TAT226 antibody is provided. In certain embodiments, host cells comprising such vectors are provided. In another aspect of the invention, a composition comprising an anti-TAT226 antibody or a polynucleotide encoding an anti-TAT226 antibody is provided. In certain embodiments, the compositions of the invention are pharmaceutical formulations for the treatment of cell proliferative disorders such as those recited herein. Detailed descriptions of exemplary anti-TAT226 antibodies are as follows: 1. Specific Examples of Anti-TAT226 Antibodies In one aspect, the invention provides at least one, two, three, four, five or six selected from the group consisting of Antibody to HVR: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NChl or SEQ ID NO: 4; (b) HVR- comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: H2; (c) comprising 11 ¥11-113 selected from the group consisting of 8 ugly (5 10>^0:3 and 6-11 amino acid sequences; ((1) comprising the amino acid sequence of SEQ ID NO: 12 HVR-L1; (e) HVR_L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) 11 ¥11氺3 comprising an amino acid sequence selected from the group consisting of SEQ ID >^0: 14-19. In a variant, the invention provides an anti-TAT226 antibody comprising at least one, at least two or all three VH HVR sequences selected from: (a) an amino acid sequence comprising SEQ ID ΝΟ:1 or SEQ ID NO:4 HVR-H1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; and (c) comprising an amino acid sequence selected from the group consisting of SEQ ID N03 and 6.1 HVR-H3. In one aspect, the invention provides for comprising SEQ ID ΝΟ:1 or SEQ ID NO:4 An anti-TAT226 antibody of HVR_H1 of the acid sequence. In one aspect, the invention provides an antibody comprising HVR-H2 having the amino acid sequence of SEQ ID NO: 2 or 119007. doc-68 - 200813092 SEQ ID NO: 5. TAT226 antibody. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11. In one aspect, the invention provides An anti-TAT226 antibody of HVR-H3 of the amino acid sequence of SEQ ID NO: 9 or 10. In one aspect, the invention provides an HVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11 -H3 and an anti-TAT226 antibody having HVR-H1 selected from the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 4. In one aspect, the invention provides comprising comprising a SEQ ID: 6- HVR-H3 of the amino acid sequence of 11 and an anti-TAT226 antibody having HVR-H1 of SEQ ID NO: 4. In one aspect, the invention provides HVR-H3 comprising SEQ ID NO: 9 or 10 and having Anti-TAT226 antibody of HVR-H1 of SEQ ID NO:4. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having SEQ ID NO: 3 and HVR-H1 having SEQ ID ΝΟ:1. In one aspect, the invention provides HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11 and an amino acid selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: Sequence of anti-TAT226 antibody of HVR-H2. In one aspect, the invention provides an anti-ΤΑΤ226 antibody comprising HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6.11 and HVR-H2 having SEQ ID NO: 5. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having SEQ ID NO: 9 or 10 and HVR-H2 having SEQ ID NO: 5. In one aspect, the invention provides an anti-TAT226 119007.doc-69-200813092 antibody comprising HVR-H3 having SEQ ID NO: 3 and HVR-H2 having SEQ ID NO: 2. In one aspect, the invention provides an HVR-H1 comprising an amino acid sequence having SEQ ID ...1 or SEQ ID NO: 4; an amino acid sequence having SEQ ID NO: 2 or SEQ ID NO: HVR-H2; and an anti-TAT226 antibody having HVR-H3 selected from the amino acid sequences of SEQ ID ΝΟ··3 and 6-11. In one embodiment, HVR_H1 comprises the amino acid sequence of SEQ ID: 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises an amine selected from the group consisting of SEQ ID _ 6-11 Base acid sequence. In one embodiment, 'HVR-H1 comprises the amino acid sequence of SEQ ID ΝΟ: 4; HVR-H2 comprises the amino acid sequence of SEQ ID ΝΟ: 5; and HVR-H3 comprises SEQ ID ΝΟ: 9 or 10. In one embodiment, HVR-H1 comprises the amino acid sequence of SEQ ID ΝΟ: 1; HVR-H2 comprises the amino acid sequence of SEQ ID ΝΟ: 2; and HVR-Η3 comprises the amino acid sequence of SEQ ID NO: Ij. In one aspect, the invention provides an anti-TAT226 antibody comprising at least one, at least two or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-19. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-L3 having the amino acid sequence of SEQ ID NO: 17 or 18 in one aspect, the invention provides an anti-TAT226 antibody comprising: (a) 11¥11-:»3 and 119007.doc-70-200813092 (13) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11, comprising 13 £()1〇]^0:14 The amino acid sequence of -19 is 11 ¥ 1 -1 ^3. In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and (b) an amino acid sequence comprising SEQ ID NO: 14. HVR-L3. In some embodiments, the TAT226 antibody further comprises (a) HVR-H1 comprising SEQ ID ΝΟ:1 and HVR-H2 comprising SEQ ID NO:2. In one aspect, the invention provides an anti-TAT226 antibody comprising (a) HVR_H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NCh6-ll and (b) comprising an amine selected from the group consisting of SEQ ID NO: 14-19 HVR-L3 of the acid sequence. In some embodiments, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9 and 11 ¥ 1 - 1 ^ 3 comprises an 8 ugly (5 1 〇) ^ 0: 17 amino acid sequence. In some embodiments Wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 10 and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the TAT226 antibody additionally comprises an HVR having SEQ ID NO: -H1 and HVR-H2 having SEQ ID NO: 5. In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) an amino acid sequence comprising SEQ ID ΝΟ:1 or SEQ ID NO:4 HVR-H1; (b) 11乂11-112 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; ((〇 contains from 8 ugly (^10 1^0: 3 and 6) 11-11-113 of the amino acid sequence of -11; ((1) contains 8 ugly (5 10>^0:12 amino acid sequence of 11 ¥11-LI; (e) comprises SEQ ID NO: 13 HVR-L2 of the amino acid sequence; and (f) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19. In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID: 1; (b) HVR-H2 comprising the amino acid sequence of 119007.doc-71 - 200813092 SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) comprising the amino group of SEQ ID NO: Acid sequence 11¥11-]^2; and (〇 contains 8 ugly (5 10>^0:14 amino acid sequence of 11 ¥11-L3 ° In one aspect, the present invention provides the following resistance TAT226 antibody: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (c) comprising SEQ ID 选自: HVR-H3 of the amino acid sequence of 6-11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR- comprising the amino acid sequence of SEQ ID NO: L2; and (f) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19. In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) comprising SEQ ID NO: HVR-H1 of the amino acid sequence of 4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) ^^/11-B 2 comprising the amino acid sequence of SEQ ID NO: 13; and () 〇 comprising from £8 ()1〇]^0:17 amino acid sequence HVR-L3. In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) comprising the amino acid sequence of SEQ ID NO: HVR-H2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) comprising SEQ ID NO: Amino acid sequence of 13096.doc-72-200813092, HVR-L2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-TAT226 antibody is affinity matured to achieve the desired target binding affinity. In some embodiments, any one or more of the amino acids of the antibody are substituted at the HVR position (Kabat numbering): H98, H99, H100, H100B, L90, L92, L93, L96, and L97. For example, in some embodiments, any one or more of the following substitutions can be made in any combination:

在 HVR-H3(SEQ ID NO:6)中：V98I ; S99T ; R100L或 I ; GlOObA、S或 P -在 HVR-L3(SEQ ID NO:14)中·· Q90R、K、Η 或 N ; Y92V ; T93F、Ν、G或 A ; P96F ; Τ97Ι或 A SEQ ID NO:ll(HVR-H3)及 SEQ ID NO:19(HVR-L3)之一致序列涵蓋以上取代之所有可能組合。抗TAT226抗體可包含任何合適之框架可變結構域序列，其限制條件為抗體保持結合TAT226之能力。舉例而言，在一些實施例中，本發明之抗TAT226抗體包含人類子群III 重鏈框架一致序列。在該等抗體之一實施例中，重鏈框架一致序列包含位置71、73及/或78處之取代。在該等抗體之一實施例中，位置71為A、位置73為T及/或位置78為 A。在一實施例中，該等抗體包含huMAb4D5-8之重鏈可變結構域框架序列，例如SEQ ID NO:50、51、59、35(分別為 FR1、2、3、4)。huMAb4D5-8商業上稱作 HERCEPTIN®， Genentech，Inc.，South San Francisco，CA，USA ;亦於美國 119007.doc -73- 200813092 專利第 6,407,213 號 & 第 5,821，337 號及 Lee等人，J. Mol. Biol· (2004)，3 40(5)·· 1073-93中提及。在一個此實施例中，該等抗體另外包含人類κΐ輕鏈框架一致序列。在一個此實施例中，該等抗體包含huMAb4D5-8之輕鏈可變結構域框架序列。在一實施例中，抗TAT226抗體包含具有框架序列及高變區之重鏈可變結構域，其中框架序列包含選自彼等展示於圖5A及5B中之FR1-FR4序列；HVR H1包含SEQ ID NO:4之胺基酸序列；HVR-H2包含SEQ ID NO:5之胺基酸序列；且 HVR-H3包含選自SEQ ID ΝΟ:6-11之胺基酸序列。在該等抗體之一實施例中，HVR-H3包含SEQ ID NO:9或10之胺基酸序列。在一實施例中，抗TAT226抗體包含具有框架序列及高變區之輕鏈可變結構域，其中框架序列包含選自彼等展示於圖6A及6B中之FR1-FR4序列；HVR-L1包含SEQ ID NO:12之胺基酸序列；HVR-L2包含SEQ ID NChl3之胺基酸序列；且HVR-L3包含選自SEQ ID NO:14-19之胺基酸序列。在該等抗體之一實施例中，HVR-L3包含SEQ ID NO:17或18之胺基酸序列。在一實施例中，抗TAT226抗體包含具有框架序列及高變區之重鏈可變結構域，其中框架序列如圖7所示包含SEQ ID NO:50、51、59及 35之 FR1-FR4序列；HVR H1 包含 SEQ ID NO:4之胺基酸序列；HVR-H2包含SEQ ID NO:5之胺基酸序列；且HVR-H3包含選自SEQ ID ΝΟ:6-11之胺基酸序列。在該等抗體之一實施例中，HVR-H3包含SEQ ID NO:9 119007.doc -74- 200813092 或10之胺基酸序列。在一實施例中，抗TAT226抗體包含具有框架序列及高變區之輕鏈可變結構域，其中框架序列如圖 6A及 6B所示包含 SEQ ID NO:60、61、62及 63 之 FR1-FR4 序列；11¥11-1^1包含8£(^1〇]^0:12之胺基酸序列；11¥11丄2 包含SEQIDNO:13之胺基酸序列；且HVR-L3包含選自 SEQ ID NO:14-19之胺基酸序列。在該等抗體之一實施例中，HVR-L3包含SEQIDNO:17或18之胺基酸序列。在一實施例中，抗TAT226抗體包含具有框架序列及高變區之重鏈可變結構域，其中框架序列如圖8所示包含SEQ ID NO:50、51、53及 35之FR1-FR4序列；HVR H1 包含 SEQ ID NO:4之胺基酸序列；HVR-H2包含SEQ ID NO:5之胺基酸序列；且HVR-H3包含選自SEQ ID ΝΟ:6-11之胺基酸序列。在該等抗體之一實施例中，HVR-H3包含SEQ ID NO:9 或10之胺基酸序列。在一實施例中，抗TAT226抗體包含具有框架序列及高變區之輕鏈可變結構域，其中框架序列如圖 8 所示包含 SEQ ID NO:60、61、62 及 74 之 FR1-FR4 序列；HVR-L1包含SEQIDN0:12之胺基酸序列；HVR-L2包含8丑(^1〇]^0:13之胺基酸序列；且11¥11丄3包含選自8丑(5 ID NO: 14-19之胺基酸序列。在該等抗體之一實施例中， HVR-L3包含SEQIDNO:17或18之胺基酸序列。在一些實施例中，本發明提供包含具有與選自SEQ ID NO:20-25之胺基酸序列具有至少90%、91%、92%、93%、 94%、95%、96%、97%、98%或99%之序列一致性的胺基酸序列之重鏈可變結構域之抗TAT226抗體。在一些實施例 119007.doc -75- 200813092 中，具有至少 90%、91〇/〇、92%、93%、94%、95%、 96%、97%、98%或99%之序列一致性之胺基酸序列相對於參考序列含有取代、***或缺失，但包含彼胺基酸序列之抗體保持結合至TAT226之能力。在一些實施例中，在選自 SEQ ID NO:20-25之序列中總共1至10個胺基酸經取代、插入或缺失。在一些實施例中，取代、***或缺失發生於 HVR外之區域（亦即在FR中）。在一些實施例中，抗TAT226 抗體包含具有選自SEQ ID NO:20-25之胺基酸序列之重鏈可變結構域。在一些實施例中，本發明提供包含如以下SEQIDNO:31 所述之人化4D5抗體（huMAb4D5-8)之輕鏈可變結構域的抗 TAT226 抗體（HERCEPTIN®, Genentech，Inc·，South San Francisco，CA，USA)(亦於美國專利第6,407,213號及Lee等人，J. Mol· Biol· (2004)，340(5):1073-93 中提及）。 1 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Are AlaIn HVR-H3 (SEQ ID NO: 6): V98I; S99T; R100L or I; GlOObA, S or P - in HVR-L3 (SEQ ID NO: 14) · Q90R, K, 或 or N; Y92V The consensus sequence of T93F, Ν, G or A; P96F; Τ97Ι or A SEQ ID NO: ll (HVR-H3) and SEQ ID NO: 19 (HVR-L3) encompasses all possible combinations of the above substitutions. An anti-TAT226 antibody can comprise any suitable framework variable domain sequence, with the proviso that the antibody retains the ability to bind to TAT226. For example, in some embodiments, an anti-TAT226 antibody of the invention comprises a human subgroup III heavy chain framework consensus sequence. In one embodiment of such antibodies, the heavy chain framework consensus sequence comprises substitutions at positions 71, 73 and/or 78. In one embodiment of the antibodies, position 71 is A, position 73 is T, and/or position 78 is A. In one embodiment, the antibodies comprise a heavy chain variable domain framework sequence of huMAb4D5-8, such as SEQ ID NOs: 50, 51, 59, 35 (FR1, 2, 3, 4, respectively). huMAb4D5-8 is commercially known as HERCEPTIN®, Genentech, Inc., South San Francisco, CA, USA; also in the United States 119007.doc-73-200813092 Patent No. 6,407,213 & 5,821,337 and Lee et al, J Mol. Biol· (2004), 3 40(5)·· 1073-93 mentioned. In one such embodiment, the antibodies further comprise a human kappa light chain framework consensus sequence. In one such embodiment, the antibodies comprise a light chain variable domain framework sequence of huMAb4D5-8. In one embodiment, the anti-TAT226 antibody comprises a heavy chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises a FR1-FR4 sequence selected from the group consisting of Figures 5A and 5B; HVR H1 comprises SEQ ID NO: amino acid sequence of 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 6-11. In one embodiment of the antibodies, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9 or 10. In one embodiment, the anti-TAT226 antibody comprises a light chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises a FR1-FR4 sequence selected from the group consisting of Figures 6A and 6B; HVR-L1 comprises The amino acid sequence of SEQ ID NO: 12; HVR-L2 comprises the amino acid sequence of SEQ ID NChl3; and HVR-L3 comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19. In one embodiment of the antibodies, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17 or 18. In one embodiment, the anti-TAT226 antibody comprises a heavy chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises the FR1-FR4 sequences of SEQ ID NOs: 50, 51, 59 and 35 as shown in FIG. HVR H1 comprises the amino acid sequence of SEQ ID NO: 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 6-11. In one embodiment of the antibodies, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9 119007. doc-74-200813092 or 10. In one embodiment, the anti-TAT226 antibody comprises a light chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises FR1 of SEQ ID NOs: 60, 61, 62 and 63 as shown in Figures 6A and 6B FR4 sequence; 11¥11-1^1 comprises an amino acid sequence of 8 £(^1〇]^0:12; 11¥11丄2 comprises the amino acid sequence of SEQ ID NO: 13; and HVR-L3 comprises a selected from The amino acid sequence of SEQ ID NOS: 14 to 19. In one embodiment of the antibodies, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17 or 18. In one embodiment, the anti-TAT226 antibody comprises a framework a heavy chain variable domain of the sequence and the hypervariable region, wherein the framework sequence comprises the FR1-FR4 sequences of SEQ ID NOs: 50, 51, 53 and 35 as shown in Figure 8; HVR H1 comprises the amino group of SEQ ID NO: Acid sequence; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 6-11. In one of these antibodies, HVR- H3 comprises the amino acid sequence of SEQ ID NO: 9 or 10. In one embodiment, the anti-TAT226 antibody comprises a light chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence is shown in Figure 8. FR1-FR4 sequences of SEQ ID NOS: 60, 61, 62 and 74; HVR-L1 comprises the amino acid sequence of SEQ ID NO: 12; HVR-L2 comprises an amino acid sequence of 8 ugly (^1〇)^0:13 And 11 ¥11丄3 comprises an amino acid sequence selected from the group consisting of 8 id (5 ID NO: 14-19. In one embodiment of the antibodies, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17 or 18. In some embodiments, the invention provides for comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25, 97%, 98% or 99% of the sequence-consistent amino acid sequence heavy chain variable domain anti-TAT226 antibody. In some embodiments 119007.doc-75-200813092, having at least 90%, 91〇/ The amino acid sequence of 序列, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence identity contains a substitution, insertion or deletion relative to the reference sequence, but comprises a perami group. The antibody to the acid sequence retains the ability to bind to TAT226. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted or deleted in a sequence selected from the group consisting of SEQ ID NOs: 20-25. In some embodiments , substitution, insertion or deletion The outer region in the HVR (i.e., in the FR). In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25. In some embodiments, the invention provides an anti-TAT226 antibody (HERCEPTIN®, Genentech, Inc., South San Francisco) comprising a light chain variable domain of a humanized 4D5 antibody (huMAb4D5-8) as set forth in SEQ ID NO: 31 , CA, USA) (also referred to in U.S. Patent No. 6,407,213 and Lee et al., J. Mol Biol. (2004), 340(5):1073-93). 1 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Are Ala

Ser Gin Asp Val Asn Thr Ala Val Ala Trp Tyr Gin GinSer Gin Asp Val Asn Thr Ala Val Ala Trp Tyr Gin Gin

Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Ser AlaLys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Ser Ala

Ser Phe Leu Tvr Ser Gly Val Pro Ser Arg Phe Ser GlySer Phe Leu Tvr Ser Gly Val Pro Ser Arg Phe Ser Gly

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin His Tvr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg 108(SEQ ID NO:31) (HVR殘基為加下劃線的） 119007.doc -76- 200813092 在一些實施例中，huMAb4D5-8輕鏈可變結構域序列於位置30、66及91(分別為如上文以粗體/斜體所示之Asn、 Arg及His)之一或多處經修倚。在一實施例中，經修飾之 huMAb4D5-8序列包含位置30之Ser、位置66之Gly及/或位置91之Set*。因此，在一實施例中，本發明之抗體包含具有如以下SEQ ID NO:26所述序列之輕鏈可變結構域： 1 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Are AlaSer Arg Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin His Tvr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg 108 (SEQ ID NO: 31) (HVR residues are underlined) 119007.doc -76- 200813092 In some embodiments, the huMAb4D5-8 light chain variable domain sequence is at positions 30, 66 and 91 (respectively in bold as above / One or more of Asn, Arg, and His) shown in italics are repaired. In one embodiment, the modified huMAb4D5-8 sequence comprises a Ser at position 30, a Gly at position 66, and/or a Set* at position 91. Thus, in one embodiment, an antibody of the invention comprises a light chain variable domain having the sequence set forth in SEQ ID NO: 26: 1 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr lie Thr Cys Are Ala

Ser Gin Asp Val Ser Thr Ala Val Ala Trp Tyr Gin GinSer Gin Asp Val Ser Thr Ala Val Ala Trp Tyr Gin Gin

Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser GlySer Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg 108(SEQ ID NO:26) (HVR殘基為加下劃線的）關於huMAb4D5-8之經取代殘基係於上文以粗體/斜體表示。在一態樣中，本發明提供包含具有與選自SEQ ID NO:26-31之胺基酸序列具有至少90%、91%、92%、93%、 94%、95%、96%、97%、98%或99%序列一致性之胺基酸序列之輕鏈可變結構域的抗TAT226抗體。在一些實施例中，具有至少 90%、91%、92%、93%、94%、95%、 96%、97%、98%或99%之序列一致性之胺基酸序列相對於 119007.doc -77- 200813092 參考序列含有取代、添加或缺失，但包含彼胺基酸序列之抗體保持結合至TAT226之能力。在一些實施例中，在選自 SEQ ID NO:26-31之序列中總共1至10個胺基酸經取代、插入或缺失。在一些實施例中，取代、***或缺失發生於 HVR外之區域（亦即在FR中）。在一些實施例中，抗TAT226 抗體包含具有選自SEQ ID ΝΟ··26-31之胺基酸序列之輕鏈可變結構域。在一態樣中，本發明提供包含以下之抗ΤΑΤ226抗體（a) 包含與選自SEQ ID NO:20-25之胺基酸序列具有至少 90%、91%、92%、93%、94%、95%、96%、97%、98% 或 99%序列一致性之胺基酸序列的重鏈可變結構域；及（b)包含與選自SEQ ID NO:26-31之胺基酸序列具有至少90%、 91%、92% ' 93%、94%、95%、96%、97%、98% 或 99%序列一致性之胺基酸序列的輕鏈可變結構域。在一些實施例中，具有至少 90%、91%、92%、93%、94%、95%、 96%、97%、98°/。或99%序列一致性之胺基酸序列相對於參考序列含有取代、添加或缺失，但包含彼胺基酸序列之抗體保持結合至TAT226之能力。在一些實施例中，在參考序列中總共1至10個胺基酸經取代、***或缺失。在一些實施例中，取代、***或缺失發生於HVR外之區域（亦即在 FR中）。在一些實施例中，抗ΤΑΤ226抗體包含具有選自 SEQ ID ΝΟ:20-25之胺基酸序列之重鏈可變結構域及具有選自SEQ ID ΝΟ:26-31之胺基酸序列之輕鏈可變結構域。Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg 108 (SEQ ID NO: 26) (HVR residues are underlined) The substituted residues for huMAb4D5-8 are indicated above in bold/italic. In one aspect, the invention provides for comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31. An anti-TAT226 antibody of the light chain variable domain of the amino acid sequence of %, 98% or 99% sequence identity. In some embodiments, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity is relative to 119007. Doc-77-200813092 The reference sequence contains a substitution, addition or deletion, but the antibody comprising the amino acid sequence retains the ability to bind to TAT226. In some embodiments, a total of from 1 to 10 amino acids are substituted, inserted or deleted in a sequence selected from the group consisting of SEQ ID NOs: 26-31. In some embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (i.e., in the FR). In some embodiments, the anti-TAT226 antibody comprises a light chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID 26 26-31. In one aspect, the invention provides an anti-ΤΑΤ226 antibody (a) comprising at least 90%, 91%, 92%, 93%, 94% of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25 a heavy chain variable domain of an amino acid sequence of 95%, 96%, 97%, 98% or 99% sequence identity; and (b) comprising an amino acid selected from the group consisting of SEQ ID NO: 26-31 A light chain variable domain of an amino acid sequence having at least 90%, 91%, 92% '93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, there are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98°/. Or the amino acid sequence of 99% sequence identity contains a substitution, addition or deletion relative to the reference sequence, but the antibody comprising the amino acid sequence retains the ability to bind to TAT226. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted or deleted in the reference sequence. In some embodiments, substitutions, insertions or deletions occur in regions outside the HVR (i.e., in the FR). In some embodiments, the anti-ΤΑΤ226 antibody comprises a heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 20-25 and a light having an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 26-31 Chain variable domain.

在一些實施例中，抗ΤΑΤ226抗體包含具有SEQ ID 119007.doc -78 - 200813092 NO:20之胺基酸序列之重鏈可變區及具有SEQ ID NO:26之胺基酸序列之輕鏈可變區。在一些實施例中，抗TAT226抗體包含具有SEQ ID NO:21之胺基酸序列之重鏈可變區及具有SEQ ID NO:26之胺基酸序列之輕鏈可變區。在一些實施例中，抗TAT226抗體包含具有SEQ ID NO:22之胺基酸序列之重鏈可變區及具有SEQ ID NO:27之胺基酸序列之輕鏈可變區。在一些實施例中，抗TAT226抗體包含具有SEQ ID NO:23之胺基酸序列之重鏈可變區及具有SEQ ID NO:28 之胺基酸序列之輕鏈可變區。在一些實施例中，抗TAT226 抗體包含具有SEQ ID NO:24之胺基酸序列之重鏈可變區及具有SEQ ID NO :29之胺基酸序列之輕鏈可變區。在一些實施例中，抗TAT226抗體包含具有SEQ ID NO:25之胺基酸序列之重鏈可變區及具有SEQ ID NO:30之胺基酸序列之輕鏈可變區。在一態樣中，本發明提供包含以下之抗TAT226抗體： (a)選自彼等於圖2及4中展示之一個、二個或三個VH HVR ;及/或（b)選自彼等於圖3及4中展示之一個、二個或三個VL HVR。在一態樣中，本發明提供包含選自彼等於圖9及11中所展示之重鏈可變結構域及選自彼等於圖10及 12中展示之輕鏈可變結構域的抗TAT226抗體。 2.抗體片段本發明涵蓋抗體片段。抗體片段可由諸如酶消化之傳統方式或藉由重組技術產生。在特定情況下，使用抗體片段比全抗體具有優勢。較小尺寸之片段使得可快速清除且可 119007.doc -79- 200813092 導致實體腫瘤之改良獲取。關於特定抗體片段之論述，參見 Hudson等人（2003) Mei 9:129-134。已研發各種產生抗體片段之技術。傳統上，該等片段係經由完整抗體之蛋白水解消化作用而產生（參見例如In some embodiments, the anti-ΤΑΤ226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID 119007.doc -78 - 200813092 NO:20 and a light chain having the amino acid sequence of SEQ ID NO: Variable area. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30. In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) one, two or three VH HVRs selected from the group consisting of Figures 2 and 4; and/or (b) selected from the group consisting of One, two or three VL HVRs shown in Figures 3 and 4. In one aspect, the invention provides an anti-TAT226 antibody comprising a heavy chain variable domain selected from the group consisting of those shown in Figures 9 and 11 and a light chain variable domain selected from those shown in Figures 10 and 12 . 2. Antibody Fragments The present invention encompasses antibody fragments. Antibody fragments can be produced by conventional means such as enzymatic digestion or by recombinant techniques. In certain cases, the use of antibody fragments has an advantage over whole antibodies. Fragments of smaller size allow for rapid clearance and can result in improved access to solid tumors 119007.doc -79- 200813092. For a discussion of specific antibody fragments, see Hudson et al. (2003) Mei 9: 129-134. Various techniques for producing antibody fragments have been developed. Traditionally, these fragments have been produced by proteolytic digestion of intact antibodies (see for example

Morimoio 箅尺，journal 〇/ Bi〇chemical and Bi〇physical M— 24:107_117 (1992);及 Brennan 等人， 229:81 (1985))。然而，該等片段現可直接藉由重組宿主細胞而產生。Fab、Fv及ScFv抗體片段均可於大腸桿菌（Ε· coli)中表現及自其分;必，從而使得可易於產生大量該等片段。抗體片段可自上述抗體噬菌體庫分離。或者，Fab，_ SH片段可自大腸桿菌直接回收且化學偶合以形成F(ab，）2片段（Carter等人，Bio/Technology 10:163-167 (1992))。根據另一途徑，F(ab，)2片段可直接自重組宿主細胞培養物分離。包含殘餘受體（salvage receptor)結合抗原決定基殘基之具有增加的活體内半衰期之Fab及F(ab，）2片段係描述於美國專利第5,869,046號中。用於產生抗體片段之其他技術將為熟習實踐者顯而易見。在某些實施例中，抗體為單鏈 Fv 片段（scFv)。參見 WO 93/16185 ;美國專利第 5,571，894 號及第5,587,458號。Fv及scFv為具有無恆定區之完整組合位點之僅有物種；因此在活體内使用期間其適用於降低之非特異性結合。scFv融合蛋白可經建構以於〜卜之胺基或羧基端產生效應蛋白之融合。參見Borrebaeck編之 Antibody Engineerings；同上文）。例如，如美國專利第 5，641，870號中所述’抗體片段亦可為π線性抗體’’。此等線 119007.doc •80- 200813092 性抗體可為單特異性的或雙特異性的。 i人化抗艎本發明涵蓋人化抗體。此項技術中已知各種使非人類抗體人化之方法。舉例而言，人化抗體可具有自非人類來源引入其之一或多個胺基酸殘基。該等非人類胺基酸殘基通常稱作”引入”殘基，其通常係自"引入”可變結構域獲得。人化作用基本上可根據Winter及其合作者（Jones等人（1986) Nature 321:522_525 ; Riechmann 等人（1988) Nature 332:323-327 ; Verhoeyen 等人（1988)心239··1534· 1536)之方法，藉由以高變區序列取代對應之人類抗體序列來進行。因此，此等”人化，，抗體為嵌合抗體（美國專利第 4,816,5 67號），其中大體上欠完整之人類可變結構域已由來自非人類物種之對應序列取代。實際上，人化抗體通常為其中一些高變區殘基及可能之一些FR殘基係由來自齧齒動物抗體之類似位點之殘基取代的人類抗體。選擇待用於製造人化抗體之人類可變結構域（輕及重）對於降低抗原性而言可為重要的。根據所謂”最佳擬合”法，針對已知人類可變結構域序列之全庫篩檢齧齒動物抗體之可變結構域序列。接著接受最接近齧齒動物序列之人類序列作為人化抗體之人類框架（Sims等人（1993) J. /mm⑽ 151:2296; Chothia等人（1987) J· Mo/· 5h/· 196:901)。另一種方法使用衍生自輕鏈或重鏈之特定子群之所有人類抗體一致序列的特定框架。相同框架可用於若干種不同之人化抗體（Carter 等人（1992) Proc· 7V^/· 119007.doc -81- 200813092 89:4285 ; Presta 等人（1993) / i5i:2623)。一般進-步需要經人化抗體具有保留之高抗原親和力及其他有利之生物特性。為達成此目的，根據_種方法，藉由使用親本及人化序列之三維模型分析親本序列及各種概念上之人化產物之方法來製備人化抗體。三維免疫球蛋白模型通常為可用的且為熟習此項技術者所熟悉。可使用說明且呈現所選候選免疫球蛋白序列之可能三維構形結構的電腦程式。檢驗該等呈現使得可分析殘基在候選免疫球蛋白序列功能中可能之作用，亦即分析影響候選免疫球蛋白結合其抗原之能力的殘基。以此方式，可自受體及引入序列選擇FR殘基且將其組合，以使得達成所需抗體特徵，諸如對乾抗原增加之親和力。一般而言，高變區殘基直接且最大程度上涉及在影響抗原結合中。 4.人類抗艘Morimoio ,, journal 〇/ Bi〇chemical and Bi〇physical M—24:107_117 (1992); and Brennan et al., 229:81 (1985)). However, such fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed and differentiated in E. coli; so that a large number of such fragments can be easily produced. Antibody fragments can be isolated from the above-described antibody phage library. Alternatively, the Fab, _SH fragment can be directly recovered from E. coli and chemically coupled to form a F(ab,)2 fragment (Carter et al, Bio/Technology 10: 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 having increased in vivo half-life comprising a residue of a salvage receptor in combination with an epitope are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and scFv are the only species with a complete combinatorial site without a constant region; therefore, they are suitable for reduced non-specific binding during in vivo use. The scFv fusion protein can be constructed to produce a fusion of effector proteins at the amino or carboxy terminus. See Antibody Engineerings by Borrebaeck; same as above). For example, the antibody fragment can also be a π linear antibody '' as described in U.S. Patent No. 5,641,870. These lines 119007.doc •80- 200813092 Sex antibodies can be monospecific or bispecific. i Humanized Anti-Immune The present invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a non-human source. Such non-human amino acid residues are often referred to as "introduced" residues, which are typically obtained from "introduced" variable domains. Humanization can basically be based on Winter and its collaborators (Jones et al. (1986). Nature 321:522_525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Heart 239·1534·1536) by replacing the corresponding human antibody sequence with a hypervariable region sequence Thus, such "humanization", the antibody is a chimeric antibody (U.S. Patent No. 4,816,5,67), wherein the substantially incomplete human variable domain has been replaced by a corresponding sequence from a non-human species. In fact, humanized antibodies are typically human antibodies in which some of the hypervariable region residues and possibly some of the FR residues are replaced by residues from analogous sites in rodent antibodies. Selection of human variable domains (light and heavy) to be used in the manufacture of humanized antibodies can be important to reduce antigenicity. The variable domain sequence of the rodent antibody is screened against the entire library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to the rodent sequence is then accepted as the human framework for humanized antibodies (Sims et al. (1993) J. /mm(10) 151:2296; Chothia et al. (1987) J. Mo/. 5h/. 196:901) . Another approach uses a specific framework of all human antibody consensus sequences derived from a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. 7V^/. 119007.doc-81-200813092 89:4285; Presta et al. (1993) / i5i: 2623). In general, the humanized antibody requires a high antigen affinity and other advantageous biological properties. To achieve this, humanized antibodies are prepared by a method using a three-dimensional model of a parental and humanized sequence to analyze a parental sequence and various humanized products according to the method. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. A computer program that describes and presents a possible three-dimensional configuration of the selected candidate immunoglobulin sequence can be used. Examination of such presentations allows analysis of the possible role of residues in the function of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected from the receptor and introduced sequences and combined to achieve desired antibody characteristics, such as increased affinity for dry antigens. In general, hypervariable region residues are directly and to the greatest extent involved in affecting antigen binding. 4. Human resistance to the ship

本發明之人類抗TAT226抗體可藉由組合選自人類來源之嗤菌體呈現庫之Fv純系可變結構域序列與上述已知人類恆定結構域序列來建構。或者，本發明之人類單株抗TAT226 抗體可由融合瘤法來製造。用於產生人類單株抗體之人類骨髓瘤及小鼠人類雜合骨髓瘤細胞株係由例如Kozbor X 似/·，133: 3001 (1984) ; Brodeui*等人，Μ⑽％/〇如/ Antibody Production Techniques and Applications，% 51-63 頁（Marcel Dekker，Inc.，New York，1987)及 Boerner 等人， J· 147: 86 (1991)描述。目前可能產生能夠在免疫時在不存在内源性免疫球蛋白 119007.doc -82 - 200813092 產生之情況下產生人類抗體全譜系之轉殖基因動物（例如小鼠）。舉例而言，已描述嵌合及生殖系突變小鼠體内之抗體重鏈接合區（JH)基因之同型接合缺失致使對内源抗體產生之完全抑制。此等生殖系突變小鼠體内之人類生殖系免疫球蛋白基因陣列之轉移將致使在抗原挑釁時產生人類抗體。參見例如Jakobovits等人，Pn w".上以· Sc/ t/以，90: 2551 (1993) ; Jakobovits等人，心，請，362: 255 (1993) , Bruggermann 專人，/所讲⑽ο/·，7: 33 (1993) 〇基因改組亦可用以自例如齧齒動物之非人類抗體衍生人類抗體’其中人類抗體具有與起始非人類抗體類似之親和力及特異性。根據此方法（亦稱作"抗原決定基影響"），以人類V結構域基因譜系置換如本文所述之藉由噬菌體呈現技術獲得之非人類抗體片段之重鏈或輕鏈可變區，產生非人類鏈/人類鏈scFv或Fab傲合體之群體。以抗原選擇致使非人類鏈/人類鏈嵌合scFv或Fab分離，其中人類鏈回收在移除初級噬菌體呈現純系中之對應非人類鏈時損壞之抗原結合位點，亦即抗原決定基支配（影響）人類鏈搭配物之選擇。當重複此方法以置換剩餘之非人類鏈時，獲得人類抗體（參見於1993年4月1日公開之PCT W0 93/06213)。與藉由CDR移植之傳統非人類抗體人化不同，此技術提供不具有非人類來源之FR或CDR殘基之完全人類抗體。 5.雙特異性抗艘雙特異性抗體為對至少兩種不同抗原具有結合特異性之 119007.doc -83- 200813092 單株抗體。在某些實施例中，雙特異性抗體為人類或人化抗體。在某些實施例中，結合特異性中之一者係對於 TAT226且另一者係對於任何其他抗原。在某些實施例中，雙特異性抗體可結合至TAT226之兩個不同抗原決定基。雙特異性抗體亦可用以使細胞毒性劑侷限於表現TAT226之細胞中。6亥專抗體具有T AT 2 2 6結合臂及結合細胞毒性劑〔例如沙泊寧（saporin)、抗干擾素α、長春鹼類、蓖麻毒素a 鍵、甲胺蝶π令或放射性同位素半抗原）之臂。雙特異性抗體可製備為全長抗體或抗體片段之形式（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係基於兩個免疫球蛋白重鏈-輕鏈對之共表現’其中兩條重鍵具有不同特異性（Milstein及 Cuello, 305: 537 (1983))。由於免疫球蛋白重鏈及輕鏈之隨機分類，因此該等融合瘤（雜交瘤）產生1〇個不同抗體分子之可能混合物，其中僅一者具有恰當雙特異性結構。通常藉由親和層析步驟進行之恰當分子之純化相當繁複’且產率較低。類似程序係揭示於1993年5月13日公開之 WO 93/08829 及 Traunecker 等人，五細Ο J·，10: 3655 (1991)中。根據不同方法，將具有所需結合特異性（抗體_抗原組合位點）之抗體可變結構域融合至免疫球蛋白恆定結構域序列。例如，與包含至少部分鉸鏈區、CH2區及CH3區之免疫球蛋白重鏈恆定結構域融合。在某些實施例中，在融合 119007.doc -84 - 200813092 之至少-者中存在含有輕鏈結合必需位點之第—重鍵怪定區（cm)。將編碼免疫球蛋白重鍵融合及（若需要）免疫球蛋白輕鏈之舰***獨立表現載體中且經共轉染至合適宿主生物體中。在建構中使用不等比率之三個多肽鏈提供最佳產率之實施例中，其使三個多肽片段之相互比例之調節具有較大彈性當以相等比率表現至少兩條多狀鍵導致高產率時或當㈣不具有特定顯著性時，則可能在一種表現載體中插人兩個或所有三個多肽鏈之編碼序列。在該方法之—實施例中，雙特異性抗體係由-個臂中且有第-結合特異性之雜交免疫球蛋白重鏈與另一臂中雜交免疫球蛋白重鏈-輕鏈對（提供第二結合特異性）組成。發現該不對稱結構促使所需雙特異性化合物自非所需免疫球蛋白鏈組合分離’因為僅存在半數雙特異性分子之免疫球蛋白輕鏈提供輕易分離之方式。該方法係揭示於w〇 94/04690中。關於產生雙特異性抗體之其他詳述，例如參見Suresh等人，从以办〇心五似少讲〇/〇灯，12121〇 (1986)。根據另一方法，抗體分子對之間之界面可經工程設計以使自重組細胞培養物回收之雜二聚體之百分比最大化。界面包含抗體恆定結構域之至少一部分Ch3結構域。以此方法，以較大側鏈（例如酪胺酸或色胺酸）置換來自第一抗體分子界面之一或多個小胺基酸側鏈。藉由以較小者（例如丙胺酸或蘇胺酸）置換較大胺基酸側鏈，於第二抗體分子界面上產生具有與較大侧鏈相同或類似尺寸之補償性，，空穴"。此提供増加雜二聚體之產率使其高於諸如均二聚體 119007.doc -85- 200813092 之其他非所需終產物的機制。雙特異性抗體包括交聯或”雜接合"抗體。舉例而言，可 I雜接合之抗體之一偶合至抗生物素蛋白，將其他；體偶合至生物素。例如，已提出將此等抗體用以將免疫系統細胞靶向非所需細胞（美國專利第4,676,98〇號）且用於治療 HIV 感染（WO 91/00360、WO 92/00373 及 EP 03089)。雜接合抗體可使用任何便利之交聯法來製造。此項技術中已熟知合適交聯劑，且連同許多交聯技術一起揭示於美國專利第 4,676,980號中。亦已於文獻中描述由抗體片段產生雙特異性抗體之技術。舉例而言，可使用化學鍵聯製備雙特異性抗體。The human anti-TAT226 antibody of the present invention can be constructed by combining an Fv pure line variable domain sequence selected from a bacterium of human origin and a known human constant domain sequence. Alternatively, the human monoclonal anti-TAT226 antibody of the present invention can be produced by the fusion tumor method. Human myeloma and mouse human heterozygous myeloma cell lines for producing human monoclonal antibodies are, for example, Kozbor X/, 133: 3001 (1984); Brodeui* et al., Μ(10)%/〇如/ Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987) and Boerner et al, J. 147: 86 (1991). It is currently possible to produce a transgenic animal (e.g., a mouse) capable of producing a full lineage of human antibodies in the absence of endogenous immunoglobulin 119007.doc -82 - 200813092 upon immunization. For example, it has been described that homozygous ligation of the antibody re-ligated region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al., Pn w". on Sc/t/, 90: 2551 (1993); Jakobovits et al., Heart, Please, 362: 255 (1993), Bruggermann, / (10) ο/· , 7: 33 (1993) 〇 Gene shuffling can also be used to derive human antibodies from non-human antibodies such as rodents, where human antibodies have similar affinities and specificities as the starting non-human antibodies. According to this method (also known as " epitope effect"), the human V domain gene lineage is substituted for the heavy or light chain variable region of a non-human antibody fragment obtained by phage display technology as described herein. , producing a population of non-human chain/human chain scFv or Fab. Non-human chain/human chain chimeric scFv or Fab is isolated by antigen selection, wherein the human chain recovers the antigen binding site that is damaged when the corresponding non-human chain in the pure line is removed, ie, the antigenic determinant is dominant (affected The choice of human chain collocation. When this method is repeated to replace the remaining non-human strand, a human antibody is obtained (see PCT W0 93/06213 published on April 1, 1993). Unlike the humanization of traditional non-human antibodies grafted by CDRs, this technique provides fully human antibodies that do not have FR or CDR residues of non-human origin. 5. Bispecific anti-occupation The bispecific antibody is a 119007.doc-83-200813092 monoclonal antibody with binding specificity for at least two different antigens. In certain embodiments, the bispecific antibody is a human or humanized antibody. In certain embodiments, one of the binding specificities is for TAT226 and the other is for any other antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of TAT226. Bispecific antibodies can also be used to limit cytotoxic agents to cells that express TAT226. 6 Hai special antibody has T AT 2 2 6 binding arm and binding cytotoxic agent (such as saporin, anti-interferon α, vinblastine, ricin a bond, methotrexate π or radioisotope half The arm of the antigen). The bispecific antibody can be prepared in the form of a full length antibody or antibody fragment (e.g., F(ab,)2 bispecific antibody). Methods of making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy bonds have different specificities (Milstein and Cuello, 305: 537 (1983)). Due to the random classification of immunoglobulin heavy and light chains, such fusion tumors (hybridomas) produce a possible mixture of one different antibody molecule, of which only one has the proper bispecific structure. Purification of the appropriate molecule, usually by affinity chromatography steps, is quite complex and yields are low. A similar procedure is disclosed in WO 93/08829, published May 13, 1993, and by Traunecker et al., J. J., 10: 3655 (1991). The antibody variable domain having the desired binding specificity (antibody-antigen combining site) is fused to an immunoglobulin constant domain sequence according to various methods. For example, fusion with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, the CH2 region, and the CH3 region. In certain embodiments, a first-heavy bond region (cm) containing a site necessary for light chain binding is present in at least one of the fusions 119007.doc -84 - 200813092. A ship encoding an immunoglobulin heavy bond fusion and, if desired, an immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. In embodiments where three polypeptide chains of unequal ratios are used in the construction to provide optimal yield, the adjustment of the mutual ratio of the three polypeptide fragments is greater. When at least two polymorphic bonds are expressed in equal ratios, resulting in high yield. When the rate is or when (4) does not have a specific significance, it is possible to insert a coding sequence of two or all three polypeptide chains in one expression vector. In the method - the embodiment, the bispecific anti-system consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (provided Second binding specificity) composition. This asymmetric structure was found to promote the separation of the desired bispecific compound from the undesired immunoglobulin chain combination' because the immunoglobulin light chain in which only half of the bispecific molecule is present provides a means of easy separation. This method is disclosed in w〇 94/04690. For additional details on the production of bispecific antibodies, see, for example, Suresh et al., 从似〇〇〇〇〇 12 12 12 12 12 12 12 12 12 12 12 12 12 12 1986 1986 1986 1986 1986 1986 1986 1986 According to another approach, the interface between pairs of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The interface comprises at least a portion of the Ch3 domain of the antibody constant domain. In this way, one or more small amino acid side chains from the first antibody molecule interface are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensating for the same or similar size of the larger side chain at the interface of the second antibody molecule by replacing the larger amino acid side chain with a smaller one (eg, alanine or threonine), the hole &quot ; This provides a mechanism for the addition of the heterodimer to be higher than other undesirable end products such as homodimer 119007.doc -85-200813092. Bispecific antibodies include cross-linking or "hetero-adhesive" antibodies. For example, one of the antibodies that can be heterozygous is coupled to avidin, and the other is coupled to biotin. For example, it has been proposed Antibodies are used to target immune system cells to unwanted cells (U.S. Patent No. 4,676,98) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Heteroconjugate antibodies can be used with any Convenient cross-linking processes are made. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques. The production of bispecific antibodies from antibody fragments has also been described in the literature. Techniques. For example, bispecific antibodies can be prepared using chemical linkages.

Brennan等人，心229: 81 (1985)描述其中完整抗體經蛋白水解裂解以產生F(ab，）2片段之程序。該等片段可於二硫醇複合劑亞砷酸鈉存在下經還原以穩定鄰二硫醇且防止分子間形成二硫化物。接著將所產生之Fab，片段轉化為硫硝基苯甲酸酯（TNB)衍生物。接著將Fab，-TNB衍生物之一藉由以巯基乙胺還原再轉化為Fab，-硫醇且與等莫耳量之其他Fab’-TNB衍生物混合以形成雙特異性抗體。所產生之雙特異性抗體可用作使酶選擇性固定之藥劑。最近之進展促使自大腸桿菌直接回收Fab’_SH片段，該等Fab»_SH片段可經化學偶合以形成雙特異性抗體。 Shalaby等人，J.五；φ· Md·，175: 217-225 (1992)描述完全人化雙特異性抗體F(abf)2分子之產生。各Fab’片段係自大腸桿菌獨立地分泌且於活體外經受定向化學偶合以形成雙 119007.doc -86- 200813092 特異性抗體如此形成之雙特異性抗體可結合至過度表現Brennan et al., Heart 229: 81 (1985) describe procedures in which intact antibodies are proteolytically cleaved to produce F(ab,)2 fragments. These fragments can be reduced in the presence of the dithiol complex sodium arsenite to stabilize the o-dithiol and prevent the formation of disulfides between the molecules. The resulting Fab, fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab,-TNB derivatives is then converted to Fab,-thiol by reduction with mercaptoethylamine and mixed with other molar amounts of other Fab'-TNB derivatives to form bispecific antibodies. The bispecific antibody produced can be used as an agent for selectively immobilizing an enzyme. Recent advances have led to the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J. V; φ. Md., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(abf)2 molecule. Each Fab' fragment is secreted independently from E. coli and subjected to directed chemical coupling in vitro to form a double 119007.doc-86-200813092 specific antibody. The bispecific antibody thus formed can bind to overexpression.

亦已描述由重組細胞培養物直接製造且分離雙特異性抗已使用白胺酸拉鏈製造雙 Immunol·, 148(5):1547- 體片段之各種技術。舉例而言，特異性抗體。Kostelny等人， 1553 (1992)。藉由基因融合將來自F〇s及Jun蛋白之白胺酸拉鏈肽連接至具有兩個不同抗體之Fab，部分。將抗體均二聚體於鉸鏈區還原以形成單體且接著再氧化以形成抗體雜一聚體。此方法亦可用於產生抗體均二聚體。由H〇1Hnger 專人，Proc. TVa". jcad· 5W. 90:6444-6448 (1993)描述之”雙功能抗體”技術已提供用於製造雙特異性抗體片段之替代機制。該等片段包含藉由連接子連接至輕鏈可變結構域（VL)之重鏈可變結構域（VH)，該連接子過短而不可使相同鍵上之兩個結構域成對。因此，迫使一片段之VH及 VL結構域與另一片段之互補VL及vh結構域成對，藉此形成兩個抗原結合位點。亦已報導藉由使用單鏈Fv(sFv)二聚體製造雙特異性抗體片段之另一策略。參見Gruber等人， J· /mmwwo/·，152:5368 (1994) 〇涵蓋具有二價以上之抗體。舉例而言，可製備三特異性抗體0 Tutt等人/· 147: 60 (1991) 〇 6·多價抗艘藉由表現抗體所結合之抗原的細胞，多價抗體可比二價抗體更快速地内在化（及/或分解代謝）。本發明之抗體可為 119007.doc -87- 200813092 具有二個或三個以上抗原結合位點之多價抗體（不同於IgM 種類）（例如四價抗體），其可易於藉由重組表現編碼抗體多肽鏈之核酸而產生。多價抗體可包含二聚化結構域及三個或二個以上抗原結合位點。在某些實施例中，二聚化結構域包含Fc區或鉸鏈區（或由其組成）。在此情形中，抗體將包含Fc區及三個或三個以上至pc區之抗原結合位點胺基端。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（或由其組成）。在一個此實施例中，多價抗體包含四個抗原結合位點（或由其組成）。多價抗體包含至少一條多肽鏈（例如兩條多肽鏈），其中多肽鏈包含兩個或兩個以上可變結構域。舉例而言，多肽鏈可包含VD1-(XI)n - VD2-(X2)n-Fc，其中VD1為第一可變結構域，VD2為第二可變結構域，Fc為Fc區之一條多肽鏈，XI及χ2表示胺基酸或多肽，且η為0或1。舉例而言，多肽鏈可包含：vH_ CH1-彈性連接子-VH-CH1 -Fc 區鏈；或 VH_CH1-VH-CH1 _Various techniques for the direct production of recombinant cell cultures and isolation of bispecific antibodies that have been used to make dual Immunol, 148(5):1547-body fragments have also been described. For example, specific antibodies. Kostelny et al., 1553 (1992). The leucine zipper peptide from F〇s and Jun proteins was ligated to the Fab portion with two different antibodies by gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer and then reoxidized to form an antibody heteromer. This method can also be used to generate antibody homodimers. The "bifunctional antibody" technology described by H. 1 Hnger, Proc. TVa " jcad. 5W. 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) joined to the light chain variable domain (VL) by a linker that is too short to pair two domains on the same bond. 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 making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. /mmwwo/., 152:5368 (1994) 涵盖 Covering antibodies with more than two valences. For example, a trispecific antibody can be prepared. 0 Tutt et al. / 147: 60 (1991) 〇6. Multivalent anti-residue cells by expressing an antigen bound by an antibody, the multivalent antibody can be more rapidly than the bivalent antibody In (and / or catabolism). The antibody of the present invention may be a multivalent antibody (different from an IgM species) having two or more antigen-binding sites (for example, a tetravalent antibody), which can easily encode an antibody by recombinant expression, 119007.doc -87-200813092 Produced by the nucleic acid of the polypeptide chain. A multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more to the antigen binding site amine end of the pc region. In certain embodiments, the multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, the multivalent antibody comprises (or consists of) four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (e. g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain may comprise VD1-(XI)n-VD2-(X2)n-Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, and Fc is a polypeptide of the Fc region The chain, XI and χ2 represent an amino acid or polypeptide, and η is 0 or 1. For example, the polypeptide chain may comprise: vH_CH1-elastic linker-VH-CH1-Fc region chain; or VH_CH1-VH-CH1 _

Fc區鏈。本文之多價抗體可另外包含至少兩個（例如四個）輕鏈可變結構域多肽。例如，本文之多價抗體可包含約兩個至約八個輕鏈可變結構域多肽。本文所涵蓋之輕鏈可變結構域多肽包含輕鏈可變結構域且視情況進一步包含CL結構域。 7.單結構域抗艘在一些實施例中，本發明之抗體為單結構域抗體。單結構域抗體為包含抗體之所有或部分重鏈可變結構域或所有或部分輕鍵可變結構域之單一多狀鍵。在某些實施例中， 119007.doc •88- 200813092 單結構域抗體為人類單結構域抗體（Domantis，Inc.， Waltham，ΜΑ ;參見例如美國專利第6,248,516 B1號）。在一實施例中，單結構域抗體係由抗體之所有或部分重鏈可變結構域組成。 1抗髏變異艘在一些實施例中，涵蓋本文所述抗體之胺基酸序列修飾。舉例而言，可能需要改良抗體之結合親和力及/或其他生物特性。可藉由向編碼抗體之核苷酸序列中引入適當變化或藉由肽合成來製備抗體之胺基酸序列變異體。例如’此等修飾包括抗體之胺基酸序列中殘基之缺失及/或 ***及/或取代。可進行缺失、***及取代之任何組合以達成最終建構，其限制條件為最終建構具有所需特徵。可將胺基酸變化於序列生成時引入受檢者抗體胺基酸序列中。如 Cunningham及 Wells (1989) Scz244:1081-1085所述’適用於識別為突變較佳位置之特定抗體殘基或區域之方法稱作”丙胺酸掃描突變”。此處，識別殘基或靶殘基之群（例如帶電殘基（諸如ai*g、asp、his、lys及glu))且將其以中性或帶負電荷之胺基酸（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。接著藉由在取代位點處或為取代位點引入另外或其他變異體來改進對取代表現功能敏感性的彼等胺基酸位置。因此，儘管引入胺基酸序列變異之位點為預定的，但無需預定突變本身之性質。舉例而言’為分析指定位點之突變效能，於靶密碼子或靶區進行 119007.doc -89 - 200813092 ala掃描或隨機突變且篩檢經表現免疫球蛋白之所需活性。胺基酸序列***包括長度為一個殘基至含有數百或更多殘基之多肽範圍内的胺基端及/或羧基端融合，以及單一或多傭胺基酸殘基之序列内***。末端***之實例包括具有N端甲硫胺醯基殘基之抗體。抗體分子之其他***變異包括抗體N端或C端與酶（例如ADEPT)或增加抗體血清半衰期之多狀的融合。在某些實施例中，本發明之抗體經改變以增加或減低抗體糖基化之程度。多肽之糖基化通常為N連接或〇連接的。N連接係指將碳水化合物部分連接至天冬醯胺酸殘基之側鏈。三肽序列天冬醯胺酸_χ_絲胺酸及天冬醯胺酸_χ_ 蘇胺酸（其中X為除脯胺酸以外之任何胺基酸）為將碳水化合物部分酶性連接至天冬醯胺酸侧鏈之識別序列。因此，多肽中之存在該等三肽序列中之任一者均產生可能之糖基化位點〇連接之糖基化係指將糖Ν·乙醯半乳胺糖、半乳糖或木糖中之一者連接至羥胺酸，該羥胺酸最通常為絲胺酸或蘇胺酸，儘管亦可使用％羥基脯胺酸或5_羥基離胺酸。抗體糖基化位點之添加或刪除係藉由改變胺基酸序列以使得產生或移除上述三肽序列中之—或多個（用於ν連接糖基化位點）而便利地完成。改變亦可藉由在初始抗體序列 (用於〇連接糖基化位點）中添加、缺失或取代—或多個絲胺酸或蘇胺酸殘基來進行。當抗體包含Fe區時，可改變與其連接之碳水化合物。舉 119007.doc -90- 200813092 例而言，在美國專利申請案第US 2003/0157108號（Presta， L·)中描述在連接至抗體Fc區之成熟碳水化合物結構中缺少海藻糖之抗體。亦參見US 2004/0093621(Kyowa Hakko Kogyo Co·，Ltd)。Jean-Mairet 等人之 WO 2003/011878 及 Umana等人之美國專利第6,602,684號中引用在連接至·抗體 Fc區之碳水化合物中具有平分N_乙醯半乳胺糖（GlcNAc)之抗體。於Patel等人之WO 1997/30087中報導在連接至抗體 Fc區之寡醣中具有至少一個半乳糖殘基之抗體。亦參見關於具有連接至其Fc區之經改變碳水化合物之抗體的WO 1998/58964(Raju，S·)及 WO 1999/22764(Raju，S·)。亦參見關於具有經修飾糖基化之抗原結合分子之US 2005/0123546(Umana等人）〇在某些實施例中，糖基化變異體包含Fc區，其中連接至 Fc區之碳水化合物結構缺少海藻糖。此等變異體具有改良之ADCC功能。視情況而言，Fc區另外包含進一步改良 ADCC之一或多個胺基酸取代，例如於Fc區之位置298、 333及/或334處（殘基之Eu編號）取代。關於”去海藻糖化”或 π海藻糠缺乏’’抗體之公開案實例包括：US 2003/0157108 ; WO 2000/61739 ； WO 2001/29246 ； US 2003/0115614 ； US 2002/0164328 ； US 2004/0093621 ； US 2004/0132140 ； US 2004/0110704 ； US 2004/0110282 ； US 2004/0109865 ； WO 2003/085119 ； WO 2003/084570 ； WO 2005/035586 ； WO 2005/035778 ； WO 2005/053742 ； Okazaki# A/. Mol Biol. 336:1239-1249 (2004) ; Yamane-Ohnuki等人价⑼g. 119007.doc -91- 200813092 87: 614 (2004)。產生去海藻糖化抗體之細胞株實例包括在蛋白海藻化中缺乏之Lecl3 CHO細胞（Ripka等人 249:533-545 (1986);美國專利申請案第 US 2003/0157108 A1 號，Presta，L;及Adams 等人之 WO 2004/05 6312 A1，尤其在實例11中）及基因剔除細胞株，諸如α-1，6-海藻糖基轉移酶基因、基因剔除CHO細胞 (Yamane-Ohnuki等人B/oiec/z. Biowg· 87: 614 (2004)) 〇另一變異體類型為胺基酸取代變異體。該等變異體在經不同殘基置換之抗體分子中具有至少一個胺基酸殘基。用於取代型突變之所關注位點包括高變區，但亦涵蓋FR變化。保守性取代係於表1中在標題”較佳取代”下展示。若此等取代致使生物活性發生所需改變，則可引入於表1中命名為"例示性取代"或以下參考胺基酸種類進一步描述之更多實質變化且篩檢產物。表1 原始殘基例示性取代較佳取代 Ala(A) Val ； Leu ； lie Val Arg(R) Lys ； Gin ； Asn Lys Asn(N) Gin ； His ； Asp ； Lys ； Arg Gin Asp(D) Glu ； Asn Glu Cys(C) Ser ； Ala Ser Gln(Q) Asn ； Glu Asn Glu(E) Asp ; Gin Asp Gly(G) Ala Ala His(H) Asn ； Gin ； Lys ； Arg Arg Ile(I) Leu ; Val ; Met ; Ala ; Phe ;正白胺酸 Leu Leu(L) 正白胺酸；lie ; Val ; Met ; Ala ; Phe lie 119007.doc -92- 200813092 原始殘基例示性取代較佳取代 Lys(K) Arg ； Gin ； Asn Arg Met(M) Leu ； Phe ； lie Leu Phe(F) Trp ; Leu ; Val ; lie ; Ala ; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val ； Ser Ser Trp(W) Tyr ； Phe Tyr Tyr⑺ Trp ； Phe ； Thr ； Ser Phe Val⑺ lie ; Leu ; Met ; Phe ; Ala ;正白胺酸 Leu 抗體生物特性之實質改質係藉由選擇取代來完成，該等取代對維持（a)例如折疊或螺旋構形之取代區多肽主鏈結構；（b)靶位點處分子之電荷或疏水性，或（c)大量侧鏈之效應顯著不同。胺基酸可根據其側鏈特性之相似性分組 (於 A. L. Lehninger 之 Biochemistry，第 2 版，第 73-75 頁中，Worth Publishers，New York (1975)): (1) 非極性：Ala (A)、Val (V)、Leu (L)、lie (I)、Pro (P)、Phe (F)、Trp (W)、Met (M) (2) 不帶電之極性：Gly (G)、Ser (S)、Thr (T)、Cys (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，可將天然產生之殘基基於共同側鏈特性分組： (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 ; 119007.doc -93- 200813092 (5)影響鏈定向之殘基：Gly、Pro ; ⑹芳族：Trp、Tyr、Phe 〇非保守性取代將引起將該等種類中之一員交換為另一種類。亦可將此等經取代殘基引入保守性取代位點或剩餘 (非保守性）位點中。一種取代變異體類型包括取代親本抗體（例如人化或人類抗體）之一或多個高變區殘基。一般而言，為進一步研發所選擇之所得變異體相對於產生其之親本抗體將具有經改質（例如改良）之生物特性。產生此等取代型變異體之便利方式涉及使用噬菌體呈現親和力成熟。簡言之，使若干高變區位點（例如6-7位點）突變以於各位點產生所有可能之胺基酸取代。如此產生之抗體作為與封裝於各顆粒内之至少部分噬菌體鞘蛋白（例如M13之基因ΙΠ產物）之融合物自絲狀嗟菌體顆粒呈現。接著根據其生物活性（例如結合親和力）篩檢噬菌體呈現之變異體。為識別用於修飾之候選咼變區位點，可進行掃描突變（例如丙胺酸掃描）以識別顯著有助於抗原結合之高變區殘基。或者或另外，分析抗原-抗體複合物之晶體結構以識別抗體與抗原之間之接觸點可為有益的。根據此項技術中已知之技術，此等接觸殘基及相鄰殘基為供取代之候選物。一旦產生此等變異體，則使變異體板經受使用此項技術中已知技術（包括本文中所述之彼等）之筛檢且可選擇在一或多個相關檢定中具有優越特性之抗體以供進一步研發。藉由此項技術中已知之各種方法製備編碼抗體胺基酸序 119007.doc -94- 200813092 列變異體之核酸分子。該等方法包括（但不限於）自天然來源分離（在天然產生之胺基酸序列變異體之情況下）或藉由寡核苷酸介導之（或定點）突變、PCR突變及先前製備之抗體變異或非變異譯本之序列盒突變來製備。可能需要於本發明抗體之Fc區引入一或多個胺基酸修飾，藉此產生Fc區變異體。Fc區變異體可包含於包括鉸鏈半胱胺酸之一或多個胺基酸位置包含胺基酸修飾（例如取代）之人類Fc區序列（例如人類IgGl、IgG2、IgG3或IgG4 Fc 區）。Fc region chain. Multivalent antibodies herein may additionally comprise at least two (eg, four) light chain variable domain polypeptides. For example, a multivalent antibody herein can comprise from about two to about eight light chain variable domain polypeptides. A light chain variable domain polypeptide encompassed herein comprises a light chain variable domain and, as the case may be, further comprises a CL domain. 7. Single Domain Anti-Barrels In some embodiments, the antibodies of the invention are single domain antibodies. A single domain domain antibody is a single polymorphic bond comprising all or part of a heavy chain variable domain or all or part of a light bond variable domain of an antibody. In certain embodiments, the 119007.doc •88-200813092 single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, ΜΑ; see, e.g., U.S. Patent No. 6,248,516 B1). In one embodiment, the single domain anti-system consists of all or part of the heavy chain variable domains of the antibody. 1 Anti-髅 variant vessel In some embodiments, the amino acid sequence modification of the antibodies described herein is encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. For example, such modifications include deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construction with the constraint that the final construction has the desired characteristics. The amino acid change can be introduced into the subject antibody amino acid sequence upon sequence generation. The method described in Cunningham and Wells (1989) Scz 244:1081-1085 for a particular antibody residue or region identified as a preferred location for the mutation is referred to as an "alanine scanning mutation". Here, a population of residues or target residues (eg, charged residues (such as ai*g, asp, his, lys, and glu)) and neutralized or negatively charged amino acids (eg, alanine) are identified. Or polyalanine) substitution to affect the interaction of the amino acid with the antigen. These amino acid positions that are sensitive to the substitution performance function are then improved by introducing additional or other variants at the substitution site or at the substitution site. Therefore, although the site where the introduction of the amino acid sequence is changed is predetermined, the nature of the predetermined mutation itself is not required. For example, to analyze the mutational potency of a given site, the target codon or target region is subjected to 119007.doc-89 - 200813092 ala scan or random mutation and screened for the desired activity of the immunoglobulin. Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple glyceryl acid residues. Examples of terminal insertions include antibodies having N-terminal methylthioguanidine residues. Other insertional variants of the antibody molecule include fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., ADEPT) or a polymorphism that increases the serum half-life of the antibody. In certain embodiments, the antibodies of the invention are altered to increase or decrease the extent of antibody glycosylation. The glycosylation of a polypeptide is typically N-linked or hydrazone linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartic acid residue. Tripeptide sequence aspartic acid χ χ 丝丝丝丝丝丝丝丝丝丝丝苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏苏The recognition sequence of the protamine side chain. Thus, the presence of any of the tripeptide sequences in the polypeptide results in a possible glycosylation site. The glycosylation of the glycosylation refers to the glycoside, acetaminosamine, galactose or xylose. One of them is attached to hydroxylamine, which is most typically seric acid or threonine, although % hydroxyproline or 5-hydroxy lysine may also be used. Addition or deletion of an antibody glycosylation site is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences are produced or removed (for ν-linked glycosylation sites). Alterations can also be made by addition, deletion or substitution - or multiple serine or threonine residues in the initial antibody sequence (for the hydrazone linking glycosylation site). When the antibody comprises an Fe region, the carbohydrate to which it is attached can be altered. For example, an antibody lacking trehalose in a mature carbohydrate structure linked to the Fc region of an antibody is described in U.S. Patent Application No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). An antibody having an aliquot of N-acetaminogalactosamine (GlcNAc) in a carbohydrate linked to the Fc region of the antibody is cited in WO 2003/011878 to Jean-Mairet et al. and U.S. Patent No. 6,602,684 to U.S. Patent. An antibody having at least one galactose residue in an oligosaccharide linked to the Fc region of an antibody is reported in WO 1997/30087 to Patel et al. See also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) for antibodies having altered carbohydrates linked to their Fc regions. See also US 2005/0123546 (Umana et al.) for antigen-binding molecules with modified glycosylation. In certain embodiments, the glycosylation variant comprises an Fc region in which the carbohydrate structure linked to the Fc region is absent. Trehalose. These variants have improved ADCC functionality. Optionally, the Fc region additionally comprises a further modification of one or more amino acid substitutions of the ADCC, such as at positions 298, 333 and/or 334 of the Fc region (Eu numbering of residues). Examples of publications relating to "de-alginization" or π-algae deficiency "'antibody" include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; Okazaki# A/. Mol Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. (9) g. 119007.doc-91-200813092 87: 614 (2004). Examples of cell lines which produce de-alcoholized antibodies include Lecl3 CHO cells which are deficient in protein seaweed (Ripka et al. 249: 533-545 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L; Adams et al., WO 2004/05 6312 A1, especially in Example 11) and gene knockout cell lines, such as α-1,6-trehalyltransferase gene, gene knockout CHO cells (Yamane-Ohnuki et al. B/oiec /z. Biowg 87: 614 (2004)) Another variant type is an amino acid substitution variant. The variants have at least one amino acid residue in the antibody molecule displaced by a different residue. The sites of interest for substitutional mutations include hypervariable regions, but also encompass FR changes. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitution". If such substitutions result in a desired change in biological activity, it may be referred to in Table 1 as "exemplary substitution" or more substantial changes in the following reference amino acid species and screened products. Table 1 Exemplary substitutions of the original residues are preferably substituted for Ala(A) Val; Leu; lie Val Arg(R) Lys; Gin; Asn Lys Asn(N) Gin; His; Asp; Lys; Arg Gin Asp(D) Glu Asn Glu Cys(C) Ser ; Ala Ser Gln(Q) Asn ; Glu Asn Glu(E) Asp ; Gin Asp Gly(G) Ala Ala His(H) Asn ; Gin ; Lys ; Arg Arg Ile(I) Leu ; Val ; Met ; Ala ; Phe ; Leucine Leu Leu ( L ) leucine ; lie ; Val ; Met ; Ala ; Phe lie 119007.doc -92- 200813092 Illustrative substitution of the original residue is preferred to replace Lys (K) Arg ; Gin ; Asn Arg Met ( M ) Leu ; Phe ; lie Leu Phe ( F ) Trp ; Leu ; Val ; lie ; Ala ; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T Ser Ser Trp (W) Tyr ; Phe Tyr Tyr ( 7 ) Trp ; Phe ; Thr ; Ser Phe Val ( 7 ) lie ; Leu ; Met ; Phe ; Ala ; To complete, the substitution pairs maintain (a) a substituted region polypeptide backbone structure such as a folded or helical configuration; (b) the charge or hydrophobicity of the molecule at the target site, or (c) a large number of side chains The effects are significantly different. Amino acids can be grouped according to their similarity in side chain properties (in Biochemistry, AL Lehninger, 2nd edition, pages 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala (A) ), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M) (2) Uncharged polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q) (3) Acidity: Asp (D), Glu (E) (4) Alkaline: Lys ( K), Arg (R), His (H) or, can naturally group residues based on common side chain properties: (1) Hydrophobicity: n-leucine, Met, Ala, Val, Leu, lie; 2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin; (3) Acidity: Asp, Glu; (4) Qualitative: His, Lys, Arg; 119007.doc -93- 200813092 (5) Impact chain Oriented residues: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe 〇 Non-conservative substitutions will cause one of the species to be exchanged for another species. These substituted residues can also be introduced into a conservative substitution site or a remaining (non-conservative) site. One type of substitution variant includes substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the resulting variants selected for further development will have modified (e.g., improved) biological properties relative to the parent antibody from which they are produced. A convenient way to generate such substituted variants involves the use of phage to exhibit affinity maturation. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each point. The antibody thus produced is presented as a fusion with at least a portion of the phage sheath protein (e.g., the gene ΙΠ product of M13) encapsulated in each particle from the filamentous bacterium particles. Phage-presented variants are then screened for their biological activity (e. g., binding affinity). To identify candidate mutated region sites for modification, a scanning mutation (e. g., alanine scan) can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to recognize the point of contact between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to techniques known in the art. Once such variants are produced, the variant plates are subjected to screening using known techniques in the art, including those described herein, and may select antibodies with superior properties in one or more of the relevant assays. For further research and development. Nucleic acid molecules encoding antibody amino acid sequence 119007.doc-94-200813092 column variants are prepared by various methods known in the art. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutations, PCR mutations, and previous preparations. Prepare by mutation of a sequence cassette of an antibody variant or non-variant translation. It may be desirable to introduce one or more amino acid modifications in the Fc region of an antibody of the invention, thereby producing an Fc region variant. The Fc region variant may be comprised of a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising one or more amino acid positions of the hinge cysteine comprising an amino acid modification (e.g., substitution).

根據此項技術之此描述及教示，在一些實施例中涵蓋本發明之抗體與野生型對應物抗體相比可（例如）於Fc區包含一或多處變化。然而，該等抗體與其野生型對應物相比將大體上保持治療用途所需之相同特徵。舉例而言，例如 WO 99/5 1642所述，認為可於Fc區中進行產生經變化（亦即改良或減低）之Clq結合及/或補體依賴性細胞毒性（CDC)之特定變化。亦參見關於Fc區變異體之其他實例的Duncan & Winter iVaiwre 322:738-40 (1988);美國專利第 5,648,260 號；美國專利第5,624,821號；及WO 94/29351。WO 00/42072(Presta)及 WO 2004/056312(Lowman)描述具有對 FcR之改良或減低結合之抗體變異體。該等專利公開案之内容係以特定引用的方式併入本文。亦參見Shields等人价〇/. C/z·· 9(2): 6591-6604 (2001)。負責將母體 lgG 轉移至胎兒之具有增加之半衰期及對新生兒Fc區受體（FcRn)改良之結合的抗體（Guyer 等人，J· Immunol· 117:587 (1976) 119007.doc -95- 200813092In accordance with this description and teachings of the technology, antibodies encompassing the invention in some embodiments may comprise, for example, one or more changes in the Fc region as compared to a wild-type counterpart antibody. However, such antibodies will generally retain the same characteristics required for therapeutic use as their wild-type counterparts. For example, as described, for example, in WO 99/5 1642, it is believed that specific changes in Clq binding and/or complement dependent cytotoxicity (CDC) that produce a change (i.e., improved or reduced) can be made in the Fc region. See also, for other examples of Fc region variants, Duncan & Winter iVaiwre 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or reduced binding to FcR. The contents of these patent publications are hereby incorporated by reference in their entirety. See also Shields et al. Price/. C/z·· 9(2): 6591-6604 (2001). An antibody responsible for the transfer of maternal lgG to the fetus with increased half-life and improved binding to the neonatal Fc region receptor (FcRn) (Guyer et al, J. Immunol. 117:587 (1976) 119007.doc-95-200813092

及 Kim 等人，J. Immunol. 24:249 (1994))係描述於 US 2005/0014934 Al(Hinton等人）中。該等抗體包含其中具有改良Fc區與FcRn之結合的一或多個取代之卜區。具有經改變Fc區胺基酸序列及增加或減低之ciq結合能力之多肽變異體係描述於美國專利第6，194,551 B1號、WO 99/51642 中。彼等專利公開案之内容係以特定引用的方式併入本文。亦參見 Idusogie 等人 J· ⑽/ 164: 4178-4184 (2000) 〇And Kim et al., J. Immunol. 24:249 (1994)) are described in US 2005/0014934 Al (Hinton et al.). The antibodies comprise one or more substitution regions in which the binding of the modified Fc region to FcRn is achieved. A polypeptide variant having a modified Fc region amino acid sequence and an increased or decreased ciq binding ability is described in U.S. Patent No. 6,194,551 B1, WO 99/51642. The contents of their patent publications are hereby incorporated by reference in their entirety. See also Idusogie et al. J. (10)/ 164: 4178-4184 (2000) 〇

在一態樣中，本發明提供在包含以區之以多肽界面中包含修飾之抗體，其中該等修飾促使及/或促進異源二聚化該專修飾包含將突起引入第一 Fc多肽中且將空腔引入弟一 Fc夕肽中，其中该突起可定位於空腔中以促進第一與第二Fc多肽之複合。例如美國專利第5,73i，i68號中所述，此項技術中已知用於產生具有該等修飾之抗體的方法。 9 * 抗體本發明之抗體可經進一步修飾以含有此項技術易於獲侍之額外非蛋白質部分。適用於衍生抗體之部分較為欠办f生聚口物。水溶性聚合物之非限制性實例包括 (但不限於）聚乙二醇（PEG)、乙二醇/丙二醇之共聚物、叛 f 、聚乙烯料錢、聚_ #戊裒聚l3,6-二噁烷、乙烯/順丁烯二酸酐共聚物聚私基酸（均聚物或無規共聚物）及葡聚糖或聚口比咯啶酮）聚乙-酸、取工 ,一％聚丙二醇均聚物、聚氧化丙烯/氧化乙稀共聚物、聚氫r i 乳乙基化多元醇(例如甘油）、聚乙烯醇及 119007.doc -96- 200813092 其混合物。聚乙二醇丙醛因其在具有優勢。聚合物可具有任何分子旦：‘：性而在製造中 ^ , 八有仕17刀子里且可為支鏈或非支鏈的。連接至抗體之聚合物數量可不同，且若連接—種以上之聚合物’則其可為相同或不同分子。一般而t，用於衍生之聚合物的數量及/或類型可基於包括(但不限於)以下考慮因素來確^ :待改良抗體之特㈣性或功能、抗體衍生物是否將用於界定條件下之療法等。In one aspect, the invention provides an antibody comprising a modification comprising a polypeptide in a region, wherein the modification promotes and/or facilitates heterodimerization, the specific modification comprises introducing a protuberance into the first Fc polypeptide and The cavity is introduced into the Fc-peptide, wherein the neurite can be localized in the cavity to facilitate complexation of the first and second Fc polypeptides. Methods for producing antibodies having such modifications are known in the art as described in, for example, U.S. Patent No. 5,73, i. 9* Antibodies The antibodies of the invention can be further modified to contain additional non-protein portions that are readily available in the art. The part that is suitable for the derivatization of the antibody is more than a raw material. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, rebel, polyethylene, and poly-l,6-6- Dioxane, ethylene/maleic anhydride copolymer polyglycolic acid (homopolymer or random copolymer) and dextran or polypyrrolidone) poly-ethyl acid, labor, one% poly Propylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyhydrogen ri milk ethylated polyol (e.g., glycerin), polyvinyl alcohol, and a mixture thereof, 119007.doc-96-200813092. Polyethylene glycol propionaldehyde has an advantage because of it. The polymer can have any molecular denier: ‘: sex and in manufacturing ^, 八有仕17 knives and can be branched or unbranched. The amount of polymer attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, t, the amount and/or type of polymer used for derivatization can be determined based on, but not limited to, the following considerations: whether the specificity or function of the antibody to be modified, whether the antibody derivative will be used to define the condition The next treatment.

在另只把例中’提供可藉由曝露於輻射而選擇性加熱之抗體及非蛋自質部分之接合H實施射，非蛋白、質部分為碳奈米管（Kam等人’ Proc· Natl Aead sci ι〇2: 11600 11605 (2GG5))。射可具有任何波長，且包括（但不限於）不損害正常細胞但將非蛋白質部分加熱至殺死鄰近抗體_非蛋白質部分之細胞之溫度的波長。 B·製造抗體之特定方法基於特定融合瘤之方法本發明之抗TAT226單株抗體可使用首先由Kohler等人， 256:495 (1975)所述之融合瘤法來製造，或可藉由重組DNA法（美國專利第4,816,567號）製造。以融合瘤法，小鼠或諸如倉鼠之其他適當宿主動物經免疫以引出產生或能夠產生特異性結合至用於免疫之蛋白之抗體的淋巴細胞。TAT226之抗體一般係藉由多次皮下（sc) 或腹膜内（ip)注射TAT226及佐劑而於動物體内增加。可使用此項技術中所熟知之方法來製備TAT226，其中一些係於本文中進一步描述。舉例而言，TAT226可經重組產生。在 119007.doc •97- 200813092 一實施例中，動物係以含有與免疫球蛋白重鏈之Fc部分融合之TAT226細胞外部分的TAT226衍生物免疫。在一實施例中，動物係以TAT226-IgGl融合蛋白免疫。在一實施例中，動物係以與單磷醯基脂質A(MPL)/海藻糖二黴菌酸酯 (TDM)(Ribi Immunochem. Research，Inc·，Hamilton，MT)之溶液中之TAT226免疫原性衍生物免疫，且於多個部位經皮内注射溶液。兩週後激發動物。七至十四天後將動物放血且檢定血清之抗TAT226力價。激發動物直至力價達到平穩狀態。或者，可使淋巴細胞於活體外免疫。接著使用諸如聚乙二醇之合適融合劑將淋巴細胞與骨髓瘤細胞融合以形成融合瘤細胞（Goding，Monoclonal Antibodies: Principles and ，第 59-103 頁（Academic Press，1986))。使如此製備之融合瘤細胞於例如含有抑制非融合親本骨髓瘤細胞生長或存活之一或多種物質的合適培養基中接種且生長。舉例而言，若親本骨髓瘤細胞缺少次黃嘌呤鳥嘌呤磷酸核糖轉移酶（HGPRT或HPRT)，則融合瘤之培養基通常將包括次黃嘌呤、胺基喋呤及胸苷（HAT培養基），該等物質阻止缺乏HGPRT之細胞生長。在某些實施例中，骨髓瘤細胞為彼等有效融合，藉由所選產生抗體之細胞支持抗體之穩定高含量產生，且對諸如 HAT培養基之培養基敏感之細胞。例示性骨髓瘤細胞株包括（但不限於）小鼠骨髓瘤細胞株，諸如彼等衍生自可購自 Salk Institute Cell Distribution Center， San Diego, 119007.doc -98- 200813092In another example, 'providing an antibody that can be selectively heated by exposure to radiation and a non-egg self-mass moiety, the non-proteinaceous portion is a carbon nanotube (Kam et al.' Proc. Natl) Aead sci ι〇2: 11600 11605 (2GG5)). The shot can have any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein portion to the temperature at which cells adjacent to the antibody-non-protein portion are killed. B. Specific methods for producing antibodies based on specific fusion tumor methods The anti-TAT226 monoclonal antibodies of the present invention can be produced by the fusion method described first by Kohler et al., 256:495 (1975), or by recombinant DNA. Manufactured by the method (U.S. Patent No. 4,816,567). In the fusion tumor method, a mouse or other appropriate host animal such as a hamster is immunized to elicit lymphocytes which produce or are capable of producing antibodies which specifically bind to the protein for immunization. Antibodies to TAT226 are generally increased in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of TAT226 and adjuvant. TAT226 can be prepared by methods well known in the art, some of which are further described herein. For example, TAT226 can be produced recombinantly. In an embodiment of 119007.doc •97-200813092, the animal is immunized with a TAT226 derivative containing an extracellular portion of TAT226 fused to the Fc portion of the immunoglobulin heavy chain. In one embodiment, the animal is immunized with a TAT226-IgG1 fusion protein. In one embodiment, the animal is immunogenic with TAT226 in a solution with monophosphoryl lipid A (MPL) / trehalose dimylate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT). The derivative is immunized and the solution is injected intradermally at multiple sites. The animals were challenged two weeks later. Animals were bled seven to fourteen days later and serum anti-TAT226 strength was determined. Inspire the animal until the price reaches a steady state. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form a fusion tumor cell (Goding, Monoclonal Antibodies: Principles and, pages 59-103 (Academic Press, 1986)). The thus prepared fusion tumor cells are inoculated and grown, for example, in a suitable medium containing one or more substances which inhibit the growth or survival of the non-fused parent osteomyeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of the fusion tumor will typically include hypoxanthine, aminopurine and thymidine (HAT medium). These substances prevent the growth of cells lacking HGPRT. In certain embodiments, the myeloma cells are such that they are efficiently fused, by a stable, high level of cell-supporting antibody of the selected antibody-producing antibody, and to cells that are sensitive to a medium such as HAT medium. Exemplary myeloma cell lines include, but are not limited to, mouse myeloma cell lines, such as those derived from the Salk Institute Cell Distribution Center, San Diego, 119007.doc-98-200813092.

California USA之MOPC-21及MPC-11小鼠腫瘤，及可購自 American Type Culture Collection, Rockville, Maryland 1；8八之8卩-2或又63-八§8-653細胞之骨髓瘤細胞株。亦已關於產生人類單株抗體描述人類骨髓瘤及小鼠-人類雜合骨髓瘤細胞株（Kozbor，J. Immunol·, 133:3001 (1984)；California USA MOPC-21 and MPC-11 mouse tumors, and myeloma cell lines commercially available from American Type Culture Collection, Rockville, Maryland 1; 8-8 8卩-2 or 63-eight §8-653 cells . Human myeloma and mouse-human hybrid osteomyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984);

Brodeur等人，Monoclonal Antibody Production Techniques and Applications，第 51-63 頁（Marcel Dekker，Inc·，New York，1987))。為產生結合至TAT226之單株抗體，檢定融合瘤細胞生長於其中之培養基。較佳地，藉由免疫沉澱反應或藉由諸如放射免疫檢定（RIA)或酶聯結免疫吸附檢定（ELISA)之活體外結合檢定確定由融合瘤細胞產生之單株抗體的結合特異性。例如，單株抗體之結合親和力可藉由Munson等人， Anal. ，107:220 (1980)之史卡查分析（Scatchard analysis)來確定。在識別產生具有所需特異性、親和力及/或活性之抗體的融合瘤細胞之後，可使純系藉由限制性稀釋程序次選殖且藉由標準方法生長（Goding，Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). To generate a monoclonal antibody that binds to TAT226, the medium in which the fusion tumor cells are grown is assayed. Preferably, the binding specificity of the monoclonal antibodies produced by the fusion tumor cells is determined by immunoprecipitation or by a bio-in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of a monoclonal antibody can be determined by Scatchard analysis by Munson et al., Anal., 107: 220 (1980). After identifying a fusion tumor cell that produces an antibody with the desired specificity, affinity, and/or activity, the pure line can be subcultured by a limiting dilution procedure and grown by standard methods (Goding,

Principles and Practice ^ 第 59-103 頁（Academic Press， 1986))。例如，用於此目的之合適培養基包括D-MEM或 RPMI-1640培養基。此外，融合瘤細胞可如動物體内之腹水腫瘤於活體内生長。可將由次純系分泌之單株抗體藉由習知免疫球蛋白純化程序（諸如蛋白A瓊脂糖、羥基磷灰石層析、凝膠電泳、透析或親和力層析）而自培養基、腹水 119007.doc -99- 200813092 或血清合適地分離。 2.特定庫篩檢法本發明之抗TAT226抗體可藉由使用組合庫以篩檢具有所需活性之抗體來製造。舉例而言此項技術中已知用於產生噬菌體呈現庫且為具有所需結合特徵之抗體篩檢此等庫之各種方法。此等方法一般描述於Hoogenboom等人（2001)之 178:l-37(0’Brien等人編， Human Press，Totowa，NJ)中，且在某些實施例中描述於 Lee 等人（2004) J. Mo/· 340:1073-1093 中。實際上，合成抗體純系係藉由篩檢含有呈現與噬菌體鞘蛋白融合之抗體可變區（Fv)之各種片段的噬菌體之噬菌體庫來選擇。此等噬菌體庫係藉由對抗所需抗原之親和層析法而淘出。將表現可結合至所需抗原之Fv片段的純系吸附至抗原上且從而與庫中之非結合純系分離。接著使結合純系自抗原溶離，且可藉由額外抗原吸附/溶離循環而進一步富集。可藉由以下步驟獲得本發明之任何抗TAT226抗體：設計用以選擇所關注噬菌體純系之合適抗原筛檢程序，繼而使用 Kabat 等人，<9/ 〇/Principles and Practice ^ Pages 59-103 (Academic Press, 1986)). For example, suitable media for this purpose include D-MEM or RPMI-1640 medium. In addition, the fusion tumor cells can be grown in vivo as an ascites tumor in an animal. Monoclonal antibodies secreted by sub-pure lines can be cultured from the culture medium by aseptic immunoglobulin purification procedures (such as protein A agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography) from the culture medium, ascites 119007.doc -99- 200813092 or serum is suitably isolated. 2. Specific Library Screening Method The anti-TAT226 antibody of the present invention can be produced by using a combinatorial library to screen for antibodies having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are generally described in Hoogenboom et al. (2001) 178: 1-37 (edited by 0' Brien et al., Human Press, Totowa, NJ) and described in certain embodiments by Lee et al. (2004). J. Mo/· 340: 1073-1093. In fact, synthetic antibody-only lines are selected by screening a phage library containing phage displaying various fragments of the antibody variable region (Fv) fused to the phage sheath protein. These phage libraries are panned by affinity chromatography against the desired antigen. A pure line that exhibits an Fv fragment that binds to the desired antigen is adsorbed onto the antigen and thereby separated from the unbound pure line in the library. The bound pure phase is then lysed from the antigen and further enriched by additional antigen adsorption/dissolution cycles. Any of the anti-TAT226 antibodies of the invention can be obtained by the following procedure: a suitable antigen screening procedure designed to select the pure line of the phage of interest, followed by Kabat et al., <9/ 〇/

Immunological ，第五版，NIH Publication 91_ 3242，Bethesda MD (1991)，第1-3卷中描述之來自所關注噬菌體純系之Fv序列及合適恆定區（Fc)序列建構全長抗 TAT226抗體純系。在某些實施例中，抗體之抗原結合結構域係由約110個胺基酸之兩個可變（V)區形成，其各自來自輕鏈（VL)及重 119007.doc -100- 200813092 鏈（VH) ’二者均存在三個高變環（HVR)或互補判定區 (CDR)。如 Winter等人，义狐 /所廳似/，12: 433-455 (1994)中所述，可變結構域可以單鏈卜卜“幻片段（其中vh 與VL經由短、彈性肽共價連接）或Fab片段（其中其各自融合至怪定結構域且非共價地相互作用）形式功能性地呈現於噬菌體上。如本文所用之編碼噬菌體純系之scFv及編碼噬菌體純系之Fab統稱為”fv噬菌體純系”或”1^純系”。如Winter等人，J㈣·心v〜卿⑽厂，12: 433 455 (1994) 中所述，VH及VL基因之譜系可藉由聚合酶鏈反應（1>(：11)獨立地選殖且於噬菌體庫中隨機重組，接著可為抗原結合純系搜尋噬菌體庫。來自經免疫源之庫在無需建構融合瘤之 h況下提供免疫原之高親和力抗體。或者，如Griffiths等人，五MBO 乂 12: 725·734 (1993)所述，可選殖天然譜系以在無任何免疫之情況下提供廣泛範圍之非自體抗原以及自體抗原之人類抗體的單一來源。最後，如H〇〇genb〇〇m及Immunological, Fifth Edition, NIH Publication 91_3242, Bethesda MD (1991), Volumes 1-3, the Fv sequence from the phage-pure line and the appropriate constant region (Fc) sequence construct a full-length anti-TAT226 antibody line. In certain embodiments, the antigen binding domain of an antibody is formed by two variable (V) regions of about 110 amino acids, each from a light chain (VL) and weighing 119007.doc-100-200813092 (VH) 'There are three hypervariable loops (HVR) or complementary decision regions (CDRs). As described in Winter et al., fox/shovel/, 12: 433-455 (1994), the variable domain can be a single-stranded phantom fragment (where vh and VL are covalently linked via a short, elastic peptide) Or Fab fragments (wherein each fused to a strange domain and non-covalently interacting) form functionally presented on the phage. As used herein, a scFv encoding a phage-pure line and a Fab encoding a phage-pure line are collectively referred to as "fv The phage is pure or "1^ pure". As described in Winter et al., J (4), Heart v~Qing (10), 12: 433 455 (1994), the lineage of VH and VL genes can be polymerase chain reaction (1&gt) (:11) independently colonized and randomly recombined in the phage library, and then the phage library can be searched for the antigen-binding pure line. The library from the source of the immunogen provides the high-affinity antibody of the immunogen without constructing the fusion tumor. Alternatively, as described by Griffiths et al., 5 MBO 乂 12: 725. 734 (1993), the natural lineage can be selected to provide a wide range of non-autoantigen and autoantigen human antibodies without any immunization. Single source. Finally, such as H〇〇ge Nb〇〇m and

Wmter，J· Μο/·价〇/·，227: 381-388 (1992)所述，亦可藉由自幹細胞選殖未重排基因區段且使用含有隨機序列之 PCR引子以編碼高變CDR3區且完成活體外重排來合成製造天然庫。在某些實施例中，絲狀噬菌體係藉由與微量鞘蛋白ρΠΙ 融合而用以呈現抗體片段。抗體片段可呈現為單鏈Fv片段形式（其中VH及VL結構域係藉由彈性多肽間隔基連接至相同多肽鏈上，例如MarkS等人，义Μ0/·出0/·，222: 581_597 (1991)所述）或Fab片段形式（其中一條鏈融合至“η且另一 119007.doc 200813092 條經分泌入細菌宿主細胞周質中，於該周質中藉由移動一些野生型鞠蛋白來組裝呈現於噬菌體表面上之Fab勒蛋白結構，例如Hoogenboom等人，iVwc/. 及以·，19: 4133- 4137 (1991)中所述）。一般而言，編碼抗體基因片段之核酸係由自人類或動物採集之免疫細胞獲得。若需要偏利於抗TAT226純系之庫，則以TAT226使受檢者免疫以產生抗體反應，且回收脾細胞及/或循環B細胞，其他周邊血液淋巴細胞（PBL)以用於建構庫。在一較佳實施例中，藉由在載運功能人類免疫球蛋白基因陣列（且缺少功能内源性抗體產生系統）之轉殖基因小鼠體内產生抗TAT226抗體反應，以使得TAT226免疫產生對抗TAT226之產生使B細胞之人類抗體而獲得偏利於抗 TAT226純系之人類抗體基因片段庫。下文描述產生人類抗體之轉殖基因小鼠之產生。可藉由使用合適篩檢程序以分離表現TAT226特異性膜結合抗體之B細胞，例如藉由使用TAT226親和層析法之細胞分離或將細胞吸附於螢光染料標記之TAT226，繼而藉由流式活化細胞檢選（FACS)而獲得抗TAT226反應性細胞群體之額外富集。或者，使用來自未經免疫供體之脾細胞及/或B細胞或其他P B L提供可能抗體譜系之更佳代表，且亦容許使用其中 TAT226不具抗原性之任何動物（人類或非人類）物種建構抗體庫。對於併入活體外抗體基因建構之庫而言，自受檢者才木集幹細胞以k供編碼未重排抗體基因區段之核酸。所關 119007.doc -102- 200813092 注之免疫細胞可自各種動物物種獲得’諸如人類、小鼠、大鼠、兔類、狼（luprine)、犬科、貓科、豬、牛、馬及禽類等。將編碼抗體可變基因區段（包括VH及VL區段）之核酸自所關注細胞回收且擴增。在重排VH及VL基因庫之情況下，如 Orlandi 等人，iVoc· Scz·· (USA), 86: 3 833-3837 (1989)中所述，可藉由將染色體組DNA或mRNA 自淋巴細胞分離，繼而與匹配重排VH及VL基因之5’端及3’ 端之引子進行聚合酶鏈反應（PCR)獲得所需DNA，藉此製造供表現之不同V基因譜系。如Orlandi等人（1989)及Ward 等人，341: 544-546 (1989)中所述，可自 cDNA及染色體組DNA擴增V基因，其中後向引子在編碼成熟V結構域之外顯子5’端處且正向引子基於J區段内。然而，對於自 cDNA 擴增而言，如 Jones 等人，，9: 88-89 (1991)中所述，後向引子亦可基於導引外顯子内；且如等尺，Proc· Natl· Acad· Sci· (USA)，U·· 5Ί2名-5Ί32 (1989)中所述，正向引子位於恆定區内。為使互補最大化，如Orlandi等人（1989)或Sastry等人（1989)所述，可將簡併性併入引子中。在某些實施例中，例如Marks等人，*/· Mo/·价〇/.，222: 581-597 (1991)之方法中所述或Orum 等人，iVwckk Jcz·心及以·，21: 4491-4498 (1993)之方法中所述，藉由使用靶向各V基因家族之PCR引子使庫多樣性最大化，從而擴增免疫細胞核酸樣品中存在之所有可用VH 及VL·排列。為將經擴増DNA選殖入表現載體中，可將極 119007.doc -103- 200813092 少之限制位點如Orlandi等人（1989)中所述作為一端之標記或如 Clackson等人，352: 624-628 (1991)中所述藉由以經標記引子進一步PCR擴增而引入PCR引子内。合成重排之V基因譜系可活體外衍生自V基因區段。大部分人類VH基因區段已經選殖及定序（於Tomlinson等人， J. Mo/·价〇/·，227: 776-798 (1992)中報導）且繪圖（於 Matsuda等人，GMei·，3: 88-94 (1993)中報導），·如 HoogenboomA Winter, J. Mol Biol., 221 \ 381-388 (1992)t 所述，該等經選殖區段（包括HI及H2環之所有主要構形）可用以產生具有編碼不同序列及長度之H3環之PCR引子的不同 VH基因譜系。如 Barbas 等人，尸roc. TV^i/· *SW· t/a，89: 4457-4461 (1"2)中所述，亦可製造所有序列多樣性聚集於單一長度之長H3環中之VH譜系。人類Vk及νλ 區段已經選殖且定序（於Williams及Winter，jEwr. ·/· /mmw⑽/·，23: 1456-1461 (1993)中報導）且可用以製造合成輕鏈譜系。基於VH及VL折疊及L3及H3長度範圍之合成V 基因譜系將編碼具有大量結構多樣性之抗體。在編碼DNA 之V基因擴增後，可根據Hoogenboom &Winter，J.M(9/· 5M/·，227: 381-388 (1992)之方法活體外重排生殖系V基因區段。可藉由以若干方式將VH與VL基因譜系組合在一起來建構抗體片段譜系。各譜系可於不同載體中產生且將載體於活體外重組，例如Hogrefe等人，Gwe，128: 119-126 (1993)中所述，或藉由組合感染於活體内重組，例如 119007.doc -104- 200813092Wmter, J. Μο/. 〇〇 /·, 227: 381-388 (1992), can also encode high-variant CDR3 by selecting non-rearranged gene segments from stem cells and using PCR primers containing random sequences. The area is completed and the in vitro rearrangement is completed to synthesize a natural reservoir. In certain embodiments, the filamentous phage system is used to present antibody fragments by fusion with a minimal amount of sheath protein. The antibody fragment may be in the form of a single-chain Fv fragment (wherein the VH and VL domains are linked to the same polypeptide chain by an elastic polypeptide spacer, such as MarkS et al., Μ0/·0/·, 222: 581_597 (1991) ) or Fab fragment form (one of the strands is fused to "n and another 119007.doc 200813092 is secreted into the periplasm of the bacterial host cell, and is assembled on the surface of the phage by moving some wild-type prion protein in the periplasm The Fabler protein structure, for example, Hoogenboom et al., iVwc/. and I, 19: 4133-4137 (1991). In general, the nucleic acid encoding the antibody gene fragment is immunized from human or animal. The cells are obtained. If it is necessary to favor the TAT226 pure lineage, the subject is immunized with TAT226 to generate an antibody reaction, and the spleen cells and/or circulating B cells are recovered, and other peripheral blood lymphocytes (PBL) are used for constructing the library. In a preferred embodiment, the anti-TAT226 antibody is produced by transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system). In order to obtain a human antibody gene fragment library which is biased against the TAT226 pure line, the TAT226 is immunized to produce a human antibody against BAT against the production of TAT226. The production of a transgenic mouse producing a human antibody is described below. Screening procedures to isolate B cells that express TAT226-specific membrane-bound antibodies, for example, by cell separation using TAT226 affinity chromatography or by adsorption of cells to fluorescent dye-labeled TAT226, followed by flow-activated cell selection ( FACS) to obtain additional enrichment of anti-TAT226 reactive cell populations. Alternatively, use spleen cells and/or B cells from unimmunized donors or other PBLs to provide a better representation of possible antibody lineages, and also allow the use of TAT226 An antibody library is constructed from any animal (human or non-human) species that is not antigenic. For libraries incorporated into the construction of an in vitro antibody gene, the self-tester self-collects the stem cells to encode the unrearranged antibody gene segment. Nucleic acid. 119007.doc -102- 200813092 Injected immune cells can be obtained from various animal species 'such as human, mouse, rat Rabbits, luprines, canines, felines, pigs, cows, horses, birds, etc. Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered and amplified from cells of interest. In the case of rearrangement of the VH and VL gene pools, as described in Orlandi et al., iVoc. Scz. (USA), 86: 3 833-3837 (1989), by genomic DNA or mRNA from lymph The cells are isolated, and then the primers for matching the 5' and 3' ends of the rearranged VH and VL genes are subjected to polymerase chain reaction (PCR) to obtain desired DNA, thereby producing different V gene lineages for expression. The V gene can be amplified from cDNA and genomic DNA as described in Orlandi et al. (1989) and Ward et al, 341: 544-546 (1989), wherein the backward primer encodes a mature V domain exon. The 5' end and the forward reference are based on the J segment. However, for cDNA amplification, as described in Jones et al., 9: 88-89 (1991), the backward primer can also be based on the guide exon; and if it is equal, Proc·Natl· Acad·Sci· (USA), U·· 5Ί2 -5Ί32 (1989), the forward primer is located in the constant region. To maximize complementarity, degeneracy can be incorporated into the primers as described by Orlandi et al. (1989) or Sastry et al. (1989). In some embodiments, such as those described by Marks et al., */. Mo/., 〇/., 222: 581-597 (1991) or Orum et al., iVwckk Jcz·心和以, 21 : 4491-4498 (1993), by using PCR primers that target each V gene family to maximize pool diversity, thereby amplifying all available VH and VL alignments present in the immune cell nucleic acid sample. In order to select the expanded DNA into the expression vector, a restriction site of 119007.doc -103-200813092 may be used as a marker for one end as described in Orlandi et al. (1989) or as Clackson et al., 352: The PCR primer was introduced by further PCR amplification with a labeled primer as described in 624-628 (1991). The synthetic rearranged V gene lineage can be derived in vitro from the V gene segment. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mo/. 〇/, 227: 776-798 (1992)) and plotted (in Matsuda et al., GMei· , 3: 88-94 (1993), as described in Hoogenboom A Winter, J. Mol Biol., 221 \ 381-388 (1992) t, the selected colonies (including HI and H2 rings) All major configurations can be used to generate different VH gene lineages with PCR primers encoding H3 loops of different sequences and lengths. As described in Barbas et al., corpse roc. TV^i/. *SW·t/a, 89: 4457-4461 (1"2), it is also possible to create all sequence diversity in a single length of long H3 ring. VH lineage. The human Vk and νλ segments have been cloned and sequenced (reported in Williams and Winter, jEwr. ·/· /mmw(10)/·, 23: 1456-1461 (1993)) and can be used to make synthetic light chain lineages. Synthetic V gene lineages based on VH and VL folding and L3 and H3 length ranges will encode antibodies with substantial structural diversity. After amplification of the V gene encoding DNA, the germline V gene segment can be rearranged in vitro according to Hoogenboom & Winter, JM (9/· 5M/, 227: 381-388 (1992). The antibody fragment lineages are constructed by combining the VH and VL gene lineages in a number of ways. Each lineage can be produced in a different vector and the vector recombined in vitro, for example, Hogrefe et al, Gwe, 128: 119-126 (1993) Said, or by infective recombination in vivo, for example 119007.doc -104- 200813092

Waterhouse等人，A^wc/· i?a·，21: 2265-2266 (1993)中所述之loxP系統。活體内重組途徑利用Fab片段之雙鏈性質以克服大腸桿菌轉運效率對庫大小所施加之限制。將天然VH及VL譜系獨立地選殖，一個選殖入噬菌粒中且另一個選殖入嗟菌體載體中。接著將兩個庫藉由含有嗤菌粒之細菌的噬菌體感染組合，以使得各細胞含有不同組合且庫大小僅受所存在細胞數之限制（約1012個純系）。兩載體均含有活體内重組信號，以使得VH及VL·基因重組於單一複製子上且共封裝於噬菌體病毒粒子中。該等巨庫提供大量具有優良親和力（約1〇·8 Μ之Κ，1)之各種抗體。或者，可將譜系例如如Barbas等人，iVoc· #如/· dead· 5W. [/以，88: 7978-7982 (1991)中所述連續選殖入相同載體中，或由PCR組裝在一起且接著例如如Clackson等人， 352: 624-628 (1991)中所述選殖。PCR組裝亦可用以接合VH及VL DNA與編碼彈性肽間隔基之DNA以形成單鏈Fv(scFv)譜系。在另一技術中，如Embleton等人，A/^c/· dczWs 20: 3831-3837 (1992)中所述，”細胞内 PCR 組裝” 係用以在淋巴細胞内由PCR組合VH與VL基因，且接著選殖連接基因之譜系。如同上文之Winter等人（1994)所述，經天然庫（天然或合成）產生之抗體可具有適度親和力（約1〇6至1〇7 M·1之K〆），但亦可藉由建構第二庫且自其再選擇而於活體外模擬親和力成熟。舉例而言，可藉由Hawkins等人，J. Mo/· 5/(9/·， 226: 889-896 (1992)之方法及 Gram等人，Proc· Λ^ί/· Jcad· 119007.doc -105· 200813092 •Sd t/M, 89·· 3576-3580 (1992)之方法，使用易誤用之聚合酶（於Leung等人，1: 11-15 (1989)中報導）活體外隨機引入突變。此外，可藉由在所選擇之個別Fv純系中例如使用具有載運橫跨所關注CDR之隨機序列之引子的 PCR使一或多個CDR隨機突變且篩檢較高親和力純系來進行親和力成熟。WO 9607754(公開於1996年3月14日）描述用於在免疫球蛋白輕鏈之互補判定區中誘導突變以產生輕鏈基因庫之方法。如Marks等人，⑽/.，1〇: 779-783 (1992)中所述’另一種有效途徑為使由嗟菌體呈現所選擇之VH或VL結構域與自未經免疫之供體所獲得之天然產生之V結構域變異體譜系重組，且在若干回合之鏈改組中篩檢具有較高親和力者。此技術使得可產生具有1〇-9 Μ或小於ΙΟ·9 Μ之親和力的抗體及抗體片段。可藉由此項技術中已知之各種技術來完成庫之篩檢。舉例而言，ΤΑΤ226可用以塗覆吸附板之孔，表現於附著至吸附板之宿主細胞上或用於細胞檢選中，或接合至生物素以由經抗生蛋白鏈菌素（streptavidin)塗覆之珠粒俘獲或用於篩檢噬菌體呈現庫之任何其他方法中。在適用於使至少一部分嗤菌體粒子與吸附劑結合之條件下使嗟菌體庫樣本與固定化TAT226接觸。通常，選擇包括 pH值、離子強度、溫度及其類似者之條件以模擬生理條件。洗務結合至固相之噬菌體且接著例如Barbas等人，尸_·勤//· dew/· 仍尤 88: 7978_7982 (1991)所述將其以酸溶離·，或例如Marks等人，义Μ〇/出〇/，222· 581 597 119007.doc 200813092 (1991)所述以驗溶離；或例如以類似於（1：1&〇1<：8〇11等人， 352: 624-628 (1991)之抗原競爭法之程序藉由 TAT226抗原競爭溶離。噬菌體可以單回合選擇富集20-1，000倍。此外，可使經富集噬菌體生長於細菌培養物中且另外經受數回合之選擇。選擇效率視多種因素而定，包括洗滌期間之解離動力學及單一噬菌體之多個抗體片段是否可同時與抗原嚙合。可藉由在固相中使用短時間洗滌（short washes)、多價嗟菌體呈現及高抗原塗覆密度保持具有快速解離動力學（及弱結合親和力）之抗體。高密度不僅經由多價相互作用穩定噬菌體，且有利於使解離之噬菌體再結合。可藉由使用如 Bass等人，Prokzw，8: 309-314 (1990)及 W0 92/09690 中所述之長時間洗滌及單價噬菌體呈現及如Marks等人， 10: 779-783 (1992)中所述之低抗原塗覆密度促進選擇具有緩慢解離動力學（及優良結合親和力）之抗體。可能在對TAT226具有不同親和力之噬菌體抗體（即使具有略微不同之親和力）之間進行選擇。然而，所選抗體之隨機突變（例如，以一些親和力成熟技術進行之突變）可能產生多種突變體，其中大部分與抗原結合且較少部分具有較高親和力。藉由限制TAT226，競爭淘汰極少之高親和力噬菌體。為保持所有較高親和力突變體，可將噬菌體以過量經生物素標記之TAT226培育，但其中經生物素標記之 TAT226具有比TAT226之恆定靶莫耳濃度親和力低之莫耳 119007.doc -107- 200813092 濃度。接著可藉由經抗生蛋白鏈菌素塗覆之順磁性珠粒俘獲高親和力結合噬菌體。此’’平衡俘獲”藉由容許將具有低至兩倍高之親和力之突變純系與具有較低親和力之大量過量噬菌體分離的敏感性，使得可根據其結合親和力選擇抗體。亦可操控用於洗滌與固相結合之噬菌體之條件以基於解離動力學來區別。可基於活性來選擇抗TAT226純系。在某些實施例中，本發明提供結合至天然表現TAT226之活細胞的抗TAT226抗體。在一實施例中，本發明提供阻斷TAT226配位體與 TAT226之間之結合，但不阻斷TAT226配位體與第二蛋白之間之結合的抗TAT226抗體。對應於此等抗TAT226抗體之Fv純系可藉由以下方法來選擇：（1)如上所述將抗 TAT226純系自噬菌體庫分離且視情況藉由增長合適細菌宿主中之群體而擴增所分離之噬菌體純系群體；（2)選擇分別需要阻斷及非阻斷活性之TAT226及第二蛋白；（3)將抗 TAT226噬菌體純系吸附至固定TAT226 ; (4)使用過量第二蛋白以溶離識別與第二蛋白之結合決定子重疊或共用之 TAT226結合決定子的任何非所需純系；及（5)溶離步驟（4) 後保持吸附之純系。視情況而言，可將具有所需阻斷/非阻斷特性之純系藉由將本文所述之選擇程序重複一或多次而進一步富集。使用習知程序（例如藉由使用經設計以自融合瘤或喧菌體DNA模板特異性擴增所關注之重鏈及輕鏈編碼區的寡核普酸引子），易於分離且定序編碼本發明之融合瘤衍生之 119007.doc -108- 200813092 單株抗體或噬菌體呈現Fv純系的DNA。經分離後，可將 DNA置於表現載體中，接著將其轉染至不另外產生免疫球蛋白之宿主細胞（諸如大腸桿菌細胞、猿猴COS細胞、中國倉鼠卵巢（CHO)細胞或骨髓瘤細胞）中以於重組宿主細胞中獲得所需單株抗體之合成。關於在細菌中重組表現編碼抗體之DNA之綜述文章包括Skerra等人，Cwrr· Opinion in Immunol·，5: 25 6 (1993)^. Pluckthun, Immunol. Revs, 13 0: 151 (1992)。可將編碼本發明之Fv純系之DNA與編碼重鏈及/或輕鏈恆定區之已知DNA序列（例如，可自Kabat等人（同上文）獲得之適當DNA序列）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，任何同型之恆定區均可用於此目的，包括IgG、IgM、IgA、IgD及IgE恒定區，且此等恆定區可自任何人類或動物物種獲得。如本文所用之定義 "嵌合”或’’雜交”抗體中包括衍生自一種動物（諸如人類）物種之可變結構域DNA且接著與另一動物物種之恆定區DNA 融合以形成用於”雜交”全長重鏈及/或輕鏈之編碼序列的Fv 純系。在某些實施例中，將衍生自人類可變DNA之Fv純系與人類恆定區DNA融合以形成用於全長或部分長度人類重鍵及/或輕鍵之編碼序列。亦可例如藉由以人類重鏈及輕鏈恒定結構域之編碼序列代替衍生自融合瘤純系之同源鼠序列來修飾編碼本發明之衍生自融合瘤之抗TAT226抗體的DNA(例如Morrison等尺，Proc. Natl. Acad, Sci. 8 1: 685 1-6855 (1984)之方 119007.doc -109- 200813092 法）。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所有或部分編碼序列來進一步修飾編碼衍生自融合瘤或Fv純系之抗體或片段之DNA。以此方式，製備具有本發明之衍生自Fv純系或融合瘤純系之抗體的結合特異性之"嵌合”或"雜交"抗體。载艘、宿主細胞及重組方法為重組產生本發明之抗體，將編碼其之核酸分離且*** 可複製載體中以進一步選殖（擴增DNA)或表現。編碼抗體之DAN係使用習知程序（例如，藉由使用可特異性結合至編碼抗體之重鏈及輕鏈之基因的寡核苷酸探針）輕易地分離及定序。可使用多種載體。載體之選擇部分取決於待使用之宿主細胞。一般而言，宿主細胞具有原核或真核（一痛又為甫乳動物）來源。將瞭解，任何同型之怪定區均可用於此目的，包括IgG、IgM、IgA、IgD及IgE恆定區，且此等恆定區可自任何人類或動物物種獲得。 a)使用原核宿主細產生抗體： (1)載體建構可使用標準重組技術獲得編碼本發明抗體之多肽組份的聚核苦酸序列。可將所要聚核㈣序列自產生抗體之細胞 (諸如融合瘤細胞）分離且定序。或者，可使用核苦酸合成器或PCR技術合成聚核苷酸。獲得之後，將編碼多狀之序列***可於原核宿主中複製及表現異源性聚核苷酸之重組載體中it匕項技術中可用且已知之諸多載體均可用於本發月之目的適w载體之選擇將主要取決於待插人載體中之 H9007.doc •110- 200813092 尺寸及待經載體轉化之特定宿主細胞。各載體視其功能（異源性聚核苷酸之擴增或表現，或此兩者）及其與其所 :在之特定宿主細胞之相容性而含有各種組份。載體組份一般包括(但不限於)：複製起點、標記基因之選擇、啟動子、核糖體結合位點(RBS)、信號序列、異源性核酸*** 及轉錄終止序列。般而$，將含有複製子及衍生自與宿主細胞相容之物種之對照序列的質體載體與該等宿主聯合使用。載體通常攜帶複製位點，以及可在轉化細胞中提供表型選擇之標記序列舉例而S，大腸桿菌通常係使用衍生自大腸桿菌物種之質體PBR322轉化。pBR322含有編碼安比西林 (ampiC1llin)(Amp)及四環素（Tet)抗性之基因，且因此提供識別轉化細胞之簡易方式。pBR322、其衍生物或其他微生物質體或噬菌體亦可含有或經改質以含有可由微生物體用於表現内源性蛋白之啟動子。用於表現特定抗體之 PBR322衍生物之實例係詳細描述於Carter等人之美國專利第 5,648,237號中。此外，可將含有複製子及與宿主微生物相容之對照序列之噬菌體載體與該等宿主聯合用作轉化載體。舉例而言，可將諸如λΟΕΜ·ΤΜ··11之噬菌體用於製造可用以轉化諸如大腸桿菌LE392之敏感宿主細胞的重組載體。本發明之表現載體可包含編碼各多肽組份之兩個或兩個以上啟動子-順反子對。啟動子為定位於調節其表現之順反子上游（5，）之未轉譯調節序列。原核啟動子通常分為兩 119007.doc -111- 200813092 類··誘導性及組成性。誘導性啟動子為在其反應培養條件 (例如存在或不存在營養物或溫度改變）改變之控制下引發增加程度之順反子轉錄的啟動子。眾所熟知大量由各種可能之宿主細胞辨識之啟動子。藉由將啟動子經由限制酶消化作用自源DNA移除且將經分離之啟動子序列***本發明之載體中，可將所選擇之啟動子可操作性地連接至編碼輕鏈或重鏈之順反子DNA。原生啟動子序列及許多異源性啟動子可用以直接擴增及/或表現靶基因。在一些實施例中，利用異源性啟動子，因其與原生靶多肽啟動子相比一般容許經表現靶基因之更多轉錄及更南產率。適用於與原核宿主一起使用之啟動子包括PhoA啟動子、β-半乳糖苷酶及乳糖啟動子系統、色胺酸（trp)啟動子系統及諸如tac或trc啟動子之雜交啟動子。然而，在細菌中起作用之其他啟動子（諸如其他已知之細菌或噬菌體啟動子）亦為適用的。其核㈣序列已公開，藉此使得熟習此項技術者可使用冑接子或接附㈣其可操㈣地接合至編碼靶輕鏈及重鏈之順反子（Siebenlist等人（198〇) 2〇: 269) ’從而供應任何所需之限制位點。在本發明之-態樣中’重組載體中之各順反子包含引導經表現多肽橫穿膜移位之分泌信號序列組份…般而言，信號序列可為載體之崎，或其可為插人載體之㈣狀 DNA之-部分。為本發明目的所選擇之信號序列應為由宿主細胞所識別及加工（亦即藉由信號肽酶裂解）之信號序 119007.doc •112- 200813092The loxP system described in Waterhouse et al., A^wc/. i?a., 21: 2265-2266 (1993). The in vivo recombination pathway utilizes the double-stranded nature of the Fab fragment to overcome the limitations imposed by the E. coli transport efficiency on the size of the library. The natural VH and VL lines were independently selected, one was selected into the phagemid and the other was selected into the sputum carrier. The two libraries are then combined by phage infection of the bacteria containing the sputum granules such that each cell contains a different combination and the pool size is limited only by the number of cells present (about 1012 pure lines). Both vectors contain an in vivo recombination signal such that the VH and VL genes are recombined on a single copy and co-packaged in phage virions. These giant libraries provide a large number of antibodies with excellent affinities (about 1 〇 8 Μ Κ, 1). Alternatively, the lineage can be continuously ligated into the same vector, for example, as described by Barbas et al., iVoc. #如/· dead·5W. [/, 88: 7978-7982 (1991), or assembled by PCR. And then, for example, as described in Clackson et al, 352: 624-628 (1991). PCR assembly can also be used to join VH and VL DNA with DNA encoding an elastin spacer to form a single chain Fv (scFv) lineage. In another technique, as described in Embleton et al., A/^c/. dczWs 20: 3831-3837 (1992), "intracellular PCR assembly" is used to combine VH and VL genes by PCR in lymphocytes. And then the lineage of the linked gene is selected. As described by Winter et al. (1994) above, antibodies produced by natural libraries (natural or synthetic) may have moderate affinity (about 〇6 to 1〇7 M·1 K〆), but also by The second library was constructed and re-selected to simulate affinity maturation in vitro. For example, by Hawkins et al., J. Mo/. 5/(9/., 226: 889-896 (1992) and Gram et al., Proc. Λ^ί/Jcad 119007.doc -105· 200813092 • Sd t/M, 89·3576-3580 (1992), using a misused polymerase (reported in Leung et al., 1: 11-15 (1989)) to introduce mutations in vitro In addition, affinity maturation can be performed by randomly mutating one or more CDRs and screening for higher affinity pure lines in selected individual Fv lines, for example, using PCR with primers carrying random sequences spanning the CDRs of interest. WO 9607754 (published on Mar. 14, 1996) describes a method for inducing mutations in the complementarity determining regions of immunoglobulin light chains to produce a light chain gene pool. For example, Marks et al., (10)/., 1〇: 779 Another effective pathway as described in -783 (1992) is to recombine the VH or VL domain selected by the sputum cell and the naturally occurring V domain variant lineage obtained from the unimmunized donor, And screening for higher affinity in several rounds of chain reorganization. This technique allows for the production of 1〇-9 Μ or small亲·9 Μ affinity antibody and antibody fragment. The library can be screened by various techniques known in the art. For example, ΤΑΤ226 can be used to coat the pores of the adsorption plate, which is manifested by attachment to the adsorption plate. On host cells or for cell sorting, or conjugated to biotin for capture by streptavidin coated beads or any other method for screening phage display libraries. The sputum sample is contacted with the immobilized TAT 226 under conditions such that at least a portion of the bacteriophage particles are combined with the adsorbent. Typically, conditions including pH, ionic strength, temperature, and the like are selected to simulate physiological conditions. The phage that binds to the solid phase and then, for example, Barbas et al., corpse _·勤//· dew/· are still dissolved in acid as described in 88: 7978_7982 (1991), or for example, Marks et al. / 〇〇 /, 222 · 581 597 119007.doc 200813092 (1991) to test dissolution; or for example similar to (1:1 & 〇 1 <: 8 〇 11 et al, 352: 624-628 (1991) Procedure for antigen competition by TAT226 antigen The phage can be enriched 20-1,000 times in a single round. In addition, the enriched phage can be grown in bacterial culture and additionally subjected to several rounds of selection. Selection efficiency depends on a number of factors, including during washing The dissociation kinetics and whether multiple antibody fragments of a single phage can simultaneously engage the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinity) can be maintained by using short washes, multivalent sputum presentation and high antigen coating density in the solid phase. The high density not only stabilizes the phage via multivalent interactions, but also facilitates recombination of the dissociated phage. Long-term washing and monovalent phage presentation as described in Bass et al, Prokzw, 8: 309-314 (1990) and WO 92/09690 can be used and as in Marks et al, 10: 779-783 (1992) The low antigen coating density promotes the selection of antibodies with slow dissociation kinetics (and superior binding affinity). It is possible to choose between phage antibodies with different affinities for TAT226, even with slightly different affinities. However, random mutations in selected antibodies (e. g., mutations by some affinity maturation techniques) may result in a variety of mutants, most of which bind to the antigen and a small fraction have a higher affinity. By limiting TAT226, competition eliminates very few high-affinity phage. To maintain all of the higher affinity mutants, the phage can be grown in excess of biotinylated TAT226, but the biotinylated TAT226 has a lower affinity than the TAT226 constant target molar concentration of 119007.doc-107- 200813092 Concentration. The high affinity binding phage can then be captured by the streptavidin coated paramagnetic beads. This ''balance capture') allows for the selection of antibodies based on their binding affinity by allowing the sensitivity of a mutant line with affinity as low as twice as high to be separated from a large excess of phage with lower affinity. The conditions of the phage bound to the solid phase are distinguished based on the dissociation kinetics. The anti-TAT226 pure line can be selected based on the activity. In certain embodiments, the invention provides an anti-TAT226 antibody that binds to a living cell that naturally expresses TAT226. In the embodiments, the present invention provides an anti-TAT226 antibody that blocks the binding between a TAT226 ligand and TAT226, but does not block the binding between the TAT226 ligand and the second protein. The Fv corresponding to the anti-TAT226 antibody The pure line can be selected by the following methods: (1) separating the anti-TAT226 pure autophagy library as described above and amplifying the isolated phage pure line population by increasing the population in the appropriate bacterial host, as appropriate; (2) selecting respectively Requires blocking and non-blocking activity of TAT226 and second protein; (3) adsorption of anti-TAT226 phage pure line to fixed TAT226; (4) use excess second White is identified by dissolving and binding to a second protein to determine any undesired pure lines of the TAT226 binding determinant that overlap or share; and (5) the pure line that remains adsorbed after the dissolving step (4). The desired blocking/non-blocking properties are further enriched by repeating the selection procedure described herein one or more times. Using conventional procedures (eg, by using self-fused tumor or sputum DNA) Template-specific amplification of the oligonucleotides of the heavy and light chain coding regions of interest), easy to isolate and sequence encoding the fusion tumors of the invention 119007.doc -108- 200813092 Monoclonal antibodies or phage display Fv Pure DNA. After isolation, the DNA can be placed in a performance vector and then transfected into host cells that do not otherwise produce immunoglobulins (such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells or The synthesis of the desired monoclonal antibodies is obtained in recombinant host cells in myeloma cells. Review articles on recombinant expression of DNA encoding antibodies in bacteria include Skerra et al., Cwrr. Opinion in Immunol, 5: 25 6 (1993) ^. Pluckthun, Immunol. Revs, 13 0: 151 (1992). The DNA encoding the Fv pure line of the invention can be known to encode heavy and/or light chain constant regions. DNA sequences (e.g., suitable DNA sequences obtainable from Kabat et al. (supra)) are combined to form a pure line encoding a full length or partial length heavy and/or light chain. It will be appreciated that any isotype constant region can be used for this. Objectives include IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions are available from any human or animal species. As used herein, a "chimeric" or "hybridization" antibody includes variable domain DNA derived from an animal (such as a human) species and then fused to constant region DNA of another animal species to form for" An Fv pure line that hybridizes to the coding sequence for the full length heavy and/or light chain. In certain embodiments, Fv pure lines derived from human variable DNA are fused to human constant region DNA to form coding sequences for full length or partial length human heavy and/or light bonds. The DNA encoding the anti-TAT226 antibody derived from the fusionoma of the present invention may also be modified, for example, by replacing the homologous mouse sequence derived from the fusion tumor with the coding sequence of the human heavy and light chain constant domains (eg, Morrison et al. , Proc. Natl. Acad, Sci. 8 1: 685 1-6855 (1984) pp. 119007.doc -109- 200813092 Act). DNA encoding an antibody or fragment derived from a fusion tumor or Fv pure line can be further modified by covalent attachment to all or part of the coding sequence of the immunoglobulin coding sequence, non-immunoglobulin polypeptide. In this manner, a "chimeric" or "hybrid" antibody having the binding specificity of an antibody derived from an Fv pure line or a fusion line of the present invention is prepared. The carrier, host cell, and recombinant method are recombinantly produced to produce the present invention. An antibody, which is isolated and inserted into a replicable vector for further selection (amplification of DNA) or expression. The DAN encoding the antibody is carried out using conventional procedures (for example, by using a specific binding to the encoding antibody) The oligonucleotide probes of the heavy and light chain genes are easily isolated and sequenced. A variety of vectors can be used. The choice of vector depends in part on the host cell to be used. In general, the host cell has a prokaryotic or eukaryotic (a pain is also a source of suckling animals) It will be appreciated that any homozygous region can be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be derived from any human or animal Species are obtained. a) Fine production of antibodies using prokaryotic hosts: (1) Vector construction A polynucleotide sequence encoding a polypeptide component of an antibody of the present invention can be obtained using standard recombinant techniques. The nuclear (four) sequence is isolated and sequenced from the antibody-producing cells (such as fusion tumor cells). Alternatively, the polynucleotide can be synthesized using a nucleotide acid synthesizer or a PCR technique. After obtaining, the sequence encoding the polymorphism can be inserted into the pronucleus. The recombinant vector in which the heterologous polynucleotide is replicated and expressed in the host can be used for the purpose of this month. The selection of the vector will depend mainly on the vector to be inserted. H9007.doc •110- 200813092 The size and specific host cell to be transformed by the vector. Each vector depends on its function (amplification or expression of the heterologous polynucleotide, or both) and its Specific components contain various components for compatibility. Vector components generally include, but are not limited to, origin of replication, selection of marker genes, promoter, ribosome binding site (RBS), signal sequence, heterogeneity A nucleic acid insertion and transcription termination sequence. Typically, a plastid vector containing a replicon and a control sequence derived from a species compatible with the host cell is used in conjunction with the host. The vector typically carries a replication site, And a marker sequence that provides phenotypic selection in transformed cells. S, E. coli is typically transformed with a plastid PBR322 derived from an E. coli species. pBR322 contains an anti-ampicillin (ampiC1llin) (Amp) and tetracycline (Tet) antibody. Genes, and thus provide a simple means of identifying transformed cells. pBR322, its derivatives or other microbial plastids or phage may also contain or be modified to contain a promoter that can be used by microorganisms to express endogenous proteins. An example of a PBR 322 derivative that exhibits a specific antibody is described in detail in US Pat. No. 5,648,237 to Carter et al. In addition, a phage vector containing a replicon and a control sequence compatible with the host microorganism can be used in combination with the host. As a transformation vector. For example, phage such as λΟΕΜ···11 can be used to make a recombinant vector that can be used to transform sensitive host cells such as E. coli LE392. The expression vector of the present invention may comprise two or more promoter-cistronic pairs encoding each polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5,) of a cistron that regulates its expression. Prokaryotic promoters are usually divided into two types: 119007.doc -111- 200813092 class · inducibility and constitutive. An inducible promoter is a promoter that elicits an increased degree of cistronic transcription under the control of changes in its reaction culture conditions (e.g., presence or absence of nutrients or temperature changes). A large number of promoters recognized by various possible host cells are well known. The selected promoter can be operably linked to the coding light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Cistron DNA. Native promoter sequences and many heterologous promoters can be used to directly amplify and/or display a target gene. In some embodiments, a heterologous promoter is utilized as it generally permits more transcription and a more souther yield of the expressed target gene than it is to the native target polypeptide promoter. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the beta-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria, such as other known bacteria or bacteriophage promoters, are also suitable. Its nuclear (four) sequence has been published so that those skilled in the art can use the splicing or attachment (4) to operatively (4) ligate to the cistron encoding the target light and heavy chains (Siebenlist et al. (198〇) 2〇: 269) 'Therefore, any required restriction sites are supplied. In the aspect of the invention, the cistron in the recombinant vector comprises a secretion signal sequence component that directs translocation across the membrane of the expression polypeptide. In general, the signal sequence may be a vector, or it may be Insert the vector of the (four) DNA of the vector. The signal sequence selected for the purposes of the present invention shall be a signal sequence recognized and processed by the host cell (i.e., cleaved by signal peptidase) 119007.doc • 112- 200813092

列。對於不識別及加工異源性多肽原生之信號序列的原核宿主細胞而言，信號序列經原核信號序列取代，該原核信號序列係選自例如由鹼性磷酸酶、青黴素酶、Ipp或熱穩定性腸毒素 II(STII)導引子、LamB、PhoE、PelB、〇mpA 及MBP組成之群。在本發明之一實施例中，用於表現系統之兩個順反子之信號序列為STn信號序列或其變異體。在另一態樣中’根據本發明之免疫球蛋白之產生可發生於宿主細胞之細胞質中，且因此無需在各順反子中存在分泌信號序列。就此而言，免疫球蛋白輕鏈及重鏈經表現、折豐且組裝以於細胞質内形成功能性免疫球蛋白。特定宿主菌株（例如大腸桿菌trxB-菌株）提供有利於二硫鍵形成之細胞質條件，藉此容許經表現蛋白次單位之適當折疊及組裝。Proba及 Pluckthun 159:203 (1995)。本發明之抗體亦可精由使用表現系統而產生，在該表現系統中，可調節經表現多肽組份之定量比率以使本發明之經为/必及適當組裝之抗體的產率最大化。此調節至少部分藉由同時調節多肽組份之轉譯強度而完成。一種調節轉譯強度之技術係揭示於Simmons等人之美國專利第5,840,523號中。其利用順反子中之轉譯起始區 (TIR)變異體。對於指定TIR而言，可於轉譯強度範圍内產生一系列胺基酸或核酸序列變異體，藉此提供將此因素調節至特定鏈之所需表現程度的便利方式。TIR變異體可藉由導致可改變胺基酸序列之密碼子改變的習知突變技術而產生。在某些實施例中，核苷酸序列變化為靜止的。例 119007.doc -113- 200813092 如，TIR改變可包括Shine-Dalgarno序列之數量或間距之改變以及信號序列之改變。一種產生突變信號序列之方法為在編碼序列開始處產生π密碼子組"，其不改變信號序列之胺基酸序列（亦即改變為靜止的）。此可藉由改變各密碼子之第三核苷酸位置來完成；此外，一些胺基酸（諸如白胺酸、絲胺酸及精胺酸）具有多個可增加製造該組複雜性之第一及第二位置。此突變方法係詳細描述於Yansura等人 (1992) METHODS: A Companion to Methods in EnzymoL 4:151-158中。在一實施例中，以其中各順反子之TIR強度範圍產生一組載體。此限制性組提供各鏈表現程度以及所需抗體產物產率在各種TIR強度組合下之比較。如Simmons等人之美國專利第5,840,523號中所詳述，可藉由量化報導體基因之表現程度來確定TIR強度。基於轉譯強度比較，選擇所需個體TIR以於本發明之表現載體建構中組合。適用於表現本發明之抗體之原核宿主細胞包括原始細菌 (Archaebacteria)及真細菌（Eubacteria)，諸如革蘭氏陰性或革蘭氏陽性生物體。適用細菌之實例包括埃希氏菌屬 (Escherichia)(例如大腸桿菌）、桿菌屬（Bacilli)(例如枯草桿菌（B· subtilis))、腸内菌（Enterobacteria)、假單胞菌種 (Pseudomonas species)(例如綠膿桿菌（P. aeruginosa))、減毒鼠傷寒沙門氏菌（Salmonella typhimurium)、黏質沙雷氏菌（Serratia marcescans)、克雷伯氏菌（Klebsiella)、變形桿菌屬（Proteus)、志贺桿菌屬（Shigella)、根瘤菌（Rhizobia)、 119007.doc -114- 200813092 透明顫菌（Vitreoscilla)或副球菌（Paracoccus)。在一實施例中，使用革蘭氏陰性細胞。在一實施例中，將大腸桿菌細胞用作本發明之宿主。大腸桿菌菌株之實例包括菌株 W3110(Bachmann，Cellular and Molecular Biology，第 2卷 (Washington, D.C.: American Society for Microbiology, 1987)，第1190-1219頁；ATCC保藏號第27,325號）及其衍生物，包括具有基因型W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 △ompTA(nmpc_fepE) degP41 kanR之菌株 33D3(美國專利第5,639,635號）。其他菌株及其衍生物，諸如大腸桿Column. For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from alkaline phosphatase, penicillinase, Ipp or thermostability. A group consisting of enterotoxin II (STII) leader, LamB, PhoE, PelB, 〇mpA, and MBP. In one embodiment of the invention, the signal sequence used to represent the two cistrons of the system is the STn signal sequence or a variant thereof. In another aspect, the production of an immunoglobulin according to the present invention can occur in the cytoplasm of a host cell, and thus there is no need to have a secretion signal sequence in each cistron. In this regard, the immunoglobulin light and heavy chains are expressed, folded, and assembled to form functional immunoglobulins within the cytoplasm. Specific host strains (e. g., E. coli trxB-strain) provide cytoplasmic conditions that facilitate disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun 159: 203 (1995). The antibodies of the present invention can also be produced by the use of an expression system in which the quantitative ratio of the expressed polypeptide components can be adjusted to maximize the yield of the antibody of the present invention which is or is suitably assembled. This modulation is accomplished, at least in part, by simultaneously adjusting the translational strength of the polypeptide component. A technique for modulating the translational strength is disclosed in U.S. Patent No. 5,840,523 to Simmons et al. It utilizes a translation initiation region (TIR) variant in the cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be generated within the translational intensity range, thereby providing a convenient way to adjust this factor to the desired degree of performance of a particular strand. TIR variants can be produced by conventional mutation techniques that result in codon changes that alter the amino acid sequence. In certain embodiments, the nucleotide sequence changes to be static. Example 119007.doc -113- 200813092 For example, a TIR change can include a change in the number or spacing of the Shine-Dalgarno sequences and a change in the signal sequence. One method of generating a mutated signal sequence is to generate a π codon set at the beginning of the coding sequence, which does not alter the amino acid sequence of the signal sequence (i.e., changes to quiescent). This can be accomplished by altering the third nucleotide position of each codon; in addition, some amino acids (such as leucine, serine, and arginine) have multiples that increase the complexity of making the group. One and second positions. This mutation method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in EnzymoL 4:151-158. In one embodiment, a set of vectors is generated with a range of TIR intensities of the respective cistrons. This restriction set provides a comparison of the extent of performance of each strand and the desired antibody product yield under various combinations of TIR intensities. The TIR intensity can be determined by quantifying the degree of expression of the conductor gene as detailed in U.S. Patent No. 5,840,523 to Simmons et al. Based on the comparison of translational intensities, the desired individual TIRs are selected for combination in the construction of the expression vector of the present invention. Prokaryotic host cells suitable for use in the expression of the antibodies of the invention include primordial bacteria (Archaebacteria) and eubacteria (Eubacteria), such as Gram-negative or Gram-positive organisms. Examples of suitable bacteria include Escherichia (e.g., Escherichia coli), Bacilli (e.g., B. subtilis), Enterobacteria, and Pseudomonas species. (eg P. aeruginosa), attenuated Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, 119007.doc -114- 200813092 Vitreoscilla or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as a host of the invention. Examples of Escherichia coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pages 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, A strain 33D3 having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 ΔompTA (nmpc_fepE) degP41 kanR is included (U.S. Patent No. 5,639,635). Other strains and their derivatives, such as the large intestine

菌 294(ATCC 31，446)、大腸桿菌 B、大腸桿菌 X1776(ATCC 31，537)及大腸桿菌RV308(ATCC 31，608)亦為適用的。該等實例為說明性的而非限制性的。用於建構具有所定義基因型之上述任何細菌之衍生物的方法於此項技術中已知且例如描述於 Bass 等人，pr〇teins，8:309-314 (1990)中。一般需要考慮細菌細胞中複製子之可複製性來選擇適當細菌。舉例而言，當將諸如pBR322、pBR325、pACYC177或 pKN410之热知質體用以供應複製子時，可將大腸桿菌、沙雷氏菌（Serratia)或沙門氏菌種（Salm〇nella species)合適地用作宿主。宿主細胞通常應分泌最小量之蛋白水解酶，且可將額外蛋白酶抑制劑理想地併入細胞培養物中。 (2)抗體產生將宿主細胞以上述表現載體轉化，且培養於改質為適用於誘導啟動子、選擇轉化體或擴增編碼所需序列之基因習知營養培養基中。、 119007.doc -115- 200813092 轉化意谓將DNA引入原核宿主中以使得DNA作為染色體外το素或藉由染色體成分為可複製的。視所用宿主細胞而疋，使用適於此等細胞之標準枝術進行轉化。採用氣化鈣之鈣處理一般係用於含有實質細胞壁障壁之細菌細胞。另一種轉化方法採用聚乙二醇/DMS〇。所用之另一技術為電穿孔。用以產生本發明多肽之原核細胞係生長於此項技術中已知且適用於培養所選擇之宿主細胞之培養基中。合適培養基之貫例包括魯利亞肉湯（luria br〇th)(LB)加必需之營養補充物。在一些實施例中，培養基亦含有基於表現載體之建構所選擇之選擇劑，以選擇性地容許含有表現載體之原核細胞生長。舉例而言，將安比西林添加至用於使表現安比西林抗性基因之細胞生長的培養基中。除石反、氮及無機鱗酸鹽源以外，亦可以適當濃度單獨引入或以與諸如複合氮源之另一補充物或培養基之混合物的形式包括任何必需補充物。培養基可視情況含有一或多種還原劑，其係選自由麩胱甘肽、半胱胺酸、胱胺、酼乙酸酉旨、一硫赤糖醇及二硫蘇糖醇組成之群。將原核佰主細胞於合適溫度下培養。在某些實施例中，對於大腸桿菌生長而言，生長溫度為約2〇〇c至約39艺；約 25°C至約37°C，或約30°C。培養基之pH值主要視宿主生物體而疋’可為約5至約9範圍内之任何pH值。在某些實施例中，對於大腸桿菌而言，pH值為約6·8至約7.4,或約7.〇。若將誘導性啟動子用於本發明之表現載體，則在適用於 119007.doc -116- 200813092 啟動子活化之條件下誘導蛋白表現。在本發明之一態樣中，使用PhoA啟動子以控制多肽之轉錄。因此，將經轉化之宿主細胞培養於磷酸鹽限制性培養基中以用於誘導。在某些實施例中，碟酸鹽限制性培養基為crap培養基（例如參見 Simmons 等人 ’ j Immun〇1 廳^心（2〇〇2)， 263.133 147)。如此項技術中已知，根據所採用之載體建構可使用各種其他誘導物。 f 纟一實施例中，使本發明之經表現多1分泌於宿主細胞周貝中且自宿主細胞周質回收。纟白回收通常包括一般藉諸如β透衝擊、超音波處理或溶胞之方式破壞微生物。破裏、、、田胞之後，可藉由離心或過濾移除細胞碎片或全細胞可將蛋白（例如）藉由親和樹脂層析進一步純化。或者，可將蛋白傳輸至培養基中且於其中分離。將細胞自培養物移除且將培養物上清液過遽且濃縮以用於進一步純化斤產生之蛋白。可使用諸如聚丙烯醯胺凝膠電泳（page)及 ( 方墨點檢定之通常已知^法進-步分離及識別經表現之多肽。生在T發明之一態樣中，藉由醱酵法進行大量抗體之產可使用各種大規模分批饋入醱酵程序來產生重組蛋白大規权醱酵具有至少1000公升之容量，且在某些實施 :*為約1，〇〇〇至1〇〇,〇〇〇公升之容量。該等醱酵器使用攪拌葉輪以分配氧及營養物，尤其為葡萄糖（較佳為碳/能 1原）L小規模醱酵一般係指在容量不大於約100公升且可在、勺Ια升至約100公升範圍内之醱酵器中之醱酵。 119007.doc •117- 200813092 在醱酵法中，蛋白表現之誘導通常在細胞於合適條件下生長至例如約180-220之OD550之所需密度後（在此階段，細胞處於早期生長停滯期）起始。如此項技術中已知及如上文所述，可根據所採用之載體建構使用各種誘導物。在誘導之前細胞可生長較短時期。儘管可使用更長或更短之誘導時間，但細胞通常誘導約12-50小時。為改良本發明多肽之生產產率及品質，可改進各種酸酵條件。舉例而言，為改良所分泌抗體多肽之適當組裝及折疊’可使用過度表現伴隨蛋白之額外載體，諸如Dsb蛋白 (DsbA、DsbB、DsbC、DsbD及/或 DsbG)或 FkpA(具有伴隨蛋白活性之肽基脯胺醯基順反異構酶）以共轉化宿主原核細胞。已證明伴隨蛋白促進在細菌宿主細胞中所產生之異源性蛋白的適當折疊及溶解性。Chen等人（1999) J C/zem 274:19601-19605 ; Georgiou 等人之美國專利第 6,〇83,715號；Georgiou等人之美國專利第6,027,888號； Bothmann 及 Pluckthim (2000) J·价〇/· C/2ew 275:171〇〇· 17105 ; Ramm 及 Pluckthun (2000) 乂 Biol. Chem 275:17106-17113 ; Arie等人（2001) Mol. Microbiol· 39:199-210 〇為使所表現異源性蛋白之蛋白水解最小化（尤其為彼等蛋白水解敏感者）’可將缺乏蛋白水解酶之特定宿主菌株用於本發明。舉例而言，可修飾宿主細胞菌株以達成編碼已知細菌蛋白酶之基因中的基因突變，該等細菌蛋白酶諸如蛋白酶III、OmpT、DegP、Tsp、蛋白酶！、蛋白酶Mi、 119007.doc -118- 200813092 蛋白酶v、蛋白酶VI及其組合。一些大腸桿菌蛋白酶缺乏之菌株為可用的且描述於例如Joly等人（1998)(同上文）； gi〇u荨人之美國專利第5,264,365號；Georgiou等人之美國專利第5,508，192號；Hara等人，滅㈣Μα/ />叹及ahiawce，2:63-72 (1996)中。在一實施例中，將缺乏蛋白水解酶且經過度表現一或多種伴隨蛋白之質體轉化之大腸桿菌菌株用作本發明表現系統中之宿主細胞。 (3)抗體純化在一實施例中，進一步純化本文所產生之抗體蛋白以獲得大體上均質之製劑以用於進一步檢定及使用。可採用此項技術中已知之標準蛋白純化方法。以下程序為例示之合適純化程序：免疫親和性分餾或離子交換管柱、乙醇沉澱、逆相HPLC、二氧化矽層析或諸如〇ΕΑΕ之離子交換樹脂層析、層析聚焦、SDS-PAGE、硫酸銨沉澱及使用例如 SephadexG-75之凝膠過濾。在一％樣中’將固定於固相上之蛋白A用於本發明抗體產物之免疫親和性純化。蛋白A為以高親和力結合至抗體 Fc£之來自金頁色葡萄球菌似）之41 kD細胞壁蛋白。Lindmark 等人（1983) j Immunol· Meth. 62:1-13。固定蛋白a之固相可為包含玻璃或二氧化矽表面之管柱’或為經控制之孔隙玻璃管柱或石夕酸管柱。在一些應用中，以諸如甘油之試劑塗覆管柱以試圖防止污染物之非特異性黏著。 119007.doc -119- 200813092 作為純化之第一步驟，可將如上所述衍生自細胞培養物之製劑塗覆於固定蛋白A之固相上以使得所關注之抗體特異性結合至蛋白A。接著洗滌固相以移除非特異性結合至固相之5染物。最後，藉由溶離自固相回收所關注之抗體。 b)使用真核宿主細胞產生抗體·· 用於真核冑主細豸之載體一I包括以下#限制性組份之厂—或多種：信號序列、複製起點、—或多種標記基因、增強子元素、啟動子及轉錄終止序列。 (Ό信號序列組份用於真核宿主細胞之載體亦可含有在所關注之成熟蛋白或多肽之N端具有特異性裂解位點之信號序列或其他多肤所k擇之異源性信號序列可為由宿主細胞識別及加工 (亦即由信號肽酶裂解）之信號序列。在哺乳動物細胞表現中丄可使用哺乳動物信號序列以及病毒分泌性引導物，例 (如早純疱疹gD信號。此前驅物區之DNA於閱讀框架中接合至編碼抗體之DN A。 (2)複製起點一般而言’哺乳動物表現載體無需複製κ组份。舉例而言，通常可使用SV4〇起點，僅因其含有早期啟動子。 Ο)選擇基因組份表現及選耗體可含有選擇基因，亦稱為可選擇標記。 =型選擇基因編碼⑷對抗生素或其他毒素（例如安比西 I新黴素、甲胺嗓呤或四環素）賦予抗性；⑻補充營養 119007.doc 200813092 缺陷（相關時）或（C)供應不可自複合培養基獲得之標準營養物之蛋白。選擇機制之一實例利用使宿主細胞生長停滞之藥物。以異源性基因成功轉化之彼等細胞產生賦予藥物抗性且因此保全選擇方案之蛋白。此優勢選擇之實例使用藥物新黴素、黴酚酸及濕黴素。哺乳動物細胞之合適可選擇標記的另一實例為彼等使得可識別能夠吸收抗體核酸之細胞的可選擇標記，其諸如 C： DHFR;胸苷激酶；金屬硫蛋白I及II，較佳為靈長類動物金屬硫蛋白基因；腺苷脫胺酶；鳥胺酸脫羧酶等。舉例而言，在一些實施例中，首先藉由於含有甲胺喋呤 (Mtx)、DHFR之競爭性拮抗劑之培養基中培養所有轉化體來識別經DHFR選擇基因轉化之細胞。在一些實施例中，當採用野生型DHFR時，適當宿主細胞為缺乏DHFR活性之中國倉鼠卵巢（CHO)細胞株（例如ATCC CRL-9096)。 / 或者’可藉由在含有諸如胺基糖苷類抗生素（例如康黴 I . *’ 素（kanamycin)、新黴素或G418)之可選擇標記選擇劑之培養基中之細胞生長來選擇經編碼抗體之DNA序列轉化或共轉化之宿主細胞（尤其為含有内源性DHFR之野生型宿主）、野生型DHFR蛋白及另一可選擇標記，諸如胺基糖苷 3’-磷酸轉移酶（ΑΡΗ)。參見美國專利第4,965,199號。 (4)啟動子組份表現及選殖載體通常含有以宿主生物體識別且可操作性地連接至編碼所關注多肽（例如抗體）之核酸的啟動子。已 119007.doc •121- 200813092 知真核細胞之啟動子序列。舉例而言，實際上所有真核基因均具有定位於轉錄起始位點上游⑽㈣個驗基處之AT 富集區。於許多基因轉錄起始上游7〇至8〇個鹼基處發現之另-序列為其中N可為任何核普酸之CNCAat^大部分真核基因3’端為AATAAA序％ ’其可為向編碼序列3，端添加聚Α尾之信號。在某些實施例中，任何或所有該等序列均可合適地***真核表現載體中。自哺乳動物宿主細胞中之載體轉錄係(例如)藉由獲自以 :之啟動子來控制··病毒染色體組，該等病毒諸如多瘤病毒、禽痘病毒、腺病毒（諸如腺病毒2)、牛乳頭狀瘤病毒、禽肉瘤病毋、細胞巨大病毒、反轉錄病毒、B型肝炎病毒及猿猴病毒40(SV40);異源性哺乳動物啟動子，例如肌動蛋白啟動子或免疫球蛋白啟動子；熱休克啟動子，其限制條件為此等啟動子與宿主細胞系統相容。 SV40病毒之早期及晚期啟動子以亦含有8^4〇病毒複製起點之SV40限制片段形式便利地獲得。人類細胞巨大病毒之即刻早期啟動子以Hindlll E限制片段形式便利地獲得。使用牛乳頭狀瘤病毒作為載體於哺乳動物宿主中表現Dn a 之系統揭示於美國專利第4,419,446號中。此系統之修正描述於美國專利第4,601，978號中。亦參見Reyes等人， 297:598-601 (1982)，其描述人類β干擾素cDNA在來自單純疱療病毒之胸苷激酶啟動子控制下於小鼠細胞中之表現。或者’可將勞斯肉瘤病毒（r〇us Sarcoma Virus)長末端重複序列用作啟動子。 119007.doc -122- 200813092 (5)增強子元素組份藉由更高級真核細胞轉錄編碼本發明抗體之DNA通常藉由將增強子序列***載體中而增加。現已知許多增強子序列來自哺乳動物基因（血球蛋白、彈性蛋白酶、白蛋白、胎蛋白及胰島素）。然而，通常使用來自真核細胞病毒之增強子。實例包括位於複製起點後側（bp 1〇〇-27〇)之 SV40增強子；細胞巨大病毒早期啟動子增強子；複製起點後側之多瘤增強子及腺病毒增強子。亦參見描述用於活化真核啟動子之增強子元素的Yaniv，297:17_18 (1982)。可將增強子於編碼抗體多肽之序列之5，或3，位置剪接至載體中，但通常定位於啟動子之5,位點。 (6) 轉錄終止組份用於真核宿主細胞之表現載體亦含有終止轉錄及穩定 mRNA所必需之序列。此等序列通常可自真核或病毒dnA 或cDNA之5’及（偶爾）3,未轉譯區獲得。該等區域含有在編碼抗體之mRNA之未轉譯部分中轉錄為多聚腺嘌呤片段之核苷酸區段。一種適用之轉錄終止組份為牛生長激素多聚腺嗓呤區。參見WO 94/11026及其中所揭示之表現載體。 (7) 宿主細胞之選擇及轉化選殖或表現本文載體中之DNA的合適宿主細胞包括本文所述之更高級真核細胞，包括脊椎宿主細胞。脊椎動物細胞在培養物（組織培養物）中之繁殖已成為常規程序。適用之哺乳動物宿主細胞株之實例為經SV40轉化之猴腎CV1細胞株（COS-7，ATCC CRL 1651);人類胚胎腎細胞株（次選 119007.doc -123- 200813092 殖以於懸浮培養物中生長之293或293細胞，Graham等人，乂 Gw Wro/· 36:59 (1977));幼倉鼠腎細胞（BHK，ATCC CCL 10);中國倉鼠卵巢細胞/-DHFR(CHO，Urlaub等人， Pr%· #如/· dMd· 5W. 77:4216 (1980));小鼠足細胞 (TM4，Mather，5/〇/· 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，ATCC CCL 2);犬腎細胞（MDCK，ATCC CCL 34);水牛大鼠肝細胞（BRL 3 A， ATCC CRL 1442);人類肺細胞（W138, ATCC CCL 75);人類肝臟細胞（Hep G2，HB 8065);小鼠乳腺腫瘤（MMT 060562, ATCC CCL51) ; TRI細胞（Mather等人，d画心见 Z Acad. 383:44-68 (1982)) ; MRC 5細胞；FS4細胞及人類肝腫瘤細胞株（Hep G2)。將宿主細胞以上述表現或選殖載體轉化以產生抗體，且在視需要經改質之習知營養培養基中培養以用於誘導啟動子、選擇轉化體或擴增編碼所需序列之基因。 (8)培養宿主細胞可於各種培養基中培養用以產生本發明抗體之宿主細胞。市售培養基，諸如Ham’s FlO(Sigma)、最少之基本培養基（Minimal Essential Medium)((MEM)，Sigma)、RPMI-1640(Sigma)及杜貝科氏改質伊格氏培養基（(DMEM)， Sigma)適用於培養宿主細胞。此外，可將Ham等人，Mei/z. Εηζ· 58:44 (1979)，Barnes 等人，dna/· 102:255 (1980)，美國專利第4,767,704號；第4,657,866號；第 119007.doc -124- 200813092 4,927,762 ^ f 4,560,655 ^ f 5,122,469 E ； WO 90/03430; W0 87/〇〇195或美國專利Re 3〇 985中所述之任何培養基均可用作宿主細胞之培養基。任何該等培養基均可視需要補充以激素及/或其他生長因子（諸如胰島素、轉鐵蛋白或表皮生長因子）；鹽（諸如氣化鈉、鈣鹽、鎂鹽及磷酸鹽）、緩衝液（諸如HEPES)、核苷酸（諸如腺苷及胸苷）、抗生素（諸如GENTAMYCINTM藥物）、痕量元素（定義為通常以微莫耳範圍之最終濃度存在之無機化合物）及葡萄糖或等效能源。亦可以彼等熟習此項技術者已知之適當濃度包括任何其他補充物。諸如溫度、1?11值及其類似條件之培養條件為彼等先前用於為表現而選擇之宿主細胞的條件’且將為一般熟習此項技術者所顯而易見。 (9)抗體之純化當使用重組技術時，抗體可於細胞内產生或直接分泌入坨養基中。若抗體於細胞内產生而作為第一步驟，則（例如）藉由離心或超濾移除微粒碎片（宿主細胞或溶胞片段）。當抗體分泌入培養基中時，首先使用市售之蛋白濃縮過濾器（例如Amicon或Millipore pelliC0n超濾單元）濃縮來自此等表現系統之上清液。可於前述任何步驟中包括諸如 PMSF之蛋白酶抑制劑以抑制蛋白水解，且可包括抗生素以防止外來污染物之生長。由細胞製備之抗體組合物可使用例如羥基磷灰石層析、旋膠電泳法、透析及親和層析純化，其中親和層析為便利技術。蛋白A作為親和力配位體之適用性取決於抗體中所 119007.doc -125- 200813092 存在之任何免疫球蛋白Fc結構域的種類及同型。蛋白A可用以純化基於人類γΐ、γ2或γ4重鏈之抗體（Lindmark等人，乂 /mm⑽〇/· Μ以办62:1_ 13 (1983))。推薦將蛋白G用於所有小鼠同型及用於人類Y3(Guss等人，EMBO J· 5:15671575 (1986))。親和力配位體所連接之基質可為瓊脂糖，但可使用其他基質。諸如受控微孔玻璃或聚（苯乙烯二乙烯基）苯之機械穩定基質使得可獲得與瓊脂糖可達成者相比更快之流動速率及更短之加工時間。當抗體包含CH3結構域時，Bacteria 294 (ATCC 31,446), Escherichia coli B, Escherichia coli X1776 (ATCC 31,537) and Escherichia coli RV308 (ATCC 31,608) are also suitable. The examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above mentioned bacteria having the defined genotype are known in the art and are described, for example, in Bass et al, pr〇teins, 8: 309-314 (1990). It is generally necessary to consider the replicability of replicons in bacterial cells to select appropriate bacteria. For example, when a thermophile such as pBR322, pBR325, pACYC177 or pKN410 is used to supply a replicon, Escherichia coli, Serratia or Salm〇nella species can be suitably used. As a host. The host cell should normally secrete a minimal amount of proteolytic enzyme, and additional protease inhibitors can be ideally incorporated into the cell culture. (2) Antibody production The host cell is transformed with the above expression vector, and cultured in a conventional nutrient medium suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence. 119007.doc -115- 200813092 Transformation means the introduction of DNA into a prokaryotic host such that the DNA is reproducible as extrachromosomal or by chromosomal components. Depending on the host cell used, transformation is carried out using standard techniques suitable for such cells. Calcium treatment with vaporized calcium is generally used for bacterial cells containing substantial cell wall barriers. Another method of transformation uses polyethylene glycol/DMS. Another technique used is electroporation. Prokaryotic cell lines used to produce the polypeptides of the invention are grown in a medium known in the art and suitable for use in culturing selected host cells. Examples of suitable cultures include luria br〇th (LB) plus the necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to a medium for growing cells expressing an ampicillin resistance gene. In addition to the stone counter, nitrogen and inorganic sulphate sources, any necessary supplement may also be included in the appropriate concentration alone or in admixture with another supplement or medium such as a complex nitrogen source. The medium may optionally contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, indoleacetic acid, monothiol and dithiothreitol. The prokaryotic sputum primary cells are cultured at a suitable temperature. In certain embodiments, for E. coli growth, the growth temperature is from about 2 ° C to about 39 °; from about 25 ° C to about 37 ° C, or about 30 ° C. The pH of the medium will depend primarily on the host organism and may be any pH in the range of from about 5 to about 9. In certain embodiments, for E. coli, the pH is from about 6.8 to about 7.4, or about 7. If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activation of the 119007.doc-116-200813092 promoter. In one aspect of the invention, the PhoA promoter is used to control transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-restricted medium for induction. In certain embodiments, the disc acid limited medium is a crap medium (see, for example, Simmons et al. 'j Immun〇1 Hall (2〇〇2), 263.133 147). As is known in the art, various other inducers can be used depending on the vector construction employed. f In one embodiment, the expression 1 of the present invention is secreted in the host cell and recovered from the host cell periplasm. Whitening recovery typically involves destroying microorganisms generally by means such as beta penetration, ultrasonic treatment or lysis. After disruption, cell migration, the protein can be further purified, for example, by affinity resin chromatography by centrifugation or filtration to remove cell debris or whole cells. Alternatively, the protein can be delivered to and isolated from the culture medium. The cells are removed from the culture and the culture supernatant is sputum and concentrated for further purification of the protein produced by the jin. The peptides can be isolated and identified using, for example, polyacrylamide gel electrophoresis (page) and the commonly known methods of square dot assay. Born in one of the T inventions, by fermentation The method of producing a large number of antibodies can be carried out using a variety of large-scale batch feed fermentation procedures to produce recombinant protein large-scale fermentations having a capacity of at least 1000 liters, and in some implementations: * is about 1, 〇〇〇 to 1 〇〇, 〇〇〇 liter capacity. These fermenters use a stirring impeller to distribute oxygen and nutrients, especially glucose (preferably carbon / energy 1 original) L small-scale fermentation generally refers to the capacity is not greater than about 100 liters of yeast fermented in a fermenter with a range of up to about 100 liters. 119007.doc •117- 200813092 In the fermentation process, the induction of protein expression is usually carried out under conditions where the cells are grown to the appropriate conditions. For example, after the desired density of OD550 of about 180-220 (at this stage, the cells are in an early growth stagnation phase), as known in the art and as described above, various inducers can be constructed depending on the vector employed. Cells can grow shorter before induction Although a longer or shorter induction time can be used, the cells are usually induced for about 12 to 50 hours. To improve the production yield and quality of the polypeptide of the present invention, various acid fermentation conditions can be improved. For example, for improvement Appropriate assembly and folding of secreted antibody polypeptides can use additional vectors that overexpress accompanying proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (peptidyl amidoxime with accompanying protein activity) Anti-isomerase) to co-transform host prokaryotic cells. Accompanying proteins have been shown to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) JC/zem 274: 19601-19605 Georgiou et al., U.S. Patent No. 6, 〇83,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthim (2000) J. Price/·C/2ew 275:171〇〇·17105; Ramm and Pluckthun (2000) 乂Biol. Chem 275:17106-17113; Arie et al. (2001) Mol. Microbiol· 39:199-210 〇 To minimize proteolysis of heterologous proteins (especially sensitive to their proteolysis) By)' A particular host strain lacking a proteolytic enzyme can be used in the present invention. For example, a host cell strain can be modified to achieve a genetic mutation in a gene encoding a known bacterial protease such as Protease III, OmpT, DegP, Tsp, Protease!, Protease Mi, 119007.doc -118- 200813092 Protease v, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998) (supra); gi〇u, U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara Etc., extinction (four) Μα/ /> sigh and ahiawce, 2:63-72 (1996). In one embodiment, an E. coli strain lacking a proteolytic enzyme and exhibiting one or more plastid transformations with accompanying proteins is used as a host cell in the expression system of the invention. (3) Antibody Purification In one embodiment, the antibody protein produced herein is further purified to obtain a substantially homogeneous preparation for further assay and use. Standard protein purification methods known in the art can be employed. The following procedures are exemplified as suitable purification procedures: immunoaffinity fractionation or ion exchange column, ethanol precipitation, reverse phase HPLC, cerium oxide chromatography or ion exchange resin chromatography such as hydrazine, chromatofocusing, SDS-PAGE, Ammonium sulfate precipitation and gel filtration using, for example, Sephadex G-75. Protein A immobilized on a solid phase was used for immunoaffinity purification of the antibody product of the present invention in one percent. Protein A is a 41 kD cell wall protein that binds to the antibody Fc £ with high affinity like Staphylococcus aureus. Lindmark et al. (1983) j Immunol· Meth. 62:1-13. The solid phase of the immobilized protein a may be a column comprising a glass or cerium oxide surface or a controlled glass column or a sulphuric acid column. In some applications, the tubing is coated with a reagent such as glycerin in an attempt to prevent non-specific adhesion of contaminants. 119007.doc -119-200813092 As a first step of purification, a preparation derived from a cell culture as described above can be applied to the solid phase of immobilized protein A to allow the antibody of interest to specifically bind to protein A. The solid phase is then washed to remove 5 stains that are non-specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by dissolution. b) Production of antibodies using eukaryotic host cells···································································· Elements, promoters and transcription termination sequences. (The vector of the Ό signal sequence component for use in a eukaryotic host cell may also contain a signal sequence having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest or a heterologous signal sequence selected by other polypeptides. It may be a signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signal sequences as well as viral secretory guides can be used, such as the early herpes simplex gD signal. The DNA of the precursor region is ligated into the DN A encoding the antibody in the reading frame. (2) The origin of replication Generally, the mammalian expression vector does not need to replicate the κ component. For example, the SV4 〇 origin can usually be used, It contains an early promoter. Ο) Selection of genomic expression and selection of the body can contain a selection gene, also known as a selectable marker. The =type selection gene encodes (4) antibiotics or other toxins (eg, Ambishi I neomycin, methylamine)嗓呤 or tetracycline) confers resistance; (8) supplemental nutrition 119007.doc 200813092 Defects (when relevant) or (C) supply of protein of standard nutrients not available from the complex medium. An example of an alternative mechanism utilizes a drug that arrests the growth of a host cell. The cells that have been successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus preserves the selection protocol. Examples of this advantageous selection use the drug neomycin, mildew. Phenolic acid and hygromycin. Another example of a suitable selectable marker for a mammalian cell is such a selectable marker that enables recognition of a cell capable of absorbing the antibody nucleic acid, such as C: DHFR; thymidine kinase; metallothionein I And II, preferably a primate metallothionein gene; adenosine deaminase; ornithine decarboxylase, etc. For example, in some embodiments, first by virtue of containing methotrexate (Mtx), All transformants were cultured in a medium of a competitive antagonist of DHFR to identify cells transformed with the DHFR selection gene. In some embodiments, when wild-type DHFR is employed, the appropriate host cell is a Chinese hamster ovary (CHO) lacking DHFR activity. A cell line (eg, ATCC CRL-9096). / or ' can be obtained by containing an antibiotic such as an aminoglycoside (eg, kanamycin, neomycin, or G4). 18) Selecting a cell for growth in a selection agent to select a host cell transformed or co-transformed with a DNA sequence encoding the antibody (especially a wild-type host containing endogenous DHFR), a wild-type DHFR protein, and another A selectable marker, such as an aminoglycoside 3'-phosphotransferase (ΑΡΗ). See U.S. Patent No. 4,965,199. (4) Promoter component expression and selection vectors typically contain and are operably linked by the host organism. Promoter to a nucleic acid encoding a polypeptide of interest (eg, an antibody). 119007.doc • 121- 200813092 Know the promoter sequence of a eukaryotic cell. For example, virtually all eukaryotic genes have a transcription initiation site. Point the upstream (10) (four) AT-rich areas of the base. The other sequence found at 7 to 8 bases upstream of the transcription initiation of many genes is CNCAat where N can be any nucleotide acid. Most of the eukaryotic gene 3' end is AATAAA order % 'which can be At the end of the coding sequence 3, the signal of the poly tail is added. In certain embodiments, any or all of the sequences can be suitably inserted into a eukaryotic expression vector. Vector transcripts from mammalian host cells are controlled, for example, by a promoter derived from a viral genome such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2). , bovine papilloma virus, avian sarcoma sickness, giant cell virus, retrovirus, hepatitis B virus and simian virus 40 (SV40); heterologous mammalian promoter, such as actin promoter or immunoglobulin Protein promoter; a heat shock promoter whose restriction conditions are compatible with the host cell system for such promoters. The early and late promoters of the SV40 virus are conveniently obtained in the form of SV40 restriction fragments which also contain the replication origin of the 8^4 prion. The immediate early promoter of the human cell giant virus is conveniently obtained in the form of a Hindlll E restriction fragment. A system for expressing Dn a 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. 4,601,978. See also Reyes et al, 297: 598-601 (1982), which describes the expression of human beta interferon cDNA in mouse cells under the control of the thymidine kinase promoter from the herpes simplex virus. Alternatively, the long terminal repeat of r〇us Sarcoma Virus can be used as a promoter. (5) Enhancer component The DNA encoding the antibody of the present invention by higher order eukaryotic transcription is usually increased by inserting the enhancer sequence into the vector. Many enhancer sequences are known to be derived from mammalian genes (blood globulin, elastase, albumin, fetal protein, and insulin). However, enhancers from eukaryotic viruses are commonly used. Examples include the SV40 enhancer located at the back of the replication origin (bp 1〇〇-27〇); the cell giant virus early promoter enhancer; the polyoma enhancer on the posterior side of the replication origin and the adenovirus enhancer. See also Yaniv, 297:17_18 (1982), which describes the enhancer element used to activate eukaryotic promoters. The enhancer can be ligated into the vector at position 5, or 3, of the sequence encoding the antibody polypeptide, but is typically positioned at the 5, position of the promoter. (6) Transcription termination components Expression vectors for use in eukaryotic host cells also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are typically obtained from 5' and (occasionally) 3, untranslated regions of eukaryotic or viral dnA or cDNA. These regions contain nucleotide segments transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding the antibody. One suitable transcription termination component is the bovine growth hormone polyadenosine region. See WO 94/11026 and the expression vectors disclosed therein. (7) Selection and transformation of host cells Suitable host cells for the selection or expression of DNA in the vectors herein include the higher eukaryotic cells described herein, including the spinal host cells. The propagation of vertebrate cells in cultures (tissue cultures) has become a routine procedure. 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 (secondary 119007.doc-123-200813092 colonized in suspension culture) 293 or 293 cells grown, Graham et al, 乂Gw Wro/· 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al. , Pr%· #如/· dMd· 5W. 77:4216 (1980)); mouse podocytes (TM4, Mather, 5/〇/· 23:243-251 (1980)); monkey kidney cells (CV1 ATCC) CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells ( BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al, d draw heart see Z Acad. 383:44-68 (1982)); MRC 5 cells; FS4 cells and human liver tumor cell lines (Hep G2). The host cell is transformed with the above-described expression or selection vector to produce an antibody, and cultured in a conventional nutrient medium modified as needed for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence. (8) Culturing host cells Host cells for producing the antibody of the present invention can be cultured in various media. Commercially available media such as Ham's FlO (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma) and Dubecco's Modified Ig's Medium (DMEM), Sigma) is suitable for culturing host cells. In addition, Ham et al., Mei/z. Εηζ 58:44 (1979), Barnes et al., dna/. 102:255 (1980), U.S. Patent No. 4,767,704; 4,657,866; 119007.doc - 124- 200813092 4,927,762 ^ f 4,560,655 ^ f 5,122,469 E; WO 90/03430; W0 87/〇〇195 or any of the media described in U.S. Patent Re 3 985 985 can 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, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drugs), trace elements (defined as inorganic compounds typically present in the final concentration of the micromolar range), and glucose or equivalent energy. They may also be familiar to any suitable supplement known to those skilled in the art including any other supplement. Culture conditions such as temperature, 1-11, and the like are those of the host cells previously selected for performance' and will be apparent to those of ordinary skill in the art. (9) Purification of antibodies When recombinant techniques are used, antibodies can be produced intracellularly or secreted directly into the nucleus. If the antibody is produced intracellularly as a first step, particulate debris (host cells or lysed fragments) are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from the performance system is first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore pelliC0n ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants. The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, spin gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a convenient technique. The suitability of Protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody 119007.doc-125-200813092. Protein A can be used to purify antibodies based on the human γΐ, γ2 or γ4 heavy chain (Lindmark et al., 乂 /mm(10)〇/· Μ 62:1_ 13 (1983)). Protein G is recommended for all mouse isotypes and for human Y3 (Guss et al, EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached may be agarose, but other matrices may be used. Mechanically stable matrices such as controlled microporous glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times compared to agarose achievables. When the antibody comprises a CH3 domain,

Bakerbond ABXtm樹脂（J τ Baker，Phillipsburg, NJ)適用於純化。視待回收之抗體而定，亦可使用諸如離子交換管柱分餾、乙醇沉澱、逆相HPLC、二氧化矽層析、肝素 SEPHAROSETM層析、陰離子或陽離子交換樹脂層析（諸如聚天冬胺酸管柱）、層析聚焦、SDS_PAGE及硫酸銨沉澱之其他蛋白純化技術。任何初步純化步驟之後，可使包含所關注抗體及污染物之混合物例如藉由較佳於低鹽濃度（例如約0-0.25 Μ鹽）下進行之使用pH值介於約2.5-4.5之間之溶離緩衝液的低ρΗ 值疏水性相互作用層析而經受進一步純化。一般而言，此項技術中已確定用以製備用於研究、測試及臨床用途之抗體的各種方法，與上述方法一致及/或由熟習此項技術者認為適用於所關注之特定抗體。 C·免疫接合物本發明亦提供包含接合至一或多種細胞毒性劑之本發明之任何抗ΤΑΤ226抗體的免疫接合物（可交替稱作"抗體_藥 119007.doc -126- 200813092 物接合物”或"ADC")，該（等）細胞毒性劑諸如化療劑、藥物、生長抑制劑、毒素（例如細菌、真菌、植物或動物來源之酶活性毒素或其片段））或放射性同位素（亦即放射性接合物）。免疫接合物在癌症治療中可用於局部傳遞細胞毒性劑，亦即殺死或抑制腫瘤細胞生長或增殖之藥物（Syrigos及 Epenetos (1999) Anticancer Research 19:605-614 ;Bakerbond ABXtm resin (J τ Baker, Phillipsburg, NJ) is suitable for purification. Depending on the antibody to be recovered, it may also be used, for example, ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, ceria chromatography, heparin SEPHAROSETM chromatography, anion or cation exchange resin chromatography (such as polyaspartic acid). Other purification techniques for column chromatography, chromatographic focusing, SDS_PAGE, and ammonium sulfate precipitation. After any preliminary purification step, the mixture comprising the antibody of interest and the contaminant can be used, for example, by a pH of between about 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0-0.25 cesium salt). The low pH value of the dissolution buffer was subjected to further purification by hydrophobic interaction chromatography. In general, various methods for preparing antibodies for research, testing, and clinical use have been identified in the art, consistent with the above methods and/or as deemed suitable by the skilled artisan for the particular antibody of interest. C. Immunoconjugates The invention also provides immunoconjugates comprising any of the anti-ΤΑΤ226 antibodies of the invention conjugated to one or more cytotoxic agents (alternatively referred to as "antibody-drugs 119007.doc-126-200813092 conjugates "or "ADC"), a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (such as an enzymatic active toxin or a fragment thereof of bacterial, fungal, plant or animal origin) or a radioactive isotope (also That is, radioactive conjugates. Immunoconjugates can be used in the treatment of cancer for the local delivery of cytotoxic agents, ie drugs that kill or inhibit the growth or proliferation of tumor cells (Syrigos and Epenetos (1999) Anticancer Research 19:605-614;

Niculescu-Duvaz及 Springer (1997) jdv· 26:151-172;美國專利第4,975,278號）。免疫接合物可容許將藥物部分靶傳遞至腫瘤及於其中進行細胞内積累，其中全身投予未接合之藥物可對正常細胞以及待消除之腫瘤細胞產生不可接受程度之毒性（6&1(1\¥丨11等人，1^加以（1986年 3 月 1 5 曰）第 603-05 頁）；Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review，’’於 Monoclonal Antibodies ’84: Biological And Clinical (A. Pinchera等人編）第475-506頁。已報導多株抗體及單株抗體均適用於此等策略（Rowland等人， (1986) Cancer Immunol· Immunother· 21:183-87) ° 用於 it匕等方法之藥物包括道諾黴素、多柔比星、甲胺喋呤及長春地辛（同上文之Rowland等人，（1986))。用於抗體-毒素接合物之毒素包括細菌毒素，諸如白喉毒素；植物毒素，諸如蓖麻毒素；小分子毒素，諸如格爾德黴素（geldanamycin) (Mandler 等人（2000) J· of the Nat· Cancer Inst. 92(19):1573-1581 ; Mandler等人（2000) ά Mei 119007.doc -127- 200813092Niculescu-Duvaz and Springer (1997) jdv. 26:151-172; U.S. Patent No. 4,975,278). The immunoconjugate can allow for the delivery of a drug moiety to the tumor and intracellular accumulation therein, wherein systemic administration of the unconjugated drug can produce an unacceptable degree of toxicity to normal cells and tumor cells to be eliminated (6 & 1 (1 \¥丨11等, 1^被 (March 1500, 1986), pp. 603-05); Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, ''Monoclonal Antibodies' 84: Biological And Clinical (A. Pinchera et al. eds.) pp. 475-506. Multiple antibodies and monoclonal antibodies have been reported to be suitable for such strategies (Rowland et al., (1986) Cancer Immunol. Immunother 21: 183). -87) ° Drugs used in methods such as doxorubicin, doxorubicin, methotrexate and vindesine (the same as Rowland et al., (1986) above) for antibody-toxin bonding Toxins include bacterial toxins such as diphtheria toxin; phytotoxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al. (2000) J. of the Nat Cancer Inst. 92 ( 19): 1573-158 1 ; Mandler et al. (2000) ά Mei 119007.doc -127- 200813092

Chem· Letters 10:1025-1028 ; Mandler 等人（2002)Chem· Letters 10:1025-1028; Mandler et al. (2002)

Bioconjugate Chem. 13:786-791);美登鹼（maytansinoids) (EP 1391213 ; Liu等人，（1996) /Voc. 93:8618-8623)及卡奇黴素（Lode 等人（1998) Cwcer 58:2928 ； Hinman# A(1993) Cancer Res. 53:3336-3342)。該等毒素可藉由包括微管蛋白結合、DNA結合或拓樸異構酶抑制之機制發揮其細胞毒性作用。一些細胞毒性藥物當接合至大抗體或蛋白受體配位體時傾向於為惰性或低活性的。 ZEVALIN®(替伊莫單抗（ibritumomab tiuxetan)， Biogen/Idec)為抗體_放射性同位素接合物，其係由對抗在正常及惡性B淋巴細胞表面發現之CD20抗原之鼠科IgGl κ 單株抗體及以硫脲連接子-螯合劑結合之lnIn或9GY放射性同位素組成（Wiseman 等人，（2000)五wr· c/owr· iVwc/· Med. 27(7):766-77; Wiseman等人，（2002) 5/“ 99(12):4336-42; Witzig# A ^ (2002) J. Clin. Oncol. 20(10):2453-63; Witzig 等人，（2002) J. C/k· Οπα/· 20(15):3262-69)。儘管 ZEVALIN具有對抗B細胞非霍奇金氏淋巴瘤（NHL)之活性，但是投藥在大部分患者體内均導致嚴重且長期之血細胞減少症。於2000年批准一種由連接至卡奇黴素之hu CD3 抗體組成之抗體藥物接合物MYLOTARGtm(吉妥單抗 (gemtuzumab ozogamicin), Wyeth Pharmaceuticals)藉由注射來治療急性骨髓白血病（Drugs of the Future (2000) 25(7)··686 ;美國專利第4970198號；第5079233號；第 119007.doc -128 - 200813092 5585089號；第 5606040號；第 5693762號；第 5739116號；第5767285號；第5773001號）。由經由二硫連接子SPP連接至美登鹼藥物部分DM1之huC242抗體組成之抗體藥物接合物坎突單抗（Cantuzumab mertansine)(Immunogen，Inc.)進展至治療表現CanAg之癌症（諸如結腸癌、胰腺癌、胃癌及其他）之II期試驗。由連接至美登鹼藥物部分DM1之抗前列腺特異性膜抗原（PSMA)單株抗體組成之抗體藥物接合物 MLN-2740(Millennium Pharm., BZL Biologies, Immunogen ^ Inc.)處於***腫瘤的可能治療研發中。海兔毒素 (dolastatin)之合成類似物auristatin肽、auristatin E(AE)及單甲基auristatin(MMAE)係接合至篏合單株抗體cBR96(對癌瘤上之路易斯Y(Lewis Y)具有特異性）及cAC10(對血液科惡性腫瘤上之CD30具有特異性）(Doronina等人（2003) Nature BiotechnoL 21(7):778-784)^.^.^^^^^ 47 。在某些實施例中，免疫接合物包含抗TAT226抗體及化療劑或其他毒素。適用於產生免疫接合物之化療劑於本文中 ( 描述（例如上文）。亦可使用酶活性毒素及其片段且其係描述於本文中。在某些實施例中，免疫接合物包含抗TAT226抗體及一或多種小分子毒素，包括（但不限於）小分子藥物，諸如卡奇黴素、美登驗、海兔毒素、auristatin、單端孢黴烯 (trichothecene)及CC1065 ;以及具有細胞毒性活性之此等藥物的衍生物。此等免疫接合物之實例係於下文進一步詳述。 119007.doc -129- 200813092 1.例示性免疫接合物本發明之免疫接合物（或，，抗體_藥物接合物"（"adc，，））可具有下式I，其中抗TAT226抗體經由可選連接子（L)接合 (亦即共價連接）至一或多個藥物部分（D)。Bioconjugate Chem. 13: 786-791); maytansinoids (EP 1391213; Liu et al, (1996) / Voc. 93: 8618-8623) and calicheamicin (Lode et al. (1998) Cwcer 58 : 2928 ; Hinman # A (1993) Cancer Res. 53:3336-3342). These toxins exert their cytotoxic effects by a mechanism including tubulin binding, DNA binding or topoisomerase inhibition. Some cytotoxic drugs tend to be inert or less active when conjugated to large antibody or protein receptor ligands. ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate that is a murine IgG1 κ monoclonal antibody against CD20 antigen found on the surface of normal and malignant B lymphocytes. Composition of lnIn or 9GY radioisotopes bound by a thiourea linker-chelator (Wiseman et al., (2000) five wr·c/owr·iVwc/. Med. 27(7): 766-77; Wiseman et al., ( 2002) 5/" 99(12): 4336-42; Witzig# A ^ (2002) J. Clin. Oncol. 20(10): 2453-63; Witzig et al., (2002) J. C/k· Οπα /· 20(15):3262-69). Although ZEVALIN is active against B-cell non-Hodgkin's lymphoma (NHL), administration in both patients causes severe and long-term cytopenia. In 2000, an antibody drug conjugate MYLOTARGtm (gemtuzumab ozogamicin, Wyeth Pharmaceuticals) consisting of hu CD3 antibody linked to calicheamicin was approved for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7)··686; US Patent No. 4970198; No. 5079233; No. 119007.doc-128 - 200813092 No. 5,585,089; No. 5,560, 640; No. 5,693,762; No. 5,739,116; No. 5,767, 285; No. 5,773, 301. Antibody drug conjugates consisting of huC242 antibody linked to the maytansine drug moiety DM1 via a disulfide linker SPP The monoclonal antibody (Cantuzumab mertansine) (Immunogen, Inc.) progressed to a phase II trial for the treatment of cancers that express CanAg (such as colon cancer, pancreatic cancer, gastric cancer, and others). Anti-prostate specificity by DM1 linked to the maydidine drug moiety DM1 The membrane drug antigen (PSMA) monoclonal antibody antibody drug conjugate MLN-2740 (Millennium Pharm., BZL Biologies, Immunogen ^ Inc.) is in the possible therapeutic development of prostate tumors. The synthetic analog auristatin of dolastatin The peptide, auristatin E (AE) and monomethyl auristatin (MMAE) are ligated to the monoclonal antibody cBR96 (specific for Lewis Y on cancer) and cAC10 (for hematological malignancies) CD30 is specific) (Doronina et al. (2003) Nature Biotechno L 21(7): 778-784) ^.^.^^^^^ 47 . In certain embodiments, the immunoconjugate comprises an anti-TAT226 antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents suitable for use in the production of immunoconjugates are described herein (described above (eg, above). Enzymatically active toxins and fragments thereof can also be used and are described herein. In certain embodiments, the immunoconjugate comprises anti-TAT226 Antibodies and one or more small molecule toxins, including but not limited to small molecule drugs such as calicheamicin, methine, dolastatin, auristatin, trichothecene and CC1065; and cytotoxic Derivatives of such agents of activity. Examples of such immunoconjugates are described in further detail below. 119007.doc -129- 200813092 1. Exemplary immunoconjugates The immunoconjugates of the invention (or, antibody-drugs) The conjugate """adc,," can have the formula I wherein the anti-TAT226 antibody is joined (i.e., covalently linked) to one or more drug moieties (D) via an optional linker (L).

Ab-(L-D)p i 口此，抗TAT226抗體可直接或經由連接子接合至藥物。在式I中，P為每抗體藥物部分之平均數量，其可在例如每抗體約1至約20個藥物部分之範圍内，且在某些實施例中為每抗體1至約8個藥物部分之範圍内。 a)例示性連接子連接子可包含一或多個連接子組份。例示性連接子組份包括6-馬來醯亞胺己醯基（"MC”）、馬來醯亞胺丙醯基 (’’MP”）、類胺酸-瓜胺酸（"val‘”或、"）、丙胺酸_*** 酸（"ala-phe”）、對胺基节氧羰基（”PAB”）、N_琥轴醯亞胺基 4-(2-吡啶硫基）戊酸酯（”SPP”）、冰琥珀醯亞胺基4_(n_馬來醯亞胺甲基）環己烷4甲酸酯（”SMCC”）& N_琥珀醯亞胺基 (4-碘-乙醯基）胺基苯甲酸酯（”SIAB”）。此項技術中已知各種連接子組份，其中一些於下文中描述。連接子可為促進細胞中藥物釋放之"可裂解連接子"。舉例而言，可使用酸不穩定連接子（例如膝）、蛋白酶敏感（例如肽酶敏感）連接子、光不穩定連接子、二甲基連接子或含有二硫鍵之連接子（Chad等人，Cancer Reseaixh 52:127_ 131 (1992);美國專利第 5,2〇8,〇20號）。在一些實施例中，連接子組份可包含將抗體連接至另一 119007.doc -130- 200813092 連接子組份或連接至單元係展*於下文中:复：、分之”拉伸單元"。例示性拉伸位點）： ”中波浪線表示與抗體之共價連接Ab-(L-D)p i Thus, the anti-TAT226 antibody can be conjugated to the drug either directly or via a linker. In Formula I, P is the average number of drug moieties per antibody, which may range, for example, from about 1 to about 20 drug moieties per antibody, and in certain embodiments from 1 to about 8 drug moieties per antibody. Within the scope. a) Exemplary Linkers A linker can comprise one or more linker components. Exemplary linker components include 6-maleimide hexamethylene ("MC"), maleimide propyl ketone (''MP'), and arginine- citrulline ("val '",, "), alanine _ phenylalanine ("ala-phe", p-amino oxycarbonyl ("PAB"), N-succinimide 4-(2-pyridylthio) Valerate ("SPP"), ice amber quinone imine 4_(n_maleimidomethyl)cyclohexane 4carboxylate ("SMCC") & N_amber quinone imine ( 4-iodo-ethenylamino benzoate ("SIAB"). Various linker components are known in the art, some of which are described below. The linker can be a "cleavable linker" that promotes drug release in the cell. For example, an acid labile linker (eg, a knee), a protease sensitive (eg, peptidase sensitive) linker, a photolabile linker, a dimethyl linker, or a linker containing a disulfide bond can be used (Chad et al. , Cancer Reseaixh 52: 127_131 (1992); US Patent No. 5, 2, 8, 〇 20). In some embodiments, the linker component can comprise linking the antibody to another 119007.doc-130-200813092 linker component or to a cell lineage* in the following: complex:, "stretch unit" An exemplary stretching site: "The middle wavy line indicates covalent attachment to the antibody

ΟΟ

在一些貫施例中，連接子組份可包含胺基酸單元。在一個此貝施例中，胺基酸單元使得可由蛋白酶裂解連接子，藉此促進藥物在暴露於諸如溶酶體酶之細胞内蛋白酶時自免疫接合物釋放。參見例如D〇r〇nina等人（2〇〇3) A21:778-784。例示性胺基酸單元包括（但不限於）二肽、三肽、四肽及五肽。例示性二肽包括··纈胺酸_ 瓜胺酸（vc或val-cit);丙胺酸_***酸丨“或“心忡幻；苯丙胺酸-離胺酸（fk或phe-lys);或N-甲基-纈胺酸-瓜胺酸 (Me-val-cit)。例示性三肽包括：甘胺酸-纈胺酸-瓜胺酸 119007.doc •131· 200813092 (g y CU)及甘酸-甘胺酸·甘胺酸（gly-gly-gly)。胺基酸单兀可包含天然產生之胺基酸殘基，以及微量胺基酸及非天然產生之絲_似物，諸如瓜胺酸。胺基酸單元可經設計及最優化其選擇性以由特定酶（例如腫瘤相關蛋白酶組織蛋白酶B、C及D或纖溶酶蛋白酶）酶促裂解。 —在一些實施財，連接子組份可包含直接或藉由拉伸單元及/或胺基1單元將抗體連接至藥物部分之，，間隔"單元。間隔單兀可為”自毀壞性，，或，，非自毁壞性，，的。，，非自毀壞性，, 間隔單元為部分或所有間隔單元在ADC之酶促（例如蛋白水解)裂解時保持結合至藥物部分的間隔單元。非自毀壞性間隔單元之實例包括（但不限於）甘胺酸間隔單元及甘胺酸-甘胺酸間隔單元。亦涵蓋易受序列特異性酶促裂解影響之狀間隔劑之其他組合。舉例而言，藉由腫瘤細胞相關蛋白酶酶促裂解含有甘胺酸-甘胺酸間隔單元之ADC將導致自ADC之剩餘物中釋放甘胺酸-甘胺酸藥物部分。在一個此實施例中，接著使甘胺酸_甘胺酸藥物部分於腫瘤細胞中經受獨立水解步驟，從而自藥物部***解甘胺酸_甘胺酸間隔單元。自t又壞性，間隔單元使得無需獨立水解步驟即可釋放藥物部分。在某些實施例中，連接子之間隔單元包含對胺基苄基單元。在一個此實施例中，將對胺基苄醇經由醯胺鍵連接至胺基酸單元，且在苄醇與細胞毒性劑之間形成胺基甲酸酯、胺基甲酸甲酯或碳酸酯。參見例如Hamann等人 (2005)五xperi (9ρζ·π. TTier. 心（2005) 15:1087-1103。在 119007.doc -132- 200813092 一實施例中，間隔單元為對胺基苄氧羰基（PAB)。在某些實施例中，對胺基苄基單元之伸苯基部分經Qm取代，其中Q為-Ci-Cg烧基、-〇-(Ci-C8烧基）、素、-石肖基或·氣基；且m為0-4範圍内之整數。自毁壞性間隔單元之實例另外包括（但不限於）與對胺基苄醇電學相似之芳族化合物（參見例如US 2005/0256030 A1)，諸如2-胺基咪唑-5-甲醇衍生物（Hay 等人（1999) CTzem. Zeii· 9:2237)及鄰或對·胺基苄基縮醛。可使用在醯胺鍵水解時經受環化之間 ( 隔劑，諸如經取代及未經取代之4-胺基丁酸醯胺 (Rodrigues等人，1995，2，223);經適當取代之雙環[2·2·1]及雙環[2·2·2]環系統（Storm等人，X Amer. Chem· Soc·，1972，94，5 8 1 5);及 2-胺基苯基丙酸醯胺（Amsberry等人，J· C/zem·，1990，55，5867)。於甘胺酸之a位置經取代之含胺藥物的消除反應（Kingsbury等人，J· C/zern·，1984, 27，1447)亦為適用於 ADC之自毁壞性間隔劑之實例。 I 在一實施例中，間隔單元為如下所述之支鏈雙（羥甲基）苯乙烯（BHMS)，其可用以併入及釋放多種藥物。In some embodiments, the linker component can comprise an amino acid unit. In one such embodiment, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating the release of the drug from the immunoconjugate when exposed to an intracellular protease such as a lysosomal enzyme. See, for example, D〇r〇nina et al. (2〇〇3) A21:778-784. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include · valine _ citrulline (vc or val-cit); alanine _ phenylalanine 丨 "or "heart illusion; phenylalanine- lysine (fk or phe-lys); or N-methyl-proline- citrulline (Me-val-cit). Exemplary tripeptides include: glycine-proline-citrulline 119007.doc • 131· 200813092 (g y CU) and glycine-gly-gly. The amino acid monoterpene may comprise naturally occurring amino acid residues, as well as trace amounts of amino acids and non-naturally occurring filaments such as citrulline. Amino acid units can be designed and optimized for their enzymatic cleavage by specific enzymes such as the tumor associated proteinase cathepsins B, C and D or plasmin proteases. - In some implementations, the linker component can comprise an antibody, linked to the drug moiety, either directly or via an extension unit and/or an amine unit. Intervals can be "self-destructive, or, non-self-destructive," non-self-destructive, compartmentalized units for enzymatic (eg, proteolytic) cleavage of some or all of the spacer units in the ADC Maintaining a spacer unit that binds to the drug moiety. Examples of non-self-destructive spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Also encompasses susceptible sequence-specific enzymatic cleavage Other combinations of affected spacers. For example, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease will result in the release of glycine-glycine from the remainder of the ADC. The drug moiety. In one such embodiment, the glycine-glycine drug moiety is then subjected to an independent hydrolysis step in the tumor cell to cleave the glycine-glycine spacer unit from the drug moiety. The spacer unit allows the drug moiety to be released without the need for an independent hydrolysis step. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, the p-aminobenzyl alcohol Attached to the amino acid unit by a guanamine bond and form a urethane, methyl carbamate or carbonate between the benzyl alcohol and the cytotoxic agent. See, for example, Hamann et al. (2005) Five xperi (9ρζ· π. TTier. 心(2005) 15:1087-1103. In an embodiment, 119007.doc-132-200813092, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the amine group The phenyl moiety of the benzyl unit is substituted by Qm, wherein Q is -Ci-Cg alkyl, -〇-(Ci-C8 alkyl), arginyl, -schhopenyl or ke group; and m is in the range of 0-4 Examples of self-destructive spacer units include, but are not limited to, aromatic compounds that are electrically similar to aminobenzyl alcohol (see, for example, US 2005/0256030 A1), such as 2-aminoimidazole-5-methanol. Derivatives (Hay et al. (1999) CTzem. Zeii 9: 2237) and o- or p-aminobenzyl acetals can be used to undergo cyclization upon hydrolysis of the guanamine bond (separator, such as substituted and Unsubstituted 4-aminobutyric acid decylamine (Rodrigues et al., 1995, 2, 223); appropriately substituted bicyclo [2·2·1] and bicyclo[2·2·2] ring systems (Storm et al. people X Amer. Chem. Soc., 1972, 94, 5 8 1 5); and 2-aminophenyl phenyl decanoate (Amsberry et al., J. C/zem., 1990, 55, 5867). The elimination reaction of the substituted amine-containing drug at the a position of the amino acid (Kingsbury et al., J. C/Zern, 1984, 27, 1447) is also an example of a self-destructive spacer suitable for ADC. In an embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) as described below, which can be used to incorporate and release a plurality of drugs.

酶促裂解物 119007.doc -133 - 200813092 2 · Q為c! c8烷基、_0-(Ci_C8烧基）、·_素、-确基或-氛 ^為〇4範圍内之整數；η為0或1 ;且p介於丨至約2〇之範圍内。每連接子可包含以上連接子組份中之任一或多種。在某些貝施例中’連接子如以下adc式^中之支架所展示：Enzymatic lysate 119007.doc -133 - 200813092 2 · Q is c! c8 alkyl, _0-(Ci_C8 alkyl), _ _, -, or - ^ ^ is an integer in the range of 〇 4; η is 0 or 1; and p is in the range of 丨 to about 2〇. Each linker can comprise any one or more of the above linker components. In some of the examples, the 'linker' is shown in the following adj:

Ab -ffAa-Ww-Yy]_D) j j 其中A為拉伸單元且a為〇至丨之整數；w為胺基酸單元且〜為0至12之正數’ γ為間隔單元；且丫為〇、ι或2;且Ab、〇及p如上文關於式〗所定義。此等連接子之例示性實施例於以引用的方式明確併入本文之US 2005_0238649 A1中描述。例不性連接子組份及其組合於下文式II之ADC内容中展不 ·Ab - ffAa - Ww - Yy ] _ D ) jj where A is a stretching unit and a is an integer from 〇 to ;; w is an amino acid unit and ~ is a positive number of 0 to 12 ' γ is a spacer unit; and 丫 is 〇 , ι or 2; and Ab, 〇 and p are as defined above for the formula. Illustrative embodiments of such a linker are described in US 2005_0238649 A1, which is expressly incorporated herein by reference. The example of the contiguous linker component and its combination are shown in the ADC content of Equation II below.

119007.doc -134- 200813092119007.doc -134- 200813092

MC-val_cit_PAB 隔單元及胺基酸單元可諸如 US 2005-0238649 連接子組份，包括拉伸單元、間藉由此項技術中已知之方法合成， A1中所述之彼等方法。The MC-val_cit_PAB spacer unit and the amino acid unit can be, for example, a linker component of US 2005-0238649, including stretching units, which are synthesized by methods known in the art, and the methods described in A1.

b)例示性藥物部分 (1)美登素及美登鹼在-些實施例中’免疫接合物包含接合至一或多個美登鹼分子之本發明抗體。美登鹼為藉由抑制微管蛋白聚合而起作用之有絲***抑制劑。美登素首先自東非灌木齒葉美登木（east African shrub Maytenus serrata)分離（美國專利第 3,896，111號）。隨後，發現某些微生物亦產生美登鹼，諸如美登醇及C-3美登醇醋（美國專利第4，151，042號）。合成美登醇及其衍生物及類似物揭示於以下專利中，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 4,45G，254號；第 4,362,663 號及第 4’371’533b) Exemplary Drugs (1) Maytansine and Maytans In some embodiments, an immunoconjugate comprises an antibody of the invention conjugated to one or more maytansine molecules. Maytansine is a mitotic inhibitor that acts by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was found that certain microorganisms also produced maytansines such as maytansinol and C-3 maytanol vinegar (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed in, for example, 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; No. 4,308,268; 4,309,268; 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; 4,45G , No. 254; Nos. 4,362,663 and 4'371'533

119007.doc -135 - 200813092 美登鹼藥物部分為抗體-藥物接合物中具有吸收力之藥物部分，因為其：⑴相對易於藉由醱酵或化學修飾或醱= 產物之衍生作用來製備，（ii)易受與適用於經由非二硫連接子接合至抗體之官能基衍生作用的影響，（iii)於血漿中穩定；及（iv)有效對抗各種腫瘤細胞株。此項技術中已熟知適合用作美登鹼藥物部分之美登素化合物且可根據已知方法自天然來源分離或使用基因工程設計技術產生（參見Yu等人（2002) PNAS 99:7968-7973)。美登醇及美登醇類似物亦可根據已知方法合成地製備。例示性美登鹼藥物部分包括彼等具有經改質芳環者，諸如：C-19-去氯（美國專利第4256746號）（藉由安沙黴素 P2(ansamyt〇Cin P2)之氫化鋁鋰還原反應所製備）；€_2〇_羥基（或0：-20-去甲基）+/-〇19-去氯（美國專利第43 61650號及第4307016號）（藉由使用鏈黴素（strept〇myCes)或放線菌 (Actinomyces)之去甲基作用或使用LAH之脫氯作用所製備）；及C-20-去甲氧基、C-20-醯氧基（_〇c〇R)+/_去氣（美國專利第4,294,757號）（藉由使用醯基氣之醯化作用所製備）及彼等於其他位置具有改質者。例示性美登鹼藥物部分亦包括彼等具有諸如以下改質者：C-9-SH(美國專利第4424219號）（藉由美登醇與h2S或 P2Ss反應所製備）；014-烷氧基曱基（去甲氧基/CH2 OR)(US 43 31598) ; C-14-經甲基或醯氧基曱基（ch2OH或 (^20八〇)(美國專利第4450254號）（由奴卡菌屬（]^〇〇&他&)所製備）；C-15-羥基/醯氧基（US 4364866)(藉由以鏈黴素轉化 119007.doc -136- 200813092 美登醇所製備）；c-l 5-甲氧基（美國專利第43 13946號及第 4315929號）（自 Trewia nudlflora 分離）；C-18-N-去甲基（美國專利第4362663號及第4322348號）（藉由以鏈黴素使美登醇去甲基化所製備）及4,5-去惫茸me — 古乳基（US 4371533)(藉由美之三氯化鈦/LAH還原所製備）。且畔美登鹼藥物部分之例 DM1、DM3及DM4 : 示性實施例包括具有以下結構之119007.doc -135 - 200813092 The mesaconic drug moiety is an absorbent drug moiety in an antibody-drug conjugate because it: (1) is relatively easy to prepare by fermentation or chemical modification or derivatization of the product; Ii) susceptible to effects associated with functional group derivatization to the antibody via a non-disulfide linker, (iii) stable in plasma; and (iv) effective against various tumor cell lines. The maytansin compounds suitable for use as a portion of the maytansine drug are well known in the art and can be isolated from natural sources or produced using genetic engineering techniques according to known methods (see Yu et al. (2002) PNAS 99: 7968-7973). . The maytansinol and maytansinol analogs can also be prepared synthetically according to known methods. Exemplary mayaconine drug moieties include those having a modified aromatic ring, such as: C-19-dechlorinated (U.S. Patent No. 4,256,746) (lithium aluminum hydride with ansamycin P2 (ansamyt〇Cin P2) Prepared by reduction reaction; € 2 〇 hydroxy (or 0: -20-demethyl) +/- 〇 19-dechlorinated (US Patent Nos. 43 61650 and 4307016) (by using streptomycin ( Strept〇myCes) or demethylation of actinomyces or prepared by dechlorination of LAH; and C-20-desmethoxy, C-20-decyloxy (_〇c〇R) +/_ degassing (U.S. Patent No. 4,294,757) (prepared by the use of hydrazine-based gasification) and which have upgraders at other locations. Exemplary maytansine drug moieties also include those having the following modifications: C-9-SH (U.S. Patent No. 4,424,219) (prepared by the reaction of maytansinol with h2S or P2Ss); 014-alkoxyquinone (demethoxy/CH2 OR) (US 43 31598); C-14-methyl or decyloxy (ch2OH or (^20 octa) (US Patent No. 4,450,254) (by Nocardia) Genus ()^〇〇&he&)prepared; C-15-hydroxy/decyloxy (US 4364866) (prepared by conversion of streptomycin 119007.doc-136-200813092 maytansinol) ;cl 5-methoxy (US Patent Nos. 43 13946 and 4315929) (separated from Trewia nudlflora); C-18-N-desmethyl (US Patent Nos. 4362663 and 4322348) (by Streptomycin is prepared by demethylation of maytansin) and 4,5-de-scented me--an old base (US 4,371,533) (prepared by the addition of titanium trichloride/LAH). Examples of the base drug moiety DM1, DM3 and DM4: The illustrative examples include the following structures

119007.doc -137. 200813092 ch3119007.doc -137. 200813092 ch3

其中波浪線表示藥物之硫原子與抗體-藥物接合物之連接子（L)之共價連接。已報導由SMCC連接至DM1之 HERCEPTIN®(曲妥珠單抗（trastuzumab))(WO 2005/037992 ; US 2005/0276812 A1)。其他例示性美登鹼抗體-藥物接合物具有以下結構及縮寫（其中Ab為抗體且p為1至約8):The wavy line indicates the covalent attachment of the sulfur atom of the drug to the linker (L) of the antibody-drug conjugate. HERCEPTIN® (trastuzumab) linked to DM1 by SMCC has been reported (WO 2005/037992; US 2005/0276812 A1). Other exemplary maytan base antibody-drug conjugates have the following structure and abbreviations (where Ab is an antibody and p is from 1 to about 8):

一 Ab PAn Ab P

Ab-SPP-DMl 119007.doc -138- 200813092Ab-SPP-DMl 119007.doc -138- 200813092

-Ab-Ab

Ab-SMCC-DMl 其中DM1經由BMPEO連接子連接至抗體之硫醇基的例示性抗體-藥物接合物具有以下結構及縮寫：Ab-SMCC-DMl An exemplary antibody-drug conjugate in which DM1 is linked to a thiol group of an antibody via a BMPEO linker has the following structure and abbreviations:

其中Ab為抗體；η為0、1或2;且p為1、2、3或4。 / 含有美登驗之免疫接合物、製造其之方法及其治療用途揭示於以下專利中，例如美國專利第5,208,020號、第 5,416,064 號、US 2005/0276812 Α1及歐洲專利ΕΡ 0 425 235 Β1，該等揭示内容係以引用的方式明確併入本文中。 Liu等人Proc. dead· 5W· 93:8618-8623 (1996)描述包含連接至針對人類結腸直腸癌之單株抗體C242之命名為DM 1的美登驗之免疫接合物。發現接合物對經培養結腸癌細胞具有高細胞毒性’且於活體内腫瘤生長檢定中展現 119007.doc -139· 200813092 权》腫瘤活性。Wherein Ab is an antibody; η is 0, 1 or 2; and p is 1, 2, 3 or 4. / The immunoconjugate of the invention, the method of making the same, and the therapeutic use thereof are disclosed in the following patents, for example, U.S. Patent Nos. 5,208,020, 5,416,064, US 2005/0276812 Α1, and European Patent No. 0 425 235 Β1, The disclosures are expressly incorporated herein by reference. Liu et al. Proc. dead 5W 93:8618-8623 (1996) describes the immunoconjugate of Meden, which is linked to the monoclonal antibody C242 for human colorectal cancer and designated DM1. The conjugate was found to be highly cytotoxic to cultured colon cancer cells' and exhibited tumor activity in an in vivo tumor growth assay 119007.doc-139.200813092.

Chari 等人 CancerChari et al. Cancer

Research 52:127-131Research 52: 127-131

(1992)描述其中美登驗經由二硫連接子接合至與人類結腸癌細胞株上之抗原結合之鼠類抗體A7或接合至與HER· /致癌基因結合之另一鼠類單株抗體ΤΑ· 1的免疫接合物。於活體外測試1^·1·美登驗接合物對在每-細胞中表現 3 X 1 〇5 HER 9 I π ϋ rc K_2表面抗原之人類乳癌細胞株SK-BR-3的細胞毒性。藥物接合物達成與游離美登鹼藥物類似之細胞毒 f生度一可藉由增加每一抗體分子之美登鹼分子數量而增加A7美登驗接合物於小鼠體内展示低的全身細胞毒性。抗體美登鹼接合物係藉由在不顯著減低抗體或美登鹼分子之生物活性下將抗體化學連接至美登驗分子來製備。參見例如美國專利第5,2〇8,〇2〇號（其揭示内容以引用的方式明確併入本文）。每抗體分子平均接合3-4個美登鹼分子已展不增強靶細胞之細胞毒性而不對抗體之功能或溶解性產生不利影響之功效，儘管即使僅預期一個毒素/抗體分子在裸抗體使用期間增強細胞毒性。此項技術中已熟知美登鹼且可藉由已知技術合成或自天然來源分離。合適美登鹼例如揭示於美國專利第5，潰，_號及上文提及之其他專利及非專利公開案中。較佳美登鹼為美登醇及在美登醇分子之方環或其他位置改質之美登醇類似物，諸如各種美登醇 g旨。此項技術中已知許多用於製造抗體·美登鹼接合物之連接基團，包括例如於美國專利第5208020號或EP專利〇 425 235 B1 , Chari 等人 Ccrncer 52:127-131 (1992);及 119007.doc 200813092 US 2005/016993 A1中所揭示之彼等，該等揭示内容以引用的方式明確併入本文。包含連接子組份8^1(：(：之抗體-美登驗接合物可如 US 2005/0276812 Al，"Antibody-dnig conjugates and Methods”中所揭示來製備。如上述專利所揭示，連接子包含二硫基、硫醚基、酸不穩定基、光不穩定基、肽酶不穩定基或酯酶不穩定基。其他連接子於本文中描述且例示。抗體與美登鹼之接合物可使用各種雙官能性蛋白偶合劑來製造，諸如N-琥珀醯亞胺基-3-(2-吼咬二硫基）丙酸酯 (SPDP)、琥珀醯亞胺基冰馬來醯亞胺基甲基）環己烧_ 1-甲酸酯（SMCC)、亞胺基硫雜環戊烷（IT)、醯亞胺基酯之雙官能性衍生物（諸如己二醯亞胺二甲酯HC1)、活性酯（諸如辛一酸二琥珀醯亞胺基酯）、醛（諸如戊二駿）、雙疊氮基化合物（諸如雙（對疊氮基苄醯基）己二胺）、雙重氮鹽衍生物（諸如雙（對重氮苄醯基）_乙二胺）、二異氰酸酯（諸如曱苯 2,6-二異氰酸醋）及雙活性氟化合物（諸如丨，^二氟_2,4_二硝基苯）°在某些實施例中，偶合劑為琥珀醯亞胺基_3气2_ 口比咬二硫基）丙酸酯（SPDP)(Carlss〇n等人，㈣丄 173:723-737 (1978))或Ν·琥珀醯亞胺基-4_(2-吡啶硫基）戊酸酯（SPP)以提供二硫鍵。連接子可視連接類型於各種位置連接至美登鹼。舉例而言’可藉由使用習知偶合技術與羥基反應而形成酯鍵。反應可發生於具有羥基之C-3位置、經羥甲基改質之C-14位置、經經基改質之C· 15位置及具有羥基之C-20位置。在一 119007.doc -141 - 200813092 實施例中，於美登醇或美登醇類似物之C-3位置形成鍵聯。 (2)Auristatin及海兔毒素在一些實施例中，免疫接合物包含接合至海兔毒素或海兔毒性肽類似物或衍生物（例如auristatin)之本發明抗體（美國專利第5635483號；第5780588號）。已證實海免毒素及 audstatin干擾微管動力學、GTP水解及核與細胞*** (Woyke ^ A (2001) Antimicrob. Agents and Chemother, 45( 12):3 580-3 584)且具有抗癌（美國專利第5663 149號）及抗真菌活性（Pettit 等人（1998) 42:2961-2965) °海兔毒素或auristatin藥物部分可經由肽藥物部分之N(胺基）端或C(羧基）端連接至抗體（WO 02/088172)。例示性auristatin實施例包括N端連接之單曱基auristatin 藥物部分DE及DF，其揭示於2004年3月28日公開之Senter 等人，Proceedings of the American Association for Cancer Research，第45卷，文摘號623中，其揭示内容之全文係以引用的方式明確併入本文。肽藥物部分可選自以下式DE及DF :(1992) describes a murine antibody A7 which is conjugated to an antigen on a human colon cancer cell line via a disulfide linker or a murine monoclonal antibody which binds to a HER·/oncogene. 1 immunoconjugate. The cytotoxicity of human breast cancer cell line SK-BR-3 expressing 3 X 1 〇5 HER 9 I π ϋ rc K 2 surface antigen in each cell was tested in vitro. The drug conjugate achieves a similar cytotoxicity to the free meringue drug. By increasing the number of merlotine molecules per antibody molecule, the A7 methodamine exhibits low systemic cytotoxicity in mice. . The antibody maytansine conjugate is prepared by chemically linking the antibody to a Meitenberg molecule without significantly reducing the biological activity of the antibody or the memantine molecule. See, for example, U.S. Patent No. 5,2,8, the entire disclosure of which is hereby incorporated by reference. The average binding of 3-4 meringue molecules per antibody molecule has not enhanced the cytotoxicity of target cells without adversely affecting the function or solubility of the antibody, even though only one toxin/antibody molecule is expected to be used during naked antibody use. Enhances cytotoxicity. Maytansine is well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansines are disclosed, for example, in U.S. Patent No. 5, Col. No., and other patent and non-patent publications mentioned above. Preferably, the maytansine is a maytansinol and a metanol analog which is modified at the square or other position of the maytansin molecule, such as various maytansin. A number of linking groups are known in the art for the manufacture of antibody-Medogen base conjugates, including, for example, U.S. Patent No. 5,208,020 or EP Patent No. 425 235 B1, Chari et al. Ccrncer 52: 127-131 (1992) And the disclosures of which are hereby incorporated by reference. The inclusion of the linker component 8^1 (:(: antibody-Melden conjugate can be prepared as disclosed in US 2005/0276812 Al, "Antibody-dnig conjugates and Methods". As disclosed in the above patent, the connection The subunit comprises a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group or an esterase labile group. Other linkers are described and exemplified herein. It can be made using a variety of bifunctional protein couplers, such as N-succinimide-3-(2-bite dithio)propionate (SPDP), amber succinimide, ice-maleimide Bimethyl sulfonate 1-carbamate (SMCC), imidothiolane (IT), bifunctional imide of quinone imide (such as hexamethylenediamine dimethyl ester) HC1), an active ester (such as dinonyl succinimide), an aldehyde (such as pentane), a diazide compound (such as bis(p-azidobenzylidene) hexamethylenediamine), double Nitrogen salt derivatives (such as bis(p-diazepine)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and double activity Compound (such as hydrazine, difluoro-2,4-dinitrobenzene) ° In certain embodiments, the coupling agent is amber quinone imine _3 gas 2 _ bis dithio) propionate (SPDP (Carlss〇n et al., (iv) 丄 173: 723-737 (1978)) or Ν· amber imino-4-(2-pyridylthio) valerate (SPP) to provide disulfide bonds. The visible linkage type is attached to the maytansine at various positions. For example, an ester linkage can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at a C-3 position having a hydroxyl group, modified with a hydroxymethyl group. The C-14 position, the C.15 position modified by the base, and the C-20 position having a hydroxyl group. In an embodiment of 119007.doc-141 - 200813092, the C of maytansinol or maytansinoid analogue -3 position forms a linkage. (2) Auristatin and dolastatin In some embodiments, the immunoconjugate comprises an antibody of the invention that binds to a dolastatin or a haretotoxic peptide analog or derivative (eg, auristatin) (United States Patent No. 5,356, 548; No. 5,780, 588. It has been demonstrated that seaweed toxin and audstatin interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division ( Woyke ^ A (2001) Antimicrob. Agents and Chemother, 45(12): 3 580-3 584) and has anticancer (US Patent No. 5663 149) and antifungal activity (Pettit et al. (1998) 42:2961- 2965) ° The dolastatin or auristatin drug moiety can be linked to the antibody via the N (amino) or C (carboxy) terminus of the peptide drug moiety (WO 02/088172). Exemplary auristatin embodiments include N-terminally linked monothiol auristatin drug moieties DE and DF, which are disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. In 623, the entire disclosure of the disclosure is expressly incorporated by reference. The peptide drug moiety can be selected from the following formulas DE and DF:

119007.doc -142- 200813092119007.doc -142- 200813092

〇其中de及df之波浪線表示與抗體或抗艰-連接子組份之共價連接位點’且於各位置獨立： R2係選自Η及CVC8烷基； R3係選自H、CVC8烷基、C3_C8碳環、方基、C丨-c8烧基-(C3_C8碳環）、c3_c8雜環及 (C3_c8雜環）； R4係選自H、Cl-c8烷基、 —甘^ A社 8反衣方基、（VC8烷基- 方基、CVC8烧基_(c3-c8碳環）、c Γ私 (C3-C8^,)； ^ ^ 雜環及 Cl_C8 烧基. R5係選自Η及甲基；或R及R接合形成碳環泛且古 Rb,s, _ a衣且具有式-⑴尺卞^-’其中…及 R獨立地選自H、Cl-c8烷基及 .^ 3匕8石反％且11係選自2、3、斗、3及6 ; R6係選自h&c1-c8烷基； R7係選自Η、CVC8燒基、c c 芸其 r P w * 8灭每、芳基、CVC8烷基- 方土、4_(：8烧基-(c3-C8碳環） (C3-C8雜環）； 3-c8雜環及Cl-C8烧基_ 各R8係獨立地選自H、〇H、 (Ci_C8烷基）；烷基、C3-C8碳環及Ον係選自烷基； R10係選自芳基或C3-C8雜環； 119007.doc -143- 200813092 z為 Ο、s、NH或 NR12，其中1112為(：1-(：8烷基； R11係選自Η、CVCm烷基、芳基、C3-C8雜環、-(R130)m-R14或-(R13〇)m-CH(R15)2 ; m為1-1000範圍内之整數； R13為C2-C8烷基； R14為只或(^-(：8烷基；每次出現之R15獨立地為Η、COOH、-(CH2)n_N(R16)2、-(CH2)n-S03H 或-(CHdn-SOrCrCs烷基； ( 每次出現之R16獨立地為Η、CkC8烷基或-(CH2)n_ COOH ； R18係選自-C(R8)2-C(R8)2-芳基、_(：(118)2-(：(118)2-((：3-(：8雜環）及-(!；(尺8)2-〇(118)2-(〇3-(1；8碳環）；及 η為0至6範圍内之整數。在一實施例中，R3、R4及R7獨立地為異丙基或第二丁基且R5為-Η或甲基。在一例示性實施例中，R3&R4各自為異丙基，R5為-H且R7為第二丁基。 C 在又一實施例中，R2及R6各自為甲基且R9為_H。在又一實施例中，每次出現之R8為_〇ch3。在一例示性實施例中，…及尺4各自為異丙基，“及汉6各自為曱基’ R5為-H，R7為第二丁基，每次出現之 OCH3 且 R9為-H。馬、在一實施例中，Z為-〇-或_NH_。在一實施例中，R10為芳基。在一例示性實施例中，尺1〇為_苯基。 119007.doc -144- 200813092 在一例示性實施例中，當Z為_0-時，R11為-Η、甲基或第三丁基。在一實施例中，當Ζ為_ΝΗ時，R11為-CH(R15)2，其中R15 為-(CH2)n-N(R16)2，且 R16為-CrCs烧基或-(CH2)n-COOH。在另一實施例中，當Z為-NH時，R11為-CH(R15)2，其中 R15為-(CH2)n-S03H。式DE之例示性auristatin實施例為MMAE，其中波浪線表示共價連接至抗體-藥物接合物之連接子（L):波浪 The wavy line of de and df indicates a covalent attachment site to the antibody or the anti-hard-linker component' and is independent at each position: R2 is selected from hydrazine and CVC8 alkyl; R3 is selected from H, CVC8 alkane a group, a C3_C8 carbocyclic ring, a aryl group, a C丨-c8 alkyl group-(C3_C8 carbocyclic ring), a c3_c8 heterocyclic ring and a (C3_c8 heterocyclic ring); R4 is selected from the group consisting of H, Cl-c8 alkyl, and - Anti-clothing group, (VC8 alkyl-square group, CVC8 alkyl group _(c3-c8 carbon ring), c Γ Γ (C3-C8^,); ^ ^ heterocyclic ring and Cl_C8 alkyl group. R5 is selected from Η And methyl; or R and R join to form a carbocyclic and ancient Rb, s, _ a clothing and have the formula - (1) 卞 ^ - ' where ... and R are independently selected from H, Cl-c8 alkyl and . 3匕8石反% and 11 is selected from 2, 3, bucket, 3 and 6; R6 is selected from h&c1-c8 alkyl; R7 is selected from fluorene, CVC8 alkyl, cc 芸, r P w * 8 extinction of each, aryl, CVC8 alkyl-square earth, 4_(:8 alkyl-(c3-C8 carbocyclic) (C3-C8 heterocyclic); 3-c8 heterocyclic and Cl-C8 alkyl _ each R8 Is independently selected from H, 〇H, (Ci_C8 alkyl); alkyl, C3-C8 carbocyclic and Ον are selected from alkyl; R10 is selected from aryl or C3-C8 heterocyclic; 119007.doc -143 - 200813092 z is Ο, s, NH or NR12, wherein 1112 is (: 1-(:8 alkyl; R11 is selected from fluorene, CVCm alkyl, aryl, C3-C8 heterocycle, -(R130)m-R14 or -(R13〇) m-CH(R15)2; m is an integer in the range of 1-1000; R13 is C2-C8 alkyl; R14 is only or (^-(:8 alkyl; each occurrence of R15 is independently Η, COOH, -(CH2)n_N(R16)2, -(CH2)n-S03H or -(CHdn-SOrCrCs alkyl; (each occurrence of R16 is independently hydrazine, CkC8 alkyl or -(CH2)n_COOH; R18 is selected from the group consisting of -C(R8)2-C(R8)2-aryl, _(:(118)2-(:(118)2-((:3-(:8)heterocycle) and -(! (foot 8) 2-〇(118)2-(〇3-(1;8 carbocyclic); and η is an integer in the range of 0 to 6. In one embodiment, R3, R4 and R7 are independently Isopropyl or a second butyl group and R5 is -Η or methyl. In an exemplary embodiment, R3&R4 are each isopropyl, R5 is -H and R7 is a second butyl. In an embodiment, each of R2 and R6 is methyl and R9 is _H. In yet another embodiment, each occurrence of R8 is _〇ch3. In an exemplary embodiment, ... and ruler 4 are each isopropyl Base, "and Han 6 are each sulfhydryl" R5 is -H, and R7 is the second butyl, each occurrence of O CH3 and R9 are -H. In one embodiment, Z is -〇- or _NH_. In one embodiment, R10 is an aryl group. In an exemplary embodiment, the ruler 1 is _phenyl. 119007.doc -144- 200813092 In an exemplary embodiment, when Z is _0-, R11 is -Η, methyl or a third butyl group. In one embodiment, when Ζ is ΝΗ, R11 is -CH(R15)2, wherein R15 is -(CH2)nN(R16)2, and R16 is -CrCs alkyl or -(CH2)n-COOH . In another embodiment, when Z is -NH, R11 is -CH(R15)2, wherein R15 is -(CH2)n-S03H. An exemplary auristatin embodiment of the formula DE is MMAE, wherein the wavy line indicates a linker (L) covalently linked to the antibody-drug conjugate:

式DF之例示性auristatin實施例為MMAF，其中波浪線表示共價連接至抗體-藥物接合物之連接子（L)(參見US 2005/0238649 及 Doronina 等人（2006) Bioconjugate Chem. 17:114-124)：An exemplary auristatin embodiment of formula DF is MMAF, wherein the wavy line indicates a linker (L) covalently linked to the antibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006) Bioconjugate Chem. 17:114- 124):

其他藥物部分包括以下MMAF衍生物，其中波浪線表示與抗體-藥物接合物之連接子（L)之共價連接：Other drug moieties include the following MMAF derivatives, wherein the wavy line indicates covalent attachment to the linker (L) of the antibody-drug conjugate:

119007.doc -145- 200813092119007.doc -145- 200813092

119007.doc -146- 200813092119007.doc -146- 200813092

在一態樣中，可將如上所示包括（但不限於）三乙二醇酯 (TEG)之親水性基團於R11處連接至藥物部分。在不受任何特定理論約束下，親水性基團有助於藥物部分之内化作用及不凝聚。包含auristatin/海兔毒素或其衍生物之式I之ADC的例示性實施例係描述於以引用的方式明確併入本文之US 2005-023 8649 A1 及 Doronina 等人(2006) Bioconjugate Chem· 17:114-124中。包含MMAE或MMAF及各種連接子組份之式I之ADC的例示性實施例具有以下結構及縮寫（其中nAb" 為抗體；p為1至約8 ; nVal-Cit"為纈胺酸-瓜胺酸二肽且nSn 119007.doc •147- 200813092 為硫原子）：In one aspect, a hydrophilic group including, but not limited to, triethylene glycol ester (TEG) as shown above can be attached to the drug moiety at R11. Without being bound by any particular theory, the hydrophilic group contributes to the internalization and non-coagulation of the drug moiety. Illustrative examples of ADCs of Formula I comprising auristatin/conine toxin or a derivative thereof are described in US 2005-023 8649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17, which is expressly incorporated herein by reference. 114-124. Illustrative examples of ADCs of Formula I comprising MMAE or MMAF and various linker components have the following structures and abbreviations (where nAb" is an antibody; p is from 1 to about 8; nVal-Cit" is valine-guaramine Acid dipeptide and nSn 119007.doc •147- 200813092 is a sulfur atom):

Ab-MC-vc-PAB-MMAEAb-MC-vc-PAB-MMAE

7 P7 P

Ab-MC-MMAEAb-MC-MMAE

Ab-MC-MMAF 包含MMAF及各種連接子組份之式1之ADC的例示性實施例另外包括Ab-MC-PAB-MMAF及Ab-PAB-MMAF。有趣的是，已展示包含由不可蛋白水解裂解之連接子連接至抗體之MMAF的免疫接合物具有可與包含藉由可蛋白水解裂解之連接子連接至抗體之MMAF的免疫接合物相比之活性。參見 Doronina等人（2006) 17:114-124 〇 119007.doc -148- 200813092 在此等情況下，咸信藥物釋放係藉由細胞中之抗體降解而實現。同上文。以肽為主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來製備。此等肽鍵例如可根據肽化學領域中所熟知之液相合成法（參見E. Schr6der及K.Ab-MC-MMAF Exemplary embodiments of the ADC of Formula 1 comprising MMAF and various linker components additionally include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Interestingly, immunoconjugates comprising MMAF linked to an antibody by a non-proteolytically cleavable linker have been shown to have activity comparable to immunoconjugates comprising MMAF linked to the antibody by proteolytic cleavage of the linker . See Doronina et al. (2006) 17: 114-124 〇 119007.doc -148- 200813092 In these cases, the release of the drug is achieved by degradation of the antibody in the cell. Same as above. The peptide-based drug moiety can generally be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be, for example, based on liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schr6der and K.

Ltibke，’’The Peptides·’，第 i 卷，第 76-136 頁，1965, Academic Press)來製備。Auristatin/海兔毒素藥物部分可根據以下文獻中之方法來製備：US 2005-0238649 A1、美國專利第5635483號；美國專利第5780588號；Pettit等人 (1989) J· dm· C/zem· Sm· 111:5463-5465 ; Pettit等人（1998) dWz-C⑽Drwg Daz.gn 13:243-277 ; Pettit, G.R.等人办以；2以^，1996，719-725 ; Pettit 等人（1996) J· C/zem·心c. Perkin Trans· 1 5:859-863 ；及 Doronina (2003) Nat.Ltibke, '’The Peptides·’, Vol. i, pp. 76-136, 1965, Academic Press). The Auristatin/Hytotoxin drug moiety can be prepared according to the methods in the following documents: US 2005-0238649 A1, U.S. Patent No. 5,634,843; U.S. Patent No. 5,780,588; Pettit et al. (1989) J.dm.C/zem. Sm 111: 5463-5465; Pettit et al. (1998) dWz-C (10) Drwg Daz.gn 13: 243-277; Pettit, GR et al; 2 to ^, 1996, 719-725; Pettit et al. (1996) J · C/zem·heart c. Perkin Trans· 1 5:859-863 ; and Doronina (2003) Nat.

Biotechnol· 2\(^Υ·ΊΊ込。詳言之，式DF之auristatin/海兔毒素藥物部分，諸如 MMAF及其衍生物可使用 US 2005-0238649 A1 及 Doronina 專尺Bioconjugate Chem. 17:114-124 中所述之方法來製備。式DE之Auristatin/海兔毒素藥物部分，諸如 MMAE及其衍生物可使用 Doronina等人（2003) iVai· 21:778-784中所述之方法來製備。藥物連接子部分]\40· MMAF、MC-MMAE、MC-vc-PAB-MMAF 及 MC-vc，PAB_ MMAE 可藉由例如 Doronina 等人（2003) Nat· Biotech· 21:778-784及專利申請公開案第US 2005/0238649 A1號中所述之常規方法便利地合成，且接著接合至所關注抗體。 119007.doc -149- 200813092 (3)卡奇黴素在其他實施例中，免疫接合物包含接合至一或多個卡奇黴素分子之本發明抗體。抗生素之卡奇黴素家族能夠在皮莫耳以下之濃度下產生雙鏈DNA斷裂。對於卡奇黴素家族之接合物的製備而言，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、第 5,877,296 號（均屬於 American Cyanamid Company)。可用之卡奇黴素之結構類似物包括（但不限於）γΐ1、CX21、CX31、N-乙醯基-γι1、PSAG 及 GSCHinman 等人，Cawcer 53:3336-3342 (1993)， Lode 等人，Cancer 58:2925-2928 (1998)，且屬於American Cyanamid之上述美國專利）。可接合抗體之另一種抗腫瘤藥物為QFA，其為抗葉酸劑（antifolate)。卡奇黴素及QFA均具有細胞内作用位點且不易橫穿質膜。因此，該等藥劑經由抗體介導之内化作用吸收細胞大幅地增強其細胞毒性效應。 c)其他細胞毒性劑可接合至本發明抗體之其他抗腫瘤劑包括BCNU、鏈脲黴素（streptozocin)、長春新驗及5-氣脲嘴咬’總稱作LL-E33288複合物之藥劑家族（描述於美國專利第5,053,394 號、第5,770,710號中）以及艾司匹拉黴素（esperamicin)(美國專利第5,877,296號）。可用之酶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（來自綠膿桿菌（Pseudomonas 119007.doc -150- 200813092 aeruginosa))、蓖麻毒素A鏈、相思子鹼a鏈、莫迪素a鏈 (modeccin A chain)、帚麴菌素（alpha_sarcin)、油酮 (Aleurites fordii)蛋白、康乃馨蛋白、洋商陸蛋白（ρΑρι、 PAPII 及 PAP S)、古瓜抑制劑（m〇m〇rdica charantia inhibitor)、麻楓樹蛋白（curcin)、巴豆素（cr〇tin)、葡萄纈草（sapaonaria 〇fficinalis)抑制劑、介樂寧（gel〇nin)、有絲 ***素、侷限麴菌素（restrict〇cin)、酚黴素（phen〇mycin)、伊諾黴素（enomycin)及黴菌毒素。參見例如1993年1〇月28 日公開之WO 93/21232。本發明進一步涵蓋在抗體與具有核分解活性之化合物之間形成之免疫接合物（例如核糖核酸酶或DnA核酸内切酶，諸如去氧核糖核酸酶；DNase)。在某些實施例中，免疫接合物可包含高度放射性原子。可使用各種放射性同位素用於產生放射性接合之抗體。實例包括 At2U、I131、I125、γ9〇、Re186、Rei88、^153、Biotechnol· 2\(^Υ·ΊΊ込. In particular, the auristatin/conine toxin drug moiety of the formula DF, such as MMAF and its derivatives, can be used with US 2005-0238649 A1 and Doronina Bioconjugate Chem. 17:114- Prepared by the method described in 124. The Auristatin/Hytotoxin drug moiety of formula DE, such as MMAE and its derivatives, can be prepared using the method described in Doronina et al. (2003) iVai 21:778-784. Linker moiety]\40· MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc, PAB_MMAE can be disclosed by, for example, Doronina et al. (2003) Nat Biotech 21: 778-784 and patent application The conventional method described in US 2005/0238649 A1 is conveniently synthesized and then ligated to the antibody of interest. 119007.doc -149- 200813092 (3) Kazimycin In other embodiments, the immunoconjugate comprises An antibody of the invention conjugated to one or more calicheamicin molecules. The antibiotic calicheamicin family is capable of producing double-stranded DNA breaks at concentrations below the picomole. For the preparation of conjugates of the calicheamicin family For example, see U.S. Patent Nos. 5,712,374 and 5,714,586. , 5, 739, 116, 5, 767, 285, 5, 770, 701, 5, 770, 710, 5, 773, 001, 5, 877, 296 (all belonging to the American Cyanamid Company). Structural analogs of the available calicheamicin include (but are not limited to) Ϊ́ΐ1, CX21, CX31, N-ethylidene-γι1, PSAG and GSCHinman et al, Cawcer 53: 3336-3342 (1993), Lode et al, Cancer 58: 2925-2928 (1998), and belonging to the above-mentioned American Cyanamid US patent). Another anti-tumor drug that can bind antibodies is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and are not easily traversing the plasma membrane. Thus, the uptake of such agents via antibody-mediated internalization greatly enhances their cytotoxic effects. c) Other cytotoxic agents Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozocin, Changchunxin, and 5-aerocarbazone bites, a family of agents collectively referred to as LL-E33288 complexes. (described in U.S. Patent Nos. 5,053,394, 5,770,710) and esperamicin (U.S. Patent No. 5,877,296). Useful enzyme-active toxins and fragments thereof include diphtheria A bond, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas 119007.doc -150-200813092 aeruginosa), ricin A chain, acacia Subbase a chain, modeccin A chain, alpha_sarcin, Aleurites fordii protein, carnation protein, foreign commercial protein (ρΑρι, PAPII and PAP S), ancient melon Inhibitor (m〇m〇rdica charantia inhibitor), curcin, cr〇tin, sapaonaria 〇fficinalis inhibitor, gel〇nin, mitogen It is limited to restrict〇cin, phen〇mycin, enomycin and mycotoxins. See, for example, WO 93/21232, published January 28, 1993. The invention further encompasses immunoconjugates (e.g., ribonucleases or DnA endonucleases, such as deoxyribonuclease; DNase) formed between the antibody and a compound having nuclear cleavage activity. In certain embodiments, the immunoconjugate can comprise a highly radioactive atom. Various radioisotopes can be used to generate radioconjugated antibodies. Examples include At2U, I131, I125, γ9〇, Re186, Rei88, ^153,

Bi 、P 、Pb及Lu2放射性同位素。當將免疫接合物用於偵測時，其可包含用於閃爍掃描研究之放射性原子，例如“^或P23，或用於核磁共振（NMR)成像（亦稱作磁共振成像，mri)之自旋標記，諸如碘·123、碘_131、銦-111、氟-19、碳-13、氮-15、氧-π、釓、錳或鐵。可經已知方式將放射性標記或其他標記併入免疫接合物中。舉例而言，肽可經生物合成或可藉由使用合適胺基酸前驅體之化學胺基酸合成（例如包括以氟_19代替氫）而合成。諸如沈99111或1123、Re186、Rei88^ T h匕访人 Ke及h 之標記可經由肽 119007.doc -151 - 200813092 中之半胱胺酸殘基連接。釔-90可經由離胺酸殘基連接。 IODOGEN 方法（Fraker 等人（1978)价及以· C㈣m⑽· 80: 49-57)可用以併入碘-123。"Monoclonal Antibodies in Immunoscintigraphy’’(Chatal，CRC Press 1989)詳細描述其他方法。在某些實施例中，免疫接合物可包含接合至將前藥（例如肽基化療劑，參見WO 8 1/01145)轉化為活性藥物（諸如抗癌藥物）之前藥活化酶之本發明的抗TAT226抗體。此等免疫接合物適用於抗體依賴性酶介導之前藥療法 ("ADEPT”）。可接合至本發明之抗TAT226抗體之酶包括（但不限於）驗性磷酸酯酶，其適用於將含有填酸酯之前藥轉化為游離藥物；芳基硫酸酯酶，其適用於將含有硫酸酯之鈿藥轉化為游離藥物；胞β密淀去胺酶，其適用於將非毒性 5敦基胞’咬轉化為抗癌藥物5_氟尿喷咬；蛋白酶，諸如沙雷氏菌蛋白酶、嗜熱菌蛋白酶、枯草桿菌蛋白酶、羧肽酶及組織蛋白酶（諸如組織蛋白酶Β及L)，其適用於將含肽前藥轉化為游離藥物；D_丙胺醯基羧肽酶，其適用於轉化含有D-胺基酸取代基之前藥；碳水化合物_裂解酶，諸如 β-半乳糖苷酶及神經胺糖酸苷酶，其適用於將糖基化前藥轉化為游離藥物；β_内醯胺酶，其適用於將以卜内醯胺衍生之藥物轉化為游離藥物；及盤尼西林醯胺酶⑶⑶卜山匕 amidase) ’諸如盤尼西林v醯胺酶及盤尼西林g醯胺酶，其適用於將以苯氧基乙醯基或苯基乙醯基於其胺氮處衍生之藥物分別轉化為游離藥物。可藉由此項技術中所熟知之重 H9007.doc -152- 200813092 組DNA技術將酶共價結合至本發明之抗TAT226抗體。參見例如 Neuberger等人，JVaiwre 3 12:604_608 (1984)。 d)載藥率載藥率以p表示’其為式I分子中每抗體之藥物部分的平均數。載藥率可在每抗體1至20個藥物部分（D)之範圍内。式I之ADC包括與大量藥物部分（1至20個）接合之抗體集合。由接合反應製備ADC中每抗體之藥物部分的平均數可由諸如質譜分析、ELISA檢定及HPLC之習知方式來表徵。亦可根據p確定ADC之定量分佈。在一些情況下，可藉由諸如逆相HPLC或電泳之方式達成均質ADC(其中p為特定值）自具有其他載藥率之ADC的分離、純化及表徵。對於一些抗體_藥物接合物而言，p可受抗體上之連接位點數之限制。舉例而言，如在上文例示性實施例中，當連接為半胱胺酸硫醇時，抗體可僅具有一或若干個半胱胺酸硫醇基，或可僅具有一或若干個經由其可連接連接子之充 =反應性硫醇基。在某些實施例中，例如p > 5之較高載藥率可引起某些抗體-藥物接合物之聚集、不溶、毒性或細胞滲透性損失。在某些實施例中，本發明之ADC之载藥率在1至約8、約2至約6或約3至約5之範圍内。事實上，已證實對於特定ADC而言，每抗體藥物部分之最佳比率可小於 8且可為約2至約5。參見2〇〇5 〇238649 A1。在某些實施例中，小於理論最大值之藥物部分在接合反 :期間接合至抗體。如下文所述’抗體可含有例如不與藥連接子中間物或連接子試劑反應《離胺酸殘基。一般 119007.doc -153- 200813092 而言，抗體不含有許多可連接至藥物部分之游離及反應性半胱胺酸硫醇基；事實上，抗體中之大部分半胱胺酸硫醇殘基以二硫橋之形式存在。在某些實施例中，抗體可於部分或完全還原條件下經諸如二硫蘇糖醇（DTT)或三羰基乙基膦（TCEP)之還原劑還原以產生反應性半胱胺酸硫醇基。在某些實施例中，抗體經受變性條件以暴露諸如離胺酸或半胱胺酸之反應性親核基團。 ADC之載率（藥物/抗體比率）可以不同方式控制：例如藉由：（i)限制藥物-連接子中間物或連接子試劑相對於抗體莫耳過量；（ii)限制接合反應時間或溫度；及（iii)部分或限制半胱胺酸硫醇改質之還原條件。應瞭解，當一個以上之親核性基團與藥物-連接子中間物或連接子試劑反應，繼而與藥物部分試劑反應時，則所得產物為具有一或多個藥物部分連接至抗體之分佈的ADC 化合物之混合物。可藉由雙重ELISA抗體檢定由混合物計算每抗體之藥物平均數，該平均數特異於抗體且特異於藥物。個別ADC分子可藉由質譜分析而於混合物中識別且藉由例如疏水性相互作用層析之HPLC分離（參見例如 Hamblett， K.J.等人 ’’Effect of drug loading on the pharmacology，pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate，” 文摘號 624， American Association for Cancer Research, 2004 Annual Meeting, 2004年 3 月 27-31 日，Proceedings of the AACR，第 45 卷， 2004 年 3 月；Alley，S.C.等人’’Controlling the location of 119007.doc -154- 200813092 drug attachment in antibody-drug conjUgates，” 文摘號 627,Bi, P, Pb and Lu2 radioisotopes. When an immunoconjugate is used for detection, it may comprise a radioactive atom for scintillation scanning studies, such as "^ or P23, or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) Spin label, such as iodine 123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxy-π, hydrazine, manganese or iron. Radioactive labels or other labels may be known in a known manner. Into the immunoconjugate. For example, the peptide can be synthesized by biosynthesis or can be synthesized by chemical amino acid synthesis using a suitable amino acid precursor (for example, including hydrogen instead of hydrogen). Such as Shen 99111 or 1123 The markers of Re186, Rei88^Th visitor Ke and h can be linked via a cysteine residue in peptide 119007.doc-151 - 200813092. 钇-90 can be linked via an lyophilic acid residue. Fraker et al. (1978) and C(tetra)m(10)·80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy'' (Chatal, CRC Press 1989). In an example, the immunoconjugate can comprise a prodrug (eg, peptidated) For the agent, see WO 8 1/01145) an anti-TAT226 antibody of the invention which is converted to an active drug (such as an anticancer drug) prodrug activating enzyme. These immunoconjugates are suitable for antibody-dependent enzyme-mediated prodrug therapy (" ADEPT"). An enzyme that can be conjugated to an anti-TAT226 antibody of the invention includes, but is not limited to, an assay phosphatase suitable for converting a drug containing a pre-esterate to a free drug; an arylsulfatase enzyme suitable for containing sulfuric acid The ester of the ester is converted into a free drug; the cell β-dendritic deaminase is suitable for converting a non-toxic 5 basal cell bite into an anticancer drug 5_ fluoropurine bite; a protease such as Serratia protease, a hobby Thermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsin and L), which are suitable for converting peptide-containing prodrugs into free drugs; D_alaninyl carboxypeptidase, which is suitable for transformation containing a D-amino acid substituent prodrug; a carbohydrate-lyase, such as a beta-galactosidase and a neuraminidase, which is suitable for converting a glycosylated prodrug into a free drug; beta-endoamine An enzyme suitable for converting a drug derived from indoleamine into a free drug; and penicillin glutaminase (3) (3) Buzan amidase) such as penicillin v-prolinease and penicillin g-prolinase, which are suitable for use in benzene Oxydiethyl or Acetyl-derived group-based drug were at their amine nitrogens into free drugs. The enzyme can be covalently bound to the anti-TAT226 antibody of the invention by the heavy DNA technique of H9007.doc-152-200813092, well known in the art. See, for example, Neuberger et al., JVaiwre 3 12:604_608 (1984). d) Drug loading rate The drug loading rate is expressed in p, which is the average number of drug portions per antibody in the molecule of formula I. The drug loading rate can be in the range of 1 to 20 drug portions (D) per antibody. The ADC of Formula I includes a collection of antibodies conjugated to a large number of drug moieties (1 to 20). The average number of drug moieties per antibody prepared by the ligation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the ADC can also be determined from p. In some cases, separation, purification, and characterization of a homogeneous ADC (where p is a particular value) can be achieved from an ADC having other drug loading rates, such as by reverse phase HPLC or electrophoresis. For some antibody-drug conjugates, p can be limited by the number of attachment sites on the antibody. For example, as in the above exemplary embodiments, when linked to a cysteine thiol, the antibody may have only one or several cysteine thiol groups, or may have only one or several via It can be linked to a charge-reactive thiol group of the linker. In certain embodiments, a higher loading rate, e.g., p > 5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the ADC of the present invention has a drug loading rate in the range of from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In fact, it has been demonstrated that for a particular ADC, the optimal ratio per drug portion of the antibody can be less than 8 and can range from about 2 to about 5. See 2〇〇5 〇238649 A1. In certain embodiments, a portion of the drug that is less than the theoretical maximum is conjugated to the antibody during the conjugation: As described below, an antibody may contain, for example, no reaction with a drug linker intermediate or a linker reagent. In general, 119007.doc -153- 200813092, the antibody does not contain many free and reactive cysteine thiol groups that can be attached to the drug moiety; in fact, most of the cysteine thiol residues in the antibody are The form of disulfide bridge exists. In certain embodiments, the antibody can be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to produce a reactive cysteine thiol group. . In certain embodiments, the antibody is subjected to denaturing conditions to expose a reactive nucleophilic group such as an amine acid or a cysteine. The loading rate of the ADC (drug/antibody ratio) can be controlled in different ways: for example by (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the time or temperature of the ligation reaction; And (iii) partial or limiting reduction conditions for cysteine thiol upgrading. It will be appreciated that when more than one nucleophilic group is reacted with a drug-linker intermediate or linker reagent, and then with a drug moiety reagent, the resulting product is one in which one or more drug moieties are linked to the antibody. A mixture of ADC compounds. The average number of drugs per antibody can be calculated from the mixture by a double ELISA antibody assay, which is specific for the antibody and specific for the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, for example, Hamblett, KJ et al. 'Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, SC, etc. ''Controlling the location of 119007.doc -154- 200813092 drug attachment in antibody-drug conjUgates,” Abstract No. 627,

American Association for Cancer Research, 2004 Annual Meeting，2004年 3 月 27-31 日，Proceedings 〇f the AACR，第45卷，2004年3月）。在某些實施例中，可藉由電泳或層析將具有單一載率值之均質ADC自接合混合物分離。 e)製備免疫接合物之特定方法式I之ADC可藉由採用彼等熟習此項技術者已知之有機化學反應、條件及試劑之若干途徑來製備，包括：（1)抗體之親核性基團與二價連接子試劑反應以經由共價鍵形成 Ab-L，繼而與藥物部分D反應；及（2)藥物部分之親核性基團與二價連接子試劑反應以經由共價鍵形成D-L，繼而與抗體之親核性基團反應。經由後一途徑製備式I之ADC之例示性方法描述於以引用的方式明確併入本文之US 2005_ 0238649 A1 中。抗體之親核性基團包括（但不限於）：（i)N端胺基；（ii)側鏈胺基，例如離胺酸；（iii)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、硫醇及羥基為親核性的且能夠反應以與包括以下之連接子部分及連接子試劑上之親電子基團形成共價鍵：⑴活性酯，諸如NHS酯、 HOBt酯、鹵曱酸酯及酸鹵化物；（ii)烷基及苄基鹵化物，諸如鹵乙醯胺；（iii)醛類、酮類、羧基及馬來醯亞胺基。特定抗體具有可還原之鏈間二硫鍵，亦即半胱胺酸橋。可藉由經諸如DTT(二硫蘇糖醇）或三羰基乙基膦（TCEP)之還原劑處理而使抗體具有與連接子試劑接合之反應性，以使 119007.doc -155- 200813092 得抗體完全或部分還原。因此，各半胱胺酸橋在理論上將幵y成兩個反應性硫醇親核體。可經由例如藉由使離胺酸殘基與2-亞胺基硫雜環戊烷（Traut氏試劑）反應改質離胺酸殘基將額外親核性基團引入抗體中，從而將胺轉化為硫醇。可藉由引入一個、兩個、三個、四個或四個以上半胱胺酸殘基（例如藉由製備包含一或多個非原生半胱胺酸胺基酸殘基之變異抗體）而將反應性硫醇基引入抗體中。本發明之抗體-藥物接合物亦可藉由抗體上之親電子基團（諸如酸或酮羰基）與連接子試劑或藥物上之親核性基團之間反應而產生。連接子試劑上適用之親核性基團包括 (但不限於）：醯肼、肟、胺基、肼、縮胺基硫脲、羧酸肼及芳基醢肼。在一實施例中，抗體經改質以引入能夠與連接子試劑或藥物上之親核性取代基反應之親電子部分。在另一貫施例中，糖基化抗體之糖可例如經過埃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之醛基或酮基。所得亞胺Schiff鹼基可形成穩定鍵聯，或可例如經侧氫化物試劑還原以形成穩定胺鍵。在一實施例中，糖基化抗體之碳水化合物部分與半乳糖氧化酶或偏過碘酸納之反應可產生可與藥物上之適當基團反應之抗體中的羰 (盤及酮）基（Hermanson，Bioconjugate Techniques)。在另一實施例中，含有N端絲胺酸或蘇胺酸殘基之抗體可與偏過蛾酸鈉反應，導致產生醛代替第一胺基酸（Ge〇ghegan &American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings 〇f the AACR, Vol. 45, March 2004). In certain embodiments, a homogeneous ADC having a single carrier value can be separated from the conjugated mixture by electrophoresis or crystallization. e) Specific Methods for Preparing Immunoconjugates The ADCs of Formula I can be prepared by employing several routes of organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) nucleophilic groups of antibodies The group reacts with the divalent linker reagent to form an Ab-L via a covalent bond, which in turn reacts with the drug moiety D; and (2) the nucleophilic group of the drug moiety reacts with the divalent linker reagent to form via a covalent bond DL, which in turn reacts with the nucleophilic group of the antibody. An exemplary method of preparing an ADC of Formula I via the latter route is described in US 2005- 0238649 A1, which is expressly incorporated herein by reference. The nucleophilic group of the antibody includes, but is not limited to, (i) an N-terminal amine group; (ii) a side chain amine group such as an amide acid; (iii) a side chain thiol group such as cysteine and (iv) a glycosyl or amine group to which the antibody is glycosylated. The amine, thiol and hydroxyl group are nucleophilic and are capable of reacting to form a covalent bond with an electrophilic group comprising a linker moiety and a linker reagent: (1) an active ester such as an NHS ester, a HOBt ester, a hydrazine Acid esters and acid halides; (ii) alkyl and benzyl halides such as haloacetamide; (iii) aldehydes, ketones, carboxyl groups and maleimine groups. A particular antibody has a reducible interchain disulfide bond, ie a cysteine bridge. The antibody can be reacted with a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) to obtain an antibody against 119007.doc-155-200813092 Restore completely or partially. Thus, each cysteine bridge theoretically will 幵y into two reactive thiol nucleophiles. The amine can be converted by introducing an additional nucleophilic group into the antibody by, for example, reacting an oleic acid residue with a 2-iminothiolane (Traut's reagent) to modify the amine group to introduce an additional nucleophilic group. It is a thiol. By introducing one, two, three, four or more cysteine residues (for example by preparing a variant antibody comprising one or more non-native cysteine amino acid residues) A reactive thiol group is introduced into the antibody. The antibody-drug conjugate of the present invention can also be produced by reaction between an electrophilic group on an antibody (such as an acid or a ketone carbonyl group) and a linker reagent or a nucleophilic group on a drug. Suitable nucleophilic groups for use on linker reagents include, but are not limited to, hydrazine, hydrazine, amine, hydrazine, amino thiourea, carboxylic acid hydrazine, and aryl hydrazine. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on a linker reagent or drug. In a further embodiment, the glycosylated antibody saccharide can be oxidized, for example, by an acid oxidizing reagent to form an aldehyde or ketone group reactive with the linker reagent or the amine moiety of the drug moiety. The resulting imine Schiff base can form a stable linkage or can be reduced, for example, by a side hydride 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 metaperiodate produces a carbonyl (disc and keto) group in an antibody reactive with a suitable pharmaceutically acceptable group ( Hermanson, Bioconjugate Techniques). In another embodiment, an antibody comprising an N-terminal serine acid or a threonine residue can be reacted with sodium molybdate to produce an aldehyde instead of the first amino acid (Ge〇ghegan &

Stroh，（1992) C〜m· 3:138-146 ; US 5362852)。此駿可與藥物部分或連接子親核體反應。 119007.doc •156· 200813092 藥物部分上之親核性基團包括（但不限於）··能夠反應以與連接子部分及連接子試劑上之親電子基團形成共價鍵之胺、硫醇、羥基、醯肼、肟、肼、縮胺基硫脲、羧酸肼及芳基醯肼基，該等連接子部分及連接子試劑包括：⑴活性酯，諸如NHS酯、HOBt酯、鹵甲酸酯及酸鹵化物；（ii)烷基及苄基鹵化物，諸如鹵乙醯胺；（iii)醛類、酮類、羧基及馬來醯亞胺基。本發明之化合物明確涵蓋（但不限於）由以下交聯試劑製備之 ADC : BMPS、EMCS、GMBS、HBVS、LC-SMCC、 MBS、ΜΡΒΗ、SBAP、SIA、SIAB、SMCC、SMPB、 SMPH、磺基-EMCS、磺基-GMBS、磺基-KMUS、磺基-MBS、磺基-SIAB、磺基-SMCC及磺基-SMPB及SVSB(琥珀醯亞胺基-(4-乙烯基砜）苯甲酸酯），其為市售的（例如可購自 Pierce Biotechnology，Inc.，Rockford, IL·，U.S.A ;參見例如2003-2004申請手冊及目錄第467-498頁）。包含抗體及細胞毒性劑之免疫接合物亦可使用各種雙官能性蛋白偶合劑來製造，諸如N-琥珀醯亞胺基-3-(2_吡啶二硫基）丙酸酯（SPDP)、琥珀醯亞胺基-4-(N-馬來醯亞胺基甲基）環己烷-1-甲酸酯（SMCC)、亞胺基硫雜環戊烷（IT)、醯亞胺基酯之雙官能性衍生物（諸如己二醯亞胺二曱酯 HC1)、活性酯（諸如辛二酸二琥珀醯亞胺基酯）、醛（諸如戊二醛）、雙疊氮基化合物（諸如雙（對疊氮基苄醯基）己二胺）、雙重氮衍生物（諸如雙（對重氮苄醯基）-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 119007.doc -157- 200813092 1，5-一氟-2,4-二硝基苯）。舉例而言，蓖麻毒素免疫毒素可如乂加批等人，心_(^ 238:1098 (1987)中所述來製備。麫碳-14標記之1_異硫氰基苄基_3_甲基二乙烯三胺戊乙酸 (MX-DTPA)為用於將放射性核苷酸接合至抗體之例示性螯合劑。參見WO 94/11026。或者，可例如藉由重組技術或肽合成來製造包含抗體及細胞毒性劑之融合蛋白。重組DNA*子可包含編碼抗體之區域及彼此相鄰或由不破壞接合物所需特性之編碼連接子肽之區域分離之接合物的細胞毒性部分。在又一實施例中，可將抗體接合至”受體”（諸如抗生蛋白鏈菌素）以用於腫瘤預靶向中，其中將抗體·受體接合物投予患者’繼而使用清除劑將未結合之接合物自循環移除且接著技予接合至細胞毒性劑（例如放射性核苦酸）之，，配位體，， (例如抗生物素蛋白）。 D·醫藥調配物在一態樣中，本發明進一步提供包含至少一種本發明之抗TAT226抗體及/或其至少一種免疫接合物之醫藥調配物。在一些實施例中，醫藥調配物包含抗體及 /或其免疫接合物，及2)醫藥學上可接受之載劑。在一些實施例中，醫藥調配物包含抗體及/或其免疫接合物’及視情況之2)至少一種其他治療劑。其他治療劑包括（但不限於）於下文E.2部分中所述之彼等。包含本發明之抗體或免疫接合物之醫藥調配物係藉由將具有所需純度之抗體或免疫接合物與可選之生理學上可接 119007.doc -158- 200813092 又之載M賦形劑或穩定劑（Remingtonts Phar_c^ ☆/^以第16版，〇s〇i，八.編（198〇))混合而以水溶液或凍乾或其他乾燥調配物形式製備以供儲存。所採用劑量及濃度之可接受之載劑、賦形劑或穩定劑對受體而言為非毒性的且其包括緩衝齊！，諸如磷酸鹽、摔檬酸鹽、、组胺酸及盆他有機酸；抗氧化劑，包括抗壞血酸及甲硫胺酸；防腐劑 (諸如十八基二甲基苄基氯化銨、氣化六羥季銨、氯化苯甲=錢^索氯録）；盼、丁醇或节醇；對經基苯甲酸燒基醋如對經基苯甲酸甲醋或對經基苯甲酸丙醋；兒茶紛；間苯二紛；環己醇；3_戊醇及間甲紛；低分子量㈠、於約10個殘基）多肽；蛋白，諸如血清白蛋白、明膠或免疫球蛋白’親水性聚合物’諸如聚乙烯°叫㈣；胺基酸， ^ 胺駚麩醯胺酸、天冬醯胺酸、組胺酸、精胺酸或Stroh, (1992) C~m·3:138-146; US 5362852). This can react with the drug moiety or the linker nucleophile. 119007.doc •156· 200813092 The nucleophilic group on the drug moiety includes, but is not limited to, amines, thiols that are capable of reacting to form covalent bonds with the linker moiety and the electrophilic group on the linker reagent. , hydroxy, hydrazine, hydrazine, hydrazine, amino thiourea, carboxylic acid hydrazine and aryl fluorenyl, the linker moiety and linker reagents include: (1) an active ester such as NHS ester, HOBt ester, halogenated Acid esters and acid halides; (ii) alkyl and benzyl halides such as haloacetamide; (iii) aldehydes, ketones, carboxyl groups and maleimine groups. The compounds of the present invention specifically encompass, but are not limited to, ADCs prepared from the following crosslinking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, hydrazine, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo -EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (amber succinimide-(4-vinyl sulfone) benzene Acid esters, which are commercially available (for example, available from Pierce Biotechnology, Inc., Rockford, IL., USA; see, for example, the 2003-2004 application manual and catalogue pages 467-498). Immunoconjugates comprising antibodies and cytotoxic agents can also be made using various bifunctional protein coupling agents, such as N-succinimido-3-(2-pyridinedithio)propionate (SPDP), amber醯imino-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), quinone imide Bifunctional derivatives (such as hexamethylene diamine dioxime ester HC1), active esters (such as dinonyl succinimide), aldehydes (such as glutaraldehyde), diazido compounds (such as double (p-azidobenzylhydrazine) hexamethylenediamine), a double nitrogen derivative (such as bis(p-diabenzamide)-ethylenediamine), a diisocyanate (such as toluene 2,6-diisocyanate) and a double Active fluorine compound (such as 119007.doc -157- 200813092 1,5-monofluoro-2,4-dinitrobenzene). For example, a ricin toxin immunotoxin can be prepared as described in PCT/A/CH 238:1098 (1987). 麫Carbon-14-labeled 1-isothiocyanatobenzyl_3_ Methyldiethylenetriamine pentyl acetic acid (MX-DTPA) is an exemplary chelating agent for the attachment of radionucleotides to antibodies. See WO 94/11026. Alternatively, it can be made, for example, by recombinant techniques or peptide synthesis. A fusion protein of an antibody and a cytotoxic agent. The recombinant DNA* may comprise a region encoding the antibody and a cytotoxic portion of the conjugate that is adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate. In one embodiment, an antibody can be conjugated to a "receptor" (such as streptavidin) for use in tumor pre-targeting, wherein the antibody-receptor conjugate is administered to a patient' and then the scavenger will be used The conjugate is removed from the cycle and then conjugated to a cytotoxic agent (eg, radionuclide), a ligand, (eg, avidin). D. Pharmaceutical formulation, in one aspect, The invention further provides at least one invention comprising Anti-TAT226 antibody and / or at least one immune engage the pharmaceutical formulation thereof. In some embodiments, the pharmaceutical formulation comprising the antibody and / or immune conjugate, and 2) a pharmaceutically acceptable carrier SOCIETY. In some embodiments, the pharmaceutical formulation comprises an antibody and/or an immunoconjugate thereof' and optionally 2) at least one other therapeutic agent. Other therapeutic agents include, but are not limited to, those described in Section E.2 below. A pharmaceutical formulation comprising an antibody or immunoconjugate of the invention is obtained by culturing an antibody or immunoconjugate of the desired purity with an optional physiologically acceptable 119007.doc-158-200813092 Or a stabilizer (Remingtonts Phar_c^ ☆/^ is prepared in 16th edition, 〇s〇i, VIII. (198〇)) in the form of an aqueous solution or lyophilized or other dry formulation for storage. Acceptable carriers, excipients or stabilizers in the dosages and concentrations employed are non-toxic to the recipient and include buffering! Such as phosphate, citrate, histidine and potted organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, gasification six Hydroxy quaternary ammonium, chlorinated benzoic acid = money ^ chlorinated chlorinated); expectant, butanol or stilbene; p-alkyl benzoic acid ketone such as p-aminobenzoic acid or propyl benzoic acid; Tea; isophthalic acid; cyclohexanol; 3-pentanol and m-methyl; low molecular weight (a), about 10 residues) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin 'hydrophilic polymerization ''such as polyethylene ° (4); amino acid, ^ amine glutamic acid, aspartic acid, histidine, arginine or

=胺酸；單畴、雙酿及其他碳水化合物’包括葡萄糖、甘 ::或糊精；聲合劑，諸如EDTA;糖，諸如薦糖、甘露 r、海屬糖或山梨糖醇；形成鹽之抗衡離子，諸如鈉；錯口物（例如Zn_蛋白錯合物）及/或㈣子性界面活性 :’諸如 TWEENTM、PLUR0NICSTM或聚乙二醇㈣G 配物，…。一“ a r* ;=藥物傳遞系統（例如脂質體、白蛋白微球體、微 :俘獲二!粒：及奈米膠囊)或在***液中，活性成份亦中，二二如藉由凝聚技術或藉由界面聚合所製備之微囊 ,分別為經甲基纖維素或明膠·微囊及聚·（甲基丙烯 119007.doc -159- 200813092 酸甲醋）微囊。此等技術揭示於裳 16版，Osol，A·編（1980)中。可製備持續釋放型製劑。持續釋放型製劑之合適實例包括含有本發明之抗體或免疫接合物之固體疏水性聚合物的半透性基質，該等基質為成形物品之形式，例如薄膜或微囊。持續釋放型基質之實例包括聚酯；水凝膠（例如，聚 (2-羥乙基-甲基丙烯酸脂）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；不可降解之乙烯-乙酸乙烯酯；不可降解之乳酸-乙醇酸共聚物，諸如LUPRON DEP〇Ttm(由乳酸-乙醇酸共聚物及乙酸亮丙瑞林組成之可注射微球體）；及聚基丁酸。儘管諸如乙稀-乙酸乙烯酯及乳酸_乙醇酸之聚合物使得分子可釋放100天以上，但某些水凝膠仍釋放蛋白歷時較短時期。當經囊封之抗體或免疫接合物在體内保持較長時間時’其可因在37°c下暴露於水份而變性或凝集，導致生物活性損失及可能之免疫原性變化。可視所涉及之機制設計用於穩定作用之合理策略。舉例而言，若發現凝集機制為經由硫·二硫鍵互換之分子間S §鍵形成，則可藉由改質硫氫基殘基，自酸性溶液凍乾，控制水份含量，使用適當添加劑且研發特異性聚合物基質組合物來達成穩定作用。 E·使用抗TAT226抗體及免疫接合物之方法 1·診斷方法及偵測方法在一態樣中’本發明之抗1^丁226抗體及免疫接合物適用 119007.doc -160- 200813092 於偵測生物樣本中TAT226之存在。如本文所用之術語’’偵測π涵蓋定量或定性偵測。在某些實施例中’生物樣本包含細胞或組織，諸如圖13中列出之組織。在某些實施例中，此等組織包括以相對於其他組織而言較高之程度表現 ΤΑΤ226之正常及/或癌組織，例如卵巢、腎、腦、子宮内膜、腎上腺、骨骼、肺、皮膚及軟組織。在一態樣中，本發明提供一種偵測生物樣本中ΤΑΤ226存在之方法。在某些實施例中，該方法包含在容許將抗 ΤΑΤ226抗體結合至ΤΑΤ226之條件下使生物樣本與抗 ΤΑΤ226抗體接觸，且偵測在抗ΤΑΤ226抗體與ΤΑΤ226之間是否形成複合物。在一態樣中，本發明提供一種診斷與ΤΑΤ226之表現增加相關聯之病症的方法。在某些實施例中’該方法包含使測試細胞與抗ΤΑΤ226抗體接觸；藉由偵測抗ΤΑΤ226抗體與 ΤΑΤ226之結合確定測試細胞對ΤΑΤ226之表現程度（定量或定性）；且比較測試細胞對ΤΑΤ226之表現程度與對照細胞對ΤΑΤ226之表現程度（對照細胞例如為與測試細胞相同組織來源之正常細胞，或以可與此正常細胞相比之程度表現 ΤΑΤ226之細胞），其中與對照細胞相比測試細胞對ΤΑΤ226 之較高表現程度表示存在與增加之ΤΑΤ226表現相關聯之病症。在某些實施例中，測試細胞獲自懷疑患有與ΤΑΤ226之增加表現相關聯之病症的個體。在某些實施例中’該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細胞增殖性病症包括 119007.doc -161- 200813092 癌症病況，諸如腫瘤，例如癌瘤（上皮腫瘤）及母細胞瘤（胚胎組織衍生之腫瘤）；且在某些實施例中為卵巢癌、子宮癌（包括子宮内膜癌）及腎癌，包括腎胚細胞瘤（例如威爾姆氏腫瘤）。詳言之，卵巢癌涵蓋自卵巢衍生之惡性腫瘤的異質群。大約90%之惡性卵巢腫瘤為上皮來源的；其餘為生殖細胞腫瘤及基質腫瘤。上皮卵巢腫瘤分為以下組織學亞型：漿液性腺癌（佔上皮卵巢腫瘤之約50%);子宮内膜樣腺癌（約20%);黏液性腺癌（約10%);透明細胞癌（約5-10%);布倫納氏（Brenner)(移行細胞）腫瘤（相對不常見）。第六種最常見之女性癌症卵巢癌之預後通常較差，其中五年存活率在5-30%之範圍内。關於卵巢癌之論述參見卩⑽等人（2002) ’’Pathology of epithelial ovarian cancer，’’ 在 C⑽ cer 第 9章（Jacobs等人編，Oxford University Press，New York)中；Morin等人（2001) ’’Ovarian Cancer"在〇/ Ca加，第 654-656 頁（Schwab 編，Springer-Verlag，New York)中。本發明涵蓋診斷或治療上述任何上皮卵巢腫瘤亞型（且詳言之為漿液性腺癌亞型）之方法。在某些實施例中，諸如上述彼等之診斷或偵測方法包含偵測抗TAT226抗體與於細胞表面上表現或在自其表面上表現TAT226之細胞獲得之膜製劑中表現之TAT226的結合。在某些實施例中，該方法包含在容許將抗TAT226抗體結合至TAT226之條件下使細胞與抗TAT226抗體接觸，且偵測在抗TAT226抗體與細胞表面上之TAT226之間是否形成複 119007.doc -162- 200813092 合物。用於偵測抗TAT226抗體與於細胞表面上表現 TAT226之TAT226之結合的例示性檢定為"FACS”檢定，諸如下文實例D中所述者。某些其他方法可用以偵測抗TAT226抗體與TAT226之結合。此等方法包括（但不限於）此項技術中所熟知之抗原結合檢定，諸如西方墨點法、放射性免疫檢定、ELISA(酶聯免疫吸附檢定）、”夾層”免疫檢定、免疫沉澱檢定、螢光免疫檢定、蛋白A免疫檢定及免疫組織化學（IHC)。在某些實施例中，抗TAT226抗體為經標記的。標記包括 (但不限於）經直接偵測之標記或部分（諸如螢光標記、發色標記、電子緻密標記、化學發光標記及放射性標記）以及例如經由酶促反應或分子相互作用間接偵測之部分，諸如酶或配位體。例示性標記包括（但不限於）放射性同位素 32P、14C、1251、3H及1311 ;螢光團，諸如稀土螯合劑或螢光素及其衍生物、若丹明（rhodamine)及其衍生物、丹醯基、繳酮、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））、螢光素；2,3_二氫酞嗪二酉同’辣根過乳化物轉（HRP)，驗性麟酸酶；β -半乳糖苦酶；葡萄糖澱粉酶；溶菌酶；醣氧化酶（例如葡萄糖氧化酶、半乳糖氧化酶及葡萄糖-6-磷酸鹽脫氫酶）；雜環氧化酶，諸如尿酸酶及黃嘌呤氧化酶，其與採用過氧化氣以氧化染料前驅體之酶（諸如HRP、乳過氧化物酶或微過氧化物酶）偶合；生物素/抗生物素蛋白；自旋標記、噬菌體標記、穩定自由基及其類似物。 119007.doc •163- 200813092 在某些實施例中，抗TAT226抗體固定於不溶性基質上。固定需要將抗TAT226抗體自在溶液中保持游離之任何 TAT226分離。其習知係藉由在檢定程序之前如藉由吸附至水不溶性基質或表面（丑61111丨(：11等人，1；.8.3,720,760)或藉由共價偶合（例如使用戊二醛交聯）使抗TAT226抗體不溶’ 或藉由在抗TAT226抗體與TAT226之間形成複合物之後例如藉由免疫沉澱使抗TAT226抗體不溶來完成。任何上述診斷或偵測實施例均可使用代替抗TAT226抗體或除抗TAT226抗體以外之本發明之免疫接合物來進行。治療方法例如，本發明之抗體或免疫接合物可用於活體外、離體及活體内治療方法中。在一態樣中，本發明提供活體内或活體外抑制細胞生長或增殖之方法，該方法包含在容許將免疫接合物結合至TAT226之條件下，將細胞暴露於抗 TAT226抗體或其免疫接合物。”抑制細胞生長或增殖”意謂將細胞生長或增殖減低至少10%、20%、30%、40%、 50%、60%、70%、80%、90%、95%或 100%，且包括誘導細胞死亡。在某些實施例中，細胞為腫瘤細胞。在某些實施例中，細胞為卵巢腫瘤細胞、子宮腫瘤細胞、腦腫瘤細胞或腎腫瘤細胞。在某些實施例中，例如本文所例示，細胞為異種移植物。在一態樣中，將本發明之抗體或免疫接合物用以治療或預防細胞增殖性病症。在某些實施例中，細胞增殖性病症與TAT226之增加表現及/或活性相關聯。舉例而言，在某 119007.doc -164- 200813092 些實施例中，細胞增殖性病症與TAT226於細胞表面上增加之表現相關聯。在某些實施例中，細胞增殖性病症為腫瘤或^癌症°欲以本發明之抗體或免疫接合物治療之細胞增殖性病症的實例包括（但不限於）癌性病況，諸如腫瘤，例如癌瘤（上皮腫瘤）及母細胞瘤（由胚胎組織衍生之腫瘤）且在某些實施例中為卵巢癌；子宮癌，包括子宮内膜癌；腦腫瘤（例如，星形細胞瘤，涵蓋晚期神經膠質瘤，亦稱作多形牲膠質母細胞瘤）；及腎癌，包括腎胚細胞瘤（例如威爾姆氏腫瘤）。在一態樣中，本發明提供用於治療細胞增殖性病症之方法’其包含向個體投予有效量之抗TAT226抗體或其免疫接合物。在某些實施例中，治療細胞增殖性病症之方法包含向個體投予有效量之醫藥調配物，該醫藥調配物包含抗 TAT226抗體及視情況之至少一種其他治療劑，諸如下文提供之彼等。在某些實施例中，治療細胞增殖性病症之方法包含向個體投予有效量之醫藥調配物，該醫藥調配物包含 1)包含抗TAT226抗體及細胞毒性劑之免疫接合物；及視情況之2)至少一種其他治療劑，諸如下文提供之彼等。在一態樣中，至少一些本發明之抗體或免疫接合物可結合來自除人類以外之物種的TAT226。因此，本發明之抗體或免疫接合物可用以例如在含有TAT226之細胞培養物中，於人類體内或於具有與本發明之抗體或免疫接合物交又反應之TAT226的其他哺乳動物（例如黑猩猩 '狒狒 '狨猿、食蟹猴及獼猴、豬或小鼠）體内結合TAT226。在一實施例 119007.doc -165- 200813092 中，抗TAT226抗體或免疫接合物可用於藉由使抗體或免疫接合物與TAT226接觸以使得抑制TAT226活性來抑制 TAT226活性。在一實施例中，TAT226為人類TAT226。在一實施例中，抗TAT226抗體或免疫接合物可用於在患有與增加之TAT226表現及/或活性相關聯之病症的個體中結合TAT226的方法中，該方法包含向個體投予抗體或免疫接合物，以便結合個體中之TAT226。在一實施例中， TAT226為人類TAT226，且個體為人類個體。或者，個體可為結合抗TAT226抗體之表現TAT226的哺乳動物。此外，個體可為其中引入TAT226(例如藉由投予TAT226或藉由表現編碼TAT226之轉殖基因）之哺乳動物。可因治療目的向人類投予抗TAT226抗體或免疫接合物。此外，可因獸醫學目的或作為人類疾病之動物模型向表現與抗體交叉反應之TAT226之非人類哺乳動物（例如靈長類動物、豬、大鼠或小鼠）投予抗TAT226抗體或免疫接合物。關於後者，此等動物模型可適用於評估本發明之抗體或免疫接合物之治療功效（例如測試投藥之劑量及時程）。本發明之抗體或免疫接合物在治療中可單獨或與其他組合物組合使用。舉例而言，本發明之抗體或免疫接合物可與至少一種其他治療劑及/或佐劑共投予。在某些實施例中，其他治療劑為細胞毒性劑、化療劑或生長抑制劑。在此等實施例之一中，化療劑為用於治療卵巢癌之藥劑或藥劑組合，諸如鉑化合物（例如順鉑或卡波鉑）；紫杉烷（例如紫杉醇或多烯紫杉醇）；拓朴替康；蒽環黴素（例如多柔比 119007.doc -166- 200813092 星（ADRIAMYCIN®)或脂質多柔比星（D〇XIL(g));吉西他濱；環磷醯胺；長春瑞濱（NAVELBINE®);六甲蜜胺；異環磷醯胺及依託泊苷。在此等實施例之另一者中，化療劑為用於治療子宮癌或子宮内膜癌之藥劑或藥劑組合，諸如順鉑、卡波鉑、多柔比星、紫杉醇、甲胺喋呤、氟尿嘧啶及甲經孕嗣。在此等實施例之另一者中，化療劑為用於治療腦腫瘤之藥劑或藥劑組合，諸如亞硝基脲（例如卡莫司 € 汀或洛莫司汀）；細胞毒性劑（例如伊立替康或替莫唑胺 * (temozolamide));抗血管生成劑（例如沙立度胺、TNp_ 470、血小板因子4、干擾素及内皮抑制素）；分化劑（例如類視色素、苯基丁酸酯、苯基乙酸酯及抗新普拉通 neoplaston));抗侵入劑（例如基質金屬蛋白酶抑制劑，諸如馬立馬司他（marimastat));信號轉導調節劑（例如他莫昔芬、苔蘚抑素及0-6节基鳥嘌呤）；拓樸異構酶抑制劑如伊立替康或拓朴替康）及生長因子抑制劑（例如酪胺酸激 L 酶抑制劑）。在此等實施例之另一者中，化療劑為用於治療腎癌（例如威爾姆氏腫瘤）之藥劑或藥劑組合，諸如長春新鹼、放射菌素D、阿黴素（adriamycin)、多柔比星、環磷醯胺、異環磷醯胺、依託泊苷及卡波鉑。在某些實施例中，本發明之抗體可與消炎劑及/或防腐劑組合。上述此等组合療法涵蓋組合投予（其中在相同或獨立調配物中包括兩種或兩種以上治療劑）及獨立投予，在誃十主況下，本發明之抗體或免疫接合物之投予可於投予其他^ 療劑及/或佐劑之前、同時及/或之後發生。本發明之抗體 119007.doc -167- 200813092 或免疫接合物亦可與放射療法組合使用。 f發明之抗體或免疫接合物（及任何其他治療劑或佐劑）可精由任何合適之方式投予，包括非經腸、皮下、腹膜内肺内及鼻内投予，且若需要局部治療，則病灶内投予非、、、二腸輸/主包括肌肉内、靜脈内、動脈内、腹膜内或皮下技予此外，抗體或免疫接合物係藉由尤其以降低之抗體或免疫接合物劑量脈動式輸注（pulse infusi〇n)而合適地投予。部分視短暫或長期投藥而定，可藉由任何合適途徑給藥，例如藉由注射，諸如靜脈内或皮下注射。當結合靶位於腦中時，本發明之某些實施例提供抗體或免疫接合物以橫穿血腦障壁。存在若干此項技術中已知之方式以轉運分子橫穿血腦障壁，包括（但不限於）物理方法、基於脂質之方法、基於幹細胞之方法及基於受體及通道之方法。轉運抗體或免疫接合物橫穿血腦障壁之物理方法包括 (但不限於）完全包圍血腦障壁或藉由在血腦障壁上產生開口。包圍法包括（但不限於）直接向腦中注射（參見例如= Aminic acid; monodomain, double brewed and other carbohydrates 'including glucose, glycerol:: or dextrin; sonicating agents such as EDTA; sugars such as sucrose, mannose, sucrose or sorbitol; Counter ions, such as sodium; slopherms (such as Zn_protein complex) and/or (iv) sub-interface activity: 'such as TWEENTM, PLUR0NICSTM or polyethylene glycol (tetra) G ligand, .... An "ar*; = drug delivery system (such as liposome, albumin microspheres, micro: capture two! particles: and nanocapsules) or in a macroemulsion, active ingredients, such as by coagulation technology or The microcapsules prepared by interfacial polymerization are respectively methylcellulose or gelatin microcapsules and poly(methacrylic acid 119007.doc-159-200813092 acid methyl vinegar) microcapsules. Edition, Osol, A. ed. (1980). A sustained release formulation can be prepared. Suitable examples of sustained release formulations include semipermeable matrices comprising a solid hydrophobic polymer of an antibody or immunoconjugate of the invention, such The matrix is in the form of a shaped article, such as a film or microcapsule. Examples of sustained release matrix include polyester; hydrogel (eg, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol; polylactic acid) Lactide (U.S. Patent No. 3,773,919); copolymer of L-glutamic acid with γ-ethyl-L-glutamate; non-degradable ethylene-vinyl acetate; non-degradable lactic acid-glycolic acid copolymer, such as LUPRON DEP〇Ttm (from lactic acid-glycolic acid copolymer and Injectable microspheres composed of leuprolide acetate; and polybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow molecules to be released for more than 100 days, some hydrogels remain The protein is released for a short period of time. When the encapsulated antibody or immunoconjugate is kept in the body for a long time, it can be denatured or agglomerated by exposure to water at 37 ° C, resulting in loss of biological activity and possible Immunogenicity change. A reasonable strategy for stabilizing the effect can be designed according to the mechanism involved. For example, if the agglutination mechanism is found to be an intermolecular S § bond formation via a sulfur-disulfide bond exchange, the sulfur can be modified by Hydrogen-based residues, freeze-dried from acidic solutions, control moisture content, use appropriate additives and develop specific polymer matrix compositions to achieve stabilization. E·Methods using anti-TAT226 antibody and immunoconjugates 1. Diagnostic methods and Detection Method In one aspect, the anti-1 butyl 226 antibody of the present invention and the immunoconjugate are used in the detection of the presence of TAT226 in a biological sample 119007.doc -160-200813092. The term 'as used herein' Detecting π encompasses quantitative or qualitative detection. In certain embodiments, a 'biological sample comprises cells or tissue, such as the tissue listed in Figure 13. In some embodiments, such tissue is included with respect to other tissues. Higher levels of performance ΤΑΤ 226 normal and / or cancerous tissue, such as ovary, kidney, brain, endometrium, adrenal gland, bone, lung, skin and soft tissue. In one aspect, the invention provides a biological sample for detection The method of the presence of the sorghum 226. In certain embodiments, the method comprises contacting the biological sample with an anti-ΤΑΤ226 antibody under conditions that permit binding of the anti-ΤΑΤ226 antibody to the ΤΑΤ226, and detecting whether an anti-ΤΑΤ226 antibody is formed between the anti-ΤΑΤ226 antibody and the ΤΑΤ226 Complex. In one aspect, the invention provides a method of diagnosing a condition associated with increased performance of sputum 226. In certain embodiments, the method comprises contacting the test cell with an anti-ΤΑΤ226 antibody; determining the degree of expression (quantitative or qualitative) of the test cell for the ΤΑΤ226 by detecting binding of the anti-ΤΑΤ226 antibody to ΤΑΤ226; and comparing the test cell to ΤΑΤ226 The degree of performance is compared with the degree of performance of the control cells against ΤΑΤ226 (the control cells are, for example, normal cells of the same tissue source as the test cells, or cells expressing ΤΑΤ226 to a degree comparable to the normal cells), wherein the test is compared with the control cells. The higher degree of performance of the cells for ΤΑΤ226 indicates the presence of a condition associated with increased sputum 226 performance. In certain embodiments, the test cells are obtained from an individual suspected of having a condition associated with increased performance of sputum 226. In certain embodiments the condition is a cell proliferative disorder, such as a cancer or tumor. Exemplary cell proliferative disorders that can be diagnosed using the antibodies of the invention include 119007.doc-161-200813092 cancer conditions, such as tumors, such as carcinomas (epithelial tumors) and blastomas (embryonic tissue-derived tumors); In some embodiments are ovarian cancer, uterine cancer (including endometrial cancer), and renal cancer, including nephroblastoma (eg, Wilm's tumor). In particular, ovarian cancer encompasses a heterogeneous population of malignant tumors derived from the ovary. Approximately 90% of malignant ovarian tumors are of epithelial origin; the rest are germ cell tumors and stromal tumors. Epithelial ovarian tumors are classified into the following histological subtypes: serous adenocarcinoma (about 50% of epithelial ovarian tumors); endometrioid adenocarcinoma (about 20%); mucinous adenocarcinoma (about 10%); clear cell carcinoma ( About 5-10%); Brenner (transitional cell) tumors (relatively uncommon). The sixth most common form of cancer in women with ovarian cancer is usually poor prognosis, with a five-year survival rate in the range of 5-30%. For a discussion of ovarian cancer, see 卩(10) et al. (2002) ''Pathology of epithelial ovarian cancer,'' in C(10) cer Chapter 9 (Jacobs et al., Oxford University Press, New York); Morin et al. (2001) ''Ovarian Cancer" in 〇/Ca 加, pp. 654-656 (edited by Schwab, Springer-Verlag, New York). The invention encompasses methods of diagnosing or treating any of the above epithelial ovarian tumor subtypes (and in particular serous adenocarcinoma subtypes). In certain embodiments, methods of diagnosis or detection such as those described above comprise detecting binding of an anti-TAT226 antibody to TAT226 expressed on a cell surface or expressed in a membrane preparation obtained from cells expressing TAT226 on its surface. In certain embodiments, the method comprises contacting the cell with an anti-TAT226 antibody under conditions permitting binding of the anti-TAT226 antibody to TAT226, and detecting whether a complex 119007 is formed between the anti-TAT226 antibody and TAT226 on the cell surface. Doc -162- 200813092 Compound. An exemplary assay for detecting binding of an anti-TAT226 antibody to TAT226 that expresses TAT226 on the cell surface is a "FACS" assay, such as described in Example D below. Certain other methods can be used to detect anti-TAT226 antibodies and Combination of TAT 226. These methods include, but are not limited to, antigen binding assays well known in the art, such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunization Precipitation assay, fluorescent immunoassay, protein A immunoassay, and immunohistochemistry (IHC). In certain embodiments, the anti-TAT226 antibody is labeled. The label includes, but is not limited to, a directly detected label or portion (such as fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels, and radioactive labels) and portions that are indirectly detected, for example, via enzymatic or molecular interactions, such as enzymes or ligands. Exemplary labels include (but Not limited to) radioisotopes 32P, 14C, 1251, 3H and 1311; fluorophores such as rare earth chelating agents or luciferins and their derivatives, rhodamine (rhodamine) and its derivatives, tanshinyl, ketone, luciferase (such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin; 2,3_2 Hydroquinone dioxime with 'horseradish perfusate transfer (HRP), phytase, beta-galactosidase; glucoamylase; lysozyme; sugar oxidase (eg glucose oxidase, galactose oxidase) And glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, and enzymes that use peroxygen gas to oxidize dye precursors (such as HRP, lactoperoxidase or micro Peroxidase) coupling; biotin/avidin; spin labeling, phage labeling, stable free radicals, and the like. 119007.doc • 163- 200813092 In certain embodiments, the anti-TAT226 antibody is immobilized in insoluble Immobilization Any anti-TAT226 antibody needs to be separated from any TAT226 that remains free from solution. It is known by adsorption to a water insoluble matrix or surface prior to the assay procedure (ug. 61911(:11 et al., 1 ;.8.3,720,760) or borrow This is accomplished by covalent coupling (eg, cross-linking with glutaraldehyde) to render the anti-TAT226 antibody insoluble or by insolubilizing the anti-TAT226 antibody, eg, by immunoprecipitation, after complex formation between the anti-TAT226 antibody and TAT226. Alternatively, the detection examples can be carried out using an immunoconjugate of the invention in place of or in addition to the anti-TAT226 antibody. The method of treatment, for example, the antibody or immunoconjugate of the invention can be used in vitro, ex vivo and in vivo. In a method of treatment, in one aspect, the invention provides a method of inhibiting cell growth or proliferation in vivo or in vitro, the method comprising: exposing the cell to an anti-TAT226 antibody or allowing the immunoconjugate to bind to TAT226 Its immunoconjugate. "Inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and This includes inducing cell death. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cells are ovarian tumor cells, uterine tumor cells, brain tumor cells, or kidney tumor cells. In certain embodiments, such as exemplified herein, the cells are xenografts. In one aspect, an antibody or immunoconjugate of the invention is used to treat or prevent a cell proliferative disorder. In certain embodiments, the cell proliferative disorder is associated with increased performance and/or activity of TAT226. For example, in some embodiments 119007.doc-164-200813092, cell proliferative disorders are associated with increased performance of TAT226 on the cell surface. In certain embodiments, the cell proliferative disorder is a tumor or cancer. Examples of cell proliferative disorders to be treated with an antibody or immunoconjugate of the invention include, but are not limited to, a cancerous condition, such as a tumor, such as a cancer. Tumors (epithelial neoplasms) and blastomas (tumors derived from embryonic tissue) and in some embodiments ovarian cancer; uterine cancer, including endometrial cancer; brain tumors (eg, astrocytoma, covering advanced nerves) Glioma, also known as polymorphic glioblastoma; and renal cancer, including nephroblastoma (eg, Wilm's tumor). In one aspect, the invention provides methods for treating a cell proliferative disorder' comprising administering to an individual an effective amount of an anti-TAT226 antibody or immunoconjugate thereof. In certain embodiments, a method of treating a cell proliferative disorder comprises administering to a subject an effective amount of a pharmaceutical formulation comprising an anti-TAT226 antibody and, optionally, at least one other therapeutic agent, such as those provided below . In certain embodiments, a method of treating a cell proliferative disorder comprises administering to a subject an effective amount of a pharmaceutical formulation comprising: 1) an immunoconjugate comprising an anti-TAT226 antibody and a cytotoxic agent; and optionally 2) At least one other therapeutic agent, such as those provided below. In one aspect, at least some of the antibodies or immunoconjugates of the invention can bind to TAT226 from species other than humans. Thus, an antibody or immunoconjugate of the invention can be used, for example, in a cell culture containing TAT226, in a human or in other mammals having a TAT226 that reacts with an antibody or immunoconjugate of the invention (eg, a chimpanzee) '狒狒'狨猿, cynomolgus monkey and macaque, pig or mouse) bind TAT226 in vivo. In an embodiment 119007.doc-165-200813092, an anti-TAT226 antibody or immunoconjugate can be used to inhibit TAT226 activity by contacting the antibody or immunoconjugate with TAT226 such that it inhibits TAT226 activity. In an embodiment, the TAT226 is a human TAT226. In one embodiment, an anti-TAT226 antibody or immunoconjugate can be used in a method of binding TAT226 in an individual having a disorder associated with increased TAT226 expression and/or activity, the method comprising administering to the individual an antibody or immunizing The conjugate is incorporated into the TAT226 in the individual. In one embodiment, TAT226 is human TAT226 and the individual is a human individual. Alternatively, the individual can be a mammal that expresses TAT226 in combination with an anti-TAT226 antibody. In addition, the individual can be a mammal into which TAT226 is introduced (e.g., by administering TAT226 or by expressing a transgene encoding TAT226). Anti-TAT226 antibodies or immunoconjugates can be administered to humans for therapeutic purposes. In addition, anti-TAT226 antibodies or immunoconjugates can be administered to non-human mammals (eg, primates, pigs, rats, or mice) that express TAT226 that cross-reacts with antibodies for veterinary purposes or as an animal model of human disease. Things. With regard to the latter, such animal models can be adapted to assess the therapeutic efficacy of the antibodies or immunoconjugates of the invention (e.g., to test the dosage and schedule of administration). The antibodies or immunoconjugates of the invention may be used alone or in combination with other compositions in therapy. For example, an antibody or immunoconjugate 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 a cytotoxic agent, a chemotherapeutic agent, or a growth inhibitory agent. In one of these embodiments, the chemotherapeutic agent is a medicament or combination of agents for treating ovarian cancer, such as a platinum compound (eg, cisplatin or carbopol); a taxane (eg, paclitaxel or docetaxel); topography Anthracycline; anthracycline (eg, doxorubicin 119007.doc -166-200813092 star (ADRIAMYCIN®) or lipid doxorubicin (D〇XIL (g)); gemcitabine; cyclophosphamide; vinorelbine ( NAVELBINE®); hexamethyl melamine; ifosfamide and etoposide. In the other of these embodiments, the chemotherapeutic agent is a combination of agents or agents for the treatment of uterine or endometrial cancer, such as Platinum, carbopol, doxorubicin, paclitaxel, methotrexate, fluorouracil, and gestational gestation. In the other of these embodiments, the chemotherapeutic agent is a combination of agents or agents for treating brain tumors, Such as nitrosourea (such as carovustine or lomustine); cytotoxic agents (such as irinotecan or temozolamide); anti-angiogenic agents (such as thalidomide, TNp_ 470, platelets) Factor 4, interferon and endostatin); differentiation agent (example Retinoids, phenylbutyrate, phenyl acetate and anti-nepraplastne); anti-invasive agents (eg matrix metalloproteinase inhibitors such as marimastat); signal transduction regulation Agents (eg tamoxifen, bryostatin and 0-6 knot guanine); topoisomerase inhibitors such as irinotecan or topotecan) and growth factor inhibitors (eg tyrosine L) Enzyme inhibitor). In another of these embodiments, the chemotherapeutic agent is a pharmaceutical or pharmaceutical combination for treating kidney cancer (eg, Wilm's tumor), such as vincristine, actinomycin D, adriamycin, Doxorubicin, cyclophosphamide, ifosfamide, etoposide and carboplatin. In certain embodiments, the antibodies of the invention may be combined with anti-inflammatory agents and/or preservatives. Such combination therapies described above encompass combination administration (wherein two or more therapeutic agents are included in the same or separate formulations) and are administered separately, in the case of 誃10, the antibody or immunoconjugate of the present invention It may occur before, simultaneously with, and/or after administration of other therapeutic agents and/or adjuvants. The antibodies of the invention 119007.doc -167- 200813092 or immunoconjugates can also be used in combination with radiation therapy. The antibody or immunoconjugate (and any other therapeutic or adjuvant) of the invention may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal intranasal and intranasal administration, and if local treatment is desired Intralesional injection of non-, /, digestive / main including intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous techniques. In addition, antibodies or immunoconjugates are particularly reduced antibody or immunoconjugate Dose pulsation infusion (pulse infusi〇n) and appropriate administration. Partially depending on the administration of the drug for short or long term administration, it can be administered by any suitable route, for example by injection, such as intravenous or subcutaneous injection. Certain embodiments of the invention provide antibodies or immunoconjugates to traverse the blood brain barrier when the binding target is in the brain. There are several ways known in the art to transport molecules across the blood brain barrier including, but not limited to, physical methods, lipid based methods, stem cell based methods, and receptor based and channel based methods. Physical methods for transporting antibodies or immunoconjugates across the blood-brain barrier include, but are not limited to, completely surrounding the blood-brain barrier or by creating an opening in the blood-brain barrier. Surrounding methods include, but are not limited to, direct injection into the brain (see for example

Papanastassiou等人，9: 398-406 (2002));間質輸注/對流增強型傳遞（參見例如B〇b〇等人，pr〇c心以 5W.仍」91·· 2076-2080 (1994))及於腦内植入傳遞裝置（參見例如 Gill等人，A/W· 9: 589-595 (2003);及 Gliadel Wafers™，Guildford Pharmaceutical)。於障壁中產生開口之方法包括（但不限於）超音（參見例如美國專利公開案第2002/0038086號），渗透塵（例如藉由投予高渗性甘露 119007.doc -168 - 200813092 糖醇）（Neuwelt，E. A·，Implication of the Blood-Brain Barrier and its Manipulation，第 1 &2 卷，Plenum Press， N.Y. (1989));藉由例如緩激肽或滲透劑A-7滲透（參見例如美國專利第5，112,596號、第5,268，164號、第5,506,206號及第5,686,416號）及以含有編碼抗體之基因之載體轉染跨越血腦障壁之神經元（參見例如美國專利公開案第 2003/0083299號）° 轉運抗體或免疫接合物橫穿血腦障壁之基於脂質之方法包括（但不限於）將抗體或免疫接合物囊封於偶合至與血腦障壁之血管内皮上之受體結合之抗體結合片段的脂質體中 (例如參見美國專利申請公開案第200200253 13號）且以低密度脂蛋白顆粒（參見例如美國專利申請公開案第 200402043 54號）或脂蛋白元E(參見例如美國專利申請公開案第20040131692號）塗覆抗體或免疫接合物。轉運抗體或免疫接合物橫穿血腦障壁之基於幹細胞之方法需要基因工程設計神經祖細胞（NPC)以表現所關注之抗體或免疫接合物且接著將幹細胞植入待治療個體之腦中。參見 Behrstock 等人（2005) 772er· 2005 年 12 月 15 曰高級線上公開案（報導當植入齧齒動物及靈長類動物模型之腦中時，NPC經基因工程設計以表現帕金森病（Parkinson disease)之神經營養因子GDNF降低之症狀）。轉運抗體或免疫接合物橫穿血腦障壁之基於受體及通道之方法包括（但不限於）使用糖皮質激素阻斷劑以增加血腦障壁之滲透性（參見例如美國專利申請公開案第 119007.doc -169- 200813092 2002/0065259 號、第 2003/0162695 號及第 2005/0124533 號），活化钟通道（參見例如美國專利申請公開案第 2005/0089473號）；抑制ABC藥物轉運體（參見例如美國專利申請公開案第2003/0073713號）；以轉鐵蛋白塗覆抗體或免疫接合物且調節一或多種轉鐵蛋白受體之活性（參見例如美國專利申請公開案第2003/0129186號）且使抗體或免疫接合物陽離子化（參見例如美國專利第5,〇〇4,697號）。本發明之抗體或免疫接合物將以與優良藥學實踐一致之方式調配、給藥及投予。在此上下文中考慮之因素包括治療之特定病症、治療之特定哺乳動物、個別患者之臨床病況、病症誘因、藥劑傳遞部位、投藥方法、投藥時程及醫學實踐者已知之其他因素。抗體或免疫接合物無需（但視十月況）與目前用以預防或治療所論及病症之一或多種藥劑調配。此等其他藥劑之有效量視調配物中所存在之抗體或免疫接合物之量、病症或治療類型及上述其他因素而定。其一般係以與上文所述相同之劑量及投藥途徑，或以上文所述之約1至99%之劑量，或以經驗/臨床上確定為適當之任何劑量及任何途徑來使用。為預防或治療疾病，本發明抗體或免疫接合物之適當劑量（當單獨或與諸如化療劑之一或多種其他額外治療劑組合使用時）將視待治療之疾病類型、抗體或免疫接合物類型、疾病嚴重性及進程、為預防或治療目的是否投予抗體或免疫接合物、先前療法、患者臨床病史及對抗體或免疫接合物之反應及主治醫師之判斷而定。抗體或免疫接合物 119007.doc -170- 200813092 適合-次性或經-系列治療投予至患者。視疾病類型及嚴重性而定，約i叩/kg至15 mg/kg(例如〇」mg/kg i〇叫㈣之抗體或免疫接合物彳為向患者投藥之候選起始劑量，無論例如藉由-或多次獨立投藥或藉由連接輸注。視上述因素而定，一種典型每曰劑量可在約i叫/kg至1〇〇 mg/kg* 更大之範圍内。對於經若干天或更長時間之重複投藥而言，視病況而$，-般將持續治，療直至發生對疾病症狀之所需抑制作用。抗體或免疫接合物之—種例示性劑量在約〇.〇5 mg/kg至約10 mg/kg之範圍内。因此，可向患者投予約〇.5 mg/kg、2.0 mg/kg、4.0 mg/kg4l〇 mg/kg(或其任何組合）之一或多種劑量。此等劑量可例如以每週或每三週間歇性投予（例如，以便患者接收約二至約二十，或例如約六次劑量之抗體或免疫接合物）。可投予較高之起始負載劑量，繼而投予一或多次較低劑量。例示性給藥方案包含投予約4 mg/kg之起始負載劑量，繼而為約2 mg/kg抗體之每週維持劑量。然而，其他給藥方案可適用。此治療進程易於由習知技術及檢定來監控。 3 *檢定本發明之抗TAT226抗體及免疫接合物可藉由此項技術中已知之各種檢定來表徵其物理/化學特性及/或生物活性。 ^活性檢定在一悲樣中’提供識別具有生物活性之抗TAT226抗體或其免疫接合物之檢定。生物活性可包括例如抑制細胞生長或增殖之能力（例如，，細胞殺死”活性）或誘導細胞死亡，包 119007.doc -171 - 200813092 括漸進式細胞死亡（細胞凋亡）之能力。亦提供在活體内及/ 或活體外具有此生物活性之抗體或免疫接合物。在某些實施例中，測試抗TAT226抗體或其免疫接合物於活體外抑制細胞生長或增殖之能力。此項技術中熟知用於抑制細胞生長或增殖之檢定。以本文所述之π細胞殺死，，檢定例示之用於細胞增殖的特定檢定量測細胞生存力。一種此檢定為可購自 Promega(Madison，WI)之 CellTiter-Gl〇TMS 光細胞生存力檢定。彼檢定基於對所存在ATP之定量來測定培養物中可生存細胞之數量，其為對代謝活性細胞之指示。參見Crouch 等人（1993) /mmwwo/· Mei/z. 160:81-88，美國專利第6602677號。該檢定可以96或384孔格式進行，使其受到自動高產量篩檢（HTS)之影響。參見Cree等人 (1995) dw/C⑽ cer Drwgs 6:3 98-404。檢定程序包括將單一試劑（CellTiter-Glo^^ij)直接添加至經培養細胞中。此導致細胞溶解且產生由螢光素酶反應所產生之發光信號。發光“號與所存在ATP之量成比例，所存在Ατρ之量直接與培養物中所存在之可生存細胞的數量成比例。可藉由光度計或CCD攝影成像裝置記錄資料。將發光輸出表示為相對光單位（RLU)。，另一種細胞增殖檢定為"MTT"檢定，其為量測由粒線體還原酶將溴化3_(4,5_二甲基噻唑_2_基）_2,5_二苯基四唑鹽，化為甲臢之比色檢^。如同CeUTiter_G1()TM檢定，此: 疋才日不細胞培養⑯中所存在之代謝活性細㈤的數量。參見例如 M〇Smaim (1983) “卿卿/. Μ敲 65:55_63及Zhang 119007.doc -172- 200813092 等人（2005) 65:3877-3882。在一態樣中，測試抗TAT226抗體活體外誘導細胞死亡之能力。此項技術中熟知誘導細胞死亡之檢定。在一些實施例中，此等檢定量測例如由碘化丙錠（PI)、錐蟲藍（參見 ]\4〇〇代等人（1995)(^>^(9化(：/2/26>/6^少，17:1-11)或7八八0吸收所指示之膜完整性損失。在例示性PI吸收檢定中，將細胞培養於補充以10%熱失活FBS(Hyclone)及2 mM L-麩醯胺酸之杜貝科氏經改質伊格培養基（D-MEM):Ham氏F-12(50:50) 中。因此，在不存在補體及免疫效應細胞之情況下進行此檢定。將細胞以每盤3 X 106之密度接種於100 X 20 mm之盤中且使得隔夜附著。將培養基移除且以單獨之新鮮培養基或含有各種濃度之抗體或免疫接合物之培養基替換。將細胞培育3天之時期。處理之後，將單層以PBS洗滌且藉由胰蛋白酶作用分離。接著將細胞於4°C下以1200 rpm離心5 分鐘，將離心塊再懸浮於3 ml冷Ca2+結合缓衝劑（10 mM Hepes，pH 7.4，140 mM NaCl，2.5 mM CaCl2)中且等分入以3 5 mm濾網覆蓋之12 x 7 5 mm管中（每管1 ml，每處理組 3支管）以移除細胞塊。接著使管接收PI(l〇 pg/ml)。使用 FACSCANTNHll 式細胞儀及 FACSCONVERTtm CellQuest軟體（Becton Dickinson)分析樣本。因此識別如由PI吸收測定之誘導統計學上顯著程度之細胞死亡的抗體或免疫接合物。在一態樣中，測試抗TAT226抗體或免疫接合物活體外誘導細胞凋亡（漸進式細胞死亡）之能力。用於誘導細胞凋亡 119007.doc -173 - 200813092 之抗體或免疫接合物之例示性檢定為膜聯蛋白結合檢定。在例示性膜聯蛋白結合檢定中，將細胞如前段所述於盤中培養且接種。將培養基移除且以單獨之新鮮培養基或含有 0.001至10 pg/ml抗體或免疫接合物之培養基替換。三天培育期之後，將單層以PBS洗滌且藉由胰蛋白酶作用分離。接著如前段所述將細胞離心，再懸浮於Ca2+結合緩衝劑中且等分入管中。接著使管接收經標記之膜聯蛋白（例如膜聯蛋白V_FITC)(1 pg/ml)。使用FACSCANTM流式細胞儀及 FACSCONVERTtm CellQuest軟體（BD Biosciences)分析樣本。因此，識別相對於對照組而言誘導統計學上顯著程度之膜聯蛋白結合之抗體或免疫接合物。誘導細胞凋亡之抗體或免疫接合物的另一種例示性檢定為用於偵測染色體組 DNA之核小體間降解之組蛋白DNA ELISA比色檢定。可使用例如細胞死亡偵測ELISA套組（Roche，Palo Alto，CA)來進行此檢定。用於上述任何活體外檢定之細胞包括天然表現TAT226或已經工程設計以表現TAT226之細胞或細胞株。此等細胞包括相對於相同組織來源之正常細胞過度表現TAT226之腫瘤細胞。此等細胞亦包括表現TAT226之細胞株（包括腫瘤細胞株）及非正常表現TAT226而已經編碼TAT226之核酸轉染之細胞株。本文所提供之用於上述任何活體外檢定之例示性細胞株包括表現TAT226之OVCAR3人類卵巢癌細胞株及經編碼TAT226之核酸轉染之HCT116人類結腸癌細胞株。在一態樣中，測試抗TAT226抗體或其免疫接合物活體内 119007.doc -174- 200813092 抑制細胞生長或增殖之能力。在某些實施例中，測試抗 TAT226抗體或其免疫接合物活體内抑制腫瘤生長之能力。活體内模型系統（諸如異種移植物模型）可用於此測試。在例示性異種移植物系統中，可將人類腫瘤細胞引入例如無胸腺”裸π小鼠之適當免疫功能不足之非人類動物體内。可將本發明之抗體或免疫接合物投予動物。量測抗體或免疫接合物抑制或減低腫瘤生長之能力。在以上異種移植物系統之某些實施例中，人類腫瘤細胞為來自人類患者之腫瘤細胞。此等異種移植物模型可購自Oncotest GmbH(Frieberg，Germany)。在某些實施例中，如本文所例示，人類腫瘤細胞為來自人類腫瘤細胞株之細胞，諸如 0VCAR3細胞。在某些實施例中，藉由皮下注射或藉由移植入合適部位（諸如乳腺脂肪墊）將人類腫瘤細胞引入適當免疫功能不足的非人類動物體内。 b)結合檢定及其他檢定在一態樣中，測試抗TAT226抗體之抗原結合活性。舉例而言，在某些實施例中，測試抗TAT226抗體與於細胞表面上表現之TAT226結合之能力。諸如實例D中所述之FACS檢定可用於此測試。在一態樣中，競爭檢定可用以識別與YW0.32、YW0.49、 YWO_49.B7、YWO.49.C9、YWO.49.H2 或 YWO.49.H6競爭結合TAT226之單株抗體。在某些實施例中，此競爭抗體結合至由 YW0.32、YW0.49、YWO.49.B7、YWO.49.C9、 YWO.49.H2或YWO.49.H6結合之相同抗原決定基（例如線 119007.doc -175- 200813092 性或構形抗原決定基）。例示性競爭檢定包括（但不限於）諸Papanastassiou et al, 9: 398-406 (2002)); interstitial infusion/convection enhanced transmission (see, for example, B〇b〇 et al., pr〇c heart is 5W. still) 91·· 2076-2080 (1994) And implanting a delivery device in the brain (see, for example, Gill et al, A/W 9: 589-595 (2003); and Gliadel WafersTM, Guildford Pharmaceutical). Methods of creating an opening in a barrier include, but are not limited to, supersonic (see, e.g., U.S. Patent Publication No. 2002/0038086), infiltrating dust (e.g., by administering hypertonic nectar 119007.doc -168 - 200813092 sugar alcohol (Neuwelt, E. A., Implication of the Blood-Brain Barrier and its Manipulation, Vol. 1 & 2, Plenum Press, NY (1989)); permeation by, for example, bradykinin or penetrant A-7 ( See, for example, U.S. Patent Nos. 5,112,596, 5,268,164, 5,506,206 and 5,686,416) and transfecting neurons across the blood-brain barrier with a vector containing the gene encoding the antibody (see, for example, U.S. Patent Publication No. 2003/0083299) ° Lipid-based methods of transporting antibodies or immunoconjugates across the blood-brain barrier include, but are not limited to, encapsulation of antibodies or immunoconjugates into receptors coupled to the vascular endothelium of the blood-brain barrier The liposome of the antibody-binding fragment is bound (see, for example, U.S. Patent Application Publication No. 200200253 13) and is a low-density lipoprotein particle (see, for example, U.S. Patent Application Publication No. 2004020 An antibody or immunoconjugate is coated with a lipoprotein element E (see, e.g., U.S. Patent Application Publication No. 20040131692). A stem cell-based method of transporting antibodies or immunoconjugates across the blood-brain barrier requires genetic engineering of neural progenitor cells (NPCs) to express the antibody or immunoconjugate of interest and then implant the stem cells into the brain of the individual to be treated. See Behrstock et al. (2005) 772er· December 15, 2005 曰 Advanced Online Publication (Reporting that when implanted in the brains of rodent and primate models, NPC is genetically engineered to show Parkinson's disease (Parkinson disease) ) The neurotrophic factor GDNF reduces symptoms). Receptor- and channel-based methods of transporting antibodies or immunoconjugates across the blood-brain barrier include, but are not limited to, the use of glucocorticoid blockers to increase permeability of the blood-brain barrier (see, for example, U.S. Patent Application Publication No. 119,007) .doc - 169-200813092 2002/0065259, 2003/0162695 and 2005/0124533), activating a bell channel (see, e.g., U.S. Patent Application Publication No. 2005/0089473); inhibiting ABC drug transporters (see for example U.S. Patent Application Publication No. 2003/0073713; coating an antibody or immunoconjugate with transferrin and modulating the activity of one or more transferrin receptors (see, e.g., U.S. Patent Application Publication No. 2003/0129186) The antibody or immunoconjugate is cationized (see, e.g., U.S. Patent No. 5, No. 4,697). The antibodies or immunoconjugates of the invention will be formulated, administered and administered in a manner consistent with good pharmaceutical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the schedule of administration, and other factors known to the medical practitioner. The antibody or immunoconjugate is not required (but subject to the October condition) to be formulated with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other agents will depend on the amount of antibody or immunoconjugate present in the formulation, the condition or type of treatment, and other factors mentioned above. It is generally administered in the same dosages and routes of administration as described above, or from about 1 to 99% of the dosages described above, or any dosage and any route which is empirically/clinically determined to be appropriate. For the prevention or treatment of a disease, the appropriate dose of an antibody or immunoconjugate of the invention (when used alone or in combination with one or more other therapeutic agents such as a chemotherapeutic agent) will depend on the type of disease, antibody or immunoconjugate type to be treated. , severity and progression of the disease, whether to administer antibodies or immunoconjugates for prophylactic or therapeutic purposes, prior therapy, clinical history of the patient, and response to antibodies or immunoconjugates and judgment of the attending physician. Antibody or immunoconjugate 119007.doc -170- 200813092 A suitable-sub- or via-series treatment is administered to a patient. Depending on the type and severity of the disease, an antibody or immunoconjugate 约 about i叩/kg to 15 mg/kg (eg, 〇mg/kg i ( (4) is a candidate starting dose for administration to a patient, regardless of, for example, Depending on the above factors, a typical dose per dose may range from about i/kg to 1 〇〇mg/kg*, depending on the above factors. For several days or For longer periods of repeated administration, depending on the condition, it will continue to be treated until the desired inhibition of the symptoms of the disease occurs. An exemplary dose of antibody or immunoconjugate is about 〇.〇5 mg From /kg to about 10 mg/kg. Therefore, one or more doses of about 5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 41 mg/kg (or any combination thereof) can be administered to the patient. Such doses may, for example, be administered intermittently weekly or every three weeks (eg, such that the patient receives from about two to about twenty, or such as about six doses of antibody or immunoconjugate). The initial loading dose, followed by one or more lower doses. An exemplary dosing regimen consists of the initiation of administration of about 4 mg/kg. The loading dose, which in turn is a weekly maintenance dose of about 2 mg/kg of antibody. However, other dosing regimens are applicable. This course of treatment is readily monitored by conventional techniques and assays. 3 * Characterization of the anti-TAT226 antibody of the present invention and immunization The conjugate can be characterized by various assays known in the art for its physical/chemical properties and/or biological activity. ^ Activity assay provides a biologically active anti-TAT226 antibody or immunoconjugate thereof in a sad sample The biological activity may include, for example, the ability to inhibit cell growth or proliferation (e.g., cell killing activity) or induce cell death, including the ability of progressive cell death (apoptosis) 119007.doc-171 - 200813092. Antibodies or immunoconjugates having such biological activity in vivo and/or in vitro are also provided. In certain embodiments, the ability of an anti-TAT226 antibody or immunoconjugate thereof to inhibit cell growth or proliferation in vitro is tested. A assay for inhibiting cell growth or proliferation is well known in the art. With the π cell killing described herein, the assay exemplifies the specificity for cell proliferation. Quantitative measurement of cell viability. One such assay is the CellTiter-Gl〇TMS photocell viability assay available from Promega (Madison, WI). The assay is based on the quantification of the presence of ATP to determine viable cells in culture. Quantity, which is an indication of metabolically active cells. See Crouch et al. (1993) / mmwwo/. Mei/z. 160:81-88, US Patent No. 6602677. This assay can be performed in 96 or 384 well formats. Subject to automatic high throughput screening (HTS). See Cree et al. (1995) dw/C(10) cer Drwgs 6:3 98-404. The assay procedure involves the addition of a single reagent (CellTiter-Glo^^ij) directly to the cultured cells. This causes the cells to dissolve and produce a luminescent signal produced by the luciferase reaction. The luminescence number is proportional to the amount of ATP present, and the amount of Ατρ present is directly proportional to the number of viable cells present in the culture. The data can be recorded by a photometer or a CCD photographic imaging device. For relative light units (RLU), another cell proliferation assay is the "MTT" assay, which measures brominated 3_(4,5-dimethylthiazole_2-yl)_2 by mitochondrial reductase , 5_diphenyltetrazolium salt, converted to colorimetric detection of formazan. As with the CeUTiter_G1()TM assay, this: The number of metabolically active fines (fi) present in cell culture 16 is not shown. See, for example, M 〇Smaim (1983) “Qing Qing/. Μ 65:55_63 and Zhang 119007.doc -172- 200813092 et al. (2005) 65:3877-3882. In one aspect, the ability of the anti-TAT226 antibody to induce cell death in vitro was tested. A test for inducing cell death is well known in the art. In some embodiments, such assays are, for example, from propidium iodide (PI), trypan blue (see)\4〇〇代, et al. (1995) (^>^(9)(:/2/ Loss of membrane integrity as indicated by 26>/6^min, 17:1-11) or 7880 absorption. In an exemplary PI uptake assay, cells were cultured supplemented with 10% heat-inactivated FBS (Hyclone) And 2 mM L-glutamic acid in Dubecco's modified Ig medium (D-MEM): Ham F-12 (50:50). Therefore, in the absence of complement and immune effector cells This assay was performed. Cells were seeded at a density of 3 X 106 per dish in a 100 X 20 mm dish and allowed to attach overnight. The medium was removed and either alone in fresh medium or containing various concentrations of antibody or immunoconjugate Substitution of the medium. The cells were incubated for a period of 3 days. After the treatment, the monolayer was washed with PBS and separated by trypsinization. The cells were then centrifuged at 1200 rpm for 5 minutes at 4 ° C, and the pellet was resuspended in 3 Mol cold Ca2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and aliquoted into 12 x 7 5 mm tubes covered with 35 mm mesh (per Tube 1 ml, 3 tubes per treatment group) to remove the cell mass. The tube was then subjected to PI (l〇pg/ml). The sample was analyzed using the FACSCANTNHll cytometer and the FACSCONVERTtm CellQuest software (Becton Dickinson). Absorbing an antibody or immunoconjugate that induces a statistically significant degree of cell death. In one aspect, the ability of an anti-TAT226 antibody or immunoconjugate to induce apoptosis (progressive cell death) in vitro is tested. An exemplary assay for an antibody or immunoconjugate that induces apoptosis 119007.doc -173 - 200813092 is an annexin binding assay. In an exemplary annexin binding assay, cells are cultured and inoculated in a dish as described in the previous paragraph. The medium was removed and replaced with fresh medium alone or medium containing 0.001 to 10 pg/ml of antibody or immunoconjugate. After a three-day incubation period, the monolayer was washed with PBS and separated by trypsinization. The cells are centrifuged as described in the previous paragraph, resuspended in Ca2+ binding buffer and aliquoted into the tube. The tube is then received with labeled annexin (eg Connexin V_FITC) (1 pg/ml). Samples were analyzed using FACSCANTM flow cytometry and FACSCONVERTtm CellQuest software (BD Biosciences). Thus, antibodies that induce a statistically significant degree of annexin binding relative to the control group were identified. Or immunoconjugate. Another exemplary assay for an antibody or immunoconjugate that induces apoptosis is a histone DNA ELISA colorimetric assay for detecting nucleosome degradation of genomic DNA. This assay can be performed using, for example, a cell death detection ELISA kit (Roche, Palo Alto, CA). Cells for use in any of the above in vitro assays include cells or cell lines that naturally express TAT226 or have been engineered to express TAT226. Such cells include tumor cells that overexpress TAT226 relative to normal cells of the same tissue source. These cells also include cell lines that express TAT226 (including tumor cell lines) and cell lines that have been transfected with a nucleic acid encoding TAT226 that normally expresses TAT226. Exemplary cell lines provided herein for use in any of the above in vitro assays include the OVCAR3 human ovarian cancer cell line expressing TAT226 and the HCT116 human colon cancer cell line transfected with a nucleic acid encoding TAT226. In one aspect, the anti-TAT226 antibody or its immunoconjugate is tested for its ability to inhibit cell growth or proliferation in vivo 119007.doc -174-200813092. In certain embodiments, the ability of an anti-TAT226 antibody or immunoconjugate thereof to inhibit tumor growth in vivo is tested. In vivo model systems, such as xenograft models, can be used for this test. In an exemplary xenograft system, human tumor cells can be introduced into a non-human animal of a suitable immune function, such as athymic "naked pi mice. The antibody or immunoconjugate of the invention can be administered to an animal. The ability of an antibody or immunoconjugate to inhibit or reduce tumor growth is measured. In certain embodiments of the above xenograft system, the human tumor cells are tumor cells from a human patient. Such xenograft models are commercially available from Oncotest GmbH ( Frieberg, Germany). In certain embodiments, as exemplified herein, human tumor cells are cells from a human tumor cell line, such as 0VCAR3 cells. In certain embodiments, by subcutaneous injection or by transplantation Sites (such as mammary fat pads) introduce human tumor cells into non-human animals with inadequate immune function. b) Binding assays and other assays test the antigen binding activity of anti-TAT226 antibodies in one aspect. For example, In certain embodiments, the ability of an anti-TAT226 antibody to bind to TAT226 expressed on the surface of a cell is tested, such as in Example D. The FACS assay described can be used for this test. In one aspect, a competition assay can be used to identify YW0.32, YW0.49, YWO_49.B7, YWO.49.C9, YWO.49.H2 or YWO.49. H6 competes for binding to a monoclonal antibody of TAT226. In certain embodiments, this competing antibody binds to YW0.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO. 49. H6 binds to the same epitope (eg, line 119007.doc-175-200813092 sex or conformation epitope). Exemplary competition assays include, but are not limited to,

Harlow^ Lane (1988) Antibodies: A Laboratory Manual 第 14 章（Cold Spring Harbor Laboratory， Cold Spring Harbor，NY)中所提供之彼等常規檢定。繪製結合抗體之抗原決定基的詳細例示性方法係於h Mo/ecw/ar 少第 66 卷（Humana Press，Totowa，NJ)中之 Morris (1996) ’’Epitope Mapping Protocols"中提供。據稱若兩種抗體各自阻斷另一者50%或更多之結合，則二者均結合至相同抗原決定基。在例示性競爭檢定中，於包含結合至TAT226之第一經標記抗體（例如 YW0.32、YW0.49、YWO.49.B7、YWO.49.C9、 YWO.49.H2或YWO.49.H6)及經測試其與第一抗體競爭結合TAT226之能力之第二未經標記抗體的溶液中培育固定 TAT226。第二抗體可存在於融合瘤上清液中。作為對照組，於包含第一經標記抗體但不包含第二未經標記抗體之溶液中培育固定TAT226。於容許第一抗體結合至TAT226 之條件下培育後，移除過量未結合之抗體且量測與固定 TAT226相關聯之標記量。若測試樣本中與固定TAT226相關聯之標記量相對於對照樣本大體上降低，則其表示第二抗體與第一抗體競爭結合至TAT226。在某些實施例中，固定TAT226存在於細胞表面上或存在於自於其表面表現 TAT226之細胞所獲得之膜製劑中。在一態樣中，經純化抗TAT226抗體可由一系列檢定進一步表徵，該等檢定包括（但不限於）N端定序、胺基酸分 119007.doc -176- 200813092 析、非變性尺寸排阻高壓液相層析（HPLC)、質譜分析、離子交換層析及番木瓜酶消化。在一實施例中，本發明涵蓋具有一些而非所有效應功能之經改變抗體，此使其在抗體之活體内半衰期為重要的，而某些效應功能（諸如補體及ADCC)為非必需或有害之諸多應用中為理想候選物。在某些實施例中，量測抗體之Fc 活性以確保僅保持所需特性。可進行活體外及/或活體内細胞毒性檢定以確認降低/耗盡CDC及/或ADCC活性。舉例Harlow^ Lane (1988) Antibodies: A Laboratory Manual These routine assays are provided in Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). A detailed exemplary method for mapping the antibody-binding epitopes is provided in Morris (1996) ''Epitope Mapping Protocols" in h Mo/ecw/ar, vol. 66 (Humana Press, Totowa, NJ). It is stated that if the two antibodies each block 50% or more of the binding of the other, both bind to the same epitope. In an exemplary competition assay, the first labeled antibody that binds to TAT226 is included (eg, YW0.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, or YWO.49. H6) and fixed TAT226 were incubated in a solution of a second unlabeled antibody that was tested for its ability to compete with the first antibody for binding to TAT226. The second antibody can be present in the supernatant of the fusion tumor. As a control group, immobilized TAT226 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing the first antibody to bind to TAT226, excess unbound antibody was removed and the amount of label associated with immobilized TAT226 was measured. If the amount of label associated with the immobilized TAT226 in the test sample is substantially reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to TAT226. In certain embodiments, the immobilized TAT226 is present on the surface of the cell or in a film preparation obtained from cells whose surface exhibits TAT226. In one aspect, the purified anti-TAT226 antibody can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid fraction 119007.doc-176-200813092, non-denaturing size exclusion High pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion. In one embodiment, the invention encompasses altered antibodies having some, but not all, of the effector functions, which makes it important in the in vivo half-life of the antibody, while certain effector functions (such as complement and ADCC) are non-essential or harmful. It is an ideal candidate for many applications. In certain embodiments, the Fc activity of the antibody is 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. Example

^ 而言，可進行Fc受體（FcR)結合檢定以確保抗體缺少FcyR 結合（因此可能缺少ADCC活性），但保持FcRn結合能力。用於介導ADCC之初級細胞’即NK細胞僅表現FcXRIII ’而單核細胞表現FcXRI、FcXRII及FcXRIII。造血細胞上之FcR 表現係概述於Ravetch及Kinet，jwww· 9:457- 92 (1991)第464頁之表3中。評估所關注分子之ADCC活性之活體外檢定的實例描述於美國專利第5,500,362號或第 5,821，337號中。適用於此等檢定之效應細胞包括周邊血液 / U 單核細胞（PBMC)及天然殺死（NK)細胞。或者，或另外，可於活體内，例如於諸如Clynes等人户见4夕95:652-656 (1998)中揭示之動物模型中評估所關注分子之ADCC活性。亦可進行Clq結合檢定以確認抗體不能夠結合Clq且因此缺少CDC活性。為評估補體活化作用，可進行例如 Gazzano-Santoro 等人，J. 厂 202:163 (1996)中所述之CDC檢定。亦可使用此項技術中已知之方法進行FcRn結合及活體内清除/半衰期測定。 119007.doc -177- 200813092 F·製品、在本發明之另一態樣中，提供含有適用於治療、預防及/ 或0斷上述病症之物質的製品。製品包含容器及容器上或與谷器相關聯之標記或包裝插頁。合適容器包括例如瓶子彳瓶針筒等。容器可由諸如玻璃或塑料之各種材料幵y成"亥合器固持獨立或與有效治療、預防及/或診斷病況^另一種組合物組合之組合物，且可具有無菌入口（例如谷器可為靜脈内溶液袋或具有可由皮下注射針刺穿之塞子的j瓶）組合物中至少一種活性劑為本發明之抗體或免疫接合物。標記或包裝插頁指示組合物係用於治療所選擇之病況。此外，製品可包含（a)具有組合物含於其中之第一容器，其中組合物包含本發明之抗體或免疫接合物；及 (b)具有組合物含於其中之第二容器，纟中組合物包含其他細胞毒性劑或治療劑。本發明之此實施例中之製品可另外包含指示組合物可用以治療特定病況之包裝插頁。或者，或另外，製品可另外包含第二（或第三）容器，其包含醫藥學上可接爻之緩衝劑，諸如注射用抑菌水（BWFI)、經磷酸鹽緩衝之生理食鹽水、林格氏溶液（Ringer，s s〇luti〇匀及右方疋糖溶液。其可進一步包括就商業或使用者觀點而言所需之其他物質’包括其他緩衝劑、稀釋劑、過遽器、針及針筒。 IV·實例以下為本發明之方法及組合物之實例。應瞭解，在給定上文提供之一般描述下可實踐各種其他實施例。 119007.doc -178- 200813092 Α· TAT226基因表現之分析使用含有基因表現資訊之專屬資料庫（GeneExpress⑧，In conclusion, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcyR binding (and thus may lack ADCC activity), but retains FcRn binding ability. The primary cells used to mediate ADCC, i.e., NK cells, exhibit only FcXRIII' and monocytes exhibit FcXRI, FcXRII, and FcXRIII. The FcR expression lines on hematopoietic cells are summarized in Table 3 on page 464 of Ravetch and Kinet, jwww. 9:457-92 (1991). An example of an in vitro assay for assessing the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Effector cells suitable for such assays include peripheral blood/U monocytes (PBMC) and natural kill (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed by Clynes et al., 4:95:652-656 (1998). A Clq binding assay can also be performed to confirm that the antibody is unable to bind Clq and thus lacks CDC activity. To assess complement activation, a CDC assay such as that described in Gazzano-Santoro et al., J. Plant 202: 163 (1996) can be performed. FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art. 119007.doc -177- 200813092 F. Article, In another aspect of the invention, an article of manufacture containing a substance suitable for treating, preventing, and/or eroding the above conditions is provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottle and bottle syringes and the like. The container may be a composition that may be independently or in combination with an effective therapeutic, prophylactic and/or diagnostic condition, another composition, such as a glass or plastic material, and may have a sterile inlet (eg, a barn may be At least one active agent in the composition of the intravenous solution bag or the j bottle having a stopper pierceable by a hypodermic needle is the antibody or immunoconjugate of the present invention. The marker or package insert indicates that the composition is used to treat the selected condition. Additionally, the article of manufacture may comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody or immunoconjugate of the invention; and (b) a second container having the composition contained therein, in combination Contains other cytotoxic or therapeutic agents. The article of manufacture of this embodiment of the invention may additionally comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may additionally comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, forest Grignard solution (Ringer, ss〇luti and right sugar solution. It may further include other substances required from a commercial or user point of view) including other buffers, diluents, filters, needles and Syringes. IV. Examples The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments can be practiced in the general description provided above. 119007.doc -178- 200813092 Α· TAT226 Gene Performance The analysis uses a proprietary database containing gene expression information (GeneExpress 8,

Gene Logic Inc” Gaithersburg，MD)分析人類TAT226基因表現。使用微陣列分佈檢視器進行GeneExpress®資料庫之圖解分析。圖13為各種組織中TAT226基因表現之圖示，其於左側列出。橫穿圖頂部之刻度指示基於雜交信號強度之基因表現程度。線上方及下方所呈現之點與各自之所列組織相鄰。線上方呈現之點表示正常組織中之基因表現，且線下方呈現之點表示腫瘤或病變組織中之基因表現。圖i 3 展示相對於其正常對應物，腫瘤或病變組織中趨向增加 TAT226基因表現。詳言之，TAT226在腫瘤及病變卵巢中相對於正常卵巢展示實質上之過度表現，且在威爾姆氏腫瘤中相對於正常腎展示實質上之過度表現。相對於正常組織在腫瘤或病變組織中展示過度表現之其他組織包括子宮内膜、腎上腺、骨骼、肺 '皮膚及軟組織。此外，TAT226 於正常腦組織（諸如嗅腦、海馬及基底神經節）中及腫瘤或病變腦組織（諸如神經膠質瘤）中教強表現。Gene Logic Inc" Gaithersburg, MD) analyzed human TAT226 gene expression. Graphical analysis of the GeneExpress® database using a microarray distribution viewer. Figure 13 is a graphical representation of the TAT226 gene expression in various tissues, listed on the left. The scale at the top of the figure indicates the degree of gene expression based on the intensity of the hybridization signal. The points presented above and below the line are adjacent to the respective listed tissues. The point above the line indicates the gene expression in normal tissues and the point below the line. Representing gene expression in tumor or diseased tissues. Figure i3 shows the tendency to increase TAT226 gene expression in tumors or diseased tissues relative to its normal counterpart. In detail, TAT226 is substantially relative to normal ovarian display in tumors and diseased ovaries. Excessive performance and substantial overexpression in Wilms' tumors relative to normal kidneys. Other tissues showing excessive expression in tumors or diseased tissues relative to normal tissues include endometrium, adrenal gland, bone, lung' Skin and soft tissue. In addition, TAT226 is in normal brain tissue (such as olfactory brain, hippocampus and basal Verses) and tumor or lesion in the brain tissue (such as glioma) strong performance in teaching.

GeneExpress®資料庫亦用以分析正常卵巢；正常輸卵管；透明細胞、黏液性及漿液性囊腺癌亞型之卵巢癌；轉移性卵巢癌及其他類型卵巢癌中之人類TAT226基因表現。結果於圖14中圖解報導，其中特定組織類型在圖下方表示。圖中y軸之刻度指示基於雜交信號強度之基因表現程度。漿液性囊腺癌及轉移性卵巢癌相對於正常卵巢展示 TAT226之較強過度表現。透明細胞及黏液性亞型展示與正 119007.doc -179· 200813092 常卵巢相當之表現。正常輸卵管亦展示TAT226之實質性表現。應注意，卵巢癌之漿液性亞型與輸卵管上皮組織極為類似，且卵巢及輸卵管均衍生自相同胚胎組織。參見Fox 等人（2002) ’’Pathology 〇f epithelial ovarian cancer，’’於 (9varz·⑽ Cancer第 9章（Jacobs等人編，Oxford University Press，New York)中。 Β·抗TAT226抗體之產生藉由以包含SEQ ID ΝΟ:75之胺基酸1·115及C端聚組胺酸標記之重組nTAT226_His"融合蛋白篩檢噬菌體呈現庫而產生TAT226之抗體。噬菌體呈現庫為使用Fab’-zip-噬菌體系統所產生之合成（Fab’)2庫。參見Lee等人（2004) J· /mmw⑽/. 284:119-132。庫包含huMAb4D5-8重鍵可變區（參見圖5A及5B，第二受體"B”，SEQ ID NO:50、 51、57、3 5)與如8丑(^1〇>^0:26所示之固定1111]\4八54〇5-8輕鏈可變區之框架中的重鏈HVR庫。使用噬菌體ELIS A針對 TAT226-His篩檢使用噬菌體呈現所選擇之純系（參見例如 Sidhu 等人（2004) J. Mo/· 5b/· 338:299-310)。選擇純系 YW0.32及YWO.49用於進一步分析。為改良YW0.49之親和力，在YW0.49背景中以靶向軟體隨機化之HVR-H3及HVR-L3產生噬菌體呈現庫，其中使指定HVR中所選擇之胺基酸殘基保持恆定，而其他殘基則經受突變。藉由噬菌體ELISA篩檢所選擇之純系。選擇命名為 YWO.49.B7、YWO.49.C9、YWO.49.H2及 YWO.49.H6之親和力成熟抗體用於進一步分析。如圖9及10所示，確定 119007.doc •180- 200813092 YWO.32 、 YWO.49 、 YWO.49.B7 、 YWO.49.C9 、 YWO.49.H2及YWO.49.H6之VH及VL區之核苦酸及經編碼之多肽序列。YWO.32、YWO.49、YWO.49.B7、 YWO.49.C9、YWO.49.H2 及 YWO.49.H6 之重鏈及輕鏈 HVR 序列展示於圖2_4中。衍生自YWO.49、YWO.49.B7、 YWO.49.C9、YWO.49.H2 及 YWO.49.H6 之一致 HVR-H3 及 HVR-L3序列亦展示於圖4中。將YWO.49、YWO.49.B7、 YWO.49.C9、YWO.49.H2 及 YWO.49.H6藉由使用重組技術將Fab’片段移植於適當恆定區上而"重排”為全長IgG。使用重排抗體進行下文所述之實驗。 C.重組抗原結合親和力之表徵藉由使用 BIACORE® 3000 系統（Biacore，Inc·，Piscataway， NJ)之表面電漿共振量測確定YWO.49、YWO.49.B7、 YWO.49.C9、YWO.49.H2 及 YWO.49.H6對重組抗原之結合親和力。簡而言之，根據供應商說明，以乙基w-(3-二甲胺基丙基）-碳化二醯亞胺鹽酸鹽（EDC)及iV-羥基琥珀醯亞胺（NHS)活化羧甲基化葡聚糖生物感應晶片（CM5, BIAcore Inc)。在以每分鐘5 μΐ之流動速率注射之前，將抗丁八丁226抗體以卩114.8之1〇1111^乙酸鈉稀釋至5 4§/1111以達成大約500個反應單位（RU)之偶合抗體。其次，注射1 Μ乙醇胺以阻斷未反應之基團。對於動力學量測而言，於25°C下以25 μΐ/min之流動速率將兩倍連續稀釋之TAT226_His(0.7 nM至500 nM)注射至具有0.05%吐溫20之PBS中。使用簡單一對一朗缪爾結合模型（one-to-one Langmuir binding 119007.doc -181 - 200813092 model)(BIA評估軟體3.2版）計算締合速率（kQn)及解離速率 (koff)。以koff/kQn之比率計算平衡解離常數（Kd)。此實驗結果展示於下表2中。表2 純系 k〇n/105 koff/10·4 Kd(nM) YW0.49 0.074 3.49 47.16 YWO.49.B7 0.27 <0.05 <0.18 YWO.49.C9 1.53 <0.05 <0.03 YWO.49.H2 0.22 0.05 0.23 YWO.49.H6 1.86 0.09 0.05 D·結合至細胞表面TAT226之抗體的表徵檢驗抗TAT226抗體結合至在〇VCAR3(人類卵巢癌細胞株）表面上所表現之TAT226的能力。在存在及不存在 YW0.49 、YWO.49.B7、YWO.49.C9、YWO.49.H2 或 YWO.49.H6下，於0VCAR3細胞上進行螢光活化細胞揀選 (FACS)。簡而言之，將經分離細胞以5 pg/ml初級抗體於冰上培育一小時，洗滌且以第二抗體（接合至藻紅素之抗人類IgG)於冰上培育30分鐘。使用FACScanTM流式細胞儀 (BD Biosciences，San Jose，CA)進行 FACS。對 YW0.49、YWO.49.H2 及 YW0.H6 之 FACS 分析結果展示於圖1 5中。各圖左侧之峰值表示"背景’’結合，亦即僅二級抗體之結合。各圖右侧之峰值表示指定抗TAT226抗體之結合。由FACS可見，YWO.49.B7不顯著結合至OVCAR3，即使其以關於YW0.49及其他親和力成熟抗體所觀測之Kd 範圍内（參見上表2中BIAC0RE®分析之結果）之Kd結合至 119007.doc • 182 - 200813092 重組抗原。YWO.49.C9之結合可相當於關於YWO.49.H2及 YWO.H6所觀測之結合。 E.與細胞表面抗原之結合親和力之表徵使用競爭檢定檢驗YWO.49.H2及YWO.49.H6對於 OVCAR3細胞表面上所表現之TAT226之結合親和力。簡而言之，使經標記（碘化）之YWO.49.H2或YWO.49.H6在未經標記抗體之存在下結合至OVCAR3細胞。根據Munson等人，4似/· 5沁107:220 (1980)中初始描述之斯卡查德分析法（Scatchard analysis methodology)確定抗體之結合親和力。此實驗結果展示於下表3中。表3 純系 Kd〇ftM) YWO.49.H2 0.348 YWO.49.H6 0.404 與重組 TAT226-His 相比，YWO.49.H2 及 YWO.49.H6 對於 OVCAR3細胞表面所表現之TAT226的Kd較高（比較表2及3 中 YWO.49.H2 及 YWO.49.H6 之 Kd)，此指示 YWO.49.H2 及 YWO.49.H6以比結合至於OVCAR3細胞表面所表現之 TAT226稍微較高之親和力結合至重組TAT226-His。 F· TAT226 mRNA及蛋白表現使用51核酸酶（TaqMan®)檢定及即時定量PCR分析 TAT226 mRNA於OVCAR3細胞及卵巢癌樣本板中之表現。圖16中命名為”HF ####’’之卵巢癌樣本為冷凍組織切片。將RNA自組織切片分離，使用Ambion’s Message Amp II套 119007.doc -183 - 200813092 組（Ambion，Austin，TX)擴增且逆相轉錄為cDNA。將RNA 自OVCAR3細胞分離且逆相轉錄為cDNA。在對擴增產物具有特異性之不可延伸性報導體探針存在下，藉由即時 PCR擴增TAT226 cDNA。測定臨限週期或nCt”（自報導體探針裂解所產生之信號超過背景值之週期）且將其用以計算起始TAT226 mRNA含量。如圖16之條形圖所示，相對於 Ο VC AR3細胞中之TAT226 mRNA含量表示卵巢癌樣本板中之 TAT226 mRNA含量。如下使用免疫組織化學（IHC)分析OVCAR3細胞及上述卵巢癌樣本板中之TAT226蛋白表現。將印巢癌樣本之組織切片（冷凍或嵌埋有番木瓜酶）固定於丙酮/乙醇中歷時5分鐘。將切片於PBS中洗滌，以抗生物素蛋白及生物素 (Vector Laboratories，Inc., Burlingame, CA)各自阻斷 l〇分鐘且再次於PBS中洗滌。接著將切片以10%血清阻斷20分鐘且洗乾以移除過量血清。接著將初級抗體（YWO.49.H2 或YWO.49.H6)以10 pg/ml之濃度添加至切片中歷時1小時。接著將切片以PB S洗滌。將經生物素標記之二級抗人類抗體添加至切片中歷時30分鐘，且接著以PBS洗滌切片。接者將切片暴露於Vector ABC套組（Vector Laboratories， Inc·，Burlingame，CA)之試劑歷時30分鐘且接著於PBS中洗滌。接著將切片暴露於二胺基聯苯胺（Pierce)歷時5分鐘且接著於PBS中洗條。接著將切片以Mayers蘇木精對比染色，以蓋玻片覆蓋且觀測。除首先將細胞粒化、冷凍且接著切片以外，使用相同實驗方案於OVCAR3細胞上進行 119007.doc -184- 200813092 IHC。接著使切片經受以上實驗方案。將結果定性報導於圖16中，表現程度分類為或” + ”。一般而言，在TAT226 mRNA表現程度與OVCAR3 細胞表面上之TAT226蛋白表現之間存在總體相關性。IHC 實驗亦確定識別細胞表面上之TAT226之抗體。卵巢癌細胞板中各細胞之組織學亦於圖16中報導，其中縮寫 ’’adenoca."表示 π腺癌”。 G.抗ΤΑΤ226 ADC之產生藉由將YWO.49.H2及YWO.49.H6接合至以下藥物-連接子部分而產生抗 ΤΑΤ226 ADC : MC-vc-PAB-MMAE、MC-vc-PAB-MMAF及 MC-MMAF，其在上文第 III部分 C.l.b.2 中描述。在接合之前，根據WO 2004/010957 A2中所述之方法，使用標準方法將抗體以TCEP部分還原。根據Doronina # Λ(2003) Nat. Biotechnol. 21:778-784 A US 2005/0238649 A1中所述之方法，使用標準方法將經部分還原之抗體接合至以上藥物-連接子部分。簡而言之，將經部分還原之抗體與藥物連接子部分組合以使得該等部分與半胱胺酸殘基接合。中止接合反應且純化ADC。藉由HPLC測定各ADC 之載藥率（每抗體之藥物部分平均數），如下： ADC 載藥率 YW0.49.H2-MC-vc-PAB-MMAE 3.8 YW0.49.H2-MC-vc-PAB-MMAF 4.7 YW0.49.H2-MC-MMAF 4.9 YW0.49.H6-MC-VC-PAB-MMAE 4.4 YW0.49.H6-MC-VC-PAB-MMAF 4.4 YW0.49.H6-MC-MMAF 4.1 119007.doc -185- 200813092 Η·細胞殺死檢定在以下活體外及活體内細胞殺死檢定中，測試抗體-藥物接合物（ADC)抑制表現ΤΑΤ226之細胞增殖之能力。 1. OVCAR3活體外細胞殺死檢定測試 YWO.49.H2及 YWO.49.H6 ADC抑制 OVCAR3 細胞增殖之能力。將OVCAR3細胞接種於具有20% FBS之RPMI中之96孔板中。如圖17所示，以不同濃度之ADC培育密度為每孔3000個細胞之OVCAR3細胞。將接合至MC-vc-PAB-]\41^八£之抗^41；(：16/0八125抗體用作正性對照。]^1；(：16/ CA125為已知卵巢癌抗原。參見例如Yin等人（2001) J. Biol. Chem. 276:27371-27375。將接合至 MC-vc-PAB-MMAE之抗IL-8抗體用作負性對照。培育5天後，根據製造商說明使用CellTiter-GloTM發光細胞生存力檢定（Promega， Madison，WI)量測細胞生存力。圖17中y軸上之刻度表示來自螢光素酶發光之相對光單位或’’RLU"，其為對於細胞生存力之量測。圖17展示，類似於正性對照，YW0.49.H2-MC-vc-PAB· MMAF 及 YW0.49.H6_MC-vc_PAB-MMAF 具有顯著細胞殺死活性，尤其在約0.01及0.1 pg/ml之濃度時更是如此。 YWO.49.H2-MC-VC-PAB-MMAE及 YW0.49.H6-MC-vc-PAB-MMAE亦具有細胞殺死活性，但其程度低於以YWO.49.H2-MC-vc-PAB-MMAF 及 YW0.49.H6-MC-VC-PAB-MMAF 所見之程度。YW0.49.H2-MC-vc-PAB_MMAF 及 YWO.49.H6-MC-vc-PAB-MMAF 之 IC5〇為約 0·005 nM，且 YWO.49.H2- 119007.doc -186- 200813092 MC-vc-PAB-MMAE 及 YW0.49.H6-MC-vc-PAB-MMAE 之 IC5〇為約 0.2 nM。游離 MMAE 之 IC5〇為約〇·1 nM。 YW0.49.H2-MC-MMAF及 YW0.49.H6-MC-MMAF在此檢定中不表現顯著之細胞殺死活性。在該特定檢定系統中，應注意由於MMAE及MMAF之總體高濃度，因此在高濃度之 ADC(包括負性對照ADC)下，細胞生存力大體上減低。 2.使用經HCT116轉染細胞之活體外細胞殺死檢定測試YWO.49.H2及YWO.49.H6 ADC抑制經編碼人類 TAT226之核苷酸穩定轉染之HCT116細胞（即結腸癌細胞株）增殖的能力。未經轉染之HCT116細胞通常對游離（未接合）MMAE之敏感性比對OVCAR3細胞低約5-6倍。簡而言之，HCT116細胞經如下轉染。在哺乳動物表現載體 pcDNA3.1(Invitrogen，Carlsbad，CA)中建構編碼經抗原決定基標記之人類TAT226的核苷酸。抗原決定基標記由單醇疱疹病毒1型糖蛋白D(”gD”標記）之胺基酸1-53組成，其於人類TAT226之N端置換來自胺基酸1-22之信號序列。根據製造商實驗方案，使用Lipofectamine200(Invitr*c)geii；) 將重組載體轉染至HCT116細胞中。將經轉染之HCT116細胞培養於具有10% FBS及0.4 mg/ml G418之McCoy氏5a培養基中。將細胞使用抗gD抗體染色且藉由FACS楝選以選擇表現重組gD:人類TAT226融合蛋白之個別純系。選擇命名為HCT116#9-4之純系之一以用於進一步分析。為進行細胞殺死檢定，將HCT116#9-4細胞接種於96孔板中。如圖18所示以不同濃度之ADC培育密度為每孔1〇〇〇 119007.doc -187- 200813092 個細胞之HCT116#9-4細胞。將接合至MC-vc-PAB-MMAE 之抗gp 120抗體用作負性對照。培育3天後，根據製造商說明使用CellTiter-GloTM發光細胞生存力檢定（Promega， Madison，WI)量測細胞生存力。圖 18展示 YW0.49.H2-MC-vc-PAB-MMAF及 YWO.49.H6-MC-vc-PAB-MMAF具有顯著細胞殺死活性，尤其在約0.01 pg/ml及高達所測試最高濃度時更是如此。YWO.49.H2-MC-vc_PAB-MMAF 及 YW0.49.H6-MC-vc-PAB-MMAF 之 IC5〇為約0·05 nM，且游離MMAE之IC5〇為約0·9 nM。在此特定檢定中，相對於負性對照，YW0.49.H2-MC-VC-PAB-MMAE 及 YW0.49.H6-MC_vc-PAB-MMAE 不具有實質性細胞殺死活性。與OVCAR3細胞殺死檢定（以上）相比，此檢定中 YW0.49.H2-MC-vc-PAB-MMAE與 YW0.49.H6-MC-vc-PAB-MMAE之細胞殺死活性之間的差異可歸因於各種因素，例如細胞密度及/或藥物敏感性之差異。應注意，對於所測試之通常表現TAT226 mRNA或蛋白之一些其他細胞株而言，YWO.49.H2及YWO.49.H6 ADC不展示顯著之細胞殺死活性。其可歸因於各種因素，例如細胞類型特異性效應、細胞表面TAT226表現程度及/或藥物敏感性差異。 3.使用HCT116#9-4異種移植物之活艘内檢定活體内異種移植物模型係用以測試未經接合及經接合 YWO.49.H6活體内抑制表現TAT226之腫瘤細胞增殖的能力。藉由向小鼠背側中經皮下注射約5 X 106 HCT116#9-4 119007.doc -188 - 200813092 細胞而於無胸腺裸"nu-nu"小鼠體内誘導腫瘤。使腫瘤生長直至其達到200 mm3之平均腫瘤體積。將此時間點指定為 ’’第0天”。如圖19所示，小鼠於第0天、第7天及第16天接收3 mg/kg之指定未經接合抗體或ADC的靜脈注射。將未經接合及經接合抗豚草抗體（Ab)用作負性對照。於第3 天、第7天、第10天、第16天及第21天量測平均腫瘤體積。如圖19所示，如以平均腫瘤體積所量測，在此特定異種移植物模型中與抗豚草Ab-MC-vc-PAB-MMAF相比， YWO.49.H6-MC-VC-PAB-MMAF展示顯著之腫瘤細胞殺死活性。在此異種移植物模型中，相對於抗豚草Ab-MC-vc-PAB-MMAE，YW0.49.H6-MC-vc-PAB-MMAE 不展示顯著之腫瘤細胞殺死活性。然而，由於各種因素，例如由於異種移植物腫瘤微環境中之細胞表面TAT226之藥物敏感性或表現程度的差異，此異種移植物模型不能反映在活體外所觀測之YW0.49.H6-MC_vc-PAB-MMAE之細胞殺死活性。 4.其他異種移植物模型其他異種移植物模型可用以測試未經接合及經接合抗 TAT226抗體活體内抑制表現TAT226之腫瘤細胞增殖的能力。舉例而言，卵巢及腦腫瘤之異種移植物模型可由諸如 Oncotest GmbH(Frieberg，Germany)及 Southern Research Institute (Birmingham, AL)之公開來源提供。詳言之， Oncotest模型係由在免疫缺陷裸小鼠體内生長患者腫瘤而形成。表現TAT226 mRNA及/或蛋白之異種移植物可適用於活體内表現抗TAT226抗體之細胞殺死活性。 119007.doc -189- 200813092 於Oncotest模型OVXF1023中測試經接合YWO.49.H6於活體内抑制卵巢腫瘤細胞增殖之能力。Oncotest模型 OVXF1 023係來源於癌轉移之不良分化乳頭狀漿液性腺瘤卵巢癌（Ml階段）。以圖20中所示之ADC處理OVXF1023小鼠。在圖20中，將YWO.49.H6命名為"H6";將抗豚草（對照）抗體命名為nRW"且將連接子_MC-vc-PAB-縮寫為”vc”。於圖20指定之日且以此濃度投予ADC。圖20中所示之結果指示相對於其他 ADC， YW0.49.H6_MC-vc-PAB-MMAF及 YW0.49.H6-MC-MMAF顯著降低腫瘤體積。在類似條件下，但改變劑量及對照ADC，以OVXF1023 重複上述實驗。在重複實驗中，以較高劑量（5 mg/kg)之 YW0.49.H6-MC-vc-PAB_MMAE 及較低劑量（5 mg/kg)之 YW0.49.H6_MC-vc-PAB-MMAF 及 YW0.49.H6-MC-MMAF 處理小鼠。結果表明H6 ADC(且尤其為YW0.49.H6-MC· vc-PAB-MMAE)展示相對於其各自之對照ADC(6.4 mg/kg之抗 gpl20_MC_vc-PAB-MMAE ; 7.2 mg/kg 之抗 gpl20_MC-vc-PAB-MMAF;及 5.4 mg/kg 之抗 gpl20-MC-MMAF)降低之腫瘤體積，儘管H6 ADC與其各自之對照ADC之間的功效差異對於一些資料點而言在統計學上不顯著。（資料未圖示）。該等結果與第一 0VXF1023實驗中所獲結果之間的差異可歸因於H6 ADC之劑量差異及所觀測到之對照抗gP120 ADC在降低腫瘤體積中展示意料之外之活性。此外，亦於另一 Oncotest模型OVXF899中測試H6 ADC，該模型係來源於經適度分化之乳頭狀漿液性印巢癌（原發 119007.doc •190- 200813092 性腫瘤）。在此模型中H6 ADC不降低腫瘤體積。（資料未圖示）。然而，此特定Oncotest模型展示TAT226之較低表現，其可說明所觀測之結果。 I. ThioMAb 產生經半胱胺酸工程設計之抗體或nthioMAbn，其中所選擇之YWO.49.H6殘基經半胱胺酸取代以提供用於接合連接子-藥物部分之額外位點。特定言之，在YWO.49.H6之重鏈中進行A118C取代（EU編號），或在YWO.49.H6之輕鏈中進行V205C取代（Kabat編號）。接著將所得A118C thioMAb接合至MC-MMAF，且接著將所得V205C thioMAb 接合至 MC-MMAF 或 MC-vc-PAB-MMAE。由 FACS 分析可見，所有thioMAb均可結合至OVCAR3細胞。（資料未圖示）。儘管為清楚理解之目的，已以說明及實例之方式詳細描述本發明，但不應將描述及實例理解為對本發明範疇之限制。本文所引用之所有專利及科學文獻之揭示内容全文以引用的方式明確併入本文。【圖式簡單說明】圖1展示來自人類、食蟹猴（ncynon)、小鼠及大鼠之TAT226 的對準。加陰影之殘基在各物種中相同。未加陰影之殘基於四個物種之至少兩者之間不同。來自人類、食蟹猴、小鼠及大鼠之TAT226序列之間之胺基酸一致性百分比係展示於對準下方之表中。使用ClustalW程式計算一致性百分比0 119007.doc -191 - 200813092 圖2展示如實例B中所述命名為YW0.32及YWO.49之抗 TAT226單株抗體之HI、H2及H3重鏈高變區（HVR)序列。根據如下所述之Kabat編號系統對胺基酸位置進行編號。圖3展示如實例B中所述命名為YWO.32及YW0.49之抗 TAT226單株抗體之LI、L2及L3輕鏈HVR序列。根據如下所述之Kabat編號系統對胺基酸位置進行編號。圖4展示如實例B中所述藉由使用HVR-H3及HVR-L3軟體隨機化庫對YW0.49進行親和力成熟而產生之YW0.49及 ( YWO.49.B7、YWO.49.C9、YWO.49.H2 及 YWO.49.H6 的 HVR-H3及HVR-L3序列。亦展示一致之HVR-H3及HVR-L3 序列。圖5 A及5B展示用於實踐本發明之具有如下序列識別符之例示性受體人類可變重（VH)—致框架序列： -人類VH子群I一致框架’’A”減去Kabat CDR(SEQ ID NO:32、33、34、35)。 -人類VH子群I一致框架、’fCn及nDn減去擴展之高變 V 區（SEQ ID NO:36、37、34、35 ; SEQ ID NO:36、 37、38、35 ;及 SEQ ID NO:36、37、39、35)。人類VH子群II一致框架減去Kabat CDR(SEQ ID NO:40、41、42、35)。 -人類VH子群II一致框架nB’’、”C”及減去擴展之高變區（SEQ ID NO:43、44、42、35 ; SEQ ID NO:43、 44、45、35 ;及 SEQ ID NO:43、44、46及 35)。The GeneExpress® database is also used to analyze normal ovaries; normal fallopian tubes; ovarian cancers of clear cells, mucinous and serous cystadenocarcinoma subtypes; and human TAT226 gene expression in metastatic ovarian cancer and other types of ovarian cancer. The results are reported in Figure 14, where a particular tissue type is represented below the figure. The scale on the y-axis in the graph indicates the degree of gene expression based on the intensity of the hybridization signal. Serous cystadenocarcinoma and metastatic ovarian cancer show a strong overexpression of TAT226 relative to normal ovaries. The expression of clear cells and mucinous subtypes is comparable to that of ovary 119007.doc -179· 200813092. The normal fallopian tube also shows the substantial performance of TAT226. It should be noted that the serous subtype of ovarian cancer is very similar to that of the fallopian tube epithelial tissue, and both the ovary and the fallopian tube are derived from the same embryonic tissue. See Fox et al. (2002) ''Pathology 〇f epithelial ovarian cancer,'' (9varz·(10) Cancer Chapter 9 (Jacobs et al., ed. Oxford University Press, New York). Β·Anti-TAT226 antibody production The TAT226 antibody was generated by screening the phage display library with the recombinant nTAT226_His" fusion protein labeled with amino acid 1·115 of SEQ ID NO: 75 and C-terminal polyhistidine. The phage display library was Fab'-zip- The synthetic (Fab') 2 library produced by the phage system. See Lee et al. (2004) J. /mmw(10)/.284:119-132. The library contains the huMAb4D5-8 heavy bond variable region (see Figures 5A and 5B, The two receptors "B", SEQ ID NO: 50, 51, 57, 3 5) are as light as 8 ugly (^1〇>^0:26 fixed 1111]\4 八54〇5-8 Heavy chain HVR library in the framework of the chain variable region. Use phage ELIS A for TAT226-His screening to display selected pure lines using phage (see, for example, Sidhu et al. (2004) J. Mo/· 5b/· 338:299 -310). Select pure lines YW0.32 and YWO.49 for further analysis. To improve the affinity of YW0.49, randomize the target software in the YW0.49 background. HVR-H3 and HVR-L3 produce a phage display library in which the selected amino acid residues in the designated HVR are kept constant while the other residues are subjected to mutations. The selected pure lines are screened by phage ELISA. Affinity matured antibodies of YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 were used for further analysis. As shown in Figures 9 and 10, 119007.doc • 180-200813092 YWO. 32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 in the VH and VL regions of the nucleotide and encoded polypeptide sequences. YWO.32, YWO The heavy and light chain HVR sequences of .49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 are shown in Figure 2-4. Derived from YWO.49, YWO.49. The HVR-H3 and HVR-L3 sequences of B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 are also shown in Figure 4. YWO.49, YWO.49.B7, YWO.49 .C9, YWO.49.H2 and YWO.49.H6 are "rearranged" to full length IgG by grafting the Fab' fragment to the appropriate constant region using recombinant techniques. The experiments described below were performed using rearranged antibodies. C. Characterization of Recombinant Antigen Binding Affinity YWO.49, YWO.49.B7, YWO.49.C9, YWO were determined by surface plasma resonance measurements using a BIACORE® 3000 system (Biacore, Inc., Piscataway, NJ). .49.H2 and YWO.49.H6 binding affinities for recombinant antigens. Briefly, carboxy is activated with ethyl w-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and iV-hydroxysuccinimide (NHS) according to the supplier's instructions. Methylated dextran biosensing wafer (CM5, BIAcore Inc). The anti-butabutan 226 antibody was diluted to 5 4 §/1111 with 卩114.8 of 1 1111 sodium acetate to achieve approximately 500 reaction units (RU) of the coupled antibody prior to injection at a flow rate of 5 μM per minute. Next, 1 Μ ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of TAT226_His (0.7 nM to 500 nM) were injected into PBS with 0.05% Tween 20 at 25 °C at a flow rate of 25 μΐ/min. The association rate (kQn) and the dissociation rate (koff) were calculated using a one-to-one Langmuir binding model (one-to-one Langmuir binding 119007.doc -181 - 200813092 model) (BIA evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio of koff/kQn. The results of this experiment are shown in Table 2 below. Table 2 Pure system k〇n/105 koff/10·4 Kd(nM) YW0.49 0.074 3.49 47.16 YWO.49.B7 0.27 <0.05 <0.18 YWO.49.C9 1.53 <0.05 <0.03 YWO.49 .H2 0.22 0.05 0.23 YWO.49.H6 1.86 0.09 0.05 D. Characterization of antibodies bound to cell surface TAT226 The ability of the anti-TAT226 antibody to bind to TAT226 expressed on the surface of 〇VCAR3 (human ovarian cancer cell line) was examined. Fluorescence activated cell sorting (FACS) was performed on 0VCAR3 cells in the presence and absence of YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6. Briefly, the isolated cells were incubated on ice for 5 hours with 5 pg/ml primary antibody, washed and incubated on ice for 30 minutes with a secondary antibody (conjugated to phycoerythrin anti-human IgG). FACS was performed using a FACScanTM flow cytometer (BD Biosciences, San Jose, CA). The FACS analysis results for YW0.49, YWO.49.H2 and YW0.H6 are shown in Figure 15. The peak on the left side of each figure indicates the "background' combination, i.e., only the binding of the secondary antibody. The peak on the right side of each figure indicates the binding of the designated anti-TAT226 antibody. As seen by FACS, YWO.49.B7 did not significantly bind to OVCAR3, even though it binds to 119007 within the Kd range observed for YW0.49 and other affinity matured antibodies (see results of BIAC0RE® analysis in Table 2 above) .doc • 182 - 200813092 Recombinant antigen. The combination of YWO.49.C9 may correspond to the combination observed for YWO.49.H2 and YWO.H6. E. Characterization of binding affinity to cell surface antigens The binding affinity of YWO.49.H2 and YWO.49.H6 for TAT226 expressed on the surface of OVCAR3 cells was tested using a competition assay. Briefly, labeled (iodinated) YWO.49.H2 or YWO.49.H6 was conjugated to OVCAR3 cells in the presence of unlabeled antibodies. The binding affinity of the antibodies was determined according to the Scatchard analysis methodology originally described in Munson et al., 4, 105, 220 (1980). The results of this experiment are shown in Table 3 below. Table 3 Pure Kd〇ftM) YWO.49.H2 0.348 YWO.49.H6 0.404 Compared with recombinant TAT226-His, YWO.49.H2 and YWO.49.H6 have higher Kd of TAT226 on the surface of OVCAR3 cells. (Compare KWO of YWO.49.H2 and YWO.49.H6 in Tables 2 and 3), this indicates that YWO.49.H2 and YWO.49.H6 are slightly higher than the TAT226 expressed on the surface of OVCAR3 cells. Affinity binds to recombinant TAT226-His. F· TAT226 mRNA and protein expression The expression of TAT226 mRNA in OVCAR3 cells and ovarian cancer sample plates was analyzed using 51 nuclease (TaqMan®) assay and real-time quantitative PCR. The ovarian cancer sample named "HF ####'' in Figure 16 is a frozen tissue section. RNA was isolated from tissue sections using Ambion's Message Amp II set 119007.doc -183 - 200813092 group (Ambion, Austin, TX) Amplification and reverse phase transcription into cDNA. RNA was isolated from OVCAR3 cells and reverse transcribed into cDNA. The TAT226 cDNA was amplified by real-time PCR in the presence of a non-extensible reporter probe specific for the amplification product. The threshold period or nCt" (the period during which the signal generated by the self-reported conductor probe cleavage exceeds the background value) is determined and used to calculate the initial TAT226 mRNA content. As shown in the bar graph of Figure 16, the TAT226 mRNA content in the ovarian cancer sample plate was expressed relative to the TAT226 mRNA content in the ARVC AR3 cells. Immunohistochemistry (IHC) was used to analyze the expression of TAT226 protein in OVCAR3 cells and the above ovarian cancer sample plates. The tissue sections of the nested cancer samples (frozen or embedded with papain) were fixed in acetone/ethanol for 5 minutes. The sections were washed in PBS, blocked with avidin and biotin (Vector Laboratories, Inc., Burlingame, CA) for 1 〇 minutes and washed again in PBS. Sections were then blocked with 10% serum for 20 minutes and washed dry to remove excess serum. The primary antibody (YWO.49.H2 or YWO.49.H6) was then added to the sections at a concentration of 10 pg/ml for 1 hour. The sections were then washed with PB S. Biotinylated secondary anti-human antibodies were added to the sections for 30 minutes and then the sections were washed with PBS. The sections were exposed to the Vector ABC kit (Vector Laboratories, Inc., Burlingame, CA) for 30 minutes and then washed in PBS. The sections were then exposed to diaminobenzidine (Pierce) for 5 minutes and then washed in PBS. The sections were then stained with Mayers hematoxylin, covered with coverslips and observed. The same experimental protocol was used to perform 119007.doc-184-200813092 IHC on OVCAR3 cells, except that the cells were first granulated, frozen, and sectioned. The sections were then subjected to the above experimental protocol. The results are qualitatively reported in Figure 16, and the degree of performance is classified as or "+". In general, there is an overall correlation between the extent of TAT226 mRNA expression and the performance of TAT226 protein on the surface of OVCAR3 cells. IHC experiments also identified antibodies that recognize TAT226 on the cell surface. The histology of each cell in the ovarian cancer cell plate is also reported in Figure 16, where the abbreviation ''adenoca." indicates π adenocarcinoma.) G. Anti-ΤΑΤ226 ADC is produced by YWO.49.H2 and YWO.49 .H6 is conjugated to the following drug-linker moiety to generate an anti-ΤΑΤ226 ADC: MC-vc-PAB-MMAE, MC-vc-PAB-MMAF and MC-MMAF, which are described in Section III, Clb2 above. Previously, antibodies were partially reduced in TCEP using standard methods according to the method described in WO 2004/010957 A2, according to Doronina # Λ (2003) Nat. Biotechnol. 21: 778-784 A US 2005/0238649 A1. Method, the partially reduced antibody is ligated to the above drug-linker moiety using standard methods. Briefly, the partially reduced antibody is combined with a drug linker moiety such that the portions are joined to a cysteine residue The ligation reaction was stopped and the ADC was purified. The drug loading rate of each ADC (the average number of drug parts per antibody) was determined by HPLC as follows: ADC drug loading rate YW0.49.H2-MC-vc-PAB-MMAE 3.8 YW0. 49.H2-MC-vc-PAB-MMAF 4.7 YW0.49.H2-MC-MMAF 4.9 YW0.49.H6-MC-VC-PAB-MMAE 4 .4 YW0.49.H6-MC-VC-PAB-MMAF 4.4 YW0.49.H6-MC-MMAF 4.1 119007.doc -185- 200813092 Η·cell killing assay in the following in vitro and in vivo cell killing assay Among them, the test antibody-drug conjugate (ADC) inhibits the ability to express cell proliferation of ΤΑΤ226. 1. OVCAR3 in vitro cell kill assay The ability of YWO.49.H2 and YWO.49.H6 ADC to inhibit OVCAR3 cell proliferation. OVCAR3 cells were seeded in 96-well plates in RPMI with 20% FBS. As shown in Figure 17, OVCAR3 cells with a density of 3000 cells per well were grown at different concentrations of ADC. Will be ligated to MC-vc-PAB-] \41^八£之抗^41;(:16/0八125 antibody was used as a positive control.]^1; (:16/CA125 is a known ovarian cancer antigen. See, for example, Yin et al. (2001) J. Biol. Chem. 276: 27371-27375. Anti-IL-8 antibody conjugated to MC-vc-PAB-MMAE was used as a negative control. After 5 days of incubation, CellTiter-GloTM luminescent cell viability assay was used according to the manufacturer's instructions. (Promega, Madison, WI) measures cell viability. The scale on the y-axis in Figure 17 represents the relative light unit or ‘'RLU" from luciferase luminescence, which is a measure of cell viability. Figure 17 shows that, similar to the positive control, YW0.49.H2-MC-vc-PAB· MMAF and YW0.49.H6_MC-vc_PAB-MMAF have significant cell killing activity, especially at about 0.01 and 0.1 pg/ml. This is especially true at concentrations. YWO.49.H2-MC-VC-PAB-MMAE and YW0.49.H6-MC-vc-PAB-MMAE also have cell killing activity, but the degree is lower than YWO.49.H2-MC-vc- The extent of PAB-MMAF and YW0.49.H6-MC-VC-PAB-MMAF. The IC5〇 of YW0.49.H2-MC-vc-PAB_MMAF and YWO.49.H6-MC-vc-PAB-MMAF is about 0·005 nM, and YWO.49.H2- 119007.doc -186- 200813092 MC The IC5 of -vc-PAB-MMAE and YW0.49.H6-MC-vc-PAB-MMAE is about 0.2 nM. The IC5〇 of the free MMAE is about 〇·1 nM. YW0.49.H2-MC-MMAF and YW0.49.H6-MC-MMAF did not show significant cell killing activity in this assay. In this particular assay system, it should be noted that due to the overall high concentration of MMAE and MMAF, cell viability is substantially reduced at high concentrations of ADCs (including negative control ADCs). 2. In vitro cell killing assay using HCT116 transfected cells YWO.49.H2 and YWO.49.H6 ADC inhibits HCT116 cells stably transfected with nucleotides encoding human TAT226 (ie colon cancer cell line) The ability to proliferate. Untransfected HCT116 cells are generally about 5-6 fold less sensitive to free (unbound) MMAE than to OVCAR3 cells. Briefly, HCT116 cells were transfected as follows. Nucleotides encoding human TAT226 labeled with an antigen determinant were constructed in the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The epitope tag consists of the amino acid 1-53 labeled with the monoclonal antibody herpesvirus type 1 glycoprotein D ("gD"), which replaces the signal sequence from the amino acid 1-22 at the N-terminus of human TAT226. The recombinant vector was transfected into HCT116 cells using Lipofectamine 200 (Invitr*c) geii;) according to the manufacturer's protocol. Transfected HCT116 cells were cultured in McCoy's 5a medium with 10% FBS and 0.4 mg/ml G418. Cells were stained with anti-gD antibody and selected by FACS to select individual lines that express recombinant gD: human TAT226 fusion protein. One of the pure lines named HCT116#9-4 was selected for further analysis. For cell killing assay, HCT116#9-4 cells were seeded in 96-well plates. As shown in Fig. 18, HCT116#9-4 cells of 1 〇〇〇 119007.doc -187-200813092 cells per well were cultured at different concentrations of ADC. An anti-gp 120 antibody conjugated to MC-vc-PAB-MMAE was used as a negative control. After 3 days of incubation, cell viability was measured using the CellTiter-GloTM Luminescent Cell Viability Assay (Promega, Madison, WI) according to the manufacturer's instructions. Figure 18 shows that YW0.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF have significant cell killing activity, especially at about 0.01 pg/ml and up to the highest concentration tested. This is especially true. The IC5〇 of YWO.49.H2-MC-vc_PAB-MMAF and YW0.49.H6-MC-vc-PAB-MMAF is about 0·05 nM, and the IC5〇 of free MMAE is about 0·9 nM. In this particular assay, YW0.49.H2-MC-VC-PAB-MMAE and YW0.49.H6-MC_vc-PAB-MMAE did not have substantial cell killing activity relative to the negative control. Between the cell killing activity of YW0.49.H2-MC-vc-PAB-MMAE and YW0.49.H6-MC-vc-PAB-MMAE in this assay compared to the OVCAR3 cell kill assay (above) The difference can be attributed to various factors such as differences in cell density and/or drug sensitivity. It should be noted that YWO.49.H2 and YWO.49.H6 ADCs do not exhibit significant cell killing activity for some other cell lines tested that typically exhibit TAT226 mRNA or protein. It can be attributed to various factors such as cell type-specific effects, degree of cell surface TAT226 expression, and/or differences in drug sensitivity. 3. In vivo assay using HCT116#9-4 xenografts The in vivo xenograft model was used to test the ability of unconjugated and ligated YWO.49.H6 to inhibit tumor cell proliferation in TAT226 in vivo. Tumors were induced in athymic nude "nu-nu" mice by subcutaneous injection of about 5 X 106 HCT116 #9-4 119007.doc -188 - 200813092 cells into the dorsal side of the mice. The tumor was allowed to grow until it reached an average tumor volume of 200 mm3. This time point was designated as ''Day 0.') As shown in Figure 19, mice received an intravenous injection of 3 mg/kg of the designated unconjugated antibody or ADC on Days 0, 7, and 16. Unconjugated and conjugated anti-ragweed antibodies (Ab) were used as negative controls. Mean tumor volumes were measured on days 3, 7, 10, 16 and 21. As shown in Figure 19 In the specific xenograft model, YWO.49.H6-MC-VC-PAB-MMAF showed significant results compared to the anti-ragweed Ab-MC-vc-PAB-MMAF, as measured by the mean tumor volume. Tumor cell killing activity. In this xenograft model, YW0.49.H6-MC-vc-PAB-MMAE does not display significant tumor cell killing relative to anti-ragweed Ab-MC-vc-PAB-MMAE Dead activity. However, due to various factors, such as differences in the drug sensitivity or degree of expression of the cell surface TAT226 in the xenograft tumor microenvironment, this xenograft model cannot be reflected in the YW0.49.H6 observed in vitro. - MC_vc-PAB-MMAE cell killing activity 4. Other xenograft models Other xenograft models can be used to test unjoined and ligated The anti-TAT226 antibody inhibits the ability of tumor cells expressing TAT226 to proliferate in vivo. For example, xenograft models of ovarian and brain tumors can be provided by public sources such as Oncotest GmbH (Frieberg, Germany) and Southern Research Institute (Birmingham, AL). In particular, the Oncotest model is formed by growing a tumor in a patient with immunodeficient nude mice. Xenografts exhibiting TAT226 mRNA and/or protein can be used to express the cell killing activity of the anti-TAT226 antibody in vivo. .doc -189- 200813092 The ability of conjugated YWO.49.H6 to inhibit ovarian tumor cell proliferation in vivo was tested in the Oncotest model OVXF1023. The Oncotest model OVXF1 023 was derived from poorly differentiated papillary serous adenoma ovarian cancer with metastasis ( Ml phase). OVXF1023 mice were treated with the ADC shown in Figure 20. In Figure 20, YWO.49.H6 was named "H6"; the anti-ragweed (control) antibody was named nRW" Sub_MC-vc-PAB- is abbreviated as "vc". The ADC is administered at this concentration on the date specified in Figure 20. The results shown in Figure 20 indicate relative to other ADCs. YW0.49.H6_MC-vc-PAB-MMAF and YW0.49.H6-MC-MMAF significantly reduced tumor volume. The above experiment was repeated with OVXF1023 under similar conditions, but with varying doses and control ADC. In repeated experiments, higher dose (5 mg/kg) of YW0.49.H6-MC-vc-PAB_MMAE and lower dose (5 mg/kg) of YW0.49.H6_MC-vc-PAB-MMAF and Mice were treated with YW0.49.H6-MC-MMAF. The results indicate that the H6 ADC (and especially YW0.49.H6-MC· vc-PAB-MMAE) showed anti-gpl20_MC against 7.2 mg/kg of anti-gpl20_MC_vc-PAB-MMAE compared to its respective control ADC (6.4 mg/kg; -vc-PAB-MMAF; and 5.4 mg/kg anti-gpl20-MC-MMAF) reduced tumor volume, although the difference in efficacy between the H6 ADC and its respective control ADC was statistically insignificant for some data points . (The information is not shown). The difference between these results and the results obtained in the first 0VXF1023 experiment can be attributed to the dose difference of the H6 ADC and the observed anti-gP120 ADC exhibiting unexpected activity in reducing tumor volume. In addition, H6 ADC was also tested in another Oncotest model, OVXF899, which was derived from moderately differentiated papillary serous colon cancer (primary 119007.doc •190-200813092 tumor). The H6 ADC does not reduce tumor volume in this model. (The information is not shown). However, this particular Oncotest model demonstrates the lower performance of TAT226, which illustrates the observed results. I. ThioMAb produces a cysteine engineered antibody or nthioMAbn in which the selected YWO.49.H6 residue is substituted with cysteine to provide additional sites for conjugating the linker-drug moiety. Specifically, the A118C substitution (EU numbering) is carried out in the heavy chain of YWO.49.H6, or the V205C substitution (Kabat numbering) is carried out in the light chain of YWO.49.H6. The resulting A118C thioMAb was then ligated to MC-MMAF and the resulting V205C thioMAb was then ligated to MC-MMAF or MC-vc-PAB-MMAE. As seen by FACS analysis, all thioMAbs were able to bind to OVCAR3 cells. (The information is not shown). The present invention has been described in detail by way of illustration and example, and the description The disclosures of all of the patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the alignment of TAT226 from humans, cynomolgus monkeys, mice and rats. The shaded residues are identical in each species. Unshaded residues differ between at least two of the four species. The percent amino acid identity between the TAT226 sequences from humans, cynomolgus monkeys, mice and rats is shown in the table below. Percent identity was calculated using the ClustalW program. 119007.doc -191 - 200813092 Figure 2 shows the HI, H2 and H3 heavy chain hypervariable regions of the anti-TAT226 monoclonal antibodies designated YW0.32 and YWO.49 as described in Example B. (HVR) sequence. The amino acid positions are numbered according to the Kabat numbering system as described below. Figure 3 shows the LI, L2 and L3 light chain HVR sequences of the anti-TAT226 monoclonal antibodies designated YWO.32 and YW0.49 as described in Example B. The amino acid positions are numbered according to the Kabat numbering system as described below. Figure 4 shows YW0.49 and (YWO.49.B7, YWO.49.C9, produced by affinity maturation of YW0.49 using the HVR-H3 and HVR-L3 software randomization libraries as described in Example B. HVR-H3 and HVR-L3 sequences of YWO.49.H2 and YWO.49.H6. Also shown are consistent HVR-H3 and HVR-L3 sequences. Figures 5A and 5B show the following sequence recognition for practicing the present invention. Exemplary Receptor Human Variable Weight (VH)-Oriented Framework Sequence: - Human VH Subgroup I Consistent Framework ''A' minus Kabat CDRs (SEQ ID NO: 32, 33, 34, 35) - Human VH subgroup I consensus framework, 'fCn and nDn minus extended hypervariable V regions (SEQ ID NO: 36, 37, 34, 35; SEQ ID NO: 36, 37, 38, 35; and SEQ ID NO: 36 , 37, 39, 35). Human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NO: 40, 41, 42, 35) - Human VH subgroup II consensus framework nB'', "C" and subtraction Unexpanded hypervariable regions (SEQ ID NO: 43, 44, 42, 35; SEQ ID NO: 43, 44, 45, 35; and SEQ ID NO: 43, 44, 46 and 35).

-人類VH子群III一致框架減去Kabat CDR(SEQ ID 119007.doc -192- 200813092 NO:47、48、49、35)。 -人類VH子群III一致框架”B”、”Cn&’’D”減去擴展之高變區（SEQ ID NO:50、51、49、35 ; SEQ ID NO:50、 51、52、35 ;及 SEQ ID NO:50、51、53、35) 〇 -人類 VH 受體框架” An 減去 Kabat CDR(SEQ ID NO:54、 48、55、35) ° -人類VH受體框架nB"及’’C”減去擴展之高變區（SEQ ID NO:50、51、55、35 ;及 SEQ ID NO:50、51、56、 35) 〇 -人類VH受體2框架”An減去Kabat CDR(SEQ ID NO:54、48、57、35) 〇人類VH受體2框架"Bn、f’C”及nDn減去擴展之高變區 (SEQ ID NO:50、51、57、35 ; SEQ ID NO:50、51、 58、35 ;及 SEQ ID NO:50、51、59、35)。圖6A及6B展示用於實踐本發明之具有如下序列識別符之例示性受體人類可變輕（VL)—致框架序列： -人類 VLk子群 I一致框架（κνί) : SEQ ID ΝΟ··60、61、 62 、 63 ° -人類 VLk子群 II一致框架（κν2) : SEQ ID ΝΟ:64、65、 66 λ 63 〇 •人類 VLk 子群 III 一致框架（κν3) : SEQ ID ΝΟ:67、68、 69 ' 63 ° -人類 VLk 子群 IV— 致框架（κν4) : SEQ ID NO: 70、71、 72 、 63 ° 119007.doc -193 - 200813092 圖7展示huMAb4D5-8輕鏈及重鏈之框架序列。上標/粗體之數字表示根據Kabat之胺基酸位置。圖8展示具有指定修飾之huMAb4D5-8輕鏈及重鏈框架序列。上標/粗體之數字表示根據Kabat之胺基酸位置。圖 9展示 YWO.32、YW0.49、YWO.49.B7、YWO.49.C9、 YWO.49.H2及YWO.49.H6之重鏈可變區（VH)序列。HVR係加下劃線的。圖 10展示 YW0.32、YW0.49、YWO.49.B7、YWO.49.C9、 YWO.49.H2及YWO.49.H6之輕鏈可變區（VL)序列。人化單株抗體 4D5-8("huMAb4D5-8n)及，，經修飾，·ΙηιΜΑΒ405-8 之 VL序列亦分別展示於SEQ ID NOJ1及SEQ ID ΝΟ:26中。 YW0.32 及 YWO.49 具有與"經修飾"huMAb4D5-8 VL(SEQ ID NO:26)相同之VL序列，其含有與SEQ ID NO:31相關之以下取代：N30S、R66G及H91S。HVR係加下劃線的。圖 11 展示 YW0.32、YWO.49、YWO.49.B7、YWO.49.C9、 YWO.49.H2及YWO.49.H6之重鏈可變區序列之對準。HVR 係括入框中。對不同於YW0.49之HVR-H3對應殘基之 YWO.49.B7、YWO.49.C9、YWO.49.H2 及 YWO.49.H6 之 HVR-H3殘基加陰影。圖 12展示 YWO.32、YWO.49、YWO.49.B7、YWO.49.C9、 YWO.49.H2及YWO.49.H6之輕鏈可變區序列之對準。HVR 係括入框中。對不同於YW0.49之HVR-L3對應殘基之 YWO.49.B7、YWO.49.C9、YWO.49.H2 及 YWO.49.H6 之 HVR-L3殘基加陰影。 119007.doc -194- 200813092 圖13展示如實例A中所述人類TAT226基因在各種組織中之表現程度的圖示。圖14展示如實例A中所述，人類TAT226基因在正常卵巢；正常輸卵管；透明細胞、黏液性及漿液性囊腺癌亞型之卵巢癌；轉移性卵巢癌及其他類型卵巢癌中之表現程度的圖示。圖15展示如實例D中所述’在存在或不存在指定抗 TAT226抗體下，OVCAR3細胞之螢光活化細胞揀選（FACS) 結果。圖16展示如實例F中所述，如藉由對OVCAR3細胞及卵巢癌樣本板進行之5’核酸酶（TaqMan®)檢定及免疫組織化學（IHC)所確定之TAT226 mRNA及蛋白表現。圖17展示如實例Η中所述，各種YWO.49.H2及YWO.49.H6 抗體-藥物接合物（ADC)於OVCAR3細胞殺死檢定中之活體外活性。圖18展示如實例Η中所述，各種YWO.49.H2及YW0.49.H6 ADC在使用HCT116#9-4穩定轉染物之細胞殺死檢定中之活體外活性。圖19展示如實例Η中所述，使用小鼠異種移植物之 YWO.49.H6 ADC之活體内活性。圖20展示如實例Η中所述，使用衍生自人類患者腫瘤之小鼠異種移植物之YWO.49.H6 ADC的活體内活性。 119007.doc -195- 200813092 序列表 <11〇>美商建南德克公司 <120>抗丁八1226抗體及免疫接合物 <130> P2324R1 <140 096109168 <141> 2007-03-17 <150> US 60/783,746 <151〉 2006-03-17 <160〉 78 <210〉 1 <211〉 10 <212〉 PRT <213〉人工序列 <220〉- Human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID 119007.doc -192 - 200813092 NO: 47, 48, 49, 35). - Human VH subgroup III consensus framework "B", "Cn&"D" minus extended hypervariable regions (SEQ ID NO: 50, 51, 49, 35; SEQ ID NO: 50, 51, 52, 35 And SEQ ID NO: 50, 51, 53, 35) 〇-human VH receptor framework" An minus Kabat CDRs (SEQ ID NO: 54, 48, 55, 35) ° - human VH receptor framework nB" ''C' minus the extended hypervariable region (SEQ ID NO: 50, 51, 55, 35; and SEQ ID NO: 50, 51, 56, 35) 〇-human VH receptor 2 framework" An minus Kabat CDRs (SEQ ID NO: 54, 48, 57, 35) 〇 Human VH Receptor 2 Framework "Bn, f'C" and nDn minus extended hypervariable regions (SEQ ID NOs: 50, 51, 57, 35) SEQ ID NOs: 50, 51, 58, 35; and SEQ ID NO: 50, 51, 59, 35). Figures 6A and 6B show exemplary receptor human variable light (VL)-induced framework sequences having the following sequence identifiers for practicing the invention: - Human VLk subgroup I consensus framework (κνί): SEQ ID ΝΟ·· 60, 61, 62, 63 ° - human VLk subgroup II consensus framework (κν2): SEQ ID ΝΟ: 64, 65, 66 λ 63 〇 • human VLk subgroup III consensus framework (κν3) : SEQ ID ΝΟ: 67, 68, 69 ' 63 ° - human VLk subgroup IV - framework (κν4) : SEQ ID NO: 70, 71, 72, 63 ° 119007.doc -193 - 200813092 Figure 7 shows huMAb4D5-8 light and heavy chains Frame sequence. The superscript/bold numbers indicate the position of the amino acid according to Kabat. Figure 8 shows the huMAb4D5-8 light and heavy chain framework sequences with the indicated modifications. The superscript/bold numbers indicate the position of the amino acid according to Kabat. Figure 9 shows the heavy chain variable region (VH) sequences of YWO.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The HVR system is underlined. Figure 10 shows the light chain variable region (VL) sequences of YW0.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The VL sequences of humanized monoclonal antibody 4D5-8 ("huMAb4D5-8n) and, modified, ΙηιΜΑΒ405-8 are also shown in SEQ ID NOJ1 and SEQ ID ΝΟ:26, respectively. YW0.32 and YWO.49 have the same VL sequence as "modified" huMAb4D5-8 VL (SEQ ID NO: 26), which contains the following substitutions associated with SEQ ID NO: 31: N30S, R66G and H91S. The HVR is underlined. Figure 11 shows the alignment of the heavy chain variable region sequences of YW0.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The HVR is enclosed in the box. The HVR-H3 residues of YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, which are different from the corresponding residues of HVR-H3 of YW0.49, are shaded. Figure 12 shows the alignment of the light chain variable region sequences of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The HVR is enclosed in the box. The HVR-L3 residues of YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, which are different from the corresponding residues of HVR-L3 of YW0.49, are shaded. 119007.doc -194- 200813092 Figure 13 shows a graphical representation of the extent of performance of the human TAT226 gene as described in Example A in various tissues. Figure 14 shows the degree of expression of the human TAT226 gene in normal ovaries; normal fallopian tubes; ovarian cancers of clear cells, mucinous and serous cystadenocarcinoma subtypes; metastatic ovarian cancer and other types of ovarian cancer, as described in Example A. Icon. Figure 15 shows the results of fluorescent activated cell sorting (FACS) of OVCAR3 cells in the presence or absence of the designated anti-TAT226 antibody as described in Example D. Figure 16 shows TAT226 mRNA and protein expression as determined by 5' nuclease (TaqMan®) assay and immunohistochemistry (IHC) on OVCAR3 cells and ovarian cancer sample plates as described in Example F. Figure 17 shows the in vitro activity of various YWO.49.H2 and YWO.49.H6 antibody-drug conjugates (ADCs) in the OVCAR3 cell kill assay as described in the Examples. Figure 18 shows the in vitro activity of various YWO.49.H2 and YW0.49.H6 ADCs in a cell kill assay using HCT116 #9-4 stable transfectants as described in the Examples. Figure 19 shows the in vivo activity of YWO.49.H6 ADC using mouse xenografts as described in the Examples. Figure 20 shows the in vivo activity of a YWO.49.H6 ADC using mouse xenografts derived from human patient tumors as described in the Examples. 119007.doc -195- 200813092 SEQUENCE LISTING <11〇>US----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 03-17 <150> US 60/783,746 <151> 2006-03-17 <160> 78 <210> 1 <211> 10 <212> PRT <213>Artificial sequence <220>

<223> HVR-H1 <400> 1<223> HVR-H1 <400> 1

Gly Phe Thr lie Ser Ser Ser Ser lie His 5 10 <210〉 2 <211〉 18 <212〉 PRT <213〉人工序列 <220〉 <223> HVR-H2 <400> 2Gly Phe Thr lie Ser Ser Ser Ser lie His 5 10 <210> 2 <211> 18 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > HVR - H2 < 400 > 2

Ala Arg He Thr Pro Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser 15 10 15Ala Arg He Thr Pro Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser 15 10 15

Val Lys Gly <210> 3 <211〉 13 <212〉 PRT <213〉人工序列 <220〉 <223> HVR-H3 <400> 3Val Lys Gly <210> 3 <211> 13 <212> PRT < 213 > 213 > artificial sequence <220><223> HVR-H3 <400>

Cys lie Leu Cys Phe Gly Pro Leu Trp Ala Met Asp Tyr 5 10 <210> 4 <211〉 10 <212〉 PRT <213〉人工序列 <220〉 <223〉 HVR-H1 <400> 4Cys lie Leu Cys Phe Gly Pro Leu Trp Ala Met Asp Tyr 5 10 <210> 4 <211> 10 <212> PRT <213>Artificial Sequence <220> <223> HVR-H1 <400&gt ; 4

Gly Phe Thr He Thr Asn Tyr Gly lie His 5 10 <210> 5 <211> 18 <212〉 PRT <213〉人工序列 <220〉 <223〉 HVR-H2 119007-序列表.doc 200813092 <400> 5Gly Phe Thr He Thr Asn Tyr Gly lie His 5 10 <210> 5 <211> 18 <212> PRT <213>Artificial sequence <220><223> HVR-H2 119007 - Sequence Listing.doc 200813092 <400> 5

Gly Arg lie Tyr Pro Asp Ser Gly Ala Thr Tyr Tyr Ala Asp Ser 15 10 15Gly Arg lie Tyr Pro Asp Ser Gly Ala Thr Tyr Tyr Ala Asp Ser 15 10 15

Val Lys Gly <210〉 6 <211〉 11 <212〉 PRT <213〉人工序列 <220〉 <223> HVR-H3 <400> 6Val Lys Gly <210> 6 <211> 11 <212> PRT < 213 > 213 > artificial sequence <220><223> HVR-H3 <400>

Lys Leu Trp Val Ser Arg Ala Gly Met Asp Tyr 5 10 <210> 7 <211〉 11 <212〉 PRT <213〉人工序列 <220> <223〉 HVR-H3 <400〉 7Lys Leu Trp Val Ser Arg Ala Gly Met Asp Tyr 5 10 <210> 7 <211> 11 <212> PRT <213>Artificial Sequence <220><223> HVR-H3 <400〉 7

Lys Leu Trp Val Ser Leu Ala Ala Met Asp Tyr 5 10 <210〉 8 <211〉 11 <212〉 PRT <213〉人工序列 <220> <223〉 HVR-H3 <400〉 8Lys Leu Trp Val Ser Leu Ala Ala Met Asp Tyr 5 10 <210〉 8 <211> 11 <212> PRT <213>Artificial Sequence <220><223> HVR-H3 <400> 8

Lys Leu Trp Val Thr Leu Ala Ser Met Asp Tyr 5 10 <210〉 9 <211〉 11 <212〉 PRT <213〉人工序列 <220> <223> HVR-H3 <400> 9Lys Leu Trp Val Thr Leu Ala Ser Met Asp Tyr 5 10 <210> 9 <211> 11 <212> PRT <213>Artificial Sequence <220><223> HVR-H3 <400>

Lys Leu Trp Val Ser Leu Ala Pro Met Asp Tyr 5 10 <210〉 10 <211〉 11 <212〉 PRT <213〉人工序列 <220〉 <223> HVR-H3 <400> 10Lys Leu Trp Val Ser Leu Ala Pro Met Asp Tyr 5 10 <210> 10 <211> 11 <212> PRT <213>Artificial Sequence <220〉 <223> HVR-H3 <400>

Lys Leu Trp lie Ser He Ala Gly Met Asp Tyr 5 10 <210> 11 <211〉 11 <212〉 PRT <213〉人工序列 <220〉〈223>HVR-H3—致序列 -2- 119007-序列表.doc 200813092 <220〉 <221〉 Other <222> 4 <223>Xaa:Val 或lie <220> <221〉 Other <222〉 5 <223〉Xaa=Ser 或Thr <220〉 <221〉 Other <222> 6 <223〉Xaa=Arg、Leu或He <220> <221> Other <222〉 8 <223>xaa=Gly、Ala、Ser或Pro <400> 11Lys Leu Trp lie Ser He Ala Gly Met Asp Tyr 5 10 <210> 11 <211> 11 <212> PRT <213>Artificial sequence <220> <223> HVR-H3- 119007 - Sequence Listing.doc 200813092 <220> <221> Other <222> 4 <223>Xaa:Val or lie <220><221<221> Other <222> 5 <223>Xaa= Ser or Thr <220> <221> Other <222> 6 <223>Xaa=Arg, Leu or He <220><221> Other <222> 8 <223>xaa=Gly, Ala, Ser or Pro <400> 11

Lys Leu Trp Xaa Xaa Xaa Ala Xaa Met Asp Tyr 5 10Lys Leu Trp Xaa Xaa Xaa Ala Xaa Met Asp Tyr 5 10

<210〉 12 <211〉 11 <212〉 PRT <213〉人工序列 <220〉 <223〉 HVR-L1 <400〉 12<210> 12 <211> 11 <212> PRT <213>Artificial sequence <220〉 <223> HVR-L1 <400> 12

Arg Ala Ser Gin Asp Val Ser Thr Ala Val Ala 5 10 <2I0> 13 <211〉 7 <212> PRT <213〉人工序列 <220〉 <223> HVR-L2 <400> 13Arg Ala Ser Gin Asp Val Ser Thr Ala Val Ala 5 10 <2I0> 13 <211> 7 <212> PRT < 213 > 213 ><220><223> HVR-L2 <400>

Ser Ala Ser Phe Leu Tyr Ser <210〉 14 / <211〉 9Ser Ala Ser Phe Leu Tyr Ser <210> 14 / <211〉 9

\ <212> PRT <213〉人工序列 <220〉 <223> HVR-L3 <400> 14\ <212> PRT <213>Artificial Sequence <220〉 <223> HVR-L3 <400> 14

Gin Gin Ser Tyr Thr Thr Pro Pro Thr <210〉 15 <211〉 9 <212〉 PRT <213〉人工序列 <220〉 <223> HVR-L3 <400> 15Gin Gin Ser Tyr Thr Thr Pro Pro Thr <210> 15 <211> 9 <212> PRT < 213 > 213 > Artificial Sequence <220 < 223 > HVR - L3 < 400 > 15

Gin Arg Ser Val Phe Thr Pro Pro Ala <210> 16 <211> 9 119007-序列表.doc 200813092 <212> PRT <213〉入工序列 <220> <223> HVR-L3 <400〉 16Gin Arg Ser Val Phe Thr Pro Pro Ala <210> 16 <211> 9 119007 - Sequence Listing.doc 200813092 <212> PRT <213> Entry Sequence <220><223> HVR-L3 <;400> 16

Gin Lys Ser Tyr Asn Thr Pro Pro Thr <210> 17 <211> 9 <212> PRT <213〉人工序列 <220> <223> HVR-L3 <400> 17Gin Lys Ser Tyr Asn Thr Pro Pro Thr <210> 17 <211> 9 <212> PRT <213>Artificial Sequence <220><223> HVR-L3 <400>

Gin Gin Ser Tyr Gly Thr Pro Phe lieGin Gin Ser Tyr Gly Thr Pro Phe lie

<210〉 18 <211> 9 <212> PRT f <213>A工序列 \ <220〉 <223> HVR-L3 <400> 18<210> 18 <211> 9 <212> PRT f <213> A work sequence \ <220><223> HVR-L3 <400>

Gin His Ser Tyr Ala Thr Pro Phe Thr <210> 19 <211> 9 <212> PRT <213〉人工序列 <223〉HVR-L3—致序列 <220> <221〉 Other <222> 2 <223〉文aa=Arg、Lys、His、Asn或Gin <220> <221> Other <222> 4 <223>Xaa=Tyr 或 Val <220> <221〉 Other <222> 5 ^ <223〉Xaa=Thr、Phe、Asn、Gly或Ala <220> <221〉 Other <222> 8 、 <223〉Xaa=Pro 或Phe <220〉 <221> Other <222> 9 <223>Xaa=Thr ' lie或Ala <400> 19Gin His Ser Tyr Ala Thr Pro Phe Thr <210> 19 <211> 9 <212> PRT < 213 > 213 > 213 > 223 > 223 > 223 > HVR - L3 - Sequence <220 ><221 > Other &lt ;222> 2 <223> text aa=Arg, Lys, His, Asn, or Gin <220><221> Other <222> 4 <223> Xaa=Tyr or Val <220><221 〉 Other <222> 5 ^ <223>Xaa=Thr, Phe, Asn, Gly or Ala <220><221> Other <222> 8, <223>Xaa=Pro or Phe <220 〉 <221> Other <222> 9 <223>Xaa=Thr 'lie or Ala <400> 19

Gin Xaa Ser Xaa Xaa Thr Pro Xaa Xaa <210> 20 <211〉 122 <212> PRT <213〉人工序列 119007-序列表.doc 200813092 <220> <223〉YWa32之重鏈可變區 <400> 2020in lt;211> 122 <212&gt Variant <400> 20

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

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

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

Glu Trp Val Ala Arg lie Thr Pro Ser Asp Gly Thr Thr Tyr Tyr 50 55 60Glu Trp Val Ala Arg lie Thr Pro Ser Asp Gly Thr Thr 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 Cys lie Leu Cys Phe Gly Pro 95 100 105Thr Ala Val Tyr Tyr Cys Ala Arg Cys lie Leu Cys Phe Gly Pro 95 100 105

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

Ser Ser <210〉 21 <211〉 120 <212〉 PRT <213〉人工序列 <223〉YW0.49之重鏈可變區 <400> 21Ser Ser <210> 21 <211> 120 <212> PRT <213>Artificial sequence <223>YW0.49 heavy chain variable region <400> 21

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

Asn Tyr Gly He His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 35 40 45Asn Tyr Gly He His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 35 40 45

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

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

Thr Ala Val Tyr Tyr Cys Ala Arg Lys Leu Trp Val Ser Arg Ala 95 100 105Thr Ala Val Tyr Tyr Cys Ala Arg Lys Leu Trp Val Ser Arg Ala 95 100 105

Gly Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210> 22 <211> 120 <212〉 PRT <213〉人工序列 <220> <223〉YWO.49.B7之重鏈可變區 <400> 22Gly Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210> 22 <211> 120 <212> PRT <213>Artificial Sequence <220><223>YWO.49 .B7 heavy chain variable region <400> 22

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 119007·序列表.doc 200813092 15 10 15Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 119007 · Sequence Listing.doc 200813092 15 10 15

Thr Ala Val Tyr Tyr Cys Ala Arg Lys Leu Trp Val Ser Leu Ala 95 100 105Thr Ala Val Tyr Tyr Cys Ala Arg Lys Leu Trp Val Ser Leu Ala 95 100 105

Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210〉 23Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210> 23

<211> 120 / <212〉 PRT \ <213〉人工序列 <220> <223〉YWO.49.C9之重鏈可變區 <400> 23<211> 120 / <212> PRT \ <213>Artificial sequence <220><223>YWO.49. Heavy chain variable region of C9 <400>

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

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

Glu Trp Val Gly Arg He Tyr Pro Asp Ser Gly Ala Thr Tyr Tyr 50 55 60Glu Trp Val Gly Arg He Tyr Pro Asp Ser Gly Ala Thr Tyr Tyr 50 55 60

Lys 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 Lys Leu Trp Val Thr Leu Ala V 95 100 105Lys 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 Lys Leu Trp Val Thr Leu Ala V 95 100 105

Ser Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210> 24 <211> 120 <212> PRT <213〉人工序列 <220> <223〉YWO.49.H2之重鏈可變區 <400> 24Ser Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210> 24 <211> 120 <212> PRT <213>Artificial Sequence <220><223>YWO.49 .H2 heavy chain variable region <400> 24

Glu Trp Val Gly Arg He Tyr Pro Asp Ser Gly Ala Thr Tyr Tyr -6- 119007·序列表.doc 200813092 50 55 60Glu Trp Val Gly Arg He Tyr Pro Asp Ser Gly Ala Thr Tyr Tyr -6- 119007 · Sequence Listing.doc 200813092 50 55 60

Pro Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210〉 25 <211> 120 <212> PRT <213〉人工序列 <220〉 <223〉YWO.49.H6之重鏈可變區 <400> 25Pro Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210> 25 <211> 120 <212> PRT <213>Artificial Sequence <220> <223>YWO.49 .H6 heavy chain variable region <400> 25

Thr Ala Val Tyr Tyr Cys Ala Arg Lys Leu Trp lie Ser lie Ala 95 100 105Thr Ala Val Tyr Tyr Cys Ala Arg Lys Leu Trp lie Ser lie Ala 95 100 105

Gly Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210〉 26 <211> 108 <212> PRT <213〉人工序列 <220〉 <223〉YW0.32、YW0.49及經修飾huMAb4D5-8之輕鏈可變區 <400〉 26Gly Met Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 120 <210> 26 <211> 108 <212> PRT <213>Artificial Sequence <220> <223>YW0.32 , YW0.49 and modified light chain variable region of huMAb4D5-8 <400> 26

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 15 10 15Asp He 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 65 70 75Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 65 70 75

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

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 119007-序列表.doc 200813092 95 100 105 lie Lys Arg <210〉 27 <211〉 108 <212〉 PRT <213〉人工序列 <220〉 <223>YW〇.49.B7之輕鏈可變區 <400> 27Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 119007 - Sequence Listing.doc 200813092 95 100 105 lie Lys Arg <210> 27 <211> 108 <212> PRT <213>Artificial Sequence <;220><223>YW〇.49.B7 light chain variable region <400> 27

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

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

Ser Val Phe Thr Pro Pro Ala Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210〉 28 <211〉 108 <212〉 PRT <213〉人工序列 <220> <223〉YWO.49.C9之輕鏈可變區 <400〉 28Ser Val Phe Thr Pro Pro Ala Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210> 28 <211> 108 <212> PRT < 213 > Artificial Sequence <220><223> Light chain variable region of YWO.49.C9 <400> 28

Leu Leu He Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Leu Leu He 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 He 65 70 75Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He 65 70 75

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

Ser Tyr Asn Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys ArgSer Tyr Asn Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg

<210〉 29 <211> 108 <212〉 PRT <213〉人工序列· 119007-序列表.doc 200813092 <220〉 <223〉YWO.49.H2之輕鏈可變區 <400> 29<210> 29 <211> 108 <212> PRT <213> artificial sequence·119007-SEQ ID NO: doc 200813092 <220> <223>YWO.49. Light chain variable region of H2<400> 29

Ser Tyr Gly Thr Pro Phe He Phe Gly Gin Gly Thr Lys Val Glu 95 100 105Ser Tyr Gly Thr Pro Phe He Phe Gly Gin Gly Thr Lys Val Glu 95 100 105

lie Lys Arg C210> 30 (211> 108 (212> PRT (213>人工序列 (223> YWO.49.H6之輕鏈可變區 C400> 30Lie Lys Arg C210> 30 (211> 108 (212> PRT (213> artificial sequence (223> YWO.49.H6 light chain variable region C400> 30

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15

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

Ser Tyr Ala Thr Pro Phe Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210〉 31 <211> 108 <212> PRT <213〉人工序列 <223〉huMAb05_8之輕鏈可變區 <400> 31Ser Tyr Ala Thr Pro Phe Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210> 31 <211> 108 <212> PRT <213>Artificial Sequence <223>huMAb05_8 Light Chain Variable Area <400> 31

Gly Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp Val Asn 9- 119007·序列表.doc 200813092 20 25 30Gly Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp Val Asn 9- 119007 · Sequence Listing.doc 200813092 20 25 30

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

His Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210〉 32 <211> 30 <212〉 PRT <213〉人工序列 / <220〉 <223〉序列係經合成的 <400> 32His Tyr Thr Thr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu 95 100 105 lie Lys Arg <210> 32 <211> 30 <212> PRT <213>Artificial Sequence / <220〉 <223 〉Sequence is synthesized <400> 32

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

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210〉 33 <211〉 14 <212> PRT <213〉人工序列 <220〉〈223〉序列係經合成的 <400> 33Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 33 <211> 14 <212> PRT <213>Artificial Sequence <220> <223> Sequence Synthesis <400> 33

Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met Gly 5 10 <210> 34 <211> 32 <212〉 PRT 〈213>人工序列 <220〉 <223〉序列係經合成的 <400> 34Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met Gly 5 10 <210> 34 <211> 32 <212> PRT <213>Artificial Sequence<220> <223>Sequence Synthetic <;400> 34

Arg Val Thr lie Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met 1 5 10 15Arg Val Thr lie Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met 1 5 10 15

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30

Ala Arg <210〉 35 <211> Π <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400〉 35Ala Arg <210> 35 <211> Π <212> PRT < 213> artificial sequence <220> <223> sequence is synthesized <400> 35

Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser -10- 119007·序列表.doc 200813092 5 10 <210〉 36 <211> 25 <212〉 PRT <213〉人工序列 <223〉序列係經合成的 <400〉 36Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser -10- 119007 · Sequence Listing.doc 200813092 5 10 <210> 36 <211> 25 <212> PRT < 213 > Artificial Sequence < 223 > Synthesized <400> 36

Ala Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210〉 37 <211〉 13 <212> PRT <213〉人工序列 <220> <223〉序列係經合成的 f ' <400> 37 \ Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 5 10 <210〉 38 <211> 31 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 38Ala Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 37 <211> 13 <212> PRT <213>Artificial Sequence <220><223> Sequence is synthesized f ' <400> 37 \ Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 5 10 <210> 38 <211> 31 <212> PRT <213>Artificial Sequence <220> <223>Sequence Synthesized <400> 38

Arg Val Thr lie Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met 15 10 15Arg Val Thr lie Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met 15 10 15

Ala <210〉 39 <211〉 30 <212〉 PRT <213〉人工序列 <223〉序列係經合成的 <400> 39Ala <210> 39 <211> 30 <212> PRT < 213> artificial sequence <223> sequence is synthesized <400> 39

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> 40 <211> 30 <212> PRT <213〉人工序列 <220> <223〉序列係經合成的 <400> 40Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> 40 <211> 30 <212> PRT <213>Artificial Sequence <220><223> Synthesized <400> 40

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

Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser -11 - 119007-序列表.doc 200813092 20 25 30 <210> 41 <211〉 14 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 41Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser -11 - 119007 - Sequence Listing.doc 200813092 20 25 30 <210> 41 <211> 14 <212> PRT <213>Artificial Sequence <;220><223> sequence is synthesized <400> 41

Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie Gly 5 10Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie Gly 5 10

<210> 42 <211〉 32 <212〉 PRT <213〉人工序列 <220> <223〉序列係經合成的 <400> 42<210> 42 <211>32 <212> PRT <213> artificial sequence <220><223> sequence is synthesized <400>

Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu 15 10 15Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu 15 10 15

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 30Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 30

Ala Arg <210〉 43 <211〉 25 <212〉 PRT <213〉人工序列 <223〉序列係經合成的 <400> 43Ala Arg <210> 43 <211> 25 <212> PRT < 213> artificial sequence <223> sequence system synthesized <400>

Gin Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 44 <211〉 13 <212> PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 44Gin Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 44 <211> 13 <212> PRT <213>Artificial Sequence <220><223>Sequence Synthesized <400> 44

Trp He Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp He 5 10 <210〉 45 <211〉 31 <212> PRT <213〉人工序列 <220> <223〉序列係經合成的 <400〉 45Trp He Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp He 5 10 <210> 45 <211> 31 <212> PRT <213>Artificial Sequence <220><223>Sequence Synthetic <;400〉 45

Ala -12- 119007-序列表.doc 200813092 <210〉 46 <211〉 30 <212> PRT <213〉人工序列 <223〉序列係經合成的 <400> 46Ala -12-119007-Sequence Table.doc 200813092 <210> 46 <211> 30 <212> PRT <213>Artificial Sequence <223>Sequence Synthesized <400> 46

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210〉 47 <211> 30 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 47Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> 47 <211> 30 <212> PRT <213>Artificial Sequence <220> <223>Sequence Synthesized <400> 47

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 48 <211> 14 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 48Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 48 <211> 14 <212> PRT <213>Artificial Sequence <220> <223>Sequence Synthesized <400> 48

Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 5 10 <210> 49 <211〉 32 <212> PRT <213〉人工序列 <220> <223>序列係經合成的 <400> 49Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 5 10 <210> 49 <211> 32 <212> PRT <213>Artificial Sequence <220><223><400> 49

Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 15 10 15Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 15 10 15

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30

Ala Arg <2i0> 50 <211〉 25 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 50Ala Arg <2i0> 50 < 211 > 25 < 212 > PRT < 213 > 213 > artificial sequence < 220 < 223 > 223 > sequence synthesized <400 > 50

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 1 5 10 15Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 13- 119007·序列表.doc 200813092 20 25 <210〉 51 <211> 13 <212〉 PRT <213〉人工序列 <220> <223〉序列係經合成的 <400> 51Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 13- 119007· Sequence Listing.doc 200813092 20 25 <210> 51 <211> 13 <212> PRT <213>Artificial Sequence <220><223> The sequence is synthesized <400> 51

Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 5 10 <210> 52 <211> 31 <212〉 PRT <213〉人工序列 <223〉序列係經合成的 <400> 52Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 5 10 <210> 52 <211> 31 <212> PRT <213>Artificial Sequence <223>Sequence Synthesized <400>

Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 1 5 10 15 / l Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 1 5 10 15 / l Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30

Ala <210〉 53 <211〉 30 <212> PRT <213〉人工序列 <220> <223〉序列係經合成的 <400〉 53Ala <210> 53 <211> 30 <212> PRT <213>Artificial sequence <220><223>Sequence synthesized <400> 53

<210> 54 <211〉 30 <212> PRT <213〉人工序列<210> 54 <211> 30 <212> PRT <213>

<220> <223〉序列係經合成的 <400〉 54<220><223> sequence is synthesized <400> 54

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn lie Lys 20 25 30 <210> 55 <211〉 32 <212> PRT <213〉人工序列 <223〉序列係經合成的 <400〉 55Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn lie Lys 20 25 30 <210> 55 <211> 32 <212> PRT <213>Artificial Sequence <223>Sequence Synthesized < 400> 55

Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu i 5 10 15Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu i 5 10 15

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys -14- 119007·序列表.doc 200813092 20 25 30Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys -14- 119007 · Sequence Listing.doc 200813092 20 25 30

Ser Arg <210> 56 <211〉 31 <212〉 PRT <213〉人工序列 <220> <223〉序列係經合成的 <400〉 56Ser Arg <210> 56 <211> 31 <212> PRT < 213 > artificial sequence <220><223> sequence synthesized <400> 56

Arg Phe Thr He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 1 5 10 15Arg Phe Thr He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 1 5 10 15

Ser <210〉 57 <211> 32 <212> PRT <213〉人工序列 <223〉序列係經合成的 <400〉 57Ser <210> 57 <211> 32 <212> PRT <213>Artificial sequence <223>Sequence synthesized <400> 57

Arg Phe Thr He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 15 10 15Arg Phe Thr He Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 15 10 15

Ala Arg <210〉 58 <211〉 31 <212> PRT <213〉入工序列 <220> <223〉序列係經合成的 <400〉 58Ala Arg <210> 58 <211> 31 <212> PRT < 213 > entry sequence <220><223> sequence is synthesized <400> 58

AlaAla

<2i0> 59 <211> 30 <212> PRT _ r <213〉人工序歹丨J <220〉 <223〉序列係經合成的 <400〉 59<2i0> 59 <211> 30 <212> PRT _ r < 213 > Human Process 歹丨 J < 220 < 223 > 223 > Sequence Synthesized <400> 59

Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 15 10 15Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 15 10 15

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210〉 60 -15- 119007-序列表.doc 200813092 <211> 23 <212> PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 60Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> 60 -15- 119007 - Sequence Listing.doc 200813092 <211> 23 <212> PRT <213>Artificial Sequence <;220><223> sequence is synthesized <400> 60

Gly Asp Arg Val Thr lie Thr Cys 20 <2i0> 61 <211> 15 <212〉 PRT <213〉人工序列 <223〉序列係經合成的 <400> 61Gly Asp Arg Val Thr lie Thr Cys 20 <2i0> 61 <211> 15 <212> PRT <213>Artificial sequence <223>Sequence synthesized <400>

Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr 15 10 15 - <210〉 62 <211> 32 <212> PRT <213〉人工序列 <220> <223〉序列係經合成的 <400> 62Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr 15 10 15 - <210> 62 <211> 32 <212> PRT <213>Artificial Sequence <220><223> Synthesized <400> 62

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 15 10 15Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 15 10 15

Thr Leu Thr He Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr 20 25 30Thr Leu Thr He Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr 20 25 30

Tyr CysTyr Cys

<210> 63 <211〉 10 <212> PRT <213〉入土序列 <220〉 / <223〉序列係經合成的 i <400〉 63<210> 63 <211> 10 <212> PRT < 213 > In-situ sequence <220> / <223> sequence is synthesized i <400> 63

Phe Gly Gin Gly Thr Lys Val Glu lie Lys 5 10 <210> 64 <211〉 23 <212〉 PRT <213〉人工序列 <223〉序列係經合成的 <400> 64Phe Gly Gin Gly Thr Lys Val Glu lie Lys 5 10 <210> 64 <211> 23 <212> PRT <213>Artificial sequence <223>Sequence synthesized <400> 64

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

Gly Glu Pro Ala Ser lie Ser Cys 20 <210〉 65 <211> 15 <212〉 PRT <213〉人工序列 16- 119007-序列表.doc 200813092 <220〉 <223〉序列係經合成的 <400> 65Gly Glu Pro Ala Ser lie Ser Cys 20 <210> 65 <211> 15 <212> PRT < 213 > Artificial Sequence 16 - 119007 - Sequence Listing.doc 200813092 <220> <223> Synthesized <400> 65

Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu He Tyr 15 10 15 <210〉 66 <211> 32 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 66Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu He Tyr 15 10 15 <210> 66 <211> 32 <212> PRT <213>Artificial Sequence <220> <223> Synthesized <400> 66

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 15 10 15Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 15 10 15

Thr Leu Lys lie Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 20 25 30Thr Leu Lys lie Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 20 25 30

Tyr CysTyr Cys

<210〉 67 <211〉 23 <212> PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 67<210> 67 <211> 23 <212> PRT <213> artificial sequence <220><223> sequence is synthesized <400>

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

Gly Glu Arg Ala Thr Leu Ser Cys 20 <210〉 68 <211〉 15 <212〉 PRT <213〉人工序列 <220> <223〉序列係經合成的 <400> 68Gly Glu Arg Ala Thr Leu Ser Cys 20 <210> 68 <211> 15 <212> PRT <213>Artificial Sequence <220><223>Sequence Synthesized <400>

Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie Tyr 15 10 15 <210〉 69 <211〉 32 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 69Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie Tyr 15 10 15 <210> 69 <211> 32 <212> PRT <213>Artificial Sequence <220> <223>Sequence Synthesized <400> 69

Gly lie Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 15 10 15Gly lie Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 15 10 15

Thr Leu Thr He Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr 20 25 30Thr Leu Thr He Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr 20 25 30

Tyr CysTyr Cys

<210〉 70 <211> 23 <212> PRT <213〉人工序列 17- 119007-序列表.doc 200813092 <220〉 <223〉序列係經合成的 <400〉 70<210> 70 <211> 23 <212> PRT <213> Artificial sequence 17-119007 - Sequence Listing.doc 200813092 <220> <223> Sequences synthesized <400> 70

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

Gly Glu Arg Ala Thr lie Asn Cys 20 <210〉 71 <211〉 15 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 71Gly Glu Arg Ala Thr lie Asn Cys 20 <210> 71 <211> 15 <212> PRT <213>Artificial sequence <220> <223>Sequence synthesized <400>

Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Tyr 15 10 15Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Tyr 15 10 15

<210> 72 <211〉 32 <212> PRT <213>人工序列 <220〉 <223〉序列係經合成的 <400> 72<210> 72 <211>32 <212> PRT <213> artificial sequence <220><223> sequence is synthesized <400>

Thr Leu Thr He Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr 20 25 30Thr Leu Thr He Ser Ser Leu Gin Ala Glu Asp Val Ala Val Tyr 20 25 30

Tyr Cys <210> 73 <211〉 32 <212> PRT <213〉人工序列 <223〉序列係經合成的 <400〉 73Tyr Cys <210> 73 <211> 32 <212> PRT < 213 > 213 > artificial sequence <223 > sequence is synthesized <400 > 73

Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe 15 10 15Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe 15 10 15

Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr 20 25 30Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr 20 25 30

Tyr Cys <210〉 74 <211〉 10 <212〉 PRT <213〉人工序列 <220〉 <223〉序列係經合成的 <400> 74Tyr Cys <210> 74 <211> 10 <212> PRT <213>Artificial sequence <220> <223>Sequence synthesized <400> 74

Phe Arg Gin Gly Thr Lys Val Glu He Lys 5 10 <210〉 75 <211〉 141 <212〉 PRT <213〉人類 18- 119007-序列表.doc 200813092 <400> 75Phe Arg Gin Gly Thr Lys Val Glu He Lys 5 10 <210> 75 <211> 141 <212> PRT <213> Human 18-119007 - Sequence Listing.doc 200813092 <400>

Met Trp Val Leu Gly He Ala Ala Thr Phe Cys Gly Leu Phe Leu 15 10 15Met Trp Val Leu Gly He Ala Ala Thr Phe Cys Gly Leu Phe Leu 15 10 15

Leu Pro Gly Phe Ala Leu Gin lie Gin Cys Tyr Gin Cys Glu Glu 20 25 30Leu Pro Gly Phe Ala Leu Gin lie Gin Cys Tyr Gin Cys Glu Glu 20 25 30

Phe Gin Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe lie Val Asn 35 40 45Phe Gin Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe lie Val Asn 35 40 45

Cys Thr Val Asn Val Gin Asp Met Cys Gin Lys Glu Val Met Glu 50 55 60Cys Thr Val Asn Val Gin Asp Met Cys Gin Lys Glu Val Met Glu 50 55 60

Gin Ser Ala Gly He Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala 65 70 75Gin Ser Ala Gly He Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala 65 70 75

Ala Cys Leu lie Ala Ser Ala Gly Tyr Gin Ser Phe Cys Ser Pro 80 85 90Ala Cys Leu lie Ala Ser Ala Gly Tyr Gin Ser Phe Cys Ser Pro 80 85 90

Gly Lys Leu Asn Ser Val Cys lie Ser Cys Cys Asn Thr Pro Leu 95 100 105Gly Lys Leu Asn Ser Val Cys lie Ser Cys Cys Asn Thr Pro Leu 95 100 105

Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser Ser Ala Ser Ala 110 115 120Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser Ser Ala Ser Ala 110 115 120

Leu Arg Pro Gly Leu Arg Thr Thr lie Leu Phe Leu Lys Leu Ala 125 130 135Leu Arg Pro Gly Leu Arg Thr Thr lie Leu Phe Leu Lys Leu Ala 125 130 135

Leu Phe Ser Ala His Cys 140 <210〉 76 <211> 141 <212> PRT <213〉食蟹猴 <400〉 76Leu Phe Ser Ala His Cys 140 <210> 76 <211> 141 <212> PRT <213> cynomolgus monkey <400> 76

Met Trp Val Leu Gly He Ala Ala Thr Phe Cys Gly Leu Phe Leu 1 5 10 15Met Trp Val Leu Gly He Ala Ala Thr Phe Cys Gly Leu Phe Leu 1 5 10 15

Leu Arg Pro Gly Leu Pro Thr Thr He Leu Leu Leu Lys Leu Ala 125 130 135Leu Arg Pro Gly Leu Pro Thr Thr He Leu Leu Leu Lys Leu Ala 125 130 135

Leu Phe Ser Ala His Cys 140 <210> 77 <211〉 141 <212> PRT <213〉小家鼠 <400> 77Leu Phe Ser Ala His Cys 140 <210> 77 <211> 141 <212> PRT <213> Mus musculus <400> 77

Met Trp Val Leu Gly lie Ala Ala Thr Phe Cys Gly Leu Phe Trp -19· 119007-序列表.doc 200813092 15 10 15Met Trp Val Leu Gly lie Ala Ala Thr Phe Cys Gly Leu Phe Trp -19· 119007-Sequence List.doc 200813092 15 10 15

Leu Pro Gly Leu Ala Leu Gin lie Gin Cys Tyr Gin Cys Glu Glu 20 25 30Leu Pro Gly Leu Ala Leu Gin lie Gin Cys Tyr Gin Cys Glu Glu 20 25 30

Phe Gin Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe He Val Asn 35 40 45Phe Gin Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe He Val Asn 35 40 45

Gin Ser Ala Gly lie Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala 65 70 75Gin Ser Ala Gly lie Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala 65 70 75

Gly Lys Leu Asn Ser Val Cys He Ser Cys Cys Asn Thr Pro Leu 95 100 105Gly Lys Leu Asn Ser Val Cys He Ser Cys Cys Asn Thr Pro Leu 95 100 105

Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser Ser Ala Ser Ala 110 115 120 lie Arg Pro Gly Leu Leu Thr Thr Leu Leu Phe Phe His Leu Ala 125 130 135 f 、 Leu Cys Leu Ala His Cys 140 <210> 78 <211> 141 <212> PRT <213〉褐家鼠 <400〉 78Arg Pro Gly Leu Leu Thr Thr Leu Leu Phe Phe His Leu Ala 125 130 135 f , Leu Cys Leu Ala His Cys 140 <210> 78 <211> 141 <212> PRT < 213 > brown rat < 400 > 78

Met Trp Val Leu Gly lie Ala Ala Thr Phe Cys Gly Leu Phe Trp 15 10 15Met Trp Val Leu Gly lie Ala Ala Thr Phe Cys Gly Leu Phe Trp 15 10 15

Gin Ser Ala Gly He Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala 65 70 75 C Ala Cys Leu lie Ala Ser Ala Gly Tyr Gin Ser Phe Cys Ser Pro 80 85 90Gin Ser Ala Gly He Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala 65 70 75 C Ala Cys Leu lie Ala Ser Ala Gly Tyr Gin Ser Phe Cys Ser Pro 80 85 90

Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser Ser Ala Ser Ala 110 115 120 lie Arg Pro Gly Leu Leu Thr Thr Leu Leu Phe Phe His Leu Ala 125 130 135Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser Ser Ala Ser Ala 110 115 120 lie Arg Pro Gly Leu Leu Thr Thr Leu Leu Phe Phe His Leu Ala 125 130 135

Leu Cys Leu Ala His Cys 140 -20- 119007-序列表.docLeu Cys Leu Ala His Cys 140 -20- 119007 - Sequence Listing.doc

Claims

200813092 十、申請專利範圍： 1. 一種結合至TAT226之抗體，其中該抗體包含（a)包含與 SEQ ID ΝΟ:11之一致序列相符之胺基酸序列的HVR-H3 及（b)至少一個、兩個、三個、四個或五個選自以下之 HVR ： (1) 包含SEQ ID NO:4之胺基酸序列之HVR-H1 ; (2) 包含SEQ ID NO:5之胺基酸序列之HVR-H2 ; (3) 包含SEQIDN0:12之胺基酸序列之HVR·L1; (4) 包含SEQ ID NChl3之胺基酸序列之HVR-L2 ;及 (5) 包含與SEQ ID NO: 19之一致序列相符之胺基酸序列之 HVR-L3。 2. 如請求項1之抗體，其包含具有與SEQ ID NO:19之一致序列相符之胺基酸序列的HVR-L3。 3. 如請求項2之抗體，其另外包含具有SEQ ID NO:4之胺基酸序列之HVR-H1及具有SEQ ID NO:5之胺基酸序列之 HVR-H2。 4. 如請求項3之抗體，其另外包含具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 5. 如請求項1之抗體，其中該抗體包含具有選自SEQ ID NO··6-10之胺基酸序列之HVR-H3。 6. 如請求項5之抗體，其包含具有選自SEQ ID NO: 14· 18之胺基酸序列之HVR-L3。 7. 如請求項6之抗體，其另外包含具有SEQ ID NO:4之胺基 119007.doc 200813092 酸序列之HVR-H1及具有SEQ ID ΝΟ··5之胺基酸序列之 HVR-H2。 8. 如請求項7之抗體，其另外包含具有SEQ ID NO: 12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 9. 如請求項6之抗體，其中該HVR-H3包含SEQ ID NO:9之胺基酸序列，且該11¥1^3包含8丑(5 10>^0:17之胺基酸序列。 10. 如請求項9之抗體，其另外包含具有SEQ ID NO:4之胺基酸序列之HVR-H1及具有SEQ ID NO:5之胺基酸序列之 HVR-H2。 11. 如請求項10之抗體，其另外包含具有SEQ ID ΝΟ:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 12. 如請求項6之抗體，其中該HVR-H3包含SEQ ID NChlO之胺基酸序列，且該HVR-L3包含SEQ ID NO:18之胺基酸序列。 13. 如請求項12之抗體，其另外包含具有SEQ ID ΝΟ··4之胺基酸序列之HVR-H1及具有SEQ ID ΝΟ:5之胺基酸序列之 HVR-H2。 14. 如請求項13之抗體，其另外包含具有SEQ ID NO: 12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 15. 如請求項1之抗體，其另外包含至少一個選自VH子群III 119007.doc 200813092 一致框架及VL子群I 一致框架之框架。 16· —種結合至TAT226之抗體，其中該抗體包含與選自SEQ ID NO:21-25之胺基酸序列具有至少90%序列一致性的重鏈可變結構域。 17.如請求項16之抗體，其中該抗體另外包含與選自SEQ ID NO:26-3 1之胺基酸序列具有至少90%序列一致性的輕鏈可變結構域。 18·如請求項16之抗體，其中該抗體包含與SEQ ID NO:24之胺基酸序列具有至少90%序列一致性的重鏈可變結構域。 19. 如請求項18之抗體，其另外包含與SEQ ID NO:29之胺基酸序列具有至少90%序列一致性之輕鏈可變結構域。 20. 如請求項19之抗體，其中該重鏈可變結構域包含SEQ ID NO:24之胺基酸序列，且該輕鏈可變結構域包含SEQ ID NO:29之胺基酸序列。 21. 如請求項16之抗體，其中該抗體包含與SEQ ID NO:25之胺基酸序列具有至少90%序列一致性的重鏈可變結構域。 22. 如請求項21之抗體，其另外包含與SEQ ID NO:30之胺基酸序列具有至少90%序列一致性之輕鏈可變結構域。 23. 如請求項22之抗體，其中該重鏈可變結構域包含SEQ ID NO:25之胺基酸序列，且該輕鏈可變結構域包含SEQ ID NO:30之胺基酸序列。 24. —種編碼如請求項16之抗體之聚核苷酸。 119007.doc 200813092 25. —種包含如請求項24之聚核苷酸之載體。 26. —種包含如請求項25之載體之宿主細胞。 27. 如請求項26之宿主細胞，其中該宿主細胞為真核的。 28. 如請求項27之宿主細胞，其中該宿主細胞為CHO細胞。 29. —種製造抗TAT226抗體之方法，其中該方法包含a)於適合表現編碼該抗體之聚核苷酸的條件下培養如請求項26 之宿主細胞，及b)分離該抗體。 30. —種結合於細胞表面上表現之TAT226的抗體。 1 31.如請求項30之抗體，其中該抗體結合位於來自SEQ ID NO:75之胺基酸21-115之丁八丁226區中的抗原決定基。 32.如請求項30之抗體，其中該細胞為癌細胞。 3 3.如請求項32之抗體，其中該癌細胞為卵巢癌細胞、腦腫瘤細胞或威爾姆氏腫瘤細胞（Wilm*s tumor cell)。 34.如請求項1、16或30中任一項之抗體，其中該抗體為單株抗體。 3 5.如請求項34之抗體，其中該抗體為選自Fab、Fab’-SH、卩¥、80?¥或（？汪13')2片段之抗體片段。 3 6.如請求項34之抗體，其中該抗體為人化的。 3 7.如請求項34之抗體，其中該抗體為人類的。 3 8.如請求項34之抗體，其中該抗體結合至與選自YW0.32、 YW0.49、YWO.49.B7、YWO.49.C9、YWO.49.H2 及 YWO.49.H6之抗體相同之抗原決定基。 39. —種偵測生物樣本中TAT226存在之方法，該方法包含在容許該抗體結合TAT226之條件下使該生物樣本與如請求 119007.doc 200813092 項1、16或3 0由>r 干任一項之抗體接觸，且偵測在該抗體與 TAT226之間是否形成複合物。 40·如请求項39之方法，其中該生物樣本包含卵巢腫瘤細胞、胸腫瘤細胞或威爾姆氏腫瘤細胞。 41. 一種活體外診斷與増加之1^丁226表現相關聯之細胞增殖性病症的方法’該方法包含使測試細胞與如請求項1、 16或30中任一項之抗體接觸；藉由偵測該抗體與之結合而判定該測試細胞之TAT226表現程度；且比較該測试細胞之TAT226表現程度與對照細胞之TAT226表現程度，其中與該對照細胞相比，該測試細胞之較高TAT226 表現程度指示存在與增加之TAT226表現相關聯之細胞增殖性病症。 42. 如請求項41之方法，其中該測試細胞為來自懷疑患有細胞增殖性病症之患者的細胞。 43·如請求項41之方法，其中該細胞增殖性病症係選自卵巢癌及威爾姆氏腫瘤。 44·如請求項41之方法，其中該方法包含判定TAT226於該測試細胞表面上之表現程度且比較TAT226於該測試細胞表面上之表現程度與TAT226於該對照細胞表面上之表現程度。 45. —種免疫接合物，其包含共價連接細胞毒性劑之如請求項1、16或30中任一項之結合τΑΤ226的抗體。 46·如請求項45之免疫接合物，其中該細胞毒性劑係選自毒素' 化療劑、抗生素、放射性同位素及核分解酶。 119007.doc 200813092 47. —種具有式Ab-(L-D)p之免疫接合物，其中： (a) Ab為結合TAT226且包含1)包含與SEQ ID ΝΟ]1之一致序列相符之胺基酸序列的HVR-H3及2)至少一個、兩個、三個、四個或五個選自以下之HVR的抗體： (i) 包含SEQ ID NO:4之胺基酸序列之HVR-H1 ; (ii) 包含SEQ ID NO:5之胺基酸序列之HVR-H2 ; (iii) 包含SEQIDNO:12之胺基酸序列之HVR_Ll; 1 (iv)包含SEQIDNO:13之胺基酸序列之HVR-L2 ;及 (v)包含與SEQ ID ΝΟ··19之一致序列相符之胺基酸序列之HVR-L3 ; (b) L為一連接子； (c) D為式DE或DF之藥物200813092 X. Patent Application Range: 1. An antibody that binds to TAT226, wherein the antibody comprises (a) HVR-H3 and (b) at least one comprising an amino acid sequence conforming to the sequence identical to SEQ ID NO: 11. Two, three, four or five HVRs selected from the group consisting of: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (2) amino acid sequence comprising SEQ ID NO: HVR-H2; (3) HVR·L1 comprising the amino acid sequence of SEQ ID NO: 12; (4) HVR-L2 comprising the amino acid sequence of SEQ ID NChl3; and (5) comprising SEQ ID NO: 19 HVR-L3 of the amino acid sequence of the consistent sequence. 2. The antibody of claim 1, which comprises HVR-L3 having an amino acid sequence conforming to the sequence identical to SEQ ID NO: 19. 3. The antibody of claim 2, which additionally comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4 and HVR-H2 having the amino acid sequence of SEQ ID NO: 5. 4. The antibody of claim 3, which additionally comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. 5. The antibody of claim 1, wherein the antibody comprises HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NO. 6. The antibody of claim 5, which comprises HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 14·18. 7. The antibody of claim 6, which additionally comprises HVR-H1 having the amino acid group 119007.doc 200813092 of SEQ ID NO: 4 and HVR-H2 having the amino acid sequence of SEQ ID. 8. The antibody of claim 7, which additionally comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. 9. The antibody of claim 6, wherein the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9, and the 11¥1^3 comprises an 8 ugly (5 10>^0:17 amino acid sequence. 10. The antibody of claim 9, which additionally comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4 and HVR-H2 having the amino acid sequence of SEQ ID NO: 5. 11. An antibody further comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. 12. The antibody of claim 6, wherein the HVR -H3 comprises the amino acid sequence of SEQ ID NChlO, and the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18. 13. The antibody of claim 12, which additionally comprises an amine having SEQ ID ΝΟ·4 HVR-H1 of the acid sequence and HVR-H2 having the amino acid sequence of SEQ ID NO: 5. 14. The antibody of claim 13, which additionally comprises HVR- having the amino acid sequence of SEQ ID NO: And a VR subgroup I A framework for binding to TAT226, wherein the antibody comprises a heavy chain variable domain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-25. The antibody of claim 16, wherein the antibody further comprises a light chain variable domain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 26-3 1. 18. An antibody, wherein the antibody comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24. 19. The antibody of claim 18, additionally comprising SEQ ID NO: 29 The amino acid sequence having at least 90% sequence identity. The antibody of claim 19, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 24, and The light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29. 21. The antibody of claim 16, wherein the antibody comprises at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 25. The heavy chain variable domain. 22. The antibody of claim 21, which additionally comprises SEQ ID NO: The amino acid sequence has a light chain variable domain with at least 90% sequence identity. 23. The antibody of claim 22, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:30. 24. A polynucleotide encoding an antibody of claim 16. 119007.doc 200813092 25. A vector comprising the polynucleotide of claim 24. 26. A host cell comprising the vector of claim 25. 27. The host cell of claim 26, wherein the host cell is eukaryotic. 28. The host cell of claim 27, wherein the host cell is a CHO cell. 29. A method of making an anti-TAT226 antibody, wherein the method comprises a) cultivating a host cell as claimed in claim 26 under conditions suitable for performing a polynucleotide encoding the antibody, and b) isolating the antibody. 30. An antibody that binds to TAT226 expressed on the surface of a cell. The antibody of claim 30, wherein the antibody binds to an epitope located in the Dingba 226 region of the amino acid 21-115 of SEQ ID NO: 75. 32. The antibody of claim 30, wherein the cell is a cancer cell. 3. The antibody according to claim 32, wherein the cancer cell is an ovarian cancer cell, a brain tumor cell or a Wilm*s tumor cell. The antibody of any one of claims 1, 16 or 30, wherein the antibody is a monoclonal antibody. 3. The antibody of claim 34, wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, 卩¥, 80? ¥ or (?? Wang's 2). 3. The antibody of claim 34, wherein the antibody is humanized. 3. The antibody of claim 34, wherein the antibody is human. 3. The antibody according to claim 34, wherein the antibody binds to and is selected from the group consisting of YW0.32, YW0.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 The same epitope of the antibody. 39. A method of detecting the presence of TAT226 in a biological sample, the method comprising: subjecting the antibody to TAT226, allowing the biological sample to be served by >r as requested in 119007.doc 200813092, item 1, 16, or 30 One of the antibodies contacts and detects whether a complex is formed between the antibody and TAT226. 40. The method of claim 39, wherein the biological sample comprises ovarian tumor cells, thoracic tumor cells, or Wilm's tumor cells. 41. A method for in vitro diagnostic diagnosis of a cell proliferative disorder associated with a spleen 226 expression comprising: contacting the test cell with an antibody of any one of claims 1, 16 or 30; The antibody was assayed to determine the degree of TAT226 expression of the test cells; and the degree of TAT226 expression of the test cells was compared with the degree of TAT226 expression of the control cells, wherein the test cells had higher TAT226 expression than the control cells. The degree indicates the presence of a cell proliferative disorder associated with increased TAT226 performance. 42. The method of claim 41, wherein the test cell is a cell from a patient suspected of having a proliferative disorder. The method of claim 41, wherein the cell proliferative disorder is selected from the group consisting of ovarian cancer and Wilm's tumor. 44. The method of claim 41, wherein the method comprises determining the extent of expression of TAT226 on the surface of the test cell and comparing the extent of expression of TAT226 on the surface of the test cell to the extent of TAT226 on the surface of the control cell. An immunoconjugate comprising an antibody that binds to a cytotoxic agent, such as the binding of τΑΤ226, according to any one of claims 1, 16 or 30. 46. The immunoconjugate of claim 45, wherein the cytotoxic agent is selected from the group consisting of a toxin chemotherapeutic agent, an antibiotic, a radioisotope, and a nucleolytic enzyme. 119007.doc 200813092 47. An immunoconjugate having the formula Ab-(LD)p, wherein: (a) Ab is a binding amino acid sequence comprising TAT226 and comprising 1) a sequence identical to the sequence of SEQ ID ΝΟ]1 HVR-H3 and 2) at least one, two, three, four or five antibodies selected from the following HVRs: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (iii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; 1 (iv) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; And (v) HVR-L3 comprising an amino acid sequence conforming to the sequence identical to SEQ ID ΝΟ·19; (b) L is a linker; (c) D is a drug of the formula DE or DF

且其中R2及R6各自為曱基，R3及R4各自為異丙基，R7為第二丁基，各R8係獨立地選自CH3、〇-CH3、OH及Η ; R9為Η ; R10為芳基；2為_〇_或-NH_ ; R11為 H、CrCs烷基或-(CH2)2_0_(CH2)2_〇-(CH2)2-〇-CH3 ;且 R18 為-C(R8)2-C(R8)2-芳基；且 119007.doc -6- 200813092 (d) p在約1至8之範圍内。 48. 如請求項47之免疫接合物，其中該抗體包含具有與SEQ ID NO:19之一致序列相符之胺基酸序列之HVR-L3。 49. 如請求項48之免疫接合物，其中該抗體包含具有SEQ ID NO:9之胺基酸序列之HVR-H3及具有SEQ ID NOH7之胺基酸序列之HVR-L3。 50. 如請求項49之免疫接合物，其中該抗體另外包含具有 SEQ ID NO:4之胺基酸序列之HVR-H1 ;具有SEQ ID NO:5之胺基酸序列之HVR-H2;具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 51. 如請求項48之免疫接合物，其中該抗體包含具有SEQ ID NO:10之胺基酸序列之HVR-H3及具有SEQIDNO:18之胺基酸序列之HVR-L3。 52. 如請求項5 1之免疫接合物，其中該抗體另外包含具有 SEQ ID NO:4之胺基酸序列之HVR-H1 ;具有SEQ ID NO:5之胺基酸序列之HVR-H2;具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2 〇 53. 如請求項47之免疫接合物，其中該抗體包含與選自SEQ ID NO:2 1-25之胺基酸序列具有至少90%序列一致性之重鏈可變區及與選自SEQ ID NO:26_31之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 54. 如請求項53之免疫接合物，其中該抗體包含與SEQ ID 119007.doc 200813092 ΝΟ··24之胺基酸序列具有至少90%序列一致性之重鏈可變區及與SEQ ID ΝΟ:29之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 55·如請求項54之免疫接合物，其中該抗體包含具有SEQ ID NO:24之胺基酸序列之重鏈可變區及具有SEQ ID NO:29 之胺基酸序列之輕鏈可變區。 56·如請求項53之免疫接合物，其中該抗體包含與SEQ ID NO:25之胺基酸序列具有至少90%序列一致性之重鏈可 ( 變區及與SEQ ID NO:30之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 57·如請求項56之免疫接合物，其中該抗體包含具有SEQ ID NO:25之胺基酸序列之重鏈可變區及具有SEQ ID NO:30 之胺基酸序列之輕鏈可變區。 5 8 ·如請求項4 7之免疫接合物’其具有活體外或活體内細胞殺死活性。 59·如請求項47之免疫接合物，其中該連接子係經由該抗體 I" 上之硫醇基連接至該抗體。 6 0 ·如清求項4 7之免疫接合物’其中该連接子可由蛋白酶聲解。 61.如請求項60之免疫接合物，其中該連接子包含val_cit: 肽。 62·如請求項47之免疫接合物，其中該連接子包含對胺基节基單元。 63.如請求項47之免疫接合物’其中該連接子包含6_馬來酿 119007.doc 200813092 亞胺己醯基。 64.如請求項47之免疫接合物，其中該藥物係選自MMAE及 MMAF。 65·如請求項64之免疫接合物，其中該藥物為MMAE。 66_如請求項64之免疫接合物，其中該藥物為MMAF。 67. 如請求項64之免疫接合物，其中該連接子可由蛋白酶裂解。 68. 如請求項67之免疫接合物，其中該連接子包含val-cit二肽。 69. 如請求項67之免疫接合物，其中該連接子包含對胺基苄基單元。 70. 如請求項69之免疫接合物，其中該對胺基苄基單元為對胺基苄氧羰基（PAB)。 71. 如請求項67之免疫接合物，其中該連接子包含6-馬來醯亞胺己醯基。 72. 如請求項65之免疫接合物，其中該免疫接合物具有下式And wherein R2 and R6 are each a fluorenyl group, R3 and R4 are each an isopropyl group, and R7 is a second butyl group, and each R8 is independently selected from the group consisting of CH3, 〇-CH3, OH, and hydrazine; R9 is Η; R10 is aryl 2; is _〇_ or -NH_; R11 is H, CrCs alkyl or -(CH2)2_0_(CH2)2_〇-(CH2)2-〇-CH3; and R18 is -C(R8)2- C(R8)2-aryl; and 119007.doc -6- 200813092 (d) p is in the range of about 1 to 8. 48. The immunoconjugate of claim 47, wherein the antibody comprises HVR-L3 having an amino acid sequence consistent with the sequence of SEQ ID NO: 19. 49. The immunoconjugate of claim 48, wherein the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 9 and HVR-L3 having the amino acid sequence of SEQ ID NO: 7. 50. The immunoconjugate of claim 49, wherein the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4; HVR-H2 having the amino acid sequence of SEQ ID NO: 5; ID NO: HVR-L1 of the amino acid sequence of 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. 51. The immunoconjugate of claim 48, wherein the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 10 and HVR-L3 having the amino acid sequence of SEQ ID NO: 18. 52. The immunoconjugate of claim 51, wherein the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4; HVR-H2 having the amino acid sequence of SEQ ID NO: 5; HVR-L1 of the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. The immunoconjugate of claim 47, wherein the antibody comprises and is selected from the group consisting of ID NO: 2 1-25 amino acid sequence having a heavy chain variable region with at least 90% sequence identity and a light chain having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 26-31 Variable area. 54. The immunoconjugate of claim 53, wherein the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID 119007.doc 200813092 及.24 and SEQ ID: The amino acid sequence of 29 has a light chain variable region with at least 90% sequence identity. 55. The immunoconjugate of claim 54, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: . 56. The immunoconjugate of claim 53, wherein the antibody comprises a heavy chain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 25 (variant region and amino group of SEQ ID NO: 30) The light chain variable region of the acid sequence of claim 56, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and having The light chain variable region of the amino acid sequence of SEQ ID NO: 30. The immunoconjugate of claim 47 has an in vitro or in vivo cell killing activity. 59. Immunization according to claim 47 A conjugate, wherein the linker is linked to the antibody via a thiol group on the antibody I"60<>>, as in the immunoconjugate of '4', wherein the linker can be sonicated by a protease. The immunoconjugate of claim 60, wherein the linker comprises a val_cit: peptide. 62. The immunoconjugate of claim 47, wherein the linker comprises a pair of amino-based units. 63. The immunoconjugate of claim 47 Where the linker contains 6_Malay stuffed 119007.doc 200813092 imipenem 64. The immunoconjugate of claim 47, wherein the drug is selected from the group consisting of MMAE and MMAF. 65. The immunoconjugate of claim 64, wherein the drug is MMAE. 66_ The immunoconjugate of claim 64 The drug is MMAF. 67. The immunoconjugate of claim 64, wherein the linker is cleaved by a protease. 68. The immunoconjugate of claim 67, wherein the linker comprises a val-cit dipeptide. The immunoconjugate of claim 67, wherein the linker comprises a p-aminobenzyl unit. 70. The immunoconjugate of claim 69, wherein the p-aminobenzyl unit is p-aminobenzyloxycarbonyl (PAB) 71. The immunoconjugate of claim 67, wherein the linker comprises 6-maleimine hexamethylene. 72. The immunoconjugate of claim 65, wherein the immunoconjugate has the formula

其中S為硫原子，且p在2至5之範圍内。 73. 如請求項72之免疫接合物，其中該抗體包含具有與SEQ 10 1^0:19之一致序列相符之胺基酸序列的11¥11丄3。 74. 如請求項73之免疫接合物，其中該抗體包含具有SEQ ID NO:9之胺基酸序列之HVR-H3及具有SEQ ID NO:17之胺基酸序列之HVR-L3。 119007.doc 200813092 75. 如請求項74之免疫接合物，其中該抗體另外包含具有 SEQ ID NO:4之胺基酸序列之HVR-H1 ;具有SEQ ID ΝΟ··5之胺基酸序列之HVR-H2;具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 76. 如請求項73之免疫接合物，其中該抗體包含具有SEQ ID NO: 10之胺基酸序列之HVR-H3及具有SEQ ID NO: 18之胺基酸序列之HVR-L3。 77. 如請求項76之免疫接合物，其中該抗體另外包含具有 SEQ ID NO:4之胺基酸序列之HVR-H1 ;具有SEQ ID NO:5之胺基酸序列之HVR-H2;具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 78. 如請求項72之免疫接合物，其中該抗體包含與選自SEQ ID NO:21-25之胺基酸序列具有至少90%序列一致性之重鏈可變區及與選自SEQ ID NO:26_31之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 79. 如請求項78之免疫接合物，其中該抗體包含與SEQ ID NO:24之胺基酸序列具有至少90%序列一致性之重鏈可變區及與SEQ ID NO:29之胺基酸序列具有至少90%序列一致性之輕鍵可變區。 80. 如請求項79之免疫接合物，其中該抗體包含具有SEQ ID NO:24之胺基酸序列之重鏈可變區及具有SEQ ID NO:29 之胺基酸序列之輕鏈可變區。 119007.doc -10- 200813092 81. 如請求項78之免疫接合物，其中該抗體包含與SEQ ID NO:25之胺基酸序列具有至少90%序列一致性之重鏈可變區及與SEQ ID NO:30之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 82. 如請求項81之免疫接合物，其中該抗體包含具有SEQ ID NO:25之胺基酸序列之重鏈可變區及具有SEQ ID NO:30 之胺基酸序列之輕鏈可變區。 83. 如請求項66之免疫接合物，其中該免疫接合物具有下式Wherein S is a sulfur atom and p is in the range of 2 to 5. 73. The immunoconjugate of claim 72, wherein the antibody comprises 11¥11丄3 having an amino acid sequence consistent with the sequence SEQ 10 1^0:19. 74. The immunoconjugate of claim 73, wherein the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 9 and HVR-L3 having the amino acid sequence of SEQ ID NO: 17. The immunoconjugate of claim 74, wherein the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4; HVR having the amino acid sequence of SEQ ID ΝΟ·5 -H2; HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. 76. The immunoconjugate of claim 73, wherein the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 10 and HVR-L3 having the amino acid sequence of SEQ ID NO: 18. 77. The immunoconjugate of claim 76, wherein the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4; HVR-H2 having the amino acid sequence of SEQ ID NO: 5; ID NO: HVR-L1 of the amino acid sequence of 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. 78. The immunoconjugate of claim 72, wherein the antibody comprises a heavy chain variable region having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-25 and is selected from the group consisting of SEQ ID NO The amino acid sequence of 26_31 has a light chain variable region with at least 90% sequence identity. 79. The immunoconjugate of claim 78, wherein the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 and an amino acid of SEQ ID NO: The sequence has a light bond variable region with at least 90% sequence identity. 80. The immunoconjugate of claim 79, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: . The immunoconjugate of claim 78, wherein the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 25 and SEQ ID NO: NO: The amino acid sequence of 30 has a light chain variable region with at least 90% sequence identity. 82. The immunoconjugate of claim 81, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: . 83. The immunoconjugate of claim 66, wherein the immunoconjugate has the formula

其中S為硫原子，且p在2至5之範圍内。 84. 如請求項83之免疫接合物，其中該抗體包含具有與SEQ 10 1^0:19之一致序列相符之胺基酸序列的11¥11-1^。 85. 如請求項84之免疫接合物，其中該抗體包含具有SEQ ID NO:9之胺基酸序列之HVR-H3及具有SEQ ID NO:17之胺基酸序列之HVR-L3。 86. 如請求項85之免疫接合物，其中該抗體另外包含具有 SEQ ID ΝΟ··4之胺基酸序列之HVR-H1 ;具有SEQ ID NO:5之胺基酸序列之HVR-H2;具有SEQ ID NChl2之胺基酸序列之HVR-L1及具肴SEQ ID NO:13之胺基酸序列之 HVR-L2。 87. 如請求項84之免疫接合物，其中該抗體包含具有SEQ ID ΝΟ··10之胺基酸序列之HVR-H3及具有SEQ ID ΝΟ··18之胺基酸序列之HVR-L3。 119007.doc 11 200813092 88. 如請求項87之免疫接合物，其中該抗體另外包含具有 SEQ ID NO:4之胺基酸序列之HVR-H1 ;具有SEQ ID NO:5之胺基酸序列之HVR-H2;具有SEQ ID NO:12之胺基酸序列之HVR-L1及具有SEQ ID NO:13之胺基酸序列之 HVR-L2。 89. 如請求項83之免疫接合物，其中該抗體包含與選自SEQ ID NOJ1-25之胺基酸序列具有至少90%序列一致性之重鏈可變區及與選自SEQ ID NO:26-31之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 90. 如請求項89之免疫接合物，其中該抗體包含與SEQ ID ΝΟ··24之胺基酸序列具有至少90%序列一致性之重鏈可變區及與SEQ ID ΝΟ:29之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 91. 如請求項90之免疫接合物，其中該抗體包含具有SEQ ID NO:24之胺基酸序列之重鏈可變區及具有SEQ ID NO:29 之胺基酸序列之輕鏈可變區。 92. 如請求項89之免疫接合物，其中該抗體包含與SEQ ID NO:25之胺基酸序列具有至少90%序列一致性之重鏈可變區及與SEQ ID NO:30之胺基酸序列具有至少90%序列一致性之輕鏈可變區。 93. 如請求項92之免疫接合物，其中該抗體包含具有SEQ ID NO:25之胺基酸序列之重鏈可變區及具有SEQ ID NCh30 之胺基酸序列之輕鏈可變區。 94. 一種醫藥組合物，其包含如請求項47、75、77、80、 119007.doc -12· 200813092 82、86、88、91或93中任一項之免疫接合物及醫藥學上可接受之載劑。 95. —種活體外抑制細胞增殖之方法，其包含在容許免疫接合物與TAT226結合之條件下將細胞暴露於如請求項47、 75、77、80、82、86、88、91或93中任一項之免疫接合物。 96·如請求項95之方法，其中該細胞為腫瘤細胞。 97·如請求項96之方法，其中該腫瘤細胞為卵巢腫瘤細胞、子宮腫瘤細胞、腦腫瘤細胞或威爾姆氏腫瘤細胞。 98·如請求項96之方法，其中該細胞為異種移植物。 99· 一種如請求項 47、75、77、80、82、86、88、91 或 93 中任一項之免疫接合物的用途，其係用於製備用以治療細胞增殖性病症之藥劑。 100·如請求項99之用途，其中該細胞增殖性病症係選自卵巢癌、子宮癌、腦腫瘤及威爾姆氏腫瘤。 101·如請求項99之用途，其中該細胞增殖性病症係與ΤΑΤ226 於細胞表面上増加之表現相關聯。 119007.doc 13-Wherein S is a sulfur atom and p is in the range of 2 to 5. 84. The immunoconjugate of claim 83, wherein the antibody comprises 11¥11-1^ having an amino acid sequence consistent with the sequence SEQ 10 1^0:19. 85. The immunoconjugate of claim 84, wherein the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 9 and HVR-L3 having the amino acid sequence of SEQ ID NO: 17. 86. The immunoconjugate of claim 85, wherein the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID ΝΟ; 4; HVR-H2 having the amino acid sequence of SEQ ID NO: 5; HVR-L1 of the amino acid sequence of SEQ ID NChl2 and HVR-L2 of the amino acid sequence of SEQ ID NO: 13. 87. The immunoconjugate of claim 84, wherein the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID 1010 and HVR-L3 having the amino acid sequence of SEQ ID ΝΟ18. The immunoconjugate of claim 87, wherein the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4; HVR having the amino acid sequence of SEQ ID NO: -H2; HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. 89. The immunoconjugate of claim 83, wherein the antibody comprises a heavy chain variable region having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 1-25 and is selected from the group consisting of SEQ ID NO:26 The amino acid sequence of -31 has a light chain variable region with at least 90% sequence identity. 90. The immunoconjugate of claim 89, wherein the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID 及24 and an amino group of SEQ ID NO:29 The acid sequence has a light chain variable region with at least 90% sequence identity. 91. The immunoconjugate of claim 90, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: . 92. The immunoconjugate of claim 89, wherein the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 25 and an amino acid of SEQ ID NO: A sequence has a light chain variable region with at least 90% sequence identity. 93. The immunoconjugate of claim 92, wherein the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NCh30. 94. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 47, 75, 77, 80, 119007.doc -12. 200813092 82, 86, 88, 91 or 93 and pharmaceutically acceptable Carrier. 95. A method of inhibiting cell proliferation in vitro, comprising exposing a cell to a condition such as claim 47, 75, 77, 80, 82, 86, 88, 91 or 93 under conditions permitting binding of the immunoconjugate to TAT226 Any of the immunoconjugates. 96. The method of claim 95, wherein the cell is a tumor cell. The method of claim 96, wherein the tumor cell is an ovarian tumor cell, a uterine tumor cell, a brain tumor cell, or a Wilm's tumor cell. 98. The method of claim 96, wherein the cell is a xenograft. 99. Use of an immunoconjugate according to any one of claims 47, 75, 77, 80, 82, 86, 88, 91 or 93 for the preparation of a medicament for the treatment of a cell proliferative disorder. 100. The use of claim 99, wherein the cell proliferative disorder is selected from the group consisting of ovarian cancer, uterine cancer, brain tumor, and Wilm's tumor. 101. The use of claim 99, wherein the cell proliferative disorder is associated with an expression of ΤΑΤ226 on the cell surface. 119007.doc 13-