TW201206466A

TW201206466A - Antibodies with pH dependent antigen binding

Info

Publication number: TW201206466A
Application number: TW100108288A
Authority: TW
Inventors: Jaume Pons; Jeffrey Raymond Chabot; Riggers Javier Fernando Chaparro; Bruce Charles Gomes; Hong Liang; Kapil Mayawala; Jerome Thomas Ii Mettetal; Arvind Rajpal; David Louis Shelton
Original assignee: Rinat Neuroscience Corp; Pfizer
Priority date: 2010-03-11
Filing date: 2011-03-11
Publication date: 2012-02-16

Abstract

The present invention relates to antibodies with pH dependent binding to its antigen such that the affinity for antigen binding at physiological pH (i.e., pH 7.4) is greater than at endosomal pH (i.e., pH 6.0 or 5.5). In other words, the KD or koff ratio at pH 5.5/ pH 7.4 or at pH 6.0/ pH 7.4 is more than, or ranges between, 2, 3, 4, 8, 10, 16, 20, 30, 40, or 100 or more. Such pH dependent antibodies preferentially dissociate from the antigen in the endosome. This can increase antibody half life, as compared to antibodies with equivalent KDs at pH 7.4 but with no pH dependent binding, when the antigen is one that undergoes antigen-mediated clearance (e.g., PCSK9). Antibodies with pH dependent binding can decrease total antigen half life when the antigen undergoes reduced clearance when bound to antibody (e.g., IL6). Antibodies with pH dependent binding can also prolong the decrease in antigen which is not antibody-bound. This can be important when antagonizing a target antigen typically present at high levels (e.g., IgE, DKK1, C5 and SOST). In addition, such antibodies can increase antigen half life when the antigen is a receptor and the receptor has increased clearance when bound to antibody (e.g, GMCSF receptor).

Description

201206466 pH下與FcRn之結合，進而成功地延長半衰期。在一項非人靈長動物之藥物動力學試驗中，IgGl之T250Q/M428L取代顯示35天之半衰期，顯著延長相較於野生型igGl之14天之半衰期（H i n t ο n e t a 1 ，J I m m u n ο 1 1 7 6 : 3 4 6 - 3 5 6，2 0 0 6 )0 雖然恆定區之取代可顯著改善治療性IgG抗體之功能 ’但在嚴格保守性之恆定區的取代可能具有在人產生免疫原性之風險（Presta，supra，2008; De Groot and Martin, Clin Immunol 131 : 189-201，2009)，而在高度多變性之可變區序列的取代可能較不具免疫原性。關於可變區之報告包括工程化該CDR殘基以改善抗體與抗原之結合親和性 (Rothe et al., Expert 0 p i n Biol Ther 6 : 1 77- 1 87，2006; Bostrom et al., Methods Mol Biol 525 : 3 53-3 76，2009; Thie et al., Methods Mol Biol 525 ： 309-322，2009 ) ' 工程化該CDR及骨架殘基以改善抗體之穩定性（W0rn and Pluckthun, J Mol Biol 305 : 989- 1 0 1 0，200 1; Ewert et al., Methods 34: 1 84-1 99，2004 )及減少抗體免疫原性之風險 (De Groot and Martin, supra, 2009; Jones et al., Methods Mol Bio 525 : 405-423，xiv，2009 )。這些報告指出，藉由利用隨機庫之嗾菌體或核糖體展示之親和性成熟可達到改善之對抗原之親和性。經改善之穩定性可合理地自序列基底及結構基底之合理設計獲得。降低免疫原性風險（去免疫化）可藉由各種人化方法及移除T細胞表位完成，此可利用電腦技術預測或於活體外試驗測定。此外，可變區 201206466 可經工程化以降低pi。在這些抗體觀察到相較於野生型抗體較長之半衰期，雖然具有可相比之FCRn結合力（lgawa et a 1., PEDS， Advance Access, doi : 1 0.1 093/ protein/gzq009, 2010) ° 本發明關於工程化或選擇呈PH依賴性抗原結合之抗體以修飾抗體及/或抗原半衰期。如果抗原媒介之廓清機轉正常地降解當與抗原結合時之抗體，IgG2抗體之半衰期可能會被縮短。同樣地，該抗原：抗體複合物可能影響抗原之半衰期，不是藉由防止抗原受到典型之降解過程以延長半衰期，就是經由抗體媒介之降解以縮短半衰期。本發明關於在pH 7.4相較於內小體pH下（即pH 5.5至6.0)對抗原具有較高之親和性之抗體，致使在諸如pH 5.5/pH 7.4或在pH 6.0/pH 7.4下之KD比係2或2以上。本發明關於對彼之抗原呈該等pH依賴性結合之抗體及設計、製造及使用該等抗體之方法。有用之抗體實例以抗原諸如前蛋白轉化酶枯草溶菌素9 ( PCSK9 )亦稱爲 NARC-1、IgE、dickkopf-相關蛋白 1 ( DKK1 )、補體 5 ( C5)、硬化蛋白（S0ST)及GMCSF受體爲標的。 PC SK9係一在若干形式之家族性高膽固醇血症中被發現之基因突變蛋白質。PCS K9係經合成爲酶原，該酶原之特定模體在內質網中經自催化處理。族群試驗顯示，若干 PCSK9突變係「獲得功能」之突變且見於體染色體顯性高膽固醇血症之個體，然而其他「失去功能（L0F)」之突變係與血漿膽固醇減少有關。此族群之發病率及死亡率試 -8- 201206466 驗清楚地顯示，降低P c S Κ 9之功能顯著地減少心血管疾病之風險。【發明內容】本發明關於抗體類，該等抗體與彼之抗原呈PH依賴性結合，致使在生理性pH (即pH 7.4)之抗原結合親和性高於在內小體pH (即pH 6.0或5.5)之抗原結合親和性。換句話說，在pH 5.5/ρΗ 7·4或pH 6·0/ρΗ 7.4之KD或kw比係大於或介於2、 3、 4、 8、 10、 16、 20、 30、 40或 100或 100以上。該等pH依賴性抗體優先地在內小體內與抗原解離。當抗原係經抗原媒介性廓清者（例如PCSK9 )時，相較於在pH 7.4具有相等KD値但無pH依賴性結合力之抗體，該等與抗原呈pH依賴性結合之抗體可具有增加之抗體半衰期。當與抗體（例如IL6 )結合之抗原的廓清減少時，呈pH依賴性結合之抗體可降低總抗原半衰期。呈pH依賴性結合之抗體亦可延長非與抗體結合之抗原的抗體媒介性減少。此呈pH依賴性結合之抗體對於拮抗通常呈高量存在之標的抗原（例如IgE、DKK1、C5及S0ST)而言可能是重要的。此外，當抗原係受體（例如GMCSF受體）且該受體與抗體結合時之廓清增加時，該等抗體可增加抗原半衰期。本發明以下描述之任何實施態樣中，KD及kcff可於 25°C或37°C下測量。在較佳之實施態樣中，該呈pH依賴性結合之抗體在 pH 7.4相較於pH 6.0下以較高之親和性與抗原特異性結合 -9- 201206466 ’其中在pH 6.0/pH 7.4及25°C下之KD比及/或km比係大於或介於2、3、4、8、10、16或16以上’且其中當暴露於該抗原時，相較於在pH 7.4對該抗原具有類似親和性但在pH 6·0/ρΗ 7.4之相當KD及/或k^f比係小於2且不呈PH依賴性結合之抗體，該抗體具有減少之活體內血漿廓清率。較佳地該抗原不是介白素-6受體（IL6R)，或較佳地該抗體不是如 W0 20 1 0/1 068 1 2 或 W0 2009/04 1 62 1 所揭示之抗-IL6R 抗體 Fv3-m73、F v4 - m 7 3 或 Η 3 p 1/L 7 3。在另一較佳之實施態樣中，該呈pH依賴性結合之抗體在pH 7.4相較於pH 6.0下以較高之親和性與抗原特異性結合，其中在pH 6.0/pH 7.4及25°C下之KD比及/或keff比大於或介於2、3、4、8、10、16或16以上，其中該抗原於活體內具膜結合性及可溶性，且其中相較於在pH 7.4對該抗原具有類似親和性但在pH 6.0/pH 7.4之相當KD及/或1^比係小於2之抗體，該抗體媒介對細胞膜受體增加之定位。較佳地該抗原不是IL6R，或較佳地該抗體不是如W0 2010/106812 或 W0 2009/041621 所揭示之抗-IL6R 抗體Fv3-m73、Fv4-m73或H3pl/L73»在另一較佳之實施態樣中，該抗原係非傳訊誘捕體之可溶性受體。在其他較佳之實施態樣中，該呈pH依賴性結合之抗體係媒介抗體依賴性細胞媒介性細胞毒性（ADCC)及/或補體依賴性細胞毒性（ CDC)之抗體藥物共軛物》本發明包括一種呈pH依賴性結合之抗體，該抗體在 pH 7.4相較於pH 6.0下以較高之親和性與抗原特異性結合 -10- 201206466 ’其中在pH 6.0/pH 7.4及25°C下之KD比及/或k。"比係大於或介於2、3、4、8、10、16或16以上，且其中當非抗體結合之抗原暴露於該抗體時，相較於暴露在pH 7.4對該抗原具有類似親和性但在pH 6.0/pH 7.4之相當KD及/或k。^比係小於2且不呈PH依賴性結合之抗體，該抗原於活體內之量的減少係經延長。本發明提供一種呈pH依賴性結合之抗體，該抗體在 pH 7.4相較於pH 6.0下以較高之親和性與抗原特異性結合，其中在pH 6·0/ρΗ 7.4及25°C下之KD比及/或koff比係大於或介於2、3、4、8、10、16或16以上，且其中相較於在 pH 7.4對與抗體結合之抗原具有類似親和性但在pH 6.Ο/pH 7.4之相當KD及/或k^f比係小於2且不呈pH依賴性結合之抗體，該抗原於活體內之量減少。在較佳之實施態樣中，該抗原係骨橋素。本發明亦提供一種呈pH依賴性結合之促效抗體’該抗體在pH 7.4相較於pH 6.0下以較高之親和性與抗原特異性結合，其中在pH 6.0/pH 7.4及25°C下之KD比及/或koff比係大於或介於2、3、4、8、10、16或16以上，且其中該抗原係受體，且該受體當暴露於該抗體時，相較於暴露於在 pH 7.4對該受體具有類似親和性但在pH 6.Ο/pH 7.4之相當 KD及/或keff比係小於2之抗體，具有減少之活體內廓清率。在較佳之實施態樣中，該受體係GMCSF受體。在任何前述抗體之其他較佳實施態樣中’於pH 6·0/ρΗ 7.4之KD比或k〇ff比係大於或介於20、30、40或1〇〇 -11 - 201206466 或100以上。在其他較佳之實施態樣中，較佳之於pH 6.0/pH 7.4之KD比或kdf比係介於2至3、2至4、2至8、2至 10、2至16或2至20或2至20以上，或3至4、3至8、3至10、 3至16或3至20，或4至8、4至10、4至16或4至20或4至20以上，或8至10、8至16、8至20或8至20以上，或10至16、10 至20或10至20以上，或16至20或16至20以上。在其他先前所述抗體之較佳實施態樣中，該於pH 7.4 及25 °C下與抗原結合之抗體的KD係介於約0.01奈莫耳至約 1〇〇奈莫耳，或更佳地介於約0.1奈莫耳至約10奈莫耳。在其他先前描述抗體之較佳實施態樣中，該抗體與抗原於卩117.4之結合的1{(^係介於約1\1(^-4 3-1至約1\1(^-1 s-1，更佳地介於約 lxlOE-3 s-1 至約 lxlOE-1 s-1。在前述抗體之另一較佳實施態樣中，該抗原係PCS K9 。在一較佳之實施態樣中，該抗-PCSK9抗體不是PCSK9抗體H1M30 0N (見US201 0/0166768)。在其他較佳之實施態樣中，該抗原係IgE、C5或DKK1，且在較佳之實施態樣中，該KD係介於1.0奈莫耳至約10奈莫耳或介於1.0奈莫耳至約100奈莫耳。本發明亦提供一種延長投藥間隔及/或減少治療劑量以利用治療性抗體治療病患之方法，該方法包含對該病患投予治療有效量之先前所述之本發明之抗體中之任一抗體，其中相較於在pH 7.4具有類似親和性但pH 6.0/7.4及25° C下之KD比及/或kafdt係小於2之抗體，該抗體具有延長之藥物藥效學效應及/或半衰期。 -12- 201206466 本發明亦考慮藉由pH依賴性調節抗體結合親和性以製備具有延長之半衰期及/或藥物藥效學效應之抗體之方法，該方法包含選擇可使影響pKa之微環境最佳化之抗體 CDR組胺酸殘基或其他殘基，致使在pH 6.0/pH7.4下抗體抗原結合之KD比及/或k〇ff&係大於或介於2、3、4、8、10 、16或16以上。本發明亦考慮藉此方法製備之抗體，包括具有1、2、3、4、5個或更多個組胺酸取代CDR殘基以使影響pKa之微環境最佳化之抗體。在上述方法之較佳實施態樣中，該方法另包含使抗體突變以達到在25°C及pH 7.4測量時KD爲至少100奈莫耳之抗體親和性。在另一實施態樣中，本發明提供一種抗體庫，其在CDR殘基或其他殘基富含組胺酸以使影響pKa之微環境最佳化。在其他較佳之實施態樣中，本發明提供一種經分離之抗體，該抗體與PCSK9特異性結合且包含源自如SEQ ID NO : 4或5所示之重鏈可變區（VH )胺基酸序列之VH互補決定區1 ( CDR1 ) 、VH CDR2及VH CDR3或在彼等之 CDR1、CDR2及/或CDR3具有一、二、三或更多個保守性胺基酸取代之變異體。在較佳之實施態樣中，該抗體另包含如SEQ ID NO : 3所示之輕鏈可變區（VL)胺基酸序列之VL CDR1、CDR2 及CDR3或在彼等之CDR1、CDR2及/或CDR3具有一、二、三或更多個保守性胺基酸取代之變異體。201206466 Binding to FcRn at pH, which in turn successfully extends half-life. In a non-human primate pharmacokinetic assay, IgGl's T250Q/M428L substitution showed a half-life of 35 days, significantly prolonging the half-life of 14 days compared to wild-type igGl (H int ο neta 1 , JI mmun ο 1 1 7 6 : 3 4 6 - 3 5 6,2 0 0 6 )0 Although substitution of the constant region can significantly improve the function of therapeutic IgG antibodies', substitutions in the strictly conserved constant region may have immunity in humans Risk of primordiality (Presta, supra, 2008; De Groot and Martin, Clin Immunol 131: 189-201, 2009), while substitutions in highly variable, variable region sequences may be less immunogenic. Reporting on variable regions involves engineering the CDR residues to improve binding affinity of the antibody to the antigen (Rothe et al., Expert 0 pin Biol Ther 6 : 1 77- 1 87, 2006; Bostrom et al., Methods Mol Biol 525: 3 53-3 76, 2009; Thie et al., Methods Mol Biol 525 : 309-322, 2009 ) 'Engineering the CDRs and backbone residues to improve antibody stability (W0rn and Pluckthun, J Mol Biol 305: 989- 1 0 1 0,200 1; Ewert et al., Methods 34: 1 84-1 99, 2004 ) and reducing the risk of antibody immunogenicity (De Groot and Martin, supra, 2009; Jones et al. , Methods Mol Bio 525 : 405-423, xiv, 2009 ). These reports indicate that improved affinity for antigens can be achieved by affinity maturation of bacterial or ribosome display using random pools. The improved stability is reasonably achievable from the rational design of the sequence substrate and structural substrate. Reducing the risk of immunogenicity (de-immunization) can be accomplished by various humanization methods and removal of T cell epitopes, which can be predicted using computer technology or in vitro assays. In addition, variable zone 201206466 can be engineered to reduce pi. A longer half-life compared to wild-type antibodies was observed in these antibodies, although there is comparable FCRn binding (lgawa et al 1., PEDS, Advance Access, doi : 1 0.1 093/protein/gzq009, 2010) ° The present invention relates to engineering or selecting antibodies that bind to PH-dependent antigens to modify antibody and/or antigen half-life. If the antigen medium is conditioned to degrade normally, the half-life of the IgG2 antibody may be shortened when the antibody is bound to the antigen. Similarly, the antigen:antibody complex may affect the half-life of the antigen, either by preventing the antigen from undergoing a typical degradation process to extend the half-life, or by degradation of the antibody medium to shorten the half-life. The present invention relates to an antibody having a higher affinity for an antigen at pH 7.4 than at an endosome pH (i.e., pH 5.5 to 6.0), resulting in KD at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4. The ratio is 2 or more. The present invention relates to antibodies which exhibit such pH-dependent binding to their antigens, and methods of designing, manufacturing and using such antibodies. Examples of useful antibodies are antigens such as proprotein convertase subtilisin 9 (PCSK9), also known as NARC-1, IgE, dickkopf-related protein 1 (DKK1), complement 5 (C5), sclerostin (S0ST), and GMCSF. The body is the target. PC SK9 is a gene mutation protein that is found in several forms of familial hypercholesterolemia. PCS K9 is synthesized as a zymogen, and the specific motif of the zymogen is autocatalyzed in the endoplasmic reticulum. Ethnicity trials have shown that several PCSK9 mutations are "obtained as functional" mutations and are found in individuals with somatic chromosomal hypercholesterolemia, whereas other "loss of function (L0F)" mutations are associated with decreased plasma cholesterol. The morbidity and mortality test for this group -8-201206466 clearly shows that reducing the function of P c S Κ 9 significantly reduces the risk of cardiovascular disease. SUMMARY OF THE INVENTION The present invention relates to antibodies which exhibit PH-dependent binding to their antigens such that the antigen binding affinity at physiological pH (ie pH 7.4) is higher than the pH of the endosomal (ie pH 6.0 or 5.5) Antigen binding affinity. In other words, the KD or kw ratio at pH 5.5/ρΗ 7.4 or pH 6·0/ρΗ 7.4 is greater than or between 2, 3, 4, 8, 10, 16, 20, 30, 40 or 100 or More than 100. These pH dependent antibodies preferentially dissociate from the antigen in the inner body. When the antigen is antigen-mediated (eg, PCSK9), the antibody that binds to the antigen in a pH-dependent manner may have an increase compared to an antibody having an equivalent KD値 at pH 7.4 but no pH-dependent binding. Antibody half-life. When the clearance of the antigen bound to the antibody (eg, IL6) is reduced, antibodies that bind in a pH-dependent manner can reduce the total antigen half-life. Antibodies that bind in a pH-dependent manner also prolong the decrease in antibody mediatedness of antigens that are not bound to the antibody. Such pH-dependent binding antibodies may be important for antagonizing antigens (e.g., IgE, DKKl, C5, and SOST) that are normally present in high amounts. Furthermore, when an antigenic receptor (e.g., GMCSF receptor) and the receptor is increased in binding to an antibody, the antibodies increase the antigen half life. In any of the embodiments described below, KD and kcff can be measured at 25 ° C or 37 ° C. In a preferred embodiment, the pH-dependent antibody binds to the antigen with a higher affinity at pH 7.4 compared to pH 6.0. 9-201206466 'At pH 6.0/pH 7.4 and 25 The KD ratio and/or km ratio at °C is greater than or between 2, 3, 4, 8, 10, 16 or 16' and wherein when exposed to the antigen, the antigen is present at pH 7.4 An antibody having similar affinity but having a KD and/or k^f ratio of less than 2 at pH 6·0/ρΗ 7.4 and not PH-dependent binding has a reduced in vivo plasma clearance rate. Preferably, the antigen is not the interleukin-6 receptor (IL6R), or preferably the antibody is not an anti-IL6R antibody Fv3 as disclosed in WO 20 1 0/1 068 1 2 or WO 2009/04 1 62 1 -m73, F v4 - m 7 3 or Η 3 p 1/L 7 3. In another preferred embodiment, the pH-dependent antibody binds to the antigen with a higher affinity at pH 7.4 compared to pH 6.0, wherein at pH 6.0/pH 7.4 and 25 ° C. The KD ratio and/or keff ratio is greater than or equal to 2, 3, 4, 8, 10, 16 or more, wherein the antigen has membrane binding and solubility in vivo, and wherein compared to pH 7.4 The antigen has a similar affinity but at a pH of 6.0/pH 7.4, an equivalent KD and/or a ratio of less than 2, the antibody mediator increases the localization of the cell membrane receptor. Preferably, the antigen is not IL6R, or preferably the antibody is not an anti-IL6R antibody Fv3-m73, Fv4-m73 or H3pl/L73» as disclosed in WO 2010/106812 or WO 2009/041621. In the aspect, the antigen is a soluble receptor of a non-message trap. In other preferred embodiments, the pH-dependently binding anti-systemic antibody-dependent cellular vector cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) antibody drug conjugates. Included is a pH-dependent antibody that binds to the antigen with high affinity at pH 7.4 compared to pH 6.0 - 201206466466 - at pH 6.0 / pH 7.4 and 25 ° C KD ratio and / or k. "The ratio is greater than or between 2, 3, 4, 8, 10, 16 or more, and wherein when the non-antibody-bound antigen is exposed to the antibody, it has a similar affinity to the antigen compared to exposure to pH 7.4. Sexually but at a pH of 6.0/pH 7.4 is equivalent to KD and / or k. An antibody having a ratio of less than 2 and not PH-dependent binding, the amount of the antigen being reduced in vivo is prolonged. The present invention provides an antibody that binds in a pH-dependent manner, and the antibody specifically binds to an antigen with a higher affinity at pH 7.4 than pH 6.0, wherein at pH 6.1/ρΗ 7.4 and 25 ° C The KD ratio and/or koff ratio is greater than or between 2, 3, 4, 8, 10, 16 or 16 and wherein it has a similar affinity to the antigen bound to the antibody at pH 7.4 but at pH 6. An antibody having a KD and/or k^f ratio of Ο/pH 7.4 that is less than 2 and not pH-dependently binds, and the amount of the antigen in vivo is reduced. In a preferred embodiment, the antigen is osteopontin. The invention also provides an agonistic antibody that binds in a pH-dependent manner. The antibody specifically binds to the antigen with a higher affinity at pH 7.4 compared to pH 6.0, wherein the pH is 6.0/pH 7.4 and 25 ° C. The KD ratio and/or koff ratio is greater than or between 2, 3, 4, 8, 10, 16 or 16 and wherein the antigen is a receptor and the receptor is exposed to the antibody as compared to Exposure to an antibody having a similar affinity for the receptor at pH 7.4 but having a KD and/or keff ratio of less than 2 at pH 6.Ο/pH 7.4 has a reduced in vivo clearance rate. In a preferred embodiment, the receptor is a GMCSF receptor. In other preferred embodiments of any of the foregoing antibodies, the KD ratio or k〇ff ratio at pH 6·0/ρΗ 7.4 is greater than or between 20, 30, 40 or 1〇〇-11 - 201206466 or above. . In other preferred embodiments, preferably the KD ratio or kdf ratio of pH 6.0/pH 7.4 is between 2 and 3, 2 to 4, 2 to 8, 2 to 10, 2 to 16, or 2 to 20 or 2 to 20 or more, or 3 to 4, 3 to 8, 3 to 10, 3 to 16, or 3 to 20, or 4 to 8, 4 to 10, 4 to 16, or 4 to 20 or 4 to 20 or more, or 8 To 10, 8 to 16, 8 to 20 or 8 to 20 or more, or 10 to 16, 10 to 20 or 10 to 20 or more, or 16 to 20 or 16 to 20 or more. In a preferred embodiment of the other previously described antibodies, the KD line of the antibody that binds to the antigen at pH 7.4 and 25 ° C is between about 0.01 nanomolar to about 1 nanomolar, or better. The ground is between about 0.1 nanograms to about 10 nanometers. In other preferred embodiments of the previously described antibodies, the binding of the antibody to the antigen 卩117.4 is 1{(^ is between about 1\1(^-4 3-1 to about 1\1(^-1) S-1, more preferably from about lxlOE-3 s-1 to about lxlOE-1 s-1. In another preferred embodiment of the aforementioned antibody, the antigen is PCS K9. In a preferred embodiment In this manner, the anti-PCSK9 antibody is not a PCSK9 antibody H1M30 0N (see US201 0/0166768). In other preferred embodiments, the antigen is IgE, C5 or DKK1, and in a preferred embodiment, the KD The system is between 1.0 nanomolar to about 10 nanomolar or between 1.0 nanomolar to about 100 nanomolar. The invention also provides an extended dosing interval and/or a reduced therapeutic dose for treating a patient with a therapeutic antibody. Method, the method comprising administering to the patient a therapeutically effective amount of any of the antibodies of the invention described above, wherein the antibody has similar affinity at pH 7.4 but at pH 6.0/7.4 and 25 ° C An antibody having a KD ratio and/or a kafdt system of less than 2, the antibody having an extended pharmacodynamic effect and/or a half life. -12- 201206466 The present invention also contemplates A method of modulating antibody binding affinity to produce an antibody having an extended half-life and/or pharmacodynamic effect, the method comprising selecting an antibody CDR histidine residue or other such that the microenvironment affecting the pKa is optimized a residue such that the KD ratio and/or k〇ff&> of antibody antigen binding at pH 6.0/pH 7.4 is greater than or equal to 2, 3, 4, 8, 10, 16 or above. The invention also contemplates Antibodies prepared by this method include antibodies having 1, 2, 3, 4, 5 or more histamine-substituted CDR residues to optimize the microenvironment affecting the pKa. In another embodiment, the method further comprises mutating the antibody to achieve an antibody affinity of KD of at least 100 nanomolar when measured at 25 ° C and pH 7.4. In another embodiment, the invention provides an antibody library, The CDR residues or other residues are enriched in histidine to optimize the microenvironment affecting the pKa. In other preferred embodiments, the invention provides an isolated antibody that specifically binds to PCSK9 and comprises A heavy chain variable region (VH) amine group derived from SEQ ID NO: 4 or 5 The VH complementarity determining region 1 (CDR1), VH CDR2 and VH CDR3 of the sequence or variants of one, two, three or more conservative amino acid substitutions in the CDR1, CDR2 and/or CDR3 thereof. In a preferred embodiment, the antibody further comprises VL CDR1, CDR2 and CDR3 of the light chain variable region (VL) amino acid sequence set forth in SEQ ID NO: 3 or CDR1, CDR2 and/or CDR3 thereof A variant having one, two, three or more conservative amino acid substitutions.

本發明亦提供與PCSK9特異性結合且包含具有如SEQ -13- 201206466 ID NO : 6所示之胺基酸序列之重鏈可變區（VH )互補決定區1 ( CDR1 )、具有如SEQ ID NO : 7所示之胺基酸序列之VH CDR2、及/或具有如SEQ ID NO : 8所示之胺基酸序列之VH CDR3，或在彼等之CDR1、CDR2及/或CDR3具有一或多個保守性胺基酸取代之變異體之經分離之抗體，以及與PC SK9特異性結合且包含具有如SEQ ID NO : 6所示之胺基酸序列之VH CDR1、具有如SEQ ID NO : 7所示之胺基酸序列之VH CDR2、及/或具有如SEQ ID NO : 9所示之胺基酸序列之VH CDR3，或在彼等之CDR1、CDR2及/或 CDR3具有——二、三個或更多個保守性胺基酸取代之變異體之經分離之抗體。在另一實施態樣中，本發明考慮經分離之抗體，該抗體包含具有如SEQ ID NO: 10所示之胺基酸序列之輕鏈可變區（VL) CDR1、具有如SEQ ID NO: 11所示之胺基酸序列之VL CDR2及/或具有如SEQ ID NO : 12所示之胺基酸序列之VL CDR3，或在彼等之CDR1、CDR2及/或CDR3具有一、二、三個或更多個保守性胺基酸取代之變異體。在上述之較佳實施態樣中，該抗體另包含具有如SEQ ID NO: 10所示之胺基酸序列之VL CDR1、具有如SEQ ID NO: 11所示之胺基酸序列之VL CDR2及/或具有如SEQ ID NO: 12所示之胺基酸序列之VL CDR3，或在彼等之CDR1 、CDR2及/或CDR3具有一、二、三個或更多個保守性胺基酸取代之變異體，更佳地，該VH區包含SEQ ID NO: 4或 SEQ ID NO: 5且該VL區包含SEQ ID NO: 3或在彼等之 -14- 201206466 SEQ ID NO : 4、SEQ ID NO : 5及/或 SEQ ID NO : 3具有一、二、三個或更多個保守性胺基酸取代之變異體。在本發明之PCSK9抗體之另一較佳實施態樣中，該抗體具有一或多個Fc突變，較佳地N434S、N434H、M428L-N434H 雙突變、M428L-N434A 雙突變、T250Q-M428L 雙突變或M428L-N434S雙突變。在另一實施態樣中，本發明提供抗體或彼之抗原結合部分，該抗體或彼之抗原結合部分係由寄存於美國菌種保存中心（American Type Culture Collection)且具有 ATCC 登記號PTA-10547或PTA_10548及/或PTA-10549之質體所編碼6 本發明亦考慮醫藥組成物，該等組成物包含治療有效量之任何前述之抗體、重組產製任何前述抗體之宿主細胞、編碼任何前述抗體之經分離之核酸及編碼任何前述抗體之經分離之核酸。本發明亦考慮一種減少有需要之個體的血液LDL膽固醇之量之方法，該方法包含對該個體投予治療有效量之以 PCS K9抗原爲標的之本發明之任何抗體。本發明之詳細說明本發明關於抗體類，該等抗體與彼之抗原呈pH依賴性結合’致使在生理性p Η (即p Η 7 · 4 )之抗原結合親和性高於在內小體pH (即pH 6.0或5.5 )之抗原結合親和性。換句話說，在 pH 5.5/pH 7.4 或 pH 6·0/ρΗ 7.4 之 KD 或 k。"比 -15- 201206466 係大於或介於 2、3、4、8、10、16、20、30、40 或 100 或 100以上。該等pH依賴性抗體優先地在內小體內與抗原解離。當抗原係經抗原媒介性廓清者（例如PC SK9 )時，相較於在pH 7.4具有相等KD値但無pH依賴性結合力之抗體，該等與抗原呈pH依賴性結合之抗體可具有增加之抗體半衰期。當與抗體（例如IL6 )結合之抗原的廓清減少時，呈pH依賴性結合之抗體可降低平均總抗原半衰期。呈pH 依賴性結合之抗體亦可延長非與抗體結合之抗原的減少。此呈pH依賴性結合之抗體對於拮抗通常呈高量存在之標的抗原（例如IgE、DKK1、C5及SOST)而言可能是重要的。此外，當抗原係受體（例如GMCSF受體）且該受體與抗體結合時之廓清增加時，該等抗體可增加抗原半衰期。若抗原媒介標的媒介性（target-mediated)降解，那麼當該抗原舉例來說進行標的媒介性廓清時（例如PCSK9 )，利用該等呈p Η依賴性結合之抗體達成在內小體內之解離可增加該抗體之藥物藥效學效應。該呈pH依賴性結合之抗體自該抗原解離後不再受抗原媒介性降解，其可經 FcRn結合後再循環至細胞外，且相較於在pH 7.4具有類似 KD但無pH依賴性結合力之抗體，該呈pH依賴性結合之抗體將具有更長之半衰期。當可溶性抗原（例如IgE、C5、DKK1或SOST)以高濃度存在時，使用該等呈pH依賴性結合之抗體亦具有治療效用。當抗體與內小體內之抗原解離且抗原在溶小體內降解時，該抗體可再循環至血漿以與其他游離抗原結合， -16- 201206466 相較於在pH 7.4具有類似KD但無pH依賴性結合力之抗體，此抗體可延長非抗體結合之抗原的減少且可減少該所需之治療劑量。此外，當抗原係以膜結合和可溶性二種形式存在時，例如受體，使用呈pH依賴性結合之抗體可有效地增進抗體與該膜結合形式抗原之結合。藉由與該可溶性形式抗原解離，抗體有更多與膜形式抗原再結合之機會，使更多抗體位在細胞膜附近。若抗體與膜形式抗原經雙價結合，經由親和力（avidity )之作用，可能使該抗體之有效親和性變高，或使該有效解離速率變慢。此可被應用於使用抗體藥物共軛物（ADC )標的以膜結合及可溶性二種形式存在之抗原。在包含FcRn之內皮細胞中，可溶性抗原將被再循環至血漿區室內之ADC清除，使抗體得以與膜結合抗原結合。使用呈pH依賴性結合之抗體時，增加與膜結合形式之抗原的雙價或單價性結合，將造成增加之抗體與膜結合抗原之內化及細胞死亡。若與該受體以雙價結合’親合力可能增加有效親和性或減緩有效解離速率。 ADCC及補體依賴性細胞毒性（CDC)之機轉亦可利用呈pH依賴性結合之抗體加以探討。在包含FcRn之內皮細胞中’可溶性抗原將被再循環至血漿區室內之ADC清除，使抗體得以與膜結合抗原結合。自可溶性受體釋放抗體將增加接著可與膜結合抗原結合之可用游離抗體，並增加細胞死亡。 -17- 201206466 一般技術除非另外說明，本發明之實施將採用分子生物學（包括重組技術）、微生物學、細胞生物學、生物化學及免疫學之習用技術，這些技術係該領域之習知技藝。該等技術係於文獻中充分解釋，諸如Molecular Cloning: A Laboratory Manual, second edition ( Sambrook et al., 1 9 8 9 ) Cold Spring Harbor Press ; OligonucleotideThe present invention also provides a heavy chain variable region (VH) complementarity determining region 1 (CDR1) which specifically binds to PCSK9 and comprises an amino acid sequence as shown in SEQ-13-201206466 ID NO: 6, having SEQ ID The VH CDR2 of the amino acid sequence shown by NO: 7 and/or the VH CDR3 having the amino acid sequence as shown in SEQ ID NO: 8, or having CDR1, CDR2 and/or CDR3 in one or An isolated antibody of a plurality of conservative amino acid substituted variants, and a VH CDR1 that specifically binds to PC SK9 and comprises an amino acid sequence as set forth in SEQ ID NO: 6 has SEQ ID NO: The VH CDR2 of the amino acid sequence shown in 7 and/or the VH CDR3 having the amino acid sequence as shown in SEQ ID NO: 9, or the CDR1, CDR2 and/or CDR3 thereof have —— An isolated antibody of three or more conservative amino acid substituted variants. In another embodiment, the invention contemplates an isolated antibody comprising a light chain variable region (VL) CDR1 having an amino acid sequence as set forth in SEQ ID NO: 10 having SEQ ID NO: The VL CDR2 of the amino acid sequence shown in Figure 11 and/or the VL CDR3 having the amino acid sequence as shown in SEQ ID NO: 12, or the CDR1, CDR2 and/or CDR3 thereof have one, two, three One or more conservative amino acid substituted variants. In a preferred embodiment of the invention, the antibody further comprises a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 10, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11 and / or a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 12, or having one, two, three or more conservative amino acid substitutions in the CDR1, CDR2 and/or CDR3 thereof Variant, more preferably, the VH region comprises SEQ ID NO: 4 or SEQ ID NO: 5 and the VL region comprises SEQ ID NO: 3 or at -14-201206466 SEQ ID NO: 4, SEQ ID NO : 5 and/or SEQ ID NO: 3 has one, two, three or more conservative amino acid substituted variants. In another preferred embodiment of the PCSK9 antibody of the present invention, the antibody has one or more Fc mutations, preferably N434S, N434H, M428L-N434H double mutation, M428L-N434A double mutation, T250Q-M428L double mutation Or M428L-N434S double mutation. In another embodiment, the invention provides an antibody or antigen binding portion thereof, the antibody or antigen binding portion thereof being deposited in the American Type Culture Collection and having ATCC Accession No. PTA-10547 Or the plastids of PTA_10548 and/or PTA-10549. The invention also contemplates pharmaceutical compositions comprising a therapeutically effective amount of any of the foregoing antibodies, a host cell recombinantly producing any of the foregoing antibodies, encoding any of the foregoing antibodies An isolated nucleic acid and an isolated nucleic acid encoding any of the foregoing antibodies. The invention also contemplates a method of reducing the amount of blood LDL cholesterol in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any of the antibodies of the invention, which is based on the PCS K9 antigen. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antibodies which exhibit pH-dependent binding to their antigens such that the antigen binding affinity in physiological p Η (i.e., p Η 7 · 4 ) is higher than that in endosome pH. (i.e., pH 6.0 or 5.5) antigen binding affinity. In other words, KD or k at pH 5.5/pH 7.4 or pH 6·0/ρΗ 7.4. " -15- 201206466 is greater than or between 2, 3, 4, 8, 10, 16, 20, 30, 40 or 100 or more. These pH dependent antibodies preferentially dissociate from the antigen in the inner body. When the antigen is antigen-mediated (eg, PC SK9), the antibody that binds to the antigen in a pH-dependent manner may have an increase compared to an antibody that has an equivalent KD値 at pH 7.4 but no pH-dependent binding. The half-life of the antibody. When the clearance of the antigen bound to the antibody (e.g., IL6) is reduced, antibodies that bind in a pH-dependent manner reduce the average total antigen half-life. Antibodies that bind in a pH-dependent manner can also prolong the reduction of antigens that are not bound to the antibody. Such pH-dependent binding antibodies may be important for antagonizing the antigens (e.g., IgE, DKKl, C5, and SOST) that are normally present in high amounts. Furthermore, when an antigenic receptor (e.g., GMCSF receptor) and the receptor is increased in binding to an antibody, the antibodies increase the antigen half-life. If the target of the antigen is degraded by target-mediated, then when the antigen is exemplified by the mediator (for example, PCSK9), the dissociation in the inner body can be achieved by using the antibody that is p-dependently bound. Increase the pharmacodynamic effect of the antibody. The pH-dependently bound antibody is no longer subject to antigenic degradation after dissociation from the antigen, which can be recycled to the cell via FcRn binding and has a similar KD but no pH-dependent binding at pH 7.4. The antibody, the pH-dependent antibody will have a longer half-life. When a soluble antigen (e.g., IgE, C5, DKK1 or SOST) is present in a high concentration, the use of such pH-dependently bound antibodies also has therapeutic effects. When the antibody dissociates from the antigen in the endosome and the antigen degrades in the lysosomal, the antibody can be recycled to the plasma for binding to other free antigens, -16-201206466 has a similar KD but no pH dependence at pH 7.4 A binding antibody that increases the reduction of non-antibody binding antigen and reduces the therapeutic dose required. Furthermore, when the antigen is present in both membrane-bound and soluble forms, such as receptors, the use of antibodies that bind in a pH-dependent manner is effective to enhance binding of the antibody to the membrane-bound form of the antigen. By dissociating from the soluble form of the antigen, the antibody has more chance of recombining with the membrane form antigen, allowing more of the antibody to be located near the cell membrane. If the antibody is bivalently bound to the membrane-form antigen, the affinity of the antibody may be made high by the action of avidity, or the effective dissociation rate may be slowed down. This can be applied to antigens present in both membrane-bound and soluble forms using the antibody drug conjugate (ADC). In endothelial cells containing FcRn, the soluble antigen will be recycled to the ADC in the plasma compartment for clearance, allowing the antibody to bind to the membrane-bound antigen. When a pH-dependently bound antibody is used, increased bivalent or monovalent binding to the membrane-bound antigen will result in increased internalization and cell death of the antibody-bound antigen. Bivalent binding to the receptor' affinity may increase the effective affinity or slow the rate of effective dissociation. ADCC and complement dependent cytotoxicity (CDC) can also be explored using antibodies that are pH dependent. In endothelial cells containing FcRn, the soluble antigen will be recirculated to the plasma compartment for ADC clearance, allowing the antibody to bind to the membrane-bound antigen. Release of antibodies from soluble receptors will increase the available free antibodies that can then bind to membrane-bound antigens and increase cell death. -17- 201206466 General Techniques Unless otherwise stated, the practice of the present invention will employ techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well-known techniques in the art. . These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide

Synthesis ( M. J. Galt, ed., 1 9 8 4 ) ； Methods in MolecularSynthesis ( M. J. Galt, ed., 1 9 8 4 ) ; Methods in Molecular

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Culture : Laboratory Procedures ( A. Doyle, J. B. Griffiths, and D.G. Newell, eds., 1 993 - 1 998 ) J. Wiley and Sons ； Methods in Enzymology ( Academic Press, Inc. ) ;Handbook of Experimental Immunology ( D. M. Weir and C.C. Blackwell, eds.) ; Gene Transfer Vectors for Mammalian Cells ( J.M. Miller and M.P. Calos, eds., 1987 ) ；Current Protocols in Molecular Biology ( F.M.Culture : Laboratory Procedures ( A. Doyle, JB Griffiths, and DG Newell, eds., 193 - 1 998 ) J. Wiley and Sons ; Methods in Enzymology ( Academic Press, Inc. ) ; Handbook of Experimental Immunology ( DM Weir and CC Blackwell, eds.) ; Gene Transfer Vectors for Mammalian Cells ( JM Miller and MP Calos, eds., 1987 ) ; Current Protocols in Molecular Biology ( FM

Ausubel et al., eds., 1 98 7 ) ; PCR : The Polymerase Chain Reaction, ( Mullis et al., eds., 1 994 ) ； Current Protocols in Immunology ( J.E. Coligan et al., eds., 1991 -18- 201206466 )；Short Protocols in Molecular Biology ( Wiley and Sons, 19 9 9 ) ； Immunobiology ( C.A. Jane way and P. Travers, 1 9 9 7 ) ; Antibodies ( P. Finch, 1 9 9 7 );Ausubel et al., eds., 1 98 7 ) ; PCR : The Polymerase Chain Reaction, ( Mullis et al., eds., 1 994 ) ; Current Protocols in Immunology ( JE Coligan et al., eds., 1991 -18 - 201206466); Short Protocols in Molecular Biology (Wiley and Sons, 19 9 9 ); Immunobiology (CA Jane way and P. Travers, 1 9 9 7 ); Antibodies ( P. Finch, 1 9 9 7);

Antibodies : a practical approach ( D. Catty., e d., IRL Press, 1 98 8- 1 989 ) ; Monoclonal antibodies : a practical approach ( P. Shepherd and C. Dean, eds., Oxford University Press, 2 0 0 0 ) ； Using antibodies : a laboratory manual ( E. Harlow and D. Lane ( Cold Spring Harbor Laboratory Press, 1 9 9 9 ) ； The Antibodies ( M. Zanetti and J. D . Capra, e d s ., Harwood Academic Publishers, 19 9 5 定義「抗體」係免疫球蛋白分子，其可透過位於該免疫球蛋白分子之可變區的至少一個抗原辨認區特異性地與標的結合，諸如碳水化合物、多核苷酸、脂肪、多肽等。此處所使用之該用語不僅包含完整之多株或單株抗體，但亦包含彼等之任何抗原結合片段（即「抗原結合部分」）或彼等之單鏈、包含抗體之融合蛋白、及任何其他含有抗原辨認區之經修飾構型之免疫球蛋白分子，包括例如但不限於單鏈（scFv )及結構域抗體（例如人、駱駝或鯊魚結構域抗體）、大型抗體（maxibodies )、迷你抗體（ minibodies )、細胞內抗體（intrabodies )、雙價抗體、三價抗體、四價抗體、vNAR及bis-scFv (見例如Hollinger -19- 201206466 and Hudson, Nature Biotech 23 : 1 1 26- 1 1 36，2005 )。抗體包括任何類型之抗體，諸如IgG、IgA或IgM (或彼之亞型），且該抗體不需要是任何特定類型。根據彼之重鏈的恆定結構域之抗體胺基酸序列，免疫球蛋白可被分成不同類型。有五種主要的免疫球蛋白類型：IgA' IgD、IgE、 IgG及IgM，其中某些類型可進一步分成亞型（同型），例如 IgGl、IgG2、IgG3、IgG4、IgAl 和 IgA2。對應不同類型之免疫球蛋白的重鏈恆定結構域分別被稱爲α、5、 ε、r和#。不同類型之免疫球蛋白的次單位結構及三維構型係廣爲週知。抗體之「抗原結合部位」用語在此處係指完整抗體之一或多個片段，該片段保留與給定抗原（例如標的X)特異性結合之能力。抗體之抗原結合功能可藉由完整抗體之片段實現。抗體之「抗原結合片段」用語所涵蓋之結合片段之實例包括Fab、Fab1 ' F ( ab' ) 2、由VH及CH1結構域組成之Fd片段、由抗體單臂之VL及VH結構域組成之Fv片段、單一結構域抗體（dAb)片段（Ward et al.，Nature 341 :544-546，1 989 )及經分離之互補決定區（CDR)。此處所使用之「CDRj可能根據任何卡巴（Kabat)、柯西亞（Chothia)、擴展、AbM、完整及/或構形定義加以定義。在特定抗體中組成CDR之胺碁酸殘基之特性可利用該領域廣爲周知之方法加以測定。此處所使用之抗體 CDR可能被識別爲原本由卡巴等人所定義之超變異區。見例如 Kabat et al.， 1992， Sequences of Proteins of -20- 201206466Antibodies : a practical approach ( D. Catty., e d., IRL Press, 1 98 8- 1 989 ) ; Monoclonal antibodies : a practical approach ( P. Shepherd and C. Dean, eds., Oxford University Press, 2 0 0 antibodies are used in E. Harlow and D. Lane ( Cold Spring Harbor Laboratory Press, 1 9 9 9 ; The Antibodies ( M. Zanetti and J. D. Capra, eds ., Harwood Academic Publishers , 19 9 5 defines an "antibody" immunoglobulin molecule that specifically binds to a target, such as a carbohydrate, a polynucleotide, a fat, through at least one antigen recognition region located in a variable region of the immunoglobulin molecule. Polypeptide, etc. The term used herein includes not only intact multi-strain or monoclonal antibodies, but also any antigen-binding fragments thereof (ie, "antigen-binding portions") or their single-stranded, antibody-containing fusion proteins. And any other immunoglobulin molecule comprising a modified configuration of the antigen recognition region, including, for example, but not limited to, single-stranded (scFv) and domain antibodies (eg, human, camel or shark) Domain antibodies), large antibodies (maxibodies), minibodies, intrabodies, bivalent antibodies, trivalent antibodies, tetravalent antibodies, vNAR and bis-scFv (see eg Hollinger -19- 201206466 and Hudson, Nature Biotech 23 : 1 1 26- 1 1 36, 2005 ). Antibodies include any type of antibody, such as IgG, IgA or IgM (or subtypes thereof), and the antibody need not be of any particular type. The antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be divided into different types. There are five major types of immunoglobulins: IgA' IgD, IgE, IgG and IgM, some of which can be further divided into Subtypes (homotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains corresponding to different types of immunoglobulins are referred to as α, 5, ε, r, and #, respectively. The subunit structures and three-dimensional conformation systems of different types of immunoglobulins are well known. The term "antigen-binding site" of an antibody refers herein to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., the target X). The antigen binding function of the antibody can be achieved by a fragment of the intact antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include Fab, Fab1 'F (ab'), Fd fragment consisting of VH and CH1 domains, and VL and VH domains consisting of one arm of the antibody. Fv fragments, single domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1 989) and isolated complementarity determining regions (CDRs). As used herein, "CDRj may be defined in terms of any Kabat, Chothia, Extension, AbM, complete and/or conformational definitions. The properties of the amine carboxylic acid residues that make up the CDRs in a particular antibody may be utilized The field is well known in the art and the CDRs of the antibodies used herein may be identified as hypervariable regions originally defined by Kabbah et al. See, for example, Kabat et al., 1992, Sequences of Proteins of -20-201206466

Immunological Interest, 5th ed., Public Health Service, NIH，Washington D.C.。CDR之位置亦可能被識別爲最早由柯西亞等人所描述之結構性環圈結構。見例如Chothia et al., Nature 342 ： 8 77- 8 83, 1 9 8 9。其他識別 C D R 之方法包括「AbM定義」，該法爲卡巴法及柯西亞法之折衷，係源自利用牛津分子（Oxford Molecular)之AbM抗體模型軟體（現爲 Accelrys®)，或如 MacCallum et al.，J. Mol· Biol. 262: 732-745，1 996所述根據觀察到之抗原接觸之 CDR之「接觸定義」。在此處稱爲CDR之「構形定義」之另一方法中，CDR之位置可能被鑑別爲對抗原結合造成焓貢獻之殘基。見例如 Makabe et al.，Journal of Biological Chemistry，283: 1156-1166，2008。雖然其他 CDR 邊界定義可能不嚴格遵守上述之方法，但將與至少部分之卡巴 CDR重疊，不過它們可能根據特定殘基或殘基群或甚至整個CDR不顯著影響抗原結合之預測或實驗結果被縮短或延長。此處所使用之CDR可能指由任何該領域已知之方法（包括多種方法之組合）所定義之CDR。此處所使用之「單株抗體」係指自實質上同源之抗體族群獲得之抗體，意即除了可能少量存在之可能天然發生之突變以外，組成該族群之個別抗體係相同的。單株抗體具高度專一性，其係以單一抗原部位爲標的。另外，和通常包含拮抗不同決定簇（表位）之不同抗體的多株抗體製劑不同的是，各單株抗體係以抗原上之單一決定簇爲標的。修飾語「單株」表示抗體係自實質上同源之抗體族群獲 -21 - 201206466 得之特徵，不應被視爲需要藉由任何特定之方法產製該抗體。舉例來說，本發明所使用之單株抗體可藉由最早由 Kohler and Milstein，1 975，Nature 256 : 495 所描述之雜交瘤方法製備，或可藉由重組DNA方法諸如美國專利第 4,8 1 6,5 67號所述者製備。該單株抗體亦可自例如利用Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The location of the CDR may also be identified as the structural loop structure first described by Kosia et al. See, for example, Chothia et al., Nature 342: 8 77- 8 83, 1 9 8 9 . Other methods for identifying CDRs include "AbM definition," a compromise between the Kabbah and Koccia methods, derived from the use of Oxford Molecular's AbM antibody model software (now Accelrys®), or as MacCallum et al. J. Mol Biol. 262: 732-745, 1 996 "Contact definition" based on the observed CDRs of antigen contact. In another method, referred to herein as a "configuration definition" of a CDR, the position of the CDR may be identified as a residue that contributes to the antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283: 1156-1166, 2008. While other CDR boundary definitions may not strictly adhere to the above methods, they will overlap with at least a portion of the Kappa CDRs, although they may be shortened based on predictions or experimental results that do not significantly affect antigen binding based on a particular residue or group of residues or even the entire CDR. Or extended. A CDR as used herein may refer to a CDR as defined by any method known in the art, including combinations of methods. As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibody, i.e., the individual resistant systems that make up the population are identical except for mutations that may occur naturally in small amounts. Individual antibodies are highly specific and are characterized by a single antigenic site. In addition, unlike a plurality of antibody preparations which usually comprise different antibodies antagonizing different determinants (epitopes), each monoclonal antibody system is based on a single determinant on the antigen. The modifier "single plant" indicates that the anti-system is derived from a substantially homologous group of antibodies and is not considered to be required to produce the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be prepared by the hybridoma method first described by Kohler and Milstein, 1 975, Nature 256: 495, or by recombinant DNA methods such as U.S. Patent No. 4,8 Prepared as described in No. 1,6,567. The monoclonal antibody can also be utilized, for example,

McCafferty et al.，1990，Nature 348 : 5 52-5 54描述之技術所產製之噬菌體庫分離。此處所使用之「人化」抗體係指非人（例如鼠）抗體之形式，其爲包含源自非人免疫球蛋白之最少序列之嵌合性免疫球蛋白、免疫球蛋白鏈或彼等之片段（諸如Fv、 Fab、Fab’、F ( ab’）2或抗體之其他抗原結合子序列）。較佳地，人化抗體係其中源自接受者之互補決定區（CDR )的殘基被源自諸如具有該所欲特異性、親和性及能力之小鼠、大鼠或兔等非人物種（捐贈者抗體）之CDR的殘基所取代之人免疫球蛋白（接受者抗體）。在一些情況中，人免疫球蛋白之Fv骨架區（FR )殘基被對應之非人殘基取代。另外，該人化抗體可能包含不在接受者抗體或經導入之CDR或骨架序列中之殘基，但該些殘基被包括以進一步改善及最佳化抗體之表現。整體而言，該人化抗體將包含實質上所有之至少一個且通常兩個可變結構域，其中所有或實質上所有之CDR區對應非人免疫球蛋白之CDR區且所有或實質上所有之FR區係人免疫球蛋白共同序列之FR 區。該人化抗體亦將理想地包含至少部分之免疫球蛋白恆定區或結構域（Fc)，通常爲人免疫球蛋白之該部分。較 •22- 201206466 佳的是具有如WO 99/58572所述之修飾的Fc區之抗體。其他形式之人化抗體具有一或多個相對於原始抗體經改變之 CDR ( CDR LI、CDR L2、CDR L3、CDR HI、CDR H2及 / 或CDR H3 )，該等CDR亦被稱爲「源自」原始抗體之一或多個CDR的一或多個CDR。此處所使用之「人抗體」係指具有對應可由人產製及 /或利用任何該領域之技藝人士所知或此處所揭示之製造人抗體之技術所製備之抗體的胺基酸序列之胺基酸序列的抗體。此定義之人抗體包括含有至少一個人重鏈多肽或至少一個人輕鏈多肽之抗體。一個該等實例係包含鼠輕鏈及人重鏈多肽之抗體。人抗體可利用該領域已知之多種技術製備。在一實施態樣中，該人抗體係選自噬菌體庫，其中該職菌體庫表現人抗體（Vaughan et al·，1 996, Nature Biotechnology, 1 4 : 3 0 9-3 1 4; Sheets et al., 1 998, Proc.McCafferty et al., 1990, Nature 348: 5 52-5 54 Techniques for the isolation of phage libraries produced. As used herein, a "humanized" anti-system refers to a form of a non-human (eg, murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or the like that comprises minimal sequence derived from a non-human immunoglobulin. Fragments (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding sequence of antibodies). Preferably, the humanized anti-system is derived from a residue of the complementarity determining region (CDR) of the recipient derived from a non-human species such as a mouse, rat or rabbit having the desired specificity, affinity and ability. Human immunoglobulin (recipient antibody) substituted with the residue of the CDR of the (donor antibody). In some cases, the Fv framework region (FR) residue of a human immunoglobulin is replaced by a corresponding non-human residue. In addition, the humanized antibody may comprise residues that are not in the recipient antibody or in the introduced CDR or backbone sequences, but such residues are included to further improve and optimize the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin and all or substantially all of them The FR region is the FR region of the human immunoglobulin consensus sequence. The humanized antibody will also desirably comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that portion of a human immunoglobulin. More preferred from 22 to 201206466 are antibodies having a modified Fc region as described in WO 99/58572. Other forms of humanized antibodies have one or more altered CDRs (CDR LI, CDR L2, CDR L3, CDR HI, CDR H2 and/or CDR H3) relative to the original antibody, which are also referred to as "sources" One or more CDRs of one or more CDRs of the original antibody. As used herein, "human antibody" refers to an amino group having an amino acid sequence corresponding to an antibody which can be produced by humans and/or which utilizes the techniques of any of those skilled in the art or disclosed herein to produce human antibodies. An antibody to an acid sequence. Human antibodies of this definition include antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a murine light chain and a human heavy chain polypeptide. Human antibodies can be prepared using a variety of techniques known in the art. In one embodiment, the human anti-system is selected from the group consisting of phage libraries, wherein the human bacterial library exhibits human antibodies (Vaughan et al., 1996, Nature Biotechnology, 1 4 : 3 0 9-3 1 4; Sheets et Al., 1 998, Proc.

Natl. Acad. S c i. ( USA) 95 : 6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227 ： 381; Marks et al., 1991，J. Mol. Biol.，222: 581)。人抗體亦可藉由免疫接種動物加以製備，該動物體內之內源性基因座（loci )已被基因轉殖導入之人免疫球蛋白基因座所取代，例如內源性免疫球蛋白基因已被部份或完全不活化之鼠》此方法係於美國專利第 5,545,807 、 5,545,806 、 5,569,825 、 5,625，126、5,63 3,425 及 5,661，016 號中描述。或者，該人抗體可藉由永生化產製以標的抗原爲標靶之抗體的人B淋巴細胞加以製備（該B淋巴細胞可自個體收集或可能在活 -23- 201206466 體外被免疫）。見例如 Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. L i s s, p. 77，1 9 8 5 ； Boerner et al·，1991，J. Immunol.，1 47 ( 1 ) : 86-95 及美國專利第 5,75 0,3 73 號。抗體之「可變區」係指單獨或經組合之抗體輕鏈之可變區或抗體重鏈之可變區。如該領域所知，重鏈及輕鏈之可變區各由四個骨架區（FR)及連接該四個骨架區之含有超變異區之三個互補決定區（CDR )組成。各鏈中之 CDR被FR拉近在一起，並與來自其他鏈中之CD R導致形成抗體之抗原結合部位。至少有兩種技術用於決定CDR :( 1 )根據跨種序列變異性之方法（即Kabat et al. Sequences of Proteins of Immunological Interest, ( 5th ed., 1991, National Institutes of Health, Bethesda MD ) ):及（2)根據抗原-抗體複合物之結晶學試驗之方法（ Al-lazikani et a 1, 1 9 9 7, J . Μ ο 1 e c . Biol. 273 : 927-948 ) 0 此處所使用之CDR係指以任一方法或兩種方法之組合所定義之CDR。該技藝中所謂之抗體之「恆定區」係指單獨或經組合之抗體輕鏈之恆定區或抗體重鏈之恆定區。此處所使用之用語「PC SK9」係指保留至少部分之 PCSK9活性之任何形式之PCSK9及彼之變異體。除非不同地指明諸如特別指涉人PCSK9，否則PCSK9包括所有哺乳動物物種之天然序列PC SK9，例如人、犬、貓、馬及牛。一個示範性之人PCSK9係見於Uniprot登記號Q8NBP7 ( -24- 201206466 SEQ ID NO ： 16)。此處所使用之「PCSK9拮抗抗體」係指能抑制PCSK9 生物活性及/或抑制由PCSK9信號所媒介之下游途徑的抗體，包括PCSK9媒介之LDLR下調及PCSK9媒介之LDL血液清除減少。pH依賴性PC SK9拮抗抗體包含阻斷、拮抗、抑制或減少（至任何程度包括顯著地）PCS K9生物活性之抗體，包括由PCSK9信號媒介之下游途徑，諸如LDLR交互作用及/或誘發對PC SK9之細胞反應。就本發明之目的而言，應清楚了解的是，用語「PCS K9拮抗抗體」包含所有前述用以實質上廢止、降低或中和PCSK9本身、PCSK9生物活性（包括但不限於彼所媒介之與LDLR之交互作用、下調LDLR及血液LDL清除減少之任何態樣之能力）或該生物活性之後果至任何有意義之程度之確定用語、標題及功能狀態及特徵。在一些實施態樣中，pH依賴性PCSK9拮抗抗體與PCS K9結合且防止與LDLR之交互作用。本發明提供PCSK9拮抗抗體之實例。用語「多肽」、「寡肽」、「肽」及「蛋白質」在此處可交換使用以指稱任何長度之胺基酸鏈，較佳地是相對短鏈（例如10至100個胺基酸）。該鏈可爲線性或分支，其可能包含經修飾之胺基酸及/或可能被非胺基酸中斷》該等用語亦包含經天然或人爲干預修飾之胺基酸鏈；例如雙硫鍵形成' 糖基化、脂化、乙醯化、磷酸化或任何其他操縱或修飾’諸如與標記成份共輒。該定義亦包括例如包含一或多個胺基酸類似物（包括例如非天然胺基酸等）以 -25- 201206466 及該領域習知之其他修飾之多肽* 單鏈或相連之鏈存在。如該領域所知之「多核苷酸」換使用，係指任何長度之核苷酸之該核苷酸可爲去氧核糖核苷酸、核苷酸或鹼基及/或彼等之類似物， RNA聚合酶被納入鏈中之底物。多核苷酸，諸如甲基化核苷酸及彼等，對核苷酸結構之修飾可在該鏈組苷酸之序列可被非核苷酸成份中斷可進一步被修飾，諸如與標記成份包括例如「加蓋（caps)」、以類發生之核苷酸、核苷酸間修飾諸如電鍵結（例如甲基膦酸酯、磷酸三基甲酸酯等）及帶電鍵結（例如硫酯等）者、該些含有側基團者，諸酸酶、毒素、抗體、信號肽、聚離入劑（i n t e r c a 1 a t 〇 r )者（例如V U定含有螯合劑者（例如金屬、放射性等）、該些含有烷化劑者、該些具如α異位性核酸等）以及未經修飾，任何通常存在於糖類中之羥基可基取代、被標準保護基保護或被活之額外鍵結，或可能與固體支持物應了解的是該多肽可以或「核酸」在此處可交鏈，包括DNA及RNA。糖核苷酸、經修飾之核或任何可藉由DNA或核苷酸可包含經修飾之之類似物。若存在的話合之前或之後進行。核。多核苷酸在聚合之後共軛。其它類型之修飾似物取代一或多個天然舉例來說該些具有不帶酯、磷酸醯胺化物、胺代磷酸酯、二硫代磷酸如例如蛋白質（例如核胺酸等）、該些具有嵌、補骨脂素等）、該些金屬、硼、氧化性金屬有經修飾之鍵結者（例之多核苷酸形式。另外能被例如膦酸基、磷酸化以製備與其他核苷酸共軛。該5’及3’端ΟΗ可 -26- 201206466 被磷酸化或被胺類或自1至20個碳原子之有機加蓋基團取代。其他羥基亦可被衍生成爲標準保護基。多核苷酸亦可包含該領域通常已知之類似形式之核糖或去氧核糖糖類包括例如2’-0-甲基-核糖、2’-0-烯丙基核糖、2’-氟代·核糖或2’-疊氮基-核糖、碳環糖類似物、〇或々異位性糖類、差向異構體糖類諸如***糖、木糖或來蘇糖、吡喃糖糖類、呋喃糖糖類、景天酮庚糖、非環類似物及無鹼基核苷類似物諸如甲基核糖苷。一或多個磷酸二酯鍵結可被替代性連接基團取代。該些替代性連接基團包括但不限於其中磷酸鹽被P(0)s (「硫代鹽」）、p(s)s( 「二硫代鹽」）、（〇 ) NR2 (「醯胺化物」）、P(0)R、P(0 )OR’、CO或CH2(「甲縮醛」）取代之實施態樣，其中各 R或V係獨立地Η或經取代或未經取代之烷基（1至20個碳 )，該烷基可選擇地含有醚（·〇-)鍵結、芳基、烯基、環烷基、環烯基或芳烷基。在多核苷酸中之所有鍵結不需要完全相同。前面的敘述適用於此處所指涉之所有多核苷酸，包括RNA及DNA。若抗體以相較於彼與其他物質結合時更高之親和性、親合力（avidity )、更快速及/或更長時間地與標的結合時，該抗體與標的「特異性結合」或「優先地結合」。舉例來說，與PCS K9表位特異性或優先地結合之抗體係指相較於彼與其他PCSK9表位或非PCSK9表位結合時以更高之親和性、親合力、更快速及/或更長時間地與此表位結合之抗體。由閱讀此定義亦可了解，舉例來說與第一標的特 -27- 201206466 異性或優先地結合之抗體（或基團或表位）可能與第二標的或不與第二標的特異性或優先地結合。因此，「特異性結合」或「優先地結合」不一定需要（雖然其可包括）排他性結合。一般來說（但不必然），所謂的結合係指優先地結合。「非傳訊誘捕體」係隔離配體與彼之同源受體之可溶性受體異構體或結合蛋白質。此處所使用之「實質上純的」係指其爲至少5 0%純的 (也就是不含汙染物），更佳地至少9 0 %純的，更佳地至少95 %純的，甚至更佳地至少98 %純的，且最佳地至少 9 9 °/〇純的物質。「宿主細胞」包括可作爲或已作爲導入多核苷酸*** 物之載體的接受者之個別細胞或細胞培養。宿主細胞包括單一宿主細胞之後代，且該後代可能因爲天然、意外或蓄意突變而不一定與原始親代細胞完全相同（在形態學或基因學DN A互補性上）。宿主細胞包括經本發明之多核苷酸在活體內轉染之細胞。如該領域所知，用語「Fc區」係用於定義免疫球蛋白重鏈之C端區域。該「Fc區」可能爲天然序列Fc區或變異體Fc區。雖然免疫球蛋白重鏈之Fc區的邊界可能不同，人 IgG重鏈Fc區通常被定義爲從Cys226處之胺基酸殘基或從 Pr〇2 3 0延伸至彼之羧基端。Fc區中之殘基編號係如卡巴（ Kabat)所述之 EU 指數。Kabat et al.，Sequences of Proteins of Immunological Interest, 5th Ed. Public Health -28- 201206466Natl. Acad. S c i. (USA) 95 : 6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227: 381; Marks et al., 1991, J. Mol. Biol., 222: 581). Human antibodies can also be prepared by immunizing animals. The endogenous locus (loci) in the animal has been replaced by a human immunoglobulin locus introduced by gene transfer, for example, an endogenous immunoglobulin gene has been Partially or completely inactivated mice are described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,63, 3,425, and 5,661,016. Alternatively, the human antibody can be prepared by immortalization of human B lymphocytes which are labeled with the target antigen (the B lymphocytes can be collected from the individual or may be immunized in vitro at live -23-201206466). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. L iss, p. 77, 1 9 8 5; Boerner et al., 1991, J. Immunol., 1 47 (1): 86-95 and the United States Patent No. 5, 75 0, 3 73. The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FR) and three complementarity determining regions (CDRs) comprising the hypervariable regions joining the four framework regions. The CDRs in each chain are brought together by FR and result in the formation of an antigen binding site for the antibody with CD R from other chains. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (ie, Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)) And (2) a method according to the crystallographic test of the antigen-antibody complex (Al-lazikani et a 1, 1 9 9 7, J . Μ ο 1 ec . Biol. 273 : 927-948 ) 0 used herein CDR refers to a CDR as defined by either method or a combination of both methods. The "constant region" of the antibody as used in the art refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. The term "PC SK9" as used herein refers to any form of PCSK9 and variants thereof that retain at least a portion of the PCSK9 activity. Unless otherwise indicated, such as specifically referring to PCSK9, PCSK9 includes the native sequence PC SK9 of all mammalian species, such as humans, dogs, cats, horses and cattle. An exemplary human PCSK9 line is found in Uniprot Accession No. Q8NBP7 (-24-201206466 SEQ ID NO: 16). As used herein, "PCSK9 antagonist antibody" refers to an antibody that inhibits PCSK9 biological activity and/or inhibits downstream pathways mediated by PCSK9 signaling, including downregulation of LDLR by PCSK9 mediators and reduction of LDL blood clearance by PCSK9 mediators. pH-dependent PC SK9 antagonist antibodies comprise antibodies that block, antagonize, inhibit or reduce (to any extent including significantly) PCS K9 biological activity, including downstream pathways by PCSK9 signaling media, such as LDLR interactions and/or induction to PC The cellular response of SK9. For the purposes of the present invention, it is to be understood that the term "PCS K9 antagonist antibody" encompasses all of the foregoing to substantially abolish, reduce or neutralize PCSK9 itself, PCSK9 biological activity (including but not limited to the media The interaction of LDLR, the ability to down-regulate any aspect of LDLR and blood LDL clearance, or the determined term, title, and functional status and characteristics of the fruit to any meaningful degree. In some embodiments, the pH dependent PCSK9 antagonist antibody binds to PCS K9 and prevents interaction with LDLR. The present invention provides an example of a PCSK9 antagonist antibody. The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to an amino acid chain of any length, preferably a relatively short chain (eg, 10 to 100 amino acids). . The chain may be linear or branched, which may contain modified amino acids and/or may be interrupted by non-amino acids. The terms also include amino acid chains modified by natural or human intervention; for example, disulfide bonds. Forming 'glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification' such as co-labeling with labeled components. This definition also includes, for example, the presence of one or more amino acid analogs (including, for example, unnatural amino acids, etc.) in the presence of a single chain or linked chain of -25-201206466 and other modified polypeptides known in the art. As used in the art, "polynucleotide" is used interchangeably to refer to nucleotides of nucleotides of any length which may be deoxyribonucleotides, nucleotides or bases and/or the like. RNA polymerase is incorporated into the substrate in the chain. Polynucleotides, such as methylated nucleotides and the like, may be further modified by the modification of the nucleotide structure in which the sequence of the histidine acid sequence may be interrupted by non-nucleotide components, such as with labeled components including, for example, " Caps, nucleotides, internucleotide modifications such as electrojunctions (eg, methylphosphonates, trisphosphates, etc.) and charged linkages (eg, thioesters, etc.) Those having side groups, such as acidases, toxins, antibodies, signal peptides, and intercalating agents (interca 1 at 〇r) (for example, those in which VU contains a chelating agent (such as metal, radioactivity, etc.), Any one containing an alkylating agent, such as an alpha atopic nucleic acid, and unmodified, any hydroxy group which is usually present in the saccharide, protected by a standard protecting group or otherwise bonded, or possibly The solid support should be understood to be that the polypeptide or "nucleic acid" can be cross-linked here, including DNA and RNA. A sugar nucleotide, a modified core or any of the modified analogs may be included by DNA or nucleotide. If it exists, before or after. Nuclear. The polynucleotide is conjugated after polymerization. Other types of modifications may be substituted for one or more of the natural examples, such as having no ester, a guanidinium phosphate, an amine phosphate, a dithiophosphorus such as, for example, a protein (eg, nucleotide, etc.), Inserted, psoralen, etc.), the metal, boron, oxidizing metal has a modified bond (in the form of a polynucleotide. In addition, it can be phosphorylated, for example, to prepare other nucleotides Conjugation. The 5' and 3' ends are -26-201206466 phosphorylated or substituted with amines or organic capping groups from 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. Polynucleotides may also comprise ribose or deoxyribose sugars of a similar form generally known in the art including, for example, 2'-0-methyl-ribose, 2'-0-allylribose, 2'-fluororibose or 2'-azido-ribose, carbocyclic analogue, sputum or sputum ectopic saccharide, epimer saccharide such as arabinose, xylose or lyxose, pyranose saccharide, furanose saccharide, bokeh Ketone heptose, acyclic analogs and abasic nucleoside analogs such as methylribose One or more phosphodiester linkages may be substituted by an alternative linking group including, but not limited to, phosphates by P(0)s ("thio salts"), p ( Implementation of s)s ("dithio salts"), (〇) NR2 ("ammonium amide"), P(0)R, P(0)OR', CO or CH2 ("methylal") a form wherein each R or V is independently substituted or substituted or unsubstituted alkyl (1 to 20 carbons), the alkyl optionally containing an ether (·〇-) linkage, an aryl group, an alkene Any of the linkages in the polynucleotide need not be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA. The antibody "specifically binds" or "preferentially binds" to the target when it binds to the target with a higher affinity, avidity, faster and/or longer time binding to the other substance. For example, an anti-system that specifically or preferentially binds to a PCS K9 epitope refers to a higher affinity than when it binds to other PCSK9 epitopes or non-PCSK9 epitopes. An antibody that binds to this epitope in a sexual, avid, faster, and/or longer time. It is also understood by reading this definition, for example, an antibody that binds heterologously or preferentially to the first target, special -27-201206466 ( Or a group or epitope may or may not specifically or preferentially bind to the second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In general (but not necessarily), the term "binding" refers to preferential binding. "Non-communication trap" is a soluble receptor isomer or binding protein that sequesters a ligand and its cognate receptor. "Substantially pure" means that it is at least 50% pure (ie, free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, and even more preferably at least 98. % pure, and optimally at least 9 9 ° / 〇 pure substance. "Host cells" include individual cells or cell cultures that can be used or have been accepted as a vector for introduction of a polynucleotide insert. The host cell includes a single host cell progeny, and the progeny may not necessarily be identical (in morphological or genetic DN A complementarity) to the original parent cell due to natural, accidental or deliberate mutation. Host cells include cells that are transfected in vivo by the polynucleotides of the invention. As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain may differ, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 or from Pr〇2 30 to the carboxy terminus. The residue number in the Fc region is the EU index as described by Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health -28- 201206466

Service, National Institutes of Health, Bethesda, Md., 1991.免疫球蛋白之Fc區通常包含兩個恆定結構域CH2及 CH3。該領域所使用之「Fc受體」或「FcR」描述與抗體之 Fc區結合之受體。較佳之FcR係天然序列之人FcR。另外，較佳之FcR係與IgG抗體結合之受體（r受體）且包括 Fc 7 RI、Fc r RII及Fc r RIII亞型之受體，包括等位基因變異體及可選擇之該等受體之剪切形式。FC<r RII受體包括Fc r RIIA (「活化受體」）及Fc r RIIB (「抑制受體」 )，彼等具有類似之胺基酸序列但主要差異在於細胞質結構域。FcR係於 Ravetch and Kinet, 1991, Ann. Re v. Immunol ·， 9 : 457-92 C apel et al, ·, 1 994, Immunomethods， 4 ： 25-34 :及 de Haas et al., 1 995, J. Lab. Clin. Med., 126 ： 330-4 1 中回顧。「FcR」亦包括新生兒受體FcRn，該受體負責轉運母體igG至胎兒（Guyer et al·，1 976，J. Immunol.，1 1 7 : 587; and Kim et al., 1 994, J. Immunol., 24 ： 249 )。此處關於抗體所使用之用語「競爭」係指第一抗體或彼之抗原結合部分與表位結合之方式充份類似於第二抗體或彼之抗原結合部分之結合，使得第一抗體與彼之同源表位在第二抗體存在時之結合結果相較於第二抗體不存在時第一抗體之結合可偵測地降低。或者，該情況可爲但不一定是該第二抗體與彼之表位之結合亦因第一抗體之存在而可偵測地降低。也就是說，第一抗體可抑制第二抗體與彼 -29- 201206466 之表位之結合，但第二抗體不抑制該第一抗體與彼之個別表位之結合。然而，當各種抗體不論以相同、較高或較低程度可偵測地抑制另一抗體與彼之同源表位或配體結合時，該等抗體被稱爲彼此「交叉競爭」與彼等個別表位之結合。本發明包含競爭抗體及交叉競爭抗體。不論該競爭或交叉競爭所藉以發生之機轉爲何（例如空間位阻、構形變化或與共同表位或彼之部分結合），技藝人士將了解根據此處所提供之揭示，本發明包含競爭及/或交叉競爭抗體且彼等可被用於此處所揭示之方法。「功能性Fc區」具有天然序列Fc區之至少一種效應功能。示範性「效應功能」包括Clq結合、補體依賴性細胞毒性、Fc受體結合、抗體依賴性細胞媒介性細胞毒性、吞噬作用、下調細胞表面受體（例如B細胞受體）等。該等效應功能通常需要Fc區與結合結構域（例如抗體可變結構域）結合且可利用該領域已知之各種用於評估該等抗體效應功能之測定檢測。「天然序列Fc區」包含與在天然中發現之Fc區的胺基酸序列完全相同之胺基酸序列。「變異體Fc區」包含與天然序列Fc區有至少一個胺基酸修飾之差異的胺基酸序列，但仍保留該天然序列Fc區之至少一種效應功能。較佳地，該變異體Fc區相較於天然序列Fc區或親代多肽之Fc區具有至少一個胺基酸取代，例如在天然序列Fc區或親代多肽之 Fc區中自約1至約1〇個胺基酸取代，且較佳地自約1至約5 個胺基酸取代。此處之變異體Fc區將較佳地具有與天然序 -30- 201206466 列Fc區及/或與親代多肽之Fc區至少約80%之序列一致性，最佳地具有至少約90%之序列一致性，更佳地具有至少約 9 5 %、至少約9 6 %、至少約9 7 %、至少約9 8 %、至少約9 9 % 之序列一致性。「最小預期生物效應量（MABEL)」係指在人導致最小生物效應之最小預期劑量。安全因素通常被應用於自 MABEL計算人之首次劑量。MABEL之計算應利用所有相關之活體外及活體內藥物動力學及藥物藥效學資訊》此處所使用之「治療」及「治療有效性」係指得到有益或所欲臨床結果之方法。就本發明之關於pH依賴性PCSK9 拮抗抗體之目的而言，有益或所欲之臨床結果包括但不限於下列一或多項：增進LDL清除及減少發生或改善異常膽固醇及/或脂蛋白之量，該異常膽固醇及/或脂蛋白之量係因代謝及/或飮食失調所致或包括家族性高膽固醇血症、致動脈粥樣化異常脂血症、動脈粥樣硬化及更普遍地心血管疾病（CVD)。「減少發生」係指任何嚴重性之減少，其可包括減少通常用於治療該狀況之其他藥物及/或治療之需求及/或量 (例如暴露量）。如該領域之技藝人士所了解的，就個體對治療之反應而言可能有所不同，因此舉例來說，「減少發生之方法」反映出投予pH依賴性抗體係根據該投予可能造成特定個體之發生減少之合理預期。「改善」係指投予治療後一或多種症狀相較於未投予治療時減輕或改善。「改善」亦包括縮短或減少症狀之持 -31 - 201206466 續時間。此處所使用之藥物、化合物或醫藥組成物之「有效劑量」或「有效量」係指足以影響任一或多種有益或所欲結果之量。就預防性用途而言，有益或所欲結果包括消除或減少風險、減輕嚴重性或延遲疾病之開始，包括疾病之生化學、組織學及/或行爲學之症狀、彼之倂發症及在疾病發展期間所表現之中間病理學表型。就pH依賴性PCSK9拮抗抗體之治療用途而言，有益或所欲之結果包括諸如減少高膽固醇血症或一或多種異常脂血症、動脈粥樣硬化、 CVD或冠狀動脈心臟病之症狀、降低治療該疾病所需之其他藥物之劑量、增進其他藥物之效果及/或延緩病患之疾病進展之臨床結果。有效劑量可分一或多次投予。就本發明之目的而言，藥物、化合物或醫藥組成物之有效劑量係足以直接或間接完成預防性或治療性治療之量。如在臨床背景下所了解的，藥物、化合物或醫藥組成物之有效劑量可能與或可能不與另一藥物、化合物或醫藥組成物組合達到。因此，在投予一或多種治療劑之情況下考慮「有效劑量」及若單一劑與一或多種其他劑組合時考慮給予有效量，可能或係經達成所欲之結果。「個體」或「對象」係哺乳動物，更佳地人。哺乳動物亦包括但不限於農場動物、競賽動物、寵物、靈長動物、馬、犬、貓、小鼠及大鼠。此處所使用之「載體」係指建構物，其能在宿主細胞中遞送及較佳地表現一或多種感興趣之基因或序列。載體 -32- 201206466 之實例包括但不限於病毒性載體、裸DNA或RNA表現載體、質體、黏質體或噬菌體載體、與陽離子縮合劑有關之 DNA或RNA表現載體、包封於脂質體內之DNA或RNA表現載體及某些真核細胞諸如生產細胞。此處使用之「表現控制序列」係引導核酸轉錄之核酸序列。表現控制序列可爲啓動子，諸如組成性或誘導性啓動子或增強子。表現控制序列係可操作地與所欲轉錄之核酸序列連接。此處所使用之「醫藥上可接受之載劑」或「醫藥上可接受之賦形劑」包括任何當與活性成分組合時能使該成分保留生物活性且不與個體之免疫系統反應之任何物質。實例包括但不限於任何標準醫藥載劑諸如磷酸鹽緩衝鹽水溶液、水、乳液諸如油/水乳液及各種類型之潤濕劑。較佳之用於氣霧劑或非經腸投予之稀釋劑係磷酸鹽緩衝鹽水（ PBS )或生理（0.9% )鹽水。包含該等載劑之組成物係由眾所周知之習知方法調製（見例如Remington’s Pharmaceutical Sciences, 18 th edition, A. Gennaro, ed.， Mack Publishing Co., Easton, PA, 1 990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000 ) ° 此處所使用之用語「Κπ」係指抗體與抗原結合之速率常數。具體地說，該速率常數（及平衡解離常數係利用Fab抗體片段（即單價）與抗原測量。此處所使用之用語「Κ。^」係指抗體自抗體/抗原複合 -33- 201206466 物解離之速率常數。此處所使用之用語「KD」係指抗體-抗原交互作用之平衡解離常數。分別測定結合及解離速率常數1^及Kd以決定KD及kQff 比値，利用以表面電漿共振爲基底之生物感測器，在與配體結合之分析物係單價之條件下特徵化分析物/配體之交互作用，該配體係經捕捉試劑以低量固定在感測器表面上。該分析係利用如 Karlsson et al_，Anal· Biochem 349, 1 3 6- 1 47，2 006所描述之動力學滴定方法進行。在給定測試中所採用之感應晶片、捕捉試劑及測定緩衝液係按照 Myszka，J. Mol. Recognit 12，279-284, 1 999 之建議選擇以提供穩定之捕捉配體至感測器表面、最小化分析物與表面之非特異性結合及產生適用於動力學分析之分析物結合反應。該按照分析物/配體交互作用之分析物結合反應被雙重參照及帶入1: 1蘭繆爾（Langmuir )之「質量傳輸限制模型」，其中ka、Kd及Rmax爲全局參數如Myszka & Morton et al·, Biophys· Chem 64，127-137 ( 1997)所述。平衡解離常數KD係自動力學速率常數之比値推算，即KD = kd/ka 。該等測定較佳地在25°C或37°C下進行。 A.用於預防或治療疾病之方法在關於pH依賴性PCSK9拮抗抗體之一態樣中，本發明提供一種治療或預防個體之高膽固醇血症及/或異常脂血症、動脈粥樣硬化、CVD或冠狀動脈心臟病之至少一種症 -34- 201206466 狀之方法，該方法包含對該個體投予有效量之拮抗循環 PCSK9之pH依賴性PCSK9拮抗抗體。在另一態樣中，本發明提供有效量之拮抗循環PCS K9 之PH依賴性PCS K9拮抗抗體以用於治療或預防個體之高膽固醇血症及/或異常脂血症、動脈粥樣硬化、CVD或冠狀動脈心臓病之至少一種症狀。本發明另提供有效量之拮抗細胞外或循環PC SK9之pH依賴性PC SK9拮抗抗體於製造藥物以治療或預防個體之高膽固醇血症及/或異常脂血症、動脈粥樣硬化、CVD或冠狀動脈心臟病之至少一種症狀之用途》有利地，治療性投予該抗體導致較低之血液膽固醇及 /或較低之血液LDL。較佳的是，血液膽固醇及/或血液 LDL·相較於投予前至少降低約10%或15°/。。更佳地，血液膽固醇及/或血液LDL相較於投予該抗體前至少降低約20% 。仍更佳地，血液膽固醇及/或血液LDL相較於投予該抗體前至少降低30%。有利地，血 '液膽固醇及/或血液LDL相較於投予該抗體前至少降低40%。更有利地，血液膽固醇及/或血液LDL相較於投予該抗體前至少降低50%。仍更佳地，血液膽固醇及/或血液LDL相較於投予該抗體前至少降低60%。最佳地，血液膽固醇及/或血液LDL相較於投予該抗體前至少降低70%。就此處所描述之所有方法而言，提及針對任何適當抗原之pH依賴性抗體亦包括包含一或多種額外劑之組成物。該等組成物可能進一步包含適當之賦形劑，諸如醫藥上 -35- 201206466 可接受之賦形劑包括該領域眾所周知之緩衝劑。本發明可被單獨使用或與其他習知之治療方法組合使用。該pH依賴性抗體可經由任何適當之途徑對個體投予。對該領域之技藝人士顯而易見的是，此處所描述之實例不應意圖限制而是可用技術之示例。因此在一些實施態樣中，該pH依賴性抗體係根據已知方法對個體投予，諸如靜脈內投予例如快速濃注或在一段時間內連續輸注、肌肉內、腹腔內、腦脊髓腔內、經皮、皮下、關節內、舌下、滑膜內、經噴入、脊椎鞘內、經口、吸入或局部途徑。投予可爲系統性例如靜脈內投予或局部投予。自商業途徑獲得之使用液體調製劑之噴霧器包括噴射噴霧器及超音波噴霧器可被用於投予。液體調製劑可經直接噴霧，冷凍乾燥之粉末可在重構後經噴霧投予。或者，pH依賴性抗體可利用氟碳調製劑及定量吸入器加以氣霧化或以經冷凍乾燥及磨細粉末之形式吸入。在一實施態樣中，pH依賴性抗體係經定點或靶向性 .局部遞送技術投予。定點或靶向性局部遞送技術之實例包括各種pH依賴性抗體之植入式貯劑來源或局部遞送導管，諸如輸注導管、留置導管、或針頭導管、合成性移植物、外膜層包覆、分流器及支架或其他植入式裝置、定點載劑、直接注射或直接施用。見例如PCT公開號WO 00/532 1 1及美國專利第5,981，568號。 pH依賴性抗體之各種調製劑可被用於投予。在一些實施態樣中，可僅投予該pH依賴性抗體。在一些實施態 -36- 201206466 樣中，pH依賴性抗體及醫藥上可接受之賦形劑可呈各種調製劑之形式。醫藥上可接受之賦形劑係爲該領域所知，且係有助於投予藥理有效物質之相對惰性物質。舉例來說，賦形劑可提供外形或稠度，或作爲稀釋劑。適當之賦形劑包括但不限於穩定劑、潤濕劑、乳化劑、用於改變滲透性之鹽類、包封劑、緩衝劑及皮膚穿透增進劑。用於非經腸及經腸藥物遞送之賦形劑以及調製劑係闡述於 Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing ( 2000 )。該等劑可與醫藥上可接受之載劑（諸如鹽水、林格（ Ringer )氏液、葡萄糖溶液及該類似物）組合。特定投藥配方即劑量、時間及重複性將依特定個體及該個體之醫學病史而定。如此處所述，呈p Η依賴性結合之抗體亦可經吸入投予。通常在投予pH依賴性抗體時，初始候選劑量可爲約2 毫克/公斤。就本發明之目的而言，典型之每日劑量可根據上述因子介於約3微克/公斤至3 0微克/公斤至300微克/公斤至3毫克/公斤、至30毫克/公斤、至1〇〇毫克/公斤或ι00 毫克/公斤以上之任何範圍內。舉例來說，可使用約1毫克 /公斤、約2.5毫克/公斤、約5毫克/公斤、約1〇毫克/公斤及約25毫克/公斤之劑量。視狀況而重複投予數天或更久時’該治療被持續進行直到發生所欲之症狀抑制或直到達成足夠之治療量，例如減少血液LDL之量。示範性投藥配方包含投予初始劑量約2毫克/公斤之抗體，隨後每週約1 -37- 201206466 毫克/公斤之維持劑量，或隨後隔週約1毫克/公斤之維持劑量。然而，其他投藥配方可根據醫師所希望達成之藥物動力學衰減模式使用。舉例來說，在一些實施態樣中，每週投藥一至四次係經考慮。在其他實施態樣中，每月投藥一次或隔月或每三個月投藥一次係經考慮。此治療之進展可藉由習知技術及檢測加以輕易地監測。該投藥配方（包括所使用之抗體）可隨時間而異。就本發明之目的而言，該pH依賴性抗體之適當劑量將依所採用之抗體（或彼之組成物）、所欲治療之症狀的類型及嚴重性、該劑係以預防性或治療性目的投予、先前治療、該病患之臨床病史及對該劑之反應、該病患之血液抗原之量、該病患合成及清除抗原之速率、該病患清除該經投予之劑之速率及主治醫師之考量而定。通常醫師將投予pH依賴性抗體直到達到可完成所欲結果之劑量。劑量及/或頻率可依治療療程而異。經驗性考量諸如半衰期通常將影響該劑量之決定。舉例來說，可相容於人免疫系統之抗體諸如人化抗體或全人抗體可被用以延長該抗體之半衰期及防止該抗體被宿主之免疫系統攻擊。投藥頻率可根據治療之療程加以決定及調整，通常但不一定根據症狀例如髙膽固醇血症之治療及/或抑制及/或改善及/或延緩。或者，抗體之持續性連續釋放調製劑可爲適當。各種用於達。成持續釋放之調製劑及裝置係該領域所知。在一實施態樣中，拮抗抗體之劑量在已經給予一或多次拮抗抗體之個體中可憑經驗決定。個體係經給予漸增劑 -38- 201206466 量之抗體。爲了評估療效可追縱疾病之指標。根據本發明之方法投予pH依賴性抗體可爲連續性或間歇性，此依例如接受者之生理狀況、該投藥之目的係治療性或預防性及技藝人士所知之其他因素而定。PH依賴性抗體之投予可爲實質上連續—段預先決定之時間，或可爲一系列間隔劑量。在一些實施態樣中，可能存在超過一種拮抗抗體。可能存在至少一種、至少兩種、至少三種、至少四種、至少五種不同或更多種拮抗抗體及/或肽。通常，該等抗體或肽可能具有不會互相不良影響之互補活性。pH依賴性抗體亦可與其他治療劑組合使用》pH依賴性抗體亦可與其他劑一起使用，以用於增進及/或補充該等劑之有效性。〇可接受之載劑、賦形劑或穩定劑在所採用之劑量及濃度下對接受者不具毒性，可能包括例如緩衝劑諸如磷酸鹽、檸檬酸鹽及其他有機酸；鹽諸如氯化鈉：抗氧化劑包括抗壞血酸及甲硫胺酸；保存劑（諸如十八基二甲基苄基氯化銨、六甲氯胺、氯化苯甲烴銨、氯化苄乙氧銨、酚醇、丁醇、苄醇、烷基對羥苯甲酸酯類諸如對羥苯甲酸甲酯或對羥苯甲酸丙酯、兒茶酚、間苯二酚、環己醇、3-戊醇及間甲酚）：低分子量（小於約1 〇個殘基）多肽；蛋白質諸如血清白蛋白、明膠或免疫球蛋白；親水性聚合物諸如聚乙烯基吡咯烷酮；胺基酸諸如甘胺酸、麩醯胺酸、天冬醯胺酸、組胺酸、精胺酸或離胺酸；單醣、雙醣及其他碳水 -39- 201206466 化合物包括葡萄糖、甘露糖或聚葡萄糖；螯合劑諸如 EDTA ;糖類諸如蔗糖、甘露醇、海藻糖或山梨醇；鹽形成反離子諸如鈉；金屬複合物（例如鋅蛋白質複合物）：及/或非離子性界面活性劑諸如TWEENtm、pluronicstm 或聚乙二醇（PEG)。含有pH依賴性抗體之脂質體係以該領域已知之方法製備，諸如於Epstein，et al.，1985，Proc. Natl. Acad. Sci. USA 82 : 3688 ; Hwang, et al., 1 980，Proc. Natl. Acad. Sci. USA 77: 4030 及美國專利第 4,485,045 及 4,544,545 號中所述。循環時間延長之脂質體係揭露於美國專利第 5,0 1 3,5 56號。特別有用之脂質體可利用逆相蒸發方法以包含磷脂醯膽鹼、膽固醇及PEG-衍生性磷脂醯乙醇胺（ PEG-PE )之脂質組成物產製。脂質體被擠壓通過定義孔徑大小之濾網以產生具有所欲直徑之脂質體。該活性成分亦可被包封於藉由例如凝聚技術或藉由界面聚合化所製備之微膠褒中例如分別於羥甲基纖維素或明膠微膠囊及聚-(異丁烯酸甲酯）微膠癱中、於膠體藥物遞送系統中（例如脂質體、白蛋白微球、微乳化液、奈米微粒及奈米微囊）或於***化液中。該等技術係揭示於 Remington, The Science and Practice of Pharmacy, 20th Ed.，Mack Publishing ( 2000 )。可能製備持續釋放性製劑。持續釋放製劑之適當實例包括含有該抗體之固相疏水性聚合物之半透性基體，該基體係呈形狀物件之形式（例如膜或微膠囊）。持續釋放基 -40- 201206466 體之實例包括聚酯、水凝膠（例如聚（2 -羥乙基-甲基丙烯酸酯）或聚乙烯醇）、聚交酯（美國專利第3,773,9 1 9 號）、L·麩胺酸及7乙基-L-麩胺酸鹽之共聚物、不可降解之乙烯-乙酸乙烯酯、可降解之乳酸-乙醇酸共聚物諸如 LUPRON DEPOTtm (由乳酸-乙醇酸共聚物及柳菩林（ leuprolide acetate )所組成之注射型微球）、蔗糖乙酸異丁酸酯及聚-D- ( - ) -3-羥丁酸》欲用於活體內投予之調製劑必須爲無菌。此可輕易地藉由例如無菌過濾膜之過濾達成。治療性pH依賴性抗體組成物通常被置放於具有無菌接口之容器中，例如具有可被皮下注射針穿刺之塞子的靜脈溶液袋或小瓶。適當之乳液可利用自商業途徑獲得之脂肪乳液製備，諸如 IntralipidTM 、 Liposyn™ 、 Infonutrol™ 、 1^?<^1111(^111^及LipiphysanTM。該活性成分可被溶解於預先混合之乳液組成物中，或者可被溶解於油中（例如大豆油、紅花籽油、棉花籽油、芝麻油、玉米油或杏仁油）再與磷脂（例如卵磷脂、大豆磷脂或大豆卵磷脂）及水混合以形成乳液。將瞭解的是可添加其他成分例如甘油或葡萄糖以調整該乳液之張力。適當之乳液通常將含有最高20% 例如介於5至20%之油。該脂肪乳液可包含介於〇.1至1.0微米特別是0.1至0.5微米之脂肪液滴’且具有介於5.5至8.0 之pH 〇該乳液組成物可爲該些藉由混合PH依賴性抗體與 IntralipidTM或彼之成份（大丑油、卵磷脂、甘油及水） -41 - 201206466 所製備者。用於吸入或噴入之組成物包括在醫藥上可接受之水性或有機溶劑或彼等之混合物中之溶液及懸浮液和粉末。該液體或固體組成物可能包含如前述之適當之醫藥上可接受之賦形劑。在一些實施態樣中，該組成物係以經口或經鼻呼吸途徑投予以提供局部或系統性效應。在醫藥上可接受之較佳無菌溶劑中之組成物可藉由使用氣體加以噴霧化。經噴霧化之溶液可從噴霧裝置直接吸入，或該霧化裝置可與面罩、帷幕或間歇性正壓呼吸器連接。溶液、懸浮液或粉末組成物可自裝置較佳地經口或經鼻投予，該裝置以適當方式遞送調製劑。 B. pH依賴性抗體本發明所使用之抗體可包含單株抗體、多株抗體、抗體片段（例如 Fab、Fab’、F ( ab’）2、Fv、Fc等）、嵌合抗體、雙特異性抗體、異源共軛抗體、單鏈（ScFv)、彼之突變物、包含抗體部位之融合蛋白（例如結構域抗體）、人抗體、人化抗體及任何其他含有該所需特異性之抗原辨認區的免疫球蛋白分子之經修飾構型，包括抗體之糖基化變異體、抗體之胺基酸序列變異體及經共價修飾之抗體 •該抗體可爲小鼠、大鼠、人或任何其他來源（包括嵌合或人化抗體）。在一些實施態樣中，該pH依賴性抗體係單株抗體。該pH依賴性抗體亦可爲人化抗體。在其他實施態樣中， -42- 201206466 該抗體係人抗體。在一些實施態樣中，該抗體包含經修飾之恆定區，諸如免疫惰性之恆定區，亦即具有減少之引發免疫反應之能力。在一些實施態樣中，該恆定區係經如Eur. J. Immunol., 1 999，29: 26 1 3 -2624 ； PCT公開號 W099/5 8572 及/或英國專利申請號980995 1.8所述之修飾。該Fc可爲人 IgG2或人IgG4。該Fc可爲含有突變A3 3 0P331至S3 3 0S331 ( IgG^a)之人IgG2，其中該胺基酸殘基係參考野生型IgG2 序列編號。Eur· J. Immunol·，1 999，29: 26 1 3-2624。在一些實施態樣中，該抗體包含有下列突變之IgG4之恆定區（ Armour e t al., 2003，Molecular Immunology 40 5 8 5 -593 ) :E233F234L235 至 P233V234A2 35 (IgG“c)，其中該編號參考野生型IgG4。在另一實施態樣中，該Fc係人IgG4 E233F234L235 至 P233V234A235 伴隨 G236之刪除（IgG〇b )。在另一實施態樣中，該Fc係包含鉸鏈穩定突變S228至 P228 之任何人 IgG4 Fc ( IgG4、IgG4 △ b 或 IgG4 △ c )( Aalberse et al., 2002, Immunology 105, 9-19) 〇在另一實施態樣中，該Fc可爲非糖基化Fc。在一些實施態樣中，該恆定區係藉由使該寡醣連接殘基（諸如ASn297 )及/或該恆定區中屬於糖基化辨認序列之部份的側翼殘基突變而去糖基化。在一些實施態樣中，該恆定區之N-連接糖基化係經酵素去糖基化。該恆定區之 N-連接糖基化可經酵素去糖基化或藉由在糖基化缺陷之宿主細胞中表現以去糖基化。 -43- 201206466 測定抗體與抗原之結合親和性的一個方法係測量該抗體之單官能基Fab片段的結合親和性。爲了得到單官能基 Fab片段，抗體（例如IgG )可經木瓜酶剪切或經重組表現。抗體之Fab片段的親和性可藉由裝設預先固定之鏈黴抗生物素蛋白感測晶片（SA)之表面電漿共振（ Biac〇re3 000TM表面電漿共振（SPR)系統，紐澤西州皮斯卡塔維Biacore公司），使用HBS-EP流動緩衝液（0_01莫 5：HEPES- pH 7.4· 0.15 NaCl· 3*^^：EDTA· 0.005%^ 積/體積界面活性劑P20 )加以測定。生物素基化之抗原可經HBS-EP緩衝液稀釋至濃度低於0.5微克/毫升，並利用不同的接觸時間注射經過個別晶片通道以達到兩種抗原密度範圍，亦即用於詳細動力學試驗之50至200反應單位（RU )或用於篩選測定之800至1000 RU。再生試驗顯示含25毫莫耳NaOH之25體積％乙醇有效地移除經結合之Fab同時保持晶片上之抗原供超過200次注射的活性。通常，經純化之Fab樣本的連續稀釋液（自0.1至10倍預估KD之橫跨濃度 )係以1〇〇微升/分鐘注射1分鐘，並允許最高2小時之解離時間。該Fab蛋白之濃度係藉由使用已知濃度（以胺基酸分析測定）之Fab爲標準物之ELISA及/或SDS-PAGE電泳決定。使用BIAevaluation軟體將資料完整帶入1: 1蘭繆爾（ Langmuir )結合模型（Karlsson，R. Roos，Η· Fagerstam， L. Petersson, B.，1 9 9 4. Methods Enzymology 6. 99-110) 以同時得到動力學結合速率（1^η )及解離速率（koff)。平衡解離常數（KD )數値係以k^f/ku計算。此程序適合 -44- 201206466 用於測定抗體與任何抗原之結合親和性，包括人或其他哺乳動物物種（諸如小鼠、大鼠、靈長動物）之抗原。抗體之結合親和性通常在2 5 °C測量，但亦可在3 7 °C測量。該等抗體可由該領域中習知之任何方法製備。免疫接種宿主動物之途徑及時程通常與已建立及習知之抗體刺激及產製技術一致，如此處進一步描述。用於產製人及小鼠抗體之通用技術係該領域已知及/或係於此處描述。以針對PCSK9之pH依賴性抗體而言，目前製備該等抗體之較佳方法包含如此處所揭示之免疫接種PCS K9基因剔除（ PCSK9 -/-)動物。應考慮任何哺乳動物個體包括人或源自該個體之抗體產製細胞可經操縱以作爲產製哺乳動物（包括人）雜交瘤細胞系之基礎。通常，該宿主動物係經腹膜內、肌肉內、經口、皮下、腳掌內及/或皮內接種一定量之免疫原，包括如此處所述。雜交瘤可自淋巴細胞及永生化之骨髓瘤細胞製備，使用 Kohler, B. and Milstein，C·，1975，Nat-ure 256 : 495-497 之常規體細胞雜交技術或由Buck, D. W., et al.，1 9 82, In Vitro，18: 377-381所修飾之技術。可用之骨髓瘤細胞系包括但不限於X63-Ag8.653及該些來自美國加州聖地牙哥沙克硏究所細胞分布中心（Salk Institute Cell Distribution Center)之細胞可被用於雜交。通常，該技術涉及使用促融劑諸如聚乙二醇或藉由該領域之技藝人士眾所周知之電裝置使骨髓瘤細胞與淋巴樣細胞融合。在融 -45- 201206466 合後’使該等細胞自融合培養基中分離並於選擇性生長培養基諸如次黃嘌呤-胺喋呤·胸苷（hat)培養基中生長以消除未雜交之親代細胞。任何此處所描述之培養基不論有無添加血清皆可被用於培養分泌單株抗體之雜交瘤。在細胞融合技術之另一替代技術中，EBV永生化B細胞可被用於產製本發明之PCSK9單株抗體。需要時該等雜交瘤係經擴增及次選殖，上清液之抗免疫原活性藉由習知之免疫測定方法（例如放射性免疫測定、酵素免疫測定或螢光免疫測定）檢測。可被用來作爲抗體來源之雜交瘤包含所有衍生物、產製對PCSK9具特異性之單株抗體的親代雜交瘤之子細胞或彼等之部分。產製該等抗體之雜交瘤可利用已知方法在活體外或活體內生長。該等單株抗體可藉由習知之免疫球蛋白純化方法自培養基或體液中分離，諸如硫酸銨沉澱、膠體電泳、透析、色層分析及若需要之超過濾。若有非所欲之活性可被移除，藉由例如使該製劑通過由免疫原連接固相所組成之吸附劑，自該免疫原洗脫或釋放該所欲之抗體。以和蛋白質共軛之人PC SK9或含有該標的胺基酸序列之片段免疫接種宿主動物可產生抗體族群（例如單株抗體），該蛋白質對所欲免疫接種之物種具免疫原性，例如鑰孔狀帽貝血藍素、血清白蛋白、牛甲狀腺球蛋白或大豆胰蛋白酶抑制劑，該共軛係利用雙官能性或衍生劑例如順丁烯二亞胺基苯甲醯基磺酸基琥珀醯亞胺基酯（經由半胱胺酸殘基共軛 46- 201206466 )、N-羥琥珀二醯亞胺（經由離胺酸殘基）、戊二醛、琥珀酐、S0C12或I^N^sNR (其中R和R1係不同的烷基）。一旦抗體被產製或選擇，該等抗體可如此處之實施例 1所揭示以理想化pH依賴性結合力。需要時可對該感興趣之pH依賴性抗體（單株或多株 )定序，接著該多核苷酸序列可被選殖至載體以供表現或增殖。編碼該感興趣之抗體的序列可被維持於宿主細胞中之載體，接著該宿主細胞可被擴增及冷凍以供將來使用》在細胞培養中產製重組單株抗體可透過該領域已知之方法自B細胞選殖抗體基因加以進行。見例如Tiller et al.， 2008， J. Immunol. Methods 329， 112 ;美國專利第 7,3 1 4,622 號。在替代態樣中，該多核苷酸序列可能被用於基因操縱以「人化」該抗體或改善該抗體之親和性或其他特徵。舉例來說，該恆定區可經工程化（engineered)以更類似於人之恆定區，避免該抗體用於人之臨床試驗及治療時之免疫反應。基因操縱該抗體序列以得到較高之對抗原之親和性及較高之療效係爲所欲。對該領域之技藝人士顯而易見的是，可在該pH依賴性抗體進行一或多個多核苷酸改變且仍維持彼之抗原結合能力。人化單株抗體有四個常規步驟，包括：（1)測定起始抗體之輕鏈及重鏈可變結構域之核苷酸及預測之胺基酸序列；（2 )設計該人化抗體，亦即決定在人化步驟期間將使用哪一個抗體骨架區：（3)實際人化方法/技術；及 -47- 201206466 (4 )轉染及表現該人化抗體。見例如美國專利第 4,816,567 ' 5,807,715 ' 5,866,692 > 6,331,415 ' 5,530,101 、5,693,761、 5,693,762、 5,585,089及 6，180,370號》一些包含源自非人免疫球蛋白之抗原結合部位的「人化」抗體分子已被描述，包括具有齧齒動物或經修飾之齧齒動物V區及彼等之相關CDR融合至人恆定結構域之嵌合抗體。見例如 Winter et al., 1991, Nature 349: 293-299; Lobuglio et a 1., 1 98 9, Proc. Nat. Acad. Sci. USA 86 · 4220-4224; Shaw et al., 1987, J Immunol. 138 ： 4534-4538 及 Brown et al.，1 987, Cancer Res. 47: 3 577-3 5 83。其它參考文獻描述在與適當之人抗體恆定結構域融合前移植齧齒動物CDR至人支持骨架區（FR)。見例如Riechmann et al., 1 98 8, Nature 3 32 : 3 2 3 -3 27; Verhoeyen et al. ,1 98 8, Science 23 9 : 1534-1536¾ Jones et a 1., 1 98 6, Nature 321 :5 22-52 5。另一參考文獻描述經重組工程化之齧齒動物骨架區所支持之齧齒動物CDR。見例如歐洲專利公開號 05 1 95 96。該些「人化」分子係經設計以最小化對齧齒動物抗人抗體分子之非所欲之免疫反應，以免限制該些基團在人接受者之治療應用的持續期間及有效性。舉例來說，該抗體恆定區可經工程化以使其呈免疫惰性（例如不會誘發補體溶解）。見例如PCT公開號W099/58572;英國專利申請號980995 1.8。其他亦可能被利用之人化抗體之方法係由 Daugherty et al.，1991，Nucl. Acids Res· 1 9: 247 1 -2476及美國專利第 6,180,377、6,054,297、5,997,867、 -48 - 201206466 5,866,692、6,210,671 及 6,350,861 號及 PCT 公開號 WO 0 1/2 7 160所揭示。在另一替代態樣中，全人抗體可藉由使用自商業途徑獲得之小鼠得到，該小鼠經工程化以表現特定人免疫球蛋白。經設計以產生更希望或更強烈之免疫反應的基因轉殖動物亦可被用於產製人化或人抗體。該項技術之實例爲艾比基因（Abgenix )公司（加州費利蒙市）的 XenomouseTM、美德列斯（Medarex)公司（紐澤西州普林斯頓）之HuMAb-Mouse®及TC MouseTM和雷傑納龍（ Regeneron)製藥公司（紐約州泰利城）之Veloclmmune® 小鼠。在替代態樣中，抗體可利用該領域已知之任何方法重組製備及表現。在另一替代態樣中，抗體可藉由噬菌體展示技術被重組製備。見例如美國專利第5,565,3 32、 5,5 8 0,7 1 7、5,73 3,743 及 6,265，150 號及 Winter et al.，1 994, Annu. Rev. Immunol. 12 : 433-455。或者，隨菌體展示技術(McCafferty et al., 1 990，Nature 34 8 : 5 52-5 5 3 ).可自未經免疫接種之捐贈者的免疫球蛋白可變區（V)結構域基因庫以於活體外產製人抗體及抗體片段。根據此項技術，抗體V結構域基因被符合讀框地選殖至絲狀噬菌體之主要或次要外套蛋白基因諸如Ml 3或fd中，且以官能性抗體片段被展示於該噬菌體顆粒之表面上。由於該絲狀顆粒包含該噬菌體基因組之單股DN A拷貝，因此根據該抗體之官能特性所進行之篩選亦導致選擇編碼展現該等特性之抗體 -49- 201206466 的基因》因此，該噬菌體模擬B細胞之若干特性。噬菌體展示可以各種形式進行；見例如Johnson, Kevin S. and Chiswell, David J., 1 993, Current Opinion in Structural Biology 3 : 5 64-5 7 1。V基因區段之數種來源可被用於噬菌體展示。Clackson et al·，1991，Nature 3 5 2 : 624-628 自免疫接種小鼠之脾臟衍生之V基因的小型隨機組合式庫分離出各式各樣之抗噁唑啉酮抗體陣列。來自未經免疫接種之人捐贈者的V基因貯庫可被建構，對抗各類抗原陣列（包括自體抗原）之抗體可實質上依照Mark et al.，1991，J. Mol. Biol. 222 : 58 1 -597或 Griffith et al·，1 993，EMBO J. 12: 72 5-73 4所述之技術被分離》在天然之免疫反應中，抗體基因以高比例累積突變（體細胞超突變）。若干被導入之改變將給予更高的親和性，且展示高親和性表面免疫球蛋白之B細胞在後續抗原挑戰期間被優先地複製及分化。此天然方法可藉由採取稱爲「鏈洗牌（chain shuffling )」之技術模擬（Marks et al·，1 992，Bio/Technol. 10: 779-783 )。在此方法中，由噬菌體展示所得到之「初級」人抗體的親和性可被改善，以得自未經免疫接種之捐贈者的V結構域基因之天然發生變異體（庫）之貯庫連續取代重鏈及輕鏈V區基因。此技術允許產製親和性介於皮莫耳至奈莫耳範圍之抗體及抗體片段。產製非常大型之噬菌體抗體庫（亦稱爲「所有庫之母（the mother-of-all 1 i b r a r i e s )」）之策略已由'\\^161^11〇118661&1.，1 993，>111(；1· Acids Res. 21 : 2265-2266描述。基因替換亦可被用於自 -50- 201206466 齧齒動物抗體衍生人抗體，其中該人抗體具有類似該起始齧齒動物抗體之親和性及特異性。根據此亦稱爲「表位印記」之方法，由噬菌體展示技術所得到之齧齒動物抗體之重鏈或輕鏈V結構域基因被人V結構域基因庫取代，產生齧齒動物-人嵌合物。篩選抗原導致分離能恢復官能性抗原結合部位之人可變區，亦即掌管（印記）夥伴之選擇之表位。當該方法被重複以取代剩餘之齧齒動物V結構域可得到人抗體（見PCT公開號WO 93/062 1 3 )。和慣用之 CDR移植以人化嚼齒動物抗體不同的是，此項技術提供不具齧齒動物來源之骨架或CD R殘基之全人抗體。很明顯的是，雖然上述討論係關於人化抗體，但所討論之通用原則適用於客製化用於例如犬、貓、靈長動物、馬或牛之抗體。另顯而易見的是，此處所描述之人化抗體之一或多個態樣可被組合，例如CDR移植、骨架突變及 CDR突變。抗體可被重組製備，首先自宿主動物分離抗體及抗體產製細胞、取得基因序列及使用該基因序列以於宿主細胞 (例如CHO細胞）重組表現該抗體。另一可被採用之方法係於植物（例如菸草）或基因轉殖乳中表現該抗體序列。於植物或乳中重組表現抗體之方法已被揭示。見例如 Peeters, 200 1，et al. Vaccine 1 9 : 2756 ; Lonberg，N. and D. Huszar，1 995，Int_ Rev. Immunol 13 : 65及 Pollock, et al.，1999, J Immunol Methods 231 : 147。製備抗體衍生物之方法例如人化、單鏈等係爲該領域所知。 -51 - 201206466 免疫測定及流式細胞分選技術諸如螢光激活細胞分選 (FACS)亦可被用於分離對所欲抗原具特異性之抗體。該等抗體可與許多不同之載劑結合。載劑可爲活性及 /或惰性。廣爲週知之載劑實例包括聚丙烯、聚苯乙烯、聚乙烯、葡聚糖、尼龍、澱粉酶、玻璃、天然及經改質之纖維素、聚丙醯胺、洋菜糖及磁鐵礦。該載劑之性質就本發明之目的而言可爲可溶性或不可溶性。該領域之技藝人士將知道其他用於結合抗體之適當載劑，或可利用例行實驗加以査證。在一些實施態樣中，該載劑包含以心肌爲標的之基團。編碼單株抗體之DNA可利用習知方法（例如利用能與編碼該單株抗體之重鏈及輕鏈之基因特異性結合之寡核苷酸探針）輕易地分離及定序。雜交瘤細胞被用來當作該 DNA之較佳來源。一經分離後，該DN A可被置入表現載體內（.諸如PCT公開號WO 87/04462所揭示之表現載體），該表現載體接著被轉染至原本不產製免疫球蛋白之宿主細胞諸如大腸桿菌細胞、類人猿COS細胞、中國倉鼠卵巢（ CHO )細胞或骨髓瘤細胞，以在該重組宿主細胞中合成單株抗體。見例如PCT公開號W0 87/04462。該DNA亦可藉由例如以人重鏈及輕鏈恆定結構域之編碼序列取代同源鼠序列加以修飾（Morrison et al.，1984，Proc. Nat. Acad. Sci. 81= 6851)，或藉由共價連接免疫球蛋白編碼序列與非免疫球蛋白多肽之所有或部分之編碼序列加以修飾。在該方式中，「嵌合」或「雜交」抗體係經製備以具有此處 -52- 201206466 之單株抗體之結合特異性。 pH依賴性抗體及源自抗體.之多肽可利用該領域已知之方法識別或特徵化，藉此檢測及/或測量生物活性之減少、改善或中和。在一些實施態樣中，p Η依賴性抗體或多肽係藉由培養候選劑與抗原及監測結合力及/或隨之而來之生物活性減少或中和加以識別。該結合力測定可以經純化之抗原多肽進行，或以天然表現或經轉染以表現多肽之細胞進行。在一實施態樣中，該結合力測定係競爭性結合測定，其中候選抗體與已知拮抗劑競爭結合之能力係經評估。該檢測可以不同形式進行，包括ELISΑ形式。在其他實施態樣中，pH依賴性抗體係藉由培養候選劑與抗原及監測結合力與隨之而來之LDLR表現抑制及/或血液膽固醇清除加以識別。在初步鑑別之後，候選pH依賴性抗體之活性可進一步經已知用於測試該標的生物活性之生物檢測證實及精製。或者，生物檢測可被用來直接篩選候選物。若干用於識別及特徵化抗體、肽或適體之方法係詳細描述於實施例中〇呈pH依賴性結合之抗體可利用該領域眾所周知之方法特徵化。舉例來說，一個方法係識別彼所結合之表位或稱爲「表位定位」。該領域有許多已知之用於定位及特徵化蛋白質上表位之位置之方法，包括破解抗體-抗原複合物之晶體結構、競爭測定、基因片段表現測定及合成性肽基底測定’例如於Harlow and Lane, Using Antibodies, a -53- 201206466Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH3. The "Fc receptor" or "FcR" used in this field describes a receptor that binds to the Fc region of an antibody. Preferred FcR is the human FcR of the native sequence. In addition, preferred FcR lines are receptors that bind to IgG antibodies (r receptors) and include receptors for Fc 7 RI, Fc r RII and Fc r RIII subtypes, including allelic variants and, optionally, such The cut form of the body. FC <r RII receptors include Fc r RIIA ("activating receptor") and Fc r RIIB ("inhibiting receptor"), which have similar amino acid sequences but differ mainly in the cytoplasmic domain. FcR is in Ravetch and Kinet, 1991, Ann. Rev. Immunol,, 9: 457-92 C apel et al, ·, 994, Immunomethods, 4: 25-34: and de Haas et al., 1 995, Review by J. Lab. Clin. Med., 126: 330-4 1 . "FcR" also includes the neonatal receptor FcRn, which is responsible for transporting the parent igG to the fetus (Guyer et al., 1 976, J. Immunol., 1 1 7 : 587; and Kim et al., 1 994, J). Immunol., 24: 249). The term "competition" as used herein with respect to an antibody means that the binding of the first antibody or the antigen binding portion thereof to the epitope is sufficiently similar to the binding of the second antibody or the antigen binding portion thereof such that the first antibody and the other antibody The binding of the homologous epitope in the presence of the second antibody is detectably reduced in comparison to the binding of the first antibody in the absence of the second antibody. Alternatively, the condition may be, but not necessarily, the binding of the second antibody to its epitope and also detectably decreased by the presence of the first antibody. That is, the first antibody inhibits binding of the second antibody to the epitope of -29-201206466, but the second antibody does not inhibit binding of the first antibody to its individual epitope. However, when various antibodies detectably bind another antibody to its cognate epitope or ligand at the same, higher or lower degree, the antibodies are said to "cross-competition" with each other and The combination of individual epitopes. The invention encompasses competitive antibodies and cross-competing antibodies. Regardless of the circumstances under which the competition or cross-competition occurs (eg, steric hindrance, structural change, or in combination with a common epitope or part of it), those skilled in the art will appreciate that the present invention encompasses competition and / or cross-competing antibodies and they can be used in the methods disclosed herein. A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include Clq binding, complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cellular cytotoxicity, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require binding of the Fc region to a binding domain (e. g., an antibody variable domain) and can be assayed using various assays known in the art for assessing the efficacy of such antibodies. The "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from the natural sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example from about 1 to about 约 in the native sequence Fc region or the Fc region of the parent polypeptide. One of the amino acid substitutions, and preferably from about 1 to about 5 amino acids. The variant Fc region herein will preferably have at least about 80% sequence identity to the native sequence -30-201206466 Fc region and/or to the Fc region of the parent polypeptide, optimally having at least about 90% Sequence identity, more preferably, has a sequence identity of at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. The "Minimum Expected Biological Effects (MABEL)" refers to the minimum expected dose that causes the least biological effect in humans. Safety factors are often used to calculate the first dose of a person from MABEL. MABEL calculations should utilize all relevant in vitro and in vivo pharmacokinetics and pharmacodynamic information. The terms "treatment" and "treatment effectiveness" as used herein refer to methods of obtaining beneficial or desired clinical results. For the purposes of the pH-dependent PCSK9 antagonist antibody of the present invention, beneficial or desirable clinical results include, but are not limited to, one or more of the following: enhancing LDL clearance and reducing the occurrence or amelioration of abnormal cholesterol and/or lipoprotein, The amount of abnormal cholesterol and/or lipoprotein is caused by metabolic and/or eating disorders or includes familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis, and more generally cardiovascular Disease (CVD). "Reducing occurrence" means any reduction in severity, which may include reducing the need and/or amount (e.g., exposure) of other drugs and/or treatments typically used to treat the condition. As will be appreciated by those skilled in the art, the individual's response to treatment may vary, so for example, the "reduction method" reflects that administration of a pH-dependent anti-system may result in a particular Reasonable expectation of a decrease in the occurrence of an individual. "Improvement" means that one or more symptoms after treatment are reduced or improved compared to when no treatment is administered. "Improvement" also includes the reduction or reduction of symptoms -31 - 201206466 Continued time. As used herein, "effective amount" or "effective amount" of a drug, compound or pharmaceutical composition means an amount sufficient to affect one or more of the beneficial or desired results. For preventive use, beneficial or desirable outcomes include eliminating or reducing the risk, reducing the severity, or delaying the onset of the disease, including the chemistry, histology, and/or behavioral symptoms of the disease, his convulsions, and The intermediate pathological phenotype exhibited during disease development. For therapeutic use of pH-dependent PCSK9 antagonist antibodies, beneficial or desirable outcomes include, for example, reduction of hypercholesterolemia or one or more dyslipidemia, atherosclerosis, CVD or coronary heart disease symptoms, reduction The dose of other drugs needed to treat the disease, the effect of other drugs, and/or the clinical outcome of delaying the progression of the disease in the patient. The effective dose can be administered in one or more doses. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is sufficient to effect a prophylactic or therapeutic treatment, either directly or indirectly. As will be appreciated in the clinical context, an effective dose of a drug, compound or pharmaceutical composition may or may not be combined with another drug, compound or pharmaceutical composition. Thus, the consideration of an "effective dose" in the case of administration of one or more therapeutic agents and the administration of an effective amount if a single agent is combined with one or more other agents may or may achieve the desired result. "Individuals" or "objects" are mammals, and better people. Mammals also include, but are not limited to, farm animals, race animals, pets, primates, horses, dogs, cats, mice, and rats. As used herein, "vector" refers to a construct that is capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors-32-201206466 include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plastids, vesicular or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, encapsulated in liposomes DNA or RNA expression vectors and certain eukaryotic cells such as producer cells. As used herein, a "expression control sequence" is a nucleic acid sequence that directs transcription of a nucleic acid. The expression control sequence can be a promoter, such as a constitutive or inducible promoter or enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance which, when combined with the active ingredient, will retain the biological activity of the ingredient and will not react with the individual's immune system. . Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or physiological (0.9%) saline. The compositions comprising the carriers are prepared by well known methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1 990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000) ° The term "Κπ" as used herein refers to the rate constant at which an antibody binds to an antigen. Specifically, the rate constant (and the equilibrium dissociation constant are measured using a Fab antibody fragment (i.e., a unit price) and an antigen. The term "Κ.^" as used herein refers to the dissociation of an antibody from an antibody/antigen complex-33-201206466. Rate constant. The term "KD" as used herein refers to the equilibrium dissociation constant of antibody-antigen interactions. The binding and dissociation rate constants 1 and Kd are determined to determine the KD and kQff ratios, based on surface plasma resonance. The biosensor characterization of the analyte/ligand interaction under the condition of the analyte unit price combined with the ligand, the system being immobilized on the surface of the sensor by a capture reagent in a low amount. The kinetic titration method as described in Karlsson et al, Anal Biochem 349, 1 3 6- 1 47, 2 006 is used. The sensing wafer, capture reagent and assay buffer used in a given test are according to Myszka, The recommended selection of J. Mol. Recognit 12, 279-284, 1 999 to provide stable capture of the ligand to the sensor surface, minimize non-specific binding of the analyte to the surface, and produce suitable for use in moving The analyte binding reaction of the mechanical analysis. The analyte binding reaction according to the analyte/ligand interaction is double-referenced and brought into the 1:1 "mass transfer restriction model" of Langmuir, where ka, Kd and Rmax It is a global parameter such as Myszka & Morton et al., Biophys Chem 64, 127-137 (1997). The equilibrium dissociation constant KD is the ratio of the automatic mechanical rate constant, ie KD = kd/ka. Preferably, it is carried out at 25 ° C or 37 ° C. A. Method for preventing or treating diseases In one aspect regarding pH-dependent PCSK9 antagonist antibodies, the present invention provides a method for treating or preventing hypercholesterolemia in an individual. And / or at least one of dyslipidemia, atherosclerosis, CVD or coronary heart disease - 34-201206466, the method comprising administering to the individual an effective amount of antagonistic circulating PCSK9 pH dependence PCSK9 antagonizes antibodies. In another aspect, the invention provides an effective amount of a PH-dependent PCS K9 antagonist antibody antagonizing circulating PCS K9 for use in treating or preventing hypercholesterolemia and/or dyslipidemia, arteries in an individual A porridge At least one symptom of sclerosing, CVD, or coronary heart disease. The present invention further provides an effective amount of a pH-dependent PC SK9 antagonist antibody that antagonizes extracellular or circulating PC SK9 in the manufacture of a medicament for treating or preventing hypercholesterolemia in an individual and/or Use of at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease. Advantageously, therapeutic administration of the antibody results in lower blood cholesterol and/or lower blood LDL. Preferably, blood cholesterol and/or blood LDL· is reduced by at least about 10% or 15°/% prior to administration. . More preferably, blood cholesterol and/or blood LDL is reduced by at least about 20% compared to administration of the antibody. Still more preferably, blood cholesterol and/or blood LDL is reduced by at least 30% compared to administration of the antibody. Advantageously, blood 'liquid cholesterol and/or blood LDL is at least 40% lower than before administration of the antibody. More advantageously, blood cholesterol and/or blood LDL is reduced by at least 50% compared to administration of the antibody. Still better, blood cholesterol and/or blood LDL is reduced by at least 60% compared to administration of the antibody. Optimally, blood cholesterol and/or blood LDL is reduced by at least 70% compared to administration of the antibody. For all methods described herein, reference to a pH dependent antibody to any suitable antigen also includes compositions comprising one or more additional agents. Such compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including those well known in the art. The invention may be used alone or in combination with other conventional methods of treatment. The pH dependent antibody can be administered to an individual via any suitable route. It will be apparent to those skilled in the art that the examples described herein are not intended to be limiting but are examples of available techniques. Thus in some embodiments, the pH dependent anti-system is administered to an individual according to known methods, such as intravenous administration such as bolus injection or continuous infusion over a period of time, intramuscular, intraperitoneal, cerebrospinal , percutaneous, subcutaneous, intra-articular, sublingual, intrasynovial, intra-injection, intrathecal, intraoral, inhalation or topical. Administration can be systemic, e.g., intravenous or topical. A nebulizer using a liquid modulating agent obtained from a commercial route, including a jet nebulizer and an ultrasonic atomizer, can be used for administration. The liquid preparation can be directly sprayed, and the freeze-dried powder can be administered by spraying after reconstitution. Alternatively, the pH dependent antibody can be aerosolized using a fluorocarbon modulator and a metered dose inhaler or as a lyophilized and ground powder. In one embodiment, the pH dependent anti-system is administered by site-directed or targeted. Local delivery techniques. Examples of site-directed or targeted local delivery techniques include implantable reservoir sources or local delivery catheters of various pH dependent antibodies, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, outer membrane coating, Diverters and stents or other implantable devices, spotted carriers, direct injection or direct application. See, for example, PCT Publication No. WO 00/532 1 1 and U.S. Patent No. 5,981,568. Various modulators of pH dependent antibodies can be used for administration. In some embodiments, only the pH dependent antibody can be administered. In some embodiments -36-201206466, pH dependent antibodies and pharmaceutically acceptable excipients can be in the form of various modulators. Pharmaceutically acceptable excipients are known in the art and are relatively inert materials which aid in the administration of a pharmacologically effective substance. For example, the excipient can provide a shape or consistency, or as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents, emulsifiers, salts for modifying permeability, encapsulants, buffers, and skin penetration enhancers. Excipients and modulators for parenteral and enteral drug delivery are described in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000). The agents can be combined with a pharmaceutically acceptable carrier such as saline, Ringer's solution, dextrose solution, and the like. The particular dosage formulation, i.e., dosage, time, and repeatability, will depend on the particular individual and the medical history of the individual. As described herein, antibodies that bind in a p-dependent manner can also be administered by inhalation. Typically, when a pH dependent antibody is administered, the initial candidate dose can be about 2 mg/kg. For the purposes of the present invention, typical daily doses may range from about 3 micrograms/kg to 30 micrograms/kg to 300 micrograms/kg to 3 mg/kg to 30 mg/kg to 1 inch, depending on the above factors.任何mg/kg or ι00 mg/kg or more in any range. For example, a dose of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 1 mg/kg, and about 25 mg/kg can be used. When the administration is repeated for several days or longer depending on the condition, the treatment is continued until the desired symptom suppression occurs or until a sufficient therapeutic amount is reached, for example, the amount of blood LDL is reduced. An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg of antibody followed by a maintenance dose of about 1 -37 to 201206466 mg/kg per week, or a maintenance dose of about 1 mg/kg per week thereafter. However, other dosing regimens can be used depending on the pharmacokinetic decay mode that the physician desires to achieve. For example, in some embodiments, one to four administrations per week are considered. In other embodiments, administration once a month or once every three months or every three months is considered. The progress of this treatment can be easily monitored by conventional techniques and testing. The dosage formulation (including the antibodies used) can vary over time. For the purposes of the present invention, the appropriate dosage of the pH dependent antibody will depend on the antibody (or composition thereof) employed, the type and severity of the condition being treated, and the agent being prophylactic or therapeutic. The purpose of administration, prior treatment, the clinical history of the patient and the response to the agent, the amount of blood antigen of the patient, the rate of synthesis and clearance of the antigen by the patient, and the removal of the administered agent by the patient The rate and the attending physician's considerations. Typically, the physician will administer a pH dependent antibody until a dose is achieved that will achieve the desired result. The dosage and/or frequency may vary depending on the course of treatment. Empirical considerations such as half-life will generally affect the decision of the dose. For example, an antibody that is compatible with the human immune system, such as a humanized antibody or a fully human antibody, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted according to the course of treatment, usually but not necessarily based on the treatment and/or inhibition and/or improvement and/or delay of symptoms such as cholesterol. Alternatively, a sustained continuous release modulator of the antibody may be suitable. Various for use. Modulators and devices that are sustained release are known in the art. In one embodiment, the dose of the antagonist antibody can be determined empirically in an individual who has been administered one or more antagonistic antibodies. The system was given an increasing dose of -38-201206466 antibody. In order to assess the efficacy of the disease can be traced to the indicators of the disease. Administration of the pH dependent antibody in accordance with the methods of the present invention may be continuous or intermittent depending on, for example, the physiological condition of the recipient, the therapeutic or prophylactic purpose of the administration, and other factors known to those skilled in the art. The administration of the PH-dependent antibody can be substantially continuous - a predetermined period of time, or can be a series of spaced doses. In some embodiments, more than one antagonist antibody may be present. At least one, at least two, at least three, at least four, at least five different or more antagonistic antibodies and/or peptides may be present. Generally, such antibodies or peptides may have complementary activities that do not adversely affect each other. pH dependent antibodies can also be used in combination with other therapeutic agents. pH dependent antibodies can also be used with other agents to enhance and/or supplement the effectiveness of such agents. The acceptable carrier, excipient or stabilizer is not toxic to the recipient at the dosages and concentrations employed, and may include, for example, buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride: Antioxidants include ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexachlorochloramine, benzalkonium chloride, benzethonium chloride, phenol alcohol, butanol, Benzyl alcohol, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol: low Molecular weight (less than about 1 残 residue) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, glutamic acid, aspartame Aminic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates -39 - 201206466 compounds include glucose, mannose or polydextrose; chelating agents such as EDTA; sugars such as sucrose, mannitol, seaweed Sugar or sorbitol; salt formation Such as sodium; metal complexes (e.g. Zn-protein complexes): and / or nonionic surfactant such as TWEENtm, pluronicstm or polyethylene glycol (PEG). Lipid systems containing pH dependent antibodies are prepared by methods known in the art, such as in Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 3688; Hwang, et al., 1 980, Proc. A. Acad. Sci. USA 77: 4030 and U.S. Patent Nos. 4,485,045 and 4,544,545. Lipid systems with extended cycle times are disclosed in U.S. Patent No. 5,013,5,56. Particularly useful liposomes can be produced by a reverse phase evaporation method using a lipid composition comprising phospholipid choline, cholesterol and PEG-derivatized phospholipid ethanolamine (PEG-PE). The liposomes are extruded through a sieve defining the pore size to produce a liposome of the desired diameter. The active ingredient may also be encapsulated in a microcapsule prepared by, for example, agglomeration techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microgels, respectively. In sputum, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000). It is possible to prepare sustained release formulations. Suitable examples of sustained release formulations include semipermeable matrices of solid phase hydrophobic polymers containing the antibodies in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release groups -40 to 201206466 include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or polyvinyl alcohol), polylactide (U.S. Patent No. 3,773,9 1 9) No.), a copolymer of L-glutamic acid and 7-ethyl-L-glutamate, a non-degradable ethylene-vinyl acetate, a degradable lactic acid-glycolic acid copolymer such as LUPRON DEPOTtm (from lactic acid-glycolic acid) Injectable microspheres composed of copolymer and leuprolide acetate, sucrose acetate isobutyrate and poly-D-(-)-3-hydroxybutyric acid are intended to be used in the preparation of in vivo administration. Must be sterile. This can easily be achieved by filtration, for example, of a sterile filtration membrane. Therapeutic pH-dependent antibody compositions are typically placed in a container having a sterile interface, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle. Suitable emulsions can be prepared using commercially available fat emulsions such as IntralipidTM, LiposynTM, InfonutrolTM, 1^? <^1111(^111^ and LipiphysanTM. The active ingredient may be dissolved in the pre-mixed emulsion composition or may be dissolved in oil (for example, soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil) Or almond oil) is then mixed with a phospholipid (such as lecithin, soya lecithin or soy lecithin) and water to form an emulsion. It will be appreciated that other ingredients such as glycerin or glucose may be added to adjust the tension of the emulsion. Suitable emulsions will generally Containing oils up to 20%, for example between 5 and 20%. The fat emulsion may comprise fat droplets between 0.1 to 1.0 micron, especially 0.1 to 0.5 micron, and having a pH between 5.5 and 8.0. The composition may be prepared by mixing a pH-dependent antibody with IntralipidTM or a component thereof (large oil, lecithin, glycerin, and water) -41 - 201206466. The composition for inhalation or injecting is included in The pharmaceutically acceptable aqueous or organic solvents or solutions and suspensions and powders thereof. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered orally or nasally to provide a local or systemic effect. The composition in a pharmaceutically acceptable, preferred sterile solvent can be sprayed by the use of a gas. The sprayed solution can be directly inhaled from the spray device, or the atomizing device can be attached to a mask, curtain or intermittent positive pressure breathing apparatus. The solution, suspension or powder composition can be preferably orally or nasally adapted from the device. In the case of administration, the device delivers the modulator in an appropriate manner. B. pH-Dependent Antibody The antibody used in the present invention may comprise monoclonal antibodies, polyclonal antibodies, antibody fragments (for example, Fab, Fab', F (ab') 2 Fv, Fc, etc.), chimeric antibody, bispecific antibody, heteroconjugate antibody, single chain (ScFv), mutant of the same, fusion protein containing antibody site (eg, domain antibody), human antibody, humanization Modified configurations of antibodies and any other immunoglobulin molecule containing an antigen recognition region of the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalent modifications Antibody • The antibody can be mouse, rat, human or any other source (including chimeric or humanized antibodies). In some embodiments, the pH dependent anti-system monoclonal antibody. The antibody may be a humanized antibody. In other embodiments, the anti-system human antibody is -42-201206466. In some embodiments, the antibody comprises a modified constant region, such as an immuno-inert constant region, ie, has a decrease The ability to elicit an immune response. In some embodiments, the constant region is e.g., Eur. J. Immunol., 1 999, 29: 26 1 3 -2624; PCT Publication No. WO99/5 8572 and/or British Patent Modification as described in application number 980995 1.8. The Fc can be human IgG2 or human IgG4. The Fc may be human IgG2 containing the mutation A3 3 0P331 to S3 3 0S331 (IgG^a), wherein the amino acid residue is referred to the wild type IgG2 sequence number. Eur· J. Immunol·, 1 999, 29: 26 1 3-2624. In some embodiments, the antibody comprises the constant region of IgG4 with the following mutations (Armour et al., 2003, Molecular Immunology 40 5 8 5 -593 ): E233F234L235 to P233V234A2 35 (IgG "c), wherein the numbering is referenced Wild type IgG4. In another embodiment, the Fc line human IgG4 E233F234L235 to P233V234A235 is accompanied by deletion of G236 (IgG〇b). In another embodiment, the Fc line comprises any of the hinge stabilizing mutations S228 to P228 Human IgG4 Fc (IgG4, IgG4 Δb or IgG4 Δc) (Aalberse et al., 2002, Immunology 105, 9-19) 另一 In another embodiment, the Fc may be a non-glycosylated Fc. In an embodiment, the constant region is deglycosylated by mutating the oligosaccharide linking residue (such as ASn297) and/or flanking residues of the constant region that are part of the glycosylation recognition sequence. In some embodiments, the N-linked glycosylation of the constant region is deglycosylated by an enzyme. The N-linked glycosylation of the constant region can be deglycosylated by an enzyme or by a defect in glycosylation Deglycosylation in host cells. -43- 201206466 Determination of antibodies One method of binding affinity of an antigen measures the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, the antibody (eg, IgG) can be cleaved by papain or recombinantly expressed. Fab of the antibody The affinity of the fragment can be achieved by mounting a pre-fixed streptavidin-sensing wafer (SA) surface plasma resonance (Biac〇re3 000TM Surface Plasma Resonance (SPR) system, New Jersey Piska Tavi Biacore) was determined using HBS-EP flow buffer (0_01 Mo 5: HEPES-pH 7.4·0.15 NaCl·3*^^: EDTA·0.005%^ product/volume surfactant P20). The antigen can be diluted to a concentration of less than 0.5 μg/ml via HBS-EP buffer and injected through individual wafer channels with different contact times to achieve two antigen density ranges, ie 50 to detailed kinetic experiments. 200 reaction units (RU) or 800 to 1000 RU for screening assays. Regeneration experiments show that 25 vol% ethanol with 25 mM NaOH effectively removes bound Fab while maintaining antigen on the wafer for more than 200 injections Live Typically, the serial dilutions of purified Fab samples (from 0.1 to 10 times the estimated concentration across the KD) injected for 1 min at 1〇〇 based l / min, and the solution allowed to up to 2 hours from the time. The concentration of the Fab protein was determined by ELISA and/or SDS-PAGE electrophoresis using a known concentration (determined by amino acid analysis) of Fab as a standard. Use the BIAevaluation software to bring the data completely into the 1:1 Langmuir binding model (Karlsson, R. Roos, Η·Fergestam, L. Petersson, B., 1.9, 4. Methods Enzymology 6. 99-110) At the same time, the kinetic binding rate (1^η) and the dissociation rate (koff) were obtained. The equilibrium dissociation constant (KD) number is calculated as k^f/ku. This procedure is suitable for -44-201206466 for determining the binding affinity of an antibody to any antigen, including antigens of human or other mammalian species such as mice, rats, primates. The binding affinity of the antibody is usually measured at 25 ° C, but can also be measured at 37 ° C. Such antibodies can be prepared by any method known in the art. The route of immunization of the host animal is generally consistent with established and conventional antibody stimulation and production techniques, as further described herein. General techniques for the production of human and mouse antibodies are known in the art and/or are described herein. For pH dependent antibodies against PCSK9, current preferred methods for preparing such antibodies comprise immunizing PCS K9 gene knockout (PCSK9 -/-) animals as disclosed herein. It is contemplated that any mammalian subject, including a human or an antibody derived from the individual, can be manipulated to serve as a basis for producing a mammalian (including human) hybridoma cell line. Typically, the host animal is inoculated with a quantity of immunogen by intraperitoneal, intramuscular, oral, subcutaneous, intramuscular, and/or intradermal, including as described herein. Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using conventional somatic cell hybridization techniques of Kohler, B. and Milstein, C., 1975, Nat-ure 256: 495-497 or by Buck, DW, et al. ., 1 9 82, In Vitro, 18: Techniques modified by 377-381. Useful myeloma cell lines including, but not limited to, X63-Ag8.653 and such cells from the Salk Institute Cell Distribution Center in San Diego, California, USA, can be used for hybridization. Generally, the technique involves fusing a myeloma cell with a lymphoid cell using a pro-melting agent such as polyethylene glycol or by an electrical device well known to those skilled in the art. The cells are isolated from the fusion medium after thawing -45 - 201206466 and grown in a selective growth medium such as hypoxanthine-amine thymidine (hat) to eliminate unhybridized parental cells. Any of the media described herein, with or without added serum, can be used to culture hybridomas that secrete monoclonal antibodies. In another alternative technique of cell fusion technology, EBV immortalized B cells can be used to produce the PCSK9 monoclonal antibodies of the invention. When necessary, the hybridomas are expanded and subcloned, and the anti-immunogenic activity of the supernatant is detected by a conventional immunoassay (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay). Hybridomas that can be used as a source of antibodies comprise all derivatives, daughter cells of parental hybridomas that produce monoclonal antibodies specific for PCSK9, or portions thereof. Hybridomas producing these antibodies can be grown in vitro or in vivo using known methods. Such monoclonal antibodies can be isolated from the culture medium or body fluid by conventional immunoglobulin purification methods such as ammonium sulfate precipitation, colloidal electrophoresis, dialysis, chromatography, and ultrafiltration if necessary. If undesired activity can be removed, the desired antibody is eluted or released from the immunogen by, for example, passing the preparation through an adsorbent comprising a solid phase attached to the immunogen. Immunization of a host animal with a protein conjugated human PC SK9 or a fragment containing the target amino acid sequence can produce an antibody population (eg, a monoclonal antibody) that is immunogenic to the species to be immunized, eg, a key A limpetal limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor utilizing a bifunctional or derivatizing agent such as maleimide benzylidene sulfonate amber醯imino ester (conjugated via cysteine residues 46-201206466), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, S0C12 or I^N^ sNR (wherein R and R1 are different alkyl groups). Once antibodies are produced or selected, such antibodies can be as disclosed in Example 1 herein to idealize pH-dependent binding. The pH dependent antibody of interest (single or multiple plants) can be sequenced as needed, and the polynucleotide sequence can then be ligated into a vector for expression or proliferation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, which can then be expanded and frozen for future use. The production of recombinant monoclonal antibodies in cell culture can be carried out by methods known in the art. B cell selection antibody genes were carried out. See, for example, Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S. Patent No. 7, 3, 4,622. In an alternative aspect, the polynucleotide sequence may be used in gene manipulation to "humanize" the antibody or to improve the affinity or other characteristics of the antibody. For example, the constant region can be engineered to be more similar to the human constant region, avoiding the antibody being used in clinical trials and immunological reactions in humans. The gene manipulates the antibody sequence to obtain a higher affinity for the antigen and a higher therapeutic effect. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made at the pH dependent antibody while still maintaining its antigen binding ability. Humanized monoclonal antibodies have four general steps, including: (1) determining the nucleotides of the light and heavy chain variable domains of the starting antibody and the predicted amino acid sequence; (2) designing the humanized antibody That is, which antibody framework region will be used during the humanization step: (3) actual humanization methods/technologies; and -47-201206466 (4) transfection and expression of the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567 '5,807,715 ' 5,866,692 > 6,331,415 ' 5,530,101, 5,693,761, 5,693,762, 5,585,089, and 6,180,370. Some "humanized" antibody molecules comprising antigen-binding sites derived from non-human immunoglobulins have been described. Included are chimeric antibodies having a rodent or modified rodent V region and their associated CDR fusion to a human constant domain. See, for example, Winter et al., 1991, Nature 349: 293-299; Lobuglio et a 1., 1 98 9, Proc. Nat. Acad. Sci. USA 86 · 4220-4224; Shaw et al., 1987, J Immunol 138: 4534-4538 and Brown et al., 1 987, Cancer Res. 47: 3 577-3 5 83. Other references describe the transplantation of rodent CDRs into the human support framework region (FR) prior to fusion with an appropriate human antibody constant domain. See, for example, Riechmann et al., 1 98 8, Nature 3 32 : 3 2 3 -3 27; Verhoeyen et al., 1 98 8, Science 23 9 : 1534-15363⁄4 Jones et a 1., 1 98 6, Nature 321 :5 22-52 5. Another reference describes rodent CDRs supported by recombinantly engineered rodent framework regions. See, for example, European Patent Publication No. 05 1 95 96. These "humanized" molecules are designed to minimize undesired immune responses to rodent anti-human antibody molecules so as not to limit the duration and effectiveness of such groups in therapeutic applications in human recipients. For example, the antibody constant region can be engineered to render it immunologically inert (e.g., does not induce complement solubilization). See, for example, PCT Publication No. WO99/58572; British Patent Application No. 980995 1.8. Other methods of humanized antibodies that may also be utilized are by Daugherty et al., 1991, Nucl. Acids Res. 9: 247 1 - 2476 and U.S. Patents 6,180,377, 6,054,297, 5,997,867, -48 - 201206466 5,866,692, 6,210,671 and No. 6,350,861 and PCT Publication No. WO 0 1/2 7 160. In another alternative, a fully human antibody can be obtained by using a commercially available mouse that is engineered to express a particular human immunoglobulin. Gene-transgenic animals designed to produce a more desirable or more potent immune response can also be used to produce humanized or human antibodies. Examples of this technology are XenomouseTM from Abgenix (Fremont, Calif.), HuMAb-Mouse® from Continental (Princeton, New Jersey), and TC MouseTM and Regener (Regeneron) Veloclmmune® mouse from the pharmaceutical company (Terry City, NY). In an alternate aspect, the antibody can be reconstituted and expressed using any method known in the art. In another alternative, the antibody can be recombinantly produced by phage display technology. See, for example, U.S. Patent Nos. 5,565, 3 32, 5, 5 0 0, 7 1 7 , 5, 73 3, 743 and 6, 265, 150 and Winter et al., 1 994, Annu. Rev. Immunol. 12: 433-455. Alternatively, with the display technique of the cells (McCafferty et al., 1 990, Nature 34 8 : 5 52-5 5 3 ). Immunoglobulin variable region (V) domain genes from donors who are not immunized The library is used to produce human antibodies and antibody fragments in vitro. According to this technique, an antibody V domain gene is selected in-frame to a major or minor coat protein gene such as Ml 3 or fd of a filamentous phage, and is displayed on the surface of the phage particle as a functional antibody fragment. on. Since the filamentous particle comprises a single DN A copy of the phage genome, screening based on the functional properties of the antibody also results in the selection of a gene encoding the antibody-49-201206466 that exhibits these properties. Thus, the phage mimetic B Several characteristics of cells. Phage display can be performed in a variety of formats; see, for example, Johnson, Kevin S. and Chiswell, David J., 1 993, Current Opinion in Structural Biology 3: 5 64-5 7 1. Several sources of V gene segments can be used for phage display. Clackson et al., 1991, Nature 3 5 2 : 624-628 A small random combinatorial library of V genes derived from the spleen of immunized mice isolated a wide variety of anti-oxazolinone antibody arrays. V gene depots from unimmunized donors can be constructed, and antibodies against various antigen arrays (including autoantigens) can be substantially in accordance with Mark et al., 1991, J. Mol. Biol. 222: 58 1 -597 or Griffith et al., 1 993, EMBO J. 12: 72 5-73 4 The technique described is isolated. In the natural immune response, antibody genes accumulate mutations at high rates (somatic hypermutation) . Several introduced changes will give higher affinity, and B cells displaying high affinity surface immunoglobulin will be preferentially replicated and differentiated during subsequent antigen challenge. This natural method can be modeled by a technique called "chain shuffling" (Marks et al., 1992, Bio/Technol. 10: 779-783). In this method, the affinity of the "primary" human antibody obtained by phage display can be improved to obtain a continuous reservoir of the naturally occurring variant (library) of the V domain gene from the unimmunized donor. Substitution of heavy and light chain V region genes. This technique allows the production of antibodies and antibody fragments with affinity ranging from Pimol to Naim. The strategy for producing very large phage antibody libraries (also known as "the mother-of-all 1 ibraries") has been adopted by '\\^161^11〇118661&1.,1 993,&gt ; 111 (;1· Acids Res. 21 : 2265-2266. Gene replacement can also be used to derive human antibodies from -50-201206466 rodent antibody, wherein the human antibody has affinity similar to the starting rodent antibody And specificity. According to this method, which is also called "epitope imprinting", the heavy chain or light chain V domain gene of the rodent antibody obtained by the phage display technology is replaced by the human V domain gene library to produce a rodent - Human chimera. Screening for an antigen results in the isolation of a human variable region capable of restoring a functional antigen binding site, ie, the epitope selected by the partner (imprinted) partner. When the method is repeated to replace the remaining rodent V domain A human antibody is obtained (see PCT Publication No. WO 93/062 13). Unlike conventional CDR grafting, which is a humanized chewing animal antibody, this technique provides a human body that does not have a rodent-derived skeleton or CD R residue. Antibody. Obvious Although the above discussion is about humanized antibodies, the general principles discussed apply to customary antibodies such as dogs, cats, primates, horses or cattle. It is also apparent that the humanized antibodies described herein are described. One or more aspects may be combined, such as CDR grafting, backbone mutation, and CDR mutation. The antibody may be recombinantly prepared by first isolating the antibody and antibody from the host animal to produce the cell, obtaining the gene sequence, and using the gene sequence for the host. A cell (e.g., a CHO cell) recombinantly expresses the antibody. Another method that can be employed is to express the antibody sequence in a plant (e.g., tobacco) or a gene-transferred milk. A method of recombinantly expressing an antibody in a plant or milk has been disclosed. See, for example, Peeters, 200 1, et al. Vaccine 1 9 : 2756; Lonberg, N. and D. Huszar, 1 995, Int Rev. Immunol 13 : 65 and Pollock, et al., 1999, J Immunol Methods 231 : 147 Methods for preparing antibody derivatives such as humanization, single-strand, etc. are known in the art. -51 - 201206466 Immunoassays and flow cytometric sorting techniques such as fluorescence activated cell sorting (FACS) can also be used. For the isolation of antibodies specific for the desired antigen. The antibodies can be combined with a number of different carriers. The carrier can be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, Polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, canola sugar and magnetite. The nature of the carrier may be soluble or insoluble for the purposes of the present invention. Those skilled in the art will be aware of other suitable carriers for binding antibodies, or may be verified by routine experimentation. In some embodiments, the carrier comprises a group that is marked by the myocardium. The DNA encoding the monoclonal antibody can be easily separated and sequenced by a conventional method (e.g., using an oligonucleotide probe capable of specifically binding to a gene encoding a heavy chain and a light chain of the monoclonal antibody). Hybridoma cells are used as a preferred source of this DNA. Once isolated, the DN A can be placed into a performance vector (such as the expression vector disclosed in PCT Publication No. WO 87/04462), which is then transfected into a host cell that would otherwise not produce an immunoglobulin, such as E. coli cells, anthropoid COS cells, Chinese hamster ovary (CHO) cells or myeloma cells are used to synthesize monoclonal antibodies in the recombinant host cells. See, for example, PCT Publication No. WO 87/04462. The DNA may also be modified by, for example, substituting a homologous mouse sequence with a coding sequence for the human heavy and light chain constant domains (Morrison et al., 1984, Proc. Nat. Acad. Sci. 81 = 6851), or by The coding sequence consisting of covalently linked immunoglobulin coding sequences with all or part of a non-immunoglobulin polypeptide is modified. In this manner, a "chimeric" or "hybridization" anti-system is prepared to have the binding specificity of a monoclonal antibody of -52 to 201206466. The pH dependent antibodies and polypeptides derived from the antibodies can be identified or characterized using methods known in the art to thereby detect and/or measure the reduction, improvement or neutralization of biological activity. In some embodiments, the p Η dependent antibody or polypeptide is identified by culturing the candidate agent with the antigen and monitoring the binding and/or consequent reduction or neutralization of the biological activity. The binding assay can be performed on a purified antigenic polypeptide or in a cell that is naturally expressed or transfected to express the polypeptide. In one embodiment, the binding assay is a competitive binding assay in which the ability of a candidate antibody to compete for binding to a known antagonist is assessed. This test can be performed in different forms, including the ELIS format. In other embodiments, the pH-dependent anti-system is identified by culturing the candidate with the antigen and monitoring binding and subsequent LDLR expression inhibition and/or blood cholesterol clearance. After initial identification, the activity of the candidate pH-dependent antibody can be further confirmed and refined by biological assays known to be used to test the biological activity of the target. Alternatively, bioassays can be used to directly screen for candidates. Several methods for identifying and characterizing antibodies, peptides or aptamers are described in detail in the Examples. 抗体 pH-dependent binding of antibodies can be characterized using methods well known in the art. For example, one method identifies the epitope to which it binds or is referred to as "epitope localization." There are a number of methods known in the art for mapping and characterizing the position of an epitope on a protein, including disrupting the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide substrate assays, for example, in Harlow and Lane, Using Antibodies, a -53- 201206466

Laboratory Manual ( Cold Spring Harbor Laboratory Press, Cold Spring Harbor，New York，1999)中第 11章所述。在另一實例中，表位定位可被用於測定pH依賴性抗體所結合之序列。表位定位可自不同之商業來源獲得，例如 Pepscan系統（Edelhertweg 15，8219 PH Lelystad，The Netherlands )。該表位可爲線性表位，亦即包含在單一片段之胺基酸中，或由不一定包含在單一片段中之胺基酸三維交互作用所形成之構型表位。各種長度之肽（例如至少 4至6個胺基酸長）可被分離或合成（例如重組合成）及用於抗體之結合檢測。在另一實例中，與pH依賴性抗體結合之表位可於系統性篩選中，藉由使用源自抗原序列之重疊肽並測定與抗體之結合加以測定。根據基因片段表現測定，編碼該抗原之開放閱讀框係經隨機分段或按特異性基因建構分段，且抗原之表現片段與所欲測試之抗體的反應性係經測定。該基因片段可能舉例來說由PCR產製，接著於活體外在放射性胺基酸存在時被轉錄及轉譯成蛋白質。該抗體與經放射性標記片段之結合接著由免疫沉澱及膠體電泳測定。某些表位亦可藉由使用噬菌體顆粒表面所展示之隨機狀序列的大型庫（噬菌體庫）加以識別。或者，可於簡單結合測定中測試經定義之重疊肽片段庫與該檢測抗體之結合。在另一實施例中，可進行抗原結合結構域之突變形成、結構域交換實驗及丙胺酸篩選突變形成以識別表位結合所需、足夠及/或必要之殘基。舉例來說，結構域交換實驗可利用突變抗原進行，其中該多肽之各種片段已 -54- 201206466 被來自其他物種之抗原的序列或密切相關但具抗原獨特性之蛋白（諸如蛋白原轉換酶家族之其他成員）取代（交換 )。藉由檢測該抗體與突變抗原之結合力，可評估特定抗原片段對抗體結合之重要性。另一可用於特徵化pH依賴性抗體之方法係使用已知和相同抗原結合之其他抗體的競爭試驗以測定該pH依賴性抗體是否如其他抗體般與該相同表位結合。競爭試驗爲該領域之技藝人士所廣爲週知。 - 表現載體可被用於直接表現pH依賴性抗體。該領域之技藝人士通曉投予表現載體以獲得外源性蛋白於活體內之表現。見例如美國專利第6,43 6,90 8、6,41 3,942及 6,376,471號。表現載體之投予包括局部或系統性投予，包括注射、經口投予、粒子槍或經導管投予及局部投予。在另一實施態樣中，該表現載體係直接投予至交感神經幹或神經節，或投予至冠狀動脈、心房、心室或心包膜中。靶向性遞送含有表現載體之治療性組成物或次基因組多核苷酸亦可被使用。受體媒介性DN A遞送技術係描述於例如 Findeis et al·，1993，Trends Biotechnol. 11 : 202 ; Chiou et a 1., 1 994， Gene Therapeutics : Methods AndThis is described in Chapter 11 of the Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999). In another example, epitope mapping can be used to determine the sequence to which a pH dependent antibody binds. Epitope mapping can be obtained from various commercial sources, such as the Pepscan system (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope can be a linear epitope, i.e., an amino acid contained in a single fragment, or a conformational epitope formed by a three-dimensional interaction of amino acids that are not necessarily contained in a single fragment. Peptides of various lengths (e.g., at least 4 to 6 amino acid lengths) can be isolated or synthesized (e.g., recombinantly synthesized) and used for binding detection of antibodies. In another example, an epitope associated with a pH dependent antibody can be determined in a systemic screen by using an overlapping peptide derived from the antigen sequence and determining binding to the antibody. Based on the gene fragment expression assay, the open reading frame encoding the antigen is segmented by random segmentation or by specificity, and the reactivity of the antigenic expression fragment with the antibody to be tested is determined. This gene fragment may, for example, be produced by PCR and then transcribed and translated into protein in the presence of a radioactive amino acid in vitro. Binding of the antibody to the radiolabeled fragment is followed by immunoprecipitation and colloidal electrophoresis. Certain epitopes can also be identified by the use of large libraries (phage libraries) of random sequences displayed on the surface of phage particles. Alternatively, the defined pool of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In another embodiment, mutation formation of the antigen binding domain, domain exchange experiments, and alanine screening mutation formation can be performed to identify residues that are required, sufficient, and/or necessary for epitope binding. For example, domain exchange experiments can be performed using mutant antigens, wherein various fragments of the polypeptide have been sequenced from antigens of other species or closely related but antigen-specific proteins (such as the proprotein convertase family). Other members) replaced (exchange). By examining the binding of the antibody to the mutated antigen, the importance of specific antigen fragments for antibody binding can be assessed. Another method that can be used to characterize pH-dependent antibodies is to use a competition assay for other antibodies known to bind to the same antigen to determine if the pH-dependent antibody binds to the same epitope as other antibodies. Competition trials are well known to those skilled in the art. - Expression vectors can be used to directly express pH dependent antibodies. Those skilled in the art are acquainted with the performance vector to obtain the performance of the exogenous protein in vivo. See, for example, U.S. Patent Nos. 6,43, 90 8, 6, 41 3,942 and 6,376,471. The administration of the performance vector includes local or systemic administration, including injection, oral administration, particle gun or transcatheter administration, and topical administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion, or to the coronary artery, atrium, ventricle, or pericardium. Targeted delivery of therapeutic compositions or subgenomic polynucleotides comprising an expression vector can also be used. Receptor-mediated DN A delivery techniques are described, for example, in Findeis et al., 1993, Trends Biotechnol. 11 : 202 ; Chiou et a 1., 1 994, Gene Therapeutics : Methods And

Applications Of Direct Gene Transfer ( J.A. Wolff, ed.) ;Wu et al·，1 988，J. Biol. Chem. 263 : 62 1 ; Wu et al.， 1 994, J. Biol. Chem. 269 ： 542 ； Zenke et al., 1 990, Proc. Natl. Acad. Sci. USA 87 ： 3 65 5 ； Wu et al., 1991, J. Biol. Chem. 2 66 : 3 3 8。含有多核苷酸之治療性組成物係以約 -55- 201206466 100奈克至約200毫克之DNA範園投予以供基因治療計畫中之局部投予。約500奈克至約50毫克、約1微克至約2毫克、約5微克至約5 00微克及約20微克至約1〇〇微克之DNA濃度範圍亦可被用於基因治療計畫期間。該治療性多核苷酸及多肽可利用基因遞送載具遞送。該基因遞送載具可爲病毒性或非病毒性來源（通常參考Jolly，1 994，Cancer Gene Therapy 1:51; Kimura, 1 994, Human Gene Therapy 5 ： 8 4 5 ; Connelly, 1 9 9 5, Human Gene Therapy 1 : 185 及Applications Of Direct Gene Transfer (J. Wolff, ed.); Wu et al., 1 988, J. Biol. Chem. 263: 62 1 ; Wu et al., 1 994, J. Biol. Chem. 269: 542; Zenke et al., 1 990, Proc. Natl. Acad. Sci. USA 87: 3 65 5 ; Wu et al., 1991, J. Biol. Chem. 2 66 : 3 3 8. The therapeutic composition containing the polynucleotide is administered topically in a gene therapy program at a DNA of about -55 to 201206466 100 ng to about 200 mg. A DNA concentration range of from about 500 ng to about 50 mg, from about 1 microgram to about 2 milligrams, from about 5 micrograms to about 500 micrograms, and from about 20 micrograms to about 1 microgram can also be used during the gene therapy program. The therapeutic polynucleotides and polypeptides can be delivered using a gene delivery vehicle. The gene delivery vehicle can be of a viral or non-viral source (generally reference Jolly, 994, Cancer Gene Therapy 1:51; Kimura, 1 994, Human Gene Therapy 5: 8 4 5; Connelly, 1 9 9 5, Human Gene Therapy 1 : 185 and

Kaplitt, 1994, Nature Genetics 6 ： 148)。該等編碼序列之表現可利用內源性哺乳動物或異源性啓動子誘導。編碼序列之表現可爲組成性或經調節的。供遞送所欲之多核苷酸及在所欲細胞中表現之病毒基底載體係該領域所熟知。示範性病毒基底載具包括但不限於重組反轉錄病毒（見例如PCT公開號WO 90/07936; WO 94/03622 ； WO 93/25 698 ； WO 93 /2523 4 ； W0 93/ 1 1 23 0 ； W0 93/1 02 1 8 ; W0 9 1/02805 ;美國專利第 5,2 1 9,740 及 4,777，127 號；GB 專利第 2,200,65 1 號及 EP 專利第 0 345 242 號）、阿爾法病毒基底載體（例如辛得比司（Sindbis ) 病毒載體、塞姆利基森林病毒（Semliki forest virus)( ATCC VR-67 ； ATCC VR- 1 247 )、羅斯河病毒（Ross River virus) (ATCC VR-373; ATCC VR-1246)及委內瑞拉馬腦炎病毒（ATCC VR-923 ; ATCC VR- 1 2 5 0 ; ATCC VR- 1 249; ATCC VR-5 3 2 ))及腺病毒相關病毒（AAV) 載體（見例如 PCT 公開號 WO 94/12649、WO 93/03769、 -56- 201206466Kaplitt, 1994, Nature Genetics 6: 148). The expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. The performance of the coding sequence can be constitutive or regulated. Viral substrate vectors for delivery of the desired polynucleotide and expression in the desired cells are well known in the art. Exemplary viral substrate carriers include, but are not limited to, recombinant retroviruses (see, for example, PCT Publication No. WO 90/07936; WO 94/03622; WO 93/25 698; WO 93/2523 4; W0 93/1 1 2 2 0; W0 93/1 02 1 8 ; W0 9 1/02805; U.S. Patent Nos. 5,2 1,9,740 and 4,777,127; GB Patent No. 2,200,65 1 and EP Patent No. 0 345 242), alpha virus substrate carrier (eg, Sindbis virus vector, Semliki forest virus (ATCC VR-67; ATCC VR-1 247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1 2 50; ATCC VR-1 249; ATCC VR-5 3 2) and adeno-associated virus (AAV) vectors (see For example, PCT Publication No. WO 94/12649, WO 93/03769, -56-201206466

WO 93/19191 、 WO 94/2893 8 、 WO 95/1 1 984 及 WO 95/00655)。如 Curiel，1992，Hum· Gene Ther. 3 : 147 中所述之與死腺病毒連接之DN A的投予亦可被採用。非病毒遞送之載具及方法亦可被採用，包括但不限於與單獨之死腺病毒連接或未連接之聚陽離子縮合DNA (見例如 Curiel，1992，Hum. Gene Ther. 3 : 147)、與配體連接之 DNA (見例如 Wu，J_，1 98 9，Biol. Chem. 264: 1 6985 )、真核細胞遞送載具細胞（見例如美國專利第 5,8 1 4,482 號、PCT 公開號 WO 95/07994、WO 96/1 7072、 WO 95/30763及WO 97/4233 8 )及核電荷中和或與細胞膜融合》裸DNA亦可被採用。示範性裸DNA導入方法係於 PCT公開號WO 90/11092及美國專利第5,580,859號中描述。可作爲基因遞送載具之脂質體係描述於美國專利第 5,422，120 號、PCT 公開號 WO 95/ 1 3 796、WO 94/23697、 WO 9 1 / 1 4445及EP 0524968中。其他方法係描述於Philip, 1 994，Mol. Cell Biol., 14 : 24 1 1 及 Woffendiη，1 994 Proc. Natl. Acad. Sci· 91 : 1581 中。本發明包含組成物（包括醫藥組成物），該組成物包含此處所述之抗體或由此處所述之方法產製且具有此處所述之特徵之抗體。此處所使用之組成物包含一或多種pH 依賴性抗體，及/或一或多種包含編碼一或多種該等抗體之序列的多核苷酸。該等組成物可能進一步包含適當之賦形劑，諸如醫藥上可接受之賦形劑包括該領域眾所周知之緩衝劑。 ’ -57- 201206466 本發明亦提供pH依賴性抗體之CDR部分（包括柯西亞 (Chothia ) CDR 及卡巴（Kabat) CDR) 。CDR 區之測定係屬該領域所熟知之技藝。應了解在一些實施態樣中， CDR可爲卡巴及柯西亞CDR之組合（亦稱爲「組合CDR」或「延伸CDR」）。在一些實施態樣中，該CDR係卡巴 CDR。在其他實施態樣中，該CDR係柯西亞CDR。換句話說，在具有超過一個CDR之實施態樣中，該CDR可爲卡巴、柯西亞、組合CDR或彼等之組合中之任一者。本發明亦提供製備任何該等抗體或多肽之方法。本發明之抗體可利用該領域已知之方法製備。多肽可藉由抗體之蛋白水解或其他降解產製、藉由如上所述之重組方法（亦即單一或融合多肽）或藉由化學合成加以製備。該等抗體之多肽特別是最多約50個胺基酸之較短多肽係方便地藉由化學合成製備。化學合成之方法係該領域所知且可自商業途徑獲得。舉例來說，抗體可藉由採用固相方法之自動多肽合成儀產製。見例如美國專利第5,807,7 1 5、 4,8 1 6,567及6,331，415號。在另一替代態樣中，該等抗體及肽可利用該領域廣爲週知之方法重組製備。在一實施態樣中，多核苷酸包含編碼抗體4A5、5A10、6F6、7D4或L1L3之重鏈及/或輕鏈可變區之序列。編碼該感興趣之抗體的序列可被維持於宿主細胞之載體內，接著該宿主細胞可被擴增及冷凍以供將來使用。載體（包括表現載體）及宿主細胞另於此處說明。本發明亦包含本發明之抗體的scFv。單鏈可變區片段 -58- 201206466 係藉由使用短連接肽連接輕鏈及/或重鏈可變區加以製備 (Bird et al.s 1 9 8 8，S c i en c e 2 4 2 : 4 2 3 - 4 2 6 ) » 連接肽之實例係（GGGGS) 3(SEQ ID NO: 22)，其橋接一可變區之羧基端與另一可變區之胺基端之間約3.5奈米之距離。其它序列之連接子已被設計及使用（Bird et al.，198 8，同上）。連接子應爲短且可彎折之多肽，較佳地包含少於約20個胺基酸殘基。連接子可依次經修飾以得到額外功能，諸如與藥物連接或與固態支持物連接。單鏈變異體可經重組或合成產製。就scFv之合成產製而言，可使用自動化合成儀。就scFv之重組產製而言，包含編碼該scFv之多核苷酸之適當質體可被導入適當之宿主細胞中，不論是真核細胞諸如酵母菌、植物、昆蟲或哺乳動物細胞，抑或是原核細胞諸如大腸桿菌。編碼感興趣之scFv之多核苷酸可藉由例行操作加以製備，諸如連接多核苷酸。該形成之scFv 可利用該領域已知之標準蛋白質純化技術分離。其他形式之單鏈抗體諸如雙功能抗體（diabodies )亦被包含。雙價抗體係雙價、雙特異性之抗體，其中VH及 VL結構域係表現於單一多肽鏈上，但使用過短以防止該同一鏈上之兩個結構域之間配對之連接子，藉此強迫該等結構域與另一鏈之互補結構域配對並產生兩個抗原結合部位（見例如 Holliger，P·，et al.，1993，Proc. Natl. Acad Sci. USA 90 ： 6444-6448 ； Poljak, R. J., et al., 1 994, Structure 2 : 1121-1123)。舉例來說，雙特異性抗體亦即對至少兩種不同抗原具 -59- 201206466 有結合特異性之單株抗體可利用此處所揭示之抗體製備》製備雙特異性抗體之方法係該領域所知（見例如Suresh et al., 1 9 8 6, Methods in Enzymology 121 : 210)。傳統上，雙特異性抗體之重組產製係基於兩個免疫球蛋白重鏈-輕鏈對之共表現，其中該兩個重鏈具有不同之特異性（ Millstein and Cuello, 1 983, Nature 3 05, 53 7-53 9 )。根據一種製備雙特異性抗體之方法，具有所欲結合特異性之抗體可變結構域（抗體-抗原結合部位）係與免疫球蛋白恆定結構域序列融合。該融合較佳係具有免疫球蛋白重鏈恆定結構域，包含至少部份之鉸鏈區、CH2區及 CH3區。較佳地是使包含輕鏈結合所需之部位的第一重鏈恆定區（CH1)存在於該融合之至少一者。編碼免疫球蛋白重鏈融合及若需要之免疫球蛋白輕鏈之DN A被***分開之表現載體，並被共轉染至適當之宿主有機體。在使用不等比例之三個多肽鏈建構以提供最佳產量之實施態樣中，此提供調整三個多肽片段之相互比例的高度靈活性。然而，當以相等比例表現至少兩個多肽鏈導致髙產量，或該比例不具特別顯著性時，有可能在一個表現載體內***兩個或所有三個多肽鏈之編碼序列。在一態樣中，該雙特異性抗體係由一臂爲具有第一結合特異性之雜交免疫球蛋白重鏈及另一臂爲雜交免疫球蛋白重鏈-輕鏈對（提供第二結合特異性）所組成。此種免疫球蛋白輕鏈僅位於該雙特異性分子之一半的不對稱結構有利於自非所欲之免疫球蛋白鏈組合中分離該所欲之雙特 -60- 201206466 異性化合物。此方法係描述於PCT公開號WO 94/04690。包含兩個共價連接抗體之異源共軛抗體亦屬於本發明之範圍內。該等抗體已被用於使免疫系統細胞以非所欲之細胞爲標靶（美國專利第4,676,980號）及用於治療HIV感染（PCT公開號 WO 91/00360、WO 92/200373 及 EP 03089 )。異源共軛抗體可利用任何方便之交聯方法製備。適當之交聯劑及技術係該領域所廣爲週知且描述於美國專利第 4,676,980號。嵌合或雜交抗體亦可利用已知之合成蛋白質化學方法於活體外製備，包括該些涉及交聯劑之方法。舉例來說，免疫毒素可利用雙硫交換反應或藉由形成硫醚鍵加以建構。爲達此目的之適當試劑實例包括亞胺基硫醇鹽及甲基-4-毓基丁亞胺酸酯。包含抗體5A10或7D4之一或多個CDR或衍生自抗體 5A10或7D4之一或多個CDR之人化抗體可利用例如該領域已知之任何方法製備。舉例來說，四個常規步驟可被用於人化單株抗體。這些步驟爲：（Π測定起始抗體之輕鏈及重鏈可變結構域之核苷酸及預測之胺基酸序列；（2 ) 設計該人化抗體，亦即決定在人化步驟期間將使用哪一個抗體骨架區；（3)使用實際人化方法/技術；及.（4)轉染及表現該人化抗體。見例如美國專利第4,8 1 6,5 67、 5,807,715 、 5,866,692 、 6,331，415 、 5,530，101 、 5,693,761 ' 5,693,762、 5,585,089及 6，180,370號。在重組人化抗體中，該Fc部分可經修飾以避免與Fc τ -61 - 201206466 受體及補體及免疫系統之交互作用。用於技術係於WO 99/58572中描述。舉例來說工程化以更類似於人之恆定區，避免該抗試驗及治療時之免疫反應。見例如美國及 5,866,692號。包含抗體或彼之變異體之輕鏈或重鏈個CDR，或衍生自抗體或彼之變異體之一化抗體可利用該領域已知之任何方法製備人化抗體可利用該領域習知之任何方本發明包含對本發明之抗體及多肽之顯著影響彼等之性質之功能相等性抗體及活性及/或親和性之變異體。舉例來說，經突變以獲得對彼之抗原具有所欲結合親肽之修飾係該領域之例行操作，不須在此之修飾係於實施例中舉例說明。經修飾之有保守性取代之胺基酸殘基、不顯著有害性或使該多肽對彼之配體之親和性成熟（個胺基酸刪除或添加或使用化學類似物之胺基酸序列導入包括長度介於一個殘或更多殘基之多肽之胺基端及/或羧基端序列內***單一或多個胺基酸殘基。末端具有N-端甲硫胺醯基殘基之抗體或與表位。抗體分子之其它導入變異體包括在抗骨酵素或多肽以增加該抗體於血液循環中之製備該等抗體之，該恆定區可經體用於人之臨床專利第5,997,867 可變區或一或多或多個CDR之人〇法製備。修飾，包括不會具有增進或降低胺基酸序列可能和性之抗體。多詳細說明。多肽多肽實例包括具地改變該官能活增進）之一或多多肽。基至包含上百個融合，也包括在導入之實例包括標籤融合之抗體豊之N或C端融合半衰期。 -62- 201206466 取代變異體在該抗體分子中具有至少一個被移除之胺基酸殘基且在該位置導入不同之殘基。最受關注之供取代性突變形成之位置包括超變異區，但是亦考慮FR改變。保守性取代係顯示於表1中標題「保守性取代」之欄。若該等取代導致生物活性之改變，接著更顯著之改變稱爲「示範性取代」或如下參照胺基酸分類進一步描述者可被導入及篩選該產物。表1 :胺基酸取代原始殘基保守性取代示範性取代丙胺酸(A) 纈胺酸纈胺酸；白胺酸：異白胺酸精胺酸(R) 離胺酸離胺酸；麩醯胺酸；天冬醯胺酸天冬醯胺酸(N) 麩醯胺酸麩醯胺酸；組胺酸；天冬胺酸；離胺酸：精胺酸天冬胺酸(D) 麩胺酸麩胺酸；天冬醢胺酸半胱胺酸(C) 絲胺酸絲胺酸；丙胺酸麩醯胺酸(Q) 天冬醯胺酸天冬醯胺酸；麩胺酸麩胺酸(E) 天冬胺酸天冬胺酸；麩醯胺酸甘胺酸(G) 丙胺酸丙胺酸組胺酸(H) 精胺酸天冬醯胺酸；麩醯胺酸；離胺酸；精胺酸異白胺酸(I) 白胺酸白胺酸；顯胺酸；甲硫胺酸；丙胺酸；***酸 :正白胺酸白胺酸(L) 異白胺酸正白胺酸；異白胺酸；纈胺酸；甲硫胺酸；丙胺酸；***酸離胺酸(K) 精胺酸精胺酸；麩醯胺酸；天冬醯胺酸甲硫胺酸(M) 白胺酸白胺酸；***酸；異白胺酸 ***酸(F) 酪胺酸白胺酸；纈胺酸；異白胺酸；丙胺酸；酪胺酸脯胺酸(P) 丙胺酸丙胺酸絲胺酸(S) 蘇胺酸蘇胺酸蘇胺酸(T) 絲胺酸絲胺酸色胺酸(W) 酪胺酸酪胺酸；***酸酪胺酸⑺ ***酸色胺酸；***酸；蘇胺酸；絲胺酸纈胺酸(V) 白胺酸異白胺酸；正白胺酸：甲硫胺酸；***酸；丙胺酸；正白胺酸 63- 201206466 對抗體生物特性之實質修飾可藉由選擇在維持下列特性上有顯著差異之取代加以完成：（a)取代區之多肽骨架結構，例如摺板狀或螺旋構型，（b)標的部位之分子的帶電或疏水性，或（c )側鏈之主體。天然發生之殘基根據常見之側鏈性質分成下列群組：（1 )非極性：正白胺酸、甲硫胺酸（Met )、丙胺酸（Ala )、纈胺酸（Val )、白胺酸（Leu)、異白胺酸（lie) ； (2)不帶電荷之極性：半胱胺酸（Cys )、絲胺酸（Ser )、蘇胺酸（ Thr)、天冬醯胺酸（Asn)、麩醯胺酸（Gin) ; (3)酸性（負電荷）：天冬胺酸（Asp )、麩胺酸（Glu ) ； (4 )鹼性（正電荷）：離胺酸（Lys)、精胺酸（Arg);( 5 )影響側鏈方向性之殘基：甘胺酸（Gly )、脯胺酸（ Pro );及（6 )芳香性：色胺酸（Trp )、酪胺酸（Tyr ) 、***酸（Phe)、組胺酸（His)。非保守性取代係以上述分類中之一個分類的成員交換另一分類之成員達成。任何與維持抗體之適當構型無關之半胱胺酸殘基亦可被取代（通常以絲胺酸取代），以增進該分子之氧化穩定性及防止異常交聯。相反地，半胱胺酸鍵可被加入抗體以增進抗體之穩定性，特別是當該抗體係抗體片段諸如Fv片段時。胺基酸修飾可從改變或修飾一或多個胺基酸至完全重新設計一區諸如可變區。可變區之變化可改變結合親和性 -64- 201206466 及/或特異性。在一些實施態樣中，在一 CDR結構域內不發生超過一至五個保守性胺基酸取代。在其他實施態樣中，在一 CDR結構域內不發生超過一至三個保守性胺基酸取代。在其他實施態樣中，該CDR結構域係CDR H3及/或 CDR L3。修飾亦包括糖基化及非糖基化之多肽，也包括具有其他轉譯後修飾之多肽，諸如舉例來說以不同之糖進行糖基化、乙醯化及磷酸化。抗體在彼等之恆定區的保守性位置係經糖基化（Jefferis and Lund，1997，Chem. Immunol. 65 :111-128 ; Wright and Morrison, 1 997, TibTECH 15 ： 26-32)。免疫球蛋白之寡糖側鏈影響該蛋白質之功能（Boyd et al., 1 996，Mol. Immunol. 32 ： 1311-1318 ； Wittwe and Howard，1990，Biochem. 29: 4175-4180)及該糖蛋白之部份之間的分子內交互作用，該交互作用可影響該糖蛋白之構形及呈現三維表面（Jefferis and Lund，同上；Wyss and Wagner，1996，Current Opin. Biotech. 7 : 409-416)。根據特定辨認結構，寡糖亦可被用來使給定糖蛋白瞄準某些分子。也有報告指出抗體之糖基化會影響ADCC。特別是，經四環素調節表現( 1，4 ) -N-乙醯葡萄胺基轉移酶III (GnTIII )之CHO細胞被報告具有增強之ADCC活性（ Umana et al., 1 999，Nature Biotech. 1 7 : 176-180 )，WO 93/19191, WO 94/2893 8 , WO 95/1 1 984 and WO 95/00655). Administration of DN A linked to the dead adenovirus as described in Curiel, 1992, Hum Gene Ther. 3: 147 can also be employed. Vehicles and methods for non-viral delivery may also be employed, including but not limited to polycation condensation DNA linked or unligated to a separate adenovirus (see, for example, Curiel, 1992, Hum. Gene Ther. 3: 147), and Ligand-ligated DNA (see, e.g., Wu, J., 1 98 9, Biol. Chem. 264: 1 6985), eukaryotic cell delivery vehicle cells (see, e.g., U.S. Patent No. 5,814,482, PCT Publication No. WO 95/07994, WO 96/1 7072, WO 95/30763, and WO 97/4233 8) and nuclear charge neutralization or fusion with cell membranes may also be employed. An exemplary naked DNA introduction method is described in PCT Publication No. WO 90/11092 and U.S. Patent No. 5,580,859. A lipid system that can be used as a gene delivery vehicle is described in U.S. Patent No. 5,422,120, PCT Publication No. WO 95/1 3 796, WO 94/23697, WO 9 1 /1 4445, and EP 0524968. Other methods are described in Philip, 1 994, Mol. Cell Biol., 14: 24 1 1 and Woffendi η, 1 994 Proc. Natl. Acad. Sci 91: 1581. The invention comprises a composition (including a pharmaceutical composition) comprising an antibody as described herein or an antibody produced by the methods described herein and having the characteristics described herein. A composition for use herein comprises one or more pH dependent antibodies, and/or one or more polynucleotides comprising sequences encoding one or more of such antibodies. Such compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers well known in the art. The invention also provides CDR portions of pH dependent antibodies (including Chothia CDRs and Kabat CDRs). The determination of the CDR regions is well known in the art. It will be appreciated that in some embodiments, the CDRs can be a combination of Kabbah and Koccia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDR is a Kabbah CDR. In other embodiments, the CDR is a Kosia CDR. In other words, in embodiments having more than one CDR, the CDR can be either Kabbah, Kosia, a combined CDR, or a combination thereof. The invention also provides methods of making any of such antibodies or polypeptides. Antibodies of the invention can be prepared by methods known in the art. The polypeptide may be produced by proteolysis or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides) or by chemical synthesis. The polypeptides of such antibodies, particularly the shorter polypeptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be produced by automated peptide synthesizers employing a solid phase method. See, for example, U.S. Patent Nos. 5,807,7, 5, 4,8, 6,567, and 6,331,415. In another alternative, the antibodies and peptides can be recombinantly produced using methods well known in the art. In one embodiment, the polynucleotide comprises a sequence encoding a heavy chain and/or a light chain variable region of antibody 4A5, 5A10, 6F6, 7D4 or L1L3. The sequence encoding the antibody of interest can be maintained in a vector of the host cell, which can then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are also described herein. The invention also encompasses scFvs of the antibodies of the invention. The single-chain variable region fragment -58-201206466 was prepared by ligating the light chain and/or heavy chain variable region with a short linker peptide (Bird et al.s 1 9 8 8, S ci en ce 2 4 2 : 4 2 3 - 4 2 6 ) » An example of a linker peptide (GGGGS) 3 (SEQ ID NO: 22) which bridges between the carboxy terminus of one variable region and the amine terminus of the other variable region by about 3.5 nm The distance. Linkers of other sequences have been designed and used (Bird et al., 198 8, supra). The linker should be a short and bendable polypeptide, preferably containing less than about 20 amino acid residues. The linker can in turn be modified to provide additional functionality, such as attachment to a drug or attachment to a solid support. Single-stranded variants can be produced recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, the appropriate plasmid comprising the polynucleotide encoding the scFv can be introduced into a suitable host cell, whether eukaryotic cells such as yeast, plant, insect or mammalian cells, or prokaryotic Cells such as E. coli. Polynucleotides encoding the scFv of interest can be prepared by routine manipulation, such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art. Other forms of single chain antibodies such as diabodies are also included. A bivalent, bispecific, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but are used too short to prevent pairing between the two domains on the same chain. This forces the domains to pair with the complementary domains of the other strand and create two antigen binding sites (see, eg, Holliger, P., et al., 1993, Proc. Natl. Acad Sci. USA 90: 6444-6448; Poljak, RJ, et al., 1 994, Structure 2: 1121-1123). For example, bispecific antibodies, ie, monoclonal antibodies having binding specificity for at least two different antigens -59-201206466 can be prepared using the antibodies disclosed herein. Methods for preparing bispecific antibodies are known in the art. (See, for example, Suresh et al., 1 9 8 6, Methods in Enzymology 121: 210). 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 chains have different specificities (Millstein and Cuello, 1 983, Nature 3 05 , 53 7-53 9 ). According to one method of producing a bispecific antibody, an antibody variable domain (antibody-antigen binding site) having the desired binding specificity is fused to an immunoglobulin constant domain sequence. Preferably, the fusion has an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, the CH2 region, and the CH3 region. Preferably, the first heavy chain constant region (CH1) comprising the moiety required for light chain binding is present in at least one of the fusions. The DN A encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into the appropriate host organism. This provides a high degree of flexibility in adjusting the mutual ratio of the three polypeptide fragments in an embodiment using three polypeptide chains of varying proportions to provide optimal yield. However, when at least two polypeptide chains are expressed in equal proportions resulting in sputum production, or the ratio is not particularly significant, it is possible to insert a coding sequence for two or all three polypeptide chains in one expression vector. In one aspect, the bispecific anti-system consists of a hybrid immunoglobulin heavy chain with a first binding specificity and a hybrid immunoglobulin heavy chain-light chain pair with one arm (providing a second binding specificity) Sexual). The asymmetric structure of such an immunoglobulin light chain located only in one half of the bispecific molecule facilitates the separation of the desired double-specific compound from the undesired combination of immunoglobulin chains. This method is described in PCT Publication No. WO 94/04690. Heterologous conjugated antibodies comprising two covalently linked antibodies are also within the scope of the invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (PCT Publication Nos. WO 91/00360, WO 92/200373 and EP 03089). . The heterologous conjugated antibody can be prepared by any convenient cross-linking method. Suitable crosslinkers and techniques are well known in the art and are described in U.S. Patent No. 4,676,980. Chimeric or hybrid antibodies can also be prepared in vitro using known synthetic protein chemistry, including those involving crosslinkers. For example, an immunotoxin can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrate. A humanized antibody comprising one or more CDRs of antibody 5A10 or 7D4 or one or more CDRs derived from antibody 5A10 or 7D4 can be prepared, for example, by any method known in the art. For example, four general steps can be used to humanize monoclonal antibodies. These steps are: (Π determining the nucleotides of the light and heavy chain variable domains of the starting antibody and the predicted amino acid sequence; (2) designing the humanized antibody, ie determining that during the humanization step Which antibody backbone region is used; (3) using actual humanization methods/technologies; and (4) transfecting and expressing the humanized antibody. See, for example, U.S. Patents 4,8 1 6,5 67, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761 '5,693,762, 5,585,089 and 6,180,370. In recombinant humanized antibodies, the Fc portion can be modified to avoid interaction with the Fc τ -61 - 201206466 receptor and complement and immune system The technique is described in WO 99/58572. For example, it is engineered to be more similar to the human constant region, avoiding the immune response during the test and treatment. See, for example, U.S. and 5,866,692. The light chain or heavy chain CDR of the variant, or the antibody derived from the antibody or variant thereof, can be prepared by any method known in the art. Any one of the prior art can be utilized. Antibodies and polypeptides that significantly affect their functionally equivalent antibodies and variants of activity and/or affinity. For example, mutations to obtain a modification of the antigen to which the antigen is desired to bind to the field Routine operations, which are not necessarily exemplified in the examples, are modified to have conservatively substituted amino acid residues, are not significantly deleterious, or affinity matures the polypeptide to its ligand. (Amino acid deletion or addition or addition of amino acid sequence using a chemical analog, including the insertion of a single or multiple amine groups in the amino terminus and/or carboxy terminal sequence of a polypeptide having a residue of one or more residues in length An acid residue. An antibody or epitope with an N-terminal methionine thiol residue at the end. Other introduced variants of the antibody molecule include an anti-ostease or polypeptide to increase the antibody in the blood circulation to prepare the antibody. The constant region can be prepared by human use in the variable region of human clinical patent No. 5,997,867 or one or more CDRs. Modifications, including no increase or decrease in amino acid sequence possibility and The antibody is described in more detail. One example of a polypeptide polypeptide includes one or more polypeptides that modify the functional activity. The base includes N or C-end fusions comprising hundreds of fusions, including antibodies introduced in the examples including introduction of the tag fusion. The half-life. -62- 201206466 The substitution variant has at least one removed amino acid residue in the antibody molecule and introduces a different residue at this position. The most interesting position for the formation of a substitution mutation includes hypermutation Zones, but FR changes are also considered. Conservative substitutions are shown in the heading "Conservative substitutions" in Table 1. If such substitutions result in a change in biological activity, then a more significant change is referred to as "exemplary substitution" or as further described below with reference to the amino acid classification, the product can be introduced and screened. Table 1: Amino acid substitution of the original residue Conservative substitution Exemplary substitution of alanine (A) Proline glutamic acid; leucine: isoleucine arginine (R) lysine lysine; bran Proline acid; aspartic acid aspartic acid (N) glutamic acid bran acid; histidine; aspartic acid; lysine: arginine aspartate (D) bran Amino acid glutamic acid; aspartate sucrose (C) serine acid serine; alanine glutamic acid (Q) aspartic acid aspartic acid glutamine Acid (E) aspartic acid aspartate; glutamic acid glycine (G) alanine alanine histidine (H) arginine aspartic acid; glutamic acid; lysine ; arginine isoleucine (I) leucine leucine; leucine; methionine; alanine; phenylalanine: orthoamine leucine (L) isoleucine white amine Acid; isoleucine; valine; methionine; alanine; phenylalanine lysine (K) arginine arginine; glutamic acid; aspartate methionine (M Leucine leucine; phenylalanine; iso-alanine phenylalanine (F) tyrosine Amino acid; valine acid; isoleucine; alanine; tyrosine glutamic acid (P) alanine alanine serine (S) sulphate sulphate sulphate (T) leucine Serine tryptophan (W) tyrosine tyrosine; phenylalanine tyrosine (7) phenylalanine tryptophan; phenylalanine; threonine; serine valine (V) leucine Acid; leucine: methionine; phenylalanine; alanine; leucine 63- 201206466 Substantial modification of the biological properties of an antibody can be accomplished by selecting substitutions that maintain significant differences in the following properties: a) the polypeptide backbone structure of the substitution region, such as a plate-like or helical configuration, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the host of the side chain. Naturally occurring residues are classified into the following groups based on common side chain properties: (1) non-polar: noractucine, methionine (Met), alanine (Ala), proline (Val), amine Acid (Leu), isoleucine (lie); (2) Uncharged polarity: cysteine (Cys), serine (Ser), threonine (Th), aspartic acid ( Asn), glutamic acid (Gin); (3) acidic (negative charge): aspartic acid (Asp), glutamic acid (Glu); (4) basic (positive charge): lysine (Lys , arginine (Arg); (5) residues that affect the directionality of the side chain: glycine (Gly), proline (Pro); and (6) aromatic: tryptophan (Trp), cheese Amine acid (Tyr), phenylalanine (Phe), histidine (His). Non-conservative substitutions are made by members of one of the above categories exchanging members of another classification. Any cysteine residue that is unrelated to the proper configuration of the maintenance antibody can also be substituted (usually substituted with serine) to enhance the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, a cysteine bond can be added to the antibody to enhance the stability of the antibody, particularly when the anti-system antibody fragment, such as an Fv fragment. Amino acid modifications can range from changing or modifying one or more amino acids to completely redesigning a region such as a variable region. Changes in the variable region can alter binding affinity -64-201206466 and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions occur within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions occur within a CDR domain. In other embodiments, the CDR domain is CDR H3 and/or CDR L3. Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides having other post-translational modifications, such as, for example, glycosylation, acetylation, and phosphorylation with different sugars. The conserved positions of the antibodies in their constant regions are glycosylated (Jefferis and Lund, 1997, Chem. Immunol. 65: 111-128; Wright and Morrison, 1 997, TibTECH 15: 26-32). The oligosaccharide side chain of an immunoglobulin affects the function of the protein (Boyd et al., 1 996, Mol. Immunol. 32: 1311-1318; Wittwe and Howard, 1990, Biochem. 29: 4175-4180) and the glycoprotein Intramolecular interactions between the parts that affect the conformation of the glycoprotein and its three-dimensional surface (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7: 409-416) . Oligosaccharides can also be used to target a given glycoprotein to certain molecules, depending on the particular identity structure. It has also been reported that antibody glycosylation affects ADCC. In particular, CHO cells expressing (1,4)-N-acetylglucosyltransferase III (GnTIII) by tetracycline were reported to have enhanced ADCC activity (Umana et al., 1 999, Nature Biotech. 1 7 : 176-180 ),

GnTIII是一種酶催化形成平分型GlcNAc之糖基轉移酶。抗體之糖基化通常不是N·連接就是0-連接。N-連接係指碳水化合物基團連接至天冬醯胺酸殘基之側鏈。天冬醯 -65- 201206466 胺酸-X-絲胺酸、天冬醯胺酸-X-蘇胺酸及天冬醯胺酸-x-半胱胺酸之三肽序列係供碳水化合物基團與天冬醯胺酸側鏈酵素性連接之辨識序列，其中X係除脯胺酸以外之任何胺基酸。因此，多肽中有任何該等三肽序列之存在即產生可能的糖基化位置。〇·連接糖基化係指連接糖類N-乙醯半乳糖胺、半乳糖或木糖之一者於羥基胺基酸，最常見的是絲胺酸或蘇胺酸，不過5-羥基脯胺酸或5-羥基離胺酸亦可被使用。在抗體中加入糖基化位置可藉由改變胺基酸序列以使該序列包含一或多個上述之三肽序列（供N_連接糖基化位置）而方便地完成。該改變亦可藉由在原始抗體之序列加入或取代一或多個絲胺酸或蘇胺酸殘基加以完成（供0-連接糖基化位置）。抗體之糖基化模式亦可在不改變基礎核苷酸序列下加以改變。糖基化大部分取決於用於表現該抗體之宿主細胞。由於用於表現重組糖蛋白例如抗體以作爲潛在治療劑之細胞種類鮮少爲天然細胞，因此抗體之糖基化模式的差異可被預期（見例如 Hse et al.，1997，J. Biol· Chem. 272: 9062-9070)。除了宿主細胞之選擇以外，在重組產製抗體期間可影響糖基化之因素包括生長模式、培養基調製劑、培養密度、氧化、pH、純化程序及該類似因素。各種方法已被提出以改變在特定宿主生物體中達成之糖基化模式，包括導入或過度表現與寡糖產製有關之某些酵素（美國專利第 -66 - 201206466 5,047,3 3 5 ' 5,510,261及5,278,299號）》糖基化或某些類型之糖基化可自糖蛋白經酵素移除，舉例來說利用內切糖苷酶H(Endo Η) 、N-糖苷酶F、內切糖苷酶F1、內切糖苷酶F2、內切糖苷酶F3。此外，該重組宿主細胞可經基因工程化以不全地處理某些種類之多醣。這些及類似之技術係該領域所廣爲週知。其他修飾之方法包括使用該領域已知之偶合技術，包括但不限於酵素方法、氧化性取代及螯合。修飾可被用於例如連接免疫測定之標記。經修飾之多肽係利用該領域已建立之方法製備，且可利用該領域已知之標準測定篩選，這些方法中有些係於下面及實施例中描述。在本發明之一些實施態樣中，該抗體包含經修飾之恆定區，諸如呈免疫惰性或部分惰性之恆定區，例如不引發補體媒介性溶解、不刺激ADCC或不活化小神經膠質細胞 ;或在下列任一或多項具有減少之活性（相較於未經修飾之抗體）：引發補體媒介性溶解、刺激ADCC或活化小神經膠質細胞。恆定區之不同修飾可被用於達到最佳量及/ 或組合之效應功能。見例如 Μ 〇 r g a η et al., 1 995, Immunology 86 ： 319-324 ； Lund et al ·， 1996, J. Immunology 157 ： 4963-4969 ； Iduso gie et al · ,2000, J. Immunology 164 ： 4178-4184 ;Tao et al ·， 1 989， J. Immunology 143 ： 2595-2601 及.Jefferi s et al., 1 998, Immunologii :al Rev ie ws 163: 59-76 。在一些實施態樣中，該恆定區係經如 Eur. J. Immunol.，1999, 29 : 2613-2624 67- 201206466 ;PCT公開號W09 9/58572及/或英國專利申請號9809951.8 所述之修飾。在其他實施態樣中，該抗體包含人重鏈IgG2 恆定區，該恆定區包含下列突變：A3 3 0P331至S 3 3 0S331 (胺基酸編號參照野生型IgG2序列）（Eur. J. Immunol.， 1999, 29: 2613-2624)。在其他實施態樣中，該恆定區之 N-連接糖基化係經去糖基化。在一些實施態樣中，該恆定區之N連接糖基化係藉由使該糖基化胺基酸殘基或該恆定區中屬於N-糖基化辨認序列之部份的側翼殘基突變而去糖基化。舉例來說，N糖基化位置N297可經突變成A、Q、K 或 Η 〇見 Tao et al., 1 989，J. Immunology 143 : 2595-2601 及 Jefferis et al., 1 9 9 8, Immunological Reviews 1 63 : 59-76。在一些實施態樣中，該恆定區之N-連接糖基化係經去糖基化。該恆定區之N-連接糖基化可經酵素（諸如藉由酵素PNGase移除碳水化合物）去糖基化或藉由在糖基化缺陷之宿主細胞中表現以去糖基化。其他抗體修飾包括已如PCT公開號WO 99/5 8 5 72所述修飾之抗體。除了針對標的分子之結合結構域之外，該等抗體還包含具有實質上與人免疫球蛋白重鏈之恆定結構域的所有或部分同源之胺基酸序列的效應結構域。該等抗體能與標的分子結合但不引發顯著之補體依賴性溶解或細胞媒介性破壞該標的。在一些實施態樣中，該效應結構域能特異性結合FcRn及/或Fc r RHb。這些作用通常是根據衍生自兩或多個人免疫球蛋白重鏈CH2結構域之嵌合結構域。以此方式修飾之抗體特別適用於慢性抗體治療，以避免 -68- 201206466 習知抗體治療之發炎及其他不良反應》本發明包括親和性成熟實施態樣。舉例來說，親和性成熟抗體可利用該領域已知之方法產製（Marks et al.， 1 992，Bio/Technology, 10 ： 779-783 ； Barbas et al.，1 994, Proc Nat. Acad. Sci, USA 9 1 : 3 809-3 8 1 3 ； Schier et al.， 1 995， Gene, 169 ： 1 47- 1 55 ： Yelton et al.， 1 995， J. Immunol., 1 5 5 : 1994-2004 : Jackson et al., 1995， J. Immunol·, 154 ( 7) ： 3 310-9 ; Hawkins et al·，1992，J.GnTIII is a glycosyltransferase that catalyzes the formation of a bipartite GlcNAc. Glycosylation of antibodies is usually not N. linkage or 0-linkage. N-linked refers to the attachment of a carbohydrate group to the side chain of an aspartic acid residue. Asparagus-65- 201206466 Amino acid-X-serine, aspartic acid-X-threonine and aspartic acid-x-cysteine tripeptide sequence for carbohydrate groups An identification sequence linked to an aspartic acid side chain enzyme, wherein X is any amino acid other than proline. Thus, the presence of any of these tripeptide sequences in the polypeptide results in a potential glycosylation site. 〇·linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to hydroxylamine acid, the most common being serine or threonine, but 5-hydroxyguanamine Acid or 5-hydroxy lysine can also be used. The addition of a glycosylation site to an antibody can be conveniently accomplished by altering the amino acid sequence such that the sequence comprises one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). This alteration can also be accomplished by the addition or substitution of one or more serine or threonine residues in the sequence of the original antibody (for the 0-linkage glycosylation position). The glycosylation pattern of the antibody can also be altered without altering the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell types used to express recombinant glycoproteins, such as antibodies, as potential therapeutic agents are few naturally occurring, differences in glycosylation patterns of antibodies can be expected (see, for example, Hse et al., 1997, J. Biol. Chem). 272: 9062-9070). In addition to the selection of host cells, factors that can affect glycosylation during recombinant production of antibodies include growth patterns, media modulators, culture density, oxidation, pH, purification procedures, and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes associated with oligosaccharide production (US Patent No. -66 - 201206466 5,047, 3 3 5 ' 5,510,261) And 5,278,299) glycosylation or certain types of glycosylation can be removed from the glycoprotein by an enzyme, for example, using endoglycosidase H (Endo Η), N-glycosidase F, endoglycosidase F1 , endoglycosidase F2, endoglycosidase F3. In addition, the recombinant host cell can be genetically engineered to incompletely treat certain types of polysaccharides. These and similar technologies are well known in the art. Other methods of modification include the use of coupling techniques known in the art including, but not limited to, enzyme methods, oxidative substitution, and chelation. Modifications can be used, for example, to link markers of immunoassays. Modified polypeptides are prepared by methods established in the art and can be screened using standards known in the art, some of which are described below and in the Examples. In some embodiments of the invention, the antibody comprises a modified constant region, such as a constant region that is immunologically inert or partially inert, such as, for example, does not induce complement mediated lysis, does not stimulate ADCC, or does not activate microglia; or A reduced activity (as compared to an unmodified antibody) in any one or more of the following: elicits complement mediated lysis, stimulates ADCC, or activates microglia. Different modifications of the constant region can be used to achieve the optimal amount and/or combination of effect functions. See, for example, Μ 〇rga η et al., 1 995, Immunology 86: 319-324; Lund et al., 1996, J. Immunology 157: 4963-4969; Iduso gie et al., 2000, J. Immunology 164 : 4178 -4184; Tao et al, 1 989, J. Immunology 143: 2595-2601 and .Jefferi s et al., 1 998, Immunologii: al Rev ie ws 163: 59-76. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29: 2613-2624 67-201206466; PCT Publication No. WO 09 9/58572 and/or British Patent Application No. 9809951.8 . In other embodiments, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A3 3 0 P331 to S 3 3 0S331 (amino acid number reference wild-type IgG2 sequence) (Eur. J. Immunol. , 1999, 29: 2613-2624). In other embodiments, the N-linked glycosylation of the constant region is deglycosylated. In some embodiments, the N-linked glycosylation of the constant region is by mutating the glycosylated amino acid residue or a flanking residue of the constant region that is part of the N-glycosylation recognition sequence. And deglycosylation. For example, the N glycosylation position N297 can be mutated to A, Q, K or Η. See Tao et al., 1 989, J. Immunology 143: 2595-2601 and Jefferis et al., 1 9 9 8, Immunological Reviews 1 63 : 59-76. In some embodiments, the N-linked glycosylation of the constant region is deglycosylated. N-linked glycosylation of the constant region can be deglycosylated by an enzyme (such as removal of a carbohydrate by an enzyme PNGase) or by deglycosylation by host cells in a glycosylation defect. Other antibody modifications include those which have been modified as described in PCT Publication No. WO 99/5 8 5 72. In addition to the binding domain to the target molecule, the antibodies also comprise an effector domain having an amino acid sequence substantially homologous to all or part of the constant domain of the human immunoglobulin heavy chain. Such antibodies bind to the target molecule but do not elicit significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding to FcRn and/or Fc r RHb. These effects are generally based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are particularly useful for chronic antibody treatment to avoid -68-201206466 Inflammation of the conventional antibody treatment and other adverse reactions. The present invention includes an affinity matured embodiment. For example, affinity matured antibodies can be produced using methods known in the art (Marks et al., 1 992, Bio/Technology, 10: 779-783; Barbas et al., 1 994, Proc Nat. Acad. Sci , USA 9 1 : 3 809-3 8 1 3 ; Schier et al., 1 995, Gene, 169 : 1 47- 1 55 : Yelton et al., 1 995, J. Immunol., 1 5 5 : 1994- 2004 : Jackson et al., 1995, J. Immunol., 154 (7): 3 310-9; Hawkins et al., 1992, J.

Mol· Biol·，226: 889-896 及 PCT 公開號 W02004/0581 84) o 下列方法可被用於調整抗體之親和性及特徵化CDR。 —種特徵化抗.體之CDR及/或改變（諸如改善）多肽諸如抗體之結合親和性之方法被稱爲「庫掃描突變形成」。通常’庫掃描突變形成如下述進行。CDR中之一或多個胺基酸位置係經兩或多個（諸如3、4、5、6、7、8、9、1 0、 11、12、13、14、15、16、17、18、19 或 20個）胺基酸取代’使用該領域已建立之方法。如此產生克隆之小型庫（在一些實施態樣中，每個經分析之胺基酸位置有一個克隆 )’各克隆具有兩或多個成員之複雜性（若每個位置被兩或多個胺基酸取代）。通常，該庫亦包括包含天然（未經 • 取代）胺基酸之克隆。來自各庫之少數克隆例如約20至80 . 個克隆（依該庫之複雜性而定）係經篩選與該標的多肽（或其他結合標的）之結合親和性，並識別具有增加、相同 '降低或不具結合力之候選克隆。測定結合親和性之方法 -69- 201206466 係該領域所廣爲週知。結合親和性可利用Biac〇re表面電漿共振分析測定，該分析檢測約2倍或更高之結合親和性差異。Biacore特別適用於當起始抗體已經以相當高之親和性結合時，例如約10奈莫耳或更低之KD。利用Biacore 表面電漿共振篩選係描述於此處之實施例。結合親和性可利用金耐科薩（Kinexa)生物感測器、鄰近閃燦檢測、ELIS A、ORIGEN免疫測定（IGEN )、螢光淬熄、螢光轉移及/或酵母菌展示決定。結合親和性亦可利用適當之生物測定篩選。在一些實施態樣中，CDR中之每個胺基酸位置皆利用該領域已知之突變形成方法（某些方法於此處描述）以所有20個天然胺基酸取代（在一些實施態樣中，一次一個）。如此產生克隆之小型庫（在一些實施態樣中，每個經分析之胺基酸位置有一個克隆），各克隆具有20個成員之複雜性（若每個位置被所有20個胺基酸取代）。在一些實施態樣中，欲經篩選之庫包含在兩或多個位置之取代，該取代可能在相同CDR或在兩或多個CDR中。因此，該庫可能包含在一個CDR中之兩或多個位置之取代。該庫可能包含在二或多個CDR中之兩或多個位置之取代。該庫可能包含3、4、5或多個位置之取代，該等位置見於二、三、四、五或六個CDR中。該取代可利用低重複性密碼子製備。見例如 Balint et al·, 1 993，Gene 137 ( 1): 109-1 8之表 2 » 該CDR可爲CDRH3及/或CDRL3。該CDR可爲一或多個 -70- 201206466 CDRLl、CDRL2、CDRL3、CDRHl、CDRH2 及 /或 CDRH3 。該CDR可爲卡巴CDR、柯西亞CDR或延伸CDR。具有增強結合力之候選物可被定序，藉此識別導致增強親和性之CDR取代突變（亦稱爲「增強」取代）。結合之候選物亦可被定序，藉此識別保留結合力之CDR取代β 多次篩選可被進行。舉例來說，具有增強結合力之候選物（各在一或多個CDR之一或多個位置包含胺基酸取代 )亦可被用於設計在各增強之CDR位置（也就是在CDR中取代突變顯示增強之結合力的胺基酸位置）包含至少該原始及經取代之胺基酸的第二庫。此庫之製備、篩選及選擇另於下討論。庫掃描突變形成亦提供特徵化CDR之方法，因爲具有增強結合力、相同結合力、降低結合力或無結合力之克隆的頻率亦提供有關各胺基酸位置在抗體-抗原複合物穩定性上之重要性的資訊。舉例來說，如果CDR之位置被改變成所有20個胺基酸時仍保留結合力，該位置被認爲不可能是抗原結合所必需之位置。相反地，如果CDR之位置只有在低百分比之取代時保留結合力，該位置被認爲是CDR功能上重要之位置。因此，庫掃描突變形成方法產生關於可被改變成許多不同之胺基酸（包括所有20個胺基酸）之 CDR中之位置，以及無法被改變或僅能改變成少數胺基酸之CDR中之位置的資訊》具有增強親和性之候選物可被組合於第二庫中，該庫包括經增強之胺基酸、原始胺基酸，且可能視該所欲或使 -71 - 201206466 用所欲之篩選或選擇方法所允許之庫的複雜性而另外包括該位置之額外取代》此外若有需要的話，鄰近之胺基酸位置可被隨機化成至少兩或多個胺基酸。鄰近胺基酸之隨機化可能允許該突變CDR之額外構型柔韌性，這可能轉而允許或有利於導入較大數量之增強突變。該庫亦包在第一次篩選時不顯示增強親和性之取代位置。該第二庫係利用該領域已知之任何方法篩選或選擇具有增強及/或改變之結合親和性之庫成員，包括利用 Biacore表面電漿共振分析之篩選及利用該領域已知之任何用於選擇之方法選擇，包括噬菌體展示、酵母菌展示及核糖體展示。本發明亦包含融合蛋白質，該融合蛋白質包含一或多個來自本發明之抗體或多肽之片段或區域。在一實施態樣中，提供包含如SEQ ID NO: 3所示之輕鏈可變區之至少 10個相鄰胺基酸及/或SEQ ID NO: 4或5所示之重鏈可變區之至少1 〇個胺基酸的融合多肽。在其他實施態樣中，提供包含至少約1 〇個、至少約1 5個、至少約2 0個、至少約2 5個或至少約30個輕鏈可變區之相鄰胺基酸及/或至少約1〇個、至少約15個、至少約20個、至少約25個或至少約30個重鏈可變區之相鄰胺基酸的融合多肽。在其他實施態樣中，該融合多肽包含一或多個CDR。在仍其他實施態樣中，該融合多肽包含CDR H3 ( VH CDR3 )及/或CDR L3 ( VL CDR3)。就本發明之目的而言，融合蛋白包含一或多種抗體及在天然分子中未與其連接之另一胺基酸序列，例如 -72- 201206466 異源性序列或來自另一區之同源性序列。示範性異源序列包括但不限於「標籤」諸如FLAG標籤或6His標籤。標籤係該領域所廣爲周知。融合多肽可藉由該領域已知之方法產製，例如合成或重組。通常，本發明之融合蛋白係藉由使用此處所描述之重組方法使編碼彼等之多核苷酸表現加以製備，雖然彼等亦可藉由該領域所知之其他方法製備，包括例如化學合成〇本發明亦提供組成物，該等組成物包含與劑共軛（例如連接）以利與固相支持物（諸如生物素或抗生物素蛋白 )偶合之抗體或多肽。爲了簡單起見大致將以抗體爲例，但應了解該等方法適用於任何此處所述之抗原結合及/或拮抗劑實施態樣。共軛一般係指與此處所述之該等成分連接》該連接（至少供投予時相近地固定該等成分）可以數種方式達成。舉例來說，當劑與抗體各自具有能與彼此反應之取代基時，兩者間之直接反應係可能的。舉例來說，一成分上之親核基團諸如胺基或氫硫基可能能與另一成分上之含羰基基團諸如酐或酸性鹵化物，或與含有好的離去基團（例如鹵化物）之烷基反應。本發明之抗體或多肽可能與標示劑諸如螢光分子、放射性分子或任何其他該領域已知之標記連接。標記在該領域中一般係用於（直接地抑或間接地）提供信號。本發明亦提供組成物（包括醫藥組成物）及套組，該組成物及套組如此處揭示包含此處所描述之任何或所有抗 -73- 201206466 體及/或多肽。本發明亦提供經分離之編碼本發明之抗體及肽之多核苷酸，及包含該多核苷酸之載體及宿主細胞。在另一態樣中，本發明提供編碼此處所述之任何抗體（包括抗體片段 )及多肽之多核苷酸，諸如具有受損效應功能之抗體及多肽。多核苷酸可利用該領域已知之方法製備及表現。在另一態樣中，本發明提供包含本發明之任何多核苷酸之組成物（諸如醫藥組成物）。在一些實施態樣中，該組成物包含表現載體，該表現載體包含編碼此處所述之抗體之多核苷酸。在其他實施態樣中，該組成物包含表現載體，該表現載體包含編碼此處所述之任何抗體或多肽之多核苷酸。·在仍其他之實施態樣中，該組成物包含如SEQ ID NO: 23及SEQ ID NO: 24之任一或兩者所示之多核苷酸。表現載體及投予多核苷酸組成物另於此處描述。在另一態樣中，本發明提供製備此處所述之任何多核苷酸之方法。與任何該等序列互補之多核苷酸亦包含於本發明中。多核苷酸可能爲單股（編碼或反義）或雙股，且可能爲 DNA (基因組、cDNA或合成性）或RNA分子。RNA分子包括HnRNA分子及mRNA分子，該HnRNA分子包含內含子且以一對一之方式對應DNA，該mRNA分子不包含內含子。額外之編碼或非編碼序列可能但不一定存在於本發明之多核苷酸內，且多核苷酸可能但不一定與其他分子及/或支持物質連接。 -74- 201206466 #核苷酸可能包含天然序列（即編碼抗體或彼之部分 2內源性序列）或可能包含該序列之變異體。多核苷酸變異體包含一或多個取代、添加、刪除及/或***以使該編免疫活性相對於天然免疫活性分子而言不被減少 ·> Ιί _編碼多肽之免疫活性之影響一般係如此處所述檢測 «變異體較佳地展現與編碼天然抗體或彼之部分之多核苷酸序列至少約70%之一致性，更佳地至少約8〇%之一致性 ’甚至更佳地至少約90%之一致性及最佳地至少約95 %之一致性。若二個多核苷酸或多肽序列如下所述排比以得到最高對應性且該二個序列中之核苷酸或胺基酸序列相同，則該二個序列被稱爲「一致」。兩序列之間的比較通常藉由在比較窗中比較序列加以進行，以識別及比較具有序列相似性之局部區域。此處所使用之「比較窗」係指至少約20個、通常30個至約75個、或40個至約50個連續位置之區段，在該窗中一序列可與具有相同連續位置數量之參考序列在該二序列經最佳排比後比較。供比較之序列的最佳排比可利用雷斯基（Lasergene )生物資訊套裝軟體中之邁佳來（Megalign)程式（威斯康辛州麥迪遜市DNASTAR公司）以內建參數進行。此程式具體化下列參考文獻所述之數種排比計畫：Dayhoff， M.O. 1 97 8, A model of evolutionary change in proteins -Matrices for detecting distant relationships. In Dayhoff, M.O. ( ed. ) Atlas of Protein Sequence and Structure ( -75- 201206466Mol·Biol., 226: 889-896 and PCT Publication No. W02004/0581 84) o The following methods can be used to adjust the affinity of the antibody and characterize the CDRs. A method of characterizing the CDRs of an anti-body and/or altering (e.g., improving) the binding affinity of a polypeptide, such as an antibody, is referred to as "library scan mutation formation." Normally, the library scan mutation formation proceeds as follows. One or more amino acid positions in the CDR are two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) Amino acid substitutions 'Use established methods in the field. The resulting mini-library of clones (in some embodiments, one clone per each amino acid position analyzed) 'each clone has the complexity of two or more members (if each position is bound by two or more amines) Substituted by acid). Typically, the library also includes clones containing natural (unsubstituted) amino acids. A small number of clones from each pool, for example, about 20 to 80. The clones (depending on the complexity of the library) are screened for binding affinity to the target polypeptide (or other binding target) and are identified as having an increase, the same 'lower Or candidate clones that do not have binding strength. Methods for determining binding affinity -69-201206466 are well known in the art. Binding affinity can be determined using Biac(R) surface plasma resonance analysis, which detects a difference in binding affinity of about 2 fold or greater. Biacore is particularly useful when the starting antibody has been bound with a relatively high affinity, e.g., a KD of about 10 nanomolar or less. The Biacore surface plasmon resonance screening system is described herein as an example. Binding affinity can be determined using a Kinexa biosensor, proximity to flash detection, ELIS A, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinity can also be screened using appropriate bioassays. In some embodiments, each of the amino acid positions in the CDRs is substituted with all 20 native amino acids using methods known in the art for mutation formation (some methods are described herein) (in some embodiments) , one at a time). The resulting cloned mini-library (in some embodiments, one clone per analyzed amino acid position), each clone has a complexity of 20 members (if each position is replaced by all 20 amino acids) ). In some embodiments, the library to be screened comprises substitutions at two or more positions, which may be in the same CDR or in two or more CDRs. Therefore, the library may contain substitutions at two or more locations in one CDR. The library may contain substitutions at two or more positions in two or more CDRs. The library may contain substitutions of 3, 4, 5 or more positions found in two, three, four, five or six CDRs. This substitution can be made using low repetitive codons. See, for example, Balint et al., 1 993, Gene 137(1): 109-1 8 Table 2 » The CDRs can be CDRH3 and/or CDRL3. The CDR may be one or more of -70-201206466 CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and/or CDRH3. The CDR can be a Kappa CDR, a Koccia CDR or an extended CDR. Candidates with enhanced binding can be sequenced, thereby identifying CDR substitution mutations (also referred to as "enhancement" substitutions) that result in enhanced affinity. The conjugated candidate can also be sequenced, whereby the CDR-restricted beta multiple screening that recognizes retention of binding can be performed. For example, candidates with enhanced binding (each comprising one or more CDRs at one or more positions) can also be used to design at each of the enhanced CDR positions (ie, in the CDRs) The mutation exhibits an enhanced binding affinity for the amino acid position) comprising at least a second pool of the original and substituted amino acids. The preparation, screening and selection of this library are discussed below. Library scanning mutation formation also provides a method for characterizing CDRs, as the frequency of clones with enhanced binding, same binding, reduced binding or no binding also provides information on the stability of the antibody-antigen complexes for each amino acid position. Information on the importance of it. For example, if the position of the CDR is changed to all 20 amino acids, the binding force is retained, and this position is considered unlikely to be the position necessary for antigen binding. Conversely, if the position of the CDR retains binding only when replaced by a low percentage, this position is considered to be an important position on the CDR function. Thus, the library scan mutation formation method yields positions in the CDRs that can be altered to many different amino acids (including all 20 amino acids), as well as in CDRs that cannot be altered or can only be altered into a few amino acids. Information on the position" Candidates with enhanced affinity can be combined in a second library comprising an enhanced amino acid, an original amino acid, and possibly as intended or used -71 - 201206466 The complexity of the library allowed by the screening or selection method additionally includes additional substitutions at this position. Further, if desired, the adjacent amino acid sites can be randomized to at least two or more amino acids. Randomization of the adjacent amino acid may allow for additional conformational flexibility of the mutated CDRs, which may in turn allow or facilitate the introduction of a larger number of enhanced mutations. The library also includes no substitution locations for enhanced affinity at the time of the first screening. The second library utilizes any method known in the art to screen or select library members having enhanced and/or altered binding affinities, including screening using Biacore surface plasmon resonance analysis and utilizing any of the known in the art for selection. Method selection includes phage display, yeast display, and ribosome display. The invention also encompasses fusion proteins comprising one or more fragments or regions derived from an antibody or polypeptide of the invention. In one embodiment, at least 10 adjacent amino acids comprising a light chain variable region as set forth in SEQ ID NO: 3 and/or a heavy chain variable region set forth in SEQ ID NO: 4 or 5 are provided a fusion polypeptide of at least one amino acid. In other embodiments, an adjacent amino acid comprising at least about 1 、, at least about 15, at least about 20, at least about 25, or at least about 30 light chain variable regions is provided and/or Or a fusion polypeptide of at least about 1 , at least about 15, at least about 20, at least about 25, or at least about 30 of the adjacent amino acids of the heavy chain variable region. In other embodiments, the fusion polypeptide comprises one or more CDRs. In still other embodiments, the fusion polypeptide comprises CDR H3 (VH CDR3) and/or CDR L3 (VL CDR3). For the purposes of the present invention, a fusion protein comprises one or more antibodies and another amino acid sequence not linked thereto in a natural molecule, such as a heterologous sequence of -72 to 201206466 or a homologous sequence from another region. . Exemplary heterologous sequences include, but are not limited to, "tags" such as FLAG tags or 6His tags. Labels are well known in the art. Fusion polypeptides can be produced by methods known in the art, such as synthesis or recombination. In general, the fusion proteins of the invention are prepared by encoding the polynucleotides encoding them using recombinant methods described herein, although they may be prepared by other methods known in the art, including, for example, chemical synthesis. The invention also provides compositions comprising an antibody or polypeptide conjugated (e.g., linked) to a reagent for coupling to a solid support, such as biotin or avidin. For the sake of simplicity, antibodies will generally be exemplified, but it should be understood that such methods are applicable to any of the antigen binding and/or antagonist embodiments described herein. Conjugation generally refers to the attachment to the components described herein. The attachment (at least for the fixation of the components at the time of administration) can be accomplished in a number of ways. For example, when the agent and the antibody each have a substituent reactive with each other, a direct reaction between the two is possible. For example, a nucleophilic group such as an amine group or a thiol group on one component may be capable of reacting with a carbonyl group such as an anhydride or an acid halide on another component, or with a good leaving group (eg, halogenated) The alkyl reaction of the substance). The antibody or polypeptide of the invention may be linked to a labeling agent such as a fluorescent molecule, a radioactive molecule or any other label known in the art. Tags are generally used in this domain to provide signals (directly or indirectly). The invention also provides compositions (including pharmaceutical compositions) and kits, the compositions and kits as disclosed herein comprising any or all of the anti-73-201206466 bodies and/or polypeptides described herein. The invention also provides isolated polynucleotides encoding the antibodies and peptides of the invention, and vectors and host cells comprising the polynucleotides. In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides having impaired effector functions. Polynucleotides can be prepared and expressed using methods known in the art. In another aspect, the invention provides a composition (such as a pharmaceutical composition) comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises a performance vector comprising a polynucleotide encoding an antibody as described herein. In other embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In still other embodiments, the composition comprises a polynucleotide as shown in either or both of SEQ ID NO: 23 and SEQ ID NO: 24. The expression vector and the administration polynucleotide composition are also described herein. In another aspect, the invention provides a method of making any of the polynucleotides described herein. Polynucleotides complementary to any of these sequences are also encompassed by the present invention. Polynucleotides may be single stranded (coding or antisense) or double stranded and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules and mRNA molecules, which contain introns and correspond to DNA in a one-to-one manner, and the mRNA molecules do not contain introns. Additional coding or non-coding sequences may, but are not necessarily, present within the polynucleotides of the invention, and the polynucleotides may, but are not necessarily, linked to other molecules and/or support materials. -74- 201206466 #nucleotides may comprise a native sequence (ie, an antibody encoding or a portion 2 endogenous sequence thereof) or may comprise a variant of the sequence. A polynucleotide variant comprises one or more substitutions, additions, deletions and/or insertions such that the immunological activity is not reduced relative to the innate immunologically active molecule.> Ιί _ The effect of the immunological activity of the encoded polypeptide is generally As described herein, the test «variant preferably exhibits at least about 70% identity, more preferably at least about 8% homology to the polynucleotide sequence encoding the native antibody or portion thereof, even more preferably at least Approximately 90% consistency and optimally at least about 95% consistency. Two sequences are said to be "consistent" if the two polynucleotide or polypeptide sequences are aligned as described below to give the highest correspondence and the nucleotide or amino acid sequences in the two sequences are identical. Comparisons between the two sequences are typically performed by comparing the sequences in a comparison window to identify and compare local regions with sequence similarity. As used herein, "comparison window" means a segment of at least about 20, typically 30 to about 75, or 40 to about 50 consecutive locations, in which a sequence can have the same number of consecutive positions. The reference sequence is compared after the two sequences are optimally aligned. The optimal alignment of the sequences to be compared can be performed using built-in parameters using the Megalign program (DNASTAR, Madison, Wisconsin) in the Lasergene Bioinformatics package. This program exemplifies several of the following reference plans: Dayhoff, MO 1 97 8, A model of evolutionary change in proteins -Matrices for detecting distant relationships. In Dayhoff, MO ( ed. ) Atlas of Protein Sequence and Structure ( -75- 201206466

National Biomedical Research Foundation, Washington DC ),Vol. 5, Suppl. 3, pp. 3 45 -3 5 8 ί Hein J., 1 990, UnifiedNational Biomedical Research Foundation, Washington DC ), Vol. 5, Suppl. 3, pp. 3 45 -3 5 8 ί Hein J., 1 990, Unified

Approach to Alignment and Phy logenes pp. 6 2 6-645Approach to Alignment and Phy logenes pp. 6 2 6-645

Methods in Enzymology vol. 183, ( Academic Press, Inc., San Diego, CA) ； Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5 ： 15 1-153 ； Myers, E.W. and Muller W., 1 988, CABIOS 4 : 11-17 ； Robinson, E. D., 1971, Comb. Theor. 11 : 105 ; Santou, N., Nes, M., 1 9 8 7, Mol. Biol. Evol. 4 : 406-425 ; Sneath, P . H . A . and S okal, R. R., 1 9 7 3,Methods in Enzymology vol. 183, ( Academic Press, Inc., San Diego, CA) ; Higgins, DG and Sharp, PM, 1989, CABIOS 5 : 15 1-153 ; Myers, EW and Muller W., 1 988, CABIOS 4: 11-17; Robinson, ED, 1971, Comb. Theor. 11 : 105 ; Santou, N., Nes, M., 1 9 8 7, Mol. Biol. Evol. 4 : 406-425 ; Sneath, P H. A . and S okal, RR, 1 9 7 3,

Numerical Taxonomy the Principles and Practice of Numerical Taxonomy ( Freeman Press, San Francisco, CA )；Wilbur, W.J. and Lipman, D.J., 1 983, Proc. Natl.Numerical Taxonomy the Principles and Practice of Numerical Taxonomy (Freeman Press, San Francisco, CA); Wilbur, W.J. and Lipman, D.J., 1 983, Proc. Natl.

Acad. Sci. USA 80 ： 726-730 » 較佳地，「序列一致性之百分比」係藉由在至少20個位置之比較窗中比較二個經最佳排比之序列加以決定，其中在比較窗中多核苷酸或多肽序列之部分相較於供二序列最佳排比之參考序列（其不包含添加或刪除）可能包含 20%或低於20%、通常5%至15°/。、或10%至12%之添加或刪除（即缺口）。百分比之計算係藉由測定二個序列中出現相同之核酸鹼基或胺基酸殘基之位置的數目’以得到匹配位置之數目，將該匹配位置之數目除以參考序列之位置總數（即窗之大小），並將該結果乘以10 0以得到序列—致性百分比。變異體亦可能或選擇性地與天然基因或彼之部分或互 -76- 201206466 補部分實質上同源。該等多核苷酸變異體能在中度嚴謹度條件下與編碼天然抗體之天然發生之DNA序列（或互補序列）雜交。適當之「中度嚴謹度條件」包括在5倍SSC、0.5% SDS 、1.0毫莫耳EDTA(pH 8.0)之溶液中預先清洗；於5(TC 至65°C之5倍SSC中隔夜雜交；接著於65°C中各以含有0.1 % SDS之2倍、0.5倍及0.2倍SSC清洗20分鐘兩次。此處所使用之「高度嚴謹度條件」或「高嚴謹條件」係該些：（1 )採用低離子張力及高溫清洗，例如於5 0 °C 之0.0 15莫耳氯化鈉/0.001 5莫耳檸檬酸鈉/0.1 %十二基硫酸鈉；（2 )在42 °C之雜交期間採用變性劑，諸如甲醯胺，例如具有0.1%牛血清白蛋白/0.1%?丨(；〇11/0.1%聚乙烯基吡咯烷酮/含有750毫莫耳氯化鈉、75毫莫耳檸檬酸鈉之PH 6.5之50毫莫耳磷酸鈉緩衝液之50% (體積/體積）甲醯胺 •，或（3 )於42 t採用50%甲醯胺、5倍SSC ( 0.75莫耳 NaCl、0.075莫耳檸檬酸鈉）、50毫莫耳磷酸鈉（pH 6.8 )、〇·1°/。焦磷酸鈉、5倍丹哈德（Denhardt’s )溶液、經超音波化之鮭魚***DNA(50微克/毫升）、0.1%SDS及 10%硫酸葡聚糖，並於42 °C以0.2倍SSC (氯化鈉/檸檬酸鈉 )清洗及於5 5 °C以5 0%甲醯胺清洗’之後於5 5 °C以含有 EDTA之0.1倍SSC進行高嚴謹度清洗。技藝人士將知道如何必要地調整溫度、離子張力等以適應諸如探針長度及該類似之因素。該領域之一般技藝人士將瞭解的是’由於基因密碼簡 -77- 201206466 倂之結果，有許多核苷酸序列編碼此處所描述之多肽。這些多核苷酸中有些與任何天然基因之核苷酸序列具有極低之同源性。然而，本發明特別考慮因爲使用不同的密碼子而有所差異之多核苷酸。另外，包含此處所提供之多核苷酸序列之基因的等位基因係屬於本發明之範圍內》等位基因係因爲一或多個突變，諸如核苷酸之刪除、添加及/或取代而被改變之內源性基因。該形成之mRNA及蛋白質可能但不一定具有經改變之結構或功能。等位基因可利用標準技術（諸如雜交、擴增及/或資料庫序列比較）加以識別。本發明之多核苷酸可利用化學合成、重組方法或PCR 獲得。化學多核苷酸合成之方法係該領域所廣爲週知，不須在此詳細說明。該領域之技藝人士可利用此處所提供之序列及商用DNA合成儀產製所欲之DNA序列。在利用重組方法製備多核苷酸時，包含所欲序列之多核苷酸可如此處進一步所述被***適當之載體內，該載體依次可被導入適當之宿主細胞以供複製及擴增。多核苷酸可藉由該領域所知之任何方法被***宿主細胞中》細胞之轉形係藉由直接攝取、胞飲作用、轉染、F交配（F-mating )或電穿孔導入外源性多核苷酸。一旦導入後，該外源性多核苷酸可以非整合性載體（諸如質體）或整合至該宿主細胞之基因組中而被維持於該細胞內。經此放大之多核苷酸可利用該領域所熟習之方法自該宿主細胞分離。見例如 Sambrook et al.，1989 ’ 同上。 -78- 201206466 另外，PCR允許DNA序列之複製。PCR技術係該領域所廣爲週知且描述於美國專利第4,683,195、4,800,159、 4,754,065 及 4,683,202號以及 PCR : The Polymerase Chain Reaction, Mullis et al., 1 994, eds. ( Birkauswer Press, B oston, MA )。 RN A可藉由使用適當載體內經分離之DNA且將其*** 適當之宿主細胞中獲得。當細胞複製且該DNA被轉錄成爲 RNA時，該RNA即可利用該領域之技藝人士所廣爲週知之方法分離，例如於Sambrook et al.，1 989 (同上）所述。適當之選殖載體可根據標準技術建構，或可選自該領域爲數眾多之可用選殖載體。雖然該經選擇之選殖載體可能因所意圖使用之宿主細胞而異，適用之選殖載體通常具有自我複製之能力、可能具有特定限制內切酶之單一標的及/或可能帶有可用於選擇含有該載體之克隆的標誌基因。適當實例包括質體及細菌性病毒，例如PUC18、pUC19 、Bluescript (例如 pBS SK+)及其衍生物、mpl8、mpl9 、pBR322 ' pMB9、ColEl、pCRl、RP4、噬菌體 DNA 及穿梭載體諸如pSA3及pAT28。這些及許多其他選殖載體可購自商業賣方諸如伯樂（BioRad )公司、斯壯特基（ Strategene)公司及英維特基（Invitrogen)公司。表現載體通常是可複製之多核苷酸建構物，其包含本發明之多核苷酸》這暗示表現載體必需能在宿主細胞中以附加體或染色體DNA之組成部分被複製。適當之表現載體包括但不限於質體、病毒載體（包括腺病毒、腺病毒相關 •79- 201206466 病毒、反轉錄病毒）、黏質體及PCT公開號WO 87/04462 中所揭示之表現載體。載體成份通常包括但不限於下列一或多項：信號序列；複製起點；一或多種標誌基因；適當之轉錄控制元件（諸如啓動子、增強子及終止子）。就表現（即轉譯）而言，通常也需要一或多種轉譯控制元件，諸如核糖體結合位置、轉譯起始位置及終止密碼子。含有感興趣之多核苷酸之載體可藉由任何適當之方法導入宿主細胞，包括電穿孔、使用氯化鈣、氯化铷、磷酸鈣、DEAE-葡聚糖或其他物質之轉染、微彈撞擊、脂質體轉染及感染（例如該載體爲感染性劑諸如牛痘病毒）。導入載體或多核苷酸之選擇通常將視該宿主細胞之特徵而定〇本發明亦提供包含此處所述之任何多核苷酸之宿主細胞。任何能過度表現異源性DN A之宿主細胞可被使用以分離編碼該感興趣之抗體、多肽或蛋白質之基因。哺乳動物宿主細胞之非限制性實例包括但不限於COS、HeLa、NSO 及CHO細胞。亦見PCT公開號WO 87/04462。適當之非哺乳動物宿主細胞包括原核生物（諸如大腸桿菌或枯草桿菌 )及酵母菌（諸如啤酒釀母菌、***酵母（S. pombe)或乳酸克魯維酵母菌（K. lactis ))。較佳地，該宿主細胞以相較於該對應之感興趣之內源性抗體或蛋白質若存在於宿主細胞中高出約5倍、更佳爲高出10倍、甚至更佳爲高出20倍之量表現cDNA »篩選與抗原特異性結合之宿主細胞係由免疫測定或FACS進行。過度表現感興趣之抗體或 -80- 201206466 蛋白之細胞可被識別。 C.組成物用於本發明之方法中之組成物包含有效量之此處所討論之pH依賴性抗體或pH依賴性抗體衍生性多肽。該等組成物之實例以及如何調製彼等也被描述於較早章節及以下。在一實施態樣中，該組成物包含一或多種pH依賴性抗體。在其他實施態樣中，該pH依賴性抗體辨識人PCS K9。在其他實施態樣中，該pH依賴性抗體係人化抗體。在另外之實施態樣中，該pH依賴性抗體包含不引發非所需或非所欲之免疫反應（諸如抗體媒介性溶解或ADCC )之恆定區。在其他實施態樣中，該pH依賴性抗體包含抗體之一或多個CDR (諸如一、二 '三、四 '五或在一些實施態樣中所有六個CDR)。在一些實施態樣中，該pH依賴性抗體係人抗體。本發明所使用之組成物可另包含呈冷凍乾燥調製劑或水性溶液形式之醫藥上可接受之載劑、賦形劑或穩定劑（ Remington : The Science and Practice of Pharmacy 20th Ed.，2000，Lippincott Williams and Wilkins, Ed. K. E.Acad. Sci. USA 80: 726-730 » Preferably, the "percentage of sequence identity" is determined by comparing the sequences of the two best alignments in a comparison window of at least 20 locations, wherein in the comparison window The portion of the polynucleotide or polypeptide sequence may comprise 20% or less, typically 5% to 15%, compared to the reference sequence for optimal alignment of the second sequence (which does not include addition or deletion). , or 10% to 12% of additions or deletions (ie, gaps). The percentage is calculated by determining the number of positions where the same nucleobase or amino acid residue appears in the two sequences to obtain the number of matching positions, and the number of matching positions is divided by the total number of positions of the reference sequence (ie, The size of the window) and multiply the result by 10 0 to get the sequence-to-sequence percentage. The variant may also or alternatively be substantially homologous to the native gene or part of it or to the complement of the -76-201206466 complement. Such polynucleotide variants are capable of hybridizing to naturally occurring DNA sequences (or complementary sequences) encoding natural antibodies under moderate stringency conditions. Appropriate "moderate stringency conditions" include pre-washing in a solution of 5 times SSC, 0.5% SDS, 1.0 millimolar EDTA (pH 8.0); overnight hybridization at 5 (5 times SCC from TC to 65 °C; Then, it was washed twice at 65 ° C with 2 times, 0.5 times and 0.2 times SSC containing 0.1% SDS. The "high stringency conditions" or "high stringency conditions" used here are: (1) Use low ion tension and high temperature cleaning, for example, 0.015 mol sodium chloride at 50 °C / 0.001 5 mol sodium citrate / 0.1% sodium dodecyl sulfate; (2) during the hybridization period at 42 °C Using a denaturing agent such as formamide, for example with 0.1% bovine serum albumin / 0.1% 丨 (; 〇 11 / 0.1% polyvinylpyrrolidone / containing 750 mM sodium chloride, 75 mM sodium citrate 50% (vol/vol) methotrexate of PH 6.5 in 50 millimolar sodium phosphate buffer, or (3) 50% methotrexate, 5 times SSC (0.75 molar NaCl, 0.075 mo in 42 t) Sodium citrate), 50 mM sodium phosphate (pH 6.8), 〇·1°/. sodium pyrophosphate, 5 times Denhardt's solution, supersonicized salmon sperm DNA (50 μg/m ), 0.1% SDS and 10% dextran sulfate, washed with 0.2 times SSC (sodium chloride/sodium citrate) at 42 °C and with 50% methotrexate at 5 5 °C. High stringency cleaning with 0.1 times SSC containing EDTA at 5 ° C. The skilled artisan will know how to adjust the temperature, ionic tension, etc. as necessary to accommodate factors such as probe length and the like. Those of ordinary skill in the art will appreciate As a result of the genetic code simplification -77-201206466, there are a number of nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides have very low homology to the nucleotide sequence of any native gene. However, the present invention specifically contemplates polynucleotides that differ by the use of different codons. In addition, allelic genes comprising the genes of the polynucleotide sequences provided herein are within the scope of the invention. An endogenous gene that is altered by one or more mutations, such as deletions, additions, and/or substitutions of nucleotides. The formed mRNA and protein may, but need not, have altered structure or function. Identification using standard techniques such as hybridization, amplification and/or library sequence comparison. The polynucleotides of the invention can be obtained by chemical synthesis, recombinant methods or PCR. Methods for chemical polynucleotide synthesis are widely known in the art. It is well known that it is not necessary to be described in detail herein. Those skilled in the art can use the sequences and commercial DNA synthesizers provided herein to produce desired DNA sequences. When preparing a polynucleotide by recombinant methods, the desired sequence is included. The polynucleotide can be inserted into a suitable vector, as described further herein, which can in turn be introduced into a suitable host cell for replication and amplification. The polynucleotide can be inserted into a host cell by any method known in the art. The transformation of the cell is introduced into the exogenous by direct uptake, pinocytosis, transfection, F-mating or electroporation. Polynucleotide. Once introduced, the exogenous polynucleotide can be maintained in the cell by a non-integrating vector (such as a plastid) or integrated into the genome of the host cell. The amplified polynucleotide can be isolated from the host cell using methods well known in the art. See, for example, Sambrook et al., 1989 ‘Ibid. -78- 201206466 In addition, PCR allows replication of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, and PCR: The Polymerase Chain Reaction, Mullis et al., 1 994, eds. (Birkauswer Press, Boston, MA ). RN A can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as described in Sambrook et al., 1 989 (supra). Suitable selection vectors can be constructed according to standard techniques or can be selected from a wide variety of available selection vectors in this field. While the selected selection vector may vary depending on the host cell in which it is intended to be used, suitable selection vectors typically have the ability to self-replicate, may have a single restriction of the specific restriction enzyme, and/or may be available for selection. A marker gene containing a clone of the vector. Suitable examples include plastid and bacterial viruses such as PUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322 'pMB9, ColEl, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other selection vectors are commercially available from commercial vendors such as BioRad, Strategene, and Invitrogen. The expression vector is typically a replicable polynucleotide construct comprising a polynucleotide of the invention. This implies that the expression vector must be capable of being replicated in the host cell as a component of the episome or chromosomal DNA. Suitable expression vectors include, but are not limited to, plastid, viral vectors (including adenovirus, adenovirus associated • 79-201206466 virus, retrovirus), viscous bodies, and expression vectors disclosed in PCT Publication No. WO 87/04462. Vector components typically include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; appropriate transcriptional control elements (such as promoters, enhancers, and terminators). In the case of performance (i.e., translation), one or more translational control elements are also typically required, such as ribosome binding positions, translation initiation positions, and stop codons. The vector containing the polynucleotide of interest can be introduced into the host cell by any suitable method, including electroporation, transfection using calcium chloride, barium chloride, calcium phosphate, DEAE-dextran or other substances, microprojectile. Impact, lipofection, and infection (eg, the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell. The invention also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DN A can be used to isolate a gene encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, NSO, and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or Bacillus subtilis) and yeasts (such as S. cerevisiae, S. pombe or K. lactis). Preferably, the host cell is about 5 times higher, more preferably 10 times higher, or even more preferably 20 times higher than the corresponding endogenous antibody or protein of interest in the host cell. Amount of Expression cDNA » Screening of host cell lines that specifically bind to antigen is performed by immunoassay or FACS. Excessive expression of antibodies of interest or cells of the -80- 201206466 protein can be identified. C. Compositions The compositions useful in the methods of the invention comprise an effective amount of a pH dependent antibody or pH dependent antibody-derived polypeptide as discussed herein. Examples of such components and how to modulate them are also described in earlier sections and below. In one embodiment, the composition comprises one or more pH dependent antibodies. In other embodiments, the pH dependent antibody recognizes human PCS K9. In other embodiments, the pH dependent anti-systematic antibody. In still other embodiments, the pH dependent antibody comprises a constant region that does not elicit an unwanted or undesired immune response, such as antibody vector lysis or ADCC. In other embodiments, the pH dependent antibody comprises one or more CDRs of the antibody (such as one, two 'three, four 'five or all six CDRs in some embodiments). In some embodiments, the pH dependent anti-system human antibody. The composition used in the present invention may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized preparation or an aqueous solution (Remington: The Science and Practice of Pharmacy 20th Ed., 2000, Lippincott) Williams and Wilkins, Ed. KE

Hoover )。可接受之載劑、賦形劑或穩定劑在所採用之劑量及濃度下對接受者不具毒性’且可能包括緩衝劑諸如磷酸鹽、檸檬酸鹽及其他有機酸；抗氧化劑包括抗壞血酸及甲硫胺酸；保存劑（諸如十八基二甲基苄基氯化銨、六甲氯胺、氯化苯甲烴銨、氯化苄乙氧銨、酚醇、丁醇、〒醇 -81 - 201206466 、烷基對羥苯甲酸酯類諸如對羥苯甲酸甲酯或對羥苯甲酸丙酯、兒茶酚、間苯二酚、環己醇、3-戊醇及間甲酚）；低分子量（小於約10個殘基）多肽；蛋白質諸如血清白蛋白、明膠或免疫球蛋白；親水性聚合物諸如聚乙烯基吡咯烷酮；胺基酸諸如甘胺酸、麩醯胺酸、天冬醯胺酸、組胺酸、精胺酸或離胺酸；單醣、雙醣及其他碳水化合物包括葡萄糖、甘露糖或葡聚糖；螯合劑諸如EDTA :糖類諸如蔗糖、甘露醇、海藻糖或山梨醇；鹽形成反離子諸如鈉；金屬複合物（例如鋅蛋白質複合物）：及/或非離子性界面活性劑諸如TWEENtm、PLURONICSTM或聚乙二醇（ PEG)。醫藥上可接受之賦形劑另於此處說明。在一實施態樣中，該抗體係以無菌水性溶液之調製劑投予，其具有介於約5.0至約6.5之pH且包含自約1毫克/毫升至約200毫克/毫升之抗體、自約1毫莫耳至約100毫莫耳之組胺酸緩衝劑、自約0.01毫克/毫升至約10毫克/毫升之聚山梨醇酯80、自約100毫莫耳至約400毫莫耳之海藻糖及自約0.01毫莫耳至約1.0毫莫耳之二水EDTA二鈉。該pH依賴性抗體及彼之組成物亦可與其他劑一起使用，以用於增進及/或補充該等劑之有效性。 D.套組本發明亦提供用於快速方法之套組。本發明之套組包括一或多個含有此處所述之pH依賴性抗體（諸如人化抗體）或肽之容器及根據此處所述之本發明之任何方法之使 -82- 201206466 用說明。通常，這些說明包含投予pH依賴性抗體以供上述治療性治療之描述。在一些實施態樣中，該抗體係人化抗體。在一些實施態樣中，該抗體係人抗體。在其他實施態樣中，該抗體係單株抗體。有關使用pH依賴性抗體之說明通常包括該意圖治療之劑量、投藥計畫及投予途徑之資訊。該等容器可爲單位劑量、大量包裝（例如多劑量包裝）或次單位劑量。本發明之套組所提供之說明通常爲在標籤或包裝仿單上之書面說明（例如包含在套組中之紙），但機器讀取之說明（例如磁性或光學儲存磁碟上攜帶之說明）亦可被接受〇本發明之套組係經適當包裝。適當之包裝包括但不限於小瓶、瓶子、罐、可彎折之包裝（例如密封之美拉（ Mylar )或塑膠袋）及該類似物。亦考慮的是與特殊裝置組合使用之包裝，諸如吸入器、經鼻投予裝置（例如霧化器）或輸注裝置諸如小型泵。套組可能具有無菌接口（例如該容器可能爲具有可被皮下注射針穿刺之塞子的靜注溶液袋或小瓶）。該容器也可能具有無菌接口（例如該容器可能爲具有可被皮下注射針穿刺之塞子的靜注溶液袋或小瓶）。該組成物中至少有一種活性劑係pH依賴性抗體。該容器（例如預先塡充之針筒或自動注射器）可能另包含第二醫藥活性劑。套組可能可任意選擇地提供額外成份諸如緩衝劑及解說資訊。通常，該套組包含容器及在該容器上或與該容器 -83- 201206466 相關之標籤或包裝仿單。突變及修飾爲了表現本發明之抗體，編碼VH及VL區之DNA片段可先利用上述之任何方法獲得。各種修飾例如突變、刪除及/或添加亦可利用該領域之技藝人士所知之標準方法被導入該DNA序列。舉例來說，可利用標準方法誘發突變形成，諸如PCR媒介性突變形成，其中該突變之核苷酸被納入PCR引子以使該PCR產物包含所欲之突變或定點突變形成。可能被製造之一種取代舉例來說係改變抗體中一或多個半胱胺酸，其可能對另一殘基諸如但不限於丙胺酸或絲胺酸具化學反應性。舉例來說，可發生非典型半胱胺酸之取代。該取代可於抗體之可變結構域之CDR或骨架區或於恆定結構域中發生。在一些實施態樣中，該半胱胺酸係典型半胱胺酸。亦可在抗體之例如重鏈及/或輕鏈之可變結構域進行修飾，以例如改變該抗體之結合特性。舉例來說’可在一或多個CDR區製造突變以增加或降低該抗體對抗原之KD、增加或降低K。^或改變該抗體之結合特異性。定點突變形成之技術係該領域所廣爲週知。見例如s a m b r 0 0 k e t a 1 ·及 Ausubel et al ·(同上）° 修飾或突變亦可於骨架區或恆定結構域發生以增加 PH依賴性抗體之半衰期。見例如PCT公開號WO 00/095 60 -84- 201206466 。在骨架區或恆定結構域之突變亦可被創造以改變該抗體之免疫原性'提供與另一分子共價或非共價結合之位點或改變諸如補體固定、FcR結合及抗體依賴性細胞媒介性細胞毒性之該等特性。根據本發明，單一抗體可能在可變結構域之任一或多個CDR或骨架區或恆定結構域中具有突變〇在稱爲「種系化」之過程中，在VH及VL序列中之某些胺基酸可經突變以符合該些在種系VH及VL序列中天然發現者。特別是在vH及VL序列之骨架區中之胺基酸序列可經突變以符合種系序列以降低該抗體經投予時之免疫原性風險。人νΗ及VL基因之種系DNA序列係該領域所知（見例如Vbase人種系序列資料庫；亦見Kabat，E. A.，et al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition， U · S. Department of Health and Human Services, NIH Publ. No. 9 1 -3242; Tomlinson et al·，1 992, J. Mol. Biol. 227 : 776-798 及 Cox et al.，1 994，Eur. J. Immunol. 24 : 827-836 )。另一種可能被創造之胺基酸取代係移除抗體中潛在之蛋白水解位置。該位置可能出現在抗體之可變結構域之 CDR或骨架區或在恆定結構域中。取代半胱胺酸殘基及移除蛋白水解位置可能降低抗體產物之異質性之風險，因此增加彼之均質性。另一類型之胺基酸取代消除天冬醯胺酸-甘胺酸對，其藉由改變該等殘基之一或兩者以形成潛在之脫醯胺位置。在另一實例中，本發明之pH依賴性抗 -85- 201206466 體之重鏈的c端離胺酸可被剪切。在本發明之各種實施態樣中，抗體之重鏈及輕鏈可任意選擇地包括信號序列。當獲得編碼本發明之νΗ及VL段之DNA片段後，可進 —步利用標準重組DNA技術操縱該等DNA片段，例如轉換該可變區基因成爲全長抗體鏈基因、Fab片段基因或scFv 基因。在該等操縱中，或VH編碼DNA片段係可操作地與編碼另一蛋白之另一 DN A片段連接，諸如抗體恆定區或可彎折之連接子。在此上下文所使用之用語「可操作地連接j係意圖指涉兩個DNA片段係經連接以使由該兩個DNA 片段所編碼之胺基酸序列維持讀框順序》編碼VH區之經分離之DNA可藉由可操作地連接該VH 編碼DNA與另一編碼重鏈恆定區（CHI、CH2及CH3)之 DN A分子以轉換成全長重鏈基因。人重鏈恆定區基因之序列係該領域所知（見例如K a b a t, E . A.，e t a 1.，1991， Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health and Human Services, NIH Publ. No. 9 1 -3242 )且包含該些區之DNA片段可藉由標準PCR擴增獲得。該重鏈恆定區可爲IgGl、IgG2、IgG3 、IgG4、IgA、IgE、IgM或IgD恆定區，但最佳的是IgGl 或IgG2恆定區。IgG恆定區序列可爲任何已知發生在不同個體之各種等位基因或同種異型，諸如Gm(l) 、Gm(2 )、Gm ( 3 )及Gm ( 17 )。該些同種異型代表在IgG 1恆定區中天然發生之胺基酸取代。就Fab片段重鏈基因而言，該VH編碼DNA可與另一僅編碼重鏈CH1恆定區之另一 -86- 201206466 DNA分子可操作地連接。該CH1重鏈恆定區可源自任何重鏈基因。編碼VL區之經分離之DNA可藉由可操作地連接該VL 編碼DNA與另一編碼輕鏈恆定區（CL)之DNA分子以轉換成全長輕鏈基因（以及Fab輕鏈基因）。人輕鏈恆定區基因之序列係該領域所知（見例如Kabat，E. A.，et al·， 1991， Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services，NIH Publ. No. 9 1 -3 242 )且包含該些區之 DNA 片段可藉由標準PCR擴增獲得。該輕鏈恆定區可爲/c或；l恆定區。/c恆定區序列可爲任何已知發生在不同個體之各種等位基因，諸如Inv ( 1 ) 、Inv ( 2 )及Inv ( 3 ) »該Λ恆定區可源自三個λ基因之任一基因。爲了產生scFv基因，該VH及編碼DNA片段係與編碼可彎折之連接子例如編碼胺基酸序列（Gly4-Ser) 3之另一片段可操作地連接，以使該V η及V L序列可被表現爲以可彎折之連接子連接之VL及VH之連續性單鏈蛋白質（見例如 Bird et al·，1988, Science 242 · 423-426; Huston et al., 1 988, Proc. Natl. Acad. Sci. USA 8 5 ： 5 879-5 883 ; McC afferty et al., 1 990，Nature 348 '· 5 52-5 54 )。該單鏈抗體可爲單價（若僅使用單一個VH及VL)、雙價（若使用兩個或多價（若使用超過兩個vH及VL) »雙特異性或多價抗體可被產製以與抗原及另一分子特異性結合。 -87- 201206466 在另一實施態樣中，融合抗體或免疫黏附素可被製造 ’其包含與另一多肽連接之所有或部分之本發明之抗體。在另一實施態樣中’只有抗體之可變結構域係與多肽連接。在另一實施態樣中，抗體之VH結構域係與第一多肽連接’同時抗體之VL結構域係與第二多肽連接，該第二多肽與第一多肽係以使該vH及VL結構域得以彼此交互作用以形成抗原結合部位之方式相關。在另一較佳之實施態樣中 ’該νΗ結構域與乂!^結構域被連接子分開，以使該Vh&Vl 結構域可彼此交互作用。該VH-連接子-¥!^抗體接著與感興趣之多肽連接。此外，融合抗體可被產製，其中兩個（或多個）單鏈抗體係彼此相連》這可被用於若想在單一多肽鏈上產製雙價或多價抗體或想產製雙特異性抗體。在其他實施態樣中，其他經修飾之抗體可利用編碼 pH依賴性抗體之核酸分子產製。舉例來說，「/c抗體」 (111 et al., 1 997, Protein Eng. 10 ： 949-5 7 ) 、「迷你抗體」(Martin et al., 1 994，EMBO J. 1 3 : 5 3 03 -9 ) 、「雙功能抗體」（Holliger et al.，1993，Proc. Natl. Acad. Sci. USA 90: 6444-6448)或「傑魯斯抗體（janusins)」（ Traunecker et al., 1991， EMBO J. 10 ： 3 6 5 5 -3 65 9 及Hoover). Acceptable carriers, excipients or stabilizers are not toxic to the recipient at the dosages and concentrations employed and may include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methyl sulfide Aminic acid; preservative (such as octadecyl dimethyl benzyl ammonium chloride, hexachloro chloramine, benzalkonium chloride, benzethonium chloride, phenol, butanol, sterol -81 - 201206466, Alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight (less than About 10 residues) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, glutamic acid, aspartic acid, group Aminic acid, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextran; chelating agents such as EDTA: sugars such as sucrose, mannitol, trehalose or sorbitol; salt formation Counter ion such as sodium; metal complex ( The zinc protein complexes): and / or nonionic surfactants boundary TWEENtm, PLURONICSTM or polyethylene glycol (PEG such). Pharmaceutically acceptable excipients are also described herein. In one embodiment, the anti-system is administered as a sterile aqueous solution having a pH of from about 5.0 to about 6.5 and comprising from about 1 mg/ml to about 200 mg/ml of the antibody. 1 millimole to about 100 millimolar histidine buffer, polysorbate 80 from about 0.01 mg/ml to about 10 mg/ml, seaweed from about 100 millimolar to about 400 millimolar Sugar and disodium EDTA dihydrate from about 0.01 millimolar to about 1.0 millimolar. The pH dependent antibody and its components can also be used with other agents for enhancing and/or supplementing the effectiveness of such agents. D. Kits The present invention also provides kits for rapid methods. Kits of the present invention include one or more containers containing pH-dependent antibodies (such as humanized antibodies) or peptides described herein, and instructions for using any of the methods of the invention described herein -82-201206466 . Generally, these instructions include the administration of pH dependent antibodies for the description of the therapeutic treatments described above. In some embodiments, the anti-system humanized antibody. In some embodiments, the anti-system human antibody. In other embodiments, the anti-system monoclonal antibody. Instructions for the use of pH-dependent antibodies typically include information on the dosage, route of administration, and route of administration of the intended treatment. The containers may be in unit dose, in bulk (e.g., in multi-dose packages) or in sub-unit doses. The instructions provided by the kit of the present invention are typically written instructions on a label or package copy (eg, paper contained in a set), but instructions for machine reading (eg, instructions carried on a magnetic or optical storage disk) The kits that are also acceptable for the present invention are suitably packaged. Suitable packaging includes, but is not limited to, vials, bottles, cans, bendable packages (e.g., sealed Mylar or plastic bags) and the like. Also contemplated are packages for use in combination with special devices, such as inhalers, nasally administered devices (e.g., nebulizers), or infusion devices such as small pumps. The kit may have a sterile interface (e.g., the container may be a static solution bag or vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile interface (e.g., the container may be a static solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a pH dependent antibody. The container (e.g., a pre-filled syringe or auto-injector) may additionally comprise a second pharmaceutically active agent. The kit may optionally provide additional ingredients such as buffers and interpretation information. Typically, the kit contains the container and a label or package copy on or associated with the container -83-201206466. Mutations and Modifications In order to express the antibodies of the present invention, DNA fragments encoding the VH and VL regions can be obtained by any of the methods described above. Various modifications, such as mutations, deletions and/or additions, can also be introduced into the DNA sequence using standard methods known to those skilled in the art. For example, mutational formation can be induced using standard methods, such as PCR vector mutation formation, wherein the mutated nucleotide is incorporated into a PCR primer to allow the PCR product to comprise a desired mutation or site-directed mutagenesis. One substitution that may be made by way of example is to alter one or more of the cysteine in the antibody, which may be chemically reactive to another residue such as, but not limited to, alanine or a serine. For example, substitution of atypical cysteine can occur. This substitution can occur in the CDR or framework regions of the variable domains of the antibody or in a constant domain. In some embodiments, the cysteine is a typical cysteine. Modifications may also be made in the variable domains of the antibody, e.g., the heavy and/or light chains, to, for example, alter the binding properties of the antibody. For example, a mutation can be made in one or more CDR regions to increase or decrease the KD, increase or decrease K of the antibody to the antigen. ^ or change the binding specificity of the antibody. The technology of site-directed mutagenesis is well known in the art. See, for example, s a m b r 0 0 k e t a 1 · and Ausubel et al · (ibid.) ° Modifications or mutations can also occur in the framework or constant domains to increase the half-life of the PH-dependent antibody. See, for example, PCT Publication No. WO 00/095 60-84-201206466. Mutations in the framework or constant domains can also be created to alter the immunogenicity of the antibody' to provide sites that covalently or non-covalently bind to another molecule or to alter such as complement fixation, FcR binding, and antibody-dependent cells. These properties of vector cytotoxicity. According to the present invention, a single antibody may have a mutation in any one or more of the CDR or framework regions or constant domains of the variable domain, in a process known as "germline", in a VH and VL sequence These amino acids can be mutated to conform to those found in the germline VH and VL sequences. In particular, the amino acid sequence in the framework regions of the vH and VL sequences can be mutated to conform to the germline sequence to reduce the risk of immunogenicity of the antibody upon administration. The germline DNA sequences of human νΗ and VL genes are known in the art (see, for example, the Vbase human germline sequence library; see also Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U · S. Department of Health and Human Services, NIH Publ. No. 9 1 -3242; Tomlinson et al., 1 992, J. Mol. Biol. 227: 776-798 and Cox et al., 1 994, Eur. J Immunol. 24: 827-836). Another amino acid substitution that may be created removes the potential proteolytic site in the antibody. This position may occur in the CDR or backbone region of the variable domain of the antibody or in a constant domain. Substitution of cysteine residues and removal of proteolytic sites may reduce the risk of heterogeneity of the antibody product, thus increasing its homogeneity. Another type of amino acid substitution eliminates the aspartic acid-glycine pair by altering one or both of these residues to form a potential release amine position. In another example, the c-terminal amide acid of the heavy chain of the pH dependent anti-85-201206466 of the invention can be cleaved. In various embodiments of the invention, the heavy and light chains of the antibody can optionally include a signal sequence. When a DNA fragment encoding the νΗ and VL segments of the present invention is obtained, the DNA fragments can be manipulated by standard recombinant DNA techniques, for example, by converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene or an scFv gene. In such manipulations, or a VH-encoding DNA fragment is operably linked to another DN A fragment encoding another protein, such as an antibody constant region or a bendable linker. The term "operably linked in the context of </RTI> is intended to mean that two DNA fragments are ligated such that the amino acid sequence encoded by the two DNA fragments maintains the reading sequence" encoding the VH region. The DNA can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA with another DN A molecule encoding the heavy chain constant regions (CHI, CH2 and CH3). The sequence of the human heavy chain constant region gene is Known in the art (see, for example, Kabat, E. A., eta 1., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publ. No. 9 1 -3242) and DNA fragments of these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but optimally the IgGl or IgG2 constant region The IgG constant region sequence can be any of the various alleles or allotypes known to occur in different individuals, such as Gm(l), Gm(2), Gm(3), and Gm(17). These allotypes represent Naturally occurring amino acid substitution in the IgG 1 constant region. In the case of a segment heavy chain gene, the VH encoding DNA can be operably linked to another -86-201206466 DNA molecule encoding only the heavy chain CH1 constant region. The CH1 heavy chain constant region can be derived from any heavy chain gene. The isolated DNA of the region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding a light chain constant region (CL). The human light chain is constant. Sequences of region genes are known in the art (see, for example, Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publ. No. 9 1 -3 242 And the DNA fragment comprising the regions can be obtained by standard PCR amplification. The light chain constant region can be /c or; 1 constant region. The /c constant region sequence can be any known to occur in different individuals, etc. a gene, such as Inv (1), Inv (2), and Inv (3). The Λ constant region can be derived from any of the three lambda genes. To generate the scFv gene, the VH and the coding DNA fragment are encoded and encoded. a bent linker such as an amino acid Another fragment of the sequence (Gly4-Ser) 3 is operably linked such that the V η and VL sequences can be represented as a continuous single-chain protein of VL and VH linked by a bendable linker (see, for example, Bird) Et al., 1988, Science 242 · 423-426; Huston et al., 1 988, Proc. Natl. Acad. Sci. USA 8 5: 5 879-5 883; McC afferty et al., 1 990, Nature 348 '· 5 52-5 54 ). The single-chain antibody can be monovalent (if only one VH and VL are used), bivalent (if two or more valencies are used (if more than two vH and VL are used) » bispecific or multivalent antibodies can be produced In order to specifically bind to an antigen and another molecule. -87- 201206466 In another embodiment, a fusion antibody or immunoadhesin can be made 'which comprises all or part of an antibody of the invention linked to another polypeptide In another embodiment, 'only the variable domain of the antibody is linked to the polypeptide. In another embodiment, the VH domain of the antibody is linked to the first polypeptide' while the VL domain of the antibody is The second polypeptide is linked, and the second polypeptide is associated with the first polypeptide such that the vH and VL domains interact with each other to form an antigen binding site. In another preferred embodiment, the νΗ The domain is separated from the 乂!^ domain by a linker such that the Vh&Vl domains can interact with each other. The VH-linker-¥!^ antibody is then ligated to the polypeptide of interest. Furthermore, the fusion antibody can be Production, in which two (or more) single-chain antibodies Linked to each other" This can be used to produce bivalent or multivalent antibodies on a single polypeptide chain or to produce bispecific antibodies. In other embodiments, other modified antibodies can utilize coding for pH dependence. Production of nucleic acid molecules of antibodies. For example, "/c antibody" (111 et al., 1 997, Protein Eng. 10: 949-5 7), "mini-antibody" (Martin et al., 1 994, EMBO) J. 1 3 : 5 3 03 -9 ), "bifunctional antibody" (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448) or "Jarusins" ( Traunecker et al., 1991, EMBO J. 10 : 3 6 5 5 -3 65 9 and

Traunecker et al·，1 992，Int. J. Cancer ( Suppl. ) 7 ： 51-52 )可依照說明書之揭示利用標準分子生物技術製備》雙特異性抗體或抗原結合片段可藉由各種方法產製，包括雜交瘤之融合或連接Fab’片段。見例如Songsivilal & Lachmann, 1 990， Clin. Exp. Immunol. 79 : 3 1 5 -32 1, •88- 201206466Traunecker et al., 992, Int. J. Cancer (Suppl.) 7: 51-52) can be prepared using standard molecular biotechnology as disclosed in the specification. Bispecific antibodies or antigen-binding fragments can be produced by various methods. , including fusion of hybridomas or ligation of Fab' fragments. See, for example, Songsivilal & Lachmann, 1 990, Clin. Exp. Immunol. 79 : 3 1 5 -32 1, •88- 201206466

Kostelny et al·，1 992，J. Immunol. 148 : 1 547- 1 553。此外，雙特異性抗體可能被形成爲「雙功能抗體」或「傑魯斯抗體」。在一些實施態樣中，該雙特異性抗體與抗原之兩種不同的表位結合。在一些實施態樣中，上述經修飾之抗體係利用來自此處所提供之人抗體的一或多個可變結構域或CDR區製備。產製抗原特異性抗體本發明之代表性抗體的重鏈及輕鏈之DNA(5L1721H23_6L3 及5L1721H23_6L3H3)係於2009年12月22日寄存於美國菌種保存中心（ATCC )(依布達佩斯公約之規定），且被分配表2之登記號。所有如此寄存之質體的公眾取得限制將於自本發明之說明書授予專利時不可撤銷地取消》 51^721112 3_61^3之重鏈及輕鏈之抗體參考物分別爲1；(：-H5H23 及 UC-H5L 172 卜 6L3。5 L 1 7 2 1 Η 2 3 _6 L 3 Η 3 之重鏈及輕鏈之抗體參考物分別爲UC-H5H23-6H3及UC-H5L 1721-6L3。表2 抗體參照物 ATCC登記號 UC-H5H23 ΡΤΑ-10547 UC-H5H23-6H3 ΡΤΑ-10548 UC-H5L 1721-6L3 ΡΤΑ-10549 【實施方式】 -89- 201206466 實施例1 :建構抗體與PCSK9抗原之pH依賴性結合之模型電腦模型被用於預測呈pH依賴性抗原結合之抗體是否可影響抗體之半衰期，亦即PC SK9血清濃度降低之期間。爲達成此模型之目的，做出下列假設：血液含1微莫耳劑量之抗體：抗體半衰期21天（非pH依賴性）；模擬進行期間100天：以pH依賴性抗體而言，K。。= le5/M/s， Koff @中性pH = KD * Κ〇η，其pH依賴性結合係以Koff在酸性內小體內之增加爲模型；及Kw於酸性pH = R *〖。„於中性P Η » 在此模型中物種改變之時間速率係由下列微分方程式就模型參數加以具體化。通常，該模型能處理從零開始直到達到呈穩定狀態之抗體量；達到穩定狀態時，藉由重新設定自零至反映該劑量之量的[mAb]以模擬快速濃注之抗體，接著允許以下描述之模型進行。 [/*J = kp^reate ~ ^acliveuplake\.^\~ ^protein,^Ρ,ο/Γ^' + kp^/f[LDL ·p LOIR}- lk0„f„eutrBl[mAb\p]+ koff^wra,[mAb p]Kostelny et al., 992, J. Immunol. 148: 1 547- 1 553. In addition, bispecific antibodies may be formed as "bifunctional antibodies" or "Jerus antibodies". In some embodiments, the bispecific antibody binds to two different epitopes of the antigen. In some embodiments, the modified anti-system described above is prepared using one or more variable domains or CDR regions from a human antibody provided herein. Production of antigen-specific antibodies The heavy and light chain DNAs of the representative antibodies of the present invention (5L1721H23_6L3 and 5L1721H23_6L3H3) were deposited with the American Type Culture Collection (ATCC) on December 22, 2009 (in accordance with the provisions of the Budapest Convention) And the registration number of Table 2 is assigned. The public access restrictions for all such stored plastids will be irrevocably cancelled when the patent is granted from the specification of the present invention. The antibody reference for the heavy and light chains of 51^721112 3_61^3 is 1; (:-H5H23 and UC-H5L 172 卜 6L3. 5 L 1 7 2 1 Η 2 3 _6 L 3 Η 3 The heavy and light chain antibody reference materials are UC-H5H23-6H3 and UC-H5L 1721-6L3, respectively. Table 2 Antibody Reference ATCC Accession No. UC-H5H23 ΡΤΑ-10547 UC-H5H23-6H3 ΡΤΑ-10548 UC-H5L 1721-6L3 ΡΤΑ-10549 [Embodiment] -89- 201206466 Example 1: Construction of pH-dependent binding of antibody to PCSK9 antigen A model computer model was used to predict whether antibodies to pH-dependent antigen binding could affect the half-life of the antibody, ie, the period during which the PC SK9 serum concentration was reduced. For the purposes of this model, the following hypothesis was made: blood contains 1 micromolar Dosage of antibody: antibody half-life 21 days (non-pH dependent); 100 days during the simulation: K. = le5/M/s, Koff @ neutral pH = KD * Κ〇η in the case of pH-dependent antibodies , its pH-dependent binding system is modeled by the increase of Koff in the acidic inner body. And Kw at acidic pH = R * 〖 in neutral P Η » The time rate of species change in this model is specified by the following differential equations for the model parameters. Usually, the model can be processed from scratch until reaching The amount of antibody in a steady state; when the steady state is reached, the antibody is simulated by re-setting the [mAb] from zero to reflect the dose, and then the model described below is allowed. [/*J = kp ^reate ~ ^acliveuplake\.^\~ ^protein,^Ρ,ο/Γ^' + kp^/f[LDL ·p LOIR}- lk0„f„eutrBl[mAb\p]+ koff^wra,[mAb p]

-k„ua, [ntAb · 2kofftneutral[mAb P P]- 一㈦·*广一[fU \LDL[ = kL create - kL^iear[LDL]-kproteinfin [LDLR\LDL]^ kL^ [LDL · LDLR\ -kpr〇teinAP' LDLR\lDL]^ k^LDL. P. LDLR] [ldlr] = kR^rea(e - k^leaf[ldlr]-kproteitti〇n[ldlr\ldlV kL^[ldl ldlr]-kproJei^n[ldlr\p] + ' LDLRl·^ krecycie\LDL - LDLR^n-k„ua, [ntAb · 2kofftneutral[mAb PP]- one (seven)·* broad one [fU \LDL[ = kL create - kL^iear[LDL]-kproteinfin [LDLR\LDL]^ kL^ [LDL · LDLR\ -kpr〇teinAP' LDLR\lDL]^ k^LDL. P. LDLR] [ldlr] = kR^rea(e - k^leaf[ldlr]-kproteitti〇n[ldlr\ldlV kL^[ldl ldlr]-kproJei ^n[ldlr\p] + ' LDLRl·^ krecycie\LDL - LDLR^n

[mAb\ = -lkonml[mAb\p\+ koffM [mAb P\- *，£色—】· *广一[祕U + 森gmto"p/aJi：g(l · [⑽^办Itote + 成endouptake(^ -[mAb\ = -lkonml[mAb\p\+ koffM [mAb P\- *, £色—]· * Guangyi [secret U + 森gmto"p/aJi:g(l · [(10)^ Do Itote + Endouptake(^ -

V V -90- 201206466 [/Μ/ί6 · ρ\ — k〇^- neutrai\piAb · Ζ*]- ^〇n1neutrat\fn^^ ' ^^oJftneutral\f^^^ ' Ρ ' Ρ\V V -90- 201206466 [/Μ/ί6 · ρ\ — k〇^- neutrai\piAb · Ζ*]- ^〇n1neutrat\fn^^ ' ^^oJftneutral\f^^^ ' Ρ ' Ρ\

^endouptake\f^^^ ' ^endouptake^P^^ * ^Lo/·/^ , ^endouptake^S — —1 ·_' — _ _ + 丨_^endouptake\f^^^ ' ^endouptake^P^^ * ^Lo/·/^ , ^endouptake^S — —1 ·_' — _ _ + 丨_

V V ~ ^activeuptake [mAb P k0„ineutral[mAb · p\p]- 2处你，"你加. J® ·户】 ^endouptake\p^^ " ^ ' ^]- ^endouptake^P^^ * ^ ' ^Lar/y ^ endouptake^ ~~ · P · P \ate — ' _ + _1VV ~ ^activeuptake [mAb P k0„ineutral[mAb · p\p]- 2 places, " you add. J® · household] ^endouptake\p^^ " ^ ' ^]- ^endouptake^P^ ^ * ^ ' ^Lar/y ^ endouptake^ ~~ · P · P \ate — ' _ + _1

V V ~~ ^^activeuptake \p^b · P * p] [ldl - ldlr] = kproteint〇n [ldlrIldl]-kLM [ldl - ldlr]-kprotein/fn[LDL - ldlr\p] + ^1LDLR]-kin1ernaHze\LDL * LDLR] [p ldlr] = kpr0tei^0„ [ldlr\p]-kPfiff [p. ldlr]- kproteinm [p ldlr\ldl] + [ldl p LDLR]-kiniernaKze [p ldlr] [ldl . P. ldlr] = Udz . LDLR\p\-kPfiJf [ldl · P. LDLR]^ kproteinfin [p - ldlrIldl] -、碰 Ildl，f. η>υή- kt·她祕ielLDL _ P · U)LJ^ [p LDLR\nr = kinterna!ize [p · LDLR]-krecyde[p - LDLk\n [LDL · LDLR\n = kinternatize[LDL· · LDLR] - kre_[LDL. LDLR\n [LDL. P. = kinterm^XLDL. P. ΙΖ>ΙΛ]- krecycie[LDL · /> · LZ)L7?L Γ .,-] r ^endouptake ^ J ~ ^enHouptake^ ^ Larty .. Γ .,1 Γη1 ynAb^fy = - neutral ΐΡ^^Ιεαφ U* learty early ^〇/f%neutrat\p^^ * •arly · 、endouptake {\-A\mAb\ttrty ^earfy [PL·；=J户L,，v_,M _ 4 ^ earlyVV ~~ ^^activeuptake \p^b · P * p] [ldl - ldlr] = kproteint〇n [ldlrIldl]-kLM [ldl - ldlr]-kprotein/fn[LDL - ldlr\p] + ^1LDLR]- kin1ernaHze\LDL * LDLR] [p ldlr] = kpr0tei^0„ [ldlr\p]-kPfiff [p. ldlr]- kproteinm [p ldlr\ldl] + [ldl p LDLR]-kiniernaKze [p ldlr] [ldl . P. ldlr] = Udz . LDLR\p\-kPfiJf [ldl · P. LDLR]^ kproteinfin [p - ldlrIldl] -, touch Ildl, f. η>υή- kt· her secret ielLDL _ P · U)LJ^ [p LDLR\nr = kinterna!ize [p · LDLR]-krecyde[p - LDLk\n [LDL · LDLR\n = kinternatize[LDL· · LDLR] - kre_[LDL. LDLR\n [LDL. P. = Kinterm^XLDL. P. ΙΖ>ΙΛ]- krecycie[LDL · /> · LZ)L7?L Γ .,-] r ^endouptake ^ J ~ ^enHouptake^ ^ Larty .. Γ .,1 Γη1 ynAb^fy = - neutral ΐΡ^^Ιεαφ U* learty early ^〇/f%neutrat\p^^ * •arly · , endouptake {\-A\mAb\ttrty ^earfy [PL·;=J household L,, v_, M _ 4 ^ early

-Kn^rAmAb · PLw,i^U + ^off,neutral[mAb /> · p\arly - 二少U ^early = *—->·小 *—一如吨、,【祕 L"凡 v early • k〇ff ^teuiral * ^Lar(p — ^on^neutrat * ^Ler/»· \earty off ^neutral ^ * Nearly ^ ^endouptake 0 — · P^Qffy ^earfy 91 201206466 〇1 ’ kendouptakel^^b · Ρ · P\ - ken(f0UjjfaiieA\ff^b · Ρ · P 'P]earty =---7}--^on,neutral ^ early ^on.neuiral ' ^leor/i' \arly -Ik. oJT^eurra/l^146 ' P ' P\arly ~ ^endouptake^ ~ · P · •arly κ early [mAb\at； = V - 2kont〇cUic [mAb\ttle [p\ate + koffficidic [mAh · P\ale v late 灸 gwtoiij?faAe(l — ~^Xl _ 办 Ifate y^e [pL： ^endouptake ~ \ariy late lk〇n^acidic [P\afe + ^〇ff acidic - n’acidic [mAbP\ttte[p\ate + 2k, ojftacidic [ntAb P P\aie- (1_ 观咖 y,au [mAb P\at^ = + lkBn<ttcidic [mAb\ale [p\ale - koff^clik [mAb · p\tt,e -konficidic [mAb P\ate [PL + [«tAb -P- P\a(e - 凡 ^endouptake ~ ~ F^ittAb · P\a{€-Kn^rAmAb · PLw,i^U + ^off,neutral[mAb /> ·p\arly - two less U ^early = *-->·small*—as for tons, [secret L" v early • k〇ff ^teuiral * ^Lar(p — ^on^neutrat * ^Ler/»· \earty off ^neutral ^ * Nearly ^ ^endouptake 0 — · P^Qffy ^earfy 91 201206466 〇1 ' kendouptakel^ ^b · Ρ · P\ - ken(f0UjjfaiieA\ff^b · Ρ · P 'P]earty =---7}--^on,neutral ^ early ^on.neuiral ' ^leor/i' \arly - Ik. oJT^eurra/l^146 ' P ' P\arly ~ ^endouptake^ ~ · P · •arly κ early [mAb\at; = V - 2kont〇cUic [mAb\ttle [p\ate + koffficidic [mAh · P\ale v late moxibustion gwtoiij?faAe(l — ~^Xl _ Do Ifate y^e [pL: ^endouptake ~ \ariy late lk〇n^acidic [P\afe + ^〇ff acidic - n'acidic [ mAbP\ttte[p\ate + 2k, ojftacidic [ntAb PP\aie- (1_ 观咖y, au [mAb P\at^ = + lkBn<ttcidic [mAb\ale [p\ale - koff^clik [mAb · p\tt,e -konficidic [mAb P\ate [PL + [«tAb -P- P\a(e - where ^endouptake ~ ~ F^ittAb · P\a{€

[mAb.p.p\at； = ^^(l-AimAb.p.pU +‘舜―· H ~ off ^acidic [mAb P · P\ate kend〇uptak,^ - A)F[mAb · P p\ate 此 endouptake {\-Atl-F\mAbPP\ate viate y,ate y,a,e active — ~^k〇n^aciljjc\niAb\aCfive^P\lcljve + ^off^aciillc^^^^ " ^Lc/iVe — endouptake^ — — ^endouptake(} ~ active dive lacr/v£ — ott^acidic [^^^Lc//Ve ]ac//Ve ^off acidic * ^\ictive ~ ^on^acidic * ^\tctive In ^ °ff acidic \fttAb P · ^lactivg ~ ^endouptake 0 LcftVc [ntAb · ^lactivg = ^activeupiake ^ ^"^on^acidic [w*^^Lcft've Lcrivf — ^ojf ^acidic ' ^\n Y active dive ^ on ^acidic ^ ' ^lactive l?c//v£ "** off ^acidic \f^^b * P * ^L^/Vc - ^endouptake 0 ~ A^ftlAb active dive [f/L4 A · P · P\tCtive — activeuptake * P * ^on,acidic ' ^lactive Lc/zvc - ^acidic · P · P\t K active ctive -^endouptake 0 _ A\mAb P · P\t 此模型所使用之參數係於下表3中說明。在一些情況中，參數係源自其他生理相關或經測量之量；這些亦於下表中顯示。 -92- 201206466 表3 (N)=非-pH依賴性單株抗體；（AS) =PH依賴性單株抗體參數說明數値 V 血液體積 2毫升 ^earlv 早期內小體體積 0.06毫升 ^late 晚期內小體體積 0.06毫升 ^active 活性攝取內小體體積 0.3毫升 ^R, create LDLR合成之速率 0.1奈莫耳/小時 create PCSK9合成之速率 1.714奈莫耳/小時 ^R, create LDL合成之速率 10.44奈莫耳/小時 kactiveuptake 活性PCSK9攝取之一級速率 III ( 2 ) ltactiveUptake ^activeuDtake 活性PCSK9攝取之時間標度 1小時 kR, dear LDLR之一級廓清速率 0.1872小時-1 ^R, dear LDL之一級廓清速率 0.018054小時―1 kproteinon 蛋白質-蛋白質結合速率 100000莫耳-1 秒-1 h,〇ff LDL/LDLR解離速率常數 ^proiein,on K，d,L Kd.L LDL/LDLRII 和性 10奈莫耳 kp.off PCSK9/LDLR解離速率常數 kproteinon KdP Kd,p PCSK9/LDLRI見和性 170奈莫耳 ^internalize 一級LDLR內化速率 In (2) 11internalize t internalize LDLR內化之時間標度 5分鐘 krecvcle 一級LDLR循環速率 In ( 2 ) !treCyCie (recycle LDLR循環之時間標度 10分鐘 ^on, neutral 抗體/PCSK9結合速率（pH7.4) l.oixio5 莫耳_1 秒-1 (N) 6.37xl04 莫耳]秒-1 (AS) ^off,neutral 抗體/PCSK9解離速率（pH 7.4) 4.85X10"4 秒-1 (N) 5.16XH)·4秒·丨（AS) k〇rt,acidic 抗體/PCSK9結合速率（ρΗ5·5) 3.73xl05 莫耳-1 秒·】（N) l.felO5 莫耳-1 秒“（AS) acidic 抗體/PCSK9解離速率（pH 5.5 ) 1.94x10-3 秒4 (N) 0.0187 秒-1 (AS) kendoimtake 攝取至早期內小體之體積速率 8微升/分鐘 A 自動循環分率 0.7 (無尺寸） F FcRn-媒介性抗體循環效率 0.972 (無尺寸） a 活性內小體處理時間之增加（相較於液相內小體） 4(無尺寸）此模型之說明如圖1所示。內小體pH (即pH 5.5或6.0 -93- 201206466 )/生理性pH 7.4之KD比自1增加至2導致抗體濃度隨時間增加，該增加係與KD比之値相關。當KD比（或1^或1^11) 改變時’抗原游離配體之濃度（圖2)隨KD比增加而改變 (圖2) ’抗體濃度（圖3)則隨koff之增加（圖3 A )及kon 之減少（圖3 B )而改變。這些結果與指定內小體ρ η爲5 . 5 或6.0無關。熱圖顯示（圖4 )係經建構以作爲呈ρΗ依賴性結合之抗體的常規模型，此圖有效地顯示相較於非-pH依賴性抗體，呈pH依賴性結合之抗體在降低抗原之血清濃度上多出多少天。驟降（Knockdown)天數係定義爲自第〇天至第 100 天之 l-(CSerum(t) ) /(Cserum(t==0))之整數，例如血清濃度減少1 00% N天後回到治療前之量將對應N天之驟降天數。熱圖顯示基於在中性pH下之KD及KD比（R ) ，相較於對照抗體，pH依賴性抗體之驟降天數係呈增加。影響可見於所有模型之KD値，特別是介於0.01至1〇〇奈莫耳，更特別地介於0.1至10奈莫耳之KD。某些參數例如劑量大小、抗體動力學、模擬時間或其他數量之改變將導致熱圖顯示之數値的一些差異。以模型預測PCSK9抗體（不論是否呈pH依賴性結合）之表現與實際實驗結果相關良好。在圖5A中，投予抗體 5 A 1 0之後的總抗體濃度隨時間之變化如圖所示，抗體 5A10不呈pH依賴性結合且KD比爲1.1。對應LDL之量顯示於圖5B。在圖6A中，投予抗體5L1721H23_6L3H3之後的總抗體濃度隨時間之變化如圖所示，抗體 -94- 201206466 5L1721H23_6L3H3呈pH依賴性結合且KD比爲14.4。對應 LDL之量亦顯示於圖6B。在圖5及6所顯示之模型及實驗中，假設pH値爲5.5，因此實驗所得之抗體KD値係於pH 5.5 計算。相較於5A10，呈pH依賴性結合之抗體顯示經延長之總抗體濃度及經延長之抑制LDL之量。其他關於呈pH依賴性結合之抗體的常規模型之討論如實施例4所示。實施例2 :製備及篩選呈pH依賴性PC SK9結合之抗-PCSK9抗體對單株抗體h5A10 (亦稱爲5A10 hu讀框）之所有CDR 進行組胺酸掃描突變形成。此抗體源自小鼠單株抗體 5A10(亦稱爲5A10.B8)，但具有人骨架區。用來作爲起始模板之h5A10抗體之序列如下所示（CDR以粗體字型表示）：可變輕鏈： DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRY TGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSTPRTFGQGTKLEIK (SEQ ID NO: 1) 可變重鏈： QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIN PSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYAMD YWGQGTTVTVSS (SEQ ID NO: 2) 抗體h5 A 10係經選殖至細菌性表現載體，該載體允許在大腸桿菌（E. coli )之周質中表現Fab。利用引子使每個CDR位置突變成組胺酸，導致58個h5A10抗體之單一組胺酸突變物。 -95- 201206466 具有這些單一組胺酸突變之抗體在經IPTG誘發後係於大腸桿菌之周質中表現，並利用在CH1之C端上的組胺酸標籤純化。經純化之Fab的親和性係於pH 5.5及7.4下利用Biacore測量。影響pH 5.5/pH 7.4之KD比的突變可在四個不同之CDR(L1、L2、H2及H3)中發現。對KD之pH依賴性改變介於最小地減少至顯著地減少。來自三個CDR ( LI、L2及H2 )之突變增加在pH 5.5/pH 7.4下之KD比而不破壞在pH 7.4下之親和性，產製組合含有這些突變之CDR的抗體。以h5A10及所有突變物而言，重鏈 CDR1 ( H1 )序列係 GYTFTSYYMH ( SEQ ID NO: 6)。以5A10、單突變物、雙突變物、三突變物及 5L 1 72 1 H23_6H3 而言，輕鏈 CDR3 ( L3 )係 QQRYSTPRT ( SEQ ID NO : 27 )。以其他親和性成熟突變物及以L1L3爲基底之突變物而言，L3係QQRYSLWRT ( SEQ ID NO : 12 )。含有這些突變之各種組合的抗體之CDR序列如下表4 所示。序列識別號提供於括弧中。 -96 · 201206466 表4 L1 L2 H2 H3 h5A10 KASQDVSTAVA(13) SASYRYT(17) EINPSGGRTNYNEKFKS(19) ERPLYAMDY (8) 單突變物： 5L-6 KASQDHSTAVA (14) 5L1-7 KASQDVHTAVA(IO) 5L2-1 HASYRYT(11) 5L2-4 SASHRYT(18) 5H2-3 EIHPSGGRTNYNEKFKS(7) 5H3-5 ERPLHAMDY P1) 雙突變物： I 5L17 21 KASQDVHTAVA(IO) HASYRYT(11) 5L17 24 KASQDVHTAVA(IO) SASHRYT(18) 5L17H23 KASQDVHTAVA(IO) EIHPSGGRTNYNEKFKS(7) 5L21 24 HASHRYT(19) 5L21 H23 HASYRYT(11) EIHPSGGRTNYNEKFKS ⑺ 5L24 H23 SASHRVT(18) EIHPSGGRTNYNEKFKS ⑺ 三突變物： 5L17 21 H23 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) 5L17 24 H23 KASQDVHTAVA(IO) SASHRYT(18) EIHPSGGRTNYNEKFKS(7) 5L1724H35 KASQDVHTAVA(IO) SASHRYT(18) ERPLHAMDY (21) 親和性成熟突變物： 5L1721H23 6H3 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) ERPLYASDL( 9) 5L1721H23 6L3 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) 5L1724H23 6L3 KASQDVHTAVA(IO) SASHRYT(18) EIHPSGGRTNYNEKFKS(7) 5L1721H23 6L3H3 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) ERPLYASDL( 9) 5L1724H23 6L3H3 KASQDVHTAVA(IO) SASHRYT(18) EIHPSGGRTNYNEKFKS(7) ERPLYASDL( 9) ElL1L3 爲基底之突變物 L1L3 RASQGISSALA(15) SASYRYT(17) EISPFGGRTNYNEKFKS(20) ERPLYASDL( 9) 6L1721H23 RASQGIHSALA(16) HASYRYT(11) EIHPFGGRTNYNEKFKS(7) 6L1721 RASQGIHSALA(16) HASYRYT(11) 6L21H2335 HASYRYT(11) EIHPFGGRTNYNEKFKS(7) -97- 201206466 這些抗體之骨架序列相較於5A10.B8不作任何改變。舉例來說，以下提供 5L1 721H23_6L3 及 5L1721H23_6L3H3 之可變重鏈之序列。相同之可變輕鏈被用於每個這些抗體，序列亦提供於下。CDR係以粗體強調。 5L1721H23_6L3 及 5L1721H23_6L3H3之可變輕鏈： DIQMTQSPSSLSASVGDRVTITCKASQDVHTAVAWYQQKPGKAPKLLIYHASYR YTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 3) 5L1721H23_6L3之可變重鏈： QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIH PSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYAMD Y WGQGTTVTVSS (SEQ ID NO: 4) 5L1721H23_6L3H3之可變重鏈： QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIH PSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYASD L WGQGTTVTVSS (SEQ ID NO: 5) 5L1721H23_6L3 及 5L1721H23_6L3H3 之可變輕鏈（ kappa ) gatatccagatgacacagtccccatcctccctgtctgcctctgtgggcgaccgcgtcaccatcacctgcaaggcctc tcaggatgtgcatactgctgtagcctggtatcagcagaagccaggcaaagccccaaaactgctgatctaccatgc atcctaccgctacactggtgtcccatcacgcttcagtggcagtggctctggtacagatttcaccttcaccattagcagc ctgcaaccagaagatattgccacttattactgccagcaacgttatagtctgtggcgcacgttcggtcaaggcaccaa gctggagatcaaa (SEQ ID NO: 23) -98- 201206466 5L1721H23_6L3H3之可變重鏈 caggtgcagctggtgcagtctggtgctgaggtgaagaagcctggcgcttccgtgaaggtttcctgcaaagcatctg gttacacctttaccagctactatatgcactgggtgcgccaagcccctggtcaaggcctggagtggatgggcgagatt catcctagcggcggtcgtactaactacaatgagaagttcaagagccgcgtgactatgactcgcgatacctccacca gcactgtctacatggaactgagctctctgcgctctgaggacactgctgtgtattactgtgcccgcgagcgccccctgt atgctagcgacctgtggggccagggtaccacggtcaccgtctcctca (SEQ ID NO: 24)[mAb.pp\at; = ^^(l-AimAb.p.pU +'舜-· H ~ off ^acidic [mAb P · P\ate kend〇uptak,^ - A)F[mAb · P p\ Ate this endouptake {\-Atl-F\mAbPP\ate viate y,ate y,a,e active — ~^k〇n^aciljjc\niAb\aCfive^P\lcljve + ^off^aciillc^^^^ " ^Lc/iVe — endouptake^ — — ^endouptake(} ~ active dive lacr/v£ — ott^acidic [^^^Lc//Ve ]ac//Ve ^off acidic * ^\ictive ~ ^on^acidic * ^\tctive In ^ °ff acidic \fttAb P · ^lactivg ~ ^endouptake 0 LcftVc [ntAb · ^lactivg = ^activeupiake ^ ^"^on^acidic [w*^^Lcft've Lcrivf — ^ojf ^acidic ' ^\n Y active dive ^ on ^acidic ^ ' ^lactive l?c//v£ "** off ^acidic \f^^b * P * ^L^/Vc - ^endouptake 0 ~ A^ftlAb active Dive [f/L4 A · P · P\tCtive — activeuptake * P * ^on,acidic ' ^lactive Lc/zvc - ^acidic · P · P\t K active ctive -^endouptake 0 _ A\mAb P · P \t The parameters used in this model are described in Table 3. In some cases, the parameters are derived from other physiologically relevant or measured quantities; these are also shown in the table below. -92- 201206466 Table 3 (N) = non-pH-dependent monoclonal antibody; (AS) = PH-dependent monoclonal antibody parameter description number 値V blood volume 2 ml ^earlv early endosomal volume 0.06 ml ^late late endosomal volume 0.06 ml ^active Active ingestion volume 0.3 ml ^R, create LDLR synthesis rate 0.1 nm / hour create PCSK9 synthesis rate 1.714 namol / hour ^ R, create LDL synthesis rate 10.44 namol / Hour kactiveuptake active PCSK9 uptake rate III ( 2 ) ltactiveUptake ^activeuDtake active PCSK9 uptake time scale 1 hour kR, dear LDLR one grade clearance rate 0.1872 hours -1 ^R, dear LDL one grade clearance rate 0.018054 hours -1 kproteinon Protein-protein binding rate 100,000 mol-1 sec -1 h, 〇ff LDL/LDLR dissociation rate constant ^proiein, on K, d, L Kd.L LDL/LDLRII and sex 10 namol kp.off PCSK9/LDLR Dissociation rate constant kproteinon KdP Kd, p PCSK9/LDLRI see and sex 170 nanomolar ^internalize primary LDLR internalization rate In (2) 11internalize t internalize LDLR internalization time scale 5 minutes krecvcle Primary LDLR cycling rate In ( 2 ) !treCyCie (recycle LDLR cycle time scale 10 minutes ^on, neutral antibody / PCSK9 binding rate (pH 7.4) l.oixio5 Moer_1 sec-1 (N) 6.37xl04 Mo Ear]sec-1 (AS) ^off, neutral antibody/PCSK9 dissociation rate (pH 7.4) 4.85X10"4 sec-1 (N) 5.16XH)·4 sec·丨(AS) k〇rt,acidic antibody/PCSK9 Binding rate (ρΗ5·5) 3.73xl05 Mohr-1 sec·](N) l.felO5 Mohr-1 sec "(AS) acidic Antibody/PCSK9 dissociation rate (pH 5.5) 1.94x10-3 sec 4 (N) 0.0187 sec-1 (AS) kendoimtake Volume rate up to early corpuscles 8 μl/min A Automatic cycle fraction 0.7 (no size) F FcRn-mediated antibody cycle efficiency 0.972 (no size) a Active corpuscle Increase in processing time (compared to liquid phase bodies) 4 (no size) This model is illustrated in Figure 1. The increase in the KD of the endosome pH (i.e., pH 5.5 or 6.0-93-201206466)/physiological pH 7.4 from 1 to 2 results in an increase in antibody concentration over time, which is related to the KD ratio. When the KD ratio (or 1^ or 1^11) changes, the concentration of the antigen free ligand (Fig. 2) changes as the KD ratio increases (Fig. 2). The antibody concentration (Fig. 3) increases with the koff (Fig. 3). A) and kon reduction (Fig. 3B) change. These results are independent of the specified inner body ρ η of 5.5 or 6.0. The heat map (Figure 4) is constructed as a conventional model of antibodies that bind in a ρΗ-dependent manner, which effectively shows that antibodies that are pH-dependently bound are in serum-reducing serum compared to non-pH-dependent antibodies. How many more days are there in concentration. The number of days of Knockdown is defined as an integer from l-(CSerum(t) ) / (Cserum(t==0)) from day 至 to day 100, eg, serum concentration is reduced by 100% N days later The amount before the treatment will correspond to the number of days of dips in N days. The heat map shows an increase in the number of days of pH-dependent antibodies compared to the control antibody based on the KD and KD ratio (R) at neutral pH. The effect can be seen on the KD値 of all models, especially between 0.01 and 1 〇〇Noire, more particularly between 0.1 and 10 NM. Certain parameters such as dose size, antibody kinetics, simulation time, or other quantitative changes will result in some differences in the number of heat maps displayed. The performance of predicting PCSK9 antibodies (whether or not pH-dependently bound) by model is well correlated with actual experimental results. In Fig. 5A, the total antibody concentration after administration of antibody 5 A 10 0 was changed as a function of time, and antibody 5A10 was not pH-dependently bound and had a KD ratio of 1.1. The amount corresponding to LDL is shown in Figure 5B. In Fig. 6A, the total antibody concentration after administration of the antibody 5L1721H23_6L3H3 was changed as a function of time, and the antibody -94-201206466 5L1721H23_6L3H3 showed a pH-dependent binding and a KD ratio of 14.4. The amount of corresponding LDL is also shown in Figure 6B. In the models and experiments shown in Figures 5 and 6, the pH 値 was assumed to be 5.5, so the antibody KD obtained by the experiment was calculated at pH 5.5. The pH-dependently bound antibody showed an extended total antibody concentration and an extended inhibition of LDL compared to 5A10. A discussion of other conventional models for pH-dependent binding of antibodies is shown in Example 4. Example 2: Preparation and Screening of pH-Dependent PC SK9 Binding Anti-PCSK9 Antibodies Histidine scanning mutations were formed on all CDRs of the monoclonal antibody h5A10 (also known as the 5A10 hu reading frame). This antibody is derived from mouse monoclonal antibody 5A10 (also known as 5A10.B8) but has a human framework region. Used as a starting template antibody h5A10 sequence was as follows (CDR in bold font): variable light chain: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRY TGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSTPRTFGQGTKLEIK (SEQ ID NO: 1) a variable heavy chain: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIN PSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYAMD YWGQGTTVTVSS (SEQ ID NO 2) The antibody h5 A 10 is selected to a bacterial expression vector which allows expression of Fab in the periplasm of E. coli. The use of primers to mutate each CDR position to histidine resulted in a single histidine mutant of 58 h5A10 antibodies. -95-201206466 Antibodies with these single histidine mutations were expressed in the periplasm of E. coli after induction by IPTG and purified using a histidine tag on the C-terminus of CH1. The affinity of the purified Fab was measured using Biacore at pH 5.5 and 7.4. Mutations affecting the KD ratio of pH 5.5/pH 7.4 can be found in four different CDRs (L1, L2, H2 and H3). The pH dependence of KD is reduced from minimal to significant. Mutations from the three CDRs (LI, L2 and H2) increased the KD ratio at pH 5.5/pH 7.4 without disrupting the affinity at pH 7.4, producing antibodies that combined the CDRs containing these mutations. In the case of h5A10 and all mutants, the heavy chain CDR1 (H1) sequence is GYTFTSYYMH (SEQ ID NO: 6). For the 5A10, single mutant, double mutant, triple mutant and 5L 1 72 1 H23_6H3, the light chain CDR3 (L3) is QQRYSTPRT (SEQ ID NO: 27). In the case of other affinity matured mutants and L1L3-based mutants, the L3 line is QQRYSLWRT (SEQ ID NO: 12). The CDR sequences of the antibodies containing various combinations of these mutations are shown in Table 4 below. The sequence identification number is provided in parentheses. -96 · 201206466 Table 4 L1 L2 H2 H3 h5A10 KASQDVSTAVA(13) SASYRYT(17) EINPSGGRTNYNEKFKS(19) ERPLYAMDY (8) Single Mutant: 5L-6 KASQDHSTAVA (14) 5L1-7 KASQDVHTAVA(IO) 5L2-1 HASYRYT( 11) 5L2-4 SASHRYT(18) 5H2-3 EIHPSGGRTNYNEKFKS(7) 5H3-5 ERPLHAMDY P1) Double Mutant: I 5L17 21 KASQDVHTAVA(IO) HASYRYT(11) 5L17 24 KASQDVHTAVA(IO) SASHRYT(18) 5L17H23 KASQDVHTAVA( IO) EIHPSGGRTNYNEKFKS(7) 5L21 24 HASHRYT(19) 5L21 H23 HASYRYT(11) EIHPSGGRTNYNEKFKS (7) 5L24 H23 SASHRVT(18) EIHPSGGRTNYNEKFKS (7) Triple Mutant: 5L17 21 H23 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) 5L17 24 H23 KASQDVHTAVA(IO) SASHRYT(18) EIHPSGGRTNYNEKFKS(7) 5L1724H35 KASQDVHTAVA(IO) SASHRYT(18) ERPLHAMDY (21) Affinity Mature Mutant: 5L1721H23 6H3 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) ERPLYASDL( 9) 5L1721H23 6L3 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) 5L1724H23 6L3 KASQDVHTAVA(IO) SASHRYT(18) EIHPSGGRTNYNEKFKS(7) 5L1721H23 6L3 H3 KASQDVHTAVA(IO) HASYRYT(11) EIHPSGGRTNYNEKFKS(7) ERPLYASDL( 9) 5L1724H23 6L3H3 KASQDVHTAVA(IO) SASHRYT(18) EIHPSGGRTNYNEKFKS(7) ERPLYASDL( 9) ElL1L3 is the base mutant L1L3 RASQGISSALA(15) SASYRYT(17) EISPFGGRTNYNEKFKS(20) ERPLYASDL( 9) 6L1721H23 RASQGIHSALA(16) HASYRYT(11) EIHPFGGRTNYNEKFKS(7) 6L1721 RASQGIHSALA(16) HASYRYT(11) 6L21H2335 HASYRYT(11) EIHPFGGRTNYNEKFKS(7) -97- 201206466 The skeletal sequences of these antibodies are compared No changes are made in 5A10.B8. For example, the sequences of the variable heavy chains of 5L1 721H23_6L3 and 5L1721H23_6L3H3 are provided below. The same variable light chain was used for each of these antibodies and the sequences are also provided below. The CDR lines are highlighted in bold. And variable light chain 5L1721H23_6L3 5L1721H23_6L3H3 of: DIQMTQSPSSLSASVGDRVTITCKASQDVHTAVAWYQQKPGKAPKLLIYHASYR YTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEIK (SEQ ID NO: 3) of the variable heavy chain 5L1721H23_6L3: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIH PSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYAMD Y WGQGTTVTVSS (SEQ ID NO: 4) of the variable heavy chain 5L1721H23_6L3H3: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIH PSGGRTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERPLYASD L WGQGTTVTVSS (SEQ ID NO: 5) 5L1721H23_6L3 5L1721H23_6L3H3 and the variable light chain (kappa) gatatccagatgacacagtccccatcctccctgtctgcctctgtgggcgaccgcgtcaccatcacctgcaaggcctc tcaggatgtgcatactgctgtagcctggtatcagcagaagccaggcaaagccccaaaactgctgatctaccatgc atcctaccgctacactggtgtcccatcacgcttcagtggcagtggctctggtacagatttcaccttcaccattagcagc ctgcaaccagaagatattgccacttattactgccagcaacgttatagtctgtggcgcacgttcggtcaaggcaccaa gctggagatcaaa (SEQ ID NO: 23) -98- 201206466 5L1721H23_6L3H3 the variable heavy chain caggtgcagctggtgcagtctggtgctgaggtgaa gaagcctggcgcttccgtgaaggtttcctgcaaagcatctg gttacacctttaccagctactatatgcactgggtgcgccaagcccctggtcaaggcctggagtggatgggcgagatt catcctagcggcggtcgtactaactacaatgagaagttcaagagccgcgtgactatgactcgcgatacctccacca gcactgtctacatggaactgagctctctgcgctctgaggacactgctgtgtattactgtgcccgcgagcgccccctgt atgctagcgacctgtggggccagggtaccacggtcaccgtctcctca (SEQ ID NO: 24)

這些抗體係經表現及純化以測定彼等於pH 7.4下之KD 及彼等於pH 5.5/pH 7.4下之KD比，在裝設硏究級感測晶片之表面電漿共振Biacore 3000生物感測器上測量，使用 HBS-EP流動緩衝液（瑞典烏普薩拉市（Uppsala) Biacore AB公司-現爲GE醫療集團）。兔多株抗-Ms IgG係以飽和量經胺偶合至晶片上，使用標準N-羥琥珀二醯亞胺/乙基二甲基胺基丙基碳二醯亞胺（NHS/EDC )化學。該緩衝液被換成HBS-EP + 1毫克/毫升BSA + 1毫克/毫升CM-葡聚糖。全長PCSK9 IgG係經稀釋至約15微克/毫升，以5微升/分鐘捕捉1分鐘以給予每個流動池約5〇〇RU之量，留一個空白作爲參考通道。3.73-302奈莫耳hPCSK9或2.54-206奈莫耳mPCSK9係以5元（membered ) 3倍系歹IJ以100微升/分鐘注射1分鐘。監測解離5分鐘。晶片在每次滴定的最後一次注射後以二次30秒脈衝之100毫莫耳磷酸再生。緩衝液循環提供空白値以雙重參照資料，該些資料接著利用 Biaevaluation軟體v.4.1被全部套用至單純結合模型。親和性係自動力學速率常數之商數（KD = keff/kon )推斷。對人PCSK9之親和性結果顯示於表5，對小鼠PCSK9之親和性結果顯示於表6。這些資料顯示抗體可經設計及篩選以在pH 7.4下具有對人或小鼠PCSK9較高之親和性及在pH C： -99- 201206466 5.5下較低之親和性。表5.抗-PCSK9抗體在pH7_4及5.5下與人PCSK9結合之結合親和性 huPCS K9 pH 7.4 huPCS K9pH 5.5 「pH 5.5/7.4 kon (1/Ms) Koff d/s) Rmax (RU) KD (nM) kon (1/Ms) Koff (1/s) Rmax (RU) KD (nM) KD I 6L1721 3.29E+04 7.08E-05 880 2.2 1.66E+05 8.40E-04 1059 5.1 2.3 5A10,B8 4.38E+04 1.04E-04 1080 2.4 1.31E+05 3.91 E«04 1020 3.0 1.3 5L1721H23_6L3H3 4.42E+04 1.72E-04 934 3.9 8.28E+04 4.80E-03 1430 58 14.9 6L1721H23 5.02E+04 2.01 E-04 645 4.0 1.10E+05 2*61 E-03 1140 24 6 5L1721H23_6L3 3.09E+04 2.14E-04 974 6.9 6.45E+04 0.0166 1320 257 37.2 5L1721H23_6H3 3.74E+04 2.73E-04 461 7.3 1.41E+05 7.72E-03 398 55 7.5 5L1721H23 3.17E+04 3.99E-04 990 「13 1.24E+05 0.0179 1030 144 11.1 5L1724H23_6L3H3 2.23E+04 3.42E-04 1020 15 9.55E+04 7.89E-03 1500 83 5.5 5L1724H23_6L3 3.59E+04 7.90E-04 910 22 7.54E+04 0.0407 1050 540 24.5 6L21H2335 5.81 E+04 7.33E-03 892 126 6.55E+05 2.38E-01 624 363 2.9 5L1724H23 - - 20 -I 2.03E+05 7.93E-03 114 39 5L1724H35 - - 40 - - - 0 - 表6.抗-PCSK9抗體在?117.4及5.5下與小鼠1^1(；9結合之結合親和性 msPCK9 pH 7.4 msPCK9 pH 5.5 huPSCKd pH 5.5/7.4 kon (1/Ms) Koff (1/s) Rmax (RU) KD (nM) kon (1/Ms) Koff (1/s) Rmax (RU) KD (nM) KD 6L1721 8.80E+04 2.28E-04 1260 2.6 3.38E+05 1.77E-03 1120 5.2 2.0 5A10_WT 1.01 E+05 4.85E-04 1410 4.8 3.73E+05 1.94E-03 1050 5.2 1.1 5L1721H23一6L3H3 6.37E+04 5.16E-04 1630 8.1 1.60E+05 0.0187 1420 117 14.4 6L1721H23 6.48E+04 4.69E-04 1230 7.2 2.04E+05 7.29E-03 1220 36 5.0 5L1721H23_6L3 4.75E+04 676E-04 1600 14 1.05E+05 0.0428 1360 408 29.1 5L1721H23_6H3 1.13E+05 8.99E-04 615 8.0 3.07E+05 0.0341 460 111 13.9 5L1721H23 6.12E+04 1.41E-03 1380 23 1.02E+05 0.0588 1100 576 25.0 5L1724H23_6L3H3 6.31 E+04 8.27E-04 1690 13 2.11 E+05 0.0263 1520 125 9.6 5L1724H23_6L3 5.31 E+04 2.18E-03 1440 41 1.60E+05 0.121 1100 756 18.4 6L21H2335 8.99E+04 1.72E-02 1340 191 5.26E+05 0.237 502 451 2.4 5L1724H23 - - 40 - 2.73E+05 2.02E-02 91 74 5L1724H35 4.43E+04 1.53E-03 164 35 - - 0 - PCSK9 抗體 H1M300N 之體 h5A10及 5L1721H23_6L3H3以及 PCSK9抗親和性及動力學參數（見US20 1 0/0 1 66768, -100- 201206466 例如表7 )。所有實驗係於B i a c 〇 r e 2 0 0 0生物感測器上進行〇抗-人Fc感測晶片係藉由400毫莫耳EDC及100毫莫耳 NHS之1: 1 (體積/體積）混合物以10微升/分鐘之流動速率活化Biacore CM4感測晶片之所有流動池7分鐘加以製備。抗·人Fc試劑（山羊F(AB〇 2片段抗人IgG Fc，卡佩爾 (Cappel)目錄編號5 5 05 3 )係經10毫莫耳醋酸鈉pH 5.0 稀釋至60微克/毫升，並以20微升/分鐘注射至所有流動池 7分鐘。所有流動池係以1〇〇毫莫耳之乙二胺於150毫莫耳硼酸鹽緩衝液pH 8.5以10微升/分鐘封閉7分鐘。動力學分析係利用如Karlsson et al.，Anal. Biochem 349，136-147 ( 2006)所描述之動力學滴定方法進行。相同抗體（例如5L1721H23_6L3H3)以2微克/毫升被捕捉至下游流動池（流動池2、3及4 )，以1 0微升/分鐘之流動速率分別捕捉30秒、60秒及120秒至流動池2、3及4。流動池1係用來作爲參照表面。在捕捉抗體之後，P C S K 9 被以低至高濃度之注射液系列經30微升/分鐘注射至所有流動池。最高濃度爲200奈莫耳PCSK9及稀釋因子爲3倍。每次PCSK9注射係2分鐘，在200奈莫耳PCSK9注射後之解離時間爲20分鐘。以流動緩衝液取代PC SK9進行類似之注射步驟以達雙重參照之目的（雙重參照係如Myszka，J. Mol. Recognit 12，2 79-284,1 999所述）。在每次分析週期之後，所有流動池利用三次3 0秒注射75毫莫耳磷酸再生。來自流動池2及3之給定PCSK9/抗體對之感應圖被完整帶 -101 - 201206466 入單純1: 1蘭綴爾（Langmuir)之質量傳輸結合模型。這些實驗係於pH 6.0及7.4下進行，分別使用樣本及 10毫旲耳磷酸鈉、15〇毫莫耳氯化鈉、〇〇5 %吐溫（Tween )-2 0、pH 6及10毫莫耳磷酸鈉、15〇毫莫耳氯化鈉、 0.05%吐溫-20、pH 7.4之流動緩衝液。表7.pH依賴性結合抗體 PH6.0(磷酸鈉）酸鈉丨比値 K〇, kd, pH 6/ pH 6/ kd, pH 7.4 5A10 WT MM·、.1) kd(s1) U/2 (min) < Κ〇 (PM) ka (M-1s1) kd(s1) ^1/2 (min) Kd (PM) K〇, pH 7.4 3.31 E+05 <6Ε·5 193 181 2.41 E+05 < 6E-05 < 193 249 5L1721H 23 BL3H3 3.07E+05 2.86Ε-03 4.0 9316 2.42E+05 1.74E-04 66.4 719 13.0 16.4 H1M300N 4.27E+05 3.21 Ε-04 36.0 752 1.67E+05 6.92E-05 166.9 414 1.8 4.6 實施例3 : PH依賴性PC SK9結合抗體在降低膽固醇上具有經延長之藥物藥效學效應 A. pH依賴性PCSK9結合拮抗抗體在較長之時間期間降低小鼠之血清膽固醇爲了測定pH依賴性PCSK9結合拮抗抗體是否可在比非 pH依賴性抗體更長之時間期間內降低活體內膽固醇之量，當酸敏感性抗體 5L1721H23_6H3及 5L1721H23_6L3H3和非酸敏感性抗體h5A10 (僅投予10毫克/公斤）及5A10.B8 以1、3或1 0毫克/公斤注射至小鼠體內時，檢測它們對血清膽固醇之時間效應。所有四種抗體在中性pH下（pH 7.4 )均對小鼠PCSK9具有5至14奈莫耳之類似結合親和性。抗體51^172 11123_61^3及51^172 11^23_61^31^在1^5.5下分別 -102- 201206466 具有Π7奈莫耳及40 8奈莫耳之較低之親和性，然而h5Al 0 及5Α10.Β8在pH 5.5下具有和pH 7.4類似之KD(5.2奈莫耳 )。公鼠C57/bl6 ( 6至7周齡）被飼養於12小時光/暗循環中，在第7天採血以收集大約70微升之血清。拮抗PCSK9 抗體及不與任何已知之哺乳動物蛋白質結合之對照同型相符單株抗體係經靜脈注射至7周齡之C57/bl6公鼠，在注射後第5、12、19、26、61及75天收集血清樣本。以Ace Alera儀器（紐澤西州西考德威爾市（West Caldwell)阿爾法偉士曼（Alfa Wassermann)公司）分析所有血清樣本之總膽固醇、三酸甘油脂、HDL膽固醇，LDL膽固醇之量利用弗雷德瓦德（Fried ewald )公式計算。圖7顯示在注射PCS K9拮抗抗體之後，總膽固醇之量快速及劑量依賴性地減少。小鼠之LDL膽固醇量太低以致無法可靠地測量及計算。在1 0毫克/公斤之劑量下，所有四種抗體皆在第5 及12天降低35至40%之HDL-膽固醇，然而注射 5L1721H23_6H3 及 5L1721H23_6L3H3之動物直到第 61 天才恢復基準量，注射h 5 A 1 0及5 A 1 0 . B 8之動物分別在第2 6及 33天恢復至基準量。 B· pH依賴性PCSK9結合抗體在小鼠具有延長之半衰期抗體之血清濃度在實施例b所描述之相同試驗中測定 ’以決定PH敏感性抗PC SK9抗體是否導致較長之抗體半衰期。相較於與其他可溶性抗原結合之抗體，正常之抗 -103- 201206466 PCSK9抗體諸如h5A10及5A10.B6因爲PCSK9媒介性抗體/ 抗原複合物之降解作用而具有較短之劑量依賴性半衰期。如圖8A所示，PH依賴性結合特性減少抗體降解並延長抗 PCSK9抗體 5L1721H23_6H3及 5L1721H23_6L3H3之半衰期。爲了進一步證實該5L1721H23_6H3及5L1721H23_6L3H3 之延長PK係因PCSK9媒介性廓清抗體減少所致，在PCSK9 基因剔除小鼠進行類似之時間量程試驗。該小鼠體內之 PH敏感性及非敏感性抗體的血清抗體濃度及減少速率之間並無顯著差異，直到注射3毫克/公斤之人PC SK9至小鼠。在注射後，該非PH敏感性PCSK9抗體相較於PH敏感性抗體及陰性對照抗體顯示增加之降解（圖8 B )。這些結果表示，該觀察到之pH依賴性PCSK9結合PCSK9抗體之降解減少係因該抗體自PCS K9解離所致，因此防止該抗體之 PCSK9媒介性降解。 C· pH敏感性PCSK9拮抗抗體在較長之時間期間內降低猴之血清膽固醇圖9B顯示PH敏感性抗PCSK9拮抗抗體5L1721H23_6H3 及5L1721H23_6L3H3和非敏感性抗PCSK9抗體L1L3對石蟹獼猴（cynomolgus)之血清LDL-膽固醇量之影響（對照組百分比）。在第〇天經靜脈快速濃注對母石蟹獼猴投予抗體（各1.5毫克/公斤）。所有三個抗體治療組之LDL-膽固醇在一天內減少至基準量之5 0 %。雖然在投予非p Η敏感性抗體後第1〇天之LDL -膽固醇量恢復至基準量，但以ρΗ敏 -104- 201206466 感性抗體治療之猴的LDL膽固醇維持低下直到第21天。 HDL之量在抗體治療後實質上維持不變（圖9A)。圖10顯示pH敏感性抗PCSK9抗體之半衰期相較於非pH敏感性 L 1 L 3爲長。實施例4 :呈pH依賴性抗原結合之抗體之常規模型電腦模型被用於預測與彼之通用抗原呈P Η依賴性抗原結合之抗體是否可影響抗體之半衰期及/或降低抗原之量或血清濃度之期間。爲達成此模型之目的’做出下列假設：1)抗體，血液含1微莫耳劑量之抗體，抗體半衰期21 天；2 )模擬進行期間1〇〇天；抗體結合力及pH依賴性結合力，Κ〇η = le5/M/s，於中性 pH之 Koff = KD * Κοη : pH依賴性結合力係以酸性內小體內之K。^增加爲模型；於酸性 pH之 Kuf = R * 中性 pH 之 KQff。圖1 5詳細說明呈pH依賴性結合之抗體用於建構模型之穿越模型，定義該模型之公式如下： d/dt (Normal—Dose) = - k一dist*Normal一Dose d/dt (mAb) = k—dist*Normal一Dose - (mAb*kon*Normal一antigen)十(kon*KD* mAb一antigen} - k_blood_endo*mAb/ VNorm + fl* k一blood一endo*mAb一e/ VNonn + (1-fl)* f2*k_blood_endo*mAb_le/ VNorm " — d/dt(mAb_antigen) = (mAb*kon*Normal一antigen) - (kon*KD* mAh 一 antigen}-k_blood—endo*mAb—antigen/ VNorm + fl* k一blood_endo*mAb一antigen一e/ VNorm + (1-fl) * f2*k__blood_endo*cmplx__le/ VNorni d/dt (Normal一antigen) =- (mAb*kon*Normal一antigen) + (kon*KD* mAb一antigen} + In (2}/antigen一halflife*antigen—level -These anti-systems were characterized and purified to determine the KD at pH 7.4 and the KD ratio at pH 5.5/pH 7.4, on a surface-plasma resonance Biacore 3000 biosensor equipped with a graded sensing wafer. Measurements were made using HBS-EP Flow Buffer (Uppsala, Biacore AB, USA - now GE Healthcare). Multiple strains of rabbit anti-Ms IgG were coupled to the wafer with a saturation amount of amine using standard N-hydroxysuccinimide/ethyldimethylaminopropyl carbodiimide (NHS/EDC) chemistry. The buffer was exchanged for HBS-EP + 1 mg/ml BSA + 1 mg/ml CM-glucan. The full-length PCSK9 IgG was diluted to approximately 15 μg/ml and captured at 5 μl/min for 1 minute to give approximately 5 〇〇RU per flow cell, leaving a blank as a reference channel. 3.73-302 Nemo hPCSK9 or 2.54-206 Nemo mPCSK9 was injected at 100 μl/min for 1 minute at 5 members (membered) 3 times 歹 IJ. Monitor dissociation for 5 minutes. The wafer was regenerated with 100 millimoles of phosphoric acid pulsed for a second 30 seconds after the last injection of each titration. The buffer cycle provides blank 値 with double reference data, which is then fully applied to the simple binding model using Biaevaluation software v.4.1. The quotient of the automatic mechanical rate constant of the affinity system (KD = keff/kon) is inferred. The results of affinity for human PCSK9 are shown in Table 5, and the results of affinity for mouse PCSK9 are shown in Table 6. These data show that antibodies can be designed and screened to have a higher affinity for human or mouse PCSK9 at pH 7.4 and a lower affinity at pH C: -99-201206466 5.5. Table 5. Binding affinity of anti-PCSK9 antibody to human PCSK9 at pH 7_4 and 5.5 huPCS K9 pH 7.4 huPCS K9pH 5.5 "pH 5.5/7.4 kon (1/Ms) Koff d/s) Rmax (RU) KD (nM ) kon (1/Ms) Koff (1/s) Rmax (RU) KD (nM) KD I 6L1721 3.29E+04 7.08E-05 880 2.2 1.66E+05 8.40E-04 1059 5.1 2.3 5A10, B8 4.38E +04 1.04E-04 1080 2.4 1.31E+05 3.91 E«04 1020 3.0 1.3 5L1721H23_6L3H3 4.42E+04 1.72E-04 934 3.9 8.28E+04 4.80E-03 1430 58 14.9 6L1721H23 5.02E+04 2.01 E-04 645 4.0 1.10E+05 2*61 E-03 1140 24 6 5L1721H23_6L3 3.09E+04 2.14E-04 974 6.9 6.45E+04 0.0166 1320 257 37.2 5L1721H23_6H3 3.74E+04 2.73E-04 461 7.3 1.41E+05 7.72 E-03 398 55 7.5 5L1721H23 3.17E+04 3.99E-04 990 "13 1.24E+05 0.0179 1030 144 11.1 5L1724H23_6L3H3 2.23E+04 3.42E-04 1020 15 9.55E+04 7.89E-03 1500 83 5.5 5L1724H23_6L3 3.59 E+04 7.90E-04 910 22 7.54E+04 0.0407 1050 540 24.5 6L21H2335 5.81 E+04 7.33E-03 892 126 6.55E+05 2.38E-01 624 363 2.9 5L1724H23 - - 20 -I 2.03E+05 7.93 E-03 114 39 5L1724H35 - - 40 - - - 0 - Table 6. Anti-PCSK9 antibodies at ?11 7.4 and 5.5 binding affinity to mouse 1^1 (9 binding) msPCK9 pH 7.4 msPCK9 pH 5.5 huPSCKd pH 5.5/7.4 kon (1/Ms) Koff (1/s) Rmax (RU) KD (nM) kon (1/Ms) Koff (1/s) Rmax (RU) KD (nM) KD 6L1721 8.80E+04 2.28E-04 1260 2.6 3.38E+05 1.77E-03 1120 5.2 2.0 5A10_WT 1.01 E+05 4.85E- 04 1410 4.8 3.73E+05 1.94E-03 1050 5.2 1.1 5L1721H23-6L3H3 6.37E+04 5.16E-04 1630 8.1 1.60E+05 0.0187 1420 117 14.4 6L1721H23 6.48E+04 4.69E-04 1230 7.2 2.04E+05 7.29E-03 1220 36 5.0 5L1721H23_6L3 4.75E+04 676E-04 1600 14 1.05E+05 0.0428 1360 408 29.1 5L1721H23_6H3 1.13E+05 8.99E-04 615 8.0 3.07E+05 0.0341 460 111 13.9 5L1721H23 6.12E+04 1.41 E-03 1380 23 1.02E+05 0.0588 1100 576 25.0 5L1724H23_6L3H3 6.31 E+04 8.27E-04 1690 13 2.11 E+05 0.0263 1520 125 9.6 5L1724H23_6L3 5.31 E+04 2.18E-03 1440 41 1.60E+05 0.121 1100 756 18.4 6L21H2335 8.99E+04 1.72E-02 1340 191 5.26E+05 0.237 502 451 2.4 5L1724H23 - - 40 - 2.73E+05 2.02E-02 91 74 5L1724H35 4.43E+04 1.53E-03 164 35 - - 0 - PCSK9 antibody H1M300N body h5A10 and 5L1721H23_6L3H3 and PCSK9 anti-affinity and kinetic parameters (see US20 1 0/0 1 66768, -100-201206466 eg Table 7). All experiments were performed on a B iac 〇re 2000 biosensor with a 〇-human Fc sensing wafer with a 1: 1 (vol/vol) mixture of 400 mM EDC and 100 mM NHS. A flow cell of Biacore CM4 sensing wafer was activated at a flow rate of 10 microliters per minute for 7 minutes to prepare. Anti-human Fc reagent (goat F (AB〇2 fragment anti-human IgG Fc, Cappel catalog number 5 5 05 3 ) was diluted to 60 μg/ml with 10 mmol of sodium acetate pH 5.0, and 20 μl/min was injected into all flow cells for 7 minutes. All flow cells were blocked with 1 mM of ethylenediamine in 150 mM borate buffer pH 8.5 for 7 minutes at 10 μl/min. The analytical analysis was performed using a kinetic titration method as described by Karlsson et al., Anal. Biochem 349, 136-147 (2006). The same antibody (eg 5L1721H23_6L3H3) was captured to a downstream flow cell at 2 μg/ml (flow cell) 2, 3, and 4), capturing 30, 60, and 120 seconds at flow rates of 10 μl/min to flow cells 2, 3, and 4. Flow cell 1 is used as a reference surface. PCSK 9 was injected into all flow cells at a low to high concentration of injection series at 30 μl/min. The highest concentration was 200 Nemo PCSK9 and the dilution factor was 3 times. Each PCSK9 injection was 2 minutes at 200 N. The dissociation time after Moss PCSK9 injection was 20 minutes. Replace PC SK9 with flow buffer. A similar injection step is used for dual reference purposes (double reference system as described by Myszka, J. Mol. Recognit 12, 2 79-284, 1 999). After each analysis cycle, all flow cells utilize three 30 seconds. Injection of 75 mM phosphate was initiated. The induction of a given PCSK9/antibody pair from flow cells 2 and 3 was completed with a complete band of -101 - 201206466 into a mass transfer binding model of 1:1 Langmuir. The strains were applied at pH 6.0 and 7.4, respectively, using a sample and 10 mM sodium phosphate, 15 mM milliliter sodium chloride, 〇〇 5 % Tween-2 0, pH 6 and 10 mM phosphate. Sodium, 15 mM millimolar sodium chloride, 0.05% Tween-20, pH 7.4 flow buffer. Table 7. pH-dependent binding antibody PH6.0 (sodium phosphate) sodium sulphate 値K〇, kd, pH 6/pH 6/ kd, pH 7.4 5A10 WT MM·,1) kd(s1) U/2 (min) < Κ〇(PM) ka (M-1s1) kd(s1) ^1/2 ( Min) Kd (PM) K〇, pH 7.4 3.31 E+05 <6Ε·5 193 181 2.41 E+05 < 6E-05 < 193 249 5L1721H 23 BL3H3 3.07E+05 2.86Ε-03 4.0 9316 2.42E +05 1.74E-04 66.4 719 13.0 16.4 H1M300N 4.27E+05 3.21 -04 36.0 752 1.67E+05 6.92E-05 166.9 414 1.8 4.6 Example 3: PH-dependent PC SK9-binding antibody has a prolonged pharmacodynamic effect on cholesterol lowering A. pH-dependent PCSK9 binding antagonist antibody Lowering serum cholesterol in mice over a longer period of time To determine whether pH-dependent PCSK9 binding antagonist antibodies can reduce the amount of cholesterol in vivo over a longer period of time than non-pH dependent antibodies, when acid sensitive antibodies 5L1721H23_6H3 and 5L1721H23_6L3H3 When the non-acid-sensitive antibody h5A10 (10 mg/kg only) and 5A10.B8 were injected into mice at 1, 3 or 10 mg/kg, their time effects on serum cholesterol were examined. All four antibodies had similar binding affinities of 5 to 14 nanomolar to mouse PCSK9 at neutral pH (pH 7.4). Antibody 51^172 11123_61^3 and 51^172 11^23_61^31^ under 1^5.5 respectively -102- 201206466 has a lower affinity of 奈7 Nemo and 40 8 Nemo, however h5Al 0 and 5Α10 Β8 has a KD (5.2 nmer) similar to pH 7.4 at pH 5.5. Male rats C57/bl6 (6 to 7 weeks old) were housed in a 12-hour light/dark cycle, and blood was collected on day 7 to collect approximately 70 microliters of serum. Antagonistic PCSK9 antibodies and control isoforms that do not bind to any known mammalian proteins are individually administered to C57/bl6 male mice at 7 weeks of age, 5, 12, 19, 26, 61 and 75 after injection. Serum samples were collected on days. Analysis of total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol in all serum samples by Ace Alera Instruments (Alfa Wassermann, West Caldwell, New Jersey) Fried ewald formula calculation. Figure 7 shows a rapid and dose-dependent reduction in total cholesterol after injection of PCS K9 antagonist antibody. The amount of LDL cholesterol in mice is too low to be reliably measured and calculated. At the dose of 10 mg/kg, all four antibodies were reduced by 35 to 40% of HDL-cholesterol on days 5 and 12, whereas animals injected with 5L1721H23_6H3 and 5L1721H23_6L3H3 did not return to baseline until day 61, and h 5 A was injected. The animals of 1 0 and 5 A 1 0 . B 8 returned to the baseline amount on the 2nd and 6th day respectively. B. pH-dependent PCSK9-binding antibody has an extended half-life in mice The serum concentration of the antibody was determined in the same assay as described in Example b to determine whether the pH-sensitive anti-PC SK9 antibody resulted in a longer antibody half-life. Normal anti-103-201206466 PCSK9 antibodies such as h5A10 and 5A10.B6 have a shorter dose-dependent half-life due to the degradation of the PCSK9 mediator antibody/antigen complex compared to antibodies that bind to other soluble antigens. As shown in Figure 8A, the PH-dependent binding properties reduced antibody degradation and prolonged the half-life of anti-PCSK9 antibodies 5L1721H23_6H3 and 5L1721H23_6L3H3. To further confirm that the prolonged PK lines of 5L1721H23_6H3 and 5L1721H23_6L3H3 were reduced by PCSK9 vector clearance antibody, a similar time-scale test was performed in PCSK9 knockout mice. There was no significant difference between the PH sensitivity of the mouse and the serum antibody concentration and rate of decrease of the non-sensitive antibody until 3 mg/kg of PC SK9 was injected into the mouse. After injection, the non-PH-sensitive PCSK9 antibody showed increased degradation compared to the pH-sensitive antibody and the negative control antibody (Fig. 8B). These results indicate that the observed degradation of the pH-dependent PCSK9-binding PCSK9 antibody was caused by the dissociation of the antibody from PCS K9, thereby preventing PCSK9 vector degradation of the antibody. C· pH-sensitive PCSK9 antagonist antibody reduces serum cholesterol in monkeys over a longer period of time. Figure 9B shows serum-sensitive LDL of anti-PCSK9 antagonist antibodies 5L1721H23_6H3 and 5L1721H23_6L3H3 and non-sensitive anti-PCSK9 antibody L1L3 against cynomolgus. - The effect of the amount of cholesterol (% of the control group). Anti-body (1.5 mg/kg each) was administered to the mother stone crab macaques by intravenous bolus injection on the third day. LDL-cholesterol in all three antibody treatment groups was reduced to 50% of the baseline in one day. Although the amount of LDL-cholesterol on the first day after the administration of the non-p-sensitive antibody was restored to the baseline amount, the LDL cholesterol of the monkey treated with the sensitizing antibody was maintained until the 21st day. The amount of HDL remained essentially unchanged after antibody treatment (Fig. 9A). Figure 10 shows that the half-life of the pH sensitive anti-PCSK9 antibody is longer than that of the non-pH sensitive L 1 L 3 . Example 4: A conventional model computer model of a pH-dependent antigen-binding antibody is used to predict whether an antibody that binds to a universal antigen in a P-dependent antigen can affect the half-life of the antibody and/or reduce the amount of antigen or serum. The period of concentration. To achieve the goal of this model, the following hypotheses were made: 1) antibodies, blood containing 1 micromolar dose of antibody, half-life of antibody for 21 days; 2) 1 day of simulation; antibody binding and pH-dependent binding , Κ〇η = le5/M/s, at neutral pH Koff = KD * Κοη : The pH-dependent binding force is K in the acidic inner body. ^ Increased to the model; Kuf = R * neutral pH KQff at acidic pH. Figure 158 details the pH-dependent binding of the antibody to the model to model the crossing model. The formula for defining the model is as follows: d/dt (Normal-Dose) = - k-dist*Normal-Dose d/dt (mAb) = k-dist*Normal-Dose - (mAb*kon*Normal-antigen) ten (kon*KD* mAb-antigen} - k_blood_endo*mAb/ VNorm + fl* k-blood-endo*mAb-e/VNonn+ ( 1-fl)* f2*k_blood_endo*mAb_le/ VNorm " — d/dt(mAb_antigen) = (mAb*kon*Normal-antigen) - (kon*KD* mAh an antigen}-k_blood—endo*mAb—antigen/ VNorm + fl* k-blood_endo*mAb-antigen-e/VNorm + (1-fl) * f2*k__blood_endo*cmplx__le/ VNorni d/dt (Normal-antigen) =- (mAb*kon*Normal-antigen) + ( Kon*KD* mAb-antigen} + In (2}/antigen-halflife*antigen-level -

In (2) /antigen_halflife*Normal_antigen - k_blood_endo*Normal_antigen/ VNorm + fl* k__blood一endo*Endosomes一antigen一e/ VNorm d/dt (mAh一e) = k_jDlood__endo*mAb / VEndo - fl*k__blood_endo*mAb__e / VEndo -(mAb一e*kon*Endosomes一antigen一e) + ( kon*KD* mAb_antigen一e> - (1-fl) 一blood一endo *mAb一e/ VEnd0 -105- 201206466 d/dt (mAb一antigen一e> = k」Dlood一endo*mAb一antigen / VEndo - fl*k_blood_endo*mAb_antTgen_e / VEndo + <mAb一e*kon*Endosomes一antigen_e>-(kor^KD* idSd一antigen_e) - (1-fl) *k_blood—endo *mAb一antigen一e/ VEndo d/dt (Endosomes一antigen—e} = Jc一blood—endo*Normal一antigen/ VEnd0 -fl*k一blood_endo*Endosomes一antigen_e / VEndo - (mAh—e*Jcon*Endosomes一antigen_e> + (Jcon*KD* mAh一antigen一θ) - (1-fl) blood—endo *Endosomes_antigen一e / VEnd〇 d/dt (mAb_le) = - (1-fl) * f2*k一blood—endo* mAh—le/ Vlei^o + (1-fl) *k一blood一endo *mAb一θ/ VLEnd。- (mAb_le*kon*Antigen一 1θ> + (R*kon*KD* cplx__le) -Tl-fl)^(l-f2) *k_biood_endo*mAb_ie/ VLEndo d/dt <cplx一le> = - (1-fl) * f2*k一blood一endo*cplx一le/ 〜沾。+ (1-fl) *k_blood—endo *mAb__antigen_e/ + (mAb__le*kon*Antigen_le) - (R*kon*KD* cplx_le>—- (1-fl) * <T-f2> *kJ^lood一endo*cplx:le/ 〜咖一 d/dt(Antigen一le> =+ (1-fl) *k—blood一endo *Endosomes_antigen—e/ - (mAb__le*kon*Antigen_le) + (R*kon*KD* cplx_le) - (1-fl) ★k—blood endo*Antigen—le/ Vuend。此模型所使用之參數係於下表8中說明。表8 VNorm 5.6公升血液體積 VEndo 0.06公升早期內小體之體積 VLEndo 0.06公升晚期內小體之體積 kdist 0.48/无運輸至血液中之速率 kon 0.0001/ 0 莫聊單株抗體-抗原結合速率 k_blood_endo 11.5公升/ 天內小體內化之速率 fl 0.7 自早期內小體回收之組分 f2 0.95 自晚期內小體回收之單株抗體組分 KD 可變結合親和性[奈莫耳] R 可變內小體與中性pH之親和性之比抗原半衰期血液抗原之半衰期[天數] 抗原濃度可變血液抗原之量[奈莫耳] 圖4之熱圖顯示呈pH依賴性結合之抗體對抗原驟降之影響超過不呈pH依賴性結合之抗體之影響。此驟降之量 -106- 201206466 係藉由測量在1 〇〇天模擬期間該二種抗體之游離抗原曲線之間的面積加以決定。這可被想成是呈P H依賴性結合之抗體影響抗原驟降之天數超過不呈pH依賴性結合之抗體之驟降多少天，雖然這二項指標不一定完全相關。在圖上較淡之區域反應較長之時間及/或較多之游離抗原之減少。圖11之熱圖顯示若該抗原爲DKK1、IgE或C5時，呈pH 依賴性抗原結合之潛在抗體如何影響抗原驟降。以各種抗原而言，呈pH依賴性結合之抗體可延長抗原之驟降。圖12顯示對IgE呈pH依賴性結合之抗體之更詳細模型。延長IgE驟降時間發生在具有約1奈莫耳或更高之KD及在 pH 6.0/pH 7.4下之KD比（R)爲3或更高之抗體。對抗原DKK1之類似分析顯示於圖13，C5之分析顯示於圖14。以DKK1而言，KD之理想範圍係在3至30之KD比範圍內之1.0至100奈莫耳。以C5而言，KD之理想範圍亦爲在3至30之KD比範圍內之1.0至100奈莫耳。實施例5 :製備及篩選呈pH依賴性IgE結合之抗IgE抗體在抗IgE抗體5.948-H100Y之畫底線殘基之位置進行組胺酸取代，該抗體具有下列可變輕鏈（VL )及可變重鏈 (VH)胺基酸序列（CDR爲粗體字型）。 -107- 201206466In (2) /antigen_halflife*Normal_antigen - k_blood_endo*Normal_antigen/ VNorm + fl* k__blood-endo*Endosomes-antigen-e/VNorm d/dt (mAh-e) = k_jDlood__endo*mAb / VEndo - fl*k__blood_endo*mAb__e / VEndo -(mAb-e*kon*Endosomes-antigen one e) + ( kon*KD* mAb_antigen one e> - (1-fl) one blood one endo *mAb one e/VEnd0 -105- 201206466 d/dt (mAb one Antigen one e> = k"Dlood-endo*mAb-antigen / VEndo - fl*k_blood_endo*mAb_antTgen_e / VEndo + <mAb-e*kon*Endosomes-antigen_e>-(kor^KD* idSd-antigen_e) - (1 -fl) *k_blood-endo *mAb-antigen-e/VEndo d/dt (Endosomes-antigen-e} = Jc-blood-endo*Normal-antigen/VEnd0-fl*k-blood_endo*Endosomes-antigen_e / VEndo - (mAh-e*Jcon*Endosomes-antigen_e> + (Jcon*KD* mAh-antigen one θ) - (1-fl) blood-endo *Endosomes_antigen-e / VEnd〇d/dt (mAb_le) = - (1- Fl) * f2*k-blood-endo* mAh-le/ Vlei^o + (1-fl) *k-blood-endo *mAb-θ/ VLEnd.- (mAb_le*kon*Antigen-1θ> + (R *kon*KD* cplx__le) -Tl-fl)^(l-f2 *k_biood_endo*mAb_ie/ VLEndo d/dt <cplx-le> = - (1-fl) * f2*k a blood-endo*cplx-le/~ dip. + (1-fl) *k_blood-endo *mAb__antigen_e/ + (mAb__le*kon*Antigen_le) - (R*kon*KD* cplx_le>-- (1-fl) * <T-f2> *kJ^lood one Endo*cplx:le/ ~ 咖一d/dt(Antigen一le> =+ (1-fl) *k-blood-endo *Endosomes_antigen-e/ - (mAb__le*kon*Antigen_le) + (R*kon*KD * cplx_le) - (1-fl) ★k-blood endo*Antigen-le/ Vuend The parameters used in this model are described in Table 8. Table 8 VNorm 5.6 liters blood volume VEndo 0.06 liter early corpuscle Volume VLEndo 0.06 liter late volume inside the body kdist 0.48 / no transport to the blood rate kon 0.0001/ 0 Mo chat single antibody - antigen binding rate k_blood_endo 11.5 liters / day small body rate fl 0.7 from the early small Component recovered by body f2 0.95 Single antibody component recovered from late endosome KD variable binding affinity [Nemo] R The ratio of variable endosomes to neutral pH Affinity Half-life Blood antigen Half-life [days] antigen concentration variable blood antigen amount [Nemo] Figure 4 heat map showing pH-dependent binding of antibody to antigen The effect of the drop was greater than the effect of antibodies that were not pH-dependently bound. The amount of this drop -106-201206466 was determined by measuring the area between the free antigen profiles of the two antibodies during the 1 day simulation. This can be thought of as the number of days in which the PH-dependent antibody affects the sudden drop in antigen over the number of non-pH-dependent antibodies, although these two indicators are not necessarily fully correlated. The longer the regional response and/or the greater reduction in free antigen. The heat map of Figure 11 shows how potential antibodies that are pH-dependent antigen binding affect antigenic dips if the antigen is DKK1, IgE or C5. For various antigens, antibodies that bind in a pH-dependent manner can prolong the dip of the antigen. Figure 12 shows a more detailed model of the antibody that binds pH-dependently to IgE. The extended IgE sag occurs at about 1 nm or Higher KD and antibodies with a KD ratio (R) of 3 or higher at pH 6.0/pH 7.4. A similar analysis of the antigen DKK1 is shown in Figure 13, and an analysis of C5 is shown in Figure 14. In the case of DKK1, the ideal range of KD is from 1.0 to 100 nanomolar in the range of KD of 3 to 30. In the case of C5, the ideal range of KD is also 1.0 to 100 nanomolar in the KD ratio range of 3 to 30. Example 5: Preparation and screening of pH-dependent IgE-conjugated anti-IgE antibody Histamine substitution at the position of the anti-IgE antibody 5.948-H100Y bottom line residue, the antibody having the following variable light chain (VL) and A heavy chain (VH) amino acid sequence (CDR is in bold). -107- 201206466

VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHRNGYNYLDWYLQKPGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPATFG GGTKVEIK (SEQ ID NO: 25)VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHRNGYNYLDWYLQKPGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPATFG GGTKVEIK (SEQ ID NO: 25)

VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWM GWMDPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGYY DSDGYYSFSGMDVWGQGTTVTVSS (SEQ ID NO: 26) 該等抗體係於25° C下測試與IgE之結合。在親和性及動力學常數之測定方面，所有實驗皆在Biacore T2 00生物感測器上進行。藉由固定中性抗生物素蛋白製備生物素捕捉表面： Biacore CM4感測晶片之所有流動池係藉由400毫莫耳EDC 及100毫莫耳NHS之1 : 1 (體積/體積）混合物以10微升/分鐘之流動速率活化7分鐘。中性抗生物素蛋白（皮爾斯（ Pierce)公司，產品編號3 1 000 )係經10毫莫耳醋酸鈉pH 4.5稀釋至100微克/毫升，並以20微升/分鐘注射至所有流動池7分鐘。所有流動池係以1〇〇毫莫耳之乙二胺於150毫莫耳硼酸鹽緩衝液pH 8.5以10微升/分鐘封閉7分鐘。VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWM GWMDPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGYY DSDGYYSFSGMDVWGQGTTVTVSS (SEQ ID NO: 26) These anti-systems were tested for binding to IgE at 25 °C. All experiments were performed on a Biacore T2 00 biosensor in terms of affinity and kinetic constant determination. Preparation of Biotin Capture Surface by Immobilization of Neutral Avidin: All flow cells of the Biacore CM4 Sensing Wafer are 10: 1 (vol/vol) mixture of 400 millimolar EDC and 100 millimolar NHS. The flow rate of microliters per minute was activated for 7 minutes. Neutral avidin (Pierce, product number 3 1 000) was diluted to 100 μg/ml with 10 mmol of sodium acetate pH 4.5 and injected into all flow cells for 7 min at 20 μl/min. . All flow cells were blocked with 1 mM of ethylenediamine in 150 mM borate buffer pH 8.5 for 7 minutes at 10 μl/min.

IgE建構物（人IgE-FC(Ch2-Ch3-Ch4)，在實驗室內部表現及純化）係藉由將IgE與等莫耳量之EZ-Link NHS-LC-LC-生物素（賽默科技（Thermo Scientific)公司，目錄編號2 1 343 )於室溫下培養30分鐘加以最小生物素基化。游離生物素經由緩衝液交換移除。所有Biacore實驗係利用感測晶片之四個流動池當中平行的二個（1及2或3及4 )進行。下游之流動池被用來作 -108- 201206466 爲分析流動池，另一流動池則被當作參照表面。因此，經生物素基化之IgE ( B-IgE )被捕捉於分析流動池，每個經純化之F ab片段被注射至二池。來自參照流動池之感應圖係自分析流動池之感應圖減去。 B -1 g E係在即將捕捉之前以流動緩衝液稀釋至終濃度5 微克/毫升。在pH 6.0下，B-IgE係以1〇微升/分鐘注射60秒。在pH 7.4下’ B-IgE係經注射80秒以獲得類似之捕捉量。動力學分析係利用如 Karlsson et al., Anal. Biochem 349 :136-147, 200 6所描述之動力學滴定方法進行。在捕捉B -1 g E之後’經純化之F ab片段被以低至高濃度之注射液系列經3 0微升/分鐘注射至分析及參照流動池》 Fab之最高濃度從30奈莫耳至200奈莫耳，稀釋因子爲3倍。每次Fab稀釋注射2分鐘’在最後一次注射後爲3〇分鐘之最終解離時間。以流動緩衝液取代F a b進行類似之注射步驟以達雙重參照之目的（雙重參照係如Myszka，j Mol Recognit 12: 279-284，1999所述）。該雙重參照之感應圖被完整帶入單純1: 1蘭綴爾（Langmuir)之質量傳輸結合模型。适些貫驗係於p Η 6 · 0及7 · 4下進彳了’分別使用樣本及 10毫莫耳碟酸鈉、150毫莫耳氯化鈉、〇.〇5 %吐溫（Tween )-20、1毫克/毫升BSA、pH 6及10毫莫耳磷酸鈉' 150毫莫耳氯化鈉、0.05%吐溫- 20、1毫克/毫升bsa、pH 7.4之流動緩衝液。如下表9所示，在IgE抗體5.948-H100Y中之組胺酸取 -109- 201206466 代成功地導致具有在？116.0下較快之1^^及在？116.〇41^ 7.4時較高之KD及k。^比之抗體。表9.組胺酸取代對IgE抗體之抗原結合的效應 pH 6.0 pH 7.4 ka(M-1S-1) kd (s· 1) kd (min) KD (M) ka(M-1S· 1) kd (s-1) kd (min) KD fM) kd· pH6/ kd· PH7.4 KD ratio pH 6.0/pH 7.4 H100Y 1.02E+06 1.52E- 04 76.00 1.49E- 10 1.03E+06 5.01E- 05 230.5879 4.87E- 11 3.03 3.1 L38H312H100Y 3.29E+05 1.22E- 02 0.95 3.71E- 08 2.02E+05 1.08E- 03 10.69672 5.33E- 09 11.30 7.0 H25H100Y 2.98E+05 1.51E- 03 7.65 5.08E- 09 3.11E+05 1.97E- 04 58.64189 6.34H- 10 7.66 8.0 H17H25H100Y 2.23E+05 3.64E- 03 3.17 1.63E- 08 2.22E+05 4.22E- 04 27.37548 1.90E- 09 8.63 8.6 此處引述之所有參考文獻之揭示藉此以參照方式納入此處。【圖式簡單說明】圖1顯示不同時間之抗體濃度增加爲KD比之函數。圖2顯示不同時間之游離配體（抗原）減少爲KD比之函數。圖3顯示k。^及1^η改變對不同時間之抗體濃度之影響〇圖4之熱圖顯示呈pH依賴性結合之抗體降低血清抗原濃度之天數多出多少天，抗原血清濃度之降低以Kd(R) 、抗原之血清半衰期及抗原之血清濃度爲函數。R相當於在內小體P Η比生理性P Η下之K D比。圖5證實該pH依賴性抗體模型之預測性。該模型成功地預測5 A 1 0之總抗體濃度（圖5 A )。圖5 B顯示5 A 1 〇對 -110- 201206466 LDL之時間效應。圖6亦證實該pH依賴性抗體模型之預測性。該模型成功地預測5L1721H23_6L3H3(6L3H3)之總抗體濃度（圖 6A )。圖6B顯示6L3H3對LDL之時間變化效應。相較於 5A10，該pH依賴性結合抗體6L3H3延長其中LDL被降低之期間。圖7顯示投予各種PCSK9抗體對總膽固醇效應之時間量程。圖7A顯示5A10對總膽固醇之劑量依賴性效應。圖 7B顯示pH依賴性抗體5L1721H23__6H3之劑量依賴效應。此P Η依賴性抗體之效應相較於5 A 1 0之效應持久。圖8顯示呈p Η依賴性結合之抗體5 L 1 7 2 1 Η 2 3 _6 Η 3及 5L172 1H2 3_6L3H3相較於不呈pH依賴性結合之抗體具有減少之抗體降解及延長之半衰期。圖8B之圖顯示，圖8A顯示之效應係因標的媒介性降解所致。在PCS K9基因剔除鼠體內之抗體降解在注射PCSK9後顯著增加。圖9之圖顯示pH敏感性PCSK9拮抗抗體及非pH敏感性 PCSK9拮抗抗體對猴膽固醇量之效應。雖然未檢測到HDL 量之顯著變化（圖9A )，該pH敏感性抗體相較於非PH依賴性抗體L1L3媒介較長時間之LDL量之減少。圖10顯示呈pH依賴性結合之PCSK9抗體相較於非PH 依賴性抗體具有延長之活體內半衰期。圖1 1之熱圖顯示pH依賴性結合之常規模型。相較於不呈pH依賴性結合之抗體，該等以抗原DKK1、IgE或C5 爲標的之呈pH依賴性結合之抗體可顯著增加該抗原經歷 -111 - 201206466 減少量之天數。圖12顯示在投予以抗原IgE爲標的之呈pH依賴性結合之抗體後，IgE抗原濃度之時間量程模型。圖13顯示在投予以抗原DKK1爲標的之呈pH依賴性結合之抗體後，DKK1抗原濃度之時間量程模型。圖14顯示在投予以抗原C5爲標的之呈pH依賴性結合之抗體後，C 5抗原濃度之時間量程模型。圖1 5詳細說明用於建構模型之呈PH依賴性結合之抗體的穿越模型。 -112- 201206466 序列表 863461 <110>禮納特神經系統科學公司輝瑞股份有限公司 <12〇>具有pH依賴性抗原結合力之抗體類 <i4〇> TW 100108288 <Hi> 2011-03-11 <150> US 61/313,102 <151> 2010-03-11 <150> US 61/447,638 <151> 2011-02-28 <160> 27 <170> Patentln version 3.5 <210> 1 <211> 107 <212> PRT <213>智人 <400〉 1The IgE construct (human IgE-FC (Ch2-Ch3-Ch4), expressed and purified in the laboratory) is based on IgE and ML-Link NHS-LC-LC-Biotin (Momo Technology) (Thermo Scientific), catalog number 2 1 343) was incubated for 30 minutes at room temperature for minimal biotinylation. Free biotin is removed via buffer exchange. All Biacore experiments were performed using two parallel (1 and 2 or 3 and 4) of the four flow cells of the sensing wafer. The downstream flow cell was used to analyze the flow cell from -108 to 201206466 and the other flow cell was used as the reference surface. Thus, biotinylated IgE (B-IgE) was captured in the analytical flow cell and each purified Fra panel was injected into the second cell. The sensing map from the reference flow cell is subtracted from the sensing map of the analytical flow cell. The B -1 g E line was diluted with flow buffer to a final concentration of 5 μg/ml just prior to capture. At pH 6.0, B-IgE was injected at 1 μL/min for 60 seconds. At pH 7.4, 'B-IgE was injected for 80 seconds to obtain a similar capture amount. The kinetic analysis was carried out using a kinetic titration method as described by Karlsson et al., Anal. Biochem 349: 136-147, 200 6 . After capture of B -1 g E 'purified F ab fragment was injected into the analysis and reference flow cell at a low to high concentration of the injection series through 30 μl / min. The highest concentration of Fab from 30 nmer to 200 Nemo, the dilution factor is 3 times. Each Fab dilution injection was 2 minutes' after the last injection was the final dissociation time of 3 minutes. A similar injection step was carried out by substituting a flow buffer for F a b for dual reference purposes (double reference system as described by Myszka, j Mol Recognit 12: 279-284, 1999). The double-referenced sensor map was completely brought into a pure mass transfer model of 1:1 Langmuir. Appropriately tested in p Η 6 · 0 and 7 · 4 into the 'different use of sample and 10 millimolar sodium sodium, 150 millimoles sodium chloride, 〇. 〇 5% Tween (Tween) -20, 1 mg/ml BSA, pH 6 and 10 millimolar sodium phosphate '150 millimoles sodium chloride, 0.05% Tween-20, 1 mg/ml bsa, pH 7.4 flow buffer. As shown in Table 9 below, the histidine acid in the IgE antibody 5.948-H100Y was successfully produced in the -109-201206466 generation. 1^^ and faster in 116.0? 116. 〇41^ 7.4 higher KD and k. ^ Compared to antibodies. Table 9. Effect of histidine substitution on antigen binding of IgE antibodies pH 6.0 pH 7.4 ka(M-1S-1) kd (s· 1) kd (min) KD (M) ka(M-1S· 1) kd (s-1) kd (min) KD fM) kd·pH6/ kd· PH7.4 KD ratio pH 6.0/pH 7.4 H100Y 1.02E+06 1.52E- 04 76.00 1.49E- 10 1.03E+06 5.01E- 05 230.5879 4.87E- 11 3.03 3.1 L38H312H100Y 3.29E+05 1.22E- 02 0.95 3.71E- 08 2.02E+05 1.08E- 03 10.69672 5.33E- 09 11.30 7.0 H25H100Y 2.98E+05 1.51E- 03 7.65 5.08E- 09 3.11E+05 1.97E- 04 58.64189 6.34H- 10 7.66 8.0 H17H25H100Y 2.23E+05 3.64E- 03 3.17 1.63E- 08 2.22E+05 4.22E- 04 27.37548 1.90E- 09 8.63 8.6 All references cited here The disclosure of the document is hereby incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the increase in antibody concentration at different times as a function of the KD ratio. Figure 2 shows the reduction in free ligand (antigen) as a function of KD ratio at different times. Figure 3 shows k. The effect of ^ and 1^η changes on antibody concentration at different times. The heat map of Figure 4 shows how many days after the pH-dependent binding of the antibody reduces the serum antigen concentration, and the antigen serum concentration decreases by Kd(R), The serum half-life of the antigen and the serum concentration of the antigen are a function. R is equivalent to the K D ratio of the inner body P Η to the physiological P Η. Figure 5 demonstrates the predictability of this pH dependent antibody model. This model successfully predicted the total antibody concentration of 5 A 10 (Fig. 5 A ). Figure 5B shows the time effect of 5 A 1 〇 versus -110- 201206466 LDL. Figure 6 also demonstrates the predictability of this pH dependent antibody model. This model successfully predicted the total antibody concentration of 5L1721H23_6L3H3 (6L3H3) (Fig. 6A). Figure 6B shows the time-varying effect of 6L3H3 on LDL. This pH-dependent binding antibody 6L3H3 prolongs the period in which LDL is lowered compared to 5A10. Figure 7 shows the time scale of the effect of various PCSK9 antibodies on total cholesterol. Figure 7A shows the dose dependent effect of 5A10 on total cholesterol. Figure 7B shows the dose dependent effect of the pH dependent antibody 5L1721H23__6H3. The effect of this P Η dependent antibody is longer than the effect of 5 A 10 . Figure 8 shows that antibodies 5 p 1 7 2 1 Η 2 3 _6 Η 3 and 5L172 1H2 3_6L3H3 in a p Η-dependent binding have reduced antibody degradation and prolonged half-life compared to antibodies that are not pH-dependently bound. Figure 8B is a graph showing that the effect shown in Figure 8A is due to the subjective degradation of the label. Antibody degradation in PCS K9 knockout mice was significantly increased after injection of PCSK9. Figure 9 is a graph showing the effect of pH-sensitive PCSK9 antagonistic antibodies and non-pH sensitive PCSK9 antagonist antibodies on monkey cholesterol levels. Although no significant change in the amount of HDL was detected (Fig. 9A), the pH-sensitive antibody was reduced in LDL amount over a longer period of time than the non-PH-dependent antibody L1L3 medium. Figure 10 shows that PCSK9 antibodies that bind in a pH-dependent manner have an extended in vivo half-life compared to non-PH-dependent antibodies. The heat map of Figure 11 shows a conventional model of pH dependent binding. Such pH-dependent binding of antibodies to the antigen DKK1, IgE or C5 significantly increased the number of days the antigen experienced a decrease of -111 - 201206466 compared to antibodies that were not pH-dependently bound. Figure 12 shows a time-scale model of IgE antigen concentration after administration of antibody with pH-dependent binding of antigen IgE as the target. Figure 13 shows a time-scale model of DKK1 antigen concentration after administration of a pH-dependent antibody to the antigen DKK1. Figure 14 shows a time-scale model of C5 antigen concentration after administration of antibody to pH-dependent binding of antigen C5. Figure 15 illustrates in detail the traversing model of the antibody for PH-dependent binding used to construct the model. -112- 201206466 Sequence Listing 863461 <110> Renaissance Neuroscience Scientific Corporation Pfizer Inc. <12〇> Antibody with pH-dependent antigen binding force<i4〇> TW 100108288 <Hi> 2011-03-11 <150> US 61/313,102 <151> 2010-03-11 <150> US 61/447,638 <151> 2011-02-28 <160> 27 <170> Patentln version 3.5 <210> 1 <211> 107 <212> PRT <213> Homo sapiens <400>

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

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

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

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro 65 70 75 80Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr lie Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp He Ala Thr Tyr Tyr Cys Gin Gin Arg Tyr Ser Thr Pro Arg 85 90 95Glu Asp He Ala Thr Tyr Tyr Cys Gin Gin Arg Tyr Ser Thr Pro Arg 85 90 95

Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 <210> 2 <211> 118 <212> PRT <213>智人 <400> 2Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 <210> 2 <211> 118 <212> PRT <213> Homo sapiens <400> 2

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

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

Tyr Met His 丁rp Val Arg Gin Ala Pro Gly Gin Gly Leu G]u Trp Met 35 40 45Tyr Met His Ding rp Val Arg Gin Ala Pro Gly Gin Gly Leu G]u Trp Met 35 40 45

Gly Glu lie Asn Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 201206466Gly Glu lie Asn Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 201206466

Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110

Thr Val Thr Va】Ser Ser 115 <210> 3 <211> 107 <212> PRT <213>智人 <400> 3Thr Val Thr Va] Ser Ser 115 <210> 3 <211> 107 <212> PRT <213> Homo sapiens <400> 3

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

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

Tyr His Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr His Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Arg Tyr Ser Leu Trp Arg 85 90 95Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Arg Tyr Ser Leu Trp Arg 85 90 95

Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 <210> 4 <213> ]18 <212> PRT <213>智人 <400> 4Thr Phe Gly Gin Gly Gly Thr Lys Leu Glu lie Lys 100 105 <210> 4 <213> ]18 <212> PRT <213> Homo sapiens <400>

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

Tyr Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45Tyr Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45

Gly Glu He His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 -2- 201206466Gly Glu He His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 -2- 201206466

Lys Ser Arg Val Thr Met Thr Arg Asp Tlir Ser Thr Ser Thr Va] Tyr 65 70 75 80Lys Ser Arg Val Thr Met Thr Arg Asp Tlir Ser Thr Ser Thr Va] Tyr 65 70 75 80

Met GIu Leu Ser Ser Leu Arg Ser GIu Asp Thr A]a Val 丁yr Tyr Cys 85 90 95Met GIu Leu Ser Ser Leu Arg Ser GIu Asp Thr A]a Val Ding Ty Tyr Cys 85 90 95

Thr Val Thr Val Ser Ser 115Thr Val Thr Val Ser Ser 115

<210> 5 <211> 118 <212> PRT <2n>智人 <400> 5<210> 5 <211> 118 <212> PRT <2n> Homo sapiens <400> 5

Gly Glu lie His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60Gly Glu lie His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60

Ala Arg Glu Arg Pro Leu Tyr Ala Ser Asp Leu Trp Gly Gin Gly Thr loo 105 noAla Arg Glu Arg Pro Leu Tyr Ala Ser Asp Leu Trp Gly Gin Gly Thr loo 105 no

Thr Val Thr Val Ser Ser 115 <210〉 6 <211> ]〇 <212> PRT <213>智人 <400〉 6Thr Val Thr Val Ser Ser 115 <210> 6 <211> ]〇 <212> PRT <213> Homo sapiens <400> 6

Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His 1 5 10 <210> 7 <2Π> 17 <212> PRT <213>智人 <400〉 7Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His 1 5 10 <210> 7 <2Π> 17 <212> PRT <213> Homo sapiens <400> 7

Glu lie His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys -3- 201206466 15 1 5 )0Glu lie His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys -3- 201206466 15 1 5 )0

Ser <210> 8 <211> 9 <212> PRT <213>智人 <400> 8Ser <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8

Glu Arg Pro Leu Tyr Ala Met Asp Tyr <210〉 9 <211> 9 <212> PRT <213>智人 <400> 9Glu Arg Pro Leu Tyr Ala Met Asp Tyr <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400>

Glu Arg Pro Leu Tyr Ala Ser Asp Leu <210〉 10 <211> 11 <212> PRT <213>智人 <400> 10Glu Arg Pro Leu Tyr Ala Ser Asp Leu <210> 10 <211> 11 <212> PRT <213> Homo sapiens <400>

Lys Ala Ser Gin Asp Val His Thr Ala Val Ala 1 5 10 <210〉 11 <211> 7 <212> PRT <213>智人 <400> 11Lys Ala Ser Gin Asp Val His Thr Ala Val Ala 1 5 10 <210> 11 <211> 7 <212> PRT <213> Homo sapiens <400>

His Ala Ser Tyr Arg Tyr Thr <210> 12 <211> 9 <212> PRT <213>智人 <400> 12His Ala Ser Tyr Arg Tyr Thr <210> 12 <211> 9 <212> PRT <213> Homo sapiens <400>

Gin Gin Arg Tyr Ser Leu Trp Arg Thr 智 > > > > 0 12 3 22 2 2 <400> 13Gin Gin Arg Tyr Ser Leu Trp Arg Thr zhi >>>> 0 12 3 22 2 2 <400> 13

Lys Ala Ser Gin Asp Val Ser Thr Ala Val Ala 1 5 】0 <210> 14 <211> 11 -4- 201206466 <212> PRT <2I3>智人 <400> 14Lys Ala Ser Gin Asp Val Ser Thr Ala Val Ala 1 5 】0 <210> 14 <211> 11 -4- 201206466 <212> PRT <2I3> Homo sapiens <400>

Lys Ala Ser Gin Asp His Ser Thr Ala Val Ala 1 5 10Lys Ala Ser Gin Asp His Ser Thr Ala Val Ala 1 5 10

<210> 15 <211> 12 <212> PRT <213>智人 <400> 15<210> 15 <211> 12 <212> PRT <213> Homo sapiens <400>

Arg Ala Ser Gin Gly lie Ser Ser Ala Leu Ala 1 5 10Arg Ala Ser Gin Gly lie Ser Ser Ala Leu Ala 1 5 10

<210> 16 <2Π> Π <212> PRT 智人 <400> 16<210> 16 <2Π> Π <212> PRT Homo sapiens <400> 16

Arg Ala Ser Gin Gly lie His Ser Ala Leu Ala 1 5 10 <210> 17 <211> 7 <212> PRT <213>智人 <400> 17Arg Ala Ser Gin Gly lie His Ser Ala Leu Ala 1 5 10 <210> 17 <211> 7 <212> PRT <213> Homo sapiens <400>

Ser Ala Ser Tyr Arg Tyr Thr <210> IS <211> 7 <212> PRT <213>智人 <400> 18Ser Ala Ser Tyr Arg Tyr Thr <210> IS <211> 7 <212> PRT <213> Homo sapiens <400> 18

Ser Ala Ser His Arg Tyr Thr <210> 19 <211> 17 <212> PRT <213>智人 <400> 19Ser Ala Ser His Arg Tyr Thr <210> 19 <211> 17 <212> PRT <213> Homo sapiens <400>

Glu lie Asn Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15Glu lie Asn Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15

Ser <210> 20 <211> 17 <212> PRT <213>智人 <400> 20 -5- 201206466Ser <210> 20 <211> 17 <212> PRT <213> Homo sapiens <400> 20 -5- 201206466

Glu lie Ser Pro Phe Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 15 10 15Glu lie Ser Pro Phe Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 15 10 15

Ser <210> 21 <211> 9 <212> PRT <213>智人 <400> 21Ser <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21

Glu Arg Pro Leu His Ala Met Asp Tyr 1 5Glu Arg Pro Leu His Ala Met Asp Tyr 1 5

SUPRT智 ^ > > > 012 3 22ΛΖ2 < < V V <400> 22SUPRT智 ^ >>> 012 3 22ΛΖ2 << V V <400> 22

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 15 10 15 <210> 23 <211> 321 <212> DNA <213>智人 <400> 23 gatatccaga tgacacagtc cccatcctcc ctgtctgcct ctgtgggcga ccgcgtcacc 60 atcacctgca aggcctctca ggatgtgcat actgctgtag cctggtatca gcagaagcca 120 ggcaaagccc caaaactgct gatctaccat gcatcctacc gctacactgg tgtcccatca 180 cgcttcagtg gcagtggctc tggtacagat ttcaccttca ccattagcag cctgcaacca 240 gaagatattg ccacttatta ctgccagcaa cgttatagtc tgtggcgcac gttcggtcaa 300 ggcaccaagc tggagatcaa a 321 <210> 24 <211> 354 <212> DNA <213>智人 <400> 24 caggtgcagc tggtgcagtc tggtgctgag gtgaagaagc ctggcgcttc cgtgaaggtt 60 tcctgcaaag catctggtta cacctttacc agctactata tgcactgggt gcgccaagcc 120 cctggtcaag gcctggagtg gatgggcgag attcatccta gcggcggtcg tactaactac 180 aatgagaagt tcaagagccg cgtgactatg actcgcgata cctccaccag cactgtctac 240 atggaactga gctctctgcg ctctgaggac actgctgtgt attactgtgc ccgcgagcgc 300 cccctgtatg ctagcgacct gtggggccag ggtaccacgg tcaccgtctc ctca 354Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 15 10 15 <210> 23 <211> 321 <212> DNA <213> Homo sapiens <400> 23 gatatccaga tgacacagtc cccatcctcc ctgtctgcct ctgtgggcga ccgcgtcacc 60 atcacctgca aggcctctca ggatgtgcat actgctgtag cctggtatca gcagaagcca 120 ggcaaagccc caaaactgct gatctaccat gcatcctacc gctacactgg tgtcccatca 180 cgcttcagtg gcagtggctc tggtacagat ttcaccttca ccattagcag cctgcaacca 240 gaagatattg ccacttatta ctgccagcaa cgttatagtc tgtggcgcac gttcggtcaa 300 ggcaccaagc tggagatcaa a 321 < 210 > 24 < 211 > 354 < 212 > DNA < 213 > Homo sapiens < 400 > 24 caggtgcagc tggtgcagtc tggtgctgag gtgaagaagc ctggcgcttc cgtgaaggtt 60 tcctgcaaag catctggtta cacctttacc agctactata tgcactgggt gcgccaagcc 120 cctggtcaag gcctggagtg gatgggcgag attcatccta gcggcggtcg tactaactac 180 aatgagaagt tcaagagccg cgtgactatg actcgcgata cctccaccag cactgtctac 240 atggaactga gctctctgcg ctctgaggac actgctgtgt attactgtgc ccgcgagcgc 300 cccctgtatg ctagcgacct gtggggccag ggtaccacgg t Caccgtctc ctca 354

<210> 25 <2U> 113 <212> PRT <2】3>智人 <400〉 25 -6- 201206466<210> 25 <2U> 113 <212> PRT <2]3> Homo sapiens <400> 25 -6- 201206466

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

Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Arg 20 25 30Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Arg 20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45

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

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ala 85 90 95Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ala 85 90 95

Leu Gin Thr Pro Pro Ala Thr Phe Gly Gly Gly Thr Lys Val Glu He ]〇0 105 110Leu Gin Thr Pro Pro Ala Thr Phe Gly Gly Gly Thr Lys Val Glu He ]〇0 105 110

Lys <210〉 26 <211> 125 <212> PRT <2I3>智人 <400> 26Lys <210> 26 <211> 125 <212> PRT <2I3> Homo sapiens <400> 26

Ser Val Lys Val Ser Cys Lys Ala Ser Gly 丁yr Thr Phe Thr Ser Tyr 20 25 30Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ding yr Thr Phe Thr Ser Tyr 20 25 30

Asp lie Asn Trp Val Arg Gin Ala Thr Gly Gin Gly Leu Glu Trp Met 35 40 45Asp lie Asn Trp Val Arg Gin Ala Thr Gly Gin Gly Leu Glu Trp Met 35 40 45

Gly Trp Met Asp Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gin Lys Phe 50 55 60Gly Trp Met Asp Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gin Lys Phe 50 55 60

Gin Gly Arg Val Thr Met Thr Arg Asn Thr Ser lie Ser Thr Ala Tyr 65 70 75 80Gin Gly Arg Val Thr Met Thr Arg Asn Thr Ser lie Ser Thr Ala Tyr 65 70 75 80

Ala Arg Gly Tyr Tyr Asp Ser Asp Gly Tyr Tyr Ser Phe Ser Gly Met 100 105 110Ala Arg Gly Tyr Tyr Asp Ser Asp Gly Tyr Tyr Ser Phe Ser Gly Met 100 105 110

Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 27 <211> 9 <212> PRT <213>智人 -7- 201206466 <400> 27Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 27 <211> 9 <212> PRT <213> Homo sapiens -7- 201206466 <400>

Gin Gin Arg Tyr Ser Thr Pro Arg Thr -8- 201206466 863461 發明專刺說明書 (本申 ifW§式 '辦.> ※雪®Gin Gin Arg Tyr Ser Thr Pro Arg Thr -8- 201206466 863461 Invention specific instructions (this application ifW § 'do.> ※ ※ Snow®

※申請案號：100108288 ※申請曰：100年03月11曰※Application number: 100108288 ※Application曰: 100 years, March 11曰

一、發明名稱：（中文/英文）具有pH依賴性抗原結合力之抗體類 Antibodies with pH dependent antigen binding ^qq6. 1)I. Name of the invention: (Chinese/English) Antibodies with pH-dependent antigen binding. Antibodies with pH dependent antigen binding ^qq6. 1)

..l ij f · <' . : 一、〜，产 - * 〇 6~t\ ^ ^ " * ^ ,, V V V w - '；巧二、中文發明摘要： -- 本發明關於抗體類，該等抗體與彼之抗原呈PH依賴性結合’致使在生理性p Η (即p Η 7.4 )之抗原結合親和性高於在內小體pH (即ΡΗ 6.0或5.5 )之抗原結合親和性。換句話說，在 pH 5·5/ρΗ 7.4 或 pH 6.0/pH 7.4 之 KD 或 koff 比係大於或介於2、 3、 4、 8、 10、 16、 20、 30、 40或 100或 100以上。該等pH依賴性抗體優先地在內小體內與抗原解離。當抗原係經抗原媒介性廓清者（例如PC SK9 )時’相 Q 較於在pH 7.4具有相等KD値但無pH依賴性結合力之抗體，該等與抗原呈pH依賴性結合之抗體可具有增加之抗體半衰期。當與抗體（例如IL6 )結合之抗原的廓清減少時，呈PH依賴性結合之抗體可降低總抗原半衰期。呈pH依賴性結合之抗體亦可延長非與抗體結合之抗原的減少。此呈 pH依賴性結合之抗體對於拮抗通常呈高量存在之標的抗原（例如IgE、DKK1、C5及SOST)而言可能是重要的。此外’當抗原係受體（例如GMCSF受體）且該受體與抗體結合時之廓清增加時，該等抗體可增加抗原半衰期。 201206466 三、英文發明摘要：..l ij f · <' . : I, ~, production - * 〇6~t\ ^ ^ " * ^ ,, VVV w - '; Qiao Er, Chinese Abstract: - The present invention relates to antibodies The antibodies bind PH-dependently to their antigens such that the antigen binding affinity of the physiological p Η (ie p Η 7.4 ) is higher than the antigen binding affinity of the endosome pH (ie ΡΗ 6.0 or 5.5). . In other words, the KD or koff ratio at pH 5·5/ρΗ 7.4 or pH 6.0/pH 7.4 is greater than or between 2, 3, 4, 8, 10, 16, 20, 30, 40 or 100 or more. . These pH dependent antibodies preferentially dissociate from the antigen in the inner body. When the antigen is antigen-mediated (eg, PC SK9), the phase Q has an antibody that has an equivalent KD 値 but no pH-dependent binding at pH 7.4, and the antibody that binds to the antigen in a pH-dependent manner may have Increased antibody half-life. When the clearance of the antigen bound to the antibody (e.g., IL6) is reduced, antibodies that bind PH-dependently reduce the total antigen half-life. Antibodies that bind in a pH-dependent manner can also prolong the reduction of antigens that are not bound to the antibody. This pH-dependent binding of antibodies may be important to antagonize the target antigens (e.g., IgE, DKKl, C5, and SOST) that are typically present in high amounts. Furthermore, when an antigenic receptor (e.g., GMCSF receptor) and the receptor is increased in binding to an antibody, the antibodies increase the antigen half-life. 201206466 III. Abstract of English invention:

The present invention relates to antibodies with pH dependent binding to its antigen such that the affinity for antigen binding at physiological pH (i.e., pH 7.4) is greater than at endosomal pH (i.e., pH 6.0 or 5.5). In other words, the K〇 or k〇ff ratio at pH 5.5/ pH 7.4 or at pH 6.0/ pH 7.4 is more than, or ranges between, 2, 3, 4, 8, 10, 16, 20, 30, 40, or 100 or more. Such pH dependent antibodies preferentially dissociate from the antigen in the endosome. This can increase antibody half life, as compared to antibodies with equivalent K〇s at pH 7.4 but with no pH dependent binding, when the antigen is one that undergoes antigen-mediated clearance (e.g., PCSK9). Antibodies with pH dependent binding can decrease total antigen half life when the antigen undergoes reduced clearance when bound to antibody (e.g., IL6). Antibodies with pH dependent binding can also prolong the decrease in antigen which is not antibody-bound. This can be important when antagonizing a target antigen typically present at high levels (e.g., IgE, DKK1, C5 and SOST). In addition, such antibodies can increase antigen half life when the antigen is a receptor and the receptor has increased clearance when bound to antibody (e.g, GMCSF receptor).The present invention relates to antibodies with pH binding to its antigen such that the affinity for antigen binding at physiological pH (ie, pH 7.4) is greater than at endosomal pH (ie, pH 6.0 or 5.5). In other words, the K 〇or k〇ff ratio at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4 is more than, or ranges between, 2, 3, 4, 8, 10, 16, 20, 30, 40, or 100 or more. Such pH dependent antibodies preferentially dissociate from the antigen in the endosome. This can increase antibody half life, as compared to antibodies with equivalent K〇s at pH 7.4 but with no pH dependent binding, when the antigen is one of undergoes antigen-mediated clearance (eg, PCSK9). Antibodies with pH dependent can decrease total antigen half life when the antigen undergoes reduced clearance when bound to antibody (eg, IL6). Antibodies with pH dependent binding can also prolong the decrease in antigen which is not antibody- Bound. This can be important when antagonizing a target antigen ty Pically present at high levels (e.g., IgE, DKK1, C5 and SOST). In addition, such antibodies can increase antigen half life when the antigen is a receptor and the receptor has increased clearance when bound to antibody (e.g, GMCSF receptor).

Claims

201206466 1 ο.如申請專利範圍第1至8項中任一項之抗體’其中該抗體與抗原在pH 7.4之結合的KD係介於約0.01奈莫耳至約奈莫耳。 11.如申請專利範圍第10項之抗體，其中該抗體與抗原在pH 7.4之結合的KD係介於約0.1奈莫耳至約10奈莫耳 1 2.如申請專利範圍第1至8項中任一項之抗體’其中該抗體與抗原在pH 7.4之結合的Kw係介於約lxlOE-4 s-1 至約 1 X 1 0 E -1 s - 1。 1 3 .如申請專利範圍第1 2項之抗體，其中該抗體與抗原在pH 7.4之結合的Κ。^係介於約lxlOE-3 s-1至約lxlOE-1 s -1 ° 1 4.如申請專利範圍第1項之抗體，其中該抗原係 PCSK9。 1 5 ·如申請專利範圍第6項之抗體，其中該抗原係IgE 、C5或DKK1且該KD係介於1.0奈莫耳至約1〇奈莫耳或介於 1 .〇奈莫耳至約1 00奈莫耳。 1 6 · —種如申請專利範圍第丨至1 5項中任一項之抗體於製備供延長治療性抗體之投藥間隔及/或減少治療劑量的藥物之用途’其中相較於在pH 7.4具有類似親和性但在PH 6.0/pH 7.4之KD比及/或kcff比係小於2之抗體，該抗體具有延長之藥物藥效學效應及/或半衰期。 1 7. —種藉由pH依賴性調節抗體結合親和性以製備具有延長之半衰期及/或藥物藥效學效應之抗體之方法，該方法包含選擇可使影響pKa之微環境最佳化之抗體CDR組 S -3- 201206466 胺酸殘基或其他殘基，致使在pH 6.0/pH7.4下之抗體抗原結合之K D比及/或k。f f比係大於或介於2、3、4、8、1 〇、1 6 或1 6以上。 1 8 ·如申請專利範圍第1 7項之方法，該方法另包含使該抗體突變以達到在pH 7.4之KD爲至少100奈莫耳之抗體親和性》 19. —種抗體庫，其在CDR殘基或其他殘基富含組胺酸以使影響pKa之微環境最佳化。 20. —種經分離之抗體，其與PCSK9特異性結合且包含源自如SEQ ID NO: 4或5所示之重鏈可變區（VH)胺基酸序列之VH互補決定區1 ( CDR1 ) 、VH CDR2及VH CDR3。 2 1 .如申請專利範圍第20項之經分離之抗體，其另包含如SEQ ID NO : 3所示之輕鏈可變區（VL )胺基酸序列之 VL CDR1、CDR2及 CDR3。 22.如申請專利範圍第21項之經分離之抗體，其中該 VH CDR1具有如SEQ ID NO: 6所示之胺基酸序列，VH CDR2具有如SEQ ID NO : 7所示之胺基酸序列，VH CDR3 具有如SEQ ID NO: 8或9所示之胺基酸序列，VL CDR1具有如SEQ ID NO: 10所示之胺基酸序列，VL CDR2具有如 SEQ ID NO: 11所示之胺基酸序列且VL CDR3具有如SEQ ID NO : 12所示之胺基酸序列。 2 3 .如申請專利範圍第2 2項之經分離之抗體’其中該 VH CDR3具有SEQ ID NO : 9之胺基酸序列。 24.如申請專利範圍第23項之經分離之抗體，其中該 -4- 201206466 VH 包含 SEQ ID NO: 4或 SEQ ID NO: 5 且該 vl 包含 SEQ ID NO : 3。 2 5 _如申請專利範圍第2 4項之經分離之抗體，其中該 VH包含 SEQ ID NO ·· 5。 26. —種醫藥組成物，其包含治療有效量之如申請專利範圍第1至1 5及20至25項中任一項之抗體。 27·—種宿主細胞，其重組產製如申請專利範圍第1至 15及20至25項中任一項之抗體。 28. —種經分離之核酸，其編碼如申請專利範圍第丨至 1 5及20至25項中任一項之抗體。 29. —種如申請專利範圍第1至15及20至25項中任一項之抗體於製備供減少個體血液LDL膽固醇之量的藥物之用途。 201206466 863461The antibody of any one of claims 1 to 8 wherein the KD of the antibody to bind to the antigen at pH 7.4 is between about 0.01 nmole to about 1 nm. 11. The antibody of claim 10, wherein the KD of the antibody binding to the antigen at pH 7.4 is between about 0.1 nanomolar and about 10 nanomolar 1 2. As claimed in claims 1 to 8 The antibody of any one of which the binding of the antibody to the antigen at pH 7.4 is between about 1 x 1 OE-4 s-1 to about 1 X 1 0 E -1 s -1 . An antibody according to claim 12, wherein the antibody binds to an antigen at a pH of 7.4. ^ is an antibody of about 1xlOE-3 s-1 to about lxlOE-1 s -1 ° 1 4. The antibody of claim 1 wherein the antigen is PCSK9. The antibody according to claim 6, wherein the antigen is IgE, C5 or DKK1 and the KD is between 1.0 nanomolar to about 1 nanomolar or between 1. 〇Nemo to about 1 00 Nemo. 1 6 - The use of an antibody according to any one of claims 1 to 5 for the preparation of a drug for prolonging the administration interval of a therapeutic antibody and/or reducing the therapeutic dose, wherein compared to having a pH of 7.4 An antibody having similar affinity but a KD ratio and/or a kcff ratio of less than 2 at pH 6.0/pH 7.4 has an extended pharmacodynamic effect and/or half-life. 1 7. A method for preparing an antibody having an extended half-life and/or pharmacodynamic effect by pH-dependent regulation of antibody binding affinity, the method comprising selecting an antibody that optimizes the microenvironment affecting pKa CDR group S -3- 201206466 Amino acid residues or other residues, resulting in a KD ratio and/or k of antibody antigen binding at pH 6.0/pH 7.4. The f f ratio is greater than or between 2, 3, 4, 8, 1 〇, 16 or 16 or more. 1 8 · The method of claim 17, wherein the method further comprises mutating the antibody to achieve a KD of at least 100 nanomolar at a pH of 7.4. 19. An antibody library, which is in the CDR Residues or other residues are enriched in histidine to optimize the microenvironment that affects the pKa. 20. An isolated antibody which specifically binds to PCSK9 and comprises a VH complementarity determining region 1 derived from a heavy chain variable region (VH) amino acid sequence as set forth in SEQ ID NO: 4 or 5 (CDR1) ), VH CDR2 and VH CDR3. The antibody isolated according to claim 20, which further comprises the VL CDR1, CDR2 and CDR3 of the light chain variable region (VL) amino acid sequence as shown in SEQ ID NO: 3. 22. The isolated antibody of claim 21, wherein the VH CDR1 has the amino acid sequence set forth in SEQ ID NO: 6, and the VH CDR2 has the amino acid sequence set forth in SEQ ID NO: , VH CDR3 has the amino acid sequence as shown in SEQ ID NO: 8 or 9, VL CDR1 has the amino acid sequence shown in SEQ ID NO: 10, and VL CDR2 has the amine as shown in SEQ ID NO: The acid sequence and the VL CDR3 has the amino acid sequence set forth in SEQ ID NO: 12. 2 3. The isolated antibody as claimed in claim 2, wherein the VH CDR3 has the amino acid sequence of SEQ ID NO: 9. 24. The isolated antibody of claim 23, wherein the -4- 201206466 VH comprises SEQ ID NO: 4 or SEQ ID NO: 5 and the vl comprises SEQ ID NO: 3. 2 5 - The isolated antibody of claim 24, wherein the VH comprises SEQ ID NO.. 26. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 15 and 20 to 25 of the patent application. A host cell which is recombinantly produced as described in any one of claims 1 to 15 and 20 to 25. 28. An isolated nucleic acid encoding an antibody according to any one of claims 1-5 to 20 and 25. 29. Use of an antibody according to any one of claims 1 to 15 and 20 to 25 for the preparation of a medicament for reducing the amount of blood LDL cholesterol in an individual. 201206466 863461

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丨2 丁ood 丁 9Ό 丁寸0 丁CNId 丁00 (親η<)25:φ OOCSILot ST-·locvlT-0?Lo卜 sInCNI0 s- (宜麟綱麵丨2 丁丁丁 9Ό 丁寸0丁 CNId 丁00 (pro-η<)25:φ OOCSILot ST-·locvlT-0?Lo sInCNI0 s- (Yi Lin Gang