TW201215618A - Antibody compositions and methods of use - Google Patents
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201215618 扣 六、發明說明: 【發明所屬之技術領域】 本發明係關於抗複合物I及抗gH抗體及其使用方法。 相關申請案 本申請案主張均於2010年9月29曰申請之美國臨時申請 案第61/387,735號及第61/387,725號以及於2011年7月1曰申 請之美國臨時申請案第61/504,056號之權益,該等臨時申 請案皆以全文引用的方式併入本文中。 ^ 對作為正文檔案經由EFS網提交之序列表的引用 序列表作為ASCII格式化正文檔案經由EFS網與說明書 一起同時提交,槽案名稱為 「P4680Rl_Sequence Listing.TXT」,創建曰期為2011年9月一,且大小為39.9千 位元組。經由EFS網提交之序列表為說明書之一部分且據 此以全文引用的方式併入本文中。 【先前技術】 人類細胞巨大病毒(Human cytomegalovirus,HCMV)為 鲁 β-疮療病毒(herpesvirus)且亦稱為人類疮修病毒-5(HHV-5)。存在其他種類之會感染其他哺乳動物之細胞巨大病毒 (CMV),諸如鼠類 CMV(MCMV)、天竺鼠(guinea pig)CMV(GPCMV)、猿 CMV(SCCMV)、恆河猴 (rhesus)CMV(rhCMV)及黑猩猩 CMV(CCMV)。HCMV 為一 種感染接近50%美國人口之常見疱疹病毒。對於絕大多數 人類受感染個體而言,HCMV感染無症狀。然而,在疾病 及免疫抑制(例如HIV感染、移植患者中由藥物誘發之免疫 158864.doc 201215618 抑制)之情況下,HCMV再活化或原發性感染會導致多種臨 床表現,諸如單核白血球增多症(mononucleosis)、肝炎、 視網膜炎、肺炎、失明及器官衰竭。此外,在妊娠背景 下,獲得原發性CMV感染儘管對母親影響很小,但會在發 育胎兒中具有嚴重臨床影響。 先天性HCMV感染具有特別重要性,因為由在妊娠期間 受感染之母親所生之許多小孩會在子宮内受感染且會罹患 破壞性臨床疾病。在美國及歐洲,12 6,0 0 0名婦女在姓娠 期間患有原發性HCMV感染且由此等母親所生之約40,000 名嬰兒患有先天性感染。在美國,歸因於HCMV感染, 750名小孩中即有1名生來具有或顯現殘疾,包括:智力遲 鈍、聽力喪失、視力喪失、器官缺陷及生長缺陷。先天性 HCMV感染為胎兒畸形之最常見的感染性病因。在孕婦已 出現原發性感染之後,當前無核准療法可用於預防或治療 胎兒感染。因此,此項技術中極其需要發現能預防先天性 HCMV感染之組合物及方法。 在2005年,Nigro及同事公開一項向患有原發性HCMV感 染之孕婦投與人類CMV高免疫球蛋白(HIG)的研究(Nigro 等人,(2005) TVew Eng/· «/· 353:1350-1362)。在該研究 之一臂中,31名由受HCMV感染之母親所生之嬰兒中僅有 1名生來患有疾病,而7/14(50%)的由未經治療之婦女所生 之小孩生來患有HCMV疾病。同上文。 在妊娠期間,HCMV會經由胎盤自受感染之母親向胎兒 擴散。使胎兒錨定於子宮之胎盤含有特殊化上皮細胞、基 158864.doc 201215618 質纖維母細胞、内皮細胞及特殊化巨嗟細胞。HCMV病毒 表面含有各種病毒醣蛋白複合物,其已顯示為胎盤中所見 之特定細胞類型之感染所需。含有gH/gL與UL128、UL130 及UL131之CMV醣蛋白複合物(在本文中稱為「複合物I」) 特定地為感染内皮細胞、上皮細胞及巨噬細胞所需。含有 gH/gL與gO之CMV醣蛋白複合物(在本文中稱為「複合物 II」)特定地為感染纖維母細胞所需。HIG已顯示會阻斷病 毒進入易受HCMV感染之所有4種胎盤細胞中。 φ 由於製備及廣泛分配HIG存在困難以及醫師及醫學團體 反對特定言之在孕婦中使用人類血液產品,產生包含可保 護胎兒免遭先天性HCMV感染之一或多種單株抗體之組合 物將最為有益。迄今尚未開發出單株抗體組合物可用於預 防CMV之母體-胎兒傳播。Lanzavecchia及Macagno已揭示 自受感染患者之永生化B細胞分離的天然產生之抗體,其 與由UL130與UL13 1之組合、或UL128、UL130及UL131之 組合產生之構形抗原決定基結合且中和CMV傳播(美國專 # 利公開案第 2008/0213265 號及第 2009/0081230號)。Shenk 及Wang已揭示與複合物I之蛋白質結合之抗體(美國專利第 7,7〇4,510號)。Funaro等人亦在美國專利公開案第2010-0040602號中揭示CMV之中和抗體。另外,在2個患者群體 (同種異體骨髓移植接受者及患有AIDS及CMV視網膜炎之 患者)之人類中測試抗gH單株抗體MSL-109 (Drobyski等人, 51:1 190-1 196 (1991) ; Boeckh 等人, 5/ood Marrow 7>α/?5^/α«ί· 7:343-351 (2001);及 Borucki 等 158864.doc 201215618 人64:103-111 (2004)),未獲成功。 此項技術中仍然需要開發用於預防HCMV感染’包括先 天性HCMV感染之單株抗體。 【發明内容】 本發明提供與HCMV複合物I特異性結合之經分離抗體。 在某些實施例中,本發明之抗複合物I抗體包含6個HVR : ⑷包含胺基酸序列SEQ ID ΝΟ:6之HVR-H1 ; (b)包含胺基 酸序列SEQ ID NO:7之HVR-H2 ; (c)包含胺基酸序列SEQ ID NO:8之HVR-H3 ; (d)包含胺基酸序列SEQ ID NO:9之 HVR-L1 ; (e)包含選自由SEQ ID N〇:10-19組成之群之胺基 酸序列的HVR-L2 ;及(f)包含胺基酸序列SEQ ID NO:20之 HVR-L3。該等抗體可進一步包含含有胺基酸序列SEQ ID NO:43之輕鏈可變域構架FR1及含有胺基酸序列SEQ ID NO:44之FR2在其他實施例中,本發明之抗複合物I抗體包 含3個重鏈高變區(HVR-H1、HVR-H2及HVR-H3)及3個輕 鏈高變區(HVR-L1、HVR-L2及 HVR-L3),其中:(a) HVR-H1包含胺基酸序列SEQ ID NO:6 ; (b) HVR-H2包含胺基酸 序列SEQ ID NO:7 ; (c) HVR-H3包含胺基酸序列SEQ ID NO:8 ; (d) HVR-L1 包含胺基酸序列 SEQ ID NO:9 ; (f) HVR-L3包含胺基酸序列SEQ ID NO:20;且(e) HVR-L2及 輕鏈可變域構架FR3之第一胺基酸包含胺基酸序列SEQ ID NO:21。201215618 Deduction 6. Description of the Invention: [Technical Field of the Invention] The present invention relates to an anti-complex I and an anti-gH antibody and a method of using the same. RELATED APPLICATIONS This application is filed on Sep. 29, 2010, the U.S. Provisional Application Nos. 61/387,735 and 61/387,725, and the U.S. Provisional Application No. 61/504,056 filed on July 1, 2011. These patent applications are hereby incorporated by reference in their entirety. ^ The reference sequence table of the sequence table submitted as the text file via the EFS network is submitted as an ASCII formatted text file along with the specification via the EFS network. The name of the slot file is "P4680Rl_Sequence Listing.TXT", and the creation period is September 2011. One, and the size is 39.9 kilobytes. The Sequence Listing submitted via the EFS network is part of the specification and is hereby incorporated by reference in its entirety. [Prior Art] Human cytomegalovirus (HCMV) is a herpesvirus and is also known as human hemovirus-5 (HHV-5). There are other types of cell giant viruses (CMV) that infect other mammals, such as murine CMV (MCMV), guinea pig CMV (GPCMV), 猿CMV (SCCMV), rhesus (rhesus) CMV (rhCMV) ) and chimpanzee CMV (CCMV). HCMV is a common herpesvirus that infects nearly 50% of the US population. For most human infected individuals, HCMV infection is asymptomatic. However, in the case of disease and immunosuppression (eg HIV infection, drug-induced immunity 158864.doc 201215618 inhibition), HCMV reactivation or primary infection can lead to a variety of clinical manifestations, such as mononucleosis (mononucleosis), hepatitis, retinitis, pneumonia, blindness and organ failure. In addition, in the context of pregnancy, obtaining a primary CMV infection, although having little effect on the mother, has a serious clinical impact in the development of the fetus. Congenital HCMV infection is of particular importance because many children born to mothers infected during pregnancy are infected in the womb and suffer from devastating clinical conditions. In the United States and Europe, 12,600 women have a primary HCMV infection during their surname and thus about 40,000 babies born to their mothers have congenital infections. In the United States, one of 750 children was born with or developed disability due to HCMV infection, including: mental retardation, hearing loss, vision loss, organ defects, and growth defects. Congenital HCMV infection is the most common infectious cause of fetal malformations. There are currently no approved therapies to prevent or treat fetal infections after a primary infection has occurred in a pregnant woman. Therefore, there is a great need in the art to find compositions and methods that prevent congenital HCMV infection. In 2005, Nigro and colleagues published a study on the administration of human CMV hyperimmune globulin (HIG) to pregnant women with primary HCMV infection (Nigro et al., (2005) TVew Eng/· «/· 353: 1350-1362). In one of the studies, only one of the 31 babies born to mothers infected with HCMV was born with a disease, and 7/14 (50%) of the children born to untreated women Born to have HCMV disease. Same as above. During pregnancy, HCMV spreads from the infected mother to the fetus via the placenta. The placenta that anchors the fetus to the uterus contains specialized epithelial cells, nucleus fibroblasts, endothelial cells, and specialized python cells. The HCMV virus surface contains various viral glycoprotein complexes that have been shown to be required for infection of the particular cell type seen in the placenta. The CMV glycoprotein complex (referred to herein as "complex I") containing gH/gL and UL128, UL130 and UL131 is specifically required for infection of endothelial cells, epithelial cells and macrophages. The CMV glycoprotein complex (referred to herein as "complex II") containing gH/gL and gO is specifically required for infection of fibroblasts. HIG has been shown to block the entry of viruses into all four placental cells susceptible to HCMV infection. φ Due to difficulties in the preparation and widespread distribution of HIG and the use of human blood products by pregnant physicians and medical groups specifically in pregnant women, it would be most beneficial to produce a composition comprising one or more monoclonal antibodies that protect the fetus from congenital HCMV infection. . Monoclonal antibody compositions have not been developed to date to prevent maternal-fetal transmission of CMV. Lanzavecchia and Macagno have revealed naturally occurring antibodies isolated from immortalized B cells of infected patients that bind to and neutralize conformal epitopes resulting from the combination of UL130 and UL13 1 or the combination of UL128, UL130 and UL131 CMV Communication (US Specialized Publication No. 2008/0213265 and No. 2009/0081230). Shenk and Wang have disclosed antibodies that bind to the protein of complex I (U.S. Patent No. 7,7,4,510). CMV neutralizing antibodies are also disclosed in U.S. Patent Publication No. 2010-0040602. In addition, anti-gH monoclonal antibody MSL-109 was tested in humans of two patient populations (allogeneic bone marrow transplant recipients and patients with AIDS and CMV retinitis) (Drobyski et al., 51:1 190-1 196 ( 1991); Boeckh et al., 5/ood Marrow 7>α/?5^/α«ί· 7:343-351 (2001); and Borucki et al. 158864.doc 201215618 person 64:103-111 (2004)), Unsuccessful. There is still a need in the art to develop monoclonal antibodies for the prevention of HCMV infection, including congenital HCMV infection. SUMMARY OF THE INVENTION The present invention provides isolated antibodies that specifically bind to HCMV complex I. In certain embodiments, an anti-Complex I antibody of the invention comprises 6 HVRs: (4) HVR-H1 comprising the amino acid sequence SEQ ID: 6; (b) comprising the amino acid sequence SEQ ID NO: 7. HVR-H2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) comprising a fragment selected from SEQ ID N : HVR-L2 of the amino acid sequence of 10-19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20. The antibodies may further comprise a light chain variable domain framework FR1 comprising the amino acid sequence SEQ ID NO: 43 and FR2 comprising the amino acid sequence SEQ ID NO: 44. In other embodiments, the anti-complex I of the invention The antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein: (a) HVR -H1 comprises the amino acid sequence SEQ ID NO:6; (b) HVR-H2 comprises the amino acid sequence SEQ ID NO:7; (c) HVR-H3 comprises the amino acid sequence SEQ ID NO:8; (d) HVR-L1 comprises the amino acid sequence SEQ ID NO: 9; (f) HVR-L3 comprises the amino acid sequence SEQ ID NO: 20; and (e) the first amine of HVR-L2 and the light chain variable domain framework FR3 The base acid comprises the amino acid sequence SEQ ID NO:21.
在特定實施例中,抗複合物I抗體包含(a) VH,其包含胺 基酸序列 SEQ ID NO:45 或 SEQ ID NO:46 或 SEQ ID 158864.doc -6- 201215618 NO:47 ;及(b) VL,其包含胺基酸序列SEQ ID NO:48或SEQ ID NO:49。此等抗體可進一步包含含有胺基酸序列SEQ ID NO:41之輕鏈可變域構架FR3及含有胺基酸序列SEQ ID NO:42之FR4。 在一些實施例中,抗複合物I抗體包含與胺基酸序列SEQ ID NO:45 或 SEQ ID NO:46 或 SEQ ID NO:47具有至少 95%序 列一致性之VH序列及與胺基酸序列SEQ ID NO:48或SEQ ID NO:49具有至少95%序列一致性之VL序列。在一些實施 例中,抗體包含含有胺基酸序列SEQ ID NO:45或SEQ ID NO:46或SEQ ID NO:47之VH。在一些實施例中,抗複合物 I抗體包含含有胺基酸序列SEQ ID NO:48或SEQ ID NO:49 之VL。在一些實施例中,抗複合物I抗體包含VH序列SEQ ID NO:45 或 SEQ ID >10:46及 VL序列 SEQ ID NO:49。 本發明亦提供與HCMV gH特異性結合之經分離抗體。 在一些實施例中,本發明之抗gH抗體包含3個重鏈高變 區(HVR-H1、HVR-H2及HVR-H3)及3個輕鏈高變區(HVR-Ll、HVR-L2 及 HVR-L3),其中: (a) HVR-H1包含胺基酸序列SEQ ID NO:71 ; (b) HVR-H2 包含選自 SEQ ID N〇:72、SEQ ID NO:73、 8£卩10>^0:74及8£(^10]^0:93之胺基酸序列; (c) HVR-H3包含胺基酸序列SEQ ID NO:75 ; (d) HVR-L1包含胺基酸序列SEQ ID NO:76 ; (e) HVR-L2包含胺基酸序列SEQ ID NO:77 ;且 (f) HVR-L3包含胺基酸序列SEQ ID NO:78。 158864.doc 201215618 在一些實施例中,抗gH抗體包含含有胺基酸序列SEQ ID NO:93之HVR-H2,其中在SEQ ID NO:93之位置6處之胺 基酸係選自由Ser、Thr、Asn、Gin、Phe ' Met及Leu組成 之群,且在SEQ ID NO:93之位置8處之胺基酸係選自由Thr 及Arg組成之群。 在某些實施例中,抗gH抗體包含含有胺基酸序列SEQ ID NO:72、SEQ ID NO:73 或 SEQ ID NO:74之HVR-H2。 在其他實施例中,抗gH抗體包含含有胺基酸序列SEQ ID NO:94之HVR-H2,其中該序列在(SEQ ID NO:94之)位 置 54處包含選自由 Ser、Thr、Asn、Gin、Phe、Met及 Leu 組成之群之胺基酸。在一些實施例中,該抗體進一步在位 置56處包含選自由Thr及Arg組成之群之胺基酸。 本發明亦提供具有胺基酸序列與SEQ ID NO:94至少95% 一致之VH序列的抗gH抗體,其中該序列包含胺基酸 Asn54、Ser54、Thr54、Gln54、Phe54、Met54 或 Leu54及 / 或Arg56 ^在某些實施例中,抗體包含含有選自SEQ ID NO:87、SEQ ID NO:88 及 SEQ ID NO:89 之胺基酸序列之 VH。在一些實施例中,VH包含與SEQ ID NO:94 95%—致 之胺基酸序列,其中該序列在位置54處含有選自Asn54、 Ser54、Thr54、Gln54、Phe54、Met54 或 Leu54 之胺基酸及 / 或在位置56處含有Arg(Arg56);且(b)VL序列與胺基酸序 列SEQ ID NO:90具有至少95%序列一致性。在某些實施例 中,VH 包含選自 SEQ ID NO:87、SEQ ID NO:88 及 SEQ ID NO:89之胺基酸序列。在一些實施例中,VL包含胺基酸序 158864.doc 201215618 列SEQ ID NO:90。在某些實施例中,抗體包含VH序列 SEQIDNO:89及VL序列SEQIDNO:90。In a particular embodiment, the anti-Complex I antibody comprises (a) VH comprising an amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46 or SEQ ID 158864. doc -6 - 201215618 NO: 47; b) VL comprising the amino acid sequence SEQ ID NO: 48 or SEQ ID NO: 49. Such antibodies may further comprise a light chain variable domain framework FR3 comprising the amino acid sequence SEQ ID NO: 41 and FR4 comprising the amino acid sequence SEQ ID NO: 42. In some embodiments, the anti-Complex I antibody comprises a VH sequence and amino acid sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 46 or SEQ ID NO: 47 SEQ ID NO: 48 or SEQ ID NO: 49 has a VL sequence with at least 95% sequence identity. In some embodiments, the antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 45 or SEQ ID NO: 46 or SEQ ID NO: 47. In some embodiments, the anti-Complex I antibody comprises a VL comprising the amino acid sequence SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, the anti-Complex I antibody comprises the VH sequence SEQ ID NO: 45 or SEQ ID > 10:46 and the VL sequence SEQ ID NO:49. The invention also provides isolated antibodies that specifically bind to HCMV gH. In some embodiments, the anti-gH antibody of the present invention comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3), wherein: (a) HVR-H1 comprises the amino acid sequence SEQ ID NO: 71; (b) HVR-H2 comprises a mutation selected from the group consisting of SEQ ID N: 72, SEQ ID NO: 73, 8 £ 10 ; ^0: 74 and 8 £(^10]^0:93 amino acid sequence; (c) HVR-H3 comprises the amino acid sequence SEQ ID NO: 75; (d) HVR-L1 contains an amino acid sequence SEQ ID NO: 76; (e) HVR-L2 comprises the amino acid sequence SEQ ID NO: 77; and (f) HVR-L3 comprises the amino acid sequence SEQ ID NO: 78. 158864.doc 201215618 In some embodiments The anti-gH antibody comprises HVR-H2 comprising the amino acid sequence SEQ ID NO: 93, wherein the amino acid at position 6 of SEQ ID NO: 93 is selected from the group consisting of Ser, Thr, Asn, Gin, Phe 'Met and A group consisting of Leu, and the amino acid at position 8 of SEQ ID NO: 93 is selected from the group consisting of Thr and Arg. In certain embodiments, the anti-gH antibody comprises an amino acid sequence comprising SEQ ID NO: 72. SEQ ID NO: 73 or HVR-H2 of SEQ ID NO: 74. In other embodiments, the anti-gH antibody comprises an amino acid sequence comprising SEQ I D NO: 94 HVR-H2, wherein the sequence comprises an amino acid selected from the group consisting of Ser, Thr, Asn, Gin, Phe, Met and Leu at position 54 (SEQ ID NO: 94). In an embodiment, the antibody further comprises an amino acid selected from the group consisting of Thr and Arg at position 56. The invention also provides an anti-gH having a VH sequence having an amino acid sequence at least 95% identical to SEQ ID NO: 94. An antibody, wherein the sequence comprises the amino acid Asn54, Ser54, Thr54, Gln54, Phe54, Met54 or Leu54 and/or Arg56^ In certain embodiments, the antibody comprises a molecule selected from the group consisting of SEQ ID NO: 87, SEQ ID NO: 88 And VH of the amino acid sequence of SEQ ID NO: 89. In some embodiments, VH comprises an amino acid sequence of 95% identical to SEQ ID NO: 94, wherein the sequence comprises an Asn54, at position 54 The amino acid of Ser54, Thr54, Gln54, Phe54, Met54 or Leu54 and/or contains Arg(Arg56) at position 56; and (b) the VL sequence is at least 95% identical to the amino acid sequence SEQ ID NO:90 Sex. In certain embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89. In some embodiments, VL comprises an amino acid sequence 158864.doc 201215618 column SEQ ID NO:90. In certain embodiments, the antibody comprises the VH sequence SEQ ID NO: 89 and the VL sequence SEQ ID NO: 90.
在某些實施例中,本發明抗體與HCMV表面上之HCMV 複合物I特異性結合且中和HCMV,EC90為0.1 pg/ml或0.1 pg/ml以下。在某些實施例中,本發明之經分離抗複合物I 抗體與HCMV表面上之HCMV複合物I特異性結合且在以下 抗體濃度下中和 50%之HCMV : 0.05 pg/ml、0·02 pg/ml、 0.015 pg/ml、0_014 pg/ml、0.013 pg/ml、0.012 pg/ml、 0.011 pg/ml、0.010 pg/ml、0_009 pg/ml、0.008 pg/ml、 0.007 pg/ml、0.006 pg/ml、0.005 pg/ml、0.004 pg/ml、 0.003 pg/ml、0.002 pg/ml、0.001 pg/ml、0.0009 pg/ml、 0.0008 pg/ml、0.0007 pg/ml或0.0007 pg/ml以下(例如在 10-8M、 ΙΟ·9 Μ、ΙΟ·10 Μ、ΙΟ·11 Μ、ΙΟ·12 Μ、ΙΟ.13 M或 ΙΟ·13 M以下之 抗體漠度下)。 在某些實施例中,本發明之經分離抗gH抗體與HCMV gH特異性結合。該等抗體與HCMV表面上之gH結合且中和 HCMV,EC90為1 pg/ml或1 pg/ml以下。本發明之經分離 抗gH抗體與HCMV表面上之gH結合且在以下抗體濃度下中 和 50% 之 HCMV : 0· 1 pg/ml、0.09 pg/ml、0·08 pg/ml、 0.07 pg/ml、0.06 pg/ml、0.05 pg/ml、0.04 pg/ml、0.03 pg/ml、0.02 pg/ml、0.015 pg/ml、0.014 pg/ml、0.013 pg/ml、0.012 pg/ml、0.011 pg/ml、0.010 pg/ml、0.009 pg/ml、0.008 pg/ml、0.007 pg/ml、0.006 pg/ml、0.005 pg/ml、0.004 pg/ml、0.003 pg/ml、0.002 pg/ml、0.001 158864.doc • 9- 201215618 pg/ml 或 0.001 pg/ml 以下(例如在 1〇_8 μ、1(Τ9 Μ、ΙΟ·10 Μ、 10·11 Μ、ΙΟ·12 Μ、10·13 Μ或 ΙΟ·13 Μ以下之抗體濃度下)。 本發明抗體可為單株抗體,包括例如人類抗體、人類化 抗體或嵌合抗體。本發明亦提供特異性結合HCMV gH及/ 或複合物I之抗體片段。 在特定實施例中’特異性結合HCMV複合物I及/或gH之 抗體為全長IgGl抗體。 本發明亦提供編碼特異性結合HCMV複合物I及/或gH之 抗體的經分離核酸。本發明亦提供包含編碼此等抗體之核 酸之宿主細胞。 本發明進一步提供一種產生抗體之方法,其包含培養含 有編碼特異性結合複合物〗及/或之抗體之核酸的宿主細 胞以便產生該抗體。該方法可進一步包含自該宿主細胞回 收該抗體。 本發明亦提供一種包含抗複合物〗抗體、或抗gH抗體、 或抗複合物I抗體與抗gH抗體之組合及醫藥學上可接受之 載劑的醫藥調配物。各抗體之醫藥調配物可為單獨的或經 組合。醫藥調配物可進一步包含另一治療劑(例如更昔洛 早(ganciclovir)、佛斯卡耐特(f〇scarnet)、顯更昔洛韋 (valganciclovir)及西多福韋(cid〇f〇vir))。 本發明亦提供包含抗複合物〗抗體、或抗gH抗體、或抗 複合物I抗體與抗gH抗體之組合的組合物。包含各抗體之 組合物可為單獨的或經組合。組合物可進一步包含另一治 療劑(例如更昔洛韋、佛斯卡耐特、纈更昔洛韋及西多= 158864.doc 201215618 韋)。 本發明亦提供用於抑制、治療或預防HCMV感染之包含 抗複合物I抗體及/或抗gH抗體之組合物。在一些實施例 中,其係用於抑制、治療或預防先天性HCMV感染或所移 植組織、器官或供體受或已受HCMV感染之組織或器官移 植接受者之HCMV感染。其他實施例包括用於先前已受 HCMV感染且處於再活化風險中之移植接受者。在某些實 施例中,組織或器官移植接受者呈HCMV感染血清陰性。 在某些實施例中,包含結合HCMV gH之抗體之組合物與 包含結合HCMV複合物I之抗體之組合物分開。 包含本發明抗體之組合物亦可用於製造藥劑。該藥劑可 用於治療、抑制或預防HCMV感染,諸如抑制、預防或治 療先天性HCMV感染或所移植器官、組織或供體受或已受 HCMV感染之器官或組織移植接受者之HCMV感染。在其 他實施例中,移植接受者先前已受HCMV感染且處於再活 化風險中。在某些實施例中,藥劑可進一步包含另一治療 劑(例如更昔洛韋、佛斯卡耐特、纈更昔洛韋及西多福 韋)。在某些實施例中,器官或組織移植接受者呈HCMV感 染血清陰性。在某些實施例中,包含結合HCMV gH之抗 體之組合物係呈與結合HCMV複合物I之抗體分開的組合物 形式。 本發明亦提供一種治療、抑制或預防HCMV感染之方 法,其包含投與患者有效量之包含抗gH抗體、抗複合物I 抗體或其組合之組合物。本發明亦提供一種治療、抑制或 158864.doc 11 3 201215618 預防先天性HCMV感染之方法,其包含投與孕婦有效量之 包含本發明抗體或其組合之組合物。本發明亦提供一種治 療受HCMV感染之胎兒之方法,其包含投與孕婦有效量之 包含本發明抗體或其組合之組合物。本發明亦提供一種治 療受HCMV感染之嬰兒或在妊娠期間暴露於HCMV之嬰兒 的方法,其包含投與該嬰兒有效量之包含本發明抗體或其 組合之組合物。 本發明亦提供一種治療、抑制或預防器官或組織移植接 受者之HCMV感染之方法,其包含投與該移植器官或組織 接受者有效量之包含本發明抗體或其組合之組合物以治 療、抑制或預防起因於自受或已受HCMV感染之器官供體 或組織供體獲得之器官或組織的HCMV感染。其他實施例 包括移植接受者先前已受HCMV感染且處於再活化風險中 時所用之方法。治療方法可進一步包含投與患者另一治療 劑(例如更昔洛韋、佛斯卡耐特、纈更昔洛韋及西多福 韋)。 在某些實施例中,包含結合HCMV gH之抗體之組合物 係呈與包含結合HCMV複合物I之抗體之組合物分開的組合 物形式。在其他實施例中,包含結合HCMV gH之抗體之 組合物與包含結合HCMV複合物I之抗體之組合物同時、在 其之前或在其之後投與。 本發明亦提供一種用於抑制、治療或預防HCMV感染之 抗複合物I抗體及/或抗gH抗體。在一些實施例中,其係用 於抑制、治療或預防先天性HCMV感染或所移植組織、器 158864.doc •12- 201215618 官或供體受或已受HCMV感染之組織或器官移植接受者之 HCMV感染。其他實施例包括用於先前已受HCMV感染且 處於再活化風險中之移植接受者。在某些實施例中,組織 或器官移植接受者呈HCMV感染血清陰性。 本發明抗體可用於製造藥劑。該藥劑可用於治療、抑制 或預防HCMV感染,諸如抑制、預防或治療先天性HCMV 感染或所移植器官、組織或供體受或已受HCMV感染之器 官或組織移植接受者之HCMV感染。在其他實施例中,移 植接受者先前已受HCMV感染且處於再活化風險中。在某 些實施例中,藥劑可進一步包含另一治療劑(例如更昔洛 韋、佛斯卡耐特、纈更昔洛韋及西多福韋)。在某些實施 例中,器官或組織移植接受者呈HCMV感染血清陰性。 本發明亦提供一種治療、抑制或預防HCMV感染之方 法,其包含投與患者有效量之抗gH抗體、抗複合物I抗體 或其組合。本發明亦提供一種治療、抑制或預防先天性 HCMV感染之方法,其包含投與孕婦有效量之本發明抗體 或其組合。本發明亦提供一種治療受HCMV感染之胎兒之 方法,其包含投與孕婦有效量之本發明抗體或其組合。 本發明亦提供一種治療、抑制或預防器官或組織移植接 受者之HCMV感染之方法,其包含投與該移植器官或組織 接受者有效量之本發明抗體或其組合以治療、抑制或預防 起因於自受或已受HCMV感染之器官供體或組織供體獲得 之器官或組織的HCMV感染。其他實施例包括移植接受者 先前已受HCMV感染且處於再活化風險中時所用之方法。 158864.doc -13- 201215618 治療方法可進一步包含投與患者另一治療劑(例如更昔洛 韋、佛斯卡耐特、纈更昔洛韋及西多福韋)。 在某些實施例中,結合HCMV gH之抗體與結合HCMV複 合物I之抗體分開投與。在其他實施例中,結合HCMV gH 之抗體與結合HCMV複合物I之抗體同時、在其之前或在其 之後投與。 在某些實施例中,器官移植物為心臟、腎、肝、肺、胰 臟、腸或胸腺。在其他實施例中,組織移植物為手、角 膜、皮膚、臉、蘭氏小島(islets of langerhans)、骨髓、幹 細胞、全血、血小板、血清、血球、血管 '心瓣膜(heart valve)、骨、骨祖細胞、軟骨、韋刃帶、腱、肌肉内襯 (muscle lining) 〇 本發明亦提供與本發明之抗gH抗體及/或抗複合物I抗體 結合相同抗原決定基之抗體。其他實施例包括與HCMV gH之抗原決定基結合之抗體,該抗原決定基包含對應於選 自由以下組成之群之胺基酸的胺基酸:在SEQ ID NO: 1之 位置168處之色胺酸;在SEQ ID ΝΟ:1之位置446處之天冬 胺酸;在SEQ ID ΝΟ:1之位置171處之脯胺酸;及其組合。 其他實施例包括與HCMV複合物I之抗原決定基結合之抗 體,該抗原決定基包含對應於選自由以下組成之群之胺基 酸的胺基酸:在SEQ ID NO:203之位置47處之麩醯胺酸; (ii)在SEQ ID NO:203之位置51處之離胺酸;(iii)在SEQ ID NO:203之位置46處之天冬胺酸;及(iv)其組合。其他實施 例包括與HCMV複合物I之多肽結合之抗體,其中該多肽包 158864.doc -14- 201215618 含胺基酸序列 sralpdqtrykyVEqLVdLT lnyhydas (SEQ ID NO:194)。 【貫施方式】 I.定義 出於本文中之目的,「接受體人類構架」為包含源於人 類免疫球蛋白構架或人類共同構架(如下文所定義)之輕鏈 可變域(VL)構架或重鏈可變域(VH)構架之胺基酸序列的構 架。「源於」人類免疫球蛋白構架或人類共同構架之接受 ® 體人類構架可包含人類免疫球蛋白構架或人類共同構架之 相同胺基酸序列,或其可含有胺基酸序列變化。在一些實 施例中,胺基酸變化之數目為10或1〇以下、9或9以下、8 或8以下、7或7以下、6或6以下、5或5以下、4或4以下、3 或3以下、或2或2以下。在一些實施例中,VL接受體人類 構架序列與VL人類免疫球蛋白構架序列或人類共同構架 序列相同。 「親和力」係指分子(例如抗體)之單一結合位點與該分 • 子之結合搭配物(例如抗原)之間非共價相互作用總和的強 度。除非另外指示,否則如本文所用,「結合親和力」係 指反映結合對(例如抗體與抗原)成員之間丨:丨相互作用之固 有結合親和力。分子X對其搭配物Y之親和力通常可用解 離常數(Kd)表示。親和力可藉由此項技術中已知之常用方 法(包括本文所述之方法)量測。有關量測結合親和力之特 定說明性及例示性實施例描述於下文中。 「親和力成熟」抗體係指相較於在一或多個高變區 158864.doc € 201215618 (HVR)中不具有一或多個改變之親本抗體,在一或多個高 變區中具有此等改變之抗體,.此等改變使得抗體對抗原之 親和力改良。 術語「抗複合物I抗體」及「與複合物I結合之抗體」係 指能夠以足夠親和力結合複合物I之抗體,以使該抗體適 用作靶向複合物I之診斷劑及/或治療劑。在一實施例中, 抗複合物I抗體與無關非複合物I蛋白質結合之程度小於該 抗體與複合物I結合的約10 %,如例如藉由放射免疫檢定 (RIA)所量測。在某些實施例中,與複合物〗結合之抗體之 解離常數(Kd)為 $1 μΜ、$100 nM、$10 nM、$1 nM、$0.1 nM、20.01 nM或 $0.001 nM(例如 10·8 M或 10·8 M以下,例 如ΙΟ·8 Μ至ΙΟ·13 Μ,例如ΙΟ·9 Μ至1 Ο·13 Μ)。在某些實施例 中,抗複合物I抗體與複合物I之在人類CMV分離株之間保 守的抗原決定基結合。在某些實施例中,抗複合物I抗體 與複合物I之在感染不同物種之CMV病毒株之間保守的抗 原決定基結合。在某些實施例中,「抗複合物I抗體」結合 複合物I之構形抗原決定基且在某些實施例中,抗複合物J 抗體與複合物I之不為gH之個別蛋白質成員(亦即gL、 UL128、UL130或UL131)内的抗原決定基結合。 術語「抗gH抗體」及「與gH結合之抗體」係指能夠以 足夠親和力結合gH之抗體,以使該抗體適用作靶向gH之 診斷劑及/或治療劑。在一實施例中,抗gH抗體與無關非 gH蛋白質結合之程度小於該抗體與gH結合的約1 〇%,如例 如藉由放射免疫檢定(RIA)所量測。在某些實施例中,與 158864.doc •16- 201215618 gH結合之抗體之解離常數(Kd)為$1 、$100 nM、£10 nM、$1 nM、S0.1 nM、$0.01 nM或 $0.001 nM(例如 l〇·8 μ 或 l〇·8 M以下,例如 10·8 μ至 ι〇-13 Μ,例如 10·9 M至 l〇·13 Μ) °在某些實施例中,抗gH抗體與印之在人類cmv分離 株之間保守的抗原決定基結合。在某些實施例中’抗§11抗 體與gH之在感染不同物種之cmv病毒株之間保守的抗原 決定基結合。 術語「抗體」在本文中以最廣泛意義使用且涵蓋各種抗 體結構,包括(但不限於)單株抗體、多株抗體、多特異性 抗體(例如雙特異性抗體)及抗體片段,只要其展現所要抗 原結合活性即可。 「抗體片段」係指除完整抗體以外之分子,其包含完整 抗體中結合該完整抗體所結合之抗原的部分。抗體片段之 貫例包括(但不限於)Fv、Fab、Fab'、Fab'-SH、F(ab')2 ;雙 功能抗體;線抗體;單鏈抗體分子(例如scFv);及自抗體 片段形成之多特異性抗體。 與參照抗體「結合相同抗原決定基之抗體」係指在競爭 檢疋中阻斷該參照抗體與其抗原結合達5〇%或5〇%以上的 抗體且反之,5亥參照抗體在競爭檢定中阻斷該抗體與其 抗原結合達50%或50%以上。本文提供一種例示性競爭檢 定。 術語「嵌合」抗體係指重鏈及/或輕鏈之一部分源於特 疋來源或物種,而重鏈及/或輕鏈之其餘部分源於不同來 源或物種之抗體。 矣.. 158864.doc -17· 201215618 抗體之「類別」係指其重鏈所具有之恆定域或恆定區之 類型。存在5種主要抗體類別:IgA、IgD、IgE、IgG及 IgM,且若干此等類別可進一步分成子類(同型),例如 IgGi、IgG2、IgG3、IgG4、IgA!及 IgA2。對應於免疫球蛋 白之不同類別之重鏈恆定域分別被稱為α、δ、ε、γ及μ。In certain embodiments, an antibody of the invention specifically binds to HCMV complex I on the surface of HCMV and neutralizes HCMV with an EC90 of 0.1 pg/ml or less. In certain embodiments, the isolated anti-Complex I antibody of the invention specifically binds to HCMV complex I on the surface of HCMV and neutralizes 50% of HCMV at the following antibody concentrations: 0.05 pg/ml, 0·02 Pg/ml, 0.015 pg/ml, 0_014 pg/ml, 0.013 pg/ml, 0.012 pg/ml, 0.011 pg/ml, 0.010 pg/ml, 0_009 pg/ml, 0.008 pg/ml, 0.007 pg/ml, 0.006 Pg/ml, 0.005 pg/ml, 0.004 pg/ml, 0.003 pg/ml, 0.002 pg/ml, 0.001 pg/ml, 0.0009 pg/ml, 0.0008 pg/ml, 0.0007 pg/ml or 0.0007 pg/ml or less ( For example, in the antibody inferiority of 10-8M, ΙΟ·9 Μ, ΙΟ·10 Μ, ΙΟ·11 Μ, ΙΟ·12 Μ, ΙΟ.13 M or ΙΟ·13 M or less). In certain embodiments, an isolated anti-gH antibody of the invention specifically binds to HCMV gH. These antibodies bind to gH on the surface of HCMV and neutralize HCMV with an EC90 of 1 pg/ml or less. The isolated anti-gH antibody of the present invention binds to gH on the surface of HCMV and neutralizes 50% of HCMV at the following antibody concentrations: 0·1 pg/ml, 0.09 pg/ml, 0·08 pg/ml, 0.07 pg/ Ml, 0.06 pg/ml, 0.05 pg/ml, 0.04 pg/ml, 0.03 pg/ml, 0.02 pg/ml, 0.015 pg/ml, 0.014 pg/ml, 0.013 pg/ml, 0.012 pg/ml, 0.011 pg/ Ml, 0.010 pg/ml, 0.009 pg/ml, 0.008 pg/ml, 0.007 pg/ml, 0.006 pg/ml, 0.005 pg/ml, 0.004 pg/ml, 0.003 pg/ml, 0.002 pg/ml, 0.001 158864. Doc • 9- 201215618 pg/ml or less than 0.001 pg/ml (eg at 1〇_8 μ, 1 (Τ9 Μ, ΙΟ·10 Μ, 10·11 Μ, ΙΟ·12 Μ, 10.13 Μ or ΙΟ· The antibody of the present invention may be a monoclonal antibody, including, for example, a human antibody, a humanized antibody or a chimeric antibody. The present invention also provides an antibody fragment that specifically binds to HCMV gH and/or complex I. In a particular embodiment, an antibody that specifically binds to HCMV complex I and/or gH is a full-length IgG1 antibody. The invention also provides an isolated nucleic acid encoding an antibody that specifically binds to HCMV complex I and/or gH. provide A host cell comprising a nucleic acid encoding such antibodies. The invention further provides a method of producing an antibody comprising culturing a host cell comprising a nucleic acid encoding an antibody that specifically binds to the complex and/or antibody to produce the antibody. Further comprising recovering the antibody from the host cell. The invention also provides a medicament comprising an anti-complex antibody, or an anti-gH antibody, or a combination of an anti-complex I antibody and an anti-gH antibody, and a pharmaceutically acceptable carrier Formulations. Pharmaceutical formulations of each antibody may be used alone or in combination. The pharmaceutical formulation may further comprise another therapeutic agent (eg, ganciclovir, foscarnet, sensitization) Valganciclovir and cidoff〇vir. The invention also provides a combination comprising an anti-complex antibody, or an anti-gH antibody, or an anti-complex I antibody and an anti-gH antibody. The composition comprising each antibody may be used alone or in combination. The composition may further comprise another therapeutic agent (eg, ganciclovir, foscarnet, valganciclovir and Multi = 158864.doc 201215618 Wei). The present invention also provides a composition comprising an anti-Complex I antibody and/or an anti-gH antibody for use in inhibiting, treating or preventing HCMV infection. In some embodiments, it is used to inhibit, treat or prevent HCMV infection of a congenital HCMV infection or transplanted tissue, organ or donor or recipient of a tissue or organ transplant infected with HCMV. Other embodiments include transplant recipients for those who have previously been infected with HCMV and are at risk of reactivation. In certain embodiments, the tissue or organ transplant recipient is seronegative for HCMV infection. In certain embodiments, a composition comprising an antibody that binds to HCMV gH is separated from a composition comprising an antibody that binds to HCMV complex I. Compositions comprising an antibody of the invention can also be used in the manufacture of a medicament. The agent can be used to treat, inhibit or prevent HCMV infection, such as inhibiting, preventing or treating HCMV infection in a congenital HCMV infection or transplanted organ, tissue or donor, or an organ or tissue transplant recipient infected with HCMV. In other embodiments, the transplant recipient has previously been infected with HCMV and is at risk of reactivation. In certain embodiments, the agent may further comprise another therapeutic agent (e.g., ganciclovir, foscarnet, valganciclovir, and cidofovir). In certain embodiments, the organ or tissue transplant recipient is seronegative for HCMV infection. In certain embodiments, the composition comprising an antibody that binds to HCMV gH is in the form of a composition separate from the antibody that binds to HCMV complex I. The invention also provides a method of treating, inhibiting or preventing HCMV infection comprising administering to a patient an effective amount of a composition comprising an anti-gH antibody, an anti-complex I antibody, or a combination thereof. The invention also provides a method of treating, inhibiting, or preventing a congenital HCMV infection comprising administering to a pregnant woman an effective amount of a composition comprising an antibody of the invention or a combination thereof. The invention also provides a method of treating a fetus infected with HCMV comprising administering to a pregnant woman an effective amount of a composition comprising an antibody of the invention or a combination thereof. The invention also provides a method of treating an infant infected with HCMV or an infant exposed to HCMV during pregnancy comprising administering to the infant an effective amount of a composition comprising an antibody of the invention or a combination thereof. The invention also provides a method of treating, inhibiting or preventing HCMV infection in an organ or tissue transplant recipient comprising administering to the transplant organ or tissue recipient an effective amount of a composition comprising an antibody of the invention or a combination thereof for treatment, inhibition Or prevention of HCMV infection resulting from an organ or tissue obtained from an organ donor or tissue donor that has received or has been infected with HCMV. Other embodiments include methods used by transplant recipients who have previously been infected with HCMV and are at risk of reactivation. The method of treatment may further comprise administering to the patient another therapeutic agent (e.g., ganciclovir, Foscarnet, valganciclovir, and cidofovir). In certain embodiments, a composition comprising an antibody that binds to HCMV gH is in the form of a composition separate from a composition comprising an antibody that binds to HCMV complex I. In other embodiments, a composition comprising an antibody that binds to HCMV gH is administered concurrently with, prior to, or subsequent to a composition comprising an antibody that binds to HCMV complex I. The invention also provides an anti-Complex I antibody and/or an anti-gH antibody for use in inhibiting, treating or preventing HCMV infection. In some embodiments, it is used to inhibit, treat, or prevent congenital HCMV infection or transplanted tissue, or a tissue or organ transplant recipient who has or has been infected with HCMV. HCMV infection. Other embodiments include transplant recipients for those who have previously been infected with HCMV and are at risk of reactivation. In certain embodiments, the tissue or organ transplant recipient is seronegative for HCMV infection. The antibodies of the invention are useful in the manufacture of pharmaceutical agents. The agent can be used to treat, inhibit or prevent HCMV infection, such as inhibiting, preventing or treating HCMV infection in a congenital HCMV infection or transplanted organ, tissue or donor, or a transplant recipient of HCMV infection. In other embodiments, the transplant recipient has previously been infected with HCMV and is at risk of reactivation. In certain embodiments, the agent may further comprise another therapeutic agent (e.g., ganciclovir, foscarnet, valganciclovir, and cidofovir). In certain embodiments, the organ or tissue transplant recipient is seronegative for HCMV infection. The invention also provides a method of treating, inhibiting or preventing HCMV infection comprising administering to a patient an effective amount of an anti-gH antibody, an anti-Complex I antibody, or a combination thereof. The invention also provides a method of treating, inhibiting or preventing a congenital HCMV infection comprising administering to a pregnant woman an effective amount of an antibody of the invention or a combination thereof. The invention also provides a method of treating a fetus infected with HCMV comprising administering an effective amount of an antibody of the invention or a combination thereof to a pregnant woman. The invention also provides a method of treating, inhibiting or preventing HCMV infection in an organ or tissue transplant recipient comprising administering to the transplant organ or tissue recipient an effective amount of an antibody of the invention or a combination thereof for treating, inhibiting or preventing the cause HCMV infection of an organ or tissue obtained from an organ donor or tissue donor that has been or has been infected with HCMV. Other embodiments include methods used by transplant recipients who have previously been infected with HCMV and are at risk of reactivation. 158864.doc -13- 201215618 The method of treatment may further comprise administering to the patient another therapeutic agent (e.g., ganciclovir, Foscarnet, valganciclovir, and cidofovir). In certain embodiments, an antibody that binds to HCMV gH is administered separately from an antibody that binds to HCMV Complex I. In other embodiments, the antibody that binds to HCMV gH is administered concurrently with, prior to, or subsequent to the antibody that binds to HCMV complex I. In certain embodiments, the organ transplant is a heart, kidney, liver, lung, pancreas, intestine or thymus. In other embodiments, the tissue graft is a hand, a cornea, a skin, a face, islets of langerhans, bone marrow, stem cells, whole blood, platelets, serum, blood cells, blood vessels, heart valves, bones , osteoprogenitor cells, cartilage, Wei stalk, sputum, muscle lining 〇 The present invention also provides an antibody that binds to the same epitope as the anti-gH antibody and/or anti-complex I antibody of the present invention. Other embodiments include an antibody that binds to an epitope of HCMV gH, the epitope comprising an amino acid corresponding to an amino acid selected from the group consisting of: a tryptamine at position 168 of SEQ ID NO: Acid; aspartic acid at position 446 of SEQ ID NO: 1; proline at position 171 of SEQ ID NO: 1; and combinations thereof. Other embodiments include an antibody that binds to an epitope of HCMV complex I, the epitope comprising an amino acid corresponding to an amino acid selected from the group consisting of: at position 47 of SEQ ID NO:203 Branic acid; (ii) an amino acid at position 51 of SEQ ID NO: 203; (iii) aspartic acid at position 46 of SEQ ID NO: 203; and (iv) a combination thereof. Other embodiments include antibodies that bind to a polypeptide of HCMV complex I, wherein the polypeptide comprises 158864.doc -14 - 201215618 amino acid sequence sralpdqtrykyVEqLVdLT lnyhydas (SEQ ID NO: 194). [Comprehensive means] I. Definition For the purposes of this article, "acceptor human framework" is a light chain variable domain (VL) framework comprising a human immunoglobulin framework or a human common framework (as defined below). Or the framework of the amino acid sequence of the heavy chain variable domain (VH) framework. Acceptance from "human immunoglobulin frameworks or human common frameworks" The human human framework may comprise the same amino acid sequence of a human immunoglobulin framework or a human common framework, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 Or 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework sequence is identical to the VL human immunoglobulin framework sequence or the human co-framework sequence. "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and a binding partner (e.g., an antigen) of the molecule. As used herein, "binding affinity" refers to the inherent binding affinity that reflects the 丨:丨 interaction between members of a binding pair (e.g., an antibody and an antigen), unless otherwise indicated. The affinity of the molecule X for its conjugate Y is usually expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below. An "affinity mature" anti-system refers to a parent antibody that does not have one or more changes in one or more hypervariable regions 158864.doc € 201215618 (HVR), having this in one or more hypervariable regions Such altered antibodies, such changes result in improved affinity of the antibody for the antigen. The terms "anti-complex I antibody" and "antibody I bind to complex I" refer to an antibody capable of binding complex I with sufficient affinity to render the antibody useful as a diagnostic and/or therapeutic agent for targeting complex I. . In one embodiment, the anti-Complex I antibody binds to an unrelated non-complex I protein to a lesser extent than about 10% of the binding of the antibody to Complex I, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody has a dissociation constant (Kd) of $1 μΜ, $100 nM, $10 nM, $1 nM, $0.1 nM, 20.01 nM, or $0.001 nM (eg, 10·8 M or 10·). Below 8 M, for example, ΙΟ·8 Μ to ΙΟ·13 Μ, for example ΙΟ·9 Μ to 1 Ο·13 Μ). In certain embodiments, the anti-Complex I antibody binds to an epitope of the Complex I that is conserved between human CMV isolates. In certain embodiments, the anti-Complex I antibody binds to a conserved epitope of the complex I between CMV strains infected with different species. In certain embodiments, an "anti-Complex I antibody" binds to a conformational epitope of Complex I and, in certain embodiments, an anti-Complex J antibody and an individual protein member of Complex I that is not gH ( That is, the epitope binding within gL, UL128, UL130 or UL131). The terms "anti-gH antibody" and "antibody that binds to gH" refer to an antibody capable of binding gH with sufficient affinity to make the antibody suitable as a diagnostic agent and/or therapeutic agent for targeting gH. In one embodiment, the anti-gH antibody binds to an unrelated non-gH protein to a lesser extent than about 1% of the antibody binds to gH, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that binds to 158864.doc •16-201215618 gH is $1, $100 nM, £10 nM, $1 nM, S0.1 nM, $0.01 nM, or $0.001 nM (eg, L〇·8 μ or l〇·8 M or less, for example, 10·8 μ to ι〇-13 Μ, for example, 10·9 M to l〇·13 Μ) ° In some embodiments, anti-gH antibody and imprint Conserved epitope binding between human cmv isolates. In certain embodiments, the anti-§11 antibody binds to an epitope determinant of gH that is conserved between cmv strains of different species. The term "antibody" is used herein in its broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as they exhibit The desired antigen binding activity can be. "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies; line antibodies; single-chain antibody molecules (eg, scFv); A multispecific antibody is formed. An antibody that "binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 5% or more in a competitive assay and, conversely, the 5 reference antibody is blocked in the competition assay. The antibody binds to its antigen for up to 50% or more. This document provides an exemplary competition check. The term "chimeric" anti-system refers to a portion of a heavy chain and/or a light chain that is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from antibodies from different sources or species.矣.. 158864.doc -17· 201215618 The “category” of an antibody refers to the type of constant or constant region of its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgGi, IgG2, IgG3, IgG4, IgA!, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.
除非另外指示,否則如本文所用之術語「複合物I」係 指來自任何細胞巨大病毒來源,包括感染諸如靈長類動物 (例如人類)及齧齒動物(例如小鼠及大鼠)之哺乳動物之 CMV的任何天然複合物I。該術語涵蓋所有gH、gL、 UL128、UL130及UL131多肽之組合。該術語亦涵蓋複合 物I之蛋白質之天然存在的變異體,例如剪接變異體或對 偶基因變異體。例示性HCMV gH之胺基酸序列展示於SEQ ID ΝΟ:1中。例示性HCMV gL之胺基酸序列展示於SEQ ID NO:2中。例示性HCMV UL 128之胺基酸序列展示於SEQ ID NO:3中。例示性HCMV UL130之胺基酸序列展示於SEQ ID NO:4中。例示性HCMV UL 131之胺基酸序列展示於SEQ ID NO:5 中。HCMV gH、gL、UL128、UL130 及 UL131 之其他 例示性序列可見於Genbank寄存編號GU179289(Dargan等 人,Gei F/ro/. 91: 1535-1546 (2010))中,其以全文引用 的方式併入本文中,且以SEQ ID NO:206(gH)、SEQ ID NO:208(gL) 、 SEQ ID NO:205(UL 128) ' SEQ ID NO:204(UL130)及SEQ ID NO:203(UL131)包括在本文中。 除非另外指示’否則如本文所用之術語「複合物II」係 指來自任何細胞巨大病毒來源,包括感染諸如靈長類動物 •18· 158864.doc 201215618The term "complex I" as used herein, unless otherwise indicated, refers to a source of macrovirus from any cell, including mammals infected with, for example, primates (eg, humans) and rodents (eg, mice and rats). Any natural complex I of CMV. The term encompasses all combinations of gH, gL, UL128, UL130 and UL131 polypeptides. The term also encompasses naturally occurring variants of the protein of Complex I, such as splice variants or dual gene variants. The amino acid sequence of an exemplary HCMV gH is shown in SEQ ID ΝΟ:1. The amino acid sequence of an exemplary HCMV gL is shown in SEQ ID NO: 2. An exemplary amino acid sequence of HCMV UL 128 is shown in SEQ ID NO:3. An exemplary amino acid sequence of HCMV UL130 is shown in SEQ ID NO:4. An exemplary amino acid sequence of HCMV UL 131 is shown in SEQ ID NO:5. Other exemplary sequences for HCMV gH, gL, UL128, UL130, and UL131 can be found in Genbank Accession No. GU179289 (Dargan et al, Gei F/ro/. 91: 1535-1546 (2010)), which is incorporated by reference in its entirety. SEQ ID NO: 206 (gH), SEQ ID NO: 208 (gL), SEQ ID NO: 205 (UL 128) ' SEQ ID NO: 204 (UL130) and SEQ ID NO: 203 (UL131) ) is included in this article. The term "complex II" as used herein, unless otherwise indicated, refers to a source of macrovirus from any cell, including infections such as primates. 18 158864.doc 201215618
(例如人類)及齧齒動物(例如小鼠及大鼠)之哺乳動物之 CMV的任何天然複合物II。該術語涵蓋所有gH、gL及gO 之組合。該術語亦涵蓋複合物II之蛋白質之天然存在的變 異體,例如剪接變異體或對偶基因變異體。例示性HCMV gH之胺基酸序列展示於SEQ ID ΝΟ:1中。例示性HCMV gL 之胺基酸序列展示於SEQ ID NO:2中。例示性HCMV gO之 胺基酸序列展示於SEQ ID NO:209中。HCMV gH、gL及gO 之其他例示性序列可見於Genbank寄存編號GUI79289 (〇3^&11等人,乂6!烈.6>0/.91:1535-1546 (2〇1〇))中,其以 全文引用的方式併入本文中,且以SEQ ID NO,.206(gH)、 SEQ ID NO:208(gL)及 SEQ ID NO: 207(gO)包括在本文 中。 除非另外指示,否則如本文所用之術語「gH」係指來自 任何脊椎動物來源,包括諸如靈長類動物(例如人類)及齧 齒動物(例如小鼠及大鼠)之喷乳動物的任何天然gH。該術 語涵蓋「全長」未加工gH以及由在細胞中加工所產生之任 何形式的gH。該術語亦涵蓋gH之天然產生之變異體,例 如剪接變異體或對偶基因變異體。在CMV分離株之間, gH之胺基酸序列為約95%—致。例示性HCMV gH之胺基酸 序列展示於SEQ ID ΝΟ:1中。HCMV gH之另一例示性序列 可見於 Genbank 寄存編號 GUI 79289(Dargan 等人,《/. Gen. Kz>o/. 91: 1535-1546 (2010))中,其以全文引用的方式併入 本文中,且以SEQIDNO:206(gH)包括在本文中。 如本文所用之術語「細胞毒性劑」係指能抑制或阻止細 158864.doc -19· 201215618 胞功能及/或導致細胞死亡或破壞之物質。細胞毒性劑包 括(但不限於)放射性同位素(例如At2"、I131、I125、Y9〇、 Re186、Re188、Sm153、Bi212、Pi pb2^Lu之放射性同位 素);化學治療劑或藥物(例如曱胺喋呤(methotrexate)、阿 德力黴素(adriamicin)、長春花鹼(vinca alkaloid)(長春新驗 (vincristine)、長春鹼(vinblastine)、依託泊苷(etoposide))、 小紅莓(doxorubicin)、美法侖(meiphalan)、絲裂黴素 C(mitomycin C)、苯丁 酸氮芥(chl〇rambucil)、道諾徽素 (daunorubicin)或其他嵌入劑);生長抑制劑;酶及其片 段’諸如核分解酶;抗生素;毒素’諸如小分子毒素或細 菌、真菌、植物或動物來源之酶促活性毒素,包括其片段 及/或變異體;及以下揭示之各種抗腫瘤或抗癌劑。 「效應功能」係指可歸因於抗體之Fc區之彼等生物活 性’其隨抗體同型而變化。抗體效應功能之實例包括: C 1 q結合及補體依賴性細胞毒性(CDC) ; Fc受體結合;抗 體依賴性細胞介導之細胞毒性(ADCC);吞噬作用;細胞 表面受體(例如B細胞受體)之下調;及8細胞活化。 藥劑(例如醫藥調配物)之「有效量」係指在必需劑量下 且在必需時間内可有效達成所要治療或防治結果之量。 術語「Fc區」在本文中用於定義免疫球蛋白重鏈之含有 至 > 一部分恆定區之c端區。該術語包括天然序列Fc區及 變異Fc區。在—實施例中,人類IgG重鏈Fc區自ο。%或 自Pro230延伸至重鏈之缓基端。然而,區之c端離胺酸 (Lys447)可存在或可不存在。除非本文中另外規定,否則 I58864.doc 201215618Any natural complex II of CMV in mammals (e.g., humans) and rodents (e.g., mice and rats). This term covers all combinations of gH, gL and gO. The term also encompasses naturally occurring variants of the protein of Complex II, such as splice variants or dual gene variants. The amino acid sequence of an exemplary HCMV gH is shown in SEQ ID ΝΟ:1. The amino acid sequence of an exemplary HCMV gL is shown in SEQ ID NO:2. The amino acid sequence of an exemplary HCMV gO is shown in SEQ ID NO:209. Other exemplary sequences of HCMV gH, gL and gO can be found in Genbank accession number GUI79289 (〇3^&11 et al., 乂6! 烈.6>0/.91:1535-1546 (2〇1〇)) , which is incorporated herein by reference in its entirety, and is incorporated herein by reference to SEQ ID NO,.206 (gH), SEQ ID NO: 208 (gL) and SEQ ID NO: 207 (gO). The term "gH" as used herein, unless otherwise indicated, refers to any natural gH from any vertebrate source, including sprayed animals such as primates (eg, humans) and rodents (eg, mice and rats). . The term covers "full length" unprocessed gH and any form of gH produced by processing in cells. The term also encompasses naturally occurring variants of gH, such as splice variants or dual gene variants. Between the CMV isolates, the amino acid sequence of gH is about 95%. The amino acid sequence of an exemplary HCMV gH is shown in SEQ ID ΝΟ:1. Another exemplary sequence of HCMV gH can be found in Genbank Accession No. GUI 79289 (Dargan et al., /. Gen. Kz'o/. 91: 1535-1546 (2010)), which is incorporated herein by reference in its entirety. Medium, and is included herein as SEQ ID NO: 206 (gH). The term "cytotoxic agent" as used herein refers to a substance which inhibits or prevents the function of cells and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (eg, radioisotopes of At2", I131, I125, Y9〇, Re186, Re188, Sm153, Bi212, Pi pb2^Lu); chemotherapeutic agents or drugs (eg, amidoxime) Meth (methotrexate), adriamicin, vinca alkaloid (vincristine, vinblastine, etoposide), cranberry (doxorubicin), Meiphalan, mitomycin C, chl〇rambucil, daunorubicin or other intercalating agents; growth inhibitors; enzymes and fragments thereof Such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below. "Effective function" refers to the biological activity attributable to the Fc region of an antibody' which varies with the antibody isotype. Examples of antibody effector functions include: C 1 q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cells) Receptor) downregulation; and 8 cell activation. An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result at the required dosage and within the time required. The term "Fc region" is used herein to define the inclusion of an immunoglobulin heavy chain to > a portion of the constant region of the c-terminal region. The term includes native sequence Fc regions and variant Fc regions. In the examples, the human IgG heavy chain Fc region is from ο. % or extend from Pro230 to the slow base of the heavy chain. However, the c-terminus of the region may or may not be present in the amine acid (Lys447). Unless otherwise stated herein, I58864.doc 201215618
Fc區或怪定區中胺基酸殘基之編號係根據eu編號系統, 亦稱為EU索引’如 Kabat等人,o/Proie/w·? 〇/ /卿m«o/〇抑α/ /价㈣",第 5 版,pubHc Health Service,The number of amino acid residues in the Fc region or in the strange region is based on the eu numbering system, also known as the EU index 'as Kabat et al., o/Proie/w·? 〇 / / qing m «o / α α / / Price (4) ", 5th edition, pubHc Health Service,
National Institutes of Health, Bethesda,MD,1991 中所述。 「構架」或「FR」係指除高變區(Hvr)殘基以外之可變 域殘基。可變域之FR通常由4個FR域:FR1、FR2、FR3及 FR4組成。因此,HVR及FR序列在VH(或VL)中通常依以下 順序出現:FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。 術語「全長抗體」、「完整抗體」及「完全抗體」在本文 中可互換使用來指代結構實質上類似於天然抗體結構或具 有含有如本文所定義之Fc區之重鏈的抗體。 術語「宿主細胞」、「宿主細胞株」及r宿主細胞培養 物」可互換使用且指代已引入有外源性核酸之細胞,包括 此等細胞之子代。宿主細胞包括「轉型體」及「轉型細 胞」,其包括初級轉型細胞及由其獲得之子代而不考慮繼 代次數。子代在核酸含量上可能不完全與親本細胞相同, 而是可能含有突變。本文中包括具有與在原始轉型細胞中 所篩檢或選擇相同之功能或生物活性的突變子代。 「人類抗體」為胺基酸序列對應於由人類或人類細胞產 生或源於非人類來源之抗體之胺基酸序列的抗體,該非人 類來源利用人類抗體譜系或其他人類抗體編碼序列。人類 抗體之此定義明確排除包含非人類抗原結合殘基之人類化 抗體。 「人類共同構架」為代表所選人類免疫球蛋白VL或Vh 158864.doc -21 · 201215618 構架序列中最常出現之胺基酸殘基的構架。一般而言,人 類免疫球蛋白VL或VH序列選自可變域序列之子群。一般 而έ ’序列子群為如Kabat等人,National Institutes of Health, Bethesda, MD, 1991. "Framework" or "FR" refers to a variable domain residue other than a hypervariable region (Hvr) residue. The FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The terms "full length antibody", "intact antibody" and "complete antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein. The terms "host cell," "host cell strain, and r-host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and their derived progeny regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein. A "human antibody" is an antibody having an amino acid sequence corresponding to an amino acid sequence produced by a human or human cell or derived from an antibody of non-human origin, the non-human source utilizing a human antibody lineage or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. The "human common framework" is the framework representing the most frequently occurring amino acid residues in the framework of human immunoglobulin VL or Vh 158864.doc -21 · 201215618. In general, the human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. In general, the ’ _ sequence subgroup is as Kabat et al.
Immunological Interest,第 5 版,NIH 出版物 91_3242Immunological Interest, 5th edition, NIH Publication 91_3242
Bethesda MD (1991),第1-3卷中之子群。在一實施例中, 對於VL,子群為如Kabat等人(同上)中之子群κ I。在一實 施例中,對於VH ’子群為如Kabat等人(同上)中之子群 III。 「人類化」抗體係指包含來自非人類HVR之胺基酸殘基 及來自人類FR之胺基酸殘基的嵌合抗體。在某些實施例 中’人類化抗體將包含至少1個且通常2個可變域之實質上 全部’其中全部或實質上全部HVR(例如CDR)皆對應於非 人類抗體之HVR,且全部或實質上全部FR皆對應於人類抗 體之FR。人類化抗體視情況可包含源於人類抗體之抗體值 定區的至少一部分。抗體之「人類化形式」,例如非人類 抗體,係指已進行人類化之抗體。 如本文所用之術語「高變區」或「HVR」係指抗體可變 域中在序列上高度可變及/或形成結構確定之環(「高變 環」)的各區。一般而言,天然四鏈抗體包含6個HVR ; 3 個在 VH 中(HI、H2、H3),且 3個在 VL 中(LI、L2、L3)。 HVR通常包含來自高變環及/或來自「互補決定區」(cdR) 之胺基酸殘基,互補決定區具有最高序列可變性及/或參 與抗原識別。例示性高變環存在於胺基酸殘基26-32(L 1)、 50-52(L2)、91-96(L3)、26_32(H1)、53-55(H2)及 96- 158864.doc • 22- 201215618 101(H3)處。(Chothia 及 Lesk, J. Μο/· 5ζ·ο/· 196:901-917 (1987))。例示性 CDR(CDR-L1、CDR-L2、CDR-L3、CDR-HI、CDR-H2及CDR-H3)存在於LI之胺基酸殘基24-34、L2 之胺基酸殘基50-56、L3之胺基酸殘基89-97、HI之胺基酸 殘基31-35B、H2之胺基酸殘基50-65、及H3之胺基酸殘基 95-102 處。(Kabat 等人,》Segwences ο/' 〇/Bethesda MD (1991), subgroups in volumes 1-3. In one embodiment, for VL, the subgroup is a subgroup κ I as in Kabat et al. (supra). In one embodiment, the subgroup of VH' is subgroup III as in Kabat et al. (supra). A "humanized" anti-system refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In certain embodiments, a 'humanized antibody will comprise substantially all of at least 1 and typically 2 variable domains' wherein all or substantially all of the HVRs (eg, CDRs) correspond to HVRs of non-human antibodies, and all or Essentially all FRs correspond to the FR of human antibodies. The humanized antibody may optionally comprise at least a portion of the antibody value region derived from the human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized. The term "hypervariable region" or "HVR" as used herein refers to regions of the variable region of the antibody that are highly variable in sequence and/or form a structurally defined loop ("high change loop"). In general, the native four-chain antibody contains 6 HVRs; 3 in VH (HI, H2, H3) and 3 in VL (LI, L2, L3). The HVR typically comprises an amino acid residue from a hypervariable loop and/or from a "complementarity determining region" (cdR), the complementarity determining region having the highest sequence variability and/or involvement in antigen recognition. Exemplary hypervariable loops are present in amino acid residues 26-32 (L 1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-158864. Doc • 22- 201215618 101 (H3). (Chothia and Lesk, J. Μο/· 5ζ·ο/· 196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-HI, CDR-H2, and CDR-H3) are present in amino acid residues 24-34 of L, amino acid residues 50 of L2. 56. Amino acid residues 89-97 of L3, amino acid residues 31-35B of HI, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3. (Kabat et al., "Segwences ο/' 〇/
Immunological Interest,第 5 版 Public Health Service, National Institutes of Health, Bethesda, MD (1991))。除 VH 中之CDR1之外,CDR通常包含形成高變環之胺基酸殘 基。CDR亦包含作為接觸抗原之殘基之「特異性決定殘 基」或「SDR」。SDR包含於CDR之稱為簡化-CDR (abbreviated-CDR)或 a-CDR 的區域内。例示性&-匚011(&-CDR-L1、a-CDR-L2 ' a-CDR-L3 ' a-CDR-Hl、a-CDR-H2 及a-CDR-H3)存在於LI之胺基酸殘基31-34、L2之胺基酸殘 基50-55、L3之胺基酸殘基89-96、H1之胺基酸殘基31-35B、H2之胺基酸殘基50-58及H3之胺基酸殘基95-102處。 (參見 Almagro 及 Fransson, Front. Biosci. 13:1619-1633 (2008))。除非另外指示,否則HVR殘基及可變域中之其他 殘基(例如FR殘基)在本文中根據Kabat等人(同上)進行編 號。 「免疫結合物」為與一或多個異源分子,包括(但不限 於)細胞毒性劑結合之抗體。 「個體(individual/subject)」為哺乳動物。哺乳動物包 括(但不限於)馴化動物(例如母牛、綿羊、貓、狗及馬)、 158864.doc -23- 201215618 靈長類動物(例如人類及非人類靈長類動物,諸如猴)、兔 及齧齒動物(例如小鼠及大鼠)。在某些實施例中,個體為 人類。 如本文所用之「嬰兒」係指年齡自出生至不超過約!歲 之範圍内之個體且包括〇至約12個月之嬰兒。 「經分離」抗體為已與其天然環境之組分分離之抗體。 在些貫施例中,如藉由例如電泳(例如SDS-PAGE電泳、 等電聚焦(IEF)電泳、毛細管電泳)或層析(例如離子交換或 逆相HPLC)所測疋,抗體經純化至純度大於%%或μ%。 關於評估抗體純度之方法的综述,參見例如_職等人, 丄 Chromatogr. B U%:7947 (2QQ7)。 「經分離」核酸係指已與其天然環境之組分分離之核酸 分子。經分離核酸包括如下核酸分子:該核酸分子含於通 常含有該核酸分子之細胞中’但該核酸分子存在於染色體 外或不同於其天然染色體位置之染色體位置處。 「編碼抗複合物!抗體之經分離核酸」係指編碼抗體重 鏈及輕鏈(或其片段)之—或多種核酸分子 或各別載艘中之此(等)核酸分子及存在於宿主細:中= 或多個位置處之此(等)核酸分子。 編碼抗gH抗體之經分離核酸」係指編碼抗體重鍵及輕 =(或其片·Μ之一或多種核酸分子’包括單—載體或各別 體中之此(等)核酸分子及存在於宿主細胞中之—或多個 位置處之此(等)核酸分子。 如本文所用之術語「單株抗體」係指自實質上均質之抗 158864.doc -24- 201215618Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)). In addition to CDR1 in VH, CDRs typically comprise an amino acid residue that forms a hypervariable loop. The CDR also contains "specificity determining residues" or "SDR" as residues that contact the antigen. The SDR is included in the region of the CDR called the abbreviated-CDR or a-CDR. Illustrative &-匚011 (&-CDR-L1, a-CDR-L2 ' a-CDR-L3 ' a-CDR-Hl, a-CDR-H2 and a-CDR-H3) are present in the amine of LI Acidic acid residues 31-34, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50 of H2 58 and H3 amino acid residues 95-102. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (supra). An "immunoconjugate" is an antibody that binds to one or more heterologous molecules, including, but not limited to, a cytotoxic agent. "Individual/subject" is a mammal. Mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), 158864.doc -23- 201215618 primates (eg, humans and non-human primates, such as monkeys), Rabbits and rodents (eg mice and rats). In some embodiments, the individual is a human. As used herein, "infant" means age from birth to no more than about! Individuals within the age range include infants up to approximately 12 months of age. An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified by, for example, electrophoresis (eg, SDS-PAGE electrophoresis, isoelectric focusing (IEF) electrophoresis, capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC). The purity is greater than %% or μ%. For a review of methods for assessing antibody purity, see, for example, _ grade, 丄 Chromatogr. B U%: 7947 (2QQ7). "Separated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule' but the nucleic acid molecule is present at a chromosomal location that is extrachromosomic or different from its natural chromosomal location. "Isolated nucleic acid encoding an anti-complex! antibody" means a nucleic acid molecule encoding an antibody heavy and light chain (or a fragment thereof) or a plurality of nucleic acid molecules or individual carriers, and is present in the host : This (etc.) nucleic acid molecule at medium = or multiple positions. An isolated nucleic acid encoding an anti-gH antibody refers to a nucleic acid molecule encoding an antibody heavy bond and light = (or one or more of its nucleic acid molecules 'including a single vector or individual, and is present in The nucleic acid molecule at or at a plurality of locations in the host cell. The term "monoclonal antibody" as used herein refers to a substantially homogeneous anti-158864.doc -24 - 201215618
體之群體獲得的抗體,亦即除可能之變異抗體(例如含有 天然產生之突變或在產生單株抗體製_間出現之變 體,此等變異體通常以較小量存在)之外,構成該群體: 個別抗體相同及/或結合相同抗原決定基。與通常 對不同。決定子(抗原決定基)之不同抗體之多株抗體製劑不 同,早株抗體製劑之各單株抗體針對抗原上之單一決定 子。因此’修飾語「單株」#示抗體係自實質上均質:: 體群體獲得之特性,且不應_為需要藉由任何特定方= 來產生抗體。舉例而言,欲根據本發明使用之單株抗體可 藉由多種技術製備,該等技術包括(但不限於)融合瘤法、 重組DNA法、噬菌體里現法及利用含有所有或部分人類免 疫球蛋白基因座之轉殖基因動物的方法,此等方法及製備 單株抗體之其他例示性方法在本文中描述。 裸抗體」係指未與異源部分(例如細胞毒性部分)或放 射性標記結合之抗體。裸抗體可存在於醫藥調配物中。 「天然抗體」係指具有不同結構之天然產生之免疫球蛋 白分子。舉例而言,天然IgG抗體為約15〇,〇〇〇道爾頓 (dalton)之雜四聚醣蛋白,由經二硫鍵鍵結之2條相同輕鏈 及2條相同重鏈構成。自N端至(:端,各重鏈具有可變區 (VH) ’亦稱為可變重域或重鏈可變域,繼而為3個恆定域 (CHI、CH2及CH3)。類似地,自n端至C端’各輕鏈具有 可變區(VL) ’亦稱為可變輕域或輕鏈可變域,繼而為恒定 輕(CL)域。抗體之輕鏈可基於其恆定域之胺基酸序列歸為 稱為κ及λ之兩種類型之一。 158864.doc 201215618 藥品說明蚩 術 羋包f中」用於指代通常包括在治療產品之商 糸〇农T之説明書, 之適應症、用木 έ有關於與使用此等治療產品有關 警告之資訊。、劑量、投藥、組合療法、禁忌症及/或 。相料參照多肽序^「胺基酸序列—致性百分比 '義為在對準參照多肽序列與候選序列且必要時 引入二隙以達成最大序列—致性百分比之後且在不將任 何保守ft取代視為序列—致性之-部分的情況下,候選序 列中〃參照多肽序列中之胺基酸殘基一致之胺基酸殘基的 百刀比出於確定胺基酸序列一致性百分比之目的之比對 可以此項技術中之技能範圍内的各種方式達成,例如使用 可公開獲得之電腦軟體,諸如BLAST、BLAST-2、ALIGN 或Megahgn(DNASTAR)軟體。熟習此項技術者可決定適於 比對序列之參數,包括為在所比較序列之全長上達成最大 對準所高的任何演算法。然而,出於本文之目的,使用序 列比較電腦程式ALIGN-2來產生胺基酸序列一致性百分比 值。ALIGN-2序列比較電腦程式由Genentech,Inc.創作, 且源程式碼已與使用說明書一起在美國版權局(u s.An antibody obtained by a population of humans, that is, in addition to a possible variant antibody (for example, a variant containing a naturally occurring mutation or a variant produced in the production of a monoclonal antibody, which is usually present in a smaller amount) This population: Individual antibodies are identical and/or bind to the same epitope. Different from the usual pair. The multiple antibody preparations of the different antibodies of the determinant (antigenic determinant) are different, and the individual antibody of the early antibody preparation is directed against a single determinant on the antigen. Therefore, the modifier "single plant" # indicates that the resistance system is substantially homogeneous: the characteristics obtained by the body population, and should not be - required to produce antibodies by any particular party =. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, fusion knob methods, recombinant DNA methods, phage display methods, and utilization of all or part of human immunoglobulins. Methods of transgenic animal species of protein loci, such methods and other exemplary methods of making monoclonal antibodies are described herein. "Naked antibody" refers to an antibody that does not bind to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. Naked antibodies can be present in pharmaceutical formulations. "Native antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is about 15 Å, a heterotetrameric protein of dalton, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the (: terminal, each heavy chain has a variable region (VH) 'also known as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, From the n-terminus to the C-terminus, each light chain has a variable region (VL) 'also known as a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be based on its constant domain The amino acid sequence is classified into one of two types called κ and λ. 158864.doc 201215618 Drug Description 蚩 芈 f f ” ” Indications, use of hibiscus, information about warnings related to the use of such therapeutic products, dosage, dosing, combination therapy, contraindications and/or phase reference peptide sequence ^ "amino acid sequence - percentage of 'Sense' is in the case where the reference polypeptide sequence is aligned with the candidate sequence and, if necessary, the two gaps are introduced to achieve the maximum sequence--percentage percentage, and without any conservative ft substitutions as part of the sequence-sense, the candidate sequence The ratio of the amino acid residues of the amino acid reference amino acid residues in the reference polypeptide sequence is The purpose of determining the percent identity of the amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megahgn (DNASTAR) software. Those skilled in the art can determine any parameters suitable for the alignment sequence, including any algorithms that are high for achieving maximum alignment over the full length of the sequences being compared. However, for the purposes of this document, the sequence comparison computer program ALIGN is used. -2 to generate amino acid sequence identity percentage values. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been included with the instruction manual at the US Copyright Office (u s.
Copyright Office)(Washington D.C., 20559)存檔,在該版 權局其以美國版權登記號TXU510087登記。ALIGN-2程式 可自 Genentech,Inc.(South San Francisco,California)公開 獲得,或可自源程式碼編譯。ALIGN-2程式應經編譯以在 UNIX操作系統(包括數位UNIX V4.0D)上使用。所有序列 比較參數皆由ALIGN-2程式設定且不變化。 158864.doc -26 · 201215618 在採用AUGN-2進行胺基酸序列比較之情況下,如下計 鼻指定胺基酸序列A對於、#、或相對於指定胺基酸序列 B之胺基酸序列-致性%(其或者可表料指定胺基酸序列 A對於、與、或相對於指定胺基酸序列8具有或包含某一 胺基酸序列一致性:Copyright Office) (Washington D.C., 20559) is filed with the copyright office in the United States copyright registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. 158864.doc -26 · 201215618 In the case of amino acid sequence comparison using AUGN-2, the amino acid sequence A is assigned to the amino acid sequence A, #, or amino acid sequence relative to the designated amino acid sequence B as follows - % (which may or may not specify amino acid sequence A for, with, or with respect to a given amino acid sequence 8 having or comprising an amino acid sequence identity:
l〇〇x 分數 X/YL〇〇x score X/Y
其中X為由序列比對程式a L J G N _2在A與B之彼程式比對中 計分為-致匹配之胺基酸殘基的數目,且其中¥為3中胺 基酸殘基的總數。應瞭解,#胺基酸序列A之長度與胺基 酸序列B之長度不相等時,八與8之胺基酸序列一致性%將 不等於B與A之胺基酸序列一致性%。除非另外明確陳述, 否則本文中使用之所有胺基酸序列一致性%值皆使用 ALIGN-2電腦程式如前一段落中所述獲得。 術語「醫藥調配物」係指以下製劑:其呈使得允許其中 所含之活性成分之生物活性有效的形式且其不含有對該 調配物將投與之個體具有不可接受之毒性的其他組分。" 「醫藥學上可接受之載劑」係指除活性成分以外,醫藥 調配物中對個體無毒之成分。醫藥學上可接受之載劑包括 (但不限於)緩衝劑、賦形劑、穩定劑或防腐劑。 本文所用,治療」(及其語法變化形式)係指試圖改 變所治療個體之天,然病程之臨床干預,且可出於防治目的 或在臨床病理學之過程期間執行。治療之所需效果包括 (但不限於)預防疾病發生或復發、減輕症狀、減弱疾=之 任何直接或間接病理學後果、防止轉移、降低疾病進展速 158864.doc -27. 201215618 率、改善或緩和疾病病況、及缓解或改良預後。在一些實 施例中,本發明組合物用於延遲疾病發展或減緩疾病進展 或降低疾病之發病率或疾病症狀之叙重性。 術語「可變區」或「可變域」係指抗體重鏈或輕鏈中參 與抗體與抗原之結合的域。天然抗體之重鏈及輕鏈之可變 域(分別為VH及VL)通常具有類似結構’其中各域包含4個 保守構架區(FR)及3個高變區(HVR)。(參見例如Kindt等人, /所幻;,第 6版,W.H. Freeman and Co.,第 91 頁 (2007)) 單一 VH或VL域可足以賦予抗原結合特異性。此 φ 外,結合特定抗原之抗體可使用來自結合該抗原之抗體的 VH或VL·域分另ij篩檢互補VL·域或VH域之文庫進行分離。 參見例如 Portolano等人,《/· 7mm««〇/· 150:880-887 (1993); Clarkson等人,352:624-628 (1991)。 如本文所用之術語「載體」係指一種核酸分子,其能夠 使與其連接之另一核酸增殖。該術語包括呈自我複製型核 酸結構(self-replicating nucleic acid structure)形式之載體 以及併入其所引入之宿主細胞之基因組中的載體。某些載 籲 體能夠引導其可操作地連接之核酸之表現。此等載體在本 文中稱為「表現載體」。 II.組合物及方法 在一態樣中’本發明部分基於發現中和HCMV感染之感 染之單株抗體。在某些實施例中,提供與複合物!結合之 抗體U實知例中,提供與gH結合之抗體。本發明抗 體適用於例如預防、抑制及/或治療感染、先天性 158864.doc •28· 201215618 HCMV感染及由受HCMV感染之所移植組織引起的患者感 染。抗體亦可用於診斷HCMV感染。 在一態樣中,本發明亦部分基於發現包含單株抗體之組 合之組合物,該等單株抗體抑制HCMV病毒進入胎盤之以 下所有細胞類型中:内皮細胞、上皮細胞、單核細胞/巨 噬細胞及纖維母細胞,且減少及/或抑制HCMV抗性株形 成。在某些實施例中,提供使用此等組合物之方法。本發 明組合物適用於例如預防、抑制及/或治療HCMV感染、先 天性HCMV感染及由已自先前或目前受HCMV感染之患者 收集的受HCMV感染之所移植器官或組織引起的患者感 染。組合物亦可用於診斷HCMV感染。 A.例示性抗複合物I抗體 在一態樣中,本發明提供與複合物I結合之經分離抗 體。在某些實施例中,抗複合物I抗體與由UL128、 UL130、UL131與gH/gL締合產生之構形抗原決定基或在複 +合物I之個別成員内之抗原決定基特異性結合。在一些實 施例中,抗複合物I抗體中和HCMV之EC90為0.7 pg/ml、 0.5 pg/ml、0·3 pg/ml、0.1 pg/ml、0.09 pg/ml、0.08 pg/ml、0.07 pg/ml、0.06 pg/ml、0.05 pg/ml、0.04 pg/ml、0.03 pg/ml、0.02 pg/ml、0.015 pg/ml、0.012 pg/ml、0.011 pg/ml、0.010 pg/ml 或 0.010 pg/ml 以下。在 其他態樣中,抗複合物I抗體與HCVM表面上之複合物I特 異性結合且在以下抗體濃度下中和50% HCMV : 0.05 pg/ml ' 0.02 pg/ml、0.015 pg/ml、0.014 pg/ml、0.013 158864.doc -29- 201215618 pg/ml、0.012 pg/ml、0.011 pg/ml、0.010 pg/ml、0.009 pg/ml、0.008 pg/ml、0.007 pg/ml、0_006 pg/ml ' 0.005 pg/ml、0.004 pg/ml、0.003 pg/ml、0.002 pg/ml、0.001 pg/ml、0.0009 pg/ml、0.0008 pg/ml、0.0007 pg/ml 或 0.0007 pg/ml 以下(例如在 ΙΟ.8 Μ、ΙΟ—9 Μ、ΙΟ-10 M、10_11 Μ、10·12Μ、10·13Μ或l〇-13M以下之抗體濃度下)。 在一態樣中,本發明提供一種包含至少1個、2個、3 個、4個、5個或6個選自以下之HVR的抗複合物I抗體:(a) 包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包含胺基酸 序列SEQ ID NO:7之HVR-H2 ; (c)包含胺基酸序列SEQ ID NO:8之HVR-H3 ; (d)包含胺基酸序列SEQ ID NO:9之HVR-Ll ; (e)包含選自由SEQ ID NO: 10-19組成之群之胺基酸序 列之HVR-L2 ;及(f)包含胺基酸序列SEQ ID NO:20之HVR-L3。 在一態樣中,本發明提供一種包含至少1個、至少2個或 全部3個選自以下之VH HVR序列之抗體:(a)包含胺基酸序 列SEQ ID NO:6之HVR-H1 ; (b)包含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸序列SEQ ID NO:8之 HVR-H3。 在另一態樣中,本發明提供一種包含至少1個、至少2個 或全部3個選自以下之VL HVR序列之抗體:(a)包含胺基酸 序列8£(^10 1^0:9之11¥11-1^;(15)包含選自3丑9 10>10:10-19之胺基酸序列之HVR-L2 ;及(c)包含胺基酸序列SEQ ID NO:20之 HVR-L3。 158864.doc •30- 201215618 在一實施例中,抗體包含全部3個選自以下之VH HVR序 列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包含 胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸序 列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:10之HVR-L2 ;及(c)包含胺基酸序 列 SEQ ID NO:20之HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID ΝΟ:11之HVR-L2 ;及(c)包含胺基酸序 列 SEQ ID NO:20之HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQIDNO:7之HVR-H2;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:12之HVR-L2 ;及(c)包含胺基酸序 列 SEQ ID NO:20之 HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸 Ο 158864.doc -31 - 201215618 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之Vl HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:13之HVR-L2;及(c)包含胺基酸序 列 SEQ ID NO:20之 HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:14之HVR-L2;及(c)包含胺基酸序 列 SEQ ID NO:20之 HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR_L1 ; (b)包含 胺基酸序列SEQ ID NO:15之HVR-L2;及(c)包含胺基酸序 列 SEQ ID NO:20之 HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:16之HVR-L2;及(c)包含胺基酸序 158864.doc -32- 201215618 列 SEQ ID NO:20之HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:17之HVR-L2 ;及(c)包含胺基酸序 列 SEQ ID NO:20之HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:18之HVR-L2 ;及(c)包含胺基酸序 列 SEQ ID NO:20之HVR-L3。 在另一實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:19之HVR-L2 ;及(c)包含胺基酸序 列 SEQ ID NO:20之HVR-L3。 在一些實施例中,抗體包含全部3個選自以下之VH HVR 序列:(a)包含胺基酸序列SEQ ID NO:6之HVR-H1 ; (b)包 158864.doc -33- 201215618 含胺基酸序列SEQ ID NO:7之HVR-H2 ;及(c)包含胺基酸 序列SEQ ID NO:8之HVR-H3 ;及3個選自以下之VL HVR序 列:(a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b)包含 胺基酸序列SEQ ID NO:21之HVR-L2及輕鏈可變區構架 FR3之第一胺基酸;及(c)包含胺基酸序列SEQ ID NO:20之 HVR-L3。在某些實施例中,如上提供之抗複合物I抗體之 任何一或多個胺基酸在以下HVR位置處經取代:在11乂尺-L2(SEQ ID NO:10)t :位置4、5、11及12。在某些實施例 中,取代為如本文提供之保守性取代。在某些實施例中, 可以任何組合進行任何一或多個以下取代:在HVR-L2(SEQ ID NO:57)中:D4E、D4T、D4S、G5A、D11E、 D11T、D11S及G12A。以上取代之全部可能之組合由SEQ ID NO:21之共同序列涵蓋。 在任何以上實施例中,抗複合物I抗體經人類化。在一 實施例中,抗複合物I抗體包含如任何以上實施例中之 HVR,且進一步包含接受體人類構架,例如人類免疫球蛋 白構架或人類共同構架。在另一實施例中,抗複合物I抗 體包含如任何以上實施例中之HVR,且進一步包含含有 FR1 序列 SEQ ID NO:22、FR2序列 SEQ ID NO:23、FR3序 列SEQ ID NO:24及FR4序列SEQ ID NO:25之VH。在其他實 施例中,抗複合物I抗體包含如任何以上實施例中之 HVR,且進一步包含含有FR1序列SEQ ID NO:22、FR2序 列 SEQ ID NO:27、FR3序列 SEQ ID NO:28及 FR4序列 SEQ ID NO:29之VH。在其他實施例中,抗複合物I抗體包含如 158864.doc -34- 201215618 任何以上實施例中之HVR,且進一步包含含有FR1序列 SEQ ID NO:30、FR2序列 SEQ ID NO:3 1、FR3序列 SEQ ID NO:32及FR4序列SEQ ID NO:25之VH。在其他實施例中, 抗複合物I抗體包含如任何以上實施例中之HVR,且進一 步包含含有FR1序列SEQ ID NO:33、FR2序列SEQ ID NO:23、FR3 序列 SEQ ID NO:34及 FR4序列 SEQ ID NO:25 之VH。 在另一實施例中,抗複合物I抗體包含如任何以上實施 例中之HVR,且進一步包含含有FR1序列SEQ ID NO:35、 FR2序列 SEQ ID NO:36、FR3序列 SEQ ID NO:37及 FR4序 列SEQ ID NO:38之VL。在其他實施例中,抗複合物I抗體 包含如任何以上實施例中之HVR,且進一步包含含有FR1 序列 SEQ ID NO:39 ' FR2 序列 SEQ ID NO:40、FR3 序列 SEQ ID NO:41及FR4序列SEQ ID NO:42之VL。在其他實施 例中,抗複合物I抗體包含如任何以上實施例中之HVR, 且進一步包含含有FR1序列SEQ ID NO:43、FR2序列SEQ ID NO:44、FR3 序列 SEQ ID NO:41 及 FR4 序列 SEQ ID NO:42之 VL。 在任何以上抗體中,VL FR3序列可經選自SEQ ID NO:67 或SEQ ID NO:68之序列取代。 在另一態樣中,抗複合物I抗體包含與胺基酸序列SEQ ID NO:46 或 SEQ ID NO:47 具有至少 90%、91%、92%、 93%、94%、95%、96%、97%、98°/〇、99% 或 100%序列一 致性之重鏈可變域(VH)序列。在某些實施例中,相對於參 158864.doc -35- 201215618 照序列’具有至少 90%、91%、92%、93%、94¼、95°/。、 96%、97%、98%或99%—致性之VH序列含有取代(例如保 守性取代)、插入或缺失,但包含彼序列之抗複合物1抗體 保留與複合物I結合之能力。在某些實施例中,在SEQ ID NO:45 或 SEQ ID NO:46 或 SEQ ID NO:47 中,總計 1 至 10 個 胺基酸已經取代、插入及/或缺失。在某些實施例中’取 代、插入或缺失發生在HVR外部區域中(亦即在FR中)。在 一特定實施例中,VH包含1個、2個或3個選自以下之 HVR : (a)包含胺基酸序列SEQ ID NO:6之HVR-H1,(b)包 含胺基酸序列SEQ ID NO:7之HVR-H2,及(c)包含胺基酸 序列 SEQ ID NO:8之HVR-H3。 在另一態樣中,提供一種抗複合物I抗體,其中該抗體 包含與胺基酸序列SEQ ID NO:48或SEQ ID NO:49具有至 少 90%、91%、92% ' 93%、94% ' 95%、96% ' 97%、 98%、99°/◦或100%序列一致性之輕鏈可變域(VL)。在某些 實施例中,相對於參照序列,具有至少90%、91%、 92%、93%、94%、95%、960/〇、97%、98%或 99%— 致性之 VL序列含有取代(例如保守性取代)、插入或缺失,但包含 彼序列之抗複合物I抗體保留與複合物I結合之能力。在某 些實施例中:在SEQ ID NO:48或SEQ ID NO:49中,總計1 至10個胺基酸已經取代、插入及/或缺失。在某些實施例 中,取代、插入或缺失發生在HVR外部區域中(亦即在FR 中)。在一特定實施例中,VL包含1個、2個或3個選自以下 之HVR: (a)包含胺基酸序列SEQ ID NO:9之HVR-L1 ; (b) I58864.doc -36- 201215618 包含選自SEQIDNO:10-19之胺基酸序列之HVR-L2;及(c) 包含胺基酸序列SEQ ID NO:20之HVR-L3。 在另一態樣中,提供一種抗複合物I抗體,其中該抗體 包含如任何以上提供之實施例中之VH、及如任何以上提供 之實施例中之Vl。在一實施例中,抗體包含分別於SEQ ID NO:45及SEQ ID NO:49中之VH及VL序列,包括彼等序 列之轉譯後修飾。在一實施例中,抗體包含分別於SEQ ID NO:46及SEQ ID NO:49中之VH及VL序列,包括彼等序列之 轉譯後修飾。在另一實施例中,抗體包含分別於SEQ ID NO:47及SEQ ID NO:49中之VH及Vl序列,包括彼等序列之 轉譯後修飾。在另一實施例中,抗體包含分別於SEQ ID NO:45及SEQ ID NO:48中之VH及Vl序列,包括彼等序列之 轉譯後修飾。在另一實施例中,抗體包含分別於SEQ ID >10:46及8£(^10>10:48中之¥^1及乂1^序列,包括彼等序列之 轉譯後修飾。在另一實施例中,抗體包含分別於SEQ ID NO:47及SEQ ID NO:48中之VH及Vl序列,包括彼等序列之 轉譯後修飾。 在另一態樣中,本發明提供一種與本文提供之抗複合物 I抗體競爭及/或與本文提供之抗複合物I抗體結合相同抗原 決定基的抗體。舉例而言,在某些實施例中,提供一種與 抗複合物I抗體競爭及/或與抗複合物I抗體結合相同抗原決 定基之抗體,該抗複合物I抗體包含含有胺基酸序列SEQ ID NO:45-47之VH及含有胺基酸序列SEQ ID NO:48或SEQ ID NO:49之 VL。 158864.doc -37- 201215618 在另一態樣中,本發明提供一種與抗複合物1抗體結合 相同抗原決定基之抗體,該抗原決定基包含對應於選自以 下之胺基酸的胺基酸:在SEQ ID N〇:203之胺基酸位置47 處之麩醯胺酸;在把卩ID NO:203之胺基酸位置51處之離 胺酸;在SEQ ID NO:203之胺基酸位置46處之天冬胺酸及 其組合。構成抗原決定基之相應胺基酸可處於UL13 1胺基 酸序列中之近似相同位置,但可歸因於UL13 1在各種 HCMV病毒株之間的胺基酸序列差異而不同。 在另一態樣中,本發明提供一種與HCMV複合物I之多肽 結合之抗體,其中該多肽包含胺基酸序列SRALPDQTRYK YVEQLVDLTLNYHYDAS(SEQ ID NO:194)。 在另一態樣中,本發明提供一種與本文提供之抗複合物 I抗體結合相同抗原決定基的抗體。在其他態樣中,本發 明提供一種與本文提供之抗複合物I抗體結合相同抗原決 定基之抗體,EC90為 0.7 pg/ml、0.5 pg/ml、0.3 pg/ml、 0.1 pg/ml、0.09 pg/ml、0.08 pg/ml、0.07 pg/ml ' 0.06 pg/ml、0.05 pg/ml、0.04 pg/ml、0.03 pg/ml ' 0.02 pg/ml、0.015 pg/ml、0.012 pg/ml、0.011 pg/ml、0.010 0吕/1111或〇.〇1〇48/11[11以下。在其他態樣中,本發明提供一 種與本文提供之抗複合物I抗體結合相同抗原決定基且在 以下抗體濃度下中和50%之HCMV的抗體:0.05 pg/ml、 0.02 gg/ml、0.015 pg/ml、0.014 pg/ml、0.013 pg/ml、 0.012 pg/ml、0.011 pg/ml、0.010 pg/ml、0.009 pg/ml、 0.008 pg/ml、0.007 pg/ml、0.006 pg/ml、0.005 pg/ml、 158864.doc -38- 201215618 0.004 pg/ml、0.003 pg/ml、0.002 pg/ml、0.001 pg/mi、 0.0009 pg/m卜 0.0008 pg/m卜 0.0007 pg/mi或 0.0007 gg/ml 以下(例如在 ΙΟ·8 Μ、10·9 M、l〇·10 Μ、10·" M、10.12 M、 l〇_13 M或1(T13 M以下之抗體濃度下)。 在本發明之另一態樣中,根據任何以上實施例之抗複合 物I抗體為單株抗體,包括嵌合抗體、人類化抗體或人類 抗體。在一實施例中,抗複合物“充體為抗體片段,例如 Fv、Fab、Fab'、scFv、雙功能抗體或F(aly)2片段。在另一 鲁 實施例中,抗體為全長抗體,例如完整IgGl抗體或如本文 中定義之其他抗體類別或同型。 在另一態樣中,根據任何以上實施例之抗複合物〗抗體 可併有任何如以下章節丨-7中所述之特徵_的一者或任何 特徵之組合。 B·例示性抗gH抗體 在一態樣中,本發明提供與gH結合之經分離抗體。在某 些實施例中,抗gH抗體特異性結合gH之抗原決定基且中 和 HCMV ’ EC90 為 0.8 pg/nU、〇.7 μ§/ιη1、〇 6 μ§Λη1、〇 5 pg/m卜 0·4 pg/m卜 〇·3 pg/mh 〇 2 吨岫卜 〇」^/m卜 〇 〇9Wherein X is the number of amino acid residues which are scored by the sequence alignment program a L J G N _2 in the alignment of A and B, and wherein ¥ is the total number of amino acid residues in 3. It will be appreciated that when the length of the #amino acid sequence A is not equal to the length of the amino acid sequence B, the amino acid sequence identity % of eight and eight will not be equal to the % identity of the amino acid sequence of B and A. All amino acid sequence identity % values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless otherwise stated. The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain other components that have unacceptable toxicity to the individual to which the formulation will be administered. ""Pharmaceutically acceptable carrier" means a component of a pharmaceutical formulation that is not toxic to an individual other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives. As used herein, treatment (and its grammatical variants) refers to a clinical intervention in an attempt to alter the individual being treated, but may be performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating the symptoms, attenuating the disease = any direct or indirect pathological consequences, preventing metastasis, and reducing the rate of disease progression. 158864.doc -27. 201215618 rate, improvement or Alleviate disease conditions and alleviate or improve prognosis. In some embodiments, the compositions of the invention are useful for delaying the progression of a disease or slowing the progression of a disease or reducing the morbidity of a disease or the severity of a disease condition. The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that binds to an antibody and an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) typically have a similar structure' wherein each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., / Magic; 6th Edition, W.H. Freeman and Co., page 91 (2007)) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition to this φ, an antibody that binds to a specific antigen can be isolated using a library derived from a VH or VL domain of an antibody that binds to the antigen, and a complementary VL· domain or VH domain. See, for example, Portolano et al., / 7 mm « « 〇 / · 150: 880-887 (1993); Clarkson et al, 352: 624-628 (1991). The term "vector," as used herein, refers to a nucleic acid molecule that is capable of proliferating another nucleic acid to which it is linked. The term includes a vector in the form of a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it is introduced. Certain callers are capable of directing the performance of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression carriers." II. Compositions and Methods In one aspect, the present invention is based, in part, on the discovery of a single antibody that is neutralized by infection with HCMV infection. In some embodiments, provided with the complex! In the case of the combined antibody U, an antibody that binds to gH is provided. The antibodies of the present invention are useful, for example, for the prevention, inhibition, and/or treatment of infection, congenital 158864.doc •28·201215618 HCMV infection and patient infection caused by transplanted tissues infected with HCMV. Antibodies can also be used to diagnose HCMV infection. In one aspect, the invention is also based, in part, on the discovery of compositions comprising a combination of monoclonal antibodies that inhibit HCMV virus entry into all of the cell types of the placenta: endothelial cells, epithelial cells, monocytes/giant Phagocytes and fibroblasts, and reduce and/or inhibit the formation of HCMV resistant strains. In certain embodiments, methods of using such compositions are provided. The compositions of the present invention are useful, for example, for preventing, inhibiting, and/or treating HCMV infection, congenital HCMV infection, and patient infection caused by HCMV-infected transplanted organs or tissues that have been collected from patients previously or currently infected with HCMV. The composition can also be used to diagnose HCMV infection. A. Exemplary Anti-Complex I Antibodies In one aspect, the invention provides isolated antibodies that bind to Complex I. In certain embodiments, the anti-Complex I antibody specifically binds to a conformational epitope produced by association of UL128, UL130, UL131 and gH/gL or an epitope in an individual member of Recombination I . In some embodiments, the EC90 of the anti-Complex I antibody neutralizing HCMV is 0.7 pg/ml, 0.5 pg/ml, 0.3 pg/ml, 0.1 pg/ml, 0.09 pg/ml, 0.08 pg/ml, 0.07 Pg/ml, 0.06 pg/ml, 0.05 pg/ml, 0.04 pg/ml, 0.03 pg/ml, 0.02 pg/ml, 0.015 pg/ml, 0.012 pg/ml, 0.011 pg/ml, 0.010 pg/ml or 0.010 Below pg/ml. In other aspects, the anti-Complex I antibody specifically binds to Complex I on the surface of HCVM and neutralizes 50% HCMV at the following antibody concentrations: 0.05 pg/ml ' 0.02 pg/ml, 0.015 pg/ml, 0.014 Pg/ml, 0.013 158864.doc -29- 201215618 pg/ml, 0.012 pg/ml, 0.011 pg/ml, 0.010 pg/ml, 0.009 pg/ml, 0.008 pg/ml, 0.007 pg/ml, 0_006 pg/ml ' 0.005 pg/ml, 0.004 pg/ml, 0.003 pg/ml, 0.002 pg/ml, 0.001 pg/ml, 0.0009 pg/ml, 0.0008 pg/ml, 0.0007 pg/ml or 0.0007 pg/ml or less (eg in ΙΟ) .8 Μ, ΙΟ—9 Μ, ΙΟ-10 M, 10_11 Μ, 10·12Μ, 10·13Μ or l〇-13M below the antibody concentration). In one aspect, the invention provides an anti-Complex I antibody comprising at least 1, 2, 3, 4, 5 or 6 HVRs selected from the group consisting of: (a) comprising an amino acid sequence SEQ ID NO: 6 HVR-H1; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; (d) comprising an amine HVR-L1 of the acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 10-19; and (f) comprising the amino acid sequence SEQ ID NO: 20 HVR-L3. In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8. In another aspect, the invention provides an antibody comprising at least 1, at least 2 or all 3 VL HVR sequences selected from the group consisting of: (a) comprising an amino acid sequence of 8 £(^10 1^0: 9: 11 ¥ 11-1; (15) HVR-L2 comprising an amino acid sequence selected from the group consisting of 3 ugly 9 10 > 10: 10-19; and (c) comprising the amino acid sequence SEQ ID NO: 20 HVR-L3. 158864.doc • 30- 201215618 In one embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; HVR-H2 comprising the amino acid sequence SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; and 3 VL HVR sequences selected from: (a) Amino acid sequence HVR-L1 of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 20. In another embodiment, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising the amino acid sequence SEQ ID NO HVR-H2 of 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 9; (b) HVR-L2 comprising the amino acid sequence SEQ ID: 11; and (c) comprising the amino acid sequence SEQ ID NO: 20 In another embodiment, the antibody comprises all three HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising an amino acid sequence HVR-H2 of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and 3 VL HVR sequences selected from: (a) comprising the amino acid sequence SEQ ID NO: HVR-L1 of 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 12; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20. In another embodiment, The antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 7; (c) comprising aminoguanidine 158864.doc -31 - 201215618 Sequence SEQ ID NO: 8 HVR-H3; and 3 Vl HVR sequences selected from: (a) comprising an amino acid sequence SEQ ID NO: 9 HVR-L1; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 13; and (c) comprising the amino acid sequence SEQ ID NO: 20 HVR-L3. In another embodiment, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising the amino acid sequence SEQ ID NO: HVR-H2 of 7; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) comprising the amino acid sequence SEQ ID NO: HVR-L1; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 20. In another embodiment, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising the amino acid sequence SEQ ID NO: HVR-H2 of 7; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) comprising the amino acid sequence SEQ ID NO: HVR_L1; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 15; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 20. In another embodiment, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising the amino acid sequence SEQ ID NO: HVR-H2 of 7; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) comprising the amino acid sequence SEQ ID NO: HVR-L1; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 16; and (c) HVR-L3 comprising the amino acid sequence 158864.doc-32-201215618 column SEQ ID NO: 20. In another embodiment, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising the amino acid sequence SEQ ID NO: HVR-H2 of 7; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) comprising the amino acid sequence SEQ ID NO: HVR-L1; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 17; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 20. In another embodiment, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising the amino acid sequence SEQ ID NO: HVR-H2; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) comprising the amino acid sequence SEQ ID NO: HVR-L1; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 18; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 20. In another embodiment, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) comprising the amino acid sequence SEQ ID NO: HVR-H2 of 7; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) comprising the amino acid sequence SEQ ID NO: HVR-L1; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 19; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 20. In some embodiments, the antibody comprises all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6; (b) package 158864.doc -33-201215618 amine-containing HVR-H2 of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and 3 VL HVR sequences selected from the group consisting of: (a) comprising an amino acid The sequence HVR-L1 of SEQ ID NO: 9; (b) the first amino acid comprising the HVR-L2 and the light chain variable region framework FR3 of the amino acid sequence SEQ ID NO: 21; and (c) comprising an amine group Acid sequence HVR-L3 of SEQ ID NO:20. In certain embodiments, any one or more of the amino acids of the anti-Complex I antibody provided above are substituted at the following HVR position: at 11 --L2 (SEQ ID NO: 10) t: position 4, 5, 11 and 12. In certain embodiments, the substitution is a conservative substitution as provided herein. In certain embodiments, any one or more of the following substitutions can be made in any combination: in HVR-L2 (SEQ ID NO: 57): D4E, D4T, D4S, G5A, D11E, D11T, D11S, and G12A. All possible combinations of the above substitutions are encompassed by the common sequence of SEQ ID NO:21. In any of the above embodiments, the anti-Complex I antibody is humanized. In one embodiment, the anti-Complex I antibody comprises an HVR as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-Complex I antibody comprises an HVR as in any of the above embodiments, and further comprising FR1 sequence SEQ ID NO: 22, FR2 sequence SEQ ID NO: 23, FR3 sequence SEQ ID NO: 24 and FR4 sequence VH of SEQ ID NO:25. In other embodiments, the anti-Complex I antibody comprises an HVR as in any of the above examples, and further comprising FR1 sequence SEQ ID NO: 22, FR2 sequence SEQ ID NO: 27, FR3 sequence SEQ ID NO: 28 and FR4 Sequence VH of SEQ ID NO:29. In other embodiments, the anti-Complex I antibody comprises an HVR as in any of the above examples, as in 158864.doc-34-201215618, and further comprising FR1 sequence SEQ ID NO: 30, FR2 sequence SEQ ID NO: 3 1 , FR3 Sequence SEQ ID NO: 32 and FR4 sequence SEQ ID NO: 25 VH. In other embodiments, the anti-Complex I antibody comprises an HVR as in any of the above examples, and further comprising FR1 sequence SEQ ID NO: 33, FR2 sequence SEQ ID NO: 23, FR3 sequence SEQ ID NO: 34 and FR4 Sequence VH of SEQ ID NO:25. In another embodiment, the anti-Complex I antibody comprises an HVR as in any of the above embodiments, and further comprising FR1 sequence SEQ ID NO: 35, FR2 sequence SEQ ID NO: 36, FR3 sequence SEQ ID NO: 37 and VL of the FR4 sequence of SEQ ID NO:38. In other embodiments, the anti-Complex I antibody comprises an HVR as in any of the above examples, and further comprising the FR1 sequence SEQ ID NO: 39 ' FR2 sequence SEQ ID NO: 40, FR3 sequence SEQ ID NO: 41 and FR4 VL of the sequence SEQ ID NO:42. In other embodiments, the anti-Complex I antibody comprises an HVR as in any of the above examples, and further comprising FR1 sequence SEQ ID NO: 43, FR2 sequence SEQ ID NO: 44, FR3 sequence SEQ ID NO: 41 and FR4 VL of the sequence SEQ ID NO:42. In any of the above antibodies, the VL FR3 sequence may be substituted with a sequence selected from SEQ ID NO: 67 or SEQ ID NO: 68. In another aspect, the anti-Complex I antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence SEQ ID NO: 46 or SEQ ID NO: 47. %, 97%, 98°/〇, 99% or 100% sequence identity heavy chain variable domain (VH) sequences. In certain embodiments, the sequence apos has at least 90%, 91%, 92%, 93%, 941⁄4, 95°/ relative to reference 158864.doc -35 - 201215618. The 96%, 97%, 98%, or 99% homologous VH sequence contains a substitution (e.g., a conservative substitution), an insertion or a deletion, but the anti-complex 1 antibody comprising the sequence retains the ability to bind to complex I. In certain embodiments, in SEQ ID NO: 45 or SEQ ID NO: 46 or SEQ ID NO: 47, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted. In some embodiments 'replacement, insertion or deletion occurs in the outer region of the HVR (i.e., in the FR). In a specific embodiment, the VH comprises 1, 2 or 3 HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 6, and (b) comprising the amino acid sequence SEQ ID NO: 7 HVR-H2, and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 8. In another aspect, an anti-Complex I antibody is provided, wherein the antibody comprises at least 90%, 91%, 92% '93%, 94 with the amino acid sequence SEQ ID NO: 48 or SEQ ID NO: 49 % '95%, 96% '97%, 98%, 99°/◦ or 100% sequence identity of the light chain variable domain (VL). In certain embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 960/〇, 97%, 98%, or 99%-dependent VL sequences relative to a reference sequence An anti-Complex I antibody comprising a substitution (eg, a conservative substitution), an insertion or a deletion, but comprising the sequence retains the ability to bind to Complex I. In certain embodiments: in SEQ ID NO: 48 or SEQ ID NO: 49, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted. In some embodiments, a substitution, insertion or deletion occurs in the outer region of the HVR (i.e., in the FR). In a particular embodiment, the VL comprises 1, 2 or 3 HVRs selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 9; (b) I58864.doc-36- 201215618 HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-19; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 20. In another aspect, an anti-Complex I antibody is provided, wherein the antibody comprises VH as in any of the examples provided above, and Vl in any of the examples provided above. In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 45 and SEQ ID NO: 49, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences set forth in SEQ ID NO: 46 and SEQ ID NO: 49, respectively, including post-translational modifications of the sequences. In another embodiment, the antibody comprises the VH and V1 sequences of SEQ ID NO: 47 and SEQ ID NO: 49, respectively, including post-translational modifications of the sequences. In another embodiment, the antibody comprises the VH and V1 sequences of SEQ ID NO: 45 and SEQ ID NO: 48, respectively, including post-translational modifications of the sequences. In another embodiment, the antibody comprises the sequence of ¥1 and 乂1^, respectively, in SEQ ID > 10:46 and 8 £(^10>10:48, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and V1 sequences of SEQ ID NO: 47 and SEQ ID NO: 48, respectively, including post-translational modifications of the sequences. In another aspect, the invention provides An antibody against which the anti-Complex I antibody competes and/or binds to the same epitope as the anti-Complex I antibody provided herein. For example, in certain embodiments, a competition with an anti-Complex I antibody is provided and/or An antibody that binds to the same epitope as the anti-Complex I antibody, the anti-Complex I antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 45-47 and comprising an amino acid sequence of SEQ ID NO: 48 or SEQ ID NO In another aspect, the present invention provides an antibody which binds to the same epitope as an anti-Complex 1 antibody, the epitope comprising an amino group corresponding to an amine group selected from the group consisting of Acidic amino acid: glutamic acid at position 47 of the amino acid of SEQ ID N: 203;卩ID NO: an amino acid at position 51 of the amino acid of 203; aspartic acid at position 46 of SEQ ID NO: 203 and combinations thereof. The corresponding amino acid constituting the epitope may be The approximate position in the UL13 1 amino acid sequence is similar, but can be attributed to the difference in the amino acid sequence of UL 13 1 between various HCMV strains. In another aspect, the present invention provides a complex with HCMV An antibody that binds to a polypeptide of I, wherein the polypeptide comprises the amino acid sequence SRALPDQTRYK YVEQLVDLTLNYHYDAS (SEQ ID NO: 194). In another aspect, the invention provides a same epitope as the anti-Complex I antibody provided herein. In other aspects, the invention provides an antibody that binds to the same epitope as the anti-Complex I antibody provided herein, having an EC90 of 0.7 pg/ml, 0.5 pg/ml, 0.3 pg/ml, 0.1 pg/ Ml, 0.09 pg/ml, 0.08 pg/ml, 0.07 pg/ml ' 0.06 pg/ml, 0.05 pg/ml, 0.04 pg/ml, 0.03 pg/ml ' 0.02 pg/ml, 0.015 pg/ml, 0.012 pg/ Ml, 0.011 pg/ml, 0.010 0 L / 1111 or 〇.〇1〇48/11 [11 or less. In other aspects, the invention provides An antibody that binds to the same epitope as the anti-Complex I antibody provided herein and neutralizes 50% of HCMV at the following antibody concentrations: 0.05 pg/ml, 0.02 gg/ml, 0.015 pg/ml, 0.014 pg/ml, 0.013 pg/ml, 0.012 pg/ml, 0.011 pg/ml, 0.010 pg/ml, 0.009 pg/ml, 0.008 pg/ml, 0.007 pg/ml, 0.006 pg/ml, 0.005 pg/ml, 158864.doc -38 - 201215618 0.004 pg/ml, 0.003 pg/ml, 0.002 pg/ml, 0.001 pg/mi, 0.0009 pg/m b 0.0008 pg/m b 0.0007 pg/mi or 0.0007 gg/ml or less (eg in ΙΟ·8 Μ, 10·9 M, l〇·10 Μ, 10·" M, 10.12 M, l〇_13 M or 1 (at an antibody concentration of T13 M or less). In another aspect of the invention, the anti-Complex I antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody or a human antibody. In one embodiment, the anti-complex is "filled with an antibody fragment, such as an Fv, Fab, Fab', scFv, bifunctional antibody or F(aly) 2 fragment. In another embodiment, the antibody is a full length antibody, For example, a full IgGl antibody or other antibody class or isotype as defined herein. In another aspect, the anti-complex antibody according to any of the above examples may have any of the characteristics as described in Section -7 below. One or any combination of features. B. Exemplary Anti-gH Antibodies In one aspect, the invention provides isolated antibodies that bind to gH. In certain embodiments, the anti-gH antibody specifically binds to the antigen of gH And neutralize HCMV 'EC90 is 0.8 pg/nU, 〇.7 μ§/ιη1, 〇6 μ§Λη1, 〇5 pg/m Bu0·4 pg/m 〇·3 pg/mh 〇2 ton岫卜〇"^/m卜〇〇9
Kg/nd、0.08 _ml、0.07 _ml、〇 〇6 吨㈤、〇 〇5 Kg/rni、0.04 pg/ml、0.03 盹㈤、〇 〇2 ^/m丨、〇 〇1Kg/nd, 0.08 _ml, 0.07 _ml, 〇 〇 6 tons (five), 〇 〇 5 Kg/rni, 0.04 pg/ml, 0.03 盹 (five), 〇 ^ 2 ^/m丨, 〇 〇1
Pg/m卜0.015盹/⑹、〇.〇1〇 ^/m丨或〇〇1〇㈣爪丨以下。在 一些實施例中,抗gH抗體與在桿狀病毒中產生之§11/社二 聚體之抗原決定基特異性結合’ IC5(^Q ()i禮至〇 Η nM 之範圍内。在各種實施例中,IC5〇可為〇.〇ι ηΜ、〇·〇2 —ν 158864.doc •39- 201215618 nM、0.03 碰、0.04 nM、0.05 nM、0.06 nM、0.07 nM、 0.08 nM、0.09 應、0.1 nM、0.11 nM、0.12 nM、0.13 nM、0.14 囊、0.15 nM、0.16 nM或 0_17 nM。 在其他實施例中,抗體與HCMV表面上之gH結合且在以 下抗體濃度下中和50%之HCMV : 0.1 pg/ml、0.09 pg/ml、 0.08 pg/mi、ο·。? pg/ml、0.06 |ig/ml、0.05 |ig/ml、0.04 pg/ml、0.03 pg/mi、0.02 pg/ml、0.015 pg/ml、0.014 pg/ml、0.013 pg/mi、0.012 pg/ml、0.011 pg/ml、0.010 Kg/ml、0.009 pg/ml、0.008 pg/ml、0.007 pg/ml、0.006 Pg/ml、0.005 pg/ml、0.004 pg/ml、0.003 pg/ml、0.002 pg/m卜 o.ooi eg/ml4〇 0(H pg/ml以下(例如i〇·9 Μ、ΙΟ·1。μ、ΙΟ·丨1 Μ、ΙΟ·12 Μ、ΙΟ·13 M或 10·丨3 M以下之抗 體濃度下)。 在一態樣中,本發明提供一種包含至少1個、2個、3 個、4個、5個或6個選自以下之HVR之抗gH抗體:(a)包含 胺基酸序列SEQ ID NO:71之HVR-H1 ; (b)包含選自SEQ ID NO:72、73或74之胺基酸序列之HVR-H2 ; (c)包含胺基酸 序列SEQ ID NO:75之HVR-H3 ; (d)包含胺基酸序列SEQ ID NO:76之HVR-L1 ; (e)包含胺基酸序列SEQ ID NO:77之 HVR-L2 ;及(f)包含胺基酸序列SEQ ID NO:78之HVR-L3。 在一實施例中,本發明提供一種包含至少1個、至少2個 或全部3個選自以下之VH HVR序列之抗體:(a)包含胺基 酸序列SEQ ID N0:71之HVR-H1 ; (b)包含胺基酸序列SEQ ID NO:72之HVR-H2 ;及(c)包含胺基酸序列SEQ ID NO:75 158864.doc •40- 201215618 之HVR-H3。在另一實施例中,抗體包含至少1個、至少2 個或全部3個選自以下之VH HVR序列:(a)包含胺基酸序 列SEQ ID NO:71之HVR-H1 ; (b)包含胺基酸序列SEQ ID NO:73之HVR-H2 ;及(c)包含胺基酸序列SEQ ID NO:75之 HVR-H3。在另一實施例中,抗體包含至少1個、至少2個 或全部3個選自以下之VH HVR序列:(a)包含胺基酸序列 SEQ ID NO:71之 HVR-H1 ; (b)包含胺基酸序列 SEQ ID NO:74之HVR-H2 ;及(c)包含胺基酸序列SEQ ID NO:75之 HVR-H3。 在另一態樣中,本發明提供一種包含至少1個、至少2個 或全部3個選自以下之VL HVR序列之抗體:(a)包含胺基酸 序列SEQ ID NO:76之HVR-L1 ; (b)包含胺基酸序列SEQ ID NO:77之HVR-L2 ;及(c)包含胺基酸序列SEQ ID NO:78之 HVR-L3 ;及包含選自 SEQ ID NO:72、SEQ ID NO:73 或 SEQ ID NO:74之胺基酸序列之HVR-H2。 在另一態樣中,本發明提供一種抗體,其包含(a)包含至 少1個、至少2個或全部3個選自以下之VH HVR序列之VH 域:(i)包含胺基酸序列SEQIDNO:71之HVR-Hl、(ii)包含 選自 SEQ ID NO:72、SEQ ID NO:73 或 SEQ ID NO:74 之胺 基酸序列之11乂11-112、及(丨丨〇包含選自8£()10 1\[〇:75之胺 基酸序列之HVR-H3 ;及(b)包含至少1個、至少2個或全部 3個選自以下之VL HVR序列之VL域:(i)包含胺基酸序列 SEQ ID NO:76 之 HVR-L1、(ii)包含胺基酸序列 SEQ ID NO:77之HVR-L2,及(c)包含胺基酸序列SEQ ID NO:78之 c· 158864.doc •41 - 201215618 HVR-L3。 在另一態樣中,本發明提供一種包含以下之抗體:(a)包 含胺基酸序列SEQ ID NO:71之HVR-H1 ; (b)包含胺基酸序 列8£〇10 1^0:72之11乂11-112;((:)包含胺基酸序列3£(5 10 NO:75之HVR-H3 ; (d)包含胺基酸序列SEQ ID NO:76之 HVR-L1 ; (e)包含胺基酸序列SEQ ID NO:77之HVR-L2 ;及 (0包含選自3£卩1〇1^〇:78之胺基酸序列之11¥11-1^3。 在另一態樣中,本發明提供一種包含以下之抗體:(a)包 含胺基酸序列SEQ ID NO:71之HVR-H1 ; (b)包含胺基酸序 列SEQ ID NO:73之HVR-H2 ; (c)包含胺基酸序列SEQ ID NO:75之HVR-H3 ; (d)包含胺基酸序列SEQ ID NO:76之 11¥11-1^1;(6)包含胺基酸序列8£(^10 1^0:77之11¥11-1^2;及 (f)包含選自SEQ ID NO:78之胺基酸序列之HVR-L3。 在另一態樣中,本發明提供一種包含以下之抗體:(a)包 含胺基酸序列SEQ ID NO:71之HVR-H1 ; (b)包含胺基酸序 列SEQ ID NO:74之HVR-H2 ; (c)包含胺基酸序列SEQ ID NO:75之HVR-H3 ; (d)包含胺基酸序列SEQ ID NO:76之 HVR-L1 ; (e)包含胺基酸序列SEQ ID NO:77之HVR-L2 ;及 (f)包含選自SEQ ID NO:78之胺基酸序列之HVR-L3。 在某些實施例中,如上提供之抗gH抗體之任何一或多個 胺基酸在以下HVR位置處經取代:在HVR-H2(SEQ ID NO:91)中:位置6及8。在某些實施例中,取代為如本文提 供之保守性取代。在某些實施例中,可以任何組合進行任 何一或多個以下取代:在HVR-H2(SEQ ID ΝΟ··91)中: 158864.doc -42- 201215618 D6S、D6T、D6N、D6Q、D6F、D6M、D6L及 T8R。以上 取代之全部可能之組合由SEQ ID NO:93之共同序列涵蓋。 在任何以上實施例中,抗gH抗體經人類化。在一實施例 中,抗gH抗體包含如任何以上實施例中之HVR,且進—步 包含接受體人類構架,例如人類免疫球蛋白構架或人類共 同構架。在另一實施例中,抗gH抗體包含如任何以上實施 例中之HVR,且進一步包含含有FR1序列SEQ ID N〇:79、 FR2序列 SEQ ID NO:80、FR3序列 SEQ ID NO:81 及 FR4序 列SEQ ID NO:82之VH。在其他實施例中,抗gH抗體包含 如任何以上實施例中之HVR,且進一步包含含有FR1序列 SEQ ID NO:83、FR2序列 SEQ ID NO:84、FR3序列 SEQ ID NO:85及 FR4序列 SEQ ID NO:86之 VL。 在另一態樣中,本發明之抗gH抗體包含與胺基酸序列 SEQ ID NO:92具有至少 90°/〇、91%、92%、93%、94%、 95%、96%、97%、98%、99%或100%序列一致性但其中位 置54處之胺基酸為Asn(N)及/或其中位置56處之胺基酸為 Asn(R)的重鏈可變域(VH)序列。在某些實施例中,相對於 參照序列,具有至少90%、91%、92%、93%、94%、 95%、96%、97%、98%或99%—致性之VH序列含有取代 (例如保守性取代)、插入或缺失,但包含彼序列之抗gH抗 體保留與gH結合之能力。在某些實施例中,在SEQ ID NO:92中,總計1至10個胺基酸已經取代、插入及/或缺 失,但其中位置54處之胺基酸為Asn(N)及/或其中位置56 處之胺基酸為Asn(R)。在某些實施例中,取代、插入或缺 158864.doc -43· 201215618 失發生在HVR外部區域中(亦即在FR中)。視情況而定,抗 吕11抗體包含於8£(^1〇]^0:87、8£(5 1〇]^0:88或8丑(^1〇 NO:89中之VH序列,包括彼序列之轉譯後修飾。在一特定 實施例中,VH包含1個、2個或3個選自以下之HVR : (a)包 含胺基酸序列SEQ ID NO:71之HVR-H1,(b)包含選自SEQ 10 1^0:72、8£(^10 1^0:73及8丑(5 10 1^0:74之胺基酸序列 之HVR-H2,及(c)包含胺基酸序列SEQIDNO:75之HVR-H3。 在另一態樣中,本發明之抗gH抗體包含與胺基酸序列 SEQ ID NO:90 具有至少 90%、91%、92%、93%、94%、 95%、96%、97%、98%、99%或100%序列一致性之輕鏈可 變域(VL)。在某些實施例中,相對於參照序列,具有至少 90%、91%、92%、93%、94%、95%、96%、97% ' 98% 或 99% —致性之VL序列含有取代(例如保守性取代)、插入或 缺失,但包含彼序列之抗gH抗體保留與gH結合之能力。 在某些實施例中,在SEQ ID NO:90中,總計1至10個胺基 酸已經取代、插入及/或缺失。在某些實施例中,取代、 插入或缺失發生在HVR外部區域中(亦即在FR中)。視情況 而定,抗gH抗體包含於SEQ ID NO:90中之VL序歹ij,包括 彼序列之轉譯後修飾。在一特定實施例中,VL包含1個、 2個或3個選自以下之HVR : (a)包含胺基酸序列SEQ ID NO:76之HVR-L1 ; (b)包含胺基酸序列SEQ ID NO:77之 HVR-L2 ;及(c)包含胺基酸序列SEQ ID NO:78之HVR-L3。 在另一態樣中,本發明之抗gH抗體包含如任何以上提供 158864.doc -44- 201215618 之實施例中之VH及如任何以上提供之實施例中之VL。在 一實施例中,抗體包含分別於SEQ ID NO:87及SEQ ID NO:90中之VH及VL序列,包括彼等序列之轉譯後修飾。 在另一實施例中,抗體包含分別於SEQ ID NO:88及SEQ ID NO:90中之VH及VL序列,包括彼等序列之轉譯後修 飾。 在另一實施例中,抗體包含分別於SEQ ID NO:89及SEQ ID NO:90中之VH及VL序列,包括彼等序列之轉譯後修 飾。 在另一態樣中,本發明提供一種與本文提供之抗gH抗體 競爭及/或與本文提供之抗gH抗體結合相同抗原決定基的 抗體。舉例而言,在某些實施例中,提供一種與抗gH抗體 競爭及/或與抗gH抗體結合相同抗原決定基之抗體,該抗 gH抗體包含含有胺基酸序列SEQ ID NO:87、88或89之VH 及含有胺基酸序列SEQ ID NO:90之VL。 在另一態樣中,本發明提供一種與抗gH抗體結合相同抗 原決定基之抗體,該抗原決定基包含對應於選自以下之胺 基酸的胺基酸:在SEQ ID ΝΟ:1之胺基酸位置168處之色胺 酸;在SEQ ID ΝΟ:1之胺基酸位置446處之天冬胺酸;在 SEQ ID ΝΟ:1之胺基酸位置171處之脯胺酸及其組合。構成 抗原決定基之相應胺基酸可處於gH胺基酸序列中之近似相 同位置,但可歸因於gH在各種HCMV病毒株之間的胺基酸 序列差異而不同。 在其他態樣中,本發明提供一種與本文提供之抗gH抗體 158864.doc -45- 201215618 結合相同杬原決定基之抗體,〇1 nM至〇 17 nM之 範圍内。在各種實施例中,lew可為017 „肘或017 ηΜα 下’例如 0.16 ηΜ、0·15 nM、〇.l4 ηΜ、〇 13 ηΜ、0.12 ηΜ、0.11 ηΜ、0.10 ηΜ、0.09 ηΜ、(j og ηΜ、〇 〇7 囊、 0·06 ηΜ、0,05 ηΜ、0.G4 ηΜ、〇·〇3 ηΜ、0.02 ηΜ、0.01 ηΜ或0.01 ηΜ以下。舉例而言,在某些實施例中,提供一 種與HB1(—種包含VH序列SEQIE)N0:89及VL序列SEQID ΝΟ:90之抗gH抗體)結合相同抗原決定基且1(:5〇為〇 17 ηΜ 或 0.17 ηΜ以下(例如 0.16 ηΜ、0.15 ηΜ、0.14 ηΜ、0.13 ηΜ、0.12 ηΜ、0.11 ηΜ、0.10 ηΜ、〇·〇9 ηΜ、0.08 ηΜ、 0.07 ηΜ ' 0.06 ηΜ ' 0.05 ηΜ ' 〇.〇4 ηΜ ' 0.03 ηΜ > 〇.〇2 ηΜ、0.01 ηΜ或〇·〇1 ηΜ以下)或中和HCMV感染之EC90為 0.8 pg/ml、0.7 pg/m 卜 〇·6 pg/nU、〇·5 pg/m 卜 0.4 pg/ml、 0.3 pg/ml、0.2 pg/ml、0.1 pg/ml、〇 〇9 μ8/ηι1、〇 〇8 pg/ml、0·07 pg/ml、0.06 pg/mi、〇·〇5 pg/ml、〇 〇4 pg/ml、0.03 pg/ml、0.02 pg/ml、0·01 μ§/πιι、〇 〇15 pg/ml、0.010 pg/ml或 0.010 pg/mi 以下的抗體。 在其他態樣中,本發明提供一種與本文提供之抗gH抗體 結合相同抗原決定基且在以下抗體濃度下中和5〇%之 HCMV的抗體:0.1 pg/mi、〇.〇9 pg/mi、〇,〇8 pg/ml、0.07 pg/ml、0_06 pg/ml、〇·〇5 pg/ml、〇.〇4 pg/ml、0.〇3 pg/rril、0.02 pg/ml、0.015 pg/ml、0.014 pg/ml、Ο.Ο13 pg/ml、0.012 pg/ml、0.011 pg/ml、(K010 pg/mi、〇.〇〇9 pg/ml、0.008 pg/ml、0.007 pg/ml、0.006 pg/ml、0.005 I58864.doc -46- 201215618 pg/ml、0.004 pg/ml、0.003 pg/ml、〇·〇〇2 pg/ml、0.001 pg/ml或 0.001 pg/ml以下(例如在 1〇·8μ、10-9M、10·10Μ、 1 (Γ11 Μ、1 (Γ12 Μ、1 〇 13 Μ或10—13 Μ以下之抗體濃度下)。 在本發明之另一態樣中,根據任何以上實施例之抗gH抗 體為單株抗體,包括嵌合抗體、人類化抗體或人類抗體。 在一實施例中,抗gH抗體為抗體片段,例如Fv、Fab、 Fab·、scFv、雙功能抗體或F(ab,)2片段。在另一實施例 中’抗體為全長抗體,例如完整IgG 1抗體或如本文中定義 之其他抗體類別或同型。 在另一態樣中’根據以上實施例之抗gH抗體可併有任何 如以下章節1 -7中所述之特徵中的一者或任何特徵之組 合。 抗體親和力 在某些實施例中,如本文提供之本發明抗體之解離常數 (Kd)為 $1 μΜ、$100 nM、S10 nM、SI nM、$0.1 nM、 50.01 nM或 50.001 nM(例如 i〇-8 ^^或 1〇-8 M以下,例如自 10 8 Μ至 10 13 Μ,例如自 1〇·9 μ至 1 〇·13 Μ)。 在貫施例中’藉由用相關抗體之Fab形式及其如關於 以下檢疋所述之抗原進行的經放射性標記之抗原結合檢定 (RIA)來量測Kd。藉由在未經標記之抗原的滴定系列存在 下用最小濃度之(Ι25ι)標記抗原使Fab平衡,隨後用經抗Fab 抗體塗佈之板捕捉結合之抗原來量測Fab對抗原之溶液結 合親和力(參見例如Chen等人,j M〇/价〇/ 293:865 881 (1999))。為了確立檢定條件,將micr〇titer®多孔板 201215618 (Thermo Scientific)用含 5 pg/ml 捕捉抗 Fab 抗體(Cappel Labs)之50 mM碳酸鈉(pH 9.6)塗佈隔夜,且隨後在室溫(約 23°C )下用含2%(w/v)牛血清白蛋白之PBS阻斷2至5小時。 在非吸附板(Nunc #269620)中,將 1〇〇 pM或26 pM [1251]-抗 原與相關Fab之連續稀釋液混合(例如與presta等人,Cawcer Res. 57:4593-4599 (1997)中評估抗 VEGF 抗體 Fab-12 — 致)。接著培育相關F ab隔夜;然而,培育可持續較長時期 (例如約65小時)以確保達到平衡。此後,將混合物轉移至 捕捉板以在室溫下培育(例如1小時)。接著移除溶液且用含 0.1%聚山梨醇酯20(TWEEN-20®)之PBS將板洗滌8次。當板 已乾燥時,添加每孔150 μΐ閃爍體(MICROSCINT-20tm ; Packard)’ 且在TOPCOUNTtm γ計數器(Packard)上對板計 數10分鐘。選擇各Fab之產生小於或等於最大結合之20% 的濃度以用於競爭性結合檢定。 根據另一實施例,Kd係使用表面電漿子共振檢定, 使用 BIACORE®-2000 或 BIACORE®-3000 (BIAcore, Inc., Piscataway,NJ),在25°C下,用固定抗原CM5晶片,在約 10個反應單位(RU)下量測。簡言之,根據供應商之說明書 用,乙基-iT-(3-二曱基胺基丙基)-碳化二亞胺鹽酸鹽(EDC) 及,羥基丁二醯亞胺(NHS)活化羧曱基化葡聚糖生物感測 器晶片(CM5,BIACORE,Inc.)。抗原用1 0 mM乙酸鈉(pH 4.8)稀釋至5 pg/ml(約0.2 μΜ),隨後以5微升/分鐘之流速 注射以達成約10個反應單位(RU)之偶合蛋白質。在注射抗 原之後.,注射1 Μ乙醇胺以阻斷未反應之基團。在動力學 158864.doc -48 - 201215618 量測中,在25°C下以約25 μΐ/min之流速注射於含0.05%聚 山梨醇酯20(TWEEN-20tm)界面活性劑之PBS(PBST)中兩倍 連續稀釋的Fab(0.78 nM至500 nM)。使用簡單一對一朗繆 爾結合模型(Langmuir binding model)(BIACORE®評估軟體 第 3.2版(BIACORE® Evaluation Software version 3.2))藉由 同時擬合締合及解離感測器圖譜來計算締合速率(kQn)及解 離速率汴。《)。平衡解離常數(Kd)係計算為比率kcff/ku。參 見例如 Chen 等人,J. Μο/. 5ζ·ο/· 293:865-881 (1999)。若藉 由以上表面電漿子共振檢定測得締合速率超過106 M·1 s·1, 則締合速率可藉由使用螢光淬滅技術測定,該技術量測在 遞增濃度之抗原存在下,在25°(:下於?68化117.2)中之20 nM抗抗原抗體(Fab形式)之螢光發射強度(激發=295 nm ; 發射=340 nm,16 nm帶通)的增加或減小,如在光譜計, 諸如配備停流之光譜光度計(stop-flow equipped spectrophometer)(Aviv Instruments)或具有經授拌比色管之 8000 系列 SLM-AMINCOtm 分光光度計(ThermoSpectronic) 中所量測6 2.抗體片段 在某些實施例中,本文提供之抗體為抗體片段。抗體片 段包括(但不限於)Fab、Fab'、Fab'-SH、F(ab')2、Fv及 scFv 片段及下文所述之其他片段。關於某些抗體片段之综述, 參見 Hudson等人,Mel 9:129-134 (2003)。關於scFv片 段之综述,參見例如 Pluekthiin,The Pharmacology of Monoclonal Antibodies,第 11 3 卷,Rosenburg 及 Moore 編, 158864.doc -49- 201215618 (Springer-Verlag, New York),第 269-3 1 5 頁(1994);亦參見 WO 93/16185 ;及美國專利第 5,571,894號及第 5,587,458 號。關於包含救助受體結合抗原決定基殘基且具有增加之 活體内半衰期之Fab及F(ab’)2片段的論述,參見美國專利 第 5,869,046號。 雙功能抗體為具有2個抗原結合位點之可具有二價或雙 特異性的抗體片段。參見例如EP 404,097 ; WO 1993/01161 ; Hudson等人,Med. 9:129-134 (2003);及 Hollinger等人,Pr〇c· Natl. Acad. Sci. USA 90: 6444-6448 (1993)。三功能抗體及四功能抗體亦描述於Hudson等人, 9:129-134 (2003)中。 單域抗體為包含抗體之全部或部分重鏈可變域或全部或 部分輕鏈可變域的抗體片段。在某些實施例中,單域抗體 為人類單域抗體(Domantis,Inc.,Waltham, MA;參見例如 美國專利第6,248,516 B1號)。 抗體片段可藉由各種技術製備,包括(但不限於)蛋白水 解消化完整抗體以及藉由重組宿主細胞(例如大腸桿菌(五 co/z·)或噬菌體)產生,如本文所述。 、散合及人類化抗體 在某些實施例中,本文提供之抗體為嵌合抗體。某些嵌 合抗體例如於美國專利第4,8丨6,567號;及Morrison等人, 5W· t/a,81:6851-6855 (1984)中描述。 在一實例中,嵌合抗體包含非人類可變區(例如源於小 鼠、大氣、倉鼠、兔或非人類靈長類動物(諸如猴)之可變 158864.doc 201215618 品)人類匣疋區。在另一實例中,嵌合抗體為「類別轉 換」抗體’其中類別或子類已自親本抗體之類別或子類改 支。攸合抗體包括其抗原結合片段。 在某些實施例中,嵌合抗體為人類化抗體。通常,非人 類杬體經人類化以降低對人類之免疫原性,同時保留親本 非人類机體之特異性及親和力。一般而言,人類化抗體包 含一或多個可變域’其中例如CDr之HVR(或其部分)源於 非人類抗體,且FR(或其部分)源於人類抗體序列。人類化 抗體視情況亦將包含人類恆定區之至少一部分。在一些實 施例中’人類化抗體中之一些殘基經來自非人類抗體 (例如HVR殘基所源於之抗體)之相應殘基取代例如以恢復 或改良抗體特異性或親和力。 人類化抗體及製備其之方法例如於Almagro及Fransson, Front. 13:1619-1633 (2008)中綜述,且例如於以下 中進一步描述:Riechmann 等人,iVaiwre 332:323-329 (1988) ; Queen 等人,#加,/ deal 5W. 86:10029-Pg/mbu 0.015盹/(6), 〇.〇1〇 ^/m丨 or 〇〇1〇(4) below the claw. In some embodiments, the anti-gH antibody specifically binds to the epitope of §11/dimer produced in baculovirus 'IC5(^Q()i to 〇ΗnM. In the embodiment, IC5〇 can be 〇.〇ι ηΜ, 〇·〇2 —ν 158864.doc •39- 201215618 nM, 0.03 touch, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 should 0.1 nM, 0.11 nM, 0.12 nM, 0.13 nM, 0.14 vesicle, 0.15 nM, 0.16 nM or 0-17 nM. In other embodiments, the antibody binds to gH on the surface of HCMV and neutralizes 50% of HCMV at the following antibody concentrations : 0.1 pg/ml, 0.09 pg/ml, 0.08 pg/mi, ο·.? pg/ml, 0.06 | ig/ml, 0.05 | ig/ml, 0.04 pg/ml, 0.03 pg/mi, 0.02 pg/ml , 0.015 pg/ml, 0.014 pg/ml, 0.013 pg/mi, 0.012 pg/ml, 0.011 pg/ml, 0.010 Kg/ml, 0.009 pg/ml, 0.008 pg/ml, 0.007 pg/ml, 0.006 Pg/ml , 0.005 pg/ml, 0.004 pg/ml, 0.003 pg/ml, 0.002 pg/m, o.ooi eg/ml4〇0 (H pg/ml or less (for example, i〇·9 Μ, ΙΟ·1.μ, ΙΟ · 丨1 Μ, ΙΟ·12 Μ, ΙΟ·13 M or 10·丨3 M below the antibody concentration). In one aspect, the invention provides an anti-gH antibody comprising at least 1, 2, 3, 4, 5 or 6 HVRs selected from the group consisting of: (a) comprising an amino acid sequence SEQ ID NO: HVR-H1 of 71; (b) HVR-H2 comprising an amino acid sequence selected from SEQ ID NO: 72, 73 or 74; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 75; d) HVR-L1 comprising the amino acid sequence SEQ ID NO: 76; (e) HVR-L2 comprising the amino acid sequence SEQ ID NO: 77; and (f) comprising the amino acid sequence SEQ ID NO: 78 HVR-L3. In one embodiment, the invention provides an antibody comprising at least 1, at least 2 or all 3 VH HVR sequences selected from: (a) comprising an amino acid sequence SEQ ID NO: 71 HVR-H1; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 72; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 75 158864. doc • 40-201215618. In another embodiment, the antibody comprises at least 1, at least 2 or all 3 VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 71; (b) comprising Amino acid sequence HVR-H2 of SEQ ID NO: 73; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO:75. In another embodiment, the antibody comprises at least 1, at least 2 or all 3 VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 71; (b) comprising Amino acid sequence HVR-H2 of SEQ ID NO: 74; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 75. In another aspect, the invention provides an antibody comprising at least 1, at least 2 or all 3 VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 76 (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 77; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 78; and comprising SEQ ID NO: 72, SEQ ID NO: 73 or HVR-H2 of the amino acid sequence of SEQ ID NO: 74. In another aspect, the invention provides an antibody comprising (a) a VH domain comprising at least 1, at least 2 or all 3 VH HVR sequences selected from: (i) comprising an amino acid sequence SEQ ID NO HVR-H1 of 71; (ii) 11乂11-112 comprising an amino acid sequence selected from SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74, and (丨丨〇 comprises a selected from 8£()10 1\[〇: HVR-H3 of the amino acid sequence of 75; and (b) a VL domain comprising at least 1, at least 2 or all 3 VL HVR sequences selected from: (i HVR-L1 comprising the amino acid sequence SEQ ID NO: 76, (ii) HVR-L2 comprising the amino acid sequence SEQ ID NO: 77, and (c) comprising the amino acid sequence SEQ ID NO: 78 158864.doc •41 - 201215618 HVR-L3. In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 71; (b) comprising Amino acid sequence 8 〇 10 1 ^ 0: 72 of 11 乂 11-112; ((:) contains amino acid sequence 3 £ (5 10 NO: 75 of HVR-H3; (d) contains amino acid sequence HVR-L1 of SEQ ID NO: 76; (e) HVR-L2 comprising the amino acid sequence SEQ ID NO: 77; and (0 comprising selected from 3 卩1 1^〇: The amino acid sequence of 78 is 11¥11-1^3. In another aspect, the invention provides an antibody comprising: (a) an HVR comprising the amino acid sequence SEQ ID NO: 71 - (H) HVR-H2 comprising the amino acid sequence SEQ ID NO: 73; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 75; (d) comprising the amino acid sequence SEQ ID NO : 76 of 11 ¥ 11-1 ^ 1; (6) contains amino acid sequence 8 £ (^10 1 ^ 0: 77 of 11 ¥ 11-1 ^ 2; and (f) comprises selected from SEQ ID NO: 78 In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 71; (b) comprising an amine group Acid sequence SEQ ID NO: 74 HVR-H2; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 75; (d) HVR-L1 comprising the amino acid sequence SEQ ID NO: 76; ) HVR-L2 comprising the amino acid sequence SEQ ID NO: 77; and (f) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 78. In certain embodiments, any one or more of the amino acids of the anti-gH antibody provided above are substituted at the HVR position: in HVR-H2 (SEQ ID NO: 91): positions 6 and 8. In certain embodiments, the substitution is a conservative substitution as provided herein. In certain embodiments, any one or more of the following substitutions can be made in any combination: in HVR-H2 (SEQ ID ΝΟ··91): 158864.doc -42- 201215618 D6S, D6T, D6N, D6Q, D6F, D6M, D6L and T8R. All possible combinations of the above substitutions are encompassed by the common sequence of SEQ ID NO:93. In any of the above embodiments, the anti-gH antibody is humanized. In one embodiment, the anti-gH antibody comprises an HVR as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-gH antibody comprises HVR as in any of the above examples, and further comprising FR1 sequence SEQ ID N〇:79, FR2 sequence SEQ ID NO:80, FR3 sequence SEQ ID NO:81 and FR4 Sequence VH of SEQ ID NO:82. In other embodiments, the anti-gH antibody comprises HVR as in any of the above examples, and further comprising SEQ ID NO: 83, FR2 sequence SEQ ID NO: 84, FR3 sequence SEQ ID NO: 85 and FR4 sequence SEQ ID NO: VL of 86. In another aspect, the anti-gH antibody of the invention comprises at least 90°/〇, 91%, 92%, 93%, 94%, 95%, 96%, 97 with the amino acid sequence SEQ ID NO:92. %, 98%, 99% or 100% sequence identity but wherein the amino acid at position 54 is Asn(N) and/or the heavy chain variable domain in which the amino acid at position 56 is Asn(R) ( VH) sequence. In certain embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the VH sequence is contained relative to the reference sequence. Substitutions (eg, conservative substitutions), insertions or deletions, but the anti-gH antibodies comprising the sequence retain the ability to bind to gH. In certain embodiments, in SEQ ID NO: 92, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted, but wherein the amino acid at position 54 is Asn(N) and/or The amino acid at position 56 is Asn(R). In some embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Depending on the case, the anti-Lu 11 antibody is included in the VH sequence of 8 £(^1〇]^0:87, 8 £(5 1〇]^0:88 or 8 ugly (^1〇NO:89, including Post-translational modification of the sequence. In a particular embodiment, the VH comprises 1, 2 or 3 HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 71, (b) ) comprising HVR-H2 selected from the group consisting of SEQ 10 1^0:72, 8 £(^10 1^0:73 and 8 ugly (5 10 1^0:74 amino acid sequence, and (c) comprising an amine group The acid sequence SEQ ID NO: 75 of HVR-H3. In another aspect, the anti-gH antibody of the invention comprises at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence SEQ ID NO: a light chain variable domain (VL) of 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, at least 90%, 91% relative to a reference sequence , 92%, 93%, 94%, 95%, 96%, 97% '98% or 99% of the VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion, but contains the anti-gH of the sequence. The antibody retains the ability to bind to gH. In certain embodiments, in SEQ ID NO: 90, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted. In some embodiments, the substitution, insertion or deletion occurs in the outer region of the HVR (ie, in the FR). As the case may be, the anti-gH antibody is included in the VL sequence 歹 ij in SEQ ID NO: 90, including the sequence Post-translational modification. In a particular embodiment, VL comprises 1, 2 or 3 HVRs selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 76; (b) comprising an amine HVR-L2 of the SEQ ID NO: 77; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78. In another aspect, the anti-gH antibody of the invention comprises as provided above 158864.doc -44-201215618 The VH of the embodiment and the VL of any of the above provided examples. In one embodiment, the antibody comprises VH of SEQ ID NO: 87 and SEQ ID NO: 90, respectively. VL sequences, including post-translational modifications of their sequences. In another embodiment, the antibodies comprise the VH and VL sequences of SEQ ID NO: 88 and SEQ ID NO: 90, respectively, including post-translational modifications of the sequences. In another embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 89 and SEQ ID NO: 90, respectively, including post-translational modifications of the sequences. Aspect, the present invention provides an anti-gH provided herein, one kind of antibody that competes with and / or provided with an anti-gH antibody herein binds to the same epitope of an antibody. For example, in certain embodiments, an antibody that competes with an anti-gH antibody and/or binds to an anti-gH antibody comprising an amino acid sequence comprising SEQ ID NO: 87, 88 is provided. Or a VH of 89 and a VL comprising the amino acid sequence SEQ ID NO:90. In another aspect, the invention provides an antibody that binds to the same epitope as an anti-gH antibody, the epitope comprising an amino acid corresponding to an amino acid selected from the group consisting of the amine of SEQ ID NO: The tryptophan acid at position 168; the aspartic acid at position 446 of the amino acid of SEQ ID NO: 1; the proline at position 171 of the amino acid of SEQ ID NO: 1, and combinations thereof. The corresponding amino acid constituting the epitope may be at approximately the same position in the gH amino acid sequence, but may be attributable to differences in the amino acid sequence of gH between the various HCMV strains. In other aspects, the invention provides an antibody that binds to the same sputum determinant as the anti-gH antibody 158864.doc-45-201215618 provided herein, in the range of 〇1 nM to 〇17 nM. In various embodiments, lew can be 017 „ elbow or 017 ηΜα under 'eg, 0.16 ηΜ, 0·15 nM, 〇.l4 ηΜ, 〇13 ηΜ, 0.12 ηΜ, 0.11 ηΜ, 0.10 ηΜ, 0.09 ηΜ, (j og ηΜ, 〇〇7 囊, 0·06 ηΜ, 0,05 ηΜ, 0.G4 ηΜ, 〇·〇3 ηΜ, 0.02 ηΜ, 0.01 ηΜ or 0.01 ηΜ or less. For example, in some embodiments, An anti-gH antibody that binds to HB1 (the VH sequence comprising SEQIE) N0:89 and VL sequence SEQ ID:90) and 1 (:5〇 is 〇17 ηΜ or 0.17 ηΜ or less (eg 0.16 ηΜ, 0.15 ηΜ, 0.14 ηΜ, 0.13 ηΜ, 0.12 ηΜ, 0.11 ηΜ, 0.10 ηΜ, 〇·〇9 ηΜ, 0.08 ηΜ, 0.07 ηΜ ' 0.06 ηΜ ' 0.05 ηΜ ' 〇.〇4 ηΜ ' 0.03 ηΜ > 〇.〇2 The EC90 of ηΜ, 0.01 ηΜ or 〇·〇1 ηΜ) or neutralizing HCMV infection is 0.8 pg/ml, 0.7 pg/m 〇·6 pg/nU, 〇·5 pg/m 卜 0.4 pg/ml, 0.3 Pg/ml, 0.2 pg/ml, 0.1 pg/ml, 〇〇9 μ8/ηι1, 〇〇8 pg/ml, 0·07 pg/ml, 0.06 pg/mi, 〇·〇5 pg/ml, 〇〇 4 pg/ml, 0.03 pg/ml, 0.02 pg/ml An antibody of 0. 01 μ§/πιι, 〇〇15 pg/ml, 0.010 pg/ml or 0.010 pg/mi or less. In other aspects, the invention provides the same antigenic binding as the anti-gH antibody provided herein. An antibody that neutralizes 5 % of HCMV at the following antibody concentrations: 0.1 pg/mi, 〇.〇9 pg/mi, 〇, 〇8 pg/ml, 0.07 pg/ml, 0_06 pg/ml, 〇· 〇5 pg/ml, 〇.〇4 pg/ml, 0.〇3 pg/rril, 0.02 pg/ml, 0.015 pg/ml, 0.014 pg/ml, Ο.Ο13 pg/ml, 0.012 pg/ml, 0.011 Pg/ml, (K010 pg/mi, 〇.〇〇9 pg/ml, 0.008 pg/ml, 0.007 pg/ml, 0.006 pg/ml, 0.005 I58864.doc -46- 201215618 pg/ml, 0.004 pg/ml , 0.003 pg/ml, 〇·〇〇2 pg/ml, 0.001 pg/ml or 0.001 pg/ml or less (for example, at 1〇·8μ, 10-9M, 10·10Μ, 1 (Γ11 Μ, 1 (Γ12 Μ , 1 〇 13 Μ or 10-13 Μ below the antibody concentration). In another aspect of the invention, the anti-gH antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody or a human antibody. In one embodiment, the anti-gH antibody is an antibody fragment, such as an Fv, Fab, Fab, scFv, bifunctional antibody or F(ab,) 2 fragment. In another embodiment the antibody is a full length antibody, such as an intact IgG 1 antibody or other antibody class or isotype as defined herein. In another aspect, the anti-gH antibody according to the above examples may be combined with any one of the features described in Sections 1-7 below or any combination of features. Antibody Affinity In certain embodiments, the dissociation constant (Kd) of an antibody of the invention as provided herein is $1 μΜ, $100 nM, S10 nM, SI nM, $0.1 nM, 50.01 nM or 50.001 nM (eg i〇-8 ^ ^ or 1〇-8 M or less, for example, from 10 8 10 to 10 13 Μ, for example, from 1〇·9 μ to 1 〇·13 Μ). In the examples, Kd was measured by the Fab format of the relevant antibody and its radiolabeled antigen binding assay (RIA) as described for the antigens described below. Fab is equilibrated by labeling the antigen with a minimum concentration (Ι25ι) in the presence of a titration series of unlabeled antigens, and then the bound antigen is captured by an anti-Fab antibody coated plate to measure the solution binding affinity of the Fab to the antigen. (See, for example, Chen et al., j M〇/price 〇 / 293:865 881 (1999)). To establish the assay conditions, micr〇titer® multiwell plate 201215618 (Thermo Scientific) was coated overnight with 50 mM sodium carbonate (pH 9.6) containing 5 pg/ml anti-Fab antibody (Cappel Labs), and then at room temperature ( Block with 2% (w/v) bovine serum albumin in PBS for about 2 to 5 hours at about 23 °C. In a non-adsorbing plate (Nunc #269620), mix 1 μpM or 26 pM [1251]-antigen with serial dilutions of the relevant Fab (eg with Presta et al, Cawcer Res. 57: 4593-4599 (1997) The anti-VEGF antibody Fab-12 was evaluated. The relevant F ab is then incubated overnight; however, the incubation can last for a longer period of time (e.g., about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution was then removed and the plate was washed 8 times with PBS containing 0.1% polysorbate 20 (TWEEN-20®). When the plate was dry, 150 μΐ scintillator (MICROSCINT-20tm; Packard)' per well was added and the plate was counted for 10 minutes on a TOPCOUNTtm gamma counter (Packard). The concentration at which each Fab is produced is less than or equal to 20% of the maximum binding is selected for competitive binding assays. According to another embodiment, the Kd system uses a surface plasmon resonance assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C with a fixed antigen CM5 wafer, Measured under about 10 reaction units (RU). Briefly, according to the supplier's instructions, ethyl-iT-(3-didecylaminopropyl)-carbodiimide hydrochloride (EDC) and hydroxybutylimine (NHS) activation Carboxydenylated dextran biosensor wafer (CM5, BIACORE, Inc.). The antigen was diluted to 5 pg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μl/min to achieve about 10 reaction units (RU) of the coupled protein. After the injection of the antigen, 1 Μ ethanolamine was injected to block the unreacted group. In the kinetics 158864.doc -48 - 201215618, PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20tm) surfactant was injected at 25 ° C at a flow rate of about 25 μΐ / min. Two-fold serial dilutions of Fab (0.78 nM to 500 nM). The association rate was calculated by simultaneously fitting the association and dissociation sensor maps using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). kQn) and the dissociation rate 汴. "). The equilibrium dissociation constant (Kd) is calculated as the ratio kcff/ku. See, for example, Chen et al., J. Μο/. 5ζ·ο/· 293:865-881 (1999). If the association rate exceeds 106 M·1 s·1 as determined by the above surface plasmon resonance assay, the association rate can be determined by using a fluorescence quenching technique which measures the presence of increasing concentrations of antigen. Increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) at 25° (: 6868) , as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or an 8000 Series SLM-AMINCOtm spectrophotometer (ThermoSpectronic) with a mixed colorimetric tube. 2. Antibody Fragments In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al, Mel 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluekthiin, The Pharmacology of Monoclonal Antibodies, Vol. 13 3, Rosenburg and Moore, 158864.doc -49-201215618 (Springer-Verlag, New York), pp. 269-3 1 5 ( 1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising rescue receptor binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. A bifunctional antibody is an antibody fragment which has a bivalent or bispecificity with two antigen binding sites. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Med. 9: 129-134 (2003); and Hollinger et al, Pr〇c. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al, 9: 129-134 (2003). A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). Antibody fragments can be produced by a variety of techniques including, but not limited to, protein hydrolyzing intact antibodies and by recombinant host cells (e.g., E. coli (five co/z) or phage), as described herein. , Dispersing and Humanizing Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Some of the chimeric antibodies are described, for example, in U.S. Patent No. 4,8,6,567; and Morrison et al, 5W.t/a, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (eg, variable 158864.doc 201215618 derived from a mouse, atmosphere, hamster, rabbit, or non-human primate (such as a monkey)) human sputum region . In another example, the chimeric antibody is a "class swap"antibody' wherein the class or subclass has been modified from a class or subclass of the parent antibody. Chelating antibodies include antigen-binding fragments thereof. In certain embodiments, the chimeric antibody is a humanized antibody. Generally, non-human steroids are humanized to reduce their immunogenicity to humans while preserving the specificity and affinity of the parental non-human organism. Generally, a humanized antibody comprises one or more variable domains' wherein the HVR (or portion thereof), e.g., CDr, is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies will also include at least a portion of the human constant region as appropriate. In some embodiments, some of the residues in the 'humanized antibody are substituted, e.g., by the corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residue is derived), for example, to restore or improve antibody specificity or affinity. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. 13: 1619-1633 (2008) and are further described, for example, in Riechmann et al, iVaiwre 332:323-329 (1988); Queen Etc., #加, / deal 5W. 86:10029-
10033 (1989);美國專利第 5,821,33 7號、第 7,527,791 號、 第 6,982,321 號及第 7,087,409 號;Kashmiri 等人,Me/办〇心 36:25-34 (2005)(描述 SDR(a-CDR)移植);Padlan,Mo/· Immunol. 28:489-498 (1991)(描述「表面重塑 (resurfacing)」);Dall’Acqua 等人,Methods 36:43-60 (2005)(描述「FR改組」);及 Osbourn等人,36:61-68 (2005)及 Klimka 等人,Br· J. Cancer, 83:252-260 (2000)(描述FR改組之「導向選擇(guided selection)」方 158864.doc -51 - 201215618 法)。 可用於人類化之人類構架區包括(但不限於):使用「最 佳配合(best-fit)」方法選擇之構架區(參見例如Sims等人, J. /wmwwo/. 151:2296 (1993));源於特定輕鏈或重鏈可變 區子群之人類抗體之共同序列的構架區(參見例如Carter等 k, Proc. Natl. Acad. Sci. USA, 89:4285 (1992);及 Presta 等人,乂化所《«W·,151:2623 (1993));人類成熟(體細胞 突變)構架區或人類生殖系構架區(參見例如Almagro及10033 (1989); U.S. Patent Nos. 5,821,33, 7, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Me/Office Hearts 36:25-34 (2005) (Describe SDR (a-CDR) transplantation); Padlan, Mo/. Immunol. 28:489-498 (1991) (description "resurfacing"); Dall'Acqua et al, Methods 36: 43-60 (2005) (Description of "FR Reorganization"); and Osbourn et al., 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (description of FR reorganization "guided selection (guided selection) Selection)" 158864.doc -51 - 201215618 law). Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" approach (see, for example, Sims et al., J. /wmwwo/. 151:2296 (1993) a framework region derived from the common sequence of human antibodies of a particular light chain or heavy chain variable region subgroup (see, eg, Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta Et al., "WW, 151: 2623 (1993)); human maturation (somatic mutation) framework or human germline framework (see for example Almagro and
Fransson,Fro价.价仍cz.· 13:1619-1633 (2008));及由篩檢 FR文庫產生之構架區(參見例如Baca等人,乂 5b/. c/zem. 272:10678-10684 (1997)及 Rosok 等人,J. C/2㈣. 271:22611-22618 (1996))-4.人類抗體 在某些實施例中,本文提供之抗體為人類抗體。人類抗 體可使用此項技術中已知之各種技術產生。人類抗體一般 地描述於 van Dijk 及 van de Winkel, Cwrr. Opk.尸/^所此〇/ 5: 368-74 (2001)及 Lonberg, Ci/rr.办以· /所則„〇/· 20:45〇_ 459 (2008)中。 人類抗體可藉由向轉殖基因動物投與免疫原來製備,該 轉殖基因動物已經改進以回應於抗原攻毒來產生完整人類 抗體或具有人類可變區之完整抗體。此等動物通常含有置 換内源性免疫球蛋白基因座或存在於染色體外或隨機整合 入動物染色體中之全部或部分人類免疫球蛋白基因座。在 此等轉殖基因小鼠中,内源性免疫球蛋白基因座通常不活 I58864.doc -52- 201215618 化。關於自轉殖基因動物獲得人類抗體之方法的綜述,參 見 Lonberg,23:1117-1 125 (2005)。亦參見例 如描述XENOMOUSEtm技術之美國專利第6,075,181號及第 6,150,584號;描述HUMAB®技術之美國專利第5,770,429 號;描述K-M MOUSE®技術之美國專利第7,041,870號及 描述VELOCIMOUSE®技術之美國專利申請公開案第US 2007/0061900號。來自由此等動物產生之完整抗體之人類 可變區可例如藉由與不同人類恆定區組合來進一步改進。Fransson, Fro Price. Price still cz. 13:1619-1633 (2008)); and the framework regions produced by screening FR libraries (see for example Baca et al., 乂 5b/. c/zem. 272: 10678-10684 (1997) and Rosok et al, J. C/2 (iv). 271:22611-22618 (1996))-4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Cwrr. Opk. The corpse / 5: 368-74 (2001) and Lonberg, Ci/rr. :45〇_ 459 (2008). Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or have human variable regions in response to antigen challenge. Intact antibodies. These animals usually contain all or part of the human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosome. In these transgenic mice The endogenous immunoglobulin locus usually does not live. I58864.doc -52 - 201215618. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, 23: 1117-1 125 (2005). See also eg U.S. Patent Nos. 6,075,181 and 6,150,584, the disclosure of which are incorporated herein by reference to U.S. Patent No. 5,770, 429, to the disclosure of U.S. Patent No. 5,770,429, to the disclosure of U.S. Patent No. 7, 041, 870, to KM MOUSE®, and to the beauty of VELOCIMOUSE® technology. U.S. Patent Application Publication No. US 2007/0061900. Human variable regions derived from intact antibodies produced by such animals can be further improved, for example, by combining with different human constant regions.
人類抗體亦可藉由基於融合瘤之方法製備。已描述用於 產生人類單株抗體之人類骨髓瘤及小鼠-人類異源骨髓瘤 細胞株。(參見例如 Kozbor ·/· Tmmwwo/.,133: 3001 (1984) ; Brodeur 等人,Monoclonal Antibody Production Techniques and Applications,第 5 1-63 頁(Marcel Dekker, Inc.,New York,1987);及 Boerner 等人,·/_ 147: 86 (1991))。經由人類B細胞融合瘤技術產生之人類抗體亦 描述於Li等人,iVoc. iVa". JcW. 5W. C/Sd,103:3557-3562 (2006)中。其他方法包括例如於以下中描述之方法:美國 專利第7,189,826號(描述自融合瘤細胞株產生單株人類18]^1 抗體)及 Ni,26(4):265-268 (2006)(描述 人類-人類融合瘤)。人類融合瘤技術(三體雜交瘤(Trioma) 技術)亦描述於以下中:Vollmers 及 Brandlein,//Vsio/og;; 20(3):927-937 (2005)以及 Vollmers及Human antibodies can also be prepared by fusion-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor ·/· Tmmwwo/., 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 5 1-63 (Marcel Dekker, Inc., New York, 1987); and Boerner Et al., /_ 147: 86 (1991)). Human antibodies produced by human B cell fusion tumor technology are also described in Li et al, iVoc. iVa " JcW. 5W. C/Sd, 103: 3557-3562 (2006). Other methods include, for example, the methods described in U.S. Patent No. 7,189,826 (which describes the production of a single human 18]^1 antibody from a fusion tumor cell line) and Ni,26(4):265-268 (2006) ( Describe human-human fusion tumors). Human fusion tumor technology (Trioma technology) is also described below: Vollmers and Brandlein, //Vsio/og;; 20(3): 927-937 (2005) and Vollmers and
Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3)·Λ85-91 (2005)。 % 158864.doc -53- 201215618 人類抗體亦可藉由分離選自人類源性噬菌體呈現文庫之 Fv純系可變域序列產生。此等可變域序列可接著與所要人 類恆定域組合。下文描述自抗體文庫選擇人類抗體之技 術。 5.自文庫獲得之抗體 本發明組合物中之抗體可藉由篩檢組合文庫中具有所要 活性之抗體來分離。舉例而言,此項技術中已知用於產生 噬菌體呈現文庫及筛檢此等文庫中具有所要結合特徵之抗 體的多種方法。此等方法例如於Hoogenboom等人, Methods Mo/ecw/ar Bz’o/ogy 178:1-37 (O'Brien 等人編, Human Press, Totowa, NJ,2001)中综述,且例如於以下中 進一步描述:McCafferty 等人,iVaiMre 348:552-554 ; Clackson等人,iVaiwre 352: 624-628 (1991) ; Marks等人,乂 Mol. Biol. 222: 581-597 (1992) ; Marks 及 Bradbury,Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3)·Λ85-91 (2005). % 158864.doc -53- 201215618 Human antibodies can also be produced by isolating Fv pure lineage variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. 5. Antibodies obtained from the library The antibodies in the compositions of the invention can be isolated by screening for antibodies having the desired activity in the combinatorial library. For example, various methods are known in the art for generating phage display libraries and screening for antibodies having the desired binding characteristics in such libraries. Such methods are reviewed, for example, in Hoogenboom et al, Methods Mo/ecw/ar Bz'o/ogy 178: 1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, 2001) and are for example in the following Further description: McCafferty et al, iVaiMre 348:552-554; Clackson et al, iVaiwre 352: 624-628 (1991); Marks et al, 乂Mol. Biol. 222: 581-597 (1992); Marks and Bradbury,
Mei/zoi/s /w Mo/ecw/ar 248:161-175 (Lo編,HumanMei/zoi/s /w Mo/ecw/ar 248:161-175 (edited by Lo, Human)
Press,Totowa, NJ, 2003) ; Sidhu等人,Mo/.价〇/. 338(2): 299-310 (2004) ; Lee 等人,/· Mo/. 340(5): 1073-1093 (2004) ; Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004);及 Lee 等人,J- Immunol. Methods 284(1-2): 119-132 (2004)。 在某些噬菌體呈現方法中,VH及VL基因之譜系係藉由 聚合酶鏈反應(PCR)分別選殖且隨機重組於噬菌體文庫 中’可接著如 Winter等人,乂狀.及ev. /mmwwo/.,12: 433-455 (1 994)中所述篩檢該等噬菌體文庫中之抗原結合噬菌體。 158864.doc -54- 201215618 噬菌體通常呈現呈單鏈Fv(scFv)片段形式或呈Fab片段形式 之抗體片段。來自經免疫來源之文庫提供對免疫原具有高 親和力之抗體而無需構築融合瘤。或者,天然譜系可經選 殖(例如自人類選殖)以提供針對廣泛範圍之非自體抗原以 及自體抗原之單一來源的抗體而不進行任何免疫,如由 Griffiths等人,五乂 12: 725-734 (1993)所述。最後,亦 可藉由自幹細胞選殖未重排之V基因區段且使用含有隨機 序列之PCR引子以編碼高度可變CDR3區且達成活體外重 排來合成製備天然文庫,如由Hoogenboom及Winter, «/. Mo/.价〇/., 227: 381-388 (1992)所述。描述人類抗體噬菌 體文庫之專利公開案包括例如:美國專利第5,750,373號、 及美國專利公開案第2005/0079574號、第2005/01 19455 號、第 2005/0266000 號、第 2007/0117126 號、第 2007/ 0160598 號、第 2007/0237764 號、第 2007/0292936 號及第 2009/0002360號。 自人類抗體文庫分離之抗體或抗體片段在本文中視為人 類抗體或人類抗體片段。 6.多特異性抗體 在某些實施例中,本文提供之抗體為多特異性抗體,例 如雙特異性抗體。多特異性抗體為對至少2個不同位點具 有結合特異性之單株抗體。在某些實施例中,結合特異性 之一係針對複合物I或gH且另一結合特異性係針對任何其 他抗原。在某些實施例中,結合特異性之一係針對複合物 I且另一結合特異性係針對gH。在某些實施例中,雙特異 158864.doc •55- 201215618 性抗體可與複合物I或gH之2個不同抗原決定基結合。雙特 異性抗體亦可用於將細胞毒性劑定位於在細胞表面上具有 複合物I或gH之細胞。雙特異性抗體可製備成全長抗體或 抗體片段。 製備多特異性抗體之技術包括(但不限於)重組共表現具 有不同特異性之兩個免疫球蛋白重鍵-輕鍵對(參見Mil stein 及 Cuello,305: 537 (1983)) ; WO 93/08829 ;及Press, Totowa, NJ, 2003); Sidhu et al., Mo/. Price/. 338(2): 299-310 (2004); Lee et al., /. Mo/. 340(5): 1073-1093 ( 2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J- Immunol. Methods 284(1-2): 119-132 (2004). In some phage display methods, the lineages of the VH and VL genes are separately selected by polymerase chain reaction (PCR) and randomly recombined into a phage library, which can then be followed by Winter et al., 乂. and ev. /mmwwo /., 12: Screening of antigen-binding phage in such phage libraries as described in 433-455 (1 994). 158864.doc -54- 201215618 Phage typically present in the form of a single-chain Fv (scFv) fragment or an antibody fragment in the form of a Fab fragment. Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need to construct fusion tumors. Alternatively, the natural lineage can be selected (eg, from human selection) to provide antibodies against a wide range of non-autoantigen and autologous antigens from a single source without any immunization, as by Griffiths et al., 乂12: 725-734 (1993). Finally, natural libraries can also be synthesized by colonizing unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieving in vitro rearrangement, such as by Hoogenboom and Winter. , «/. Mo/. Price/., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/01 19455, 2005/0266000, 2007/0117126, 2007 / 0160598, 2007/0237764, 2007/0292936 and 2009/0002360. An antibody or antibody fragment isolated from a human antibody library is considered herein to be a human antibody or a human antibody fragment. 6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificity for at least two different sites. In certain embodiments, one of the binding specificities is for Complex I or gH and the other binding specificity is for any other antigen. In certain embodiments, one of the binding specificities is for Complex I and the other binding specificity is for gH. In certain embodiments, the bispecific 158864.doc • 55-201215618 antibody binds to two different epitopes of complex I or gH. Bispecific antibodies can also be used to localize cytotoxic agents to cells having complex I or gH on the cell surface. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Techniques for preparing multispecific antibodies include, but are not limited to, recombinantly co-presenting two immunoglobulin heavy-key linkages with different specificities (see Milstein and Cuello, 305: 537 (1983)); WO 93/ 08829; and
Traunecker等人,五M50 «/· 10: 3655 (1991));及「孔中紐 (knob-in-hole)」工程改造(參見例如美國專利第5 73 1 168 號)。多特異性抗體亦可藉由以下方式製備:工程改造靜 電牽引(electrostatic steering)效應以製備抗體以雜二聚分 子(WO 2009/089004A1);交聯兩個或兩個以上抗體咬片段 (參見例如美國專利第4,676,980號,及Brennan等人 229: 81 (1985));使用白胺酸拉鏈以產生雙特異 性抗體(參見例如Kostelny 等人,J. 148(5).1547- 1553 (1992));使用「雙功能抗體」技術以製備雙特異性 抗體片段(參見例如Hollinger等人,5^·. 90:6444-6448 (1993));及使用單鍵 Fv(sFv)二聚體(參 見例如Gruber等人,《/· /wwwwo/·,152:5368 (1994));及如例 如Tutt等人,《/. 147: 60 (1991)中所述製備三特異 性抗體。 具有3個或3個以上功能性抗原結合位點之經工程改造抗 體(包括「章魚抗體(Octopus antibody)」)亦包括在本文中 (參見例如 US 2006/0025576A1)。 158864.doc -56- 201215618 本文之抗體或片段亦包括包含與複合物I或gH以及另一 不同抗原結合之抗原結合位點的「雙重作用FAb」或 「DAF」(參見你J 士aUS 2008/0069820)。 7.抗體變異體 在某些實施例中’涵蓋本文提供之抗體之胺基酸序列變 異體。舉例而言,可能需要改良抗體之結合親和力及/或 其他生物性質。抗體之胺基酸序列變異體可藉由將適當修 飾引入編碼該抗體之核苷酸序列中或藉由肽合成加以製 備。此等修飾包括例如在抗體之胺基酸序列内缺失及/或 插入及/或取代殘基。可進行缺失、插入及取代之任何組 合以獲得最終構築體,其限制條件為最終構築體具有所要 特徵’例如抗原結合。 a)取代、插入及缺失變異體 在某些實施例中,提供具有一或多個胺基酸取代之抗體 變異體。取代突變誘發之相關位點包括HVr及Fr。保守性 取代展示於表1中之標題「保守性取代」下。更實質性變 化提供於表1中之標題「例示性取代」下且如下文關於胺 基酸側鏈類別進一步描述。胺基酸取代可引入相關抗體中 且篩檢具有所要活性,例如保留/改良之抗原結合、降低 之免疫原性或改良之ADCC或CDC的產物。 158864.doc •57· 201215618 表1 原始殘基 例示性取代 較佳取代 Ala(A) Val ; Leu ; lie Val Arg(R) Lys ; Gin ; Asn Lys Asn(N) Gin ; His ; Asp,Lys ; Arg Gin Asp(D) Glu ; Asn Glu Cys(C) Ser ; Ala Ser Gln(Q) Asn ; Glu Asn Glu(E) Asp ; Gin Asp Gly(G) Ala Ala His(H) Asn ; Gin ; Lys ; Arg Arg Ile(I) Leu ; Val ; Met ; Ala ; Phe ;正白胺酸 Leu Leu(L) 正白胺酸;lie ; Val ; Met ; Ala ; Phe lie Lys(K) Arg ; Gin ; Asn Arg Met(M) Leu ; Phe ; lie Leu Phe(F) Trp ; Leu ; Val ; lie ; Ala ; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val ; Ser Ser Trp(W) Tyr ; Phe Tyr Tyr(Y) Trp ; Phe ; Thr ; Ser Phe Val(V) lie ; Leu ; Met ; Phe ; Ala ;正白胺酸 Leu 胺基酸可根據共有側鏈性質分組: (1) 疏水性:正白胺酸、Met、Ala、Val、Leu、lie ; (2) 中性親水性:Cys、Ser、Thr、Asn、Gin ; (3) 酸性:Asp、Glu ; (4) 驗性:His、Lys、Arg ; (5) 影響鏈定向之殘基:Gly ' Pro ; (6) 芳族:Trp、Tyr、Phe。 非保守性取代將導致此等類別之一之一個成員轉換成另 一類別。 一種類型之取代變異體涉及取代親本抗體(例如人類化 或人類抗體)之一或多個高變區殘基。一般而言,選擇用 -58- 158864.doc 201215618 於進—步研究之所得變異體相對於親本抗體將在某些生物 I1生貧(例如增加之親和力、降低之免疫原性)方面具有改進 (例如改良)及/或將具有親本抗體之實質上經保留之某些生 物性質。一種例示性取代變異體為親和力成熟抗體,其可 例如使用基於噬菌體呈現之親和力成熟技術(諸如本文所 述之技術)方便地產生。簡言之,突變一或多個殘基 且在噬菌體上呈現變異抗體並篩檢具有特定生物活性(例 如結合親和力)之變異抗體。 可在HVR中進行改變(例如取代)例如以改良抗體親和 力。此等改變可在HVR「熱點(h〇tsp〇t)」(亦即由在體細胞 成熟過程期間經受高頻率突變之密碼子編碼之殘基)(參見 例如 Chowdhury, Mei/zoA Μο/. Βζ·ο/· 207:179-196 (2008))及/ 或SDR(a-CDR)中進行,且測試所得變異結合親 和力。已例如於Hoogenboom等人, 心/⑽ m:1-37 (〇,Brien等人編,Human Press,T〇t〇wa, NJ,(2001))中描述藉由構築並自二級文庫再選擇來進行親 和力成熟。在親和力成熟之一些實施例中,藉由任何多種 方法(例如易出錯PCR、鏈改組或寡核苷酸定點突變誘發) 將多樣性引入選擇進行成熟之可變基因中。接著產生二級 文庫。接著篩檢文庫以鑑別具有所要親和力之任何抗體變 異體。引入多樣性之另一方法涉及HVR定向方法,其中若 干HVR殘基(例如一次4-6個殘基)經隨機化。參與抗原結合 之HVR殘基可例如使用丙胺酸掃描突變誘發或模型化來特 定鑑別。CDR-H3及CDR-L3尤其常被靶向。 158864.doc -59· 201215618 在某些實施例中,取代、插入或缺失可發生在一或多個 HVR内,只要此等改變不實質上降低抗體結合抗原之能力 即可。舉例而t ’可在題中進行不實質上降低結合親和 力之保守性改變(例如如本文提供之保守性取代)。此等改 變可在HVR「熱點」或SDR外部,在以上提供之變異vh& VL序列之某#實施财’各HVR未改變或含有至多!個、 2個或3個胺基酸取代。 一種適用於鑑別抗體之可供突變誘發靶向之殘基或區域 的方法稱為「丙胺酸掃描突變誘發」,如藉由 及Wells (1989) 244:1〇81-1〇85所述。在此方法 中,一個殘基或一組目標殘基(例如帶電荷殘基,諸如 arg、asp、his、lys及gh〇經鑑別且經中性或帶負電荷胺基 酸(例如丙胺酸或聚丙胺酸)置換以確定抗體與抗原之相互 作用是否受影響ϋ取代可在對初始取代顯示功能敏 感性之胺基酸位置處引入。或者或另外,抗原-抗體複合 物之晶體結構用以鑑別抗體與抗原之間的接觸點。此等接 觸殘基及鄰近殘基可作為取代候選物經靶向或消除。可篩 檢變異體以確定其是否含有所要性質。 胺基酸序列插入物包括長度在一個殘基至含有一百個或 一百個以上殘基之多肽之範圍内的胺基及/或羧基端融合 物' 以及具有單一或多個胺基酸殘基之序列内插入物。末 端插入物之實例包括具有Ν端曱硫胺醯基殘基之抗體。抗 體分子之其他插入變異體包括抗體端或c端與酶(例如 對於ADEPT而言)或增加抗體之血清半衰期之多肽的融合 158864.doc •60- 201215618 物。 b)糖基化變異體 在某些實施例中,本文提供之抗體經改變以增加或減小 抗體糖基化之程度。在抗體上添加糖基化位點或使抗體缺 失糖基化位點可藉由改變胺基酸序列以便產生或移除一或 多個糖基化位點來方便地達成。 當抗體包含Fc區時,可改變與其連接之碳水化合物。由 哺乳動物細胞產生之天然抗體通常包含分支雙觸角 (biantennary)寡醣,其通常藉由N_鍵聯連接至Fc區之ch2 域的 Asn297。參見例如 Wright 等人,7757^(7// 15:26-32 (1997)。寡醣可包括各種碳水化合物,例如甘露糖、N_乙 酿基葡糖胺(GlcNAc)、半乳糖及唾液酸,以及與雙觸角寡 耱結構之「主幹」中之GlcNAc連接的海藻糖。在一些實 施例中,可對本發明抗體中之寡醣進行修飾以產生具有某 些經改良性質之抗體變異體。 在一實施例中,提供具有缺乏與Fc區連接(直接或間接) 之海藻糖之碳水化合物結構的抗體變異體。舉例而言,此 抗體中之海藻糖之量可為1%至80%、1%至65%、5%至65% 或20%至40%。如例如WO 2008/077546中所述,藉由相對 於Asn 297所連接所有醣結構(例如複合、雜交及高甘露糖 結構)之總和計算Asn297處糖鏈内海藻糖的平均量來測定 海藻糖之量,如藉由MALDI-TOF質譜所量測。Asn297係 指位於Fc區中約位置297(Fc區殘基之Eu編號)處之天冬醯 胺殘基;然而,歸因於抗體中之微小序列變化,Asn297亦 三' 158864.doc •61 - 201215618Traunecker et al., M. M50 «/· 10: 3655 (1991)); and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,73, 168). Multispecific antibodies can also be prepared by engineering an electrostatic steering effect to prepare antibodies to heterodimeric molecules (WO 2009/089004 A1); cross-linking two or more antibody bit fragments (see for example U.S. Patent No. 4,676,980, and Brennan et al. 229:81 (1985)); use of a leucine zipper to produce a bispecific antibody (see, e.g., Kostelny et al, J. 148 (5). 1547 - 1553 (1992)) The use of "bifunctional antibody" technology to prepare bispecific antibody fragments (see, eg, Hollinger et al, 5^.. 90:6444-6448 (1993)); and the use of single-bond Fv (sFv) dimers (see for example Gruber et al., "/· /wwwwo/., 152:5368 (1994)); and preparation of trispecific antibodies as described, for example, in Tutt et al., /. 147: 60 (1991). Engineered antibodies (including "Octopus antibodies") having three or more functional antigen binding sites are also included herein (see, for example, US 2006/0025576 A1). 158864.doc -56- 201215618 The antibodies or fragments herein also include "dual-acting FAb" or "DAF" comprising an antigen binding site that binds to complex I or gH and another different antigen (see your J. aUS 2008/ 0069820). 7. Antibody Variants In certain embodiments, the amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. The amino acid sequence variant of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, with the proviso that the final construct has the desired characteristics 'e.g., antigen binding. a) Substitution, Insertion, and Deletion Variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Related sites induced by substitution mutations include HVr and Fr. Conservative substitutions are shown under the heading "Conservative substitutions" in Table 1. More substantial variations are provided under the heading "Exemplary Substitutions" in Table 1 and are further described below with respect to the amino acid side chain classes. Amino acid substitutions can be introduced into the relevant antibodies and screened for products having the desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. 158864.doc •57·201215618 Table 1 Exemplary substitutions of the original residues preferred substitutions Ala(A) Val; Leu; lie Val Arg(R) Lys; Gin; Asn Lys Asn(N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu ; Asn Glu Cys ( C ) Ser ; Ala Ser Gln ( Q ) Asn ; Glu Asn Glu ( E ) Asp ; Gin Asp Gly ( G ) Ala Ala His ( H ) Asn ; Gin ; Lys ; Arg Arg Ile(I) Leu ; Val ; Met ; Ala ; Phe ; Leucine Leu Leu ( L ) leucine ; lie ; Val ; Met ; Ala ; Phe lie Lys ( K ) Arg ; Gin ; Asn Arg Met(M) Leu ; Phe ; lie Leu Phe ( F ) Trp ; Leu ; Val ; lie ; Ala ; Tyr Tyr Pro ( P ) Ala Ala Ser ( S ) Thr Thr Thr ( T ) Val ; Ser Ser Trp ( W ) Tyr; Phe Tyr Tyr(Y) Trp ; Phe ; Thr ; Ser Phe Val(V) lie ; Leu ; Met ; Phe ; Ala ; ortho-leucine Leu Amino acid can be grouped according to the shared side chain properties: (1) Hydrophobic Sex: leucine, Met, Ala, Val, Leu, lie; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin; (3) Acidity: Asp, Glu; (4) Qualitative: His, Lys, Arg; (5) Residues affecting chain orientation: Gly 'Pro; (6) Aromatic: Trp, Tyr, PheA non-conservative substitution will result in the conversion of one of these categories into another category. One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the variants obtained with the -58-158864.doc 201215618 step-by-step study will improve in some organisms I1 (such as increased affinity, reduced immunogenicity) relative to the parent antibody. (eg, improved) and/or will have certain biological properties that are substantially retained by the parent antibody. An exemplary substitution variant is an affinity matured antibody that can be conveniently produced, for example, using phage-based affinity maturation techniques, such as the techniques described herein. Briefly, one or more residues are mutated and a variant antibody is presented on the phage and screened for a variant antibody having a particular biological activity (e. g., binding affinity). Alterations (e. g., substitutions) can be made in the HVR, e.g., to improve antibody affinity. These changes can be made in the HVR "hot spot (h〇tsp〇t)" (ie, the residue encoded by the codon that is subjected to high frequency mutations during the somatic maturation process) (see for example Chowdhury, Mei/zoA Μο/. Βζ • ο/· 207: 179-196 (2008)) and/or SDR (a-CDR) were performed and the resulting variation was tested for binding affinity. It has been described, for example, in Hoogenboom et al., Heart/(10) m: 1-37 (〇, Brien et al., ed., Human Press, T〇t〇wa, NJ, (2001)) by constructing and reselecting from a secondary library. Come to affinity maturity. In some embodiments of affinity maturation, diversity is introduced into a variable gene selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide site-directed mutagenesis. A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR targeting approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutation induction or modeling. CDR-H3 and CDR-L3 are especially targeted. 158864.doc -59· 201215618 In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs as long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, t' can be made in the subject to not substantially reduce the conservative change in binding affinity (e.g., conservative substitution as provided herein). These changes can be made outside the HVR "hotspot" or SDR. In the variant vh& VL sequence provided above, the implementation of each HVR has not changed or contains at most! One, two or three amino acid substitutions. One method suitable for identifying residues or regions of an antibody that are susceptible to mutation-induced targeting is known as "alanine scanning mutation induction" as described by Wells (1989) 244:1, 81-1,85. In this method, a residue or a set of target residues (eg, charged residues such as arg, asp, his, lys, and gh) are identified and neutralized or negatively charged with an amino acid (eg, alanine or The polyalanine) substitution determines whether the interaction of the antibody with the antigen is affected. The substitution can be introduced at the position of the amino acid which shows a functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify The point of contact between the antibody and the antigen. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. The variant can be screened to determine if it contains the desired properties. Amino acid sequence inserts include length An amino- and/or carboxyl-terminal fusion in the range of one residue to a polypeptide comprising one hundred or more residues and an intrasequence insert having a single or multiple amino acid residues. Examples of inserts include antibodies having a thiol sulfhydryl residue at the terminal. Other insertion variants of the antibody molecule include antibody or c-terminus with an enzyme (for example, for ADEPT) or increase serum half-life of the antibody. . B) the extent of glycosylation of antibody glycosylation variants In certain embodiments, provided herein the antibody is altered to increase or decrease the fusion polypeptide 158864.doc • 60- 201215618 thereof. Addition of a glycosylation site to an antibody or deletion of a glycosylation site by an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites. When the antibody comprises an Fc region, the carbohydrate to which it is attached can be altered. Native antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide, which is typically joined to the Asn297 of the ch2 domain of the Fc region by an N-linkage. See, for example, Wright et al, 7757(7//15:26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-ethyl glucosamine (GlcNAc), galactose, and sialic acid. And trehalose linked to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to produce antibody variants having certain improved properties. In one embodiment, an antibody variant having a carbohydrate structure lacking trehalose linked (directly or indirectly) to the Fc region is provided. For example, the amount of trehalose in the antibody can range from 1% to 80%, 1 % to 65%, 5% to 65% or 20% to 40%. As described, for example, in WO 2008/077546, all sugar structures (eg, complex, hybrid, and high mannose structures) are attached by attachment to Asn 297. The average amount of trehalose in the sugar chain at Asn297 was calculated to determine the amount of trehalose as measured by MALDI-TOF mass spectrometry. Asn297 means located at about position 297 in the Fc region (Eu number of the Fc region residue) Aspartate residue; however, due to minor sequence changes in the antibody , Asn297 also three '158864.doc •61 - 201215618
可能位於位置297之上游或下游約土3個胺基酸處,亦即介 於位置294與300之間。此等海藻糖基化變異體可具有改良 之ADCC功能。參見例如美國專利公開案第US 2003/ 0157108 號(Presta,L.);第 US 2004/0093621 號(Kyowa Hakko Kogyo Co., Ltd)。與「去海藻糖基化」或「海蕩糖 缺乏」抗體變異體相關之公開案之實例包括:US 2003/0157108 ; WO 2000/61739 ; WO 2001/29246 ; US 2003/0115614 ; US 2002/0164328 ; US 2004/0093621 ; US 2004/0132140 ; US 2004/0110704 ; US 2004/01 10282 ; US 2004/0109865 ; WO 2003/085119 ; WO 2003/084570 ; WO 2005/035586 ; WO 2005/035778 ; W02005/053 742 ; W02002/031140 ; Okazaki 等人,乂 尬/. 5,.〇/· 336:1239-1249 (2004) ; Yamane-Ohnuki 等人,Biotech. Bioeng. 87: 614 (2004)。能夠產生去海藻糖基化抗體之細胞株之實例 包括缺乏蛋白質海藻糖基化之Lecl3 CHO細胞(Ripka等人, Jrc/z. 249:533-545 (1986);美國專利申It is possible to locate approximately 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such trehalylation variants may have improved ADCC function. See, for example, U.S. Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "trehaloselation" or "hypoglycemic" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328 US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/01 10282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053 742; W02002/031140; Okazaki et al., 乂尬/. 5,.〇/· 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing de-fucosylated antibodies include Lecl3 CHO cells lacking protein fucosylation (Ripka et al, Jrc/z. 249: 533-545 (1986); US patent application
請案第 US 2003/0157108 A1 號,Presta, L ;及 WO 2004/056312 Al,Adams等人,尤其在實例11)及基因剔除 細胞株,諸如α-1,6-海藻糖基轉移酶基因基因剔除 CHO細胞(參見例如 Yamane-Ohnuki等人, 87: 614 (2004) ; Kanda,Υ·等人,Bioeng., 94(4):680-688 (2006);及 W02003/085107)。 進一步提供具有對分寡醣之抗體變異體,例如其中與抗 體之Fc區連接之雙觸角寡醣由GlcNAc對分。此等抗體變 158864.doc •62- 201215618 異體可具有降低之海藻糖基化及/或改良之ADCC功能。此 等抗體變異體之實例例如描述於wo 2003/011878(Jean-Mairet等人);美國專利第6,602,684號(Umana等人);及US 2005/0123546(Umana等人)中。亦提供寡醣中至少1個半乳 糖殘基連接至Fc區之抗體變異體。此等抗體變異體可具有 改良之CDC功能。此等抗體變異體例如描述於WO 1997/30087(Patel等人);WO 1998/58964(Raju,S.);及 WO 1999/22764(Raju, S.)中。 • c)Fc區變異體 在某些實施例中,一或多個胺基酸修飾可引入本文提供 之抗體之Fc區中,藉此產生Fc區變異體。Fc區變異體可包 含在一或多個胺基酸位置處包含胺基酸修飾(例如取代)之 人類Fc區序列(例如人類IgGl、IgG2、IgG3或IgG4 Fc區)。 在某些實施例中,本發明涵蓋具有一些而非所有效應功 能之抗體變異體,該等效應功能使該抗體成為合乎多種應 用之需要之候選物,在該等應用中,活體内抗體半衰期較 ® 重要,而某些效應功能(諸如補體及ADCC)為不必要或有 害的。可進行活體外及/或活體内細胞毒性檢定以確認 CDC及/或ADCC活性之降低/消減。舉例而言,可進行Fc受 體(FcR)結合檢定以確保抗體缺乏FcYR結合(由此可能缺乏 ADCC活性)但保留FcRn結合能力。介導ADCC之初級細胞 NK細胞僅表現Fc(RIII,而單核細胞表現Fc(RI、Fc(RII及 Fc(RIII。FcR在造血細胞上之表現概述於Ravetch及Kinet, Annu· Rev. Immunol. 9:457-492 (1991)之第 464 頁上之表 3 二· 158864.doc -63- 201215618 中。評估相關分子之ADCC活性之活體外檢定的非限制性 實例描述於美國專利第5,500,362號(參見例如Hellstrom, I. 等人,Proc. t/M 83:7059-7063 (1986))及Requests US 2003/0157108 A1, Presta, L; and WO 2004/056312 Al, Adams et al, especially in Example 11) and gene knockout cell lines, such as the α-1,6-trehalyltransferase gene CHO cells are knocked out (see, for example, Yamane-Ohnuki et al, 87: 614 (2004); Kanda, Υ· et al, Bioeng., 94(4): 680-688 (2006); and W02003/085107). Further provided are antibody variants having a oligosaccharide, such as a biantennary oligosaccharide in which the Fc region of the antibody is ligated by GlcNAc. Such antibodies may be 158864.doc • 62- 201215618 Allogeneic may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants in which at least one galactose residue in the oligosaccharide is attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). • c) Fc region variants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region). In certain embodiments, the invention encompasses antibody variants having some, but not all, of the effector functions that make the antibody a candidate for a variety of applications in which the half-life of the antibody is greater in vivo. ® is important, and certain effect functions such as complement and ADCC are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcYR binding (and thus may lack ADCC activity) but retains FcRn binding ability. Primary cells that mediate ADCC NK cells exhibit only Fc (RIII, whereas monocytes exhibit Fc (RI, Fc (RII and Fc (RIII. The expression of FcR on hematopoietic cells is outlined in Ravetch and Kinet, Annu Rev. Immunol. Non-limiting examples of in vitro assays for assessing ADCC activity of related molecules are described in U.S. Patent No. 5,500,362 (Table 3, pp. 464, pp. 464, pp. 464864.doc -63-201215618). See, for example, Hellstrom, I. et al., Proc. t/M 83:7059-7063 (1986)) and
Hellstrom,I等人,5W. CAS^ 82:1499-1502 (1985) ; 5,821,337(參見Bruggemann, M.等人,《/. Mec?· 166:1351-1361 (1987))中。或者,可採用非放射性檢定方 法(參見例如用於流動式細胞測量術之ACTITM非放射性細 胞毒性檢定(CellTechnology, Inc,Mountain View,CA);及 CytoTox 96®非放射性細胞毒性檢定(Promega,Madison, WI))。適用於此等檢定之效應細胞包括周邊血液單核細胞 (PBMC)及自然殺手(NK)細胞。或者或另外,相關分子之 ADCC活性可例如於動物模型,諸如Clynes等人,ZVoc. Nat'l Acad. Sci. USA 95:652-656 (1998)中揭示之動物模 型中進行活體内評估。亦可進行Clq結合檢定以確認抗體 不能結合Clq且因此缺乏CDC活性。參見例如WO 2006/029879 及 WO 2005/100402 中之 Clq 及 C3c 結合 ELISA。為評估補體活化,可進行CDC檢定(參見例如 Gazzano-Santoro 等人,/. TrnmMwo/· Mei/zoc/i 202:163 (1996); Cragg, M.S·等人,101:1045-1052 (2003); 及 Cragg, M.S.及 M.J. Glennie, Blood 103:273 8-2743 (2004))。亦可使用此項技術中已知之方法進行FcRn結合及 活體内清除率/半衰期測定(參見例如Petkova,S.B.等人, Int'l. Immunol. 18(12):1759-1769 (2006)) ° 效應功能降低之抗體包括Fc區殘基238、265、269、 158864.doc -64· 201215618 270、297、327及329中之一或多者經取代的抗體(美國專 利第6,737,056號)。此等Fc突變體包括在胺基酸位置265、 269、270、297及3 27中之兩者或兩者以上處具有取代的Fc 突變體’包括殘基265及297取代成丙胺酸之所謂 「DANA」Fc突變體(美國專利第7,332,581號)。 與FcR之結合得到改良或減弱之某些抗體變異體已有描 述。(參見例如美國專利第6,737,056號;WO 2004/ 056312;及 Shields 等人,乂 5/0/. c/7謂.9(2): 6591-6604 (2001)) 〇 在某些實施例中,抗體變異體包含具有改良ADCC之一 或多個胺基酸取代’例如Fc區之位置298、333及/或334(殘 基之EU編號)處之取代的Fc區。 在一些實施例中’在Fc區中進行產生改變(亦即改良或 減弱)之Clq結合及/或補體依賴性細胞毒性(CDC)的改變, 例如如美國專利第6,194,551號、WO 99/51642及Idusogie 等人,*/. 164: 4178-4184 (2000)中所述。 半衰期增加且與負責將母體IgG轉移至胎兒之新生兒Fc 受體(FcRn)(Guyer 等人,乂 1 17:587 (1976)及 Kim 等人,J. /www⑽/. 24:249 (1994))之結合改良的抗體描述於 US 2005/0014934A1 (Hinton等人)中。彼等抗體包含其中 具有改良Fc區與FcRn之結合之一或多個取代的Fc區。此等 Fc變異體包括在以下Fc區殘基中之一或多者處具有取代之 Fc變異體:238、256、265、272、286、303、305、307、 311 、 312 、 317 、 340 、 356 、 360 、 362 、 376 、 378 、 380 、 158864.doc -65- 201215618 3 82、413、424或434,例如Fc區殘基434經取代(美國專利 第 7,371,826號)。 關於Fc區變異體之其他實例,亦參見Duncan及Winter, 322:738-40 (1988);美國專利第 5,648,260號;美國 專利第 5,624,821號;及\\^0 94/29351。 d) 半胱胺酸工程改造抗體變異翘 在某些實施例中,可能需要產生半胱胺酸工程改造抗 體’例如「琉基MAb(thioMAb)」,其中抗體之一或多個殘 基經半胱胺酸殘基取代。在特定實施例中,經取代之殘基 存在於抗體之可達位點處。藉由用半胱胺酸取代彼等殘 基,反應性硫醇基藉此定位於抗體之可達位點處且可用於 使抗體與其他部分(諸如藥物部分或連接子-藥物部分)結合 以產生如本文中進一步描述之免疫結合物。在某些實施例 中’任何一或多個以下殘基可經半胱胺酸取代:輕鏈之 V205(Kabat編號);重鏈之A118(EU編號);及重鏈Fc區之 S400(EU編號)。半胱胺酸工程改造抗體可如例如美國專利 第7,521,541號中所述來產生。 e) 抗體衍生物 在某些實施例中,本文提供之抗體可進一步修飾以含有 此項技術中已知且易於獲得之其他非蛋白質部分。適於衍 生化抗體之部分包括(但不限於)水溶性聚合物。水溶性聚 «物之非限制性實例包括(但不限於)聚乙二醇(PEG)、乙 一醇/丙二醇之共聚物、羧甲基纖維素、葡聚糖、聚乙烯 醇、聚乙烯吡咯啶_、聚],3_二氧雜環戊烷 '聚_1'6_三 158864.doc • 66 - 201215618 心烷乙烯/順丁烯二酸酐共聚物、聚胺基酸(均聚物或無 規/、聚物)及葡聚糖或聚(N_乙烯基吡咯啶酮)聚乙二醇、丙 7醇均聚物、聚環氧丙烧/環氧乙院共聚物、聚氧乙稀化 多元醇(例如甘油)、聚乙烯醇及其混合物。聚乙二醇丙醛 由於其於水中之穩定性而可在製造上具有優勢。聚合物可 八有彳何刀子里,且可為分支鏈或未分支鏈。與抗體連接 之聚合物之數目可變化,且若連接一個以上聚合物,則該 等聚合物可為相同或不同分子。一般而言,用於衍生化: ® 物之數目及/或類型可基於包括(但不限於)以下之考慮 因素來確定:欲改良之抗體之特定性質或功能、抗體衍生 物是否將用於確定條件下之療法等。 在另一實施例中,提供抗體與可藉由暴露於輻射來選擇 性加熱之非蛋白質部分的結合物。在一實施例中,非蛋白 質部分為碳奈米管(Kam等人,Proc· #加/· &厂C/以 102: 11600-11605 (2005))。輻射可具有任何波長,且包括 (但不限於)不損傷普通細胞但可將非蛋白質部分加熱至可 • 殺死與抗體-非蛋白質部分鄰近之細胞之溫度的波長。 C·重組方法及組合物 可使用例如如美國專利第4,816,567號中所述之重組方法 及組合物產生抗體。在一實施例中,提供本文所述之編碼 抗複合物I抗體或抗gH抗體之經分離核酸。此核酸可編碼 包含VL之胺基酸序列及/或包含抗體之VH之胺基酸序列 (例如抗體之輕鏈及/或重鏈)。在另一實施例中,提供一或 多種包含此核酸之載體(例如表現載體)。在另一實施例 158864.doc -67- 201215618 中’提供包含此核酸之宿主細胞。在一此類實施例中,宿 主細胞包含(例如已經以下轉型):⑴包含某一核酸之載 體,該核酸編碼包含抗魏之胺基酸序列及包含抗體vh 之胺基酸序列,或(2)包含編碼包含抗體VL之胺基酸序列 之核酸的第-載體·,及包含編碼包含抗體VH之胺基酸序 宿主細胞為真核細 列之核酸的第二載體。在一實施例中 胞,例如中國倉鼠卵巢(CHO)細胞或淋巴細胞(例如γ〇、 NS0、Sp20細胞)。在一實施例中,提供一種製備抗複合物 I抗體或抗gH抗體之方法,其中該方法包含在適於表現該 抗體之條件下培養如上文所提供之包含編碼該抗體之核酸 的佰主細胞,及視情況自該宿主細胞(或宿主細胞培養基) 回收該抗體。 對於重組產生抗複合物I抗體或抗gH抗體,分離編碼例 如如上所述之抗體之核酸且插入一或多個載體中以供進一 步在宿主細胞中選殖及/或表現。此核酸可易於使用習知 程序(例如藉由使用能夠與編碼抗體之重鏈及輕鏈之基因 特異性結合的寡核苷酸探針)分離及定序。 適於選殖或表現抗體編碼載體之宿主細胞包括本文所述 之原核或真核細胞。舉例而言,抗體可於細菌中產生,特 定言之當不需要糖基化及Fc效應功能時。關於在細菌中表 現抗體片段及多肽,參見例如美國專利第5,648,237號、第 5,789,199號及第 5,840,523 號。(亦參見 Charlton, in Molecular Biology,第 248 卷(B.K.C. Lo 編,Humana Press,Totowa,NJ,2003),第 245-254 頁’其描述在大腸桿 158864.doc -68- 201215618 菌中表現抗體片段)。在表現之後 p 说抗體可自細菌細胞漿 (bacterial cell paste)分離於可溶性部分中且可進一步純 化。 除原核生物外,諸如絲狀真菌或酵母之真核微生物為適 於選殖或表現抗體編碼載體之宿主,包括糖基化路徑已經 「人類化」’ &而使得所產生之抗體具有部分或完全人類 糖基化型態的真菌及酵母菌株。參見Gerngr〇ss, 22:1409-1414 (2004)及 Li 等人,紿 μ 24:210-215 (2006)。 適於表現糖基化抗體之宿主細胞亦源於多細胞生物體 (無脊椎動物及脊椎動物)。無脊椎動物細胞之實例包括植 物及昆蟲細胞。已鑑別出眾多可與昆蟲細胞聯合使用之桿 狀病毒株’特定言之用於轉染草地黏蟲(补 ffugiperda)細跑。 植物細胞培養物亦可用作宿主。參見例如美國專利第 5,959,177 號、第 6,040,498 號、第 6,42〇 548 號、第 7,125,978號及第6,417,429號(描述用於在轉瘦基因植物中 產生抗體之PLANTIBODIEStm技術)〇 脊椎動物細胞亦可用作宿主。舉例而言,適於在懸浮液 中生長之哺乳動物細胞株可為適用的。適用哺乳動物宿主 細胞株之其他實例為經SV40轉型之猴腎CV1細胞株(COS-7);人類胚腎細胞株(293或293細胞,如例如Graham等人, J. Ge« KzW. 36:59 (1977)中所述);幼小倉鼠腎細胞 (BHK);小鼠足細胞(TM4細胞’如例如Mather,5/〇/· 158864.doc -69- 201215618 23:243-251 (1980)中所述);猴腎細胞(CVI);非洲 綠猴腎細胞(VERO-76);人類子宮頸癌細胞(HELA);犬腎 細胞(MDCK);布法羅大鼠(buffalo rat)肝細胞(BRL 3 A); 人類肺細胞(W138);人類肝細胞(Hep G2);小鼠乳腺腫瘤 (MMT 060562) ; TRI細胞,如例如Mather等人,见]7. Jcad. Scz·. 383:44-68 (1982)中所述;MRC 5 細月包;及 FS4 細 胞。其他適用哺乳動物宿主細胞株包括中國倉鼠卵巢 (CHO)細胞,包括 DHFR-CHO 細胞(Urlaub 等人,Proc. iVai/· Acad. Sci. 77:42 16 (1980));及骨髓瘤細胞株,諸如 Y0、NS0及Sp2/0。關於適於抗體產生之某些哺乳動物宿 主細胞株之綜述,參見例如Yazaki及Wu, Mei/zoA Molecular Biology,第 248卷(B.K.C. Lo編,Humana Press, Totowa, NJ),第 255-268 頁(2003)。 D.檢定 本文提供之抗複合物I抗體或抗gH抗體可藉由此項技術 中已知之各種檢定針對其物理/化學性質及/或生物活性進 行鑑別、筛檢或表徵。 1.結合檢定及其他檢定 在一態樣中,例如藉由諸如ELISA、西方墨點等之已知 方法測試本發明抗體之抗原結合活性。 在另一態樣中,競爭檢定可用於鑑別與本文所述之抗複 合物I抗體競爭結合複合物I之抗體。 在另一態樣中,競爭檢定可用於鑑別與本文所述之抗gH 抗體競爭結合gH之抗體。 158864.doc -70- 201215618 在某些實施例中,該種競爭抗體與gH或複合物I之相同 抗原決定基(例如線性抗原決定基或構形抗原決定基)結 合。 用於定位抗體所結合之抗原決定基之詳述例示性方法提 供於 Morris (1996)「Epitope Mapping Protocols」’Methods k Mo/ecw/ar 价o/og_y 第 66 卷(Humana Press, Totowa, NJ) 中o 在一個例示性競爭檢定中,在包含分別與複合物I或gH 結合之第一經標記抗體以及第二未標記抗體之溶液中培育 經固定複合物I或gH,測試該第二未標記抗體與該第一抗 體競爭結合複合物I或gH之能力。第二抗體可存在於融合 瘤上清液中。作為對照,經固定複合物I或gH在包含第一 經標記抗體但不包含第二未標記抗體之溶液中培育。在允 許第一抗體與複合物I或gH結合之條件下培育之後,移除 過量未結合抗體,且量測與經固定複合物I或gH締合之標 記的量。若相對於對照樣品,測試樣品中與經固定複合物 I或gH締合之標記的量實質上降低,則指示第二抗體與第 一抗體競爭結合複合物I或gH。參見Harlow及Lane (1988) Antibodies: A Laboratory Manual % \A (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) ° 競爭檢定亦可以如上所述之方式,用使用經複合物I或 複合物II之gH及/或其他成員轉染且在細胞表面上表現該 gH及/或該等其他成員之細胞進行的FACS來進行。另外, 用gH及/或復原複合物I或複合物II進行之ELISA亦可用於 158864.doc -71 - 201215618 競爭檢定中。使用FACS及ELISA量測抗gH抗體及抗複合 物I抗體進一步描述於實例中。 2·活性檢定 在一態樣中’提供用於鑑別具有生物活性之抗複合物j 抗體之檢疋。生物活性可包括例如與由Ul 12 8、UL13 0、 UL131與gH/gL締合產生之構形抗原決定基特異性結合; 或與複合物I之單一蛋白質内之抗原決定基特異性結合; 中和 HCMV,EC9〇為 0.7 |ig/ml或 0.7 pg/ml以下。在一些實 施例中’ EC9〇為0.5 pg/ml或0.5 pg/ml以下。在其他實施例 中,ECpo為0.3 pg/ml或0.3 pg/ml以下。在其他實施例中, EC90為0.1 pg/ml或〇. 1 pg/mi以下。在其他實施例中,ε^9〇 為0.08 pg/ml或0.08 pg/ml以下。在其他實施例中,Ec9〇為 0.06 pg/ml或0.06 pg/ml以下。在其他實施例中,Ec9〇為 0.04 pg/ml或〇.〇4 pg/ml以下。在其他實施例中,EC9〇為 0.02 pg/ml或0.02 pg/ml以下。在其他實施例中,Ec9〇為 0.015 pg/ml或0.015 pg/ml以下。在其他實施例中,Ec9〇為 0.012#邑/1111或0.012 48/1111以下。在其他實施例中,^9〇為 0.011 pg/ml或0.011 pg/ml以下。在其他實施例中,Ec9〇為 0.010 pg/ml或0.010 pg/ml以下。亦提供包含具有此生物活 性之抗體之組合物。 在一態樣中,k供用於鑑別具有生物活性之抗gH抗體之 檢定。生物活性可包括例如中和HCMV,EC90為1 pg/ml、 0.9 μδ/ml、0.8 pg/ml、〇·7 ng/m卜 〇.6 μ§/ηι卜 〇 5 ^/ml、 0.4 pg/ml、0.3 pg/ml、0.2 pg/ml、〇1 μ§Λη1、〇 〇9 158864.doc •11- 201215618Hellstrom, I et al., 5W. CAS^ 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., /. Mec? 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, for example, ACTITM non-radioactive cytotoxicity assays for flow cytometry (Cell Technology, Inc, Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)). Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the relevant molecule can be assessed in vivo, for example, in an animal model, such as the animal model disclosed in Clynes et al, ZVoc. Nat'l Acad. Sci. USA 95:652-656 (1998). A Clq binding assay can also be performed to confirm that the antibody is unable to bind Clq and thus lacks CDC activity. See, for example, the Clq and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., /. TrnmMwo/. Mei/zoc/i 202: 163 (1996); Cragg, MS et al, 101: 1045-1052 (2003) ; and Cragg, MS and MJ Glennie, Blood 103:273 8-2743 (2004)). FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al, Int'l. Immunol. 18(12): 1759-1769 (2006)). Reduced antibodies include one or more substituted antibodies of Fc region residues 238, 265, 269, 158864. doc-64. 201215618 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). These Fc mutants include a Fc mutant having a substitution at two or more of the amino acid positions 265, 269, 270, 297, and 3 27 'including the substitution of residues 265 and 297 to alanine. DANA" Fc mutant (US Patent No. 7,332,581). Certain antibody variants with improved or reduced binding to FcR have been described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312; and Shields et al., 乂 5/0/.c/7. 9(2): 6591-6604 (2001)) 某些 In some embodiments, Antibody variants comprise an Fc region having a substitution at one or more of the amino acid substitutions of the ADCC, such as at positions 298, 333 and/or 334 of the Fc region (the EU numbering of the residues). In some embodiments, 'changes in Clq binding and/or complement dependent cytotoxicity (CDC) are produced in the Fc region, such as, for example, U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al., */. 164: 4178-4184 (2000). Increased half-life and the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus (Guyer et al, 乂 1 17:587 (1976) and Kim et al, J. /www(10)/. 24:249 (1994) A combination of improved antibodies is described in US 2005/0014934 A1 (Hinton et al.). The antibodies comprise an Fc region having one or more substitutions in which the modified Fc region binds to FcRn. Such Fc variants include Fc variants having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 158864.doc -65- 201215618 3 82, 413, 424 or 434, for example, Fc region residue 434 is substituted (U.S. Patent No. 7,371,826). See also, Duncan and Winter, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and \\^0 94/29351. d) Cysteine engineered antibody variants In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as "thioMAb", in which one or more residues of the antibody are half Cyst acid residue substitution. In a particular embodiment, the substituted residue is present at a reachable site of the antibody. By substituting their residues with cysteine, the reactive thiol group is thereby localized to the accessible site of the antibody and can be used to bind the antibody to other moieties such as a drug moiety or a linker-drug moiety. An immunoconjugate is produced as described further herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU) of the heavy chain Fc region Numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. Patent No. 7,521,541. e) Antibody Derivatives In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein portions known in the art and readily available. Portions suitable for the derivatization of antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymeric materials include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidine _, poly], 3_dioxolane 'poly_1'6_three 158864.doc • 66 - 201215618 a mixture of aerane ethylene/maleic anhydride, polyamino acid (homopolymer or none) Gauge/, polymer) and dextran or poly(N_vinylpyrrolidone) polyethylene glycol, propanol homopolymer, polyglycidyl/epoxy copolymer, polyoxyethylene Polyols (such as glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can be in any knife and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and/or type of derivatization: ® can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be modified, and whether the antibody derivative will be used for determination. Therapy under conditions, etc. In another embodiment, a combination of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. #加/&&&&&&&&&&&&&&&&& Radiation can have any wavelength and includes, but is not limited to, not damaging normal cells but heating the non-protein portion to a wavelength that can kill the temperature of cells adjacent to the antibody-non-protein portion. C. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions as described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-Complex I antibody or an anti-gH antibody described herein is provided. The nucleic acid may encode an amino acid sequence comprising VL and/or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. Host cells comprising this nucleic acid are provided in another embodiment 158864.doc-67-201215618. In one such embodiment, the host cell comprises (eg, has been transformed as follows): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an anti-Wei amino acid sequence and comprising an antibody vh, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH. In one embodiment, cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., γ〇, NS0, Sp20 cells). In one embodiment, a method of making an anti-Complex I antibody or an anti-gH antibody, wherein the method comprises culturing a sputum cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody And recovering the antibody from the host cell (or host cell culture medium) as appropriate. For recombinant production of an anti-Complex I antibody or an anti-gH antibody, a nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further selection and/or expression in the host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Host cells suitable for the selection or expression of an antibody-encoding vector include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effect functions are not required. For the presentation of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in the large intestine rod 158864.doc -68-201215618 bacterium ). After performance p said that the antibody can be isolated from the soluble fraction from the bacterial cell paste and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are hosts suitable for the selection or expression of antibody-encoding vectors, including glycosylation pathways that have been "humanized" & Fully human glycosylated forms of fungi and yeast strains. See Gerngr〇ss, 22: 1409-1414 (2004) and Li et al., 绐 μ 24:210-215 (2006). Host cells suitable for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. A number of baculovirus strains that can be used in conjunction with insect cells have been identified for use in transfecting grass worms (filling ffugiperda). Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,42,548, 7,125,978, and 6,417,429, the disclosure of which is incorporated herein by reference. Cells can also be used as hosts. For example, mammalian cell strains suitable for growth in suspension may be suitable. Further examples of mammalian host cell strains are SV40 transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells such as, for example, Graham et al., J. Ge« KzW. 36: 59 (1977); young hamster kidney cells (BHK); mouse podocytes (TM4 cells as in, for example, Mather, 5/〇/· 158864.doc-69-201215618 23:243-251 (1980) Said); monkey kidney cells (CVI); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells ( BRL 3 A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells, such as, for example, Mather et al., see 7. Jcad. Scz.. 383:44 -68 (1982); MRC 5 fine-moon pack; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. iVai/. Acad. Sci. 77:42 16 (1980)); and myeloma cell lines, Such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Mei/zoA Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 ( 2003). D. Assays The anti-Complex I antibodies or anti-gH antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by various assays known in the art. 1. Binding assays and other assays In one aspect, the antigen binding activity of an antibody of the invention is tested, for example, by known methods such as ELISA, Western blots and the like. In another aspect, a competition assay can be used to identify antibodies that compete with the anti-Complex I antibodies described herein for binding to Complex I. In another aspect, a competition assay can be used to identify antibodies that compete with the anti-gH antibodies described herein for binding to gH. 158864.doc -70-201215618 In certain embodiments, the competing antibody binds to the same epitope of gH or complex I (e.g., a linear epitope or a conformational epitope). A detailed exemplary method for locating the epitope to which the antibody binds is provided by Morris (1996) "Epitope Mapping Protocols" 'Methods k Mo/ecw/ar price o/og_y volume 66 (Humana Press, Totowa, NJ) In an exemplary competition assay, the immobilized complex I or gH is incubated in a solution comprising a first labeled antibody and a second unlabeled antibody that bind to complex I or gH, respectively, and the second unlabeled The ability of the antibody to compete with the first antibody for binding to complex I or gH. The second antibody can be present in the fusion tumor supernatant. As a control, the immobilized complex I or gH was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing the binding of the first antibody to complex I or gH, excess unbound antibody is removed and the amount of label associated with immobilized complex I or gH is measured. If the amount of the label associated with the immobilized complex I or gH in the test sample is substantially reduced relative to the control sample, then the second antibody is indicated to compete with the first antibody for binding to complex I or gH. See Harlow and Lane (1988) Antibodies: A Laboratory Manual % \A (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) ° Competition assays can also be used as described above, using complex I or complex II gH And/or other members transfected and expressed on the cell surface by the FACS of the gH and/or cells of the other members. Alternatively, ELISA using gH and/or reconstituted complex I or complex II can also be used in the 158864.doc -71 - 201215618 competition assay. Anti-gH antibodies and anti-complex I antibodies were further described in the Examples using FACS and ELISA. 2. Activity assays In a single aspect, a test for identifying a biologically active anti-complex j antibody is provided. Biological activity may include, for example, specific binding to a conformational epitope produced by association of Ul 12 8 , UL 13 0, UL 131 and gH/gL; or specific binding to an epitope within a single protein of Complex I; And HCMV, EC9 〇 is 0.7 | ig / ml or less than 0.7 pg / ml. In some embodiments, the 'EC9〇 is 0.5 pg/ml or less than 0.5 pg/ml. In other embodiments, the ECpo is 0.3 pg/ml or less than 0.3 pg/ml. In other embodiments, the EC90 is 0.1 pg/ml or less than 1 pg/mi. In other embodiments, ε^9〇 is 0.08 pg/ml or less and 0.08 pg/ml or less. In other embodiments, the Ec9〇 is 0.06 pg/ml or less and 0.06 pg/ml or less. In other embodiments, the Ec9〇 is 0.04 pg/ml or less than 〇4 pg/ml. In other embodiments, the EC9 〇 is 0.02 pg/ml or less than 0.02 pg/ml. In other embodiments, the Ec9〇 is 0.015 pg/ml or less than 0.015 pg/ml. In other embodiments, Ec9〇 is 0.012#邑/1111 or 0.012 48/1111 or less. In other embodiments, ^9〇 is 0.011 pg/ml or 0.011 pg/ml or less. In other embodiments, the Ec9〇 is 0.010 pg/ml or less and 0.010 pg/ml or less. Compositions comprising antibodies having such biological activity are also provided. In one aspect, k is used to identify a bioactive anti-gH antibody assay. Biological activity may include, for example, neutralization of HCMV, EC90 of 1 pg/ml, 0.9 μδ/ml, 0.8 pg/ml, 〇·7 ng/m dip. 6 μ§/ηι〇 5 ^/ml, 0.4 pg/ Ml, 0.3 pg/ml, 0.2 pg/ml, 〇1 μ§Λη1, 〇〇9 158864.doc •11- 201215618
Pg/ml ' 〇·〇8 pg/ml、〇·07 pg/ml、〇 〇6 Kg/m卜 0.04 pg/ml或 0.04 pg/mi以下。 本發明組合物中之抗gH抗體與在桿狀病毒中表現之 gH/gL—聚體結合的IC50為〇.17 nM4〇 17 nM以下,例如 〇16 爾、0.15 nM、0.14 nM、〇.13 nM、〇12 碰、〇 ιι nM、(Uo nM、〇.G9 nM、G ⑽ nM、g Q7 nM、〇篇 _、 〇_〇5 nM、0.04 nM、0.03 nM、〇 〇2 nM、〇 〇1 邊或 〇 〇iPg/ml '〇·〇8 pg/ml, 〇·07 pg/ml, 〇 K6 Kg/m Bu 0.04 pg/ml or 0.04 pg/mi or less. The IC50 of the anti-gH antibody in the composition of the present invention and the gH/gL-mer expressed in the baculovirus is 〇.17 nM4〇17 nM or less, for example, 〇16 尔, 0.15 nM, 0.14 nM, 〇.13 nM, 〇12 touch, 〇ιι nM, (Uo nM, 〇.G9 nM, G (10) nM, g Q7 nM, _ _, 〇 _ 〇 5 nM, 0.04 nM, 0.03 nM, 〇〇 2 nM, 〇〇 1 side or 〇〇i
nM以下。亦提供包含具有此活體内及/或活體外生物活性 之抗體之組合物。 在某些實施例中,測試本發明抗體之此生物活性。對於 該種檢定之例示性描述,參見實例3。 E·免疫結合物 本發明亦提供包含含有本文抗複合物丨抗體或抗§11抗體 與或夕種細胞毋性劑結合之免疫結合物之組合物,該等 細胞毒性劑諸如化學治療劑或藥物、生長抑制劑、毒素 (例如蛋白吳毒素,細菌 '真菌、植物或動物來源之酶促 活性毒素;或其片段)、或放射性同位素。 在貫施例中,免疫結合物為抗體-藥物結合物(ADC), 其中抗體與一或多種包括(但不限於)以下之藥物結合:類 美登素(maytansinoid)(參見美國專利第5 2〇8 〇2〇號、第 5,416,064號及歐洲專利EP 〇 425 235 B1);阿瑞他汀 (auristatin),諸如單曱基阿瑞他汀(m〇n〇methylauristatin) 藥物部分DE及DF(MMAE及MMAF)(參見美國專利第 5,63 5,483號及第5,780,588號及第7,498,298號);海兔毒素 158864.doc -73- 201215618 (dolastatin);卡奇黴素(calicheamicin)或其衍生物(參見美 國專利第 5,712,374號、第 5,714,586號、第 5,739,116號、 第 5,767,285 號、第 5,770,701 號、第 5,770,710 號、第 5,773,001 號及第 5,877,296 號;Hinman 等人,Cawcer 及以. 53:3336-3342 (1993);及 Lode 等人,Cancer Res. 58:2925-2928 (1998));蒽環黴素(anthracycline),諸如道諾黴素 (daunomycin)或小紅莓(doxorubicin)(參見 Kratz 等人, CwrreW Med. C/zew. 13:477-523 (2006) ; Jeffrey 等人,Below nM. Compositions comprising antibodies having such in vivo and/or in vitro biological activity are also provided. In certain embodiments, the biological activity of an antibody of the invention is tested. For an illustrative description of this assay, see Example 3. E. Immunoconjugates The invention also provides compositions comprising an immunoconjugate comprising an anti-complex ruthenium antibody or an anti-§11 antibody and an immunosuppressive agent, such as a chemotherapeutic agent or a drug , growth inhibitors, toxins (eg, protein Wuxin, bacterial 'fungi, enzymatically active toxins of plant or animal origin; or fragments thereof), or radioisotopes. In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody binds to one or more drugs including, but not limited to, the following: maytansinoid (see U.S. Patent No. 5 2 〇8 〇2〇, 5,416,064 and European patent EP 〇425 235 B1); auristatin, such as monoterpene auristatin (m〇n〇methylauristatin) drug part DE and DF (MMAE and MMAF) (See U.S. Patent Nos. 5,63 5,483 and 5,780,588 and 7,498,298); dolphin toxin 158864.doc -73-201215618 (dolastatin); calicheamicin or its derivatives (see US patent) Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cawcer et al. 53:3336-3342 (1993) And Lode et al, Cancer Res. 58: 2925-2928 (1998)); anthracycline, such as daunomycin or doxorubicin (see Kratz et al, CwrreW Med) C/zew. 13:477-523 (2006) ; Jeffr Ey et al,
Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov 等人,16:717-721 (2005) ; Nagy 等 人,/Voc. 7V"a"· 97:829-834 (2000);Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., 16:717-721 (2005); Nagy et al., /Voc. 7V"a" 97:829-834 (2000) ;
Dubowchik 等人,5/oorg. Met/· C/zew· Leiiers 12:1529-1532 (2002) ; King 等人,J. Med. Chem. 45:4336-4343 (2002);及美國專利第6,630,579號);曱胺喋呤 (methotrexate);長春花驗醢胺(vindesine);紫杉烧 (taxane),諸如歐洲紫杉醇(docetaxel)、太平洋紫杉醇 (paclitaxel)、拉諾紫杉醇(larotaxel)、特塞紫杉醇 (tesetaxel)及沃塔紫杉醇(ortataxel); 黴菌毒素 (trichothecene);及 CC1065。 在另一實施例中,免疫結合物包含如本文所述之抗體與 酶促活性毒素或其片段,包括(但不限於)白喉 (diphtheria)A鏈、白喉毒素之非結合活性片段、外毒素 (exotoxin)A鍵(來自綠腹桿菌(Pseudomonas aeruginosa))、 篦麻毒素(ricin)A鏈、相思子毒素(abrin)A鏈、莫迪素 158864.doc •74- 201215618 (modeccin)A 鏈、α-帚麴菌素(sarcin)、油桐(Aleurites fordii)蛋白質、康乃馨(dianthin)蛋白質、洋商陸 (Phytolaca americana)蛋白質(pAPI、PAPII及 PAP-S)、苦瓜 (momordica charantia)抑制劑、麻瘋樹毒蛋白(curcin)、巴 豆毋素(crotin)、肥息草(sapa〇naria 〇fficinaiis)抑制劑、白 树素(gelonin)、有絲分裂素(mit〇geiiin)、侷限麴菌素 (restdctocin)、酚黴素(phen〇mycin)、伊諾黴素(en〇mycin) 及黴菌毒素(tricothecene)結合。Dubowchik et al., 5/oorg. Met/· C/zew· Leiiers 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and US Patent No. 6,630,579 ;methotrexate; vindesine; taxane, such as docetaxel, paclitaxel, larotaxel, tesaloxime Tesetaxel) and ortataxel; trichothecene; and CC1065. In another embodiment, the immunoconjugate comprises an antibody as described herein and an enzymatically active toxin or fragment thereof, including but not limited to, a diphtheria A chain, a non-binding active fragment of diphtheria toxin, an exotoxin ( Exotoxin) A bond (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Modicin 158864.doc •74- 201215618 (modeccin)A chain, α - sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (pAPI, PAPII and PAP-S), momordica charantia inhibitor, hemp Curcin, crotin, sapa〇naria 〇fficinaiis inhibitor, gelonin, mit 〇 ii ii ii ii rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest rest gel gel Combination of phen〇mycin, en〇mycin and trichothecene.
在另貫加例中’免疫結合物包含如本文所述之抗體與 放射性原子結合以形成放射結合物。多種放射性同位素可 用於產生放射結合物。實例包括At2丨丨、f3! ' 、 9〇、In a further addition, an immunoconjugate comprises an antibody as described herein that binds to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to generate radioconjugates. Examples include At2丨丨, f3! ', 9〇,
Re186、Re188、Sm153 Βι212、p32 ' pb2i2&Lu之放射性同位 素。當放射結合物用於偵測時,其可包含用於閃爍攝影研 究之放射性原子,例如纪99〇1或1123 ;用於核磁共振(nmr) 成像(亦稱為磁共振成像,mri)之自旋標記,諸如再次碘_ 123、姨-131、姻- ill、翁 10 r 山 1, μ 1 _ ^ u 1 鼠-19、碳-13、氮-15、氧-17、 亂、猛或鐵。 抗體與細胞毒性劑之結合物可使用多種雙官能蛋白質偶 合劑製備,該等偶合劑諸如N_丁二醯亞胺基_3_(2“比咬基 一硫基)丙酸酯(SPDP)、丁二醯亞胺基_4_(N_順丁烯二醯 亞胺甲基)環&烧-1-曱酉蔓g旨(SMCC)、亞胺基硫雜環戊烧 (IT)、亞胺基酯之雙官能衍生物(諸如二亞胺代己二酸二甲 自旨鹽酸鹽)、活性醋(諸如二丁二醯亞胺基辛二酸醋)、醛 (諸如戊一醛)、雙疊氮基化合物(諸如雙(對疊氮基苯曱醯 158864.doc -75- 201215618 基)己二胺)、雙重氮衍生物(諸如雙-(對二重氮笨甲醯基)_ 乙二胺)、二異氰酸酯(諸如曱苯2,6-二異氰酸酯)、及雙活 性氟化合物(諸如I,5-二氟-2,4-二硝基苯)。舉例而言,可 如Vitetta等人,238:1098 (1987)中所述製備篦麻毒 素免疫毒素。經碳標記之1-異硫氰基苯曱基-3-甲基二 伸乙基三胺五乙酸(MX-DTPA)為一種使放射性核苷酸與抗 體結合之例示性螯合劑。參見WO94/11026。連接子可為 有助於細胞毒性藥物在細胞中釋放之「可裂解之連接 子」。舉例而言,可使用酸不穩定連接子、肽酶敏感性連 接子、光不穩定連接子、二曱基連接子或含有二硫化物之 連接子(Chari 等人,Cancer Λα. 52:127-131 (1992);美國 專利第5,208,020號)。 本文之免疫結合物或ADC明確涵蓋(但不限於)用包括(但 不限於)以下之可購得(例如自Pierce Biotechnology,Inc., Rockford, IL.,U.S.A購得)之交聯試劑製備的此等結合物: BMPS、EMCS、GMBS、HBVS、LC SMCC、MBS ' ΜΡΒΗ、SBAP、SIA、SIAB、SMCC、SMPB、SMPH、硫 基-EMCS、硫基-GMBS、硫基-KMUS、硫基·MBS、硫基-SIAB、硫基-SMCC及硫基-SMPB、及SVSB(丁二醯亞胺 基-(4-乙烯颯)笨甲酸酯)。 F.用於診斷及偵測之方法及組合物 在某些實施例中,如本文提供之任何抗複合物I抗體及/ 或抗gH抗體或包含此等抗體之組合物皆適用於偵測生物樣 品中複合物I及/或gH之存在。如本文所用之術語「偵測」 201215618 涵蓋疋董或定性偵測。在某些實施例中,生物樣品包含細 胞或組織’諸如胎盤、腎'心臟、肺、肝、胰臟、腸、胸 腺、骨、腱、角膜、皮膚、心瓣膜及靜脈。此外,包含抗 體之組合物可用於偵測内皮細胞、上皮細胞、纖維母細胞 及巨噬細胞中之HCMV。 在一實施例中’提供用於診斷或偵測方法中之抗複合物 I抗體及/或抗gH抗體。在另一態樣中,提供一種偵測生物 樣品中複合物I及/或gH之存在之方法。在某些實施例中, 方法包含使生物樣品與如本文所述之抗複合物〖抗體及/或 抗gH抗體在允許抗複合物Σ抗體與複合物I結合及/或抗 抗體與gH結合之條件下接觸,及偵測是否在抗複合物“充 體與複合物I之間及/或在抗gH抗體與gH之間形成複合物。 此方法可為活體外或活體内方法。在一實施例中,抗複合 物I抗體或抗gH抗體或抗複合物丨抗體與抗抗體之組合用 於選擇適於用抗複合物I抗體或抗抗體或抗複合物I抗體 與抗gH抗體之組合進行治療之個體,例如當複合物I及gH 為用於選擇患者的生物標記時。 可使用本發明組合物診斷之例示性病症包括HCMV感 杂’諸如來自所移植器官或組織之HCMV感染' 先天性 HCMV感染、在妊娠期間之hcmv感染、及小孩、嬰兒及 成人中之HCMV感染。 在某些實施例中’提供包含經標記之抗複合物Ϊ抗體及/ 或抗gH抗體之組合物。標記包括(但不限於)直接偵測之標 記或部分(諸如螢光標記、發色標記、電子緻密標記、化 158864.doc -77· 201215618 學發光標記及放射性標記)、以及例如經由酶促反應或分 子相互作用間接偵測之部分(諸如酶或配位體)。例示性標 記包括(但不限於)放射性同位素32p、、i25l '化及13|][; 螢光團,諸如稀土螯合物或螢光素(flu〇rescein)及其衍生 物;若丹明(rhodamine)及其衍生物;丹磺醯基(dansyi); 傘酮(umbemferone);螢光素酶(iuceriferase),例如螢火蟲 螢光素酶及細菌螢光素酶(美國專利第4,737,456號);螢光 素(luciferin) ; 2,3-二氫酞嗪二酮;辣根過氧化酶(HRp); 鹼性磷酸酯酶(alkaline phosphatase) ; β_半乳糖苷酶;葡糖 澱粉酶·’溶菌酶;醣氧化酶,例如葡萄糖氧化酶、半乳糖 氧化酶及葡萄糖-6-磷酸去氫酶;雜環氧化酶,諸如尿酸酶 及黃嘌呤氧化酶;與採用過氧化氫來氧化染料前驅體之酶 (諸如HRP、乳過氧化酶(lact〇peroxidase)或微過氧化酶)偶 合;生物素/抗生蛋白(avidin);自旋標記;噬菌體標記; 穩定自由基及其類似物。 G.醫藥調配物 如本文所述之抗複合物I抗體或抗gH抗體或抗複合物"充 體與抗gH抗體之組合的醫藥調配物藉由混合具有所要純度 之此專抗體與一或多種視情況選用之醫藥學上可接受之載 劑P/zar廳批以第 16版,〇s〇1,A 編 (1980))製備成凍乾調配物或水溶液形式。如本文所述,抗 複合物I抗體及抗gH抗體可調配成單一組合醫藥調配物或 各別醫藥調配物。醫藥學上可接受之載劑在所用劑量及濃 度下對接受者通常無毒’且包括(但不限於):緩衝劑,諸 158864.doc -78 - 201215618 如磷酸鹽、檸檬酸鹽及其他有機酸;抗氧化劑,包括抗壞 血酸及曱硫胺酸;防腐劑(諸如氣化十八烷基二甲基苯甲 基敍;氯化六經季錄(hexamethonium chloride);氣化笨甲 烴敍(benzalkonium chloride);苄索氯敍(benzethonium chloride);苯酚;丁醇或苯曱醇;對羥基苯曱酸烷酯,諸 如對羥基苯甲酸甲酯或對羥基苯甲酸丙酯;兒茶酚;間苯 二酚;環己醇;3-戊醇;及間甲酚);低分子量(少於約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,諸如聚乙烯吡咯啶酮;胺基酸,諸 如甘胺酸、麩醯胺酸、天冬醯胺、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 或糊精;螯合劑,諸如EDTA ;糖,諸如蔗糖、甘露糖 醇、海藻糖或山梨糖醇;成鹽相對離子,諸如鈉;金屬錯 合物(例如Zn-蛋白質錯合物);及/或非離子型界面活性 劑,諸如聚乙二醇(PEG)。本文之例示性醫藥學上可接 受之載劑另外包括間質藥物分散劑,諸如可溶性中性-活 性玻尿酸酶醣蛋白(sHASEGP),例如人類可溶性PH-20 玻尿酸酶醣蛋白,諸如rHuPH20(HYLENEX®,Baxter International,Inc.)。某些例示性sHASEGP及使用方法(包 括rHuPH20)描述於美國專利公開案第2005/0260186號及第 2006/0104968號中。在一態樣中,sHASEGP與一或多種其 他葡糖胺聚糖酶(glycosaminoglycanase)(諸如軟骨素酶 (chondroitinase))組合 ° 例示性凍乾抗體調配物描述於美國專利第6,267,958號 158864.doc -79- 201215618 中。水性抗體調配物包括美國專利第6,171,586號及 W02006/044908中所述之調配物,w〇2〇〇6/〇449〇8中之調 配物包括組胺酸乙酸鹽緩衝劑。 除抗複合物I抗體及/或抗gH抗體之外,本文之調配物亦 可含有為所治療之特定適應症所必需的活性成分,較佳具 有彼此不產生不利影響之補充性活性的活性成分。舉例而 言,可能需要另外提供更昔洛韋、佛斯卡耐特、纈更昔洛 韋及西多福韋。此等活性成分適合以有效達成預定目的之 量組合存在。 活性成分可包覆在例如藉由凝聚技術或藉由界面聚合製 備之微膠囊(例如分別為羥曱基纖維素或明膠微膠囊及聚_ (曱基丙烯酸甲酯)微膠囊)中;膠態藥物傳遞系統(例如脂 質體、白蛋白微球體、微乳液、奈米粒子及奈米膠囊)中 或巨乳液中。此專技術揭示於d •SWewces 第 16 版,〇s〇l,a_ 編(1980)中。 可製備持續釋放製劑。持續釋放製劑之適合實例包括含 有抗體之固體疏水性聚合物半透基質,該等基質呈成形物 品(例如膜或微膠囊)形式。 欲用於活體内投藥之調配物通常無菌。無菌性可易於例 如藉由經由無菌過濾膜過濾來達成。 H·治療性方法及組合物 本文提供之包含抗複合物Ϊ抗體及/或抗gH抗體之任何組 合物皆可用於治療方法中。 在一態樣中,提供用作藥劑之包含抗複合物“充體或抗 158864.doc -80- 201215618 gH抗體或抗複合物I抗體及抗gH抗體之組合物。在其他態 樣中,提供用於治療HCVM感染之包含抗複合物I抗體或抗 gH抗體或抗複合物I抗體及抗gH抗體之組合物。在某些實 施例中,提供用於治療方法中之包含抗複合物I抗體或抗 gH抗體或抗複合物I抗體及抗gH抗體之組合物。在某些實 施例中,本發明提供用於治療患有HCMV感染之個體之方 法中之包含抗複合物I抗體或抗gH抗體或抗複合物I抗體及 抗gH抗體的組合物,該方法包含向該個體投與有效量之包 含抗複合物I抗體及/或抗gH抗體之組合物。在其他實施例 中,本發明提供用於預防、抑制或治療先天性HCMV感染 或所移植組織、器官或供體受或已受HCMV感染之組織或 器官移植接受者之HCMV感染之方法中的組合物。在一此 類實施例中,組織或器官移植接受者呈HCMV感染血清陰 性。在其他實施例中,移植接受者或個體先前已受HCMV 感染且處於HCMV再活化及感染之風險中。在某些實施例 中,方法另外包含向個體或移植接受者投與有效量之至少 另一例如如下文所述之治療劑。在其他實施例中,本發明 亦提供用於治療受HCMV感染之嬰兒或在妊娠期間暴露於 HCMV之嬰兒之方法中之包含抗複合物I抗體或抗gH抗體 或抗複合物I抗體及抗gH抗體的組合物,該方法包含投與 該嬰兒有效量之包含本發明抗體或其組合之組合物。在其 他實施例十,本發明提供用於治療、抑制或預防處於感染 風險中之個體中之HCMV感染之包含抗複合物I抗體或抗 gH抗體或抗複合物I抗體及抗gH抗體的組合物。根據任何 158864.doc -81 - 201215618 以上實施例之「個體」較佳為人類。 在另一癌樣中,本發明提供包含抗複合物〗抗體及/或抗 gH抗體或抗複合物I抗體及抗gH抗體之組合物製造或製備 藥劑的用途。在一實施例中,藥劑係用於治療、預防或抑 制HCMV感染。在另一實施例中,藥劑係用於治療、預防 或抑制HCMV感染,包含向患有HCMV感染之個體投與有 效量之該藥劑。在其他實施例中,藥劑係用於預防、抑制 或治療先天性HCMV感染或所移植組織、器官或供體受或 已欠HCMV感染之組織或器官移植接受者之HCMV感染的 方法中。在一此類實施例中,組織或器官移植接受者呈 HCMV感染血清陰性。在其他實施例中,移植接受者或個 體先前已受HCMV感染且處於HCMV再活化及感染之風險 中。在某些實施例中,藥劑另外包含有效量之至少另一例 如如下文所述之治療劑。在另—實施财,藥劑係用於治 療、抑制或預防處於感染風險中之個體中之Hcmv感染, 包含向該個體投與有效量之藥劑以抑制或預防HCMV感 染。在其他實施例中,藥劑係用於治療受11(:]^乂感染之嬰 兒或在妊娠期間暴露於HCMV之嬰兒,包含投與該嬰兒有 效量之包含本發明抗體或其組合之組合物。根據任何以上 實施例之「個體」可為人類。 在另-態樣中’本發明提供一種治療、預防或抑制 HCM:感染之方法。在一實施例中,方法包含向個體投與 有效量之包含抗複合物〗抗體及/或抗gH抗體之組合物。在 其他實施例t,本發明提供一種預防、抑制或治療先天性 158864.doc -82 - 201215618Radioactive isotopes of Re186, Re188, Sm153 Βι212, p32 'pb2i2&Lu. When the radioconjugate is used for detection, it may contain radioactive atoms for scintigraphy studies, such as Ji 99〇1 or 1123; for nuclear magnetic resonance (nmr) imaging (also known as magnetic resonance imaging, mri) Spin label, such as again iodine _ 123, 姨-131, marriage - ill, Weng 10 r mountain 1, μ 1 _ ^ u 1 mouse-19, carbon-13, nitrogen-15, oxygen-17, chaos, fierce or iron . Combinations of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein couplers, such as N-butylenediamine _3_(2"biti-monothio)propionate (SPDP), Butadiene imino group _4_(N-m-butyleneimine methyl) ring & burned 1-曱酉 曱酉 g ( SMCC), iminothiolane (IT), sub a difunctional derivative of an amino ester (such as diimidodipic acid dihydrogenate hydrochloride), an active vinegar (such as dibutyl succinimide octane vinegar), an aldehyde (such as valeraldehyde) Bis-azido compounds (such as bis(p-azidobenzoquinone 158864.doc -75-201215618) hexamethylenediamine), double nitrogen derivatives (such as bis-(p-diazetyl) Ethylenediamine), diisocyanate (such as toluene 2,6-diisocyanate), and bis-active fluorine compound (such as I,5-difluoro-2,4-dinitrobenzene). For example, it can be like Vitetta The ricin immunotoxin is prepared as described in 238:1098 (1987). Carbon-labeled 1-isothiocyanobenzoyl-3-methyldiethylidamine pentaacetic acid (MX-DTPA) a kind of radioactive nucleotide Combination of an exemplary chelating agent. See WO94 / 11026. Linker may facilitate release of the cytotoxic drug in the cell "of the cleavable linker." For example, an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimer linker or a linker containing a disulfide can be used (Chari et al., Cancer Λα. 52:127- 131 (1992); U.S. Patent No. 5,208,020). The immunoconjugates or ADCs herein are expressly encompassed, but not limited to, prepared with cross-linking reagents including, but not limited to, the following commercially available (e.g., commercially available from Pierce Biotechnology, Inc., Rockford, IL., USA). These combinations: BMPS, EMCS, GMBS, HBVS, LC SMCC, MBS 'ΜΡΒΗ, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio- MBS, thio-SIAB, thio-SMCC and thio-SMPB, and SVSB (butyl succinimide-(4-vinyl fluorene) benzoate). F. Methods and Compositions for Diagnosis and Detection In certain embodiments, any anti-Complex I antibody and/or anti-gH antibody or composition comprising such antibodies as provided herein are suitable for use in detecting organisms The presence of complex I and/or gH in the sample. As used herein, the term "detection" 201215618 covers 疋 or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues' such as the placenta, kidney' heart, lung, liver, pancreas, intestine, thymus, bone, tendon, cornea, skin, heart valve, and vein. In addition, an antibody-containing composition can be used to detect HCMV in endothelial cells, epithelial cells, fibroblasts, and macrophages. In one embodiment, an anti-Complex I antibody and/or an anti-gH antibody for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence of complex I and/or gH in a biological sample is provided. In certain embodiments, the method comprises reacting a biological sample with an anti-complex antibody and/or an anti-gH antibody as described herein, allowing the anti-complex Σ antibody to bind to complex I and/or the anti-antibody to bind to gH Contact under conditions, and to detect whether a complex is formed between the anti-complex "fill" and complex I and/or between the anti-gH antibody and gH. This method can be an in vitro or in vivo method. In one embodiment, the anti-Complex I antibody or the anti-gH antibody or the anti-complex 丨 antibody is combined with an anti-antibody for selection for selection with an anti-Complex I antibody or an anti-antibody or anti-Complex I antibody in combination with an anti-gH antibody. Treated individuals, for example, when complexes I and gH are biomarkers for selecting a patient. Exemplary conditions that can be diagnosed using the compositions of the invention include HCMV sensitivities such as HCMV infection from the transplanted organ or tissue 'congenital HCMV infection, hcmv infection during pregnancy, and HCMV infection in children, infants, and adults. In certain embodiments, 'providing a composition comprising a labeled anti-complex Ϊ antibody and/or an anti-gH antibody. (but Not limited to) directly detected markers or moieties (such as fluorescent labels, chromogenic markers, electron-dense markers, luminescent labels and radioactive labels), and for example via enzymatic or molecular interactions Indirect detection of parts (such as enzymes or ligands). Exemplary labels include, but are not limited to, radioisotopes 32p, i25l ', and 13|] [; fluorophores, such as rare earth chelates or luciferins (flu〇rescein) and its derivatives; rhodamine and its derivatives; dansyi; umbemferone; iuceriferase, such as firefly luciferase and Bacterial luciferase (U.S. Patent No. 4,737,456); luciferin; 2,3-dihydropyridazinedione; horseradish peroxidase (HRp); alkaline phosphatase; _-galactosidase; glucoamylase · lysozyme; sugar oxidase, such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidase, such as uricase and jaundice Oxidase; with hydrogen peroxide Coupling of enzyme precursors such as HRP, lactoperoxidase or microperoxidase; biotin/avidin; spin labeling; phage labeling; stable free radicals and analogues thereof G. Pharmaceutical Formulations A pharmaceutical formulation, such as an anti-Complex I antibody or an anti-gH antibody or an anti-complex described herein, in combination with an anti-gH antibody, by mixing the specific antibody having the desired purity with a Or a variety of pharmaceutically acceptable carriers, optionally selected from the P/zar Hall, 16th edition, 〇s〇1, A (1980)), in the form of a lyophilized formulation or an aqueous solution. As described herein, the anti-complex I antibody and the anti-gH antibody can be formulated as a single combined pharmaceutical formulation or a separate pharmaceutical formulation. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to, buffers, 158864.doc -78 - 201215618 such as phosphates, citrates and other organic acids Antioxidants, including ascorbic acid and guanidine thioglycol; preservatives (such as gasified octadecyldimethylbenzylidene; hexamethonium chloride; benzalkonium chloride) Benzothonium chloride; phenol; butanol or phenyl decyl alcohol; alkyl p-hydroxybenzoate, such as methyl or propylparaben; catechol; Phenol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 residue) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as Polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartame, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, Mannose or dextrin; chelating agent, such as EDT A; a sugar such as sucrose, mannitol, trehalose or sorbitol; a salt-forming relative ion such as sodium; a metal complex (such as a Zn-protein complex); and/or a nonionic surfactant, Such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein additionally include interstitial drug dispersing agents such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as the human soluble PH-20 hyaluronan glycoprotein, such as rHuPH20 (HYLENEX®) , Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycanases (such as chondroitinase). Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958, 158,864. 79-201215618. Aqueous antibody formulations include the formulations described in U.S. Patent Nos. 6,171,586 and WO2006/044908, and the formulations of w〇2〇〇6/〇449〇8 include histidine acetate buffers. In addition to the anti-Complex I antibody and/or the anti-gH antibody, the formulations herein may also contain the active ingredients necessary for the particular indication being treated, preferably the active ingredients which do not adversely affect each other. . For example, additional ganciclovir, foscarnet, valganciclovir and cidofovir may be required. These active ingredients are suitably present in combination in an amount effective to achieve the intended purpose. The active ingredient may be coated in microcapsules prepared by, for example, coacervation techniques or by interfacial polymerization (for example, hydroxymercapto cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively); colloidal state Drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. This technique is disclosed in d•SWewces, 16th edition, 〇s〇l, a_, (1980). Sustained release formulations can be prepared. Suitable examples of sustained release formulations include solid hydrophobic polymeric semipermeable matrices containing antibodies in the form of shaped articles such as films or microcapsules. Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. H. Therapeutic Methods and Compositions Any of the compositions comprising an anti-complex Ϊ antibody and/or an anti-gH antibody provided herein can be used in a method of treatment. In one aspect, a composition comprising an anti-complex "filled or anti-158864.doc-80-201215618 gH antibody or anti-complex I antibody and an anti-gH antibody" for use as a medicament is provided. In other aspects, provided A composition comprising an anti-Complex I antibody or an anti-gH antibody or an anti-Complex I antibody and an anti-gH antibody for use in the treatment of HCVM infection. In certain embodiments, an anti-Complex I antibody comprising a therapeutic I method is provided Or a composition of an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody. In certain embodiments, the invention provides an anti-complex I antibody or anti-gH for use in a method of treating an individual having an HCMV infection A composition of an antibody or an anti-complex I antibody and an anti-gH antibody, the method comprising administering to the individual an effective amount of a composition comprising an anti-Complex I antibody and/or an anti-gH antibody. In other embodiments, the invention A composition for use in a method of preventing, inhibiting, or treating a HCMV infection of a congenital HCMV infection or a tissue or organ transplant recipient of a transplanted tissue, organ or donor, or having been infected with HCMV. In such an embodiment Organization or device The transplant recipient is seronegative for HCMV infection. In other embodiments, the transplant recipient or individual has previously been infected with HCMV and is at risk of HCMV reactivation and infection. In some embodiments, the method additionally comprises administering to the individual or transplant The recipient is administered an effective amount of at least one other therapeutic agent, such as described below. In other embodiments, the invention also provides methods for treating an infant infected with HCMV or an infant exposed to HCMV during pregnancy. A composition comprising an anti-Complex I antibody or an anti-gH antibody or an anti-Complex I antibody and an anti-gH antibody, the method comprising administering to the infant an effective amount of a composition comprising an antibody of the invention or a combination thereof. The present invention provides a composition comprising an anti-Complex I antibody or an anti-gH antibody or an anti-Complex I antibody and an anti-gH antibody for use in the treatment, inhibition or prevention of HCMV infection in an individual at risk of infection. According to any 158864. Doc -81 - 201215618 The "individual" of the above embodiment is preferably human. In another cancer sample, the invention provides the use of a composition comprising an anti-complex antibody and/or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody for the manufacture or preparation of a medicament. In one embodiment, the agent is used to treat, prevent or inhibit HCMV infection. In another embodiment, the agent is for treating, preventing, or inhibiting HCMV infection, comprising administering to the individual having the HCMV infection an effective amount of the agent. In other embodiments, the agent is for use in a method of preventing, inhibiting, or treating a HCMV infection of a congenital HCMV infection or a tissue or organ transplant recipient of a transplanted tissue, organ, or donor that has or is owed to HCMV infection. In one such embodiment, the tissue or organ transplant recipient is seronegative for HCMV infection. In other embodiments, the transplant recipient or individual has previously been infected with HCMV and is at risk of HCMV reactivation and infection. In certain embodiments, the agent additionally comprises an effective amount of at least one other therapeutic agent, such as described below. In another embodiment, the medicament is for treating, inhibiting or preventing Hcmv infection in an individual at risk of infection, comprising administering to the individual an effective amount of an agent to inhibit or prevent HCMV infection. In other embodiments, the agent is for treating an infant infected with 11 (:) infection or an infant exposed to HCMV during pregnancy, comprising administering to the infant an effective amount of a composition comprising an antibody of the invention or a combination thereof. An "individual" according to any of the above embodiments may be a human. In another aspect, the invention provides a method of treating, preventing or inhibiting HCM: Infection. In one embodiment, the method comprises administering to the individual an effective amount A composition comprising an anti-complex antibody and/or an anti-gH antibody. In other embodiments t, the invention provides a prophylactic, inhibitory or therapeutic congenital 158864.doc -82 - 201215618
HCMV感染或所移植組織、器官或供體受或已受hcMV感 染之組織或器官移植接受者之HCMV感染的方法,其包含 向個體或移植接受者投與有效量之包含抗複合物〗抗體及 抗gH抗體之組合物。在一此類實施例中,組織或器官移植 接受者呈HCMV感染血清陰性。在其他實施例中,移植接 文者或個體先前已受HCMV感染且處於HCMV再活化及感 染之風險中。在一此類實施例中,方法另外包含向個體投 與有效量之至少另-如下文所述之治療劑。在其他實施例 中,本發明提供一種治療、抑制或預防受HCMV感染之嬰 兒或在妊娠期間暴露於HCMV之嬰兒的方法,其包含投與 "亥兒有效里之包含本發明抗體或其組合之組合物。根據 任何以上實施例之「個體」可為人類。 在另-態樣中,本發明提供—種抑制或預防處於感染風 險中之個體中之HCMV感染的方法。在一實施例中,方法 包3向個體投與有效量之包含抗複合物1抗體及/或抗讲抗 體之組合物以抑制或預防HCMV感染。在_實施例中, 個體」為人類。 在其他實施例中,所移植之器官或組織可為能夠自一個 =植至第二個體之任何器官或組織。舉例而言,所移植 7另可:(但不限於)心臟、腎、肝、肺、姨臟、腸或胸 另卜,舉例而言,所移植組織可為(但不限於)手、角 膜、皮膚、臉、蘭氏小島、骨髓、幹細胞、全血、血小 =血清、血球、血管、心瓣膜、骨、骨袓細胞、軟骨' 韌帶、腱、肌肉内襯。 158864.doc •83- 201215618 在另態樣中,本發明提供例如用於任何以上治療方法 中之包含本文提供之任何抗複合物!抗體及/或抗gH抗體的 組:物及醫藥調配物。在一實施例中,醫藥調配物包含本 文提供之任何抗複合物I抗體及/或抗gH抗體及醫藥學上可 接叉之載劑。在另一實施例中,醫藥調配物包含本文提供 之任何抗複合物I抗體及/或抗gH抗體及至少另一例如如下 文所述之治療劑。 本發明組合物中之抗體可單獨或與其他藥劑組合用於療 法中。舉例而言,本發明抗體可與至少另一治療劑共投 與。在某些實施例中,另一治療劑為更昔洛韋、纈更昔洛 羊、佛斯卡耐特及/或西多福韋。在其他實施例中,另一 治療劑為另外治療性經分離抗體。 以上所述之此等組合療法涵蓋組合投藥(其中兩種或兩 種以上治療劑包括在同一或各別調配物中);及分開投 藥,在此情況下,本發明之抗體組合物之投與可在投與另 —治療劑及/或佐劑之前、同時及/或之後進行。 本發明組合物(及任何其他治療劑)可藉由任何適合方式 投與,該等方式包括非經腸、肺内及鼻内投藥,且必要時 對於,部治療,包括病灶内投藥。非經腸輸注包括肌肉 内、靜脈内、動脈内、腹膜内或皮下投藥。給藥可部分視 投藥為短期或長期而定藉由任何適當途徑進行,例如藉由 注射(諸如靜脈内或皮下注射)進行。各種給藥時程包括(但 不限於)單次投藥或歷經多個時間點多次投藥,快速投藥 (bolus administration)及脈衝輸注涵蓋於本文中。 158864.doc -84- 201215618 本發明組合物將以符合良好醫學規範 式調配、給藥 及投與。在此情形中考慮之因素包括所治 ~ ’、 σ *、之特定病症、 所治療之特定哺乳動物、個別患者之臨戾 & Λ 不瑪狀、病症之病 因、藥劑傳遞部位、投藥方法、投藥時程 j任·久谱師已知之其 他因素。組合物無需但視情況與一或多插a 4 裡虽刖用於預防或 治療所述病症之藥劑一起調配。此等其他藥劑之有效量視 存在於調配物中之抗體之量、病症類型或治療類型上 論述之其他因素而定。此等藥劑通常以相同劑量且用如本 文所述之投藥途徑使用,或以本文所述劑量之:1%至99% 使用,或以任何劑量且藉由憑經驗/臨床確定為適當的任 何途徑使用。 對於預防或治療疾病,本發明組合物中含有之抗體之適 當劑量(當單獨或與一或多種其他額外治療劑組合使用時) 將視欲治療疾病之類型、抗體類型、疾病之嚴重性及病 程、投與抗體以達成預防抑或治療目的、先前療法、患者 之臨床病史及對抗體之反應以及主治醫師之判斷而定。本 文所述之組合物中包括之抗體適合一次性或歷經一系列治 療投與患者。視疾病之類型及嚴重性而定,無論例如藉由 一或多次分開投藥或藉由連續輸注,約} μg/kg至15 mg/kg(例如(M mg/kg_1〇 mg/kg)之各抗體可為用於投與患 者之初始候選劑量。視以上提及之因素而定,一種典型每 曰劑罝可在約1 pg/kg至100 mg/kg或100 mg/kg以上的範圍 内。對於歷經若干天或更長時間的重複投藥,視病狀而 疋,治療通常將持續直至發生疾病症狀之所要抑制。各抗 158864.doc -85 - 201215618 體之一種例示性劑量在約0.05 mg/kg至約1〇 mg/kg之範圍 内。因此,可向患者投與約0.5 mg/kg、2 〇 mg/kg、4 q mg/kg或10 mg/kg(或其任何組合)之一或多個劑量。此等劑 量可間歇投與,例如每週或每3週投與(例如以使患者接受 約2至約20或例如約6劑抗體)。可投與初始較高負載劑量 (loading dose),隨後投與一或多個較低劑量。此療法之進 程谷易藉由習知技術及檢定進行監測。 應瞭解任何以上調配物或治療方法皆可使用本文所述之 抗體之免疫結合物替代抗複合物Ϊ抗體及/或抗體來執 行,或除抗複合物I抗體及/或抗§]^抗體之外亦可使用本文 所述之抗體之免疫結合物來執行。 I·製品 在本發明之另一態樣中,提供一種含有適用於治療、預 防及/或診斷上述病症之物質的製品。該製品包含容器及 位於該容器上或與該容器相關之標籤或藥品說明書。適合 容器包括例如瓶、小瓶、注射器、1¥溶液袋等。容器可由 夕種材料,諸如玻璃或塑料形成。容器容納單獨或與可有 _ 效治療、預防及/或診斷病狀之另一組合物組合的組合 物且可具有無菌進入孔(例如容器可為具有可由皮下注 射針刺穿之塞子的靜脈内溶液袋或小瓶)。、组合物中之至 ^種活性劑為本發明抗體。標籤或藥品說明書指示組合 物用於治療所選病狀。此外’製品可包含⑷内含組合物之 第4器,其中該組合物包含本發明抗體;及内含組合 之第一合器,其中該組合物包含另一細胞毒性劑或其他 158864.doc -86 - 201215618 治療劑。本發明之此實施例中之製品可進一步包含藥品說 明書,其指不組合物可用於治療特定病狀。或者或另外, 製品可進一步包含含有醫藥學上可接受之緩衝液(諸如注 射用抑菌水(BWFI)、磷酸鹽緩衝鹽水、林格氏溶液 (Ringer’s solution)及右旋糖溶液)之第二(或第三)容器。其 可進一步包括就商業及使用者觀點而言合乎需要之其他物 質’包括其他緩衝劑、稀釋劑、過濾器、針及注射器。 應瞭解任何以上製品皆可包括本文所述之抗體之免疫結 • 合物替代抗複合物1抗體及/或抗gH抗體,或除抗複合物I抗 體及/或抗gH抗體之外’亦包括本文所述之抗體之免疫結 合物。 III.實例 以下為本發明之方法及組合物之實例。應瞭解,鑒於以 上提供之一般性描述’可實施各種其他實施例。 材料及方法 病毒生長。在除在DMEM培養中之外皆按照說明在繼代 ® 7-14下培養之人類胎兒肺纖維母細胞(MRC5)(美國菌種保 存中心(American Type Culture Collection) > ATCC; Manassas,VA)中,或在按照說明在繼代4-6下培養之p〇人 類臍靜脈内皮細胞(HUVEC)(Lonza; Basel,Switzerland)中 增殖 VR1814(Ravello Lab, Fondazione IRCCS Policlinic。A method of HCMV infection or HCMV infection of a transplanted tissue, organ or donor with or with a hcMV-infected tissue or organ transplant recipient comprising administering to the individual or transplant recipient an effective amount of an antibody comprising an anti-complex and A composition of an anti-gH antibody. In one such embodiment, the tissue or organ transplant recipient is seronegative for HCMV infection. In other embodiments, the transplant recipient or individual has previously been infected with HCMV and is at risk of HCMV reactivation and infection. In one such embodiment, the method further comprises administering to the individual an effective amount of at least another therapeutic agent as described below. In other embodiments, the invention provides a method of treating, inhibiting, or preventing an infant infected with HCMV or an infant exposed to HCMV during pregnancy, comprising administering an antibody of the invention or a combination thereof Composition. An "individual" according to any of the above embodiments may be a human. In another aspect, the invention provides a method of inhibiting or preventing HCMV infection in an individual at risk of infection. In one embodiment, method package 3 administers to the individual an effective amount of a composition comprising an anti-Complex 1 antibody and/or an anti-sense antibody to inhibit or prevent HCMV infection. In the embodiment, the individual is a human. In other embodiments, the transplanted organ or tissue can be any organ or tissue that can be implanted from one to the second individual. For example, the transplanted 7 can be: (but not limited to) heart, kidney, liver, lung, sputum, intestine or chest, for example, the transplanted tissue can be (but is not limited to) the hand, the cornea, Skin, face, Lange island, bone marrow, stem cells, whole blood, small blood = serum, blood cells, blood vessels, heart valves, bones, osteophytes, cartilage 'ligaments, tendons, muscle lining. 158864.doc • 83- 201215618 In another aspect, the invention provides, for example, for use in any of the above methods of treatment comprising any of the anti-complexes provided herein! Groups of antibodies and/or anti-gH antibodies: pharmaceutical and pharmaceutical formulations. In one embodiment, the pharmaceutical formulation comprises any of the anti-Complex I antibodies and/or anti-gH antibodies and pharmaceutically acceptable carriers provided herein. In another embodiment, a pharmaceutical formulation comprises any of the anti-Complex I antibodies and/or anti-gH antibodies provided herein and at least one other therapeutic agent, such as described below. The antibodies in the compositions of the invention may be used in therapy alone or in combination with other agents. For example, an antibody of the invention can be co-administered with at least one other therapeutic agent. In certain embodiments, the additional therapeutic agent is ganciclovir, valganciclovir, Foscarnet, and/or cidofovir. In other embodiments, the additional therapeutic agent is an additional therapeutically isolated antibody. The combination therapies described above encompasses combination administration (where two or more therapeutic agents are included in the same or separate formulations); and separate administration, in which case administration of the antibody composition of the invention This can be done before, concurrently with, and/or after administration of the additional therapeutic agent and/or adjuvant. The compositions of the present invention (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and if necessary, for topical treatment, including intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out in part by any suitable route, e.g., by injection (e.g., intravenous or subcutaneous injection), depending on whether the administration is short-term or long-term. Various dosing schedules include, but are not limited to, single administration or multiple administrations over multiple time points, bolus administration and pulse infusion are encompassed herein. 158864.doc -84- 201215618 The compositions of the present invention will be formulated, administered and administered in accordance with good medical practice. Factors considered in this case include the treatment of '', σ*, the specific condition, the particular mammal being treated, the individual patient's clinical & Λ 玛 、, the cause of the disease, the site of the drug delivery, the method of administration, The time of administration is other factors known to Jiu Jiji. The composition is not required, but optionally formulated with one or more of the agents used in the prevention or treatment of the condition. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder, or other factors discussed in the type of treatment. Such agents are usually administered in the same dosages and with administration routes as described herein, or at any of the dosages described herein: from 1% to 99%, or in any dosage and by empirical/clinical determination as appropriate. use. For the prevention or treatment of a disease, the appropriate dose of the antibody contained in the composition of the invention (when used alone or in combination with one or more additional therapeutic agents) will be the type of disease to be treated, the type of antibody, the severity of the disease, and the course of the disease. Administration of the antibody to achieve prophylactic or therapeutic purposes, prior therapy, clinical history of the patient, response to the antibody, and judgment of the attending physician. The antibodies included in the compositions described herein are suitable for administration to a patient once or over a range of treatments. Depending on the type and severity of the disease, for example, by one or more separate administrations or by continuous infusion, each from about μg/kg to 15 mg/kg (eg (M mg/kg_1〇mg/kg)) The antibody can be the initial candidate dose for administration to a patient. Depending on the factors mentioned above, a typical per dose of bismuth can range from about 1 pg/kg to 100 mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will usually continue until the symptoms of the disease are inhibited. An exemplary dose of each of the anti-158864.doc -85 - 201215618 is about 0.05 mg / From kg to about 1 mg/kg. Therefore, one or more of 0.5 mg/kg, 2 mg/kg, 4 q mg/kg or 10 mg/kg (or any combination thereof) can be administered to the patient or Multiple doses. These doses may be administered intermittently, for example weekly or every 3 weeks (eg, such that the patient receives from about 2 to about 20 or, for example, about 6 doses of the antibody). The initial higher loading dose can be administered (loading Dose), followed by one or more lower doses. The process of this therapy is monitored by conventional techniques and assays. Any of the above formulations or treatments can be performed using an immunoconjugate of an antibody described herein in place of an anti-complex Ϊ antibody and/or antibody, or in addition to an anti-complex I antibody and/or an anti- §] antibody An immunoconjugate of an antibody described herein can be used. I. Preparations In another aspect of the invention, an article of manufacture comprising a substance suitable for treating, preventing, and/or diagnosing the above conditions is provided. The article comprises a container And a label or package insert located on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, 1 solution bags, etc. The containers may be formed from materials such as glass or plastic. The containers may be contained separately or together. A composition of another combination of compositions that treats, prevents, and/or diagnoses a condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The active agent in the composition is an antibody of the invention. The label or the pharmaceutical label indicates that the composition is used to treat the selected condition. Further, the article may comprise (4) A fourth device, wherein the composition comprises an antibody of the invention; and a first combiner comprising a combination, wherein the composition comprises another cytotoxic agent or other 158864.doc-86 - 201215618 therapeutic agent. The article of manufacture in this embodiment may further comprise a package insert which means that the composition is not useful for treating a particular condition. Alternatively or additionally, the article may further comprise a pharmaceutically acceptable buffer (such as bacteriostatic water for injection ( A second (or third) container of BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials that are desirable from a commercial and user standpoint' including other buffers, diluents, filters, needles, and syringes. It is to be understood that any of the above products may include an immunoconjugate of an antibody described herein in place of an anti-complex 1 antibody and/or an anti-gH antibody, or in addition to an anti-complex I antibody and/or an anti-gH antibody. An immunoconjugate of an antibody as described herein. III. Examples The following are examples of methods and compositions of the present invention. It will be appreciated that various other embodiments may be implemented in the light of the general description. Materials and Methods Virus growth. Human fetal lung fibroblasts (MRC5) cultured under subculture® 7-14, except in DMEM culture (American Type Culture Collection >ATCC; Manassas, VA) VR1814 (Ravello Lab, Fondazione IRCCS Policlinic) was propagated in p〇 human umbilical vein endothelial cells (HUVEC) (Lonza; Basel, Switzerland) cultured under subculture 4-6 as described.
San Matteo, Pavia Italy),且濃縮上清液’再懸浮於完全培 養基中,並冷凍。完全培養基由補充有10%胎牛血清、青 黴素(penicillin)/鏈黴素(streptomycin) ' L-楚酿胺酸(全部 158864.doc •87· 201215618 來自 Invitrogen; Carlsbad, CA)及 20 mM HEPES(Cellgro; Manassas,VA)之DMEM構成。於96孔板中對MRC5及 HUVEC細胞以及人類視網膜色素上皮細胞(ARPE-19)(ATCC-按照說明培養)、單核細胞源性巨噬細胞(MDM) 及作為胎盤上皮細胞之細胞滋養層細胞(cytotrophoblast)進 行檢定。按照說明使用RosetteSep人類單核細胞富集混合 物(Stemcell Technologies,Vancover,BC,Canada)自全血分 離MDM。單核細胞接著用0.1 pg/ml脂多醣(LPS) (Invivogen)刺激且在DMEM中培育隔夜。血小板及未結合 細胞在感染之前用PBS洗除。細胞滋養層細胞自19週之胎 盤(Pereira Lab, UCSF,使用來自 Librach等人,1991, «/CF 1 13:437-449之方案)分離且接種於96孔組織培養板中。檢 定細胞滋養層細胞製備物之細胞滋養層細胞標記細胞角蛋 白(cytokeratin)7(CK7)(Dako)且發現在感染開始時陽性超 過 90%。 以下 HCMV 病毒株自 Jay Nelson 博士(University of Oregon Health and Science University(OHSU); Portland, OR)獲得:Adinis、Brown、Cano、Davis、Dement、 Grunden 、Harris 、Keone 、Lysistrata 、NewRock 、 Phoebe、Powers、Salvo、Schmoe、Simpson及 Watkins ° 以 下 HCMV病毒株自 Sunwen Chou(OHSU)博士 獲得:C079、 C323、C327、C336、C352、C353及C3 59。添加經解凍病 毒至MRC5纖維母細胞中且使其生長直至1 〇〇% CPE可見 (感染後約1 〇-12天)。3天後’刮掉細胞且收集上清液並使 158864.doc -88- 201215618 用超速離心進行濃縮。各病毒株之稀釋液用於在96孔板中 感染新鮮纖維母細胞。使病毒感染18小時,此後細胞用 100°/。乙醇固定。用 Mab810( —種抗 IE 抗體)(Millipore; Billerica,ΜΑ)進彳亍染色並進行免疫螢光分析。計算效價且 用於確定為達成中和檢定之感染倍率(MOI)l所必需之病毒 量 ° 中和檢定。基本上如Abai等人,2007, J. Twww⑽/ Mei/zoc?·?,3 22:82-93中一般進行中和檢定,例外之處為檢定 在DMEM(Gibco)中進行且藉由如上所述之免疫螢光進行偵 測。簡言之,連續稀釋抗體且與在完全培養基中稀釋之病 毒混合以使當與培養基或非抑制抗體混合時最終病毒粒子 濃度致使每個細胞約1個感染性病毒(ΜΟΙ=1)。混合抗體與 病毒且在37度下培育1小時,隨後添加至匯合單層MRC5、 ARPE-19、HUVEC或單核細胞源性巨噬細胞(MDM)中。使 病毒感染1 8小時,在該時間之後細胞用100%乙醇固定。 細胞於含2% BSA之PBS中阻斷且接著用抗HCMV IE抗體 Mab810(Millipore)或兔抗 HCMV IE(Johnson Lab,Oregon Health Sciences University)染色。細胞用PBS洗蘇且與適 當 AlexaFluor 488 及 Hoechst 染色劑(Invitrogen)—起培育。 含有指定抗體濃度之一式兩份孔之資料經平均化且與在不 存在抗體下之感染(設定為100%)進行比較。使用 ImageXpress® Micro™及 MetaXpress®(Molecular Devices) 使細胞成像並計數。對資料進行對數變換,校正,繪圖且 使用 Prism(GraphPad Software, La Jolla, CA)計算 EC50及 158864.doc -89- 201215618 EC90。使用EC50曲線擬合演算法自最佳擬合曲線計算 EC90值。偵測之檢定範圍為每孔100-6.5χΙΟ5個感染性病 毒粒子。在實驗之間觀察到檢定驗證,尤其在不同病毒感 染倍率之情況下。 臨床病毒株之擴增及叢集。使臨床HCMV病毒株(來自 Oregon Health Sciences University)在纖維母細胞上生長, 收集上清液,且使用超速離心進行濃縮。使用DNeasy血液 及組織套組(Qiagen)自受感染細胞分離DNA。使用不受專 利權限制之HCMV病毒株之比對來設計HCMV基因引子。 選擇分開小於500個鹼基之保守區。來自臨床病毒株之序 列使用Sequencher®察看且使用MacVector轉譯。藉由 ClustalW且在Jalview比對編輯器中進行比對並使用最近鄰 一致性%構築樹。 由桿狀病毒表現蛋白質。為了偵測表現之蛋白質,藉由 標準程序產生兔多株抗體。各兔用對應於HCMV蛋白質之 肽免疫。各肽如下: gH_871 HPHHEYLSDLYTPCSSSGRRDHSLERLTR(SEQIDNO:195) » gH_977 CPHVWMPPQTTPHDWKGSHTTSGLHRPH(SEQ ID NO: 196) > gL_873 CGLPPELKQT1RVNLPAHSRYGPQAVDAR(SEQIDNO:197) > UL128_988VGLDQYLESVKKHKRLDVCRAKMGYMLQ(SEQIDNO:198) > UL128_989RQWHNKLTSCNYNPLYLEADGRIR(SEQIDNO:199) > UL130_892 RDYSVSRQVRLTFTEANNQTYTFCraPNCSEQ JD NO:200) > UL130_892SPWFILTANQNPSPPWSKLTYPKPHDC(SEQIDNO:201),及 UL131 993TAEKNDYYRVPHYWDACSRALPDQTRYK(SEQIDNO:202)» 158864.doc •90- 201215618 藉由在肽結合管柱上進行捕捉且隨後溶離來純化血清。 對於分泌之蛋白質,製備各個別HCMV蛋白質之細胞外域 之桿狀病毒構築體。HCVM細胞外域之各者與桿狀病毒信 號序列及C端上之6x-His標記融合。桿狀病毒表現載體用 於感染SF9或Tini昆蟲細胞。收集蛋白質並進行凝膠過 濾,且藉由丙稀醯胺凝膠電泳及庫馬斯(coomassie)染色加 以檢查。匯合含有全部5種蛋白質(gH、gL、UL128、 UL130 及 UL131)之部分。藉由使 gB S64-K115 與 Q499-E655 融合來產生gB三聚體構築體。當藉由感染昆蟲細胞進行表 現時,此構築體產生gB蛋白質,其不經膜錨定,如所預期 形成為三聚體,結合中和及非中和gB抗體,且可能呈其融 合前形式。當gH與gL共表現時,所得gH/gL蛋白質結合 HB 1及識別非構形與構形gH抗原決定基兩者之以下抗體: MSL-109、兔抗 gH、兔抗 gL(來自 David Johnson,OHSU, Ryckman 等人,/· P7ro/. 82:60-70 (2008))、兔抗 gH_977 及 兔抗gL_873。昆蟲細胞受gH、gL、UL128、UL130及 UL131之共感染產生少量結合hu8G8及非構形及構形抗體 (包括兔抗gH、抗gL、抗UL128、抗130、抗131及上述兔 多株抗體)之異質蛋白質。 構築pRK-CMV載體。對於全長病毒醣蛋白之表面表 現,構築3個各別人類表現質體。自起始至終止擴增個別 HCMV基因。自基因組DNA擴增gH、gL、gB,且自cDNA 擴增 UL128、UL130、UL131 並首先使用 PCR Blunt II TOPO(Invitrogen)進行選殖。隨後,基因選殖入具有「自 158864.doc 91 201215618 裂解 2A肽」序列(Szymczak 等人,Α^3ί· 22:589- 94 (2004))以使各HCMV基因及末個3·基因與編碼eGFP之基 因分開的Genentech哺乳動物表現載體(pRK-tk-Neo)中。構 築3個質體,1個具有gB及eGFP,1個具有gH、gL及 eGFP,且 1 個具有 UL128、UL130、UL131 及 eGFP。質體 使用脂染胺(Lipofectamine)2000(Invitrogen; Carlsbad, CA) 轉染入人類胚腎(HEK)-293T細胞(美國菌種保存中心, ATCC; Manassas,VA)中。San Matteo, Pavia Italy), and the concentrated supernatant was resuspended in complete medium and frozen. Complete medium supplemented with 10% fetal bovine serum, penicillin/streptomycin 'L-calic acid (all 158864.doc •87·201215618 from Invitrogen; Carlsbad, CA) and 20 mM HEPES ( Cellgro; Manassas, VA) DMEM. MRC5 and HUVEC cells and human retinal pigment epithelial cells (ARPE-19) (ATCC-described culture), monocyte-derived macrophages (MDM), and cytotrophoblast cells as placental epithelial cells in 96-well plates (cytotrophoblast) is tested. MDM was isolated from whole blood using the RosetteSep Human Monocyte Enrichment Mix (Stemcell Technologies, Vancover, BC, Canada) as per the instructions. Monocytes were then stimulated with 0.1 pg/ml lipopolysaccharide (LPS) (Invivogen) and grown overnight in DMEM. Platelets and unbound cells were washed with PBS prior to infection. Cytotrophoblast cells were isolated from a 19-week placenta (Pereira Lab, UCSF, using the protocol from Librach et al., 1991, «/CF 1 13:437-449) and seeded in 96-well tissue culture plates. The cytotrophoblast cell marker cytokeratin 7 (CK7) (Dako) of the cytotrophoblast cell preparation was assayed and found to be more than 90% positive at the onset of infection. The following HCMV strains were obtained from Dr. Jay Nelson (University of Oregon Health and Science University (OHSU); Portland, OR): Adinis, Brown, Cano, Davis, Dement, Grunden, Harris, Keone, Lysistrata, NewRock, Phoebe, Powers, Salvo, Schmoe, Simpson and Watkins ° The following HCMV strains were obtained from Dr. Sunwen Chou (OHSU): C079, C323, C327, C336, C352, C353 and C3 59. The thawed virus is added to MRC5 fibroblasts and allowed to grow until 1% CPE is visible (about 1 〇-12 days after infection). After 3 days, the cells were scraped off and the supernatant was collected and concentrated by ultracentrifugation at 158864.doc -88 - 201215618. Dilutions of each strain were used to infect fresh fibroblasts in 96-well plates. The virus was infected for 18 hours, after which the cells were 100°/. Ethanol fixed. Mab810 (an anti-IE antibody) (Millipore; Billerica, ΜΑ) was used for sputum staining and immunofluorescence analysis was performed. The titer is calculated and used to determine the amount of virus necessary to achieve the neutralization assay's infection rate (MOI). Basically, neutralization tests are generally performed as in Abai et al., 2007, J. Twww(10)/Mei/zoc??, 3 22:82-93, with the exception that the assay is performed in DMEM (Gibco) and by the above The immunofluorescence is described for detection. Briefly, antibodies are serially diluted and mixed with the virus diluted in complete medium such that the final virion concentration when mixed with the medium or non-inhibitory antibody results in about 1 infectious virus per cell (ΜΟΙ = 1). The antibody was mixed with virus and incubated for 1 hour at 37 degrees, followed by addition to confluent monolayers of MRC5, ARPE-19, HUVEC or monocyte-derived macrophages (MDM). The virus was infected for 18 hours, after which time the cells were fixed with 100% ethanol. Cells were blocked in PBS containing 2% BSA and then stained with anti-HCMV IE antibody Mab810 (Millipore) or rabbit anti-HCMV IE (Johnson Lab, Oregon Health Sciences University). The cells were washed with PBS and incubated with appropriate AlexaFluor 488 and Hoechst stains (Invitrogen). Data containing two wells of the indicated antibody concentration were averaged and compared to infection in the absence of antibody (set to 100%). Cells were imaged and counted using ImageXpress® MicroTM and MetaXpress® (Molecular Devices). The data was logarithmically transformed, corrected, plotted and calculated using Prism (GraphPad Software, La Jolla, CA) EC50 and 158864.doc -89- 201215618 EC90. The EC90 value was calculated from the best fit curve using the EC50 curve fitting algorithm. The detection range is 100-6.5 χΙΟ 5 infectious virus particles per well. Verification verification was observed between experiments, especially in the case of different viral infection rates. Amplification and clustering of clinical virus strains. The clinical HCMV strain (from Oregon Health Sciences University) was grown on fibroblasts, the supernatant was collected, and concentrated using ultracentrifugation. DNA was isolated from infected cells using the DNeasy blood and tissue kit (Qiagen). HCMV gene primers were designed using an alignment of HCMV strains that were not restricted by patent rights. Conserved regions separated by less than 500 bases were selected. Sequences from clinical strains were viewed using Sequencher® and translated using MacVector. The tree is constructed by ClustalW and aligned in the Jalview alignment editor and using nearest neighbor consistency %. Protein is expressed by baculovirus. In order to detect the expressed protein, rabbit polyclonal antibodies were produced by standard procedures. Each rabbit was immunized with a peptide corresponding to the HCMV protein. Each peptide is as follows: gH_871 HPHHEYLSDLYTPCSSSGRRDHSLERLTR (SEQIDNO: 195) »gH_977 CPHVWMPPQTTPHDWKGSHTTSGLHRPH (SEQ ID NO: 196) > gL_873 CGLPPELKQT1RVNLPAHSRYGPQAVDAR (SEQIDNO: 197) > UL128_988VGLDQYLESVKKHKRLDVCRAKMGYMLQ (SEQIDNO: 198) > UL128_989RQWHNKLTSCNYNPLYLEADGRIR (SEQIDNO: 199) > UL130_892 RDYSVSRQVRLTFTEANNQTYTFCraPNCSEQ JD NO: 200) > UL130_892SPWFILTANQNPSPPWSKLTYPKPHDC (SEQ ID NO: 201), and UL131 993 TAEKNDYYRVPHYWDACSRALPDQTRYK (SEQ ID NO: 202) » 158864.doc • 90-201215618 Serum was purified by capture on a peptide-binding column and subsequent dissociation. For the secreted protein, a baculovirus construct of the extracellular domain of each HCMV protein was prepared. Each of the HCVM extracellular domains was fused to the baculovirus signal sequence and the 6x-His tag on the C-terminus. The baculovirus expression vector is used to infect SF9 or Tini insect cells. Proteins were collected and subjected to gel filtration and examined by acrylamide gel electrophoresis and coomassie staining. Confluence contains all five proteins (gH, gL, UL128, UL130 and UL131). The gB trimer construct was produced by fusing gB S64-K115 with Q499-E655. When expressed by infected insect cells, the construct produces a gB protein that is not membrane anchored, as expected to form a trimer, binds to neutralizing and non-neutralizing gB antibodies, and may be in its pre-fusion format . When gH and gL are co-expressed, the resulting gH/gL protein binds to HB1 and recognizes the following antibodies that are both non-configuration and conformational gH epitopes: MSL-109, rabbit anti-gH, rabbit anti-gL (from David Johnson, OHSU, Ryckman et al., /. P7ro/. 82:60-70 (2008)), rabbit anti-gH_977 and rabbit anti-gL_873. Insect cells are co-infected with gH, gL, UL128, UL130 and UL131 to produce a small amount of binding to hu8G8 and non-configuration and conformational antibodies (including rabbit anti-gH, anti-gL, anti-UL128, anti-130, anti-131 and the above-mentioned rabbit polyclonal antibody ) a heterogeneous protein. The pRK-CMV vector was constructed. For the surface expression of the full-length viral glycoprotein, three different types of plastids were constructed. Individual HCMV genes were amplified from start to finish. gH, gL, gB were amplified from genomic DNA, and UL128, UL130, UL131 were amplified from cDNA and first cloned using PCR Blunt II TOPO (Invitrogen). Subsequently, the gene was cloned into the sequence of "cleavage of 2A peptide from 158864.doc 91 201215618" (Szymczak et al., Α^3ί 22:589-94 (2004)) to encode each HCMV gene and the last 3 gene. The gene for eGFP is isolated from the Genentech Mammalian Expression Vector (pRK-tk-Neo). Three plastids were constructed, one with gB and eGFP, one with gH, gL and eGFP, and one with UL128, UL130, UL131 and eGFP. The plastids were transfected into human embryonic kidney (HEK)-293T cells (American Type Culture Collection, ATCC; Manassas, VA) using Lipofectamine 2000 (Invitrogen; Carlsbad, CA).
Cytogam®(HIG)消減。對於HIG之會中和病毒進入上皮 細胞中之組分的檢定,HEK-293T細胞使用脂染胺 2000(Invitrogen)用 gB/eGFP 或 gH/gL/eGFP及 UL128/UL130/ UL131/eGFP或模擬質體轉染且培育48小時。細胞使用 Accutase(Sigma)解離,集結,且分成12個等分試樣。 Cytogam®於PBS及0.5°/〇牛血清白蛋白(BSA)中稀釋至20 pg/ml且於含3 X 1 07個經轉染細胞之懸浮液中培育1小時。 Cytogam®接著連續轉移至3χ 107個經轉染細胞之新鮮等分 試樣中。進行向新細胞上之繼代直至觀察到抗體之最大特 異性缺失(6個繼代)。進行ELISA檢定以偵測某些HCMV特 異性抗體之消減。Maxisorp(NUNC)板經含由桿狀病毒產 生之純化 gB、gH/gL、或 gH/gL/UL128/UL130/UL131 的 PBS及/或經轉染HEK293T細胞之溶解產物塗佈。用與辣根 過氧化酶結合之山羊抗人類IgG Fcy(Jackson Laboratory, Bar Harbor,ME)測定偵測。探測之下限為0.08 pg/ml。所 得HIG接著使用100 kD分子量截斷濃縮器(Centricon)濃縮 -92- 158864.docCytogam® (HIG) reduction. HEK-293T cells were treated with lipofectamine 2000 (Invitrogen) with gB/eGFP or gH/gL/eGFP and UL128/UL130/UL131/eGFP or mock for HIG to neutralize the components of the virus into epithelial cells. Transfected and incubated for 48 hours. Cells were dissociated using Accutase (Sigma), assembled, and divided into 12 aliquots. Cytogam® was diluted to 20 pg/ml in PBS and 0.5°/calf serum albumin (BSA) and incubated for 1 hour in a suspension containing 3×107 transfected cells. Cytogam® was then continuously transferred to 3 χ 107 fresh aliquots of transfected cells. Subculture to new cells was performed until the greatest specific loss of antibody was observed (6 passages). An ELISA assay was performed to detect the reduction of certain HCMV specific antibodies. The Maxisorp (NUNC) plate was coated with PBS containing purified gB, gH/gL, or gH/gL/UL128/UL130/UL131 produced by baculovirus and/or lysate of transfected HEK293T cells. Detection was determined by goat anti-human IgG Fcy (Jackson Laboratory, Bar Harbor, ME) in combination with horseradish peroxidase. The lower limit of detection is 0.08 pg/ml. The resulting HIG is then concentrated using a 100 kD molecular weight cutoff concentrator (Centricon) -92- 158864.doc
S 201215618 以用於如上所述之中和檢定中。 以以下方式產生親和力消減管柱。蛋白質(1-2 mg可溶 性gB或gH/gL)相對於PBS充分透析且添加至約1 ml於PBS 中平衡之Sterogene ALD Superflow樹脂中。添加氰基棚氫 化鈉(0.2 ml之1 Μ溶液)以使蛋白質與含醛樹脂化學偶合且 使反應在4°C下進行隔夜。個別樹脂裝載入小管柱中且用 PBS充分洗滌以移除任何未結合蛋白質。將2毫克 Cytogam®於800 μΐ體積中裝載於管柱上且在0.4 mL/min之 流速下用PBS洗滌。以若干0.5 ml等分試樣收集未結合蛋 白質,該等等分試樣於旋轉濃縮器中以5000道爾頓之分子 量截斷分別濃縮至約1 00 μΐ。樣品經無菌過濾且儲存在4°C 下直至檢定。 FACS。使用脂染胺2000(Invitrogen)將先前描述之pRK-CMV載體單獨或在gH/gL之比率50:50下與UL128-131—起 轉染入HEK293T細胞中。在48小時之後,使用Accutase (Sigma)解離細胞。所有一次或二次抗體(Jackson Labs)培 育及洗滌皆在PBS、2% FCS、0.2%疊氮化鈉(FACS緩衝液) 中進行。在染色之後,細胞於含2%多聚曱醛之FACS緩衝 液中固定。使用 FACS Calibur4(Beckton Dickinson)進行螢 光分析且使用FlowJo軟體(Tree Star Inc.)處理資料。 產生抗性病毒突變體。為產生對本文所述之抗體具有抗 性之病毒突變體,使HCMV病毒株VR1814在次最佳濃度之 在 Genentech合成之抗體 MSL-109(Aulitzky 等人,/./«/己<^· D/t 163:1344-47 (1991))、HB1、hu8G8存在下在上皮細胞 158864.doc -93- 201215618 上或在HB1與hu8G8之組合存在下在ARPE 19細胞(美國菌 種保存中心,ATCC; Manassas, VA)中生長。在24孔板中開 始實驗,其中在EC50、2x EC50或EC90下之抗體各自對應 3個孔且感染倍率(MOI)為0.5。每週,各孔體積之一半繼 代於新細胞上且抗體濃度增加1.5倍或保持穩定。通常, 截至約第9繼代,突變體以單一病毒斑形式出現。以使抗 體濃度遞增至l〇x EC90之最終濃度的方式,使此等病毒生 長。隨後,儲備突變體且藉由如上所述之中和檢定分析其 對HB1及hu8G8之抗性。整個過程對ARPE-19細胞各別起 始4次(每個抗體濃度3個孔)且僅用HB1及MSL 109(hu8G8 不中和MRC5細胞上之病毒)對MRC5細胞各別起始2次。 為了產生其他抗性突變,細胞外病毒用AA-乙基亞硝基 脲(ENU,Sigma-Aldrich; St. Louis, MO)或紫外線(254λ Stratalinker,Stratagene; Santa Clara CA)處理且使其在 24孔 形式(每次處理24個孔)或96孔形式(每次處理72個孔)中感 染ARPE-19或MRC5細胞。在感染(在MOI 1或2下)之後, 培養基經含HB1 (在EC100下)或hu8G8(在EC100下)或該兩 種抗體(各自在EC50下)之組合、或更昔洛韋(GCV,Sigma-Aldrich)的完全培養基置換。每週,上清液向新鮮細胞繼 代,同時遞增抗體或GCV之濃度。生長病毒在2-3個月之 後轉移至較大孔中且儲備。整個過程各別起始2次。 定序醣蛋白。自對照或突變病毒感染細胞或上清液分離 DNA(DNA血液/組織提取套組,Qiagen; Valencia, CA)。根 據可在國家生物技術資訊中心(National Center for 158864.doc -94- 201215618S 201215618 for use in the neutralization assay described above. An affinity reduction column is generated in the following manner. Protein (1-2 mg soluble gB or gH/gL) was dialyzed extensively against PBS and added to approximately 1 ml of Sterogene ALD Superflow resin equilibrated in PBS. Sodium cyanohydride (0.2 ml of a 1 Torr solution) was added to chemically couple the protein to the aldehyde containing resin and the reaction was allowed to proceed overnight at 4 °C. Individual resins were loaded into the tubule string and washed extensively with PBS to remove any unbound protein. 2 mg of Cytogam® was loaded onto the column in a volume of 800 μΐ and washed with PBS at a flow rate of 0.4 mL/min. Unbound protein was collected in several 0.5 ml aliquots and concentrated in a rotary concentrator at a molecular weight of 5000 Daltons to concentrate to approximately 100 μM, respectively. Samples were sterile filtered and stored at 4 °C until assayed. FACS. The previously described pRK-CMV vector was transfected into HEK293T cells either alone or at a ratio of gH/gL of 50:50 using UL128-131 using lipofectamine 2000 (Invitrogen). After 48 hours, cells were dissociated using Accutase (Sigma). All primary or secondary antibodies (Jackson Labs) were cultured and washed in PBS, 2% FCS, 0.2% sodium azide (FACS buffer). After staining, the cells were fixed in FACS buffer containing 2% polyfurfural. Fluorescence analysis was performed using a FACS Calibur 4 (Beckton Dickinson) and data was processed using FlowJo software (Tree Star Inc.). Resistant virus mutants are produced. To generate a viral mutant that is resistant to the antibodies described herein, the HCMV strain VR1814 was subjected to Genentech's antibody MSL-109 at a suboptimal concentration (Aulitzky et al., /./«/Here <^· D/t 163:1344-47 (1991)), in the presence of HB1, hu8G8 on epithelial cells 158864.doc -93- 201215618 or in the presence of a combination of HB1 and hu8G8 in ARPE 19 cells (American Type Culture Collection, ATCC) ; grown in Manassas, VA). The experiment was started in a 24-well plate in which the antibodies at EC50, 2x EC50 or EC90 each corresponded to 3 wells and the infection magnification (MOI) was 0.5. One week, one-half of each well volume was subcultured to new cells and the antibody concentration was increased 1.5-fold or remained stable. Typically, as of the ninth generation, the mutant appears as a single plaque. These viruses are grown in such a way that the antibody concentration is increased to the final concentration of l〇x EC90. Subsequently, the mutants were stocked and analyzed for resistance to HB1 and hu8G8 by neutralization assay as described above. The entire process was started 4 times for ARPE-19 cells (3 wells per antibody concentration) and only with HB1 and MSL 109 (hu8G8 does not neutralize the virus on MRC5 cells). In order to generate other resistance mutations, the extracellular virus was treated with AA-ethylnitrosourea (ENU, Sigma-Aldrich; St. Louis, MO) or UV (254λ Stratalinker, Stratagene; Santa Clara CA) and allowed to be at 24 ARPE-19 or MRC5 cells were infected in well form (24 wells per treatment) or 96 well format (72 wells per treatment). After infection (under MOI 1 or 2), the medium is treated with HB1 (under EC100) or hu8G8 (under EC100) or a combination of the two antibodies (each at EC50), orciclovir (GCV, Complete medium replacement of Sigma-Aldrich). Each week, the supernatant is subcultured to fresh cells while increasing the concentration of antibody or GCV. The growing virus was transferred to larger wells and stored after 2-3 months. The whole process starts twice each time. Order glycoprotein. DNA was isolated from control or mutant virus-infected cells or supernatant (DNA blood/tissue extraction kit, Qiagen; Valencia, CA). According to the National Center for Biotechnology Information (National Center for 158864.doc -94- 201215618
Biotechnology Information,NCBI)資料庫中獲得之 HCMV 病毒株 AD169、FIX、TB40E、Toledo 及 Towne 序列之比 對,設計針對跨越各基因之保守序列的引子。擴增出各臨 床病毒株之自起始密碼子至鹼基2196(僅缺乏終止密碼子) 的醣蛋白Η。擴增自起始至鹼基2686(僅缺乏終止密碼子) 之醣蛋白B。UL128、UL130及UL131各自根據自Akter等 人,《/. F/ro/· 84:1117-22 (2003)獲得之 cDNA序列自起 始至終止進行擴增。使用染料終止劑反應對聚合酶鏈反應 (PCR)產物定序且對序列進行比對及修整(定序器)。 突變之重演。修飾如上所述之含有gH/gL基因或UL128/ UL13 0/UL131基因之pRK-CMV表現質體以取代見於各抗性 突變體中之單一突變。各gH/gL質體轉染(脂染胺2000, Invitrogen; Carlsbad, CA)入 HEK 293T細胞(ATCC)中,使 其表現2天,且接著藉由如上所述之螢光活化細胞揀選 (FACS)分析評估表面表現之HCMV蛋白質結合對照抗gH抗 體10F8或HB1的能力。各UL128/UL130/UL131質體與 gH/gL質體共轉染,使其表現2天,接著評估表面表現之蛋 白質結合HB1、hu8G8及對照兔抗UL131_993抗體的能 力。使用FlowJo(Treestar; Ashland, OR)產生分析及影像。The alignment of the HCMV strains AD169, FIX, TB40E, Toledo and Towne sequences obtained in the Biotechnology Information, NCBI) database was designed to target primers spanning the conserved sequences of the genes. Glycoproteins from the start codon of each clinical virus strain to base 2196 (only the stop codon is lacking) were amplified. Glycoprotein B was amplified from the start to base 2686 (only the stop codon was lacking). UL128, UL130 and UL131 are each amplified according to the cDNA sequence obtained from Akter et al., "/. F/ro/. 84:1117-22 (2003) from the beginning to the end. The polymerase chain reaction (PCR) product is sequenced using a dye terminator reaction and the sequences are aligned and trimmed (sequencer). The re-enactment of the mutation. The pRK-CMV-expressing plastid containing the gH/gL gene or the UL128/UL13 0/UL131 gene as described above was modified to replace a single mutation found in each of the resistant mutants. Each gH/gL plastid was transfected (Lipamine 2000, Invitrogen; Carlsbad, CA) into HEK 293T cells (ATCC) for 2 days, and then by fluorescence activated cell sorting as described above (FACS) Analysis of the ability of surface-expressed HCMV proteins to bind to control anti-gH antibody 10F8 or HB1. Each UL128/UL130/UL131 plastid was co-transfected with the gH/gL plastid for 2 days, and then the ability of the surface-expressed protein to bind HB1, hu8G8 and control rabbit anti-UL131_993 antibody was evaluated. Analysis and images were generated using FlowJo (Treestar; Ashland, OR).
分析病毒進入。使抗性及對照病毒株(在不存在抗體下 在ARPE-19細胞上並行繼代)之儲備物生長且收集上清液 (純)。進行定量PCR(qPCR)以獲得pp65 DNA pp65F TCGCGCCCGAAGAGG(SEQ ID NO:189)、pp65R CGGCC GGATTGTGGATT(SEQ ID NO:190)、Taqman 探針 CACCGA 158864.doc -95- 201215618 CGAGGATTCCGACAACG(SEQ ID NO:191)。為確定複本 數,用 pp65選殖入 Zero Blunt PCR Cloning(Invitrogen)中獲 得標準曲線。使基於複本數之病毒之稀釋液感染ARPE-19 及MRC5細胞1 8小時,隨後固定及觀測。計算每個DNA複 本之感染性粒子數,相對於在無抗體下繼代之病毒株進行 校正,且使用 Excel 第 14.1.2 版(Microsoft; Redmond,WA)繪 圖。 實例1_產生抗複合物I抗體及抗gH抗體 產生抗複合物I鼠類mAb 8G8。在每隻小鼠1χ106 pfu之 濃度下用完整之經UV不活化(3000mJ)之HCMV(病毒株 VR1814)來免疫2組Balb/c小鼠(每組10隻),每週兩次,總 計SC/IP注射7次。在第一組動物中,各小鼠用RIBI佐劑灌 注且隨後用含HCMV之PBS注射。在第二組動物中,動物 未經灌注且接著用含HCMV之RIBI佐劑注射。經免疫小鼠 之測試血液經受藉由ELIS A進行之血清樣品滴定以及如上 所述之病毒中和檢定。選擇反應性位於前5名之小鼠來產 生融合瘤。使用來自胭結節及腹股溝結節之淋巴細胞及小 鼠骨髓瘤細胞株X63-Ag8.653進行2組各別融合。融合細胞 塗鋪在96孔組織培養板(58個板)中且在融合後1天開始使用 HAT培養基補充劑(Sigma,St. Louis,Μο·)進行融合瘤選 擇。使用如上所述對上皮細胞進行之病毒中和檢定篩檢總 計738個IgG+融合瘤。對各種細胞類型測試HCMV病毒株 VR1814對所得抗體之EC50(pg/ml)且與MSL-109(—種抗gH 抗體)進行比較並展示於表2中。單株抗體8G8為篩檢中鑑 158864.doc -96- 201215618 別之最強力中和抗體且被選擇進行人類化及進一步表徵。 表2Analyze virus entry. Stocks of resistant and control virus strains (parallel passage on ARPE-19 cells in the absence of antibodies) were grown and supernatants (pure) were collected. Quantitative PCR (qPCR) was performed to obtain pp65 DNA pp65F TCGCGCCCGAAGAGG (SEQ ID NO: 189), pp65R CGGCC GGATTGTGGATT (SEQ ID NO: 190), Taqman probe CACCGA 158864. doc - 95 - 201215618 CGAGGATTCCGACAACG (SEQ ID NO: 191) . To determine the number of replicates, a standard curve was obtained by pl65 into Zero Blunt PCR Cloning (Invitrogen). The dilution of the virus based on the replica number was infected with ARPE-19 and MRC5 cells for 18 hours, followed by fixation and observation. The number of infectious particles per DNA copy was calculated and compared to the virus strains that were subcultured without antibodies and plotted using Excel version 14.1.2 (Microsoft; Redmond, WA). Example 1 - Generation of anti-Complex I antibody and anti-gH antibody Anti-Complex I murine mAb 8G8 was produced. Two groups of Balb/c mice (10 per group) were immunized with intact UV-inactivated (3000 mJ) HCMV (viral strain VR1814) at a concentration of 1χ106 pfu per mouse, twice a week for a total of SC /IP injection 7 times. In the first group of animals, each mouse was infused with RIBI adjuvant and subsequently injected with PBS containing HCMV. In the second group of animals, the animals were not perfused and then injected with RIBI adjuvant containing HCMV. The test blood of the immunized mice was subjected to serum sample titration by ELIS A and virus neutralization assay as described above. The mice in the top 5 reactivity were selected to produce fusion tumors. Two groups of fusions were performed using lymphocytes from the nodules and inguinal nodules and mouse myeloma cell line X63-Ag8.653. The fused cells were plated in 96-well tissue culture plates (58 plates) and fusion tumor selection was started using HAT medium supplement (Sigma, St. Louis, Μο.) 1 day after fusion. A total of 738 IgG+ fusion tumors were screened using virus neutralization assays on epithelial cells as described above. The HCMV strain VR1814 was tested for EC50 (pg/ml) of the resulting antibody against various cell types and compared to MSL-109 (an anti-gH antibody) and is shown in Table 2. The monoclonal antibody 8G8 was screened for the most potent neutralizing antibody and was selected for humanization and further characterization. Table 2
EC50(pg/ml) : HCMV病毒株VR1814 細胞類型 目標 上皮 内皮 纖維母細胞 MSL-109 gH 0.1642 0.0764 0.3890 鼠類8G8 複合物I 0.0014 0.0007 N/A 鼠類5H3 gH 1.15 1.42 17.76 鼠類10F8 gH 0.1 0.1 1.11 鼠類15H2 gH 0.12 0.11 1 鼠類354.1 複合物I 0.03 0.07 N/A 鼠類8G8 mAb之人類化及分析。藉由使用λ3或λ4輕鏈 (圖2)及VH1、VH3或VH7重鏈構架(圖1)進行標準CDR移植 來使鼠類融合瘤8G8人類化。對於比較,λ3與λ4之共同人 類λ生殖系序列展示於圖2中。進行中和檢定,從而比較 8G8人類/鼠類嵌合抗體(QE7/C2)與8G8 VH1、VH3或VH7 人類化重鏈與8G8 λ3或λ4輕鏈的組合。發現λ4變異體但非 λ3變異體中和HCMV(圖3)。 如圖4中所示突變λ4之HVR-L2以在胺基酸50C、50D、 56處引入取代,以及在胺基酸57(FR3之第一胺基酸)(根據 Kabat編號)處引入胺基酸取代以為抗體提供穩定性。各種 突變輕鏈接著與8G8人類VH1鏈組合且在如上所述之中和 檢定中測試所得抗體。具有單一胺基酸取代之抗體皆顯示 良好中和活性(亦即Al、E1、ΤΙ、A2、E2及T2)(圖5)。同 樣,含有2個胺基酸取代之所有抗體皆顯示良好中和活性 (亦即SGSG及TGDA)。單一突變體SG作為比較對照被包 括,且其亦顯示良好中和活性。(圖6)。 158864.doc -97- 201215618 人類化8G8 λ4抗體序列展示於圖7中(hu8G8A4 FW)。圖 8展示人類化8G8 VH1序列之序列(hu8G8.VHl)而圖9展示 人類化8G8 VH3序列之序列(hu8G8.VH3)。圖10展示人類 化8G8 λ4抗體序列,其中前2個胺基酸(QP)已經修飾以使 多肽以絲胺酸開始(Q經缺失且L突變成S)且胺基酸36保留 鼠類胺基酸(Υ)。此抗體之多肽序列展示為λ4 8G8移植 物。編碼多肽之一個代表性核酸序列展示在多肽序列以 下。 抗gH抗體之親和力成熟。使用1994年8月4日公開之PCT 公開案第WO 94/1 6730號中公開且以全文引用的方式併入 本文中之MSL-109之可變重鏈及可變輕鏈序列的抗體序列 合成單株抗體MSL-109。MSL-109 VH及VL鏈之胺基酸序 列展示於圖 11 中(VL : SEQ ID NO:90 ; VH : SEQ ID NO:92)。MSL-109抗體係基於含有重鏈VH3及輕鏈Vk2之 IgGl構架。編碼此抗體之重組DNA選殖入CHO細胞中。 藉由互補決定區(CDR)之隨機化,隨後藉由噬菌體呈現 用漸進限制濃度之經生物素標記之gH/gL選擇結合物來使 抗體MSL-109經親和力成熟。藉由寡核苷酸定點突變誘發 用「NNK」密碼子使CDR之各位置隨機化,其中N為4種天 然核苷酸之任一者,且K為50%胸腺嘧啶及50%鳥嘌呤。 NNK密碼子可編碼20種天然胺基酸之任一者。分別製備輕 鏈及重鏈之文庫,且各鏈之3個CDR之各者同時經隨機 化。此舉產生在各鏈中具有0至3個隨機胺基酸變化,在各 CDR中具有至多1個突變之純系。以噬菌體Fab片段呈現載 158864.doc -98- 201215618 體且藉由標準方法製備文庫。藉由在連續數輪選擇中與1 及0·1 nM經生物素標記之gH/gL一起培育噬菌體呈現文庫 來選擇結合純系’且接著與10〇 nM gH/gL或MSL-109 IgG 競爭以降低親和力較低之純系與gH/gL的結合。在經中性 鍵親和素(neutravidin)或抗生蛋白鏈菌素(streptavidin)塗佈 之ELISA板上捕捉結合純系,洗滌且在室溫下於1〇 mM HC1中溶離1〇分鐘。溶離之噬菌體用1/1〇體積之1 μ Tris(pH 8.0)中和且用於感染大腸桿菌以達成擴增來用於下 一輪選擇。對來自第二輪選擇之純系定序以確定在所選噬 菌體中為頻繁的突變。藉由競爭噬菌體ELIS A測試具有有 利突變之純系。 在重鏈Kabat位置53及55中具有個別或組合突變之突變 MSL-109之IgG及Fab片段經表現且測試對CMV之活體外中 和。單獨或與在胺基酸55處之胺基酸取代(用R或κ置換 T55)組合的在胺基酸53處之胺基酸取代(用s、I、N、Q、 F、M、L、G、H、K、W、Y、V或A置換D53)提供中和性 能改良之抗體(圖12B及12C)。一些此等變化之示意圖展示 於圖12A中。另外,MSL-109中之胺基酸N52可經S置換。 此取代不影響效能但允許位置5 3中之S不具有位置5 2之糖 基化。重鏈可變序列在胺基酸53及/或胺基酸位置55處使 用各種胺基酸取代有89種可能之組合(SEQ ID NO:87、 88、89及96-182)。共同序列提供為SEq m n〇:94。在噬 菌體呈現ELIS A檢定中量測此等抗gH抗體之Fab片段對在 桿狀病毒中產生之gH/gL二聚體的親和力。詳言之,呈現 β 158864.doc -99- 201215618 MSLd09變異Fab片段之噬菌體純系與連續稀釋之gH/gL — 起培育且在室溫下培育1小時。藉由在室溫下培育經gH/gL 塗佈之ELISA板孔中之混合物10分鐘來偵測未結合噬菌 體。板用PBS-T洗滌,且藉由與抗M13 HRP結合物一起培 育30分鐘,隨後洗滌並用TMB受質顯色來偵測與經固定 gH/gL結合之噬菌體。藉由非線性回歸計算IC50,即噬菌 體-gH/gL混合物中之50%噬菌體為自由的所處之點。IC50 在0.01-0.1 nM之範圍内。所選變異體之個別親和力展示於 表3中。 表3 變異體 噬菌體ELIS A ICs〇(nM) H2單一突變體 WT(MSL-109) 0.53 H2-D53L 0.02 H2-D53M 0.05 H2-D53N 0.06 H2-D53S 0.1 H2-D53T 0.06 H2雙重突變體 H2-T55R 0.17 H2-D53F/T55R 0.01 H2-D53L/T55R 0.02 H2-D53M/T55R 0.07 H2-D53N/T55R(HB1) 0.05 H2-D53Q/T55R(HB2) 0.01 H2-D53S/T55R 0.03 H2-D53T/T55R 0.03 驚人地,在VH2 HVR處之胺基酸變化產生結合及中和能 力顯著較高之抗體。舉例而言,相較於MSL-109, HB1(D53N/T55R)對gH/gL之親和力增加10倍,如藉由噬菌 體ELISA所示(表3)。相較於親本MSL-109抗體,當以Fab 片段形式在大腸桿菌中表現時,HB1(D53N/T55R)對 158864.doc •100- 201215618 HCMV進入ARPE-19細胞中之抑制更強力約40倍(亦即’ EC50=0.15 nM對6.2 nM),如藉由中和檢定所示(圖12B) ° 此外,當以全長IgG形式在CHO細胞中表現時’ HB1 (D53N/T55R)對HCMV進入ARPE-19細胞中之抑制更強力 約6倍,如藉由中和檢定所示(表4,圖12C)。相較於MSL 109抗體,HB1抗體在對各種細胞類塑進行之中和檢定(1個 代表性實驗)中的EC50及EC90(pg/ml)展示於下表4中。 表4 上皮 上皮 内皮 内皮 MDM MDM 纖維 母細胞 纖維 母細胞 EC50 EC90 EC50 EC90 EC50 EC90 EC50 EC90 MSL-109 0.3 2 0.48 2.9 0.04 0.42 0.73 4.8 HB1 0.05 0.41 0.03 0.28 0.03 0.19 0.11 0.77 實例2-抗體功能研究 比較1^1(0531^/丁5 511)及1111808與1110在中和檢定中阻斷 HCMV病毒進入上皮細胞、内皮細胞、巨噬細胞及纖維母 細胞中之能力。hu8G8關於上皮細胞之EC50為0.006 pg/ml (0.2 nM),關於内皮細胞之EC50為 0.004pg/ml(0.3 nM),且 關於單核細胞之EC50為0.001 eg/ml(0.006 nM)。在中和 此等細胞類型之各者上之HCMV方面’ hu8G8比HB 1更強 力至少8倍。然而,如所預期,hu8G8不阻斷病毒進入纖維 母細胞中,而HB 1會阻斷病毒進入纖維母細胞中,EC50為 0.11 pg/ml(0.7 nm)(參見圖 13)。 已報導當向患有原發性HCMV感染之孕婦給與時’ HIG 可預防HCMV胎兒感染及/或疾病(Nigro等人,2005) ’表明 CMV特異性抗體能夠對發育胎兒賦予保護°當藉由中和檢 101 · 158864.doc 201215618 定評估時,發現HIG可中和病毒進入所有測試細胞類型 中,但效能遠小於任一單株抗體(參見圖13)。此相對較低 之效能歸因於HIG僅具有小部分具有抗CMV中和活性之蛋 白質的多株性質。 實例3-HIG消減研究 為了鑑別高免疫球蛋白中之中和抗體組分,藉由6次連 續培育,使用用gB或複合物I轉染之HEK293T細胞消減 HIG之抗 gB抗體或抗複合物 I(gH/gL/UL 128/UL130/UL 13 1) 抗體。根據上述方法進行Cytogam®(HIG)消減。對經吸附 血清之分析顯示相較於關於對照細胞之〇°/。,有>95°/。之與 用gB轉染之細胞具有反應性的抗體由此程序所吸附,如藉 由經純化gB ELISA所檢定。然而,相較於關於對照細胞之 0%,僅約45%之與用複合物I轉染之細胞具有反應性的抗 體已經吸附,如藉由用如上所述之來自經轉染HEK293T細 胞之溶解產物進行的ELISA所檢定。 經消減之HIG接著在中和檢定中用於確定消減對預防病 毒進入上皮細胞中之影響。相較於模擬吸附HIG,吸附 HIG製備物之連續稀釋液用於如上所述之中和檢定中。此 等實驗之結果展示於圖14中。針對gB之抗體似乎不顯著有 助於HIG關於上皮細胞之中和能力,而針對複合物I之抗體 似乎顯著有助於HIG之中和活性。當對上皮細胞測試時, 移除複合物I特異性抗體使HIG之中和能力(EC50)降低約 85%。 經消減HIG關於纖維母細胞之檢定不可能,因為為偵測 158864.doc -102- 201215618 中和所需之濃度極高。HIG關於此細胞類型之EC50為約 500 pg/ml。因為UL128、UL130及UL131蛋白質不為進入 纖維母細胞中所需,所以與如上所述之管柱結合之桿狀病 毒表現的gB或gH/gL用於自HIG特異性消減對此等蛋白質/ 複合物具有特異性之抗體。在抗gB抗體接近完全消減(gB 管柱上之gB抗體95%消減對gH/gL管柱上之gB抗體0%消 減)之情況下,未觀測到中和變化。然而,在大多數抗 gH/gL抗體經消減(gH/gL管柱上之gH/gL抗體84%消減對gB 管柱上之gH/gL抗體0%消減)之情況下,觀察到EC50降低 65°/。(參見圖 14)。 由此等資料推斷HIG中之主要中和抗體係針對gH/gL/ UL128/UL13 0/UL131複合物。詳言之,複合物I中和抗體 為HIG中針對上皮細胞進入之主要中和抗體。另外,HIG 中之gH/gL抗體在抑制病毒進入纖維母細胞中方面具有主 要作用。此等實驗顯示抗gB抗體在HIG中和中之作用很 小 〇 藉由使用桿狀病毒表現之gB、gH/gL及gH/gL/UL128/ UL130/UL131吸附HIG,藉由ELISA測定約1%之HIG具有 gB反應性,而約0.1-0.2%與複合物I或gH/gL具有反應性。 若暸解HIG中複合物特異性抗體之濃度,則彼等複合物特 異性抗體之中和效能可藉由根據實際上係針對導致中和之 相關複合物之IgG的百分比校正完整HIG之中和效能來計 算(例如關於纖維母細胞,810 pg/mlx 0· 1-0.2 = 0.8-1.6), 如下表5中所示。 158864.doc -103 - 201215618 表5 EC9〇bg/ml) 中和劑 目標 上皮細胞 内皮細胞 巨噬細胞 纖維母細胞 HCMV-HIGt HCMV +未知 0.01-0.02 0.01-0.02 0.004-0.01 0.8-1.6 HB1 SH 0.4 0.28 0.19 0.78 HB2 gH 0.41 0.44 0.21 0.78 HCMV-HIG HCMV +未知 8.0 11.6 3.8 810 具有VH1之人 類化8G8 複合物I 0.02 0.04 0.010 n/a 具有VH3之人 類化8G8 複合物I 0.010 0.7 0.010 n/a 卞 EC90值根據 HIG 中之 gH/gL/UL 12 8/UL13 O/UL13 1 抗體之 濃度(0.1-0.2%)進行調整。 如以上在表5中所示,HB1與人類化8G8(VH1或VH3)之 組合能夠接近於HIG關於中和檢定中測試之所有細胞類型 的中和效能。在以下HCMV感染倍率(MOI)下感染細胞: 上皮細胞ΜΟΙ=1,内皮細胞ΜΟΙ=1,巨噬細胞MOI=0.5且 纖維母細胞ΜΟΙ=1。HB1與HIG(如根據具有複合物I特異性 之HIG的量校正)就抑制對纖維母細胞之感染而言具有類似 中和效能,但不提供關於上皮細胞、内皮細胞或巨噬細胞 之足夠效能。人類化8G8(VH1)及(VH3)與HIG(如根據具有 複合物I特異性之HIG的量校正)關於上皮細胞、内皮細胞 及巨噬細胞具有類似中和效能。然而,其未能中和對纖維 母細胞之感染。因此,關於測試之所有細胞類型,抗體之 組合提供與HIG對HCMV之中和(根據複合物I特異性抗體 進行調整)類似的對HCMV之中和。 再次在中和檢定中測試HB1及具有VH1之hu8G8中和 HCMV進入所有各種細胞類型中之能力且與HIG之計算及 158864.doc -104- 201215618 實際中和效能進行比較。用以下HCMV MOI感染細胞:上 皮細胞ΜΟΙ = 1,内皮細胞MOI=0.25,巨噬細胞ΜΟΙ=0·25 且纖維母細胞ΜΟΙ=0.8。此實驗之結果以下在表6中展示。 由此實驗獲得之平均EC90及表5中所示之結果以下在表6中 之陰影框中展示。 表6 EC9〇bg/ml) 中和劑 目標 上皮細胞 内皮細胞 巨嗟細胞 纖維母細胞 HCMV-HIG 卞 HCMV 0.01-0.02 0.007-0.014 0.01-0.02 0.4-0.7 +未知 0.01-0.02 0.01-0.02 0.01-0.02 0.6-1.2 冊1 gH 0.18 0.08 0.14 0.25 0.29 0.18 0.17 0.52 HCMV-HIG HCMV 10.2 6.7 10.9 370 +未知 9.1 9.1 7.4 590 具有VH1之人 類化8G8 複合物I 0.01 0.02 0.008 0.02 0.009 0.01 n/a fHIG根據gH/gL/UL128/UL130/UL131抗體之貢獻進行調 整0 實例4-中和HCMV臨床分離株 對自 Oregon Health Sciences University之 2個實驗室獲得 之超過20個臨床分離株的gH、gL、UL128、UL130及 UL13 1基因定序且與其他可公開獲得之序列一起進行彙 編。可公開獲得之序列由來源於美國、歐洲及日本之病毒 株產生。 溶解受各病毒株感染之細胞且使用DNA血液/組織提取 套組(Qiagen; Germantown, MD)提取DNA。根據可在國家 生物技術資訊中心資料庫中獲得之AD169、FIX、 TB40E、Toledo及Towne序列之比對,設計針對保守序列 158864.doc -105- 201215618 的引子。擴增出各臨床病毒株之自起始密碼子至鹼基 2196(僅缺乏終止密碼子)的醣蛋白gH。在Genentech使用染 料終止劑反應對聚合酶鏈反應(PCR)產物定序。序列經比 對及修整(定序器)。其他gH序列藉由以下方式自NCBI資料 庫獲得:使用來自 Chou 等人,·/· Infect. Dis. 166:604-7 (1992)之1個寄存編號且接著獲得「相關序列」。總計,在 移除信號肽之後,叢集(ClustalW,歐洲生物資訊學研究所 (EBI); Cambridgeshire, England)來自 57個病毒株之膽蛋白 序列,且使用平均距離根據一致性百分比比對成樹 (JalView; Waterhouse等人,2009)。 定序結果指示各臨床分離株之間在UL128、UL130及 UL131序列方面有1%變化(在移除信號肽之後)。此研究結 果與在歐洲進行之證明UL128、UL130及UL131在患有原 發性HCMV感染之孕婦中高度保守的研究一致(Baldanti等 人,Jrc/2. K/ro/. 151:1225-33 (2006))。 在所有病毒株中,gH在蛋白質層面上至少95%—致(在 移除信號肽之後)。構築具有2個不同分支之系統發生樹(資 料未展示)。該樹與gH蛋白質序列分開成2個系統發生群之 先前報導一致(Chou, /. /77/eci_ Db. 166:604-7 (1992))。亦 根據文獻,兩個分支中之HCMV分離株不在地理上不同(亦 即在曰本分離之病毒株可見於兩個分支中)(Pignatelli,乂 Gen. Virol. 84:647-655 (2003)) ° 測試HB1(D53N/T55R)中和不同組之HCMV臨床分離株 對纖維母細胞之感染性的能力。表7展示HB 1相較於HIG之 158864.doc -106- 201215618 效用。發現HB1可與HIG(當根據對gH/gL/UL128/ UL130/UL131具有特異性之HIG的量校正時)一樣或優於 HIG中和代表最大gH序列多樣性之HCMV病毒株之各者。 在多個實驗中在各種MOI下使用HCMV病毒株Dement、 Adinis及VR1814進行之中和檢定之結果亦展示於下表7 中〇EC50(pg/ml) : HCMV strain VR1814 Cell type Target epithelial endothelial fibroblast MSL-109 gH 0.1642 0.0764 0.3890 Murine 8G8 complex I 0.0014 0.0007 N/A Rat 5H3 gH 1.15 1.42 17.76 Rodent 10F8 gH 0.1 0.1 1.11 Murine 15H2 gH 0.12 0.11 1 Murine 354.1 Complex I 0.03 0.07 N/A Humanization and analysis of murine 8G8 mAb. The murine fusion tumor 8G8 was humanized by standard CDR grafting using the λ3 or λ4 light chain (Fig. 2) and the VH1, VH3 or VH7 heavy chain framework (Fig. 1). For comparison, the common human lambda germline sequence of λ3 and λ4 is shown in Figure 2. A neutralization assay was performed to compare the combination of the 8G8 human/murine chimeric antibody (QE7/C2) with the 8G8 VH1, VH3 or VH7 humanized heavy chain and the 8G8 λ3 or λ4 light chain. The λ4 variant but not the λ3 variant was found to neutralize HCMV (Fig. 3). HVR-L2 of mutation λ4 as shown in Figure 4 introduces substitution at amino acid 50C, 50D, 56, and amine group at amino acid 57 (first amino acid of FR3) (according to Kabat numbering) Acid substitution to provide stability to the antibody. The various mutations were lightly linked in combination with the 8G8 human VH1 chain and tested for antibodies as described above and in assays. Antibodies with a single amino acid substitution showed good neutralizing activity (i.e., Al, E1, ΤΙ, A2, E2, and T2) (Fig. 5). Similarly, all antibodies containing two amino acid substitutions showed good neutralizing activity (i.e., SGSG and TGDA). A single mutant SG was included as a comparative control and it also showed good neutralizing activity. (Figure 6). 158864.doc -97- 201215618 The humanized 8G8 λ4 antibody sequence is shown in Figure 7 (hu8G8A4 FW). Figure 8 shows the sequence of the humanized 8G8 VH1 sequence (hu8G8.VH1) and Figure 9 shows the sequence of the humanized 8G8 VH3 sequence (hu8G8.VH3). Figure 10 shows the humanized 8G8 λ4 antibody sequence in which the first two amino acids (QP) have been modified to allow the polypeptide to start with serine (Q is deleted and L is mutated to S) and the amino acid 36 retains the murine amine group. Acid (Υ). The polypeptide sequence of this antibody is shown as a λ4 8G8 graft. A representative nucleic acid sequence encoding a polypeptide is shown below the polypeptide sequence. The affinity of the anti-gH antibody is mature. The synthesis of antibody sequences of the variable heavy and variable light chain sequences of MSL-109, as disclosed in PCT Publication No. WO 94/1 6730, published on Aug. 4, 1994, which is hereby incorporated by reference in its entirety. Single antibody MSL-109. The amino acid sequence of the MSL-109 VH and VL chains is shown in Figure 11 (VL: SEQ ID NO: 90; VH: SEQ ID NO: 92). The MSL-109 anti-system is based on an IgGl framework containing heavy chain VH3 and light chain Vk2. Recombinant DNA encoding this antibody was cloned into CHO cells. The antibody MSL-109 was affinity matured by randomization of the complementarity determining regions (CDRs) followed by phage display with a progressively limiting concentration of the biotinylated gH/gL selection conjugate. Localization of CDRs by randomization of oligonucleotides using the "NNK" codon, where N is any of the four natural nucleotides, and K is 50% thymine and 50% guanine. The NNK codon can encode any of the 20 natural amino acids. Libraries of light and heavy chains were prepared separately, and each of the three CDRs of each chain was simultaneously randomized. This produces a pure line with 0 to 3 random amino acid changes in each chain with up to 1 mutation in each CDR. The phage Fab fragment was presented as a 158864.doc-98-201215618 and the library was prepared by standard methods. Combining the pure line' and then competing with 10〇nM gH/gL or MSL-109 IgG to reduce by culturing a phage display library with 1 and 0·1 nM biotinylated gH/gL in successive rounds of selection The combination of pure affinity and gH/gL with lower affinity. The bound pure lines were captured on neutravidin or streptavidin coated ELISA plates, washed and lysed in 1 mM HCl for 1 hr at room temperature. The lysed phage were neutralized with 1 μl volume of 1 μ Tris (pH 8.0) and used to infect E. coli to achieve amplification for the next round of selection. The pure line sequence from the second round of selection determines the frequent mutations in the selected phage. A pure line with a favorable mutation was tested by competitive phage ELIS A. The IgG and Fab fragments of the mutant MSL-109 with individual or combined mutations in the heavy chain Kabat positions 53 and 55 were expressed and tested for in vitro neutralization of CMV. Amino acid substitution at amino acid 53 alone or in combination with an amino acid substitution at amino acid 55 (replacement of T55 with R or κ) (with s, I, N, Q, F, M, L) , G, H, K, W, Y, V or A substitution D53) provides antibodies with improved neutralizing properties (Figures 12B and 12C). A schematic of some of these variations is shown in Figure 12A. In addition, the amino acid N52 in MSL-109 can be replaced by S. This substitution does not affect potency but allows S in position 5 3 to have no glycosylation at position 5 2 . The heavy chain variable sequence has 89 possible combinations (SEQ ID NOS: 87, 88, 89 and 96-182) with various amino acid substitutions at position 55 of the amino acid 53 and/or amino acid. The common sequence is provided as SEq m n〇:94. The affinity of the Fab fragments of these anti-gH antibodies for gH/gL dimers produced in baculovirus was measured in a phage-present ELIS A assay. In particular, the phage pure lines presenting the βL 158864.doc -99-201215618 MSLd09 variant Fab fragment were incubated with serially diluted gH/gL and incubated for 1 hour at room temperature. Unbound phage were detected by incubating the mixture in the gH/gL coated ELISA plate wells for 10 minutes at room temperature. The plates were washed with PBS-T and incubated with anti-M13 HRP conjugate for 30 minutes, followed by washing and color development with TMB to detect phage bound to immobilized gH/gL. The IC50 was calculated by nonlinear regression, i.e., 50% of the phage-gH/gL mixture was free. The IC50 is in the range of 0.01-0.1 nM. The individual affinities of the selected variants are shown in Table 3. Table 3 Variant phage ELIS A ICs〇(nM) H2 single mutant WT (MSL-109) 0.53 H2-D53L 0.02 H2-D53M 0.05 H2-D53N 0.06 H2-D53S 0.1 H2-D53T 0.06 H2 double mutant H2-T55R 0.17 H2-D53F/T55R 0.01 H2-D53L/T55R 0.02 H2-D53M/T55R 0.07 H2-D53N/T55R(HB1) 0.05 H2-D53Q/T55R(HB2) 0.01 H2-D53S/T55R 0.03 H2-D53T/T55R 0.03 Amazing The amino acid change at the VH2 HVR produces antibodies with significantly higher binding and neutralization capabilities. For example, HB1 (D53N/T55R) has a 10-fold increase in affinity for gH/gL compared to MSL-109, as shown by phage ELISA (Table 3). Compared to the parental MSL-109 antibody, when expressed as a Fab fragment in E. coli, HB1 (D53N/T55R) inhibits 158864.doc •100-201215618 HCMV into ARPE-19 cells by about 40 times more potently. (ie 'EC50=0.15 nM vs. 6.2 nM), as indicated by the neutralization assay (Fig. 12B) ° In addition, when HB_ cells are expressed in full-length IgG, 'HB1 (D53N/T55R) enters ARPE for HCMV The inhibition in -19 cells was about 6 times more potent as shown by the neutralization assay (Table 4, Figure 12C). The EC50 and EC90 (pg/ml) of the HB1 antibody in the neutralization assay (1 representative experiment) for various cell types were shown in Table 4 below compared to the MSL 109 antibody. Table 4 Epithelial endothelial endothelial MDM MDM fibroblast fibroblast EC50 EC90 EC50 EC90 EC50 EC90 EC50 EC90 MSL-109 0.3 2 0.48 2.9 0.04 0.42 0.73 4.8 HB1 0.05 0.41 0.03 0.28 0.03 0.19 0.11 0.77 Example 2 - Antibody Functional Study Comparison 1 ^1 (0531^/丁5 511) and 1111808 and 1110 block the ability of HCMV viruses to enter epithelial cells, endothelial cells, macrophages, and fibroblasts in a neutralization assay. The EC50 of hu8G8 for epithelial cells was 0.006 pg/ml (0.2 nM), the EC50 for endothelial cells was 0.004 pg/ml (0.3 nM), and the EC50 for monocytes was 0.001 eg/ml (0.006 nM). In terms of HCMV in neutralizing each of these cell types, hu8G8 is at least 8-fold more potent than HB1. However, as expected, hu8G8 did not block the entry of the virus into the fibroblasts, while HB 1 blocked the virus from entering the fibroblasts with an EC50 of 0.11 pg/ml (0.7 nm) (see Figure 13). It has been reported that 'HIG can prevent HCMV fetal infections and/or diseases when administered to pregnant women with primary HCMV infection (Nigro et al., 2005)' indicates that CMV-specific antibodies can confer protection to developing fetuses. Neutralization test 101 · 158864.doc 201215618 When assessed, HIG was found to neutralize the virus into all test cell types, but the potency was much less than either monoclonal antibody (see Figure 13). This relatively low performance is attributed to the multi-plant nature of HIG with only a small fraction of proteins with anti-CMV neutralizing activity. Example 3 - HIG Subtraction Study In order to identify neutralizing antibody components in high immunoglobulin, HEG anti-gB antibody or anti-complex I was attenuated by HEK293T cells transfected with gB or complex I by 6 consecutive incubations. (gH/gL/UL 128/UL130/UL 13 1) Antibody. Cytogam® (HIG) reduction was performed according to the above method. Analysis of the adsorbed serum showed 〇°/ compared to the control cells. , there is > 95 ° /. Antibodies reactive with cells transfected with gB are adsorbed by this procedure, as determined by a purified gB ELISA. However, compared to 0% for control cells, only about 45% of antibodies reactive with cells transfected with complex I have been adsorbed, such as by solubilization from transfected HEK293T cells as described above. The product was assayed by ELISA. The subtracted HIG is then used in a neutralization assay to determine the effect of depletion on preventing the entry of the virus into epithelial cells. Serial dilutions of the adsorbed HIG preparation were used in the neutralization assay described above as compared to the simulated adsorption of HIG. The results of these experiments are shown in Figure 14. Antibodies against gB did not appear to contribute significantly to HIG's ability to neutralize epithelial cells, whereas antibodies against complex I appeared to contribute significantly to HIG neutralizing activity. Removal of the complex I-specific antibody reduced the HIG neutralizing capacity (EC50) by about 85% when tested on epithelial cells. The elimination of HIG for fibroblasts is not possible because of the extremely high concentration required to detect 158864.doc -102- 201215618. HIG has an EC50 of about 500 pg/ml for this cell type. Since UL128, UL130 and UL131 proteins are not required for entry into fibroblasts, gB or gH/gL expressed by baculovirus in combination with the column as described above is used for specific protein/complexation from HIG. An antibody with specificity. In the case where the anti-gB antibody was nearly completely attenuated (the 95% reduction of the gB antibody on the gB column was 0% reduction of the gB antibody on the gH/gL column), no neutralization change was observed. However, an EC50 reduction of 65 was observed with most anti-gH/gL antibodies reduced (84% reduction of gH/gL antibody on gH/gL column for 0% reduction of gH/gL antibody on gB column) °/. (See Figure 14). From this data, it is concluded that the major neutralizing anti-system in HIG is directed to the gH/gL/UL128/UL13 0/UL131 complex. In particular, the complex I neutralizing antibody is a major neutralizing antibody against epithelial cell entry in HIG. In addition, gH/gL antibodies in HIG have a major role in inhibiting viral entry into fibroblasts. These experiments show that the anti-gB antibody has little effect in HIG neutralization, and adsorbs HIG by using baculovirus gB, gH/gL and gH/gL/UL128/UL130/UL131, and about 1% by ELISA. The HIG has gB reactivity, while about 0.1-0.2% is reactive with complex I or gH/gL. Knowing the concentration of complex-specific antibodies in HIG, their complex-specific antibody neutralization potency can be corrected by determining the complete HIG neutralization efficiency based on the percentage of IgG actually associated with the complex that leads to neutralization. To calculate (for example, for fibroblasts, 810 pg/ml x 0· 1-0.2 = 0.8-1.6), as shown in Table 5 below. 158864.doc -103 - 201215618 Table 5 EC9〇bg/ml) Neutralizing agent Target Epithelial cells Endothelial cells Macrophage Fibroblasts HCMV-HIGt HCMV + Unknown 0.01-0.02 0.01-0.02 0.004-0.01 0.8-1.6 HB1 SH 0.4 0.28 0.19 0.78 HB2 gH 0.41 0.44 0.21 0.78 HCMV-HIG HCMV + unknown 8.0 11.6 3.8 810 Humanized 8G8 complex with VH1 0.02 0.04 0.010 n/a Humanized 8G8 complex with VH3 I 0.010 0.7 0.010 n/a 卞The EC90 value was adjusted according to the concentration (0.1-0.2%) of the gH/gL/UL 12 8/UL13 O/UL13 1 antibody in HIG. As shown above in Table 5, the combination of HB1 and humanized 8G8 (VH1 or VH3) was able to approximate the neutralizing potency of HIG for all cell types tested in the neutralization assay. Cells were infected at the following HCMV infection rate (MOI): epithelial cells ΜΟΙ = 1, endothelial cells ΜΟΙ = 1, macrophage MOI = 0.5 and fibroblasts ΜΟΙ = 1. HB1 and HIG (as corrected for the amount of HIG specific for complex I) inhibit similar potency against fibroblast infection, but do not provide sufficient efficacy for epithelial cells, endothelial cells or macrophages . Humanized 8G8 (VH1) and (VH3) and HIG (as calibrated according to the amount of HIG with complex I specificity) have similar neutralizing potency with respect to epithelial cells, endothelial cells and macrophages. However, it failed to neutralize infection with fibroblasts. Thus, for all cell types tested, the combination of antibodies provides neutralization of HCMV similar to HIG neutralization of HCMV (adjusted for complex I-specific antibodies). The ability of HB1 and hu8G8 with VH1 and HCMV to enter all of the various cell types was again tested in the neutralization assay and compared to the HIG calculations and the actual neutralizing potency of 158864.doc -104 - 201215618. Cells were infected with the following HCMV MOI: epithelial cells ΜΟΙ = 1, endothelial cells MOI = 0.25, macrophages ΜΟΙ = 0.25 and fibroblasts ΜΟΙ = 0.8. The results of this experiment are shown below in Table 6. The average EC90 obtained in this experiment and the results shown in Table 5 are shown below in the shaded boxes in Table 6. Table 6 EC9〇bg/ml) Neutralizing agent Target epithelial cells Endothelial cells Giant blast cell fibroblast HCMV-HIG 卞HCMV 0.01-0.02 0.007-0.014 0.01-0.02 0.4-0.7 +Unknown 0.01-0.02 0.01-0.02 0.01-0.02 0.6-1.2 book 1 gH 0.18 0.08 0.14 0.25 0.29 0.18 0.17 0.52 HCMV-HIG HCMV 10.2 6.7 10.9 370 + unknown 9.1 9.1 7.4 590 Humanized 8G8 complex with VH1 I 0.02 0.008 0.008 0.02 0.009 0.01 n/a fHIG according to gH/ Adjustment of the contribution of gL/UL128/UL130/UL131 antibodies 0 Example 4 - Neutralized HCMV clinical isolates of gH, gL, UL128, UL130 and more than 20 clinical isolates obtained from 2 laboratories of Oregon Health Sciences University The UL13 1 gene is sequenced and compiled with other publicly available sequences. The publicly available sequences are produced by strains derived from the United States, Europe, and Japan. The cells infected with each virus strain were dissolved and DNA was extracted using a DNA blood/tissue extraction kit (Qiagen; Germantown, MD). The primers for the conserved sequence 158864.doc -105 - 201215618 were designed based on the alignment of the AD169, FIX, TB40E, Toledo and Towne sequences available in the National Biotechnology Information Center database. The glycoprotein gH of each clinical strain from the start codon to base 2196 (only the stop codon is lacking) was amplified. The polymerase chain reaction (PCR) product was sequenced using a dye terminator reaction at Genentech. The sequences are aligned and trimmed (sequencer). Other gH sequences were obtained from the NCBI database by using a registration number from Chou et al., Infect. Dis. 166:604-7 (1992) and then obtaining a "correlation sequence". In total, after removal of the signal peptide, clusters (ClustalW, European Institute of Bioinformatics (EBI); Cambridgeshire, England) were derived from the bilirubin sequences of 57 strains, and the average distance was used to compare the percentages according to the percent identity (JalView) Waterhouse et al., 2009). The sequencing results indicated a 1% change in UL128, UL130 and UL131 sequences between clinical isolates (after removal of the signal peptide). The results of this study are consistent with the findings in Europe that UL128, UL130 and UL131 are highly conserved among pregnant women with primary HCMV infection (Baldanti et al., Jrc/2. K/ro/. 151:1225-33 ( 2006)). In all strains, gH is at least 95% at the protein level (after removal of the signal peptide). Construct a phylogenetic tree with 2 different branches (data not shown). This tree is consistent with the previous report of the gH protein sequence divided into two phylogenetic groups (Chou, /. /77/eci_Db. 166:604-7 (1992)). According to the literature, the HCMV isolates in the two branches are not geographically distinct (i.e., the virus strain isolated in the sputum is found in two branches) (Pignatelli, 乂 Gen. Virol. 84:647-655 (2003)) ° The ability of HB1 (D53N/T55R) to neutralize the infectivity of different groups of HCMV clinical isolates to fibroblasts was tested. Table 7 shows the utility of HB 1 compared to HIG 158864.doc -106 - 201215618. It was found that HB1 can be the same as or better than HIG (when corrected according to the amount of HIG specific for gH/gL/UL128/UL130/UL131) and HIG neutralizing each of the HCMV strains representing the maximum gH sequence diversity. The results of neutralization assays using HCMV strains Dement, Adinis and VR1814 at various MOIs in various experiments are also shown in Table 7 below.
表7 病毒株名稱(分支 號) HB1(D53N/T55R) E090(pg/ml) HIG EC90(pg/ml)* HIG EC90^g/ml) Cano ⑴ 0.27 0.3-0.7 355 Keone(l) 0.12 0.1-0.2 104 NewRock(l) 0.18 0.12-0.24 119 TR(1) 0.59 0.3-0.7 350 Brown ⑵ 0.76 0.4-0.9 442 Dement(2) 0.78 0.8-1.6 80 0.08 0.15-0.3 154 Adinis(2) 2.0 0.74-1.5 233 0.08 0.1-0.2 120 Grunden⑵ 0.08 0.14-0.28 137 Harris ⑵ 0.32 0.3-0.6 305 Phoebe⑵ 0.04 0.1-0.2 115 VR1814(2) 0.08 0.1-0.3 790 0.78 0.8-1.6 0.25 0.4-0.7 370 *EC90 值根據 HCMV-HIG 中之 gH/gL/UL128/UL130/ UL131 抗體之濃度(0.1-0.2%)進行調整。 實例5-抗體之特異性 評估HB1及hu8G8之抗原特異性。構築含有病毒醣蛋白 之質體以使各蛋白質藉由用「自裂解2 A肽」分開各基因而 以相等化學計量表現(Szymczak等人,Biotechnol. 22:589-94 (2004))。質體含有 gB/eGFP、gH/gL/eGFP 或 UL128/UL130/UL131/eGFP(自 cDNA選殖)之全長基因。質 158864.doc -107· -0 201215618 體使用脂染胺2000(Invitrogen; Carlsbad,CA)轉染入人類胚 腎(HEK)-293T細胞(美國菌種保存中心,ATCC; Manassas, VA)中以在其表面處表現CMV醣蛋白。在2天之後,細胞經 解離且用飽和一次抗體HB 1、hu8G8、抗gB、親和力純化 之兔抗UL131_933或親和力純化之兔抗gH_977染色。細胞 用與別藻藍蛋白(allophycocyanin)(APC , Jackson ImmunoResearch; West Grove, PA)結合之適當二次抗體染 色。使用 FACSCalibur(BD Biosciences; San Jose, CA)量測 個別細胞之營光且使用FlowJo軟體(Tree Star; Ashland, OR)進行分析。GFP陽性細胞(表現CMV轉殖基因之細胞) 經選擇性繪圖以展示抗體結合。 如圖15中所示,HB1與表現單獨gH/gL或gH/gL與 UL128/UL130/UL131之複合物的細胞起反應。Hu8G8僅與 表現gH/gL/UL128/UL130/UL131之細胞起反應(圖15)且不 與表現單獨gH/gL或gH/gL/gO之細胞起反應(資料未展 示)。兩種抗體皆不與表現gB之細胞起反應。因此,HB 1 識別存在於gH/gL複合物及複合物I中之gH上之抗原決定 基。hu8G8抗體與形成複合物I之5種包膜蛋白内之抗原決 定基結合,但不與gH/gL/gO或單獨gH/gL結合。 實例6-評估協同作用或拮抗作用 在病毒中和檢定中以組合形式測試HB 1及hu8G8以確定 當2種抗體組合時是否存在效能差異。因為抗體具有不同 目標,且推測獨立地起阻斷病毒進入之作用,所以假定 HB 1及hu8G8之效應為累加的。因此,應用布利斯獨立方 158864.doc • 108· 201215618 程式(具有效應A及B之兩種單一化合物之組合反應C為 C=A+B-AxB)。為此,HB1與hu8G8以1:1比率混合且在對 上皮細胞進行之病毒中和檢定中以一系列稀釋液進行測試 並如上所述計算EC50。HB1不增強或減弱hu8G8關於上皮 細胞之效能且1:1曲線精確重疊模擬布利斯獨立性曲線, 表明累加性而非協同作用(參見圖16及表8)。同樣,如所預 期,hu8G8不改變HB1關於纖維母細胞之效能,因為hu8G8 不阻斷HCMV進入纖維母細胞中(資料未展示)。因此,在 1:1比率下,HB 1及hu8G8不顯示任何拮抗作用或協同作 用。 表8 mAB 關於上皮細胞之 EC5〇bl/ml) 單獨HBl 0.063 單獨hu8G8 0.0080 HBl:hu8G8,在 1:1 下 0.0060 模擬布利斯獨立性 0.0069 為評估HB1及hu8G8是否在廣泛範圍之比率下顯示協同 作用或拮抗作用,跨越EC50值之一系列逐對「棋盤格」稀 釋液用於進行中和檢定。各抗體如下表9中指示進行稀 釋,而病毒濃度為恆定的。下表9中展示對於抗體濃度之 各種組合,關於上皮細胞之受感染細胞之百分比(相對於 無抗體對照進行校正): •tmi 158864.doc -109- 201215618 表9 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 HB1 10 μ^ηιΐ 2 pg/ml 0.4 pg/ml 0.08 μ^ιπΐ 0.016 μ_1 0.0032 μβ/ηιΐ 0.00064 0.000128 Ug/ml ua/ml 0 pg/ml HB1 10 pg/ml 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HB1 2 pg/ml 0.00 0.00 0.00 0.00 0.00 0.00 0.56 0.17 0.00 HB1 0.4 pg/ml 0.23 0.00 0.28 0.47 0.32 2.08 3.57 2.94 3.97 HB1 0.08 pg/ml 0.29 0.15 0.15 0.15 1.91 13.53 26.76 25.26 26.66 HB1 0.016 gg/ml 0.00 0.48 0.42 0.59 3.29 39.70 61.94 67.60 61.94 HB1 0.0032 pg/ml 0.37 0.71 1.06 0.29 7.23 46.22 80.96 93.01 97.34 HB1 0 pg/ml 0.64 0.33 0.14 0.63 5.50 53.93 83.51 91.95 100.00 陰影=存在部分中和之各抗體之濃度。 使用如上所述之中和檢定,HB1及hu8G8之EC50在0.8、 0.016、0,0032 pg/ml之另一抗體存在下測定。此等實驗之 結果展示於下表10及表11及圖17中。 表10-在不同HB1濃度下之hu8G8效能 抗體及濃度 hu8G8 之 EC50(pg/mL) HB1 ’ 在08 pg/ml下 0.0037 HB1 ’ 在 016pg/ml 下 0.0047 HB1,在0032 gg/ml下 0.0028 HBl,在0 pg/ml下 0.0031 表11-在不同hu8G8濃度下之HB1效能 抗體及濃度 HB1 之 EC5〇hg/mL) hu8G8,在0032 pg/ml下 0.035 hu8G8,在00064 pg/ml下 0.043 1111808,在000128 4§/1111下 0.041 hu8G8,在0 pg/ml下 0.030 在比較單獨各抗體之效能與在各種比率下之組合之效能 158864.doc -110· 201215618 後,不存在協同作用或拮抗作用之證據。舉例而言,在比 較hu8G8之效能曲線與hu8G8及0.08 pg/ml HB1之效能曲線 後(在不存在滴定抗體下之感染校正為1 〇〇%),吾人發現曲 線重疊(參見圖17)。又,在各種濃度之hu8G8存在下,各 HB1效能曲線重疊(相對於100%感染校正)(參見圖17)。因 此,在廣泛範圍之比率下,不存在抗體之間具有協同作用 或拮抗作用之證據。 實例7-評估產生之病毒抗性 儘管人類CMV具有相對較低之突變率(相較於肝炎病毒 C(hepatitis virus C,HCV)或人類免疫缺乏病毒(human immunodeficiency virus,HIV)),但評估病毒經由產生抗 性突變來逃避HB 1或hu8G8或兩者之中和的能力。使病毒 在次最佳濃度之HB1(或MSL-109)或hu8G8、或HB1與 hu8G8抗體兩者一起存在下生長。隨著病毒每週向新細胞 上之繼代(各輪有約50%之體積向前繼代於新細胞上),逐 漸增加各抗體之濃度。觀測到對各個別抗體具有抗性之突 變病毒,但無突變體賦予對組合之抗性。所有抗性病毒突 變體皆自單一斑塊出現。 出現賦予對由HB 1所致之中和之抗性的突變體(參見圖 18)。然而,如藉由中和檢定所示,此等突變體仍然對 hu8G8敏感,hu8G8具有類似EC50(參見圖18及表12及表 13)。 158864.doc -111 - 201215618 表12-出現之對HB1具有抗性之HCMV突變體 HB1 EC50〜g/ml) 無Ab對照 0.04 HB1-突變病毒1 0.21 HB1-突變病毒2 無中和 HB1-突變病毒3 0.07 HB1-突變病毒4 200 HB1-突變病毒5 無中和 HB1-突變病毒6 無中和 表13-HCMV HB1抗性突變體仍然對hu8G8中和敏感 hu8G8 EC50(pg/ml) 資料集1 資料集2 無Ab對照 0.008 0.0005 HB1-突變病毒1 0.010 0.0006 HB1-突變病毒2 0.008 0.006 HB1-突變病毒3 0.001 0.0001 HB1-突變病毒4 未測出 0.0001 HB1-突變病毒5 未測出 0.001 HB1-突變病毒6 未測出 0.001Table 7 Viral strain name (branched number) HB1 (D53N/T55R) E090 (pg/ml) HIG EC90 (pg/ml)* HIG EC90^g/ml) Cano (1) 0.27 0.3-0.7 355 Keone(l) 0.12 0.1- 0.2 104 NewRock(l) 0.18 0.12-0.24 119 TR(1) 0.59 0.3-0.7 350 Brown (2) 0.76 0.4-0.9 442 Dement(2) 0.78 0.8-1.6 80 0.08 0.15-0.3 154 Adinis(2) 2.0 0.74-1.5 233 0.08 0.1-0.2 120 Grunden(2) 0.08 0.14-0.28 137 Harris (2) 0.32 0.3-0.6 305 Phoebe(2) 0.04 0.1-0.2 115 VR1814(2) 0.08 0.1-0.3 790 0.78 0.8-1.6 0.25 0.4-0.7 370 *EC90 value according to HCMV-HIG The concentration of gH/gL/UL128/UL130/UL131 antibody (0.1-0.2%) was adjusted. Example 5 - Specificity of antibodies The antigen specificity of HB1 and hu8G8 was evaluated. A plastid containing a viral glycoprotein is constructed such that each protein is expressed in equal stoichiometry by separating each gene with "self-cleavage 2 A peptide" (Szymczak et al., Biotechnol. 22:589-94 (2004)). The plastid contains the full-length gene of gB/eGFP, gH/gL/eGFP or UL128/UL130/UL131/eGFP (from cDNA selection). 158864.doc -107· -0 201215618 The body was transfected into human embryonic kidney (HEK)-293T cells (American Type Culture Collection, ATCC; Manassas, VA) using lipofectamine 2000 (Invitrogen; Carlsbad, CA). The CMV glycoprotein is expressed at its surface. After 2 days, the cells were dissociated and stained with saturated primary antibody HB1, hu8G8, anti-gB, affinity purified rabbit anti-UL131_933 or affinity purified rabbit anti-gH_977. Cells were stained with appropriate secondary antibodies in combination with allophycocyanin (APC, Jackson ImmunoResearch; West Grove, PA). The camping light of individual cells was measured using a FACSCalibur (BD Biosciences; San Jose, CA) and analyzed using FlowJo software (Tree Star; Ashland, OR). GFP-positive cells (cells expressing CMV transgenes) were selectively mapped to demonstrate antibody binding. As shown in Figure 15, HB1 reacts with cells expressing a complex of gH/gL or gH/gL alone and UL128/UL130/UL131. Hu8G8 only reacted with cells expressing gH/gL/UL128/UL130/UL131 (Fig. 15) and did not react with cells expressing gH/gL or gH/gL/gO alone (data not shown). Neither antibody reacts with cells expressing gB. Therefore, HB 1 recognizes the epitopes present on the gH/gL complex and the gH in complex I. The hu8G8 antibody binds to an antigenic determinant within the five envelope proteins forming complex I, but does not bind to gH/gL/gO or gH/gL alone. Example 6 - Evaluation of Synergism or Antagonism HB 1 and hu8G8 were tested in combination in a virus neutralization assay to determine if there was a difference in potency when the two antibodies were combined. Since antibodies have different targets and are presumed to block the entry of viruses independently, it is assumed that the effects of HB 1 and hu8G8 are additive. Therefore, the application of Bliss Independent Party 158864.doc • 108· 201215618 (combination reaction C with two single compounds of effect A and B is C=A+B-AxB). To this end, HB1 and hu8G8 were mixed at a 1:1 ratio and tested in a series of dilutions in a virus neutralization assay performed on epithelial cells and the EC50 was calculated as described above. HB1 did not augment or attenuate the efficacy of hu8G8 on epithelial cells and the 1:1 curve accurately overlaps the simulated Bliss independence curve, indicating additive rather than synergistic effects (see Figure 16 and Table 8). Also, as expected, hu8G8 did not alter the efficacy of HB1 on fibroblasts because hu8G8 did not block HCMV entry into fibroblasts (data not shown). Therefore, at a 1:1 ratio, HB 1 and hu8G8 did not show any antagonistic or synergistic effects. Table 8 mAB EC5〇bl/ml for epithelial cells alone HBl 0.063 alone hu8G8 0.0080 HBl:hu8G8, 0.0060 at 1:1 simulated Bliss independence 0.0069 To assess whether HB1 and hu8G8 show synergy over a wide range of ratios For action or antagonism, a series of "checkerboard" dilutions across a range of EC50 values are used for neutralization assays. Each antibody was diluted as indicated in Table 9 below, while the virus concentration was constant. The percentage of infected cells for epithelial cells (corrected against antibody-free controls) is shown in Table 9 below for various combinations of antibody concentrations: • tmi 158864.doc -109- 201215618 Table 9 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 hu8G8 HB1 10 μ^ηιΐ 2 pg/ml 0.4 pg/ml 0.08 μ^ιπΐ 0.016 μ_1 0.0032 μβ/ηιΐ 0.00064 0.000128 Ug/ml ua/ml 0 pg/ml HB1 10 pg/ml 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HB1 2 pg/ml 0.00 0.00 0.00 0.00 0.00 0.00 0.56 0.17 0.00 HB1 0.4 pg/ml 0.23 0.00 0.28 0.47 0.32 2.08 3.57 2.94 3.97 HB1 0.08 pg/ml 0.29 0.15 0.15 0.15 1.91 13.53 26.76 25.26 26.66 HB1 0.016 gg/ml 0.00 0.48 0.42 0.59 3.29 39.70 61.94 67.60 61.94 HB1 0.0032 pg/ml 0.37 0.71 1.06 0.29 7.23 46.22 80.96 93.01 97.34 HB1 0 pg/ml 0.64 0.33 0.14 0.63 5.50 53.93 83.51 91.95 100.00 Shadow = concentration of each antibody partially neutralized. Using the neutralization assay as described above, the EC50 of HB1 and hu8G8 was determined in the presence of another antibody at 0.8, 0.016, 0,0032 pg/ml. The results of these experiments are shown in Table 10 below and Table 11 and Figure 17. Table 10 - hu8G8 potency antibody at different HB1 concentrations and EC50 (pg/mL) concentration of hu8G8 HB1 ' 0.0037 HB1 at 08 pg/ml 0.0047 HB1 at 016 pg/ml, 0.0028 HBl at 0032 gg/ml, 0.0031 at 0 pg/ml Table 11 - HB1 potency antibody at different hu8G8 concentrations and EC5 〇hg/mL for concentration HB1 hu8G8, 0.035 hu8G8 at 0032 pg/ml, 0.043 1111808 at 6064 pg/ml, at 000128 4§/1111, 0.041 hu8G8, 0.030 at 0 pg/ml. There is no evidence of synergy or antagonism after comparing the efficacy of each antibody alone with the combination of various ratios 158864.doc -110· 201215618 . For example, after comparing the performance curve of hu8G8 with the efficacy curve of hu8G8 and 0.08 pg/ml HB1 (infected with 1% of infection in the absence of titration antibody), we found that the curves overlap (see Figure 17). Also, in the presence of various concentrations of hu8G8, each HB1 potency curve overlaps (relative to 100% infection correction) (see Figure 17). Therefore, there is no evidence of synergistic or antagonistic effects between antibodies at a wide range of ratios. Example 7 - Evaluation of virus resistance produced Although human CMV has a relatively low mutation rate (compared to hepatitis virus C (HCV) or human immunodeficiency virus (HIV)), the virus was evaluated. The ability to escape HB 1 or hu8G8 or both by generating resistance mutations. The virus is grown in the presence of suboptimal concentrations of HB1 (or MSL-109) or hu8G8, or both HB1 and hu8G8 antibodies. As the virus is subcultured to new cells every week (about 50% of each round is subcultured to new cells), the concentration of each antibody is gradually increased. A mutant virus resistant to each individual antibody was observed, but no mutant conferred resistance to the combination. All resistant virus mutations occur from a single plaque. A mutant that confers resistance to neutralization caused by HB 1 appears (see Fig. 18). However, as indicated by the neutralization assay, these mutants are still sensitive to hu8G8, which has an EC50 similar (see Figure 18 and Table 12 and Table 13). 158864.doc -111 - 201215618 Table 12 - HCMV mutants with resistance to HB1 appearing HB1 EC50~g/ml) No Ab control 0.04 HB1-mutant virus 1 0.21 HB1-mutant virus 2 No neutralizing HB1-mutant virus 3 0.07 HB1-mutant virus 4 200 HB1-mutant virus 5 no neutralizing HB1-mutant virus 6 no neutralization Table 13-HCMV HB1 resistance mutant remains sensitive to hu8G8 neutralization hu8G8 EC50 (pg/ml) Dataset 1 data Set 2 No Ab control 0.008 0.0005 HB1-mutant virus 1 0.010 0.0006 HB1-mutant virus 2 0.008 0.006 HB1-mutant virus 3 0.001 0.0001 HB1-mutant virus 4 No detectable 0.0001 HB1-mutant virus 5 No detectable 0.001 HB1-mutant virus 6 0.001 not measured
此外,出現賦予對由hu8G8所致之中和之抗性的突變 體,且此等突變體仍然對HB1敏感,HB1具有類似EC50(參 見圖19及表14及表15)。 表14-出現之對hu8G8具有抗性之HCMV突變體 hu8G8 EC5〇hg/ml) 無Ab對照 0.002 hu8G8-突變病毒1 無中和 hu8G8-突變病毒2 0.25 hu8G8-突變病毒3 無中和In addition, mutants which confer resistance to neutralization by hu8G8 appear, and these mutants are still sensitive to HB1, and HB1 has an EC50 similar (see Figure 19 and Table 14 and Table 15). Table 14 - HCMV mutants appearing resistant to hu8G8 hu8G8 EC5〇hg/ml) No Ab control 0.002 hu8G8-mutant virus 1 no neutralization hu8G8-mutant virus 2 0.25 hu8G8-mutant virus 3 no neutralization
表15-HCMV hu8G8抗性突變體仍然對HB1中和敏感 HB1 EC50_ml) 無Ab對照 0.22 hu8G8-突變病毒1 0.18 hu8G8-突變病毒2 0.07 hu8G8-突變病毒3 0.06 158864.doc -112-Table 15 - HCMV hu8G8 resistant mutants are still sensitive to HB1 neutralization HB1 EC50_ml) No Ab control 0.22 hu8G8-mutant virus 1 0.18 hu8G8-mutant virus 2 0.07 hu8G8-mutant virus 3 0.06 158864.doc -112-
S 201215618 為瞭解對HB 1及hu8G8抗體之抗性之分子性質,對來自 各抗性病毒株之gB、gH、gL、UL128、UL130及UL131定 序。相較於VR1814及D1病毒株(VR1814在無抗體壓力下在 上皮細胞上並行繼代),所有對HB 1具有抗性之病毒株在 gH中皆具有單一非保守性胺基酸突變,且在其他醣蛋白中 未發現其他突變(表1 6)。產生對HB 1具有抗性之11個個別 病毒株,涵蓋僅在3個胺基酸中之5個不同核苷酸突變。在 經定序或具有公開序列之臨床病毒株中未發現此等胺基酸 經突變。 反應於hu8G8之突變出現頻率較小,僅發現3個病毒株具 有抗性。相較於VR1814及D1病毒株,所有3個對hu8G8具 有抗性之病毒株在UL131中皆具有單一非保守性胺基酸突 變,且在其他醣蛋白中未發現其他突變(參見表16)。當突 變gH及UL1 3 1序列與60個可用臨床病毒株序列進行比較 時,該等病毒株皆不攜帶此等突變。 表16-產生抗性之突變的定位S 201215618 To understand the molecular properties of resistance to HB 1 and hu8G8 antibodies, gB, gH, gL, UL128, UL130 and UL131 from each resistant strain were sequenced. Compared to the VR1814 and D1 strains (VR1814 is subcultured on epithelial cells without antibody pressure), all strains resistant to HB1 have a single non-conservative amino acid mutation in gH, and No other mutations were found in other glycoproteins (Table 16). Eleven individual strains producing resistance to HB1 were generated, covering only five different nucleotide mutations in only three amino acids. These amino acids were not found to be mutated in clinical strains that were sequenced or had published sequences. The mutations that responded to hu8G8 appeared less frequently, and only three strains were found to be resistant. Compared to the VR1814 and D1 strains, all three strains resistant to hu8G8 had a single non-conservative amino acid mutation in UL131, and no other mutations were found in other glycoproteins (see Table 16). When the mutant gH and UL1 3 1 sequences were compared with 60 available clinical strain sequences, none of the strains carried these mutations. Table 16 - Location of mutations that produce resistance
抗性病毒株 突變蛋白質 殘基變化 ΗΒ1-突變體1 gH P171H ΗΒ1-突變體2 gH W168C ΗΒ1-突變體3 gH P171S ΗΒ1-突變體4 gH D446N ΗΒ1-突變體5 gH W168C ΗΒ1-突變體6 gH W168R hu8G8-突變體1 Ul131 Q47K hu8G8-突變體2 Ul131 K51E Hu8G8-突變體3 Ul131 D46N *-上皮細胞中所產生之突變體 **-纖維母細胞中所產生之突變體 當比較HB 1抗性病毒株相對於D1病毒株感染細胞之能力 158864.doc -113- 201215618 時,此等抗性病毒株具有深度進入缺陷(效率減小多達20 倍),表明此等病毒株之活體内生長將減弱(參見圖20)。當 比較hu8G8抗性病毒株相對於D1病毒株感染細胞之能力 時’發現抗性病毒株可以同專效率感染細胞;然而,此等 病毒株生長極其緩慢,表明在產生方面具有缺陷(資料未 展示)。需要進一步分析來瞭解此減弱之機制。 為確定此等突變是否影響HB1及hu8G8分別與gH及 UL131結合之能力,定點突變誘發用於在HEK-293T細胞之 表面上短暫表現具有抗性突變之複合物I(gH/gL/UL128/ UL130/UL131)且進行抗體結合之FACS分析。P171之突變 (突變體1及3 : P171突變成Η或S)使對HB1之抗性僅增加2 至5倍且此抗體與gH/gL之結合未顯著變化。然而,在 HB 1-突變體2、5及6中所見之突變(W168突變成C或R)完全 消除HB 1之結合能力(參見圖21)。此外,此等病毒突變體 未由HB1中和(參見圖18)。突變體4(D446N)顯示中間表 型;其對由HB1所致之中和之抗性增加500倍(參見圖18), 但仍然可偵測到與HB 1結合(參見圖21)。 為確定UL131中之突變如何影響hu8G8結合,用野生型 或突變gH/gL/UL128/UL130/UL131複合物進行對HEK-293T細胞之轉染且藉由FACS分析量測與抗gH(HBl及MSL-109)、抗UL131_993及hu8G8之結合(參見圖22)。所有3個 UL131 突變皆消除 hu8G8 對 gH/gL/UL128/UL130/UL131 複 合物之結合。 HB 1抗性突變定位在HCMV醣蛋白Η之結構模型(基於 158864.doc -114- 201215618 HSV-2 gH及EBV gH之新近解析之結構)上(Backovic等人, 户见45,197:2263 5-22640 (2010))。所有突變殘基皆定位於 gH之同一面(資料未展示)。HB1 Fab模型化於gH結構上。 HB 1 Fab之佔據面積涵蓋所有突變,表明HB 1與由此等突 變界定之抗原決定基結合。類似地,hu8G8抗性突變彼此 緊密鄰近(相隔4個殘基),且儘管UL 13 1之結構未知,但兩 者均定位於推定α螺旋域且預測位於螺旋之同一面上。因 此,連同結合分析,抗性突變闡明gH/gL/UL128/ UL13 0/UL131複合物上針對HB1與hu8G8兩者之抗原決定 基。 實例8-親和力分析 HB 1與hu8G8兩者之親和力均藉由如下所述之biacore及 史卡查(Scatchard)分析分別測定。 HB1對桿狀病毒表現之可溶性gH/gL之親和力藉由 biacore分析測定且得知為1 nM。詳言之,藉由使用 BIAcore 3000儀器(GE Healthcare; Piscataway, NJ)進行表 面電漿子共振(SPR)量測(Karlsson等人,1991)來評估HB1結 合桿狀病毒表現之分泌gH/gL之能力。基於SPR之生物感 測器報導接近表面之折射率變化。當蛋白質目標(「配位 體」)共價固定在感測器晶片表面上時,SPR可用於監測在 表面上注射之結合搭配物(「分析物」)之非共價相互作 用;分析物結合之即時量測結果可用於確定相互作用之動 力學及親和力。 在分析物B與經固定配位體A結合之1:1相互作用之情況 158864.doc 115. 201215618 下,使用方程式1描述平衡: L· Λ _ ^οη Α Λ A + B^-r~>ΑΒ ^off 在方程式1中,kQn為締合速率常數,kQff為解離速率常 數,且平衡解離常數KD由KD^koff/kcn確定。使用方程式2 確定1:1結合之複合物形成的速率: ^-]=kon[Amkoff[AB] 當以SPR信號(/?)表示時,方程式2可寫成方程式3 :Resistant virus strain mutant protein residue change ΗΒ1-mutant 1 gH P171H ΗΒ1-mutant 2 gH W168C ΗΒ1-mutant 3 gH P171S ΗΒ1-mutant 4 gH D446N ΗΒ1-mutant 5 gH W168C ΗΒ1-mutant 6 gH W168R hu8G8-mutant 1 Ul131 Q47K hu8G8-mutant 2 Ul131 K51E Hu8G8-mutant 3 Ul131 D46N *- Mutant produced in epithelial cells** Mutants produced in fibroblasts when comparing HB 1 resistance The ability of the virus strain to infect cells with the D1 strain 158864.doc -113- 201215618, these resistant strains have deep entry defects (up to 20-fold reduction in efficiency), indicating that in vivo growth of these strains will Weaken (see Figure 20). When comparing the ability of hu8G8 resistant strains to cells infected with D1 strains, 'the resistant strains were found to be able to infect cells with specific efficiency; however, these strains grew extremely slowly, indicating a defect in production (data not shown) ). Further analysis is needed to understand the mechanism of this weakening. To determine whether these mutations affect the ability of HB1 and hu8G8 to bind to gH and UL131, respectively, site-directed mutagenesis induces complex I (gH/gL/UL128/UL130) for transient expression of resistant mutations on the surface of HEK-293T cells. /UL131) and FACS analysis of antibody binding was performed. Mutation of P171 (mutants 1 and 3: P171 mutated to Η or S) increased the resistance to HB1 by only 2 to 5 fold and the binding of this antibody to gH/gL did not change significantly. However, the mutations seen in HB 1-mutants 2, 5 and 6 (mutation of W168 to C or R) completely abolished the binding ability of HB 1 (see Figure 21). Furthermore, these viral mutants were not neutralized by HB1 (see Figure 18). Mutant 4 (D446N) showed an intermediate phenotype; it increased the resistance to neutralization by HB1 by a factor of 500 (see Figure 18), but binding to HB 1 was still detectable (see Figure 21). To determine how mutations in UL131 affect hu8G8 binding, transfection of HEK-293T cells with wild-type or mutant gH/gL/UL128/UL130/UL131 complexes and anti-gH (HBl and MSL) by FACS analysis -109), a combination of anti-UL131_993 and hu8G8 (see Figure 22). All three UL131 mutations eliminated the binding of hu8G8 to the gH/gL/UL128/UL130/UL131 complex. The HB 1 resistance mutation is localized to the structural model of HCMV glycoprotein (based on the recently analyzed structure of 158864.doc -114-201215618 HSV-2 gH and EBV gH) (Backovic et al., see 45, 197: 2263 5 -22640 (2010)). All mutant residues were mapped to the same side of gH (data not shown). The HB1 Fab is modeled on the gH structure. The occupied area of the HB 1 Fab covers all mutations, indicating that HB 1 binds to the epitope defined by such mutations. Similarly, the hu8G8 resistance mutations are in close proximity to each other (4 residues apart), and although the structure of UL 13 1 is unknown, both are localized to the putative alpha helix domain and predicted to be on the same side of the helix. Thus, along with the binding assay, the resistance mutations elucidated the epitopes on both the gH/gL/UL128/UL13 0/UL131 complex against both HB1 and hu8G8. Example 8 - Affinity analysis The affinity of both HB 1 and hu8G8 was determined by biacore and Scatchard analysis as described below. The affinity of HB1 for soluble gH/gL exhibited by baculovirus was determined by biacore analysis and was known to be 1 nM. In particular, surface plasmon resonance (SPR) measurements (Karlsson et al., 1991) were performed using a BIAcore 3000 instrument (GE Healthcare; Piscataway, NJ) to assess the secretion of gH/gL from HB1 binding baculovirus expression. ability. The SPR based biosensor reports a change in refractive index close to the surface. When a protein target ("ligand") is covalently immobilized on the surface of a sensor wafer, SPR can be used to monitor non-covalent interactions of binding partners ("analytes") injected on the surface; analyte binding The instantaneous measurement results can be used to determine the dynamics and affinity of the interaction. In the case of the 1:1 interaction of analyte B with immobilized ligand A 158864.doc 115. 201215618, the equilibrium is described using Equation 1: L· Λ _ ^οη Α Λ A + B^-r~> ;ΑΒ ^off In Equation 1, kQn is the association rate constant, kQff is the dissociation rate constant, and the equilibrium dissociation constant KD is determined by KD^koff/kcn. Equation 2 is used to determine the rate of formation of a 1:1 bound complex: ^-]=kon[Amkoff[AB] When expressed as an SPR signal (/?), Equation 2 can be written as Equation 3:
rjDrjD
~^ = KonCHC + koff)R 在方程式3中,C為游離分析物之濃度且Rmax為表面之最 大分析物結合容量。類似地,對於能夠在溶液中二聚化以 形成2個結合位點之分析物B,平衡可由方程式4描述。 A + B< konVkoffi >AB + B < kon2lkoff2 > AB2 藉由量測結合速率之濃度依賴性及在不存在游離分析物 下之解離速率,可確定動力學常數並用於計算KD。 使用針對非共價固定之HB 1之抗Fc捕捉方法,隨後注射 不同濃度之gH/gL來進行SPR量測以測定結合之動力學。 遵循由製造商提供之說明書對接CM5生物感測器晶片(BR 1000 14,CM5研究等級;BIAcore,Inc.),用操作緩衝液 (10 mM HEPES(pH 7.4)、150 mM NaCl及 0.01%聚山梨醇酯 20)灌注且用70%甘油校正。如下文簡要概述,小鼠單株抗 人類Fc抗體(人類抗體捕捉套組,BR-1008-39,BIAcore, 158864.doc •116- 201215618~^ = KonCHC + koff)R In Equation 3, C is the concentration of the free analyte and Rmax is the maximum analyte binding capacity of the surface. Similarly, for analyte B, which is capable of dimerization in solution to form two binding sites, the equilibrium can be described by Equation 4. A + B< konVkoffi > AB + B < kon2lkoff2 > AB2 The kinetic constant can be determined and used to calculate KD by measuring the concentration dependence of the binding rate and the dissociation rate in the absence of free analyte. The kinetics of binding were determined using an anti-Fc capture method for non-covalently immobilized HB1 followed by injection of different concentrations of gH/gL for SPR measurements. Docking CM5 biosensor wafers (BR 1000 14, CM5 Research Grade; BIAcore, Inc.) following instructions provided by the manufacturer, using an operating buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.01% Polysorbate The alcohol ester 20) was perfused and corrected with 70% glycerol. As briefly summarized below, mouse monoclonal anti-human Fc antibody (human antibody capture kit, BR-1008-39, BIAcore, 158864.doc • 116- 201215618)
Inc.)固定在CM5晶片之全部4個流量槽上。遵循由製造商 描述之方案使用N_乙基屮,_(3_二甲基胺基丙基)碳化二亞 胺鹽酸鹽及N-羥基丁二醯亞胺(NHS)(胺偶合套組,811-^00-50 ; BIAcore,Inc.)且使用7分鐘活化時間來活化流量 槽。接著藉由在10 pL/min之流速下注射60 μί於10 mM乙 酸鈉(pH 5.0)中稀釋之25 pg/mL抗體使活化基質經由胺偶 δ /、捕捉抗體反應。在偶合注射結束時,藉由在5 pL/miη 之流速下注射35 μΙ 1 Μ乙醇胺鹽酸鹽使任何剩餘未反應 之NHS基團不活化。由在偶合程序之前及之後的SPr信號 估計以此方式共價固定之捕捉抗體之量且給出在4個流量 槽上8000-9500 RU之範圍。 小於約100(任意SPR或共振單位(RU))之Rmax值通常視為 提供良好信雜比而不限制可測定之動力學常數之範圍。初 步實驗指示在30 pL/min之流速下,〇·ΐ3 pg/mi HB1之60 μί注射液導致捕捉足夠HB 1以便在用gH/gL飽和後觀測到 約50 RU之信號。因此,此HB 1濃度及注射方案用於測定 動力學常數。Inc.) is fixed on all four flow channels of the CM5 chip. Use N_ethyl hydrazine, _(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxybutylimine (NHS) (amine coupling kit) following the protocol described by the manufacturer , 811-^00-50; BIAcore, Inc.) and using a 7 minute activation time to activate the flow cell. The activated matrix was then reacted via amine δ /, capture antibody by injecting 60 μg of 25 pg/mL antibody diluted in 10 mM sodium acetate (pH 5.0) at a flow rate of 10 pL/min. At the end of the coupling injection, any remaining unreacted NHS groups were not activated by injection of 35 μΙ 1 Μ ethanolamine hydrochloride at a flow rate of 5 pL/miη. The amount of capture antibody covalently immobilized in this manner was estimated from the SPr signal before and after the coupling procedure and given a range of 8000-9500 RU on 4 flow channels. An Rmax value of less than about 100 (arbitrary SPR or resonance unit (RU)) is generally considered to provide a good signal to noise ratio without limiting the range of measurable kinetic constants. Initial experiments indicated that at a flow rate of 30 pL/min, a 60 μL injection of 〇·ΐ3 pg/mi HB1 resulted in the capture of sufficient HB 1 to observe a signal of approximately 50 RU after saturation with gH/gL. Therefore, this HB 1 concentration and injection protocol was used to determine the kinetic constant.
如上所述藉由在流量槽2上捕捉HB 1來進行結合量測, 其中流量槽1用作參照。於操作緩衝液中製備濃度以2倍增 量自0.39 nM變化至100 nM之gH/gL溶液。收集在3〇 pL/min之流速下在感測器晶片表面上注射6〇 此等溶液 之感測器圖譜。感測晶片維持在2 5 °C下且在注射结束之 後監測解離10分鐘。在結合循環之間經由注射30 3 MThe bonding measurement is performed by capturing HB 1 on the flow cell 2 as described above, wherein the flow cell 1 is used as a reference. A concentration of gH/gL solution varying from 0.39 nM to 100 nM was prepared in the operating buffer in 2 fold increments. A sensor map of 6 Å of these solutions was injected on the surface of the sensor wafer at a flow rate of 3 〇 pL/min. The sensing wafer was maintained at 25 ° C and the dissociation was monitored for 10 minutes after the end of the injection. 30 3 M via injection between the binding cycles
MgC12來再生感測器晶片表面。此注射致使任何剩餘 158864.doc -117- 4^ 201215618 HBLgH/gL複合物自捕捉抗體解離。接著在如上之流量⑴ 上捕捉HB1以進灯下—結合循環。類似地收集在感測器晶 片上注射操作緩衝液之「空白」感測器圖譜。 藉由首先減去所量測之參照槽之信號來加工觀測之感測 器圖譜以進行動力學分析。移除由曲線之再生部分獲得之 # #υ °接著藉由減去分析物注射前基線之平均ru值將感 測器圖譜調零。最後’自所獲得之注射含有讲砂之溶液 的曲線減去所量測之僅注射操作緩衝液的感測器圖譜。使 用由製造商供應之軟體,根據1:1朗繆爾(Langmuir)結合模 型或一彳貝分析物模型分析資料。 Ί SPRk唬隨gH/gL濃度增加,指示Fe捕捉之HB丨勝任抗 原結合。根據i:i朗繆爾結合模型對資料之分析指示表17 中所示之0.15 nM之表觀平衡解離常數(Kd)及各種動力學 承數’然而’計舁曲線不良好匹配觀測之感測器圖譜,具 有2.1之相對較大χ2值。觀測之感測器圖譜藉由二價分析物 KD模型較佳描述(χ2 = 0.4),該模型產生表18中所示之1 〇 nM之表觀KD及各種動力學常數。 表17-使用1:1朗繆爾結合模型計算之動力學常數 K^s·1) R/7Jitc(RU) Κ〇(ηΜ) 6.8X103 1.02X10·4 42 "oil — ~2Λ 表18-使用二價分析物KD模型計算之動力學常數 IWM-V1) Ko/yis'1) URU-Y1) Rmo^iRU) Kp(nlVi) y1 2.5χ105 2.5 χΚΓ4 0.174 0.166 65 1.0 0.4 因為biacore不可用於測定hu8G8之親和力,所以史卡省 分析用作一種替代性方法。在此方法中,硬化抗體與一系 158864.doc -118· 201215618 列未標記抗體稀釋液混合且檢定競爭。使用Munson及 Rodbard之擬合演算法將結果繪圖以確定抗體之親和力(圖 23) ° HB1之平均Kd為1.27 nM且hu8G8之平均Kd為2.03 nM(表17)。亦藉由史卡查分析測定腺病毒細胞表面表現之 gH/gL/UL128/UL130/UL131 複合物對 HB1 與 hu8G8 兩者的 親和力量測結果且得知分別為1.27 nM及2.03 nM。 特定而言,使用埃原(Iodogen)方法(Thermo-Fisher Scientific; Waltham, ΜΑ)使 HB1 及 hu8G8蛾化。藉由使用 NAP-5管柱進行凝膠過濾,自游離125I-Na純化經放射性標 記之抗體。經純化hu8G8抗體之比活性為12.30 pCi/pg且經 純化HB1抗體之比活性為14.66 pCi/pg。50 μΐ含有固定濃 度之碘化抗體及遞減濃度之未標記抗體的競爭反應混合物MgC12 is used to regenerate the surface of the sensor wafer. This injection caused any remaining 158864.doc -117- 4^201215618 HBLgH/gL complex to dissociate from the capture antibody. Then capture HB1 on the flow rate (1) above to enter the lamp-binding cycle. A "blank" sensor map of the injection buffer on the sensor wafer was similarly collected. The observed sensor map is processed for kinetic analysis by first subtracting the signal of the measured reference groove. The ##υ° obtained by the regenerated portion of the curve is removed and the sensor map is then zeroed by subtracting the average ru value of the baseline before the analyte injection. Finally, the sensor map of the injected injection buffer was subtracted from the obtained curve containing the sand solution. The software supplied by the manufacturer was used to analyze the data according to a 1:1 Langmuir binding model or a mussel analyte model. Ί SPRk唬 increases with gH/gL concentration, indicating that HB captured by Fe is competent for antigen binding. The analysis of the data according to the i:i Langmuir binding model indicates that the apparent equilibrium dissociation constant (Kd) of 0.15 nM and the various kinetic inheritances shown in Table 17 are 'but' the curve is not well matched. The map has a relatively large χ2 value of 2.1. The observed sensor map is better described by the bivalent analyte KD model (χ2 = 0.4), which produces the apparent KD of 1 〇 nM and various kinetic constants shown in Table 18. Table 17 - Kinetic constants calculated using the 1:1 Langmuir binding model K^s·1) R/7Jitc(RU) Κ〇(ηΜ) 6.8X103 1.02X10·4 42 "oil — ~2Λ Table 18- Kinetic constant IWM-V1 calculated using the bivalent analyte KD model) Ko/yis'1) URU-Y1) Rmo^iRU) Kp(nlVi) y1 2.5χ105 2.5 χΚΓ4 0.174 0.166 65 1.0 0.4 Since biacore is not available for determination The affinity of hu8G8, so Ska's analysis is used as an alternative method. In this method, the sclerosting antibody is mixed with a line of 158864.doc-118·201215618 column unlabeled antibody dilutions and assayed for competition. The results were plotted using the fit algorithm of Munson and Rodbard to determine the affinity of the antibody (Figure 23). The mean Kd of HB1 was 1.27 nM and the average Kd of hu8G8 was 2.03 nM (Table 17). Affinity measurements of both gH/gL/UL128/UL130/UL131 complexes on both surface of adenoviral cells and HB1 and hu8G8 were also determined by Skacha analysis and found to be 1.27 nM and 2.03 nM, respectively. Specifically, HB1 and hu8G8 were mothified using the Iodogen method (Thermo-Fisher Scientific; Waltham, ΜΑ). The radiolabeled antibody was purified from free 125I-Na by gel filtration using a NAP-5 column. The specific activity of the purified hu8G8 antibody was 12.30 pCi/pg and the specific activity of the purified HB1 antibody was 14.66 pCi/pg. 50 μΐ competitive reaction mixture containing a fixed concentration of iodinated antibody and a decreasing concentration of unlabeled antibody
I 置放入96孔板中。使用Sigma Accutase®溶液(Sigma-Aldrich; St. Louis,MO)使表現蛋白質複合物gH/gL/ 128/130/13 1之用腺病毒短暫轉染之ARPE-19細胞自燒瓶脫 離,用多聚甲醛固定且用結合緩衝液(含有2% FBS、50 爪]\4 11£?£8(?^[7.2)及0.1%疊氮化鈉之〇]^£1^)洗滌。將洗 滌細胞以0.2 mL結合緩衝液25,000個細胞之密度添加至含 有一式三份之50 pL競爭反應混合物的96孔板中。含有細 胞之各競爭反應物中之碘化抗體的最終濃度為1〇〇 pM且含 有細胞之競爭反應物中之未標記抗體的最終濃度不同,起 始於500 nM且接著藉由1:2倍稀釋成10個濃度遞減,且包 括零添加僅有緩衝液之樣品。在室溫下培育含有細胞之競 爭反應物2小時,接著轉移至Millipore Multiscreen過濾板 158864.doc -119- 201215618 中且用結合緩衝液洗滌4次以使游離碘化抗體與結合碘化 抗體分離。在 Wallac Wizard 1470 γ計數器(PerkinElmer Life and Analytical Sciences; Wellesley,ΜΑ)上對過遽器計 數。使用採用 Munson及 Rodbard(y4«fl/· Biochem., 7:22-39 (1980))之擬合演算法之New Ligand軟體(Genentech)評估結 合資料以確定抗體之結合親和力。 表19 HCMV抗艎 檢定 K〇(nM) 平均 KD(nM)± SDa HB1 1 1.3 1.27 ±0.06 2 1.2 3 1.3 1 1.96 Hu8G8 2 2.06 2.03 ± 0.06 3 2.07 KD=平衡解離常數;SD=標準偏差I placed in a 96-well plate. ARPE-19 cells transiently transfected with adenovirus expressing protein complex gH/gL/128/130/13 1 were detached from the flask using Sigma Accutase® solution (Sigma-Aldrich; St. Louis, MO) for multimerization Formaldehyde was fixed and washed with binding buffer (containing 2% FBS, 50 paws) \4 11 ££8 (?^[7.2) and 0.1% sodium azide]. The washed cells were added to a 96-well plate containing triplicate 50 pL of the competition reaction mixture at a density of 25,000 cells in 0.2 mL of binding buffer. The final concentration of the iodinated antibody in each of the competing reactions containing the cells is 1 〇〇 pM and the final concentration of the unlabeled antibody in the competing reaction containing the cells is different, starting at 500 nM and then by 1:2 Diluted to 10 concentrations, and included zero-added buffer-only samples. The cell-containing competitive reaction was incubated for 2 hours at room temperature, then transferred to Millipore Multiscreen filter plate 158864.doc -119-201215618 and washed 4 times with binding buffer to separate the free iodinated antibody from the bound iodinated antibody. The filter was counted on a Wallac Wizard 1470 gamma counter (PerkinElmer Life and Analytical Sciences; Wellesley, ΜΑ). Binding data were evaluated using New Ligand software (Genentech) using a fitting algorithm of Munson and Rodbard (y4 «fl/· Biochem., 7:22-39 (1980)) to determine the binding affinity of the antibodies. Table 19 HCMV anti-caries test K〇(nM) mean KD(nM)± SDa HB1 1 1.3 1.27 ±0.06 2 1.2 3 1.3 1 1.96 Hu8G8 2 2.06 2.03 ± 0.06 3 2.07 KD=equilibrium dissociation constant; SD=standard deviation
實例9-分析hu8G8與複合物I之結合Example 9 - Analysis of the combination of hu8G8 and complex I
為進一步表徵hu8G8與複合物I之結合,進行ELISA檢定 以測試hu8G8是否可結合UL13 1之含有如實例7中鑑別之抗 性突變的部分。特定言之,自位置41處之絲胺酸密碼子至 位置68處之絲胺酸密碼子(SRALPDQTRY KYVEQLVDLT LNYHYDAS(SEQ ID NO:194))擴增編碼 UL131 之 DNA 且選 殖入具有N端His6、GST及TEV裂解位點(DNA654570)之限 制非依賴性選殖(RIC)載體中。UL131之此部分形成蛋白質 之二級結構中之推定α螺旋。亦選殖具有突變Q47K之 UL131,該突變消除hu8G8與在複合物I(gH/gL/UL128/ UL130/UL131)之情形下之UL131的結合。使序列經驗證之 構築體在大腸桿菌菌株R〇setta2(DE3)中生長。使菌元培養 158864.doc -120 - S」 201215618 物在30°C下在含有50 pg/ml卡本西林(carbenicillin)之LB培 養基中生長隔夜。在〇D600 0.7下,蛋白質在1-L培養物中 之表現用0·3 mM IPTG在16°C下誘導隔夜。收集細胞且即 刻藉由於含有無EDTA蛋白酶抑制劑鍵劑(Roche)之100 mM Tris(pH 8·0)、500 mM NaCl、5%甘油(緩衝液A)中進行音To further characterize the binding of hu8G8 to complex I, an ELISA assay was performed to test whether hu8G8 binds to the portion of UL13 1 containing the resistant mutation identified in Example 7. Specifically, the serine acid codon at position 41 to the serine codon at position 68 (SRALPDQTRY KYVEQLVDLT LNYHYDAS (SEQ ID NO: 194)) amplifies the DNA encoding UL131 and is ligated into the N-terminal His6 , GST and TEV cleavage sites (DNA654570) in a restricted independent colonization (RIC) vector. This portion of UL131 forms the putative alpha helix in the secondary structure of the protein. UL131 with mutation Q47K was also selected, which abolished the binding of hu8G8 to UL131 in the case of complex I (gH/gL/UL128/UL130/UL131). The sequence verified construct was grown in E. coli strain R〇setta2 (DE3). The culture of the bacterium was carried out overnight at 30 ° C in an LB medium containing 50 pg/ml of carbenicillin. At 〇D600 0.7, protein expression in 1-L cultures was induced overnight at 16 °C with 0.3 mM IPTG. The cells were collected and immediately transcribed by 100 mM Tris (pH 8.0), 500 mM NaCl, 5% glycerol (buffer A) containing no EDTA protease inhibitor bond (Roche).
波處理及細胞破壞來溶解。在1 〇〇〇〇 rpm下短暫離心經溶 解細胞40分鐘且澄清溶解產物裝載於重力流Ni螯合親和力 管柱(Qiagen)上。管柱用1〇管柱體積緩衝液a及10管柱體 積含有50 mM咪唑之緩衝液A洗滌。蛋白質用1〇〇 mMWave treatment and cell destruction to dissolve. The solubilized cells were briefly centrifuged at 1 rpm for 40 minutes and the clarified lysate was loaded on a gravity flow Ni chelate affinity column (Qiagen). The column was washed with 1 column volume buffer a and 10 column volumes of buffer A containing 50 mM imidazole. Protein with 1 〇〇 mM
Tris(pH 8.0)、500 mM NaCl、5%甘油、500 mMp米》坐溶離 且即刻透析入50 mM Tris(pH 8.0)、200 mM NaCl及 5%甘 油中。用 25 mM Tris(pH 8.0)、200 mM NaCl及 5%甘油在 尺寸排阻層析管柱(S200 10/30,GE)上進一步純化蛋白 質。 為測定hu8G8與UL131蛋白質片段之結合,Maxsorb £[18八板在4°(:下用每孔108、2〇〇11经或4〇11§於碳酸鹽塗佈 緩衝液中之蛋白質塗佈隔夜。在用洗滌緩衝液(含有〇 〇5% 吐溫20 [Sigma Chemical]之PBS)洗滌3次之後,各孔用檢 定稀釋劑(含有 0.5% BSA [Invitrogen; Carlsbad, CA]之洗蘇 緩衝液)阻斷1小時。hu8G8在10 pg/ml或1 pg/mi下於檢定 稀釋劑中培育1小時。在3次洗務之後,各孔與過氧化酶結 合之抗人類抗體(Jackson Immunolabs,Bar Harbor, ME)在 1:5000下一起培育1小時,或與辣根過氧化酶結合之抗 5HIS(Qiagen)在1:500或1:5000下一起培育1小時。實驗結 158864.doc -121 - 201215618 果展示於圖24中且包括經200 ng蛋白質塗佈且與10 pg/ml hu8G8—起培育之ELISA板的資料。 儘管已出於清楚理解之目的藉由說明及舉例之方式相當 詳細地描述了前述發明,但該等描述及實例不應解釋為限 制本發明範疇。本文中引用之所有專利及科學文獻之揭示 内容明確地以全文引用的方式併入本文中。 【圖式簡單說明】 圖1展示鼠類mAb 8G8之重鏈可變區(VH)(SEQ ID NO:50)與所選人類重鏈可變區:VH1 FW(SEQ ID NO:52)、人類 VH3 FW(SEQ ID NO:53)及人類 VH7 FW(SEQ ID NO:54)的胺基酸序列比對。胺基酸係根據Kabat編號來 編號。對高變區(HVR)加框。圓圈指示VL-VH相互作用 (Padl an (1994) Mo/, /mm w π 〇/. 31:169);雙重星號(一個在 另一個之上)指示維尼爾(Vernier)位置(Foote及Winter (1992) J. Mol. Biol. 224:487)及 FW-CDR 相互作用(Padlan (1994) Mo/, 31:169)。在位置 47、64、66、68 處 之單一星號指示維尼爾位置(Foote&Winter(1992)J.Mol· Biol. 224:487);在位置58處之單一星號指示FW-CDR相互 作用(Padlan (1994) Mo/· 厂 31:169) 〇 圖2展示鼠類mAb 8G8之輕鏈可變區(VL)(SEQ ID NO:51)與人類輕鏈可變區:λ3 FW區(SEQ ID NO:69)及人 類λ4 FW區(SEQ ID NO:55)的胺基酸序列比對。胺基酸 係根據Kabat編號來編號。對高變區(HVR)加框。圓圈指 示 VL-VH相互作用(Padlan (1994) Mo/. 31:169); 158864.doc -122- 201215618 雙重星號(一個在另一個之上)指示維尼爾位置(!"〇0化及 Winter (1992) J. Mol. Biol. 224:487)及 FW-CDR 相互作用 (Padlan (1994) Mo/, /mmwwo/. 31:169)。在位置47、64、 66、68處之單一星號指示維尼爾位置(Foote及Winter (1992) J. Mol. Biol. 224:487);在位置 58 處之單一星號指 示 FW-CDR 相互作用(Padlan (1994) Mo/, /wmwwo/. 31:169) ° 圖3展示比較8G8k3變異體與8G8X4變異體之中和檢定的 結果。圖A :在中和檢定中,相較於小鼠/人類嵌合8G8抗 體(QE7/C2),使用具有人類VH1、VH3或VH7之人類化 8G8X3抗體。圖B :在中和檢定中,相較於小鼠/人類嵌合 8G8抗體(QE7/C2),使用具有人類VH1、VH3或VH7之人類 化8G8X4抗體。實驗之EC50值呈現於各別實驗下方。 圖4展示8G8 HVR-L2中之突變序列。所示為HVR-L2及 FR3之第一胺基酸之胺基酸序列(WT : SEQ ID NO:57 ; A1 : SEQ ID NO:58 ; El : SEQ ID NO:59 ; ΤΙ : SEQ ID NO:60 ; A2 : SEQ ID NO:61 ; E2 : SEQ ID NO:62 ; T2 : SEQ ID NO:63 ; SG : SEQ ID NO:64 ; SGSG : SEQ ID NO:65 ; TGDA : SEQ ID NO:66)。圖中之編號係基於 Kabat 編號。 圖5展示使用具有圖4所示之含有單一胺基酸取代之突變 HVR-L2區的各種人類化8G8抗體進行之中和檢定的結果。 圖A :中和檢定。HVR-L2突變抗體皆含有人類8G8 VH1 鏈。圖B :實驗之EC50值。 I58864.doc .123- 201215618 圖6展示使用具有圖4所示之含有2個胺基酸取代之突變 HVR-L2區的各種人類化8G8抗體進行之中和檢定的結果。 圖A :中和檢定。HVR-L2突變抗體皆含有人類8G8 VH1 鏈。圖B :實驗之EC50值。 圖7展示鼠類mAb 8G8之輕鏈可變區(SEQ ID NO:51)與 人類輕鏈可變區λ4 FW(SEQ ID NO:55)及λ4 FW上8G8之人 類化輕鏈可變區(hu8G8A4 FW)(SEQ ID ΝΟ:48)的胺基酸 序列比對。胺基酸係根據Kabat編號來編號。對高變區 (HVR)力口框。圓圈指示VL-VH相互作用(Padlan (1994) Mo/, /w mw«o/· 31:169);雙重星號(一個在另一個之上)指不維尼 爾位置(Foote及 Winter (1992) J. Mol. Biol. 224:487)及 FW-CDR相互作用(Padlan (1994) Mo/. 3 1:169)。在位 置47、64、66、68處之單一星號指示維尼爾位置(Foote及 Winter (1992) J. Mol. Biol. 224:487);在位置 58 處之單一 星號指示 FW-CDR相互作用(Padlan (1994) Mo/, 31:169)。 圖8展示鼠類mAb 8G8之重鏈可變區(SEQ ID NO:50)與 人類重鏈可變區VH1構架(VH1 FW)(SEQ ID N〇:52)及VH1 FW上8G8之人類化重鏈可變區(hu8G8.VHl)(SEQ ID NO:45)的胺基酸序列比對。胺基酸係根據Kabat編號來編 號。對高變區(HVR)加框。圓圈指示VL-VH相互作用 (Padlan (1994) Mo/· /wwmwo/. 31:169);雙重星號(一個在 另一個之上)指示維尼爾位置(Foote及Winter (1992) J. Mol· Biol. 224:487)及 FW-CDR相互作用(Padlan (1994) Mo/. 158864.doc -124· 201215618 /mmwnο/. 31:169)。在位置47、64、66、68處之單一星號 指示維尼爾位置(Foote& winter (!992) J. Mol. Biol. 224:487);在位置58處之單一星號指示FW-CDR相互作用 (Padlan (1994) Mo/. 31:169)。亦展示編碼 hu8G8.VHl之例示性核酸序列(SEQ ID NO:185)。Tris (pH 8.0), 500 mM NaCl, 5% glycerol, 500 mM pm were dissolved and immediately dialyzed into 50 mM Tris (pH 8.0), 200 mM NaCl, and 5% glycerin. The protein was further purified on a size exclusion chromatography column (S200 10/30, GE) with 25 mM Tris (pH 8.0), 200 mM NaCl and 5% glycerol. To determine the binding of hu8G8 to the UL131 protein fragment, Maxsorb £ [18 plates were coated overnight at 4° (with 108, 2〇〇11 per well or 4〇11§ in carbonate coating buffer). After washing 3 times with wash buffer (PBS containing 5% 5% Tween 20 [Sigma Chemical]), each well was assayed with a diluent (containing 0.5% BSA [Invitrogen; Carlsbad, CA]). Blocked for 1 hour. hu8G8 was incubated in assay diluent for 1 hour at 10 pg/ml or 1 pg/mi. After 3 washes, each well was bound to peroxidase-conjugated anti-human antibody (Jackson Immunolabs, Bar Harbor, ME) was incubated for 1 hour at 1:5000, or with horseradish peroxidase-conjugated anti-5HIS (Qiagen) for 1 hour at 1:500 or 1:5000. Experimental knot 158864.doc -121 - 201215618 The results are shown in Figure 24 and include data for ELISA plates coated with 200 ng of protein and incubated with 10 pg/ml hu8G8. Although described in considerable detail for purposes of clarity and description, by way of illustration and example The foregoing invention is not to be construed as limiting the scope of the invention. The disclosures of all of the patents and scientific literature cited are expressly incorporated herein by reference in their entirety in their entirety herein in the the the the the the the the the the the the the the the the the the the the Alignment with the amino acid sequence of the selected human heavy chain variable region: VH1 FW (SEQ ID NO: 52), human VH3 FW (SEQ ID NO: 53), and human VH7 FW (SEQ ID NO: 54). Amino acids are numbered according to Kabat numbering. Boxing of hypervariable regions (HVR). Circles indicate VL-VH interactions (Padl an (1994) Mo/, /mm w π 〇/. 31:169); double asterisks (one above the other) indicates the position of the Vernier (Foote and Winter (1992) J. Mol. Biol. 224:487) and the FW-CDR interaction (Padlan (1994) Mo/, 31:169) A single asterisk at positions 47, 64, 66, 68 indicates the Venier position (Foote & Winter (1992) J. Mol. Biol. 224: 487); a single asterisk at position 58 indicates FW-CDR interaction ( Padlan (1994) Mo/· Plant 31: 169) Figure 2 shows the light chain variable region (VL) of murine mAb 8G8 (SEQ ID NO: 51) and human light chain variable region: λ3 FW region (SEQ ID NO: 69) and human λ4 FW region (SEQ I The amino acid sequence of D NO: 55) is aligned. Amino acids are numbered according to the Kabat number. Frame the hypervariable region (HVR). Circles indicate VL-VH interactions (Padlan (1994) Mo/. 31:169); 158864.doc -122- 201215618 Double asterisks (one above the other) indicate the Venier position (!"〇0化和Winter (1992) J. Mol. Biol. 224:487) and FW-CDR interaction (Padlan (1994) Mo/, /mmwwo/. 31:169). A single asterisk at positions 47, 64, 66, 68 indicates the Venier position (Foote and Winter (1992) J. Mol. Biol. 224:487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan) (1994) Mo/, /wmwwo/. 31:169) ° Figure 3 shows the results of the neutralization assay comparing the 8G8k3 variant with the 8G8X4 variant. Panel A: In the neutralization assay, a humanized 8G8X3 antibody with human VH1, VH3 or VH7 was used compared to the mouse/human chimeric 8G8 antibody (QE7/C2). Panel B: In the neutralization assay, a humanized 8G8X4 antibody with human VH1, VH3 or VH7 was used as compared to the mouse/human chimeric 8G8 antibody (QE7/C2). The EC50 values of the experiments are presented below the respective experiments. Figure 4 shows the mutated sequence in 8G8 HVR-L2. The amino acid sequence of the first amino acid of HVR-L2 and FR3 is shown (WT: SEQ ID NO: 57; A1: SEQ ID NO: 58; El: SEQ ID NO: 59; ΤΙ: SEQ ID NO: 60; A2: SEQ ID NO: 61; E2: SEQ ID NO: 62; T2: SEQ ID NO: 63; SG: SEQ ID NO: 64; SGSG: SEQ ID NO: 65; TGDA: SEQ ID NO: 66) . The numbers in the figure are based on the Kabat number. Figure 5 shows the results of a neutralization assay using various humanized 8G8 antibodies having a mutant HVR-L2 region containing a single amino acid substitution as shown in Figure 4. Figure A: Neutralization check. The HVR-L2 mutant antibodies all contain the human 8G8 VH1 chain. Panel B: EC50 values for the experiment. I58864.doc.123-201215618 Figure 6 shows the results of a neutralization assay using various humanized 8G8 antibodies having the mutant HVR-L2 region containing the two amino acid substitutions shown in Figure 4. Figure A: Neutralization check. The HVR-L2 mutant antibodies all contain the human 8G8 VH1 chain. Panel B: EC50 values for the experiment. Figure 7 shows the light chain variable region of murine mAb 8G8 (SEQ ID NO: 51) and the human light chain variable region λ4 FW (SEQ ID NO: 55) and the humanized light chain variable region of 8G8 on λ4 FW ( The amino acid sequence alignment of hu8G8A4 FW) (SEQ ID ΝΟ: 48). Amino acids are numbered according to the Kabat number. For the hypervariable zone (HVR) force frame. Circles indicate VL-VH interactions (Padlan (1994) Mo/, /w mw«o/· 31:169); double asterisks (one above the other) refer to non-Venil positions (Foote and Winter (1992) J Mol. Biol. 224:487) and FW-CDR interaction (Padlan (1994) Mo/. 3 1:169). A single asterisk at positions 47, 64, 66, 68 indicates the Venier position (Foote and Winter (1992) J. Mol. Biol. 224:487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan) (1994) Mo/, 31:169). Figure 8 shows the heavy chain variable region of murine mAb 8G8 (SEQ ID NO: 50) and human heavy chain variable region VH1 framework (VH1 FW) (SEQ ID N〇: 52) and humanized weight of 8G8 on VH1 FW The amino acid sequence alignment of the chain variable region (hu8G8.VH1) (SEQ ID NO: 45). The amino acid is numbered according to the Kabat number. Frame the hypervariable region (HVR). The circle indicates the VL-VH interaction (Padlan (1994) Mo/· /wwmwo/. 31:169); the double asterisk (one above the other) indicates the Venier position (Foote and Winter (1992) J. Mol·Biol 224:487) and FW-CDR interaction (Padlan (1994) Mo/. 158864.doc -124· 201215618 /mmwnο/. 31:169). A single asterisk at positions 47, 64, 66, 68 indicates the Venier position (Foote & winter (!992) J. Mol. Biol. 224:487); a single asterisk at position 58 indicates FW-CDR interaction ( Padlan (1994) Mo/. 31:169). An exemplary nucleic acid sequence encoding hu8G8.VH1 (SEQ ID NO: 185) is also shown.
圖9展示鼠類mAb 8G8之重鏈可變區(SEQ ID NO:50)與 人類重鏈可變區VH3 FW(SEQ ID NO:53)及VH3 FW上8G8 之人類化重鏈可變區(hu8G8.VH3)(SEQ ID NO:46)的胺基 酸序列比對。胺基酸係根據Kabat編號來編號。對高變區 (HVR)加框。圓圈指示VL-VH相互作用(Padlan (1994) Mo/· 31:169);雙重星號(一個在另一個之上)指示維尼 爾位置(Foote及 Winter (1992) J· Mol. Biol. 224:487)及 FW-CDR相互作用(Padlan (1994) //wwwwo/. 31:169) 〇 在位 置47、64、66、68處之單一星號指示維尼爾位置(Foote及 Winter (1992) J. Mol. Biol. 224:487);在位置 58 處之單一 星號指示FW-CDR相互作用(Padlan (1994) Mo/· 31:169) 〇 圖10展示鼠類mAb 8G8 VL之輕鏈可變區(SEQ ID ΝΟ:51) 與λ4FW區之輕鏈可變區(SEQIDNO:55)及λ4FW上8G8之 人類化輕鏈可變區(λ4 8G8移植物)(其中根據Kabat編號, 在胺基酸2及36處引入胺基酸變化)(SEQ ID NO:49)的胺基 酸序列比對。胺基酸係根據Kabat編號來編號。對高變區 (HVR)加框。圓圈指示VL-VH相互作用(Padlan (1994) Μσ/· /wwwwo/. 31:169);雙重星號(一個在另一個之上)指示維尼 158864.doc -125· 201215618 爾位置(Foote及 Winter (1992) J. Mol. Biol. 224:487)及 FW-CDR相互作用(Padlan (1994) Mo/· /wimimo/· 31:169)。在位 置47、64、66、68處之單一星號指示維尼爾位置(Foote及 Winter (1992) J. Mol. Biol· 224:487);在位置 58 處之單一 星號指示 FW-CDR相互作用(Padlan (1994) Mo/, /mmwno/· 3 1:169)。亦展示編碼λ4 8G8移植物之例示性核酸序列 (SEQ ID ΝΟ:186)。 圖11展示人類抗體MSL-109與mAb ΗΒ1之胺基酸序列比 對。圖A : MSL-109 VL(SEQ ID NO:90)與親和力成熟之 HB1 VL(亦為SEQ ID N0:90(100%—致性))的比對;及圓 B :人類抗體MSL-109 VH(SEQ ID NO:92)與親和力成熟之 1^1¥11(8£()10!^0:89)的胺基酸序列比對。胺基酸係根 據Kabat編號來編號。對高變區(HVR)加框。 圖 12A 展示來自 MSL-109 之 HVR-H2(SEQ ID NO:91)及 IGHVSdPOUSEQ ID ΝΟ··93)之胺基酸序列及所作出之各 種胺基酸取代。圖12Β及圖12C展示使用含有突變HVR-H2 區之抗體進行的2個不同中和檢定之結果。有關Fab(圖 12B)及mAb(圖12C)之中和檢定均經展示。IC50以nM單位 提供。 圖13展示比較含有λ4 8G8移植物及hu8G8.VHl之抗體(下 文稱為「hu8G8」)及HB1與HIG預防上皮細胞及纖維母細 胞感染之能力之中和檢定的結果。Figure 9 shows the heavy chain variable region of murine mAb 8G8 (SEQ ID NO: 50) and the human heavy chain variable region VH3 FW (SEQ ID NO: 53) and the humanized heavy chain variable region of 8G8 on VH3 FW ( The amino acid sequence alignment of hu8G8.VH3) (SEQ ID NO: 46). Amino acids are numbered according to the Kabat number. Frame the hypervariable region (HVR). Circles indicate VL-VH interactions (Padlan (1994) Mo/· 31:169); double asterisks (one above the other) indicate Venier positions (Foote and Winter (1992) J. Mol. Biol. 224:487 And FW-CDR interactions (Padlan (1994) //wwwwo/. 31:169) 单一 A single asterisk at positions 47, 64, 66, 68 indicates the Venier position (Foote and Winter (1992) J. Mol. Biol. 224:487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan (1994) Mo/· 31:169) Figure 10 shows the light chain variable region of murine mAb 8G8 VL (SEQ ID ΝΟ: 51) and the light chain variable region of λ4FW region (SEQ ID NO: 55) and the humanized light chain variable region of 8G8 (λ4 8G8 graft) on λ4FW (wherein at amino acid 2 and 36 according to Kabat numbering) The amino acid sequence of the amino acid change (SEQ ID NO: 49) was introduced to align. Amino acids are numbered according to the Kabat number. Frame the hypervariable region (HVR). Circles indicate VL-VH interactions (Padlan (1994) Μσ/· /wwwwo/. 31:169); double asterisks (one above the other) indicate Pooh 158864.doc -125· 201215618 Location (Foote and Winter ( 1992) J. Mol. Biol. 224:487) and FW-CDR interaction (Padlan (1994) Mo/· /wimimo/· 31:169). A single asterisk at positions 47, 64, 66, 68 indicates the Venier position (Foote and Winter (1992) J. Mol. Biol. 224: 487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan) (1994) Mo/, /mmwno/· 3 1:169). An exemplary nucleic acid sequence encoding a λ4 8G8 graft (SEQ ID NO: 186) is also shown. Figure 11 shows the alignment of the human antibody MSL-109 with the amino acid sequence of mAb ΗΒ1. Panel A: Alignment of MSL-109 VL (SEQ ID NO: 90) with affinity matured HB1 VL (also SEQ ID NO: 90 (100% cis-)); and circle B: human antibody MSL-109 VH (SEQ ID NO: 92) Aligned with the amino acid sequence of affinity matured 1^1 ¥11 (8 £ () 10!^0:89). Amino acids are numbered according to the Kabat number. Frame the hypervariable region (HVR). Figure 12A shows the amino acid sequence of HVR-H2 (SEQ ID NO: 91) and IGHVSdPOUSEQ ID (93) from MSL-109 and the various amino acid substitutions made. Figure 12A and Figure 12C show the results of 2 different neutralization assays performed using antibodies containing the mutated HVR-H2 region. Both the Fab (Fig. 12B) and mAb (Fig. 12C) neutralization assays are shown. The IC50 is provided in nM units. Fig. 13 shows the results of a comparison between the ability of the antibody containing λ4 8G8 graft and hu8G8.VH1 (hereinafter referred to as "hu8G8") and HB1 and HIG to prevent infection of epithelial cells and fibroblasts.
圖14展示使用經消減高免疫球蛋白(HIG)對上皮細胞及 纖維母細胞進行之病毒中和檢定的結果。消減HIG之抗gB 158864.doc -126- 201215618 特異性抗體、抗複合物I特異性抗體、抗gH/gL抗體,或進 行模擬消減作為對照。 圖15展示旨在測定HB1及hu8G8抗體相較於已知抗gB、 抗gH及抗UL131抗體之抗原特異性的FACS分析結果。X軸 上之APC強度指示抗體結合。y軸繪出在既定強度下之細 胞的比例,其以佔任一強度下之最多細胞數目的百分比表 示。 圖16展示中和檢定之結果,在該中和檢定中,hu8G8與 HB 1以1:1比率混合且以一系列稀釋液測試其抑制HCMV感 染上皮細胞之能力。該兩種抗體之組合具有累加效應且表 現符合布利斯(Bliss)獨立方程式(具有效應A及B之兩種單 一化合物之組合反應C為C=A+B-AxB)。 圖17展示測定在不同hu8G8濃度下之HB1效能或在不同 HB1濃度下之hu8G8效能的中和檢定結果。 圖18展示有關HB 1抗性HCMV突變體之中和檢定之結 果。圖A展示使用HB 1抗體進行之中和檢定之結果。圖B展 示使用hu8G8抗體進行之中和檢定之結果。HB 1抗性 HCMV突變體仍然對hu8G8中和敏感。 圖19展示有關hu8G8抗性HCMV突變體之中和檢定之結 果。圖A展示使用HB1抗體進行之中和檢定之結果。圖B展 示使用hu8G8抗體進行之中和檢定之結果。hu8G8抗性 HCMV突變體仍然對HB 1中和敏感。 圖20展示與HCMV病毒株(WT)D1(當產生抗性病毒株時 並行生長之VR1814)相較於各種HB1抗性病毒突變體對上 158864.doc -127- 201215618 皮細胞及纖維母細胞之病毒進入相關的資料。 圖21展示HB 1抗體與細胞表面表現之在gH中含有賦予抗 性之點突變之gH/gL結合的能力,如藉由FACS分析所檢 定。不同抗gH抗體用作細胞表面表現之陽性對照。X軸為 GFP強度,其為HCMV醣蛋白表現之指標。y軸為APC信 號,其指示抗體結合。 圖22展示hu8G8抗體與細胞表面表現之在複合物I中含有 賦予抗性之點突變之複合物I結合的能力,如藉由FACS分 析所檢定。抗UL13 1抗體及抗gH抗體用作細胞表面表現之 陽性對照。X軸為GFP強度,其為HCMV醣蛋白表現之指 標。y軸為APC信號,其指示抗體結合。 圖23A及圖23B展示旨在測定hu8G8及HB1對其抗原之結 合親和力的史卡查分析結果。使用Munson及Rodbard之擬 合演算法對結果作圖。y軸繪出所結合之1251標記抗體與總 抗體之濃度比率。總抗體依125ι標記抗體及未標記抗體之 濃度計算。 圖24展示量測hu8G8及陽性對照抗體(抗HIS)與UL13 1之 肽片段(SEQ ID NO:194之胺基酸41(Ser)至胺基酸 68(Ser))(SRA-螺旋WT)或含有胺基酸取代Q47K之相應片段 (SRA-螺旋Mut)結合的ELISA檢定結果。 158864.doc •128- 201215618 序列表 <110>美商建南德克公司 <120>抗體組合物及使用方法Figure 14 shows the results of virus neutralization assays on epithelial cells and fibroblasts using depleted high immunoglobulin (HIG). HIG anti-gB 158864.doc -126- 201215618 specific antibody, anti-complex I specific antibody, anti-gH/gL antibody, or simulated subtraction was used as a control. Figure 15 shows the results of FACS analysis aimed at determining the antigen specificity of HB1 and hu8G8 antibodies compared to known anti-gB, anti-gH and anti-UL131 antibodies. APC intensity on the X-axis indicates antibody binding. The y-axis plots the proportion of cells at a given intensity, expressed as a percentage of the maximum number of cells at any intensity. Figure 16 shows the results of a neutralization assay in which hu8G8 was mixed with HB1 at a 1:1 ratio and tested for its ability to inhibit HCMV-infected epithelial cells in a series of dilutions. The combination of the two antibodies has an additive effect and exhibits a Bliss independent equation (the combined reaction C of the two single compounds with effects A and B is C = A + B - AxB). Figure 17 shows the results of neutralization assays for determining HB1 potency at different hu8G8 concentrations or hu8G8 potency at different HB1 concentrations. Figure 18 shows the results of the neutralization assay for the HB 1 resistant HCMV mutant. Panel A shows the results of neutralization assays using HB1 antibodies. Panel B shows the results of neutralization assays using the hu8G8 antibody. The HB 1 resistant HCMV mutant is still sensitive to hu8G8 neutralization. Figure 19 shows the results of the neutralization assay for the hu8G8 resistant HCMV mutant. Panel A shows the results of neutralization assays using HB1 antibodies. Panel B shows the results of neutralization assays using the hu8G8 antibody. The hu8G8 resistant HCMV mutant is still sensitive to HB 1 neutralization. Figure 20 shows that HCMV strain (WT) D1 (VR1814 which grows in parallel when a resistant strain is produced) is compared to various HB1 resistant virus mutant pairs 158864.doc -127 - 201215618 dermal cells and fibroblasts The virus enters relevant information. Figure 21 shows the ability of the HB1 antibody to bind to the gH/gL of the cell surface containing the point mutation imparting resistance in gH, as determined by FACS analysis. Different anti-gH antibodies were used as positive controls for cell surface expression. The X-axis is the GFP intensity, which is an indicator of HCMV glycoprotein performance. The y-axis is the APC signal, which indicates antibody binding. Figure 22 shows the ability of the hu8G8 antibody to bind to the complex I of the cell surface containing the point mutation conferring resistance in complex I, as determined by FACS analysis. Anti-UL13 1 antibody and anti-gH antibody were used as positive controls for cell surface expression. The X-axis is the GFP intensity, which is an indicator of HCMV glycoprotein expression. The y-axis is the APC signal, which indicates antibody binding. Figures 23A and 23B show the results of a Scala analysis aimed at determining the binding affinity of hu8G8 and HB1 for their antigens. The results were plotted using the proposed algorithm of Munson and Rodbard. The y-axis plots the concentration ratio of the bound 1251 labeled antibody to the total antibody. The total antibody was calculated based on the concentration of the 125 conjugated antibody and the unlabeled antibody. Figure 24 shows measurement of hu8G8 and positive control antibody (anti-HIS) with a peptide fragment of UL13 1 (amino acid 41 (Ser) to amino acid 68 (Ser) of SEQ ID NO: 194) (SRA-helix WT) or ELISA assay results containing the corresponding fragment of the amino acid substituted Q47K (SRA-helix Mut) binding. 158864.doc •128- 201215618 Sequence Listing <110>US-based Nandek Company <120> Antibody Composition and Method of Use
<130> P4680Ri^O <140> 100135366 <1^> 2011-09-29 <150> 61/504,056 <151> 2011-07-01 <150> 61/387,735 <151> 2010-09-29 <150> 61/387,725 <151> 2010-09-20 <160> 209 <170> Patentln version 3.5<130> P4680Ri^O <140> 100135366 <1^> 2011-09-29 <150> 61/504,056 <151> 2011-07-01 <150> 61/387,735 <151> 2010-09-29 <150> 61/387,725 <151> 2010-09-20 <160> 209 <170> Patentln version 3.5
<210> 1 <211> 743 <212> PRT <213>人類細胞巨大病毒 <400> 1<210> 1 <211> 743 <212> PRT <213> Human Cell Huge Virus <400>
Met Atg Pro Gly Leu Pro Pro Tyr Leu Thr Val Phe Thr Val Tyr Leu 1 5 10 15Met Atg Pro Gly Leu Pro Pro Tyr Leu Thr Val Phe Thr Val Tyr Leu 1 5 10 15
Leu Ser His Leu Pro Ser Gin Arg Tyr Gly Ala Asp Ala Ala Ser Glu 20 25 30Leu Ser His Leu Pro Ser Gin Arg Tyr Gly Ala Asp Ala Ala Ser Glu 20 25 30
Ala Leu Asp Pro His Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg 35 40 45Ala Leu Asp Pro His Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg 35 40 45
Pro He Arg Phe Leu Arg Glu Asn Thr Thr Gin Cys Thr Tyr Asn Ser 50 55 60Pro He Arg Phe Leu Arg Glu Asn Thr Thr Gin Cys Thr Tyr Asn Ser 50 55 60
Ser Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala lie vSer Phe Asn 65 70 75 80Ser Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala lie vSer Phe Asn 65 70 75 80
Phe Phe Gin Ser Tyr Asn Gin Tyr Tyr Val Phe His Met Fro Arg Cys 85 90 95Phe Phe Gin Ser Tyr Asn Gin Tyr Tyr Val Phe His Met Fro Arg Cys 85 90 95
Leu Phe Ala Gly Pro Leu Ala Glu Gin Phe Leu Asn Gin Val Asp Leu 100 105 110Leu Phe Ala Gly Pro Leu Ala Glu Gin Phe Leu Asn Gin Val Asp Leu 100 105 110
Thr Glu ΊΙίγ Leu Glu Arg Tyr Gin Gin Arg Leu Asn Thr Tyr Ala Leu 115 120 125Thr Glu ΊΙίγ Leu Glu Arg Tyr Gin Gin Arg Leu Asn Thr Tyr Ala Leu 115 120 125
Val Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gin Gin Leu Lys 130 135 140Val Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gin Gin Leu Lys 130 135 140
Ala Gin Asp Ser Leu Gly Gin Gin Fro Thr Thr Val Pro Pro Pro lie 145 150 155 160Ala Gin Asp Ser Leu Gly Gin Gin Fro Thr Thr Val Pro Pro Pro lie 145 150 155 160
Asp Leu Ser lie Pro His Val Trp Met Pro Pro Gin Thr Thr Pro His 165 170 175Asp Leu Ser lie Pro His Val Trp Met Pro Pro Gin Thr Thr Pro His 165 170 175
Asp Trp Lys Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe 158864-序列表 _doc 201215618 180 185 190Asp Trp Lys Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe 158864 - Sequence Listing _doc 201215618 180 185 190
Asn Gin Thr Cys 195 lie Leu Phe Asp Gly His 200Asn Gin Thr Cys 195 lie Leu Phe Asp Gly His 200
Asp Leu Leu Phe Ser Thr 205Asp Leu Leu Phe Ser Thr 205
Val Thr Pro Cys 210Val Thr Pro Cys 210
Leu His Gin Gly Phe Tyr 215Leu His Gin Gly Phe Tyr 215
Leu Met Asp Glu Leu Arg 220Leu Met Asp Glu Leu Arg 220
Tyr Val Lys lie 225Tyr Val Lys lie 225
Thr Leu Thr GJu Asp Phe 230Thr Leu Thr GJu Asp Phe 230
Phe Val Val Thr Val Ser 235 240 lie Asp Asp AspPhe Val Val Thr Val Ser 235 240 lie Asp Asp Asp
Thr Pro Met Leu Leu He 245 250Thr Pro Met Leu Leu He 245 250
Phe Gly His Leu Pro Arg 255Phe Gly His Leu Pro Arg 255
Val Lea Phe Lys 260Val Lea Phe Lys 260
Ala Pro Tyr GJn Arg Asp 265Ala Pro Tyr GJn Arg Asp 265
Asn Phe lie Leu Arg Gin 270Asn Phe lie Leu Arg Gin 270
Thr Glu Lys His 275Thr Glu Lys His 275
Glu Leu Leu Val Leu Val 2S0Glu Leu Leu Val Leu Val 2S0
Lys Lys Ala Gin Leu Asn 285Lys Lys Ala Gin Leu Asn 285
Arg His Ser Tyr 290Arg His Ser Tyr 290
Leu Lys Asp Ser Asp Phe 295Leu Lys Asp Ser Asp Phe 295
Leu Asp Ala Ala Leu Asp 300Leu Asp Ala Ala Leu Asp 300
Phe Asn Tyr Leu 305Phe Asn Tyr Leu 305
Asp Leu Ser Ala Leu Leu 310Asp Leu Ser Ala Leu Leu 310
Arg Asn Ser Phe His Arg 315 320Arg Asn Ser Phe His Arg 315 320
Tyr Ala Val AspTyr Ala Val Asp
Val Leu Lys Ser Gly Arg 325 330Val Leu Lys Ser Gly Arg 325 330
Cys Gin Met Leu Asp Arg 335Cys Gin Met Leu Asp Arg 335
Arg Thr Val Glu 340Arg Thr Val Glu 340
Met Ala Phe Ala Tyr Ala 345Met Ala Phe Ala Tyr Ala 345
Leu Ala Leu Phe Ala Ala 350Leu Ala Leu Phe Ala Ala 350
Ala Arg Gin Glu ' 355Ala Arg Gin Glu ' 355
Glu Ala Gly Thr Glu lie 360Glu Ala Gly Thr Glu lie 360
Ser lie Pro Arg Ala Leu 365Ser lie Pro Arg Ala Leu 365
Asp Arg Gin Ala 370Asp Arg Gin Ala 370
Ala Leu Leu Gin lie Gin 375Ala Leu Leu Gin lie Gin 375
Glu Phe Met lie Thr Cys 380Glu Phe Met lie Thr Cys 380
Leu Ser Gin Thr 385Leu Ser Gin Thr 385
Pro Pro Arg Thr Thr Leu 390Pro Pro Arg Thr Thr Leu 390
Leu Leu Tyr Pro Thr Ala 395 400Leu Leu Tyr Pro Thr Ala 395 400
Val Asp Leu AlaVal Asp Leu Ala
Lys Arg Ala Leu Trp Thr 405 410Lys Arg Ala Leu Trp Thr 405 410
Pro Asp Gin lie Thr Asp 415 lie Thr Ser Leu 420Pro Asp Gin lie Thr Asp 415 lie Thr Ser Leu 420
Val Arg Leu Val Tyr lie 425Val Arg Leu Val Tyr lie 425
Leu Ser Lys Gin Asn Gin 430Leu Ser Lys Gin Asn Gin 430
Gin His Leu lie 435Gin His Leu lie 435
Pro Gin Trp Ala Leu Arg 440 G】n lie A]a Asp Phe Ala 445Pro Gin Trp Ala Leu Arg 440 G】n lie A]a Asp Phe Ala 445
Leu Gin Leu His 450Leu Gin Leu His 450
Lys Thr His Leu Ala Ser 455Lys Thr His Leu Ala Ser 455
Phe Leu Ser Ala Phe Ala 460Phe Leu Ser Ala Phe Ala 460
Arg Gin Glu Leu 465Arg Gin Glu Leu 465
Tyr Leu Met Gly Ser Leu 470Tyr Leu Met Gly Ser Leu 470
Val His Ser Met Leu Val 475 480 !58864-序列表.doc -2- s 201215618Val His Ser Met Leu Val 475 480 !58864 - Sequence Listing.doc -2- s 201215618
His Thr Thr Glu Arg Arg Glu lie Phe lie Val Glu Thr Gly Leu Cys 485 490 495His Thr Thr Glu Arg Arg Glu lie Phe lie Val Glu Thr Gly Leu Cys 485 490 495
Ser Leu Ala Glu Leu Ser His Phe Thr Gin Leu Leu Ala His Pro His 500 505 510Ser Leu Ala Glu Leu Ser His Phe Thr Gin Leu Leu Ala His Pro His 500 505 510
His Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg 515 520 525His Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg 515 520 525
Arg Asp His Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr 530 535 540Arg Asp His Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr 530 535 540
Val Pro Ala Thr Val Pro Ala Ala Leu Ser lie Leu Ser Thr Met Gin 545 550 555 560Val Pro Ala Thr Val Pro Ala Ala Leu Ser lie Leu Ser Thr Met Gin 545 550 555 560
Pro Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly 565 570 575Pro Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly 565 570 575
Glu Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Val Val 580 585 590Glu Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Val Val 580 585 590
Thr Asn Gin Tyr Leu lie Lys Gly lie Ser Tyr Pro Val Ser rfhr Thr 595 600 605Thr Asn Gin Tyr Leu lie Lys Gly lie Ser Tyr Pro Val Ser rfhr Thr 595 600 605
Val Val Gly Gin Ser Leu lie lie Thr Gin Thr Asp Ser Gin Thi Lys 610 615 620Val Val Gly Gin Ser Leu lie lie Thr Gin Thr Asp Ser Gin Thi Lys 610 615 620
Cys Glu Leu Thr Arg Asn Met His Thr Thr His Ser lie Thr Ala Ala 625 630 635 640Cys Glu Leu Thr Arg Asn Met His Thr Thr His Ser lie Thr Ala Ala 625 630 635 640
Leu Asn lie Ser Leu Glu Asn Cys Ala Phe Cys Gin Ser Ala Leu Leu 645 650 655Leu Asn lie Ser Leu Glu Asn Cys Ala Phe Cys Gin Ser Ala Leu Leu 645 650 655
Glu Tyr Asp Asp Thr Gin Gly Val lie Asn lie Met Tyr Met His Asp 660 665 670Glu Tyr Asp Asp Thr Gin Gly Val lie Asn lie Met Tyr Met His Asp 660 665 670
Ser Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val 675 680 685Ser Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val 675 680 685
Ser Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly Thr Val 690 695 700Ser Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly Thr Val 690 695 700
Leu Glu Val ITir Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 720Leu Glu Val ITir Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 720
Met Met Scr Val Tyr Ala Leu Ser Ala lie lie Gly lie Tyr Leu Leu 725 730 735Met Met Scr Val Tyr Ala Leu Ser Ala lie lie Gly lie Tyr Leu Leu 725 730 735
Tyr Arg Met Leu Lys Thr Cys 740 <210> 2 <211> 278 <2]2> PR丁 <213>人類細胞巨大病毒 <400> 2Tyr Arg Met Leu Lys Thr Cys 740 <210> 2 <211> 278 <2]2> PR Ding <213> Human Cell Huge Virus <400> 2
Met Cys Arg Arg Pro Asp Cys Gly Phe Scr Phe Ser Pro Gly Pro Val 158864-序列表.doc 201215618 15 10 15Met Cys Arg Arg Pro Asp Cys Gly Phe Scr Phe Ser Pro Gly Pro Val 158864 - Sequence Listing.doc 201215618 15 10 15
Val Leu Leu Trp Cys Cys Leu Leu Leu Pro lie Val Ser Ser Val Ala 20 25 30Val Leu Leu Trp Cys Cys Leu Leu Leu Pro lie Val Ser Ser Val Ala 20 25 30
Val Ser Val Ala Pro Thx Ala Ala Glu Lys Val Pro Ala Glu Cys Pro 35 40 45Val Ser Val Ala Pro Thx Ala Ala Glu Lys Val Pro Ala Glu Cys Pro 35 40 45
Glu Leu Thr Arg Arg Cys Leu Leu Gly Glu Val Phe Gin Giy Asp Lys 50 55 60Glu Leu Thr Arg Arg Cys Leu Leu Gly Glu Val Phe Gin Giy Asp Lys 50 55 60
Tyr Glu Ser Trp Leu Arg Pro Leu Val Asn Val Thr Arg Arg Asp Gly 65 70 75 80Tyr Glu Ser Trp Leu Arg Pro Leu Val Asn Val Thr Arg Arg Asp Gly 65 70 75 80
Pro Leu Ser Gin Leu He Arg Tyr Arg Pro Val Thr Pro Glu Ala Ala 85 90 95Pro Leu Ser Gin Leu He Arg Tyr Arg Pro Val Thr Pro Glu Ala Ala 85 90 95
Asn Ser Val Leu Leu Asp Asp Ala Phe Leu Asp Thr Leu Ala Leu Leu 100 105 110Asn Ser Val Leu Leu Asp Asp Ala Phe Leu Asp Thr Leu Ala Leu Leu 100 105 110
Tyr Asn Asn Pro Asp Gin Leu Arg AJa Leu Leu Thr Leu Leu Ser Ser 115 120 125Tyr Asn Asn Pro Asp Gin Leu Arg AJa Leu Leu Thr Leu Leu Ser Ser 115 120 125
Asp ITir Ala Pro Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys 130 135 140Asp ITir Ala Pro Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys 130 135 140
Gly Asp Giy Ser Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg 145 150 155 160Gly Asp Giy Ser Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg 145 150 155 160
Gly Tyr Asp Leu Thr Arg Leu Ser Tyr Gly Arg Ser He Phe Thr Glu 165 170 175Gly Tyr Asp Leu Thr Arg Leu Ser Tyr Gly Arg Ser He Phe Thr Glu 165 170 175
His Val Leu Gly Phe Glu Leu Val Pro Pro Ser Leu Phe Asn Val Val 180 185 190His Val Leu Gly Phe Glu Leu Val Pro Pro Ser Leu Phe Asn Val Val 180 185 190
Val A1a lie Arg Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg Leu 195 200 205Val A1a lie Arg Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg Leu 195 200 205
Pro Val Ser Thr Ala Ala Ala Pro Glu Gly lie Thr Leu Phe Tyr Gly 210 215 220Pro Val Ser Thr Ala Ala Ala Pro Glu Gly lie Thr Leu Phe Tyr Gly 210 215 220
Lea Tyr Asn Ala Val Lys Glu Phe Cys Leu Arg His Gin Leu Asp Pro 225 230 235 240Lea Tyr Asn Ala Val Lys Glu Phe Cys Leu Arg His Gin Leu Asp Pro 225 230 235 240
Pro Leu Leu Arg His Leu Asp Lys Tyr Tyr Ala Gly Leu Pro Pro Glu 245 250 255Pro Leu Leu Arg His Leu Asp Lys Tyr Tyr Ala Gly Leu Pro Pro Glu 245 250 255
Leu Lys Gin Thr Arg Val Asn Leu Pro Ala His Ser Arg Tyr Gly Pro 260 265 270Leu Lys Gin Thr Arg Val Asn Leu Pro Ala His Ser Arg Tyr Gly Pro 260 265 270
Gin Ala Val Asp Ala Arg 275Gin Ala Val Asp Ala Arg 275
<210> 3 <211> 171 <212> PRT <213>人類細胞巨大病毒 -4 - 158864-序列表.doc 201215618 <400> 3<210> 3 <211> 171 <212> PRT <213> Human cell giant virus -4 - 158864 - Sequence Listing.doc 201215618 <400> 3
Met Ser Pro Lys Asn Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 15 10 15Met Ser Pro Lys Asn Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 15 10 15
Leu Gly His Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30Leu Gly His Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30
Phe lie Asn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Mei 35 40 45Phe lie Asn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Mei 35 40 45
Cys Asn Arg Phe Thr Val Ala Leu Arg Cys Fro Asp Gly Glu Val Cys 50 55 60Cys Asn Arg Phe Thr Val Ala Leu Arg Cys Fro Asp Gly Glu Val Cys 50 55 60
Tyr Ser Pro Glu Lys Thr Ala Glu lie Arg Gly lie Val Thr Thr Met 65 70 75 80Tyr Ser Pro Glu Lys Thr Ala Glu lie Arg Gly lie Val Thr Thr Met 65 70 75 80
Thr His Ser Leu Thr Arg Gin Val Val His Asn Lys Leu Thr Ser Cys 85 90 95Thr His Ser Leu Thr Arg Gin Val Val His As Lys Leu Thr Ser Cys 85 90 95
Asn Tyr Asn Pro Leu Tyr Leu Glu Ala Asp Gly Arg lie Arg Cys Gly ]00 105 110Asn Tyr Asn Pro Leu Tyr Leu Glu Ala Asp Gly Arg lie Arg Cys Gly ]00 105 110
Lys Val Asn Asp Lys Ala Gin Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125Lys Val Asn Asp Lys Ala Gin Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125
Pro Tyr Arg Trp lie Asn Leu Glu Tyr Asp Lys lie Thr Arg lie Val 130 135 140Pro Tyr Arg Trp lie Asn Leu Glu Tyr Asp Lys lie Thr Arg lie Val 130 135 140
Gly Leu Asp Gin Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160Gly Leu Asp Gin Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160
Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gin 165 170Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gin 165 170
<210> 4 <21!> 214 <212> PRT <213>人類細胞巨大病毒 <400> 4<210> 4 <21!> 214 <212> PRT <213> Human Cell Huge Virus <400> 4
Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 15 10 15Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 15 10 15
Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Ser Thr Leu Thr 20 25 30Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Ser Thr Leu Thr 20 25 30
Ala Asn Gin Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Ser Lys 35 40 45Ala Asn Gin Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Ser Lys 35 40 45
Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60
Pro Arg Ser Pro Leu Gin Phe Ser Gly Phe Gin Arg Val Ser Thr Gly 65 70 75 80Pro Arg Ser Pro Leu Gin Phe Ser Gly Phe Gin Arg Val Ser Thr Gly 65 70 75 80
Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95 158864-序列表.doc 201215618Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95 158864 - Sequence Listing.doc 201215618
Gin Thr Leu Val Glu Lys Ser Ser Thr Trp Val Lys Lys Val lie Trp 100 105 110Gin Thr Leu Val Glu Lys Ser Ser Thr Trp Val Lys Lys Val lie Trp 100 105 110
Tyr Leu Ser Gly Arg Asn Gin Thr lie Leu Gin Arg Met Pro Arg TTir 115 120 125Tyr Leu Ser Gly Arg Asn Gin Thr lie Leu Gin Arg Met Pro Arg TTir 115 120 125
Ala Ser Lys Pro Ser Asp Gly Asn Val Gin lie Ser Val Glu Asp Ala 130 135 140Ala Ser Lys Pro Ser Asp Gly Asn Val Gin lie Ser Val Glu Asp Ala 130 135 140
Lys lie Phe Gly Ala His Met Val Pro Lys Gin Thr Lys Leu Leu Arg 145 150 155 160Lys lie Phe Gly Ala His Met Val Pro Lys Gin Thr Lys Leu Leu Arg 145 150 155 160
Phe Val Val Asn Asp Gly Thr Arg Tyr Gin Met Cys Val Met Lys Leu 165 170 175Phe Val Val Asn Asp Gly Thr Arg Tyr Gin Met Cys Val Met Lys Leu 165 170 175
Glu Scr Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gin Val 180 1S5 190Glu Scr Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gin Val 180 1S5 190
Arg Leu Thr Phe Thr Glu Ala Asn Asn Gin Thr Tyr Thr Phe Cys Thr 195 200 205Arg Leu Thr Phe Thr Glu Ala Asn Asn Gin Thr Tyr Thr Phe Cys Thr 195 200 205
His Pro Asn Leu lie Val 210His Pro Asn Leu lie Val 210
<210> 5 <211> 76 <212> PRT <213>人類細胞巨大病毒 <400> 5<210> 5 <211> 76 <212> PRT <213> Human Cell Huge Virus <400>
Met Cys Met Met Ser His Asn Lys Ala Phe Phe Leu Ser Leu Gin His 15 10 15Met Cys Met Met Ser His Asn Lys Ala Phe Phe Leu Ser Leu Gin His 15 10 15
Ala Ala Val Ser Gly Val Ala Val Cys Leu Ser Val Arg Arg Gly Ala 20 25 30Ala Ala Val Ser Gly Val Ala Val Cys Leu Ser Val Arg Arg Gly Ala 20 25 30
Gly Ser Val Pro Ala Gly Asn Arg Gly Lys Lys Thr lie lie Thr Glu 35 40 45Gly Ser Val Pro Ala Gly Asn Arg Gly Lys Lys Thr lie lie Thr Glu 35 40 45
Tyr Arg lie Thr Gly Thr Arg Ala Leu Ala Arg Cys Pro Thr Lys Pro 50 55 60Tyr Arg lie Thr Gly Thr Arg Ala Leu Ala Arg Cys Pro Thr Lys Pro 50 55 60
Val llir Ser Met Trp Asn Ser Ser Trp Thr Ser Arg 65 70 75 <2)0> 6 <211> 10 <212> FRT <213>小家鼠 <400> 6Val llir Ser Met Trp Asn Ser Ser Trp Thr Ser Arg 65 70 75 <2)0> 6 <211> 10 <212> FRT <213> Mus musculus <400>
Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 <210> 7 <211> 17 <212> PRT <213>小家鼠 <400> 7 -6-Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 <210> 7 <211> 17 <212> PRT <213> Mus musculus <400> 7 -6-
158864-序列表.doc s 201215618158864-Sequence List.doc s 201215618
Trp lie Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15Trp lie Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15
Gly <210> 8 <211> 13 <212> PRT <213>小家鼠 <400> 8Gly <210> 8 <211> 13 <212> PRT <213> Mus musculus <400> 8
Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 9 <211> 12 <212> PRT <213>小家鼠 <400> 9Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 9 <211> 12 <212> PRT <213> Mus musculus <400>
Thr Leu Ser Ser Gin His Ser Thr Tyr Thr He Glu 1 5 10Thr Leu Ser Ser Gin His Ser Thr Tyr Thr He Glu 1 5 10
<210〉 10 <211> 11 <212> PRT <213>小家鼠 <400> 10<210> 10 <211> 11 <212> PRT <213> Mus musculus <400>
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp <210> 11 <211> 11 <2\2> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 <400> 11Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp <210> 11 <211> 11 <2\2> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Small Synthetic Mutation Mouse HVR-L2 peptide <400> 11
Leu Lys Lys Asp Ala Ser His Ser Thr Gly Asp 1 5 10 <210> 12 <211> 11 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 <400> 12Leu Lys Lys Asp Ala Ser His Ser Thr Gly Asp 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Mutation Mouse HVR-L2 peptide <400> 12
Leu Lys Lys Glu Gly vSer His Ser Thr Gly Asp 1 5 10 <210> 13 <211> 11 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 158864-序列表.doc -7- 201215618 <400〉 13Leu Lys Lys Glu Gly vSer His Ser Thr Gly Asp 1 5 10 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Mutation Mouse HVR-L2 peptide 158864 - Sequence Listing. doc -7- 201215618 <400> 13
Leu Lys Lys Thr G]y Ser His Ser Thr Gly Asp 1 5 10 <210> 14 <211> 11 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 <400> 14Leu Lys Lys Thr G]y Ser His Ser Thr Gly Asp 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic mutant mouse HVR-L2 peptide <400> 14
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp 1 5 10 <210> 15 <211> 11 <2i2> PRT <2i3>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp 1 5 10 <210> 15 <211> 11 <2i2> PRT <2i3> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Mutation Mouse HVR-L2 peptide
<400> 15<400> 15
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Glu I 5 3〇 <210> 16 <211> 11 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 <400> 16Leu Lys Lys Asp Gly Ser His Ser Thr Gly Glu I 5 3〇<210> 16 <211> 11 <212> PRT <213>Artificial Sequence<220><223> Description of Artificial Sequence: Synthesis Mutant Mouse HVR-L2 Peptide <400> 16
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Thr 1 5 10 <210> 17 <211> 11 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 <400> 17Leu Lys Lys Asp Gly Ser His Ser Thr Gly Thr 1 5 10 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Mutation Mouse HVR-L2 peptide <400> 17
Leu Lys Lys Ser Gly Ser His Ser Thr Gly Asp 1 5 10 <210> 18 <211> 11 <212> PRT <213>人工序列’ <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 <400> 18Leu Lys Lys Ser Gly Ser His Ser Thr Gly Asp 1 5 10 <210> 18 <211> 11 <212> PRT <213>Artificial Sequence ' <220><223> Description of Artificial Sequence: Synthesis Mutant mouse HVR-L2 peptide <400> 18
Leu Lys Lys Ser Gly Ser His Ser Thr Gly Ser i 5 10 158864-序列表.doc · 8 · s 201215618 <210> 19 <211> 11 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠HVR-L2肽 <400> 19Leu Lys Lys Ser Gly Ser His Ser Thr Gly Ser i 5 10 158864 - Sequence Listing. doc · 8 · s 201215618 <210> 19 <211> 11 <212> PRT <213> Manual Sequence <220><223> Description of artificial sequence: synthetic mutant mouse HVR-L2 peptide <400> 19
Leu Lys Lys Thr Gly Ser His Ser Thr Gly Asp 1 5 10 <210> 20 <211> 13 <212> PRT <2丨3>小家鼠 <400> 20Leu Lys Lys Thr Gly Ser His Ser Thr Gly Asp 1 5 10 <210> 20 <211> 13 <212> PRT <2丨3> Mus musculus <400> 20
Gly Val Gly Asp Thr lie Lys Glu Gin Phe Val Tyr ValGly Val Gly Asp Thr lie Lys Glu Gin Phe Val Tyr Val
<210> 21 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成共同HVR-L2序列 <220> <221> MOD—RES <222> (4)..(4) <223>任何胺基酸 <220> <221> MOD_RES <222> (5)..(5) <223>任何胺基酸 <220> <221> M0DJES <222> (11)..(11) <223>任何胺基酸 <220> <221> MOD_RES <222> (127..(12) <223>任何胺基酸 <400> 2]<210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Common HVR-L2 Sequence <220><221> MOD- RES <222> (4)..(4) <223> Any amino acid <220><221> MOD_RES <222> (5)..(5) <223> Any amino acid <220><221> M0DJES <222> (11)..(11) <223> Any amino acid <220><221> MOD_RES <222> (127..(12) <;223>any amino acid <400> 2]
Leu Lys Lys Xaa Xaa Ser His Ser Thr Gly Xaa Xaa 1 5 10 <210> 22 <211> 25 <212> PRT <213>智人 <400> 22Leu Lys Lys Xaa Xaa Ser His Ser Thr Gly Xaa Xaa 1 5 10 <210> 22 <211> 25 <212> PRT <213> Homo sapiens <400>
Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 23 <2U> 14Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 23 <2U> 14
158864-序列表.doc -9- 201215618 <212> PRT <213>智人 <400> 23158864 - Sequence Listing. doc -9- 201215618 <212> PRT <213> Homo sapiens <400> 23
Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp lie Gly 1 5 10 <210> 24 <211> 32 <212> PRT <2i3>智人 <400> 24Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp lie Gly 1 5 10 <210> 24 <211> 32 <212> PRT <2i3> Homo sapiens <400>
Arg Val Thr lie Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu 15 10 15Arg Val Thr lie Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu 15 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 25 <211> 11 <212> PRT <213>智人 <400> 25Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 25 <211> 11 <212> PRT <213> Homo sapiens <400>
Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 26 <211> 25 <212> PRT <21.3>小家鼠 <400> 26Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 26 <211> 25 <212> PRT <21.3> Mus musculus <400>
Gin lie His Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 15 10 15Gin lie His Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 15 10 15
Thr Val Lys lie Ser Cys Lys Ala Ser 20 25 <210> 27 <211> 14 <212> PRT <213>小家鼠 <400> 27Thr Val Lys lie Ser Cys Lys Ala Ser 20 25 <210> 27 <211> 14 <212> PRT <213> Mus musculus <400> 27
Trp Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met Gly 1 5 10 <2I0> 28 <211> 32 <212> PRT <213>小家鼠 <400> 28Trp Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met Gly 1 5 10 <2I0> 28 <211> 32 <212> PRT <213> Mus musculus <400>
Arg Fhe Aia Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gin 15 10 15 lie Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys Ala Arg 20 25 30 <210> 29 <231> 11 <212> PRT <213>小家鼠 -10- 158864-序列表.doc 201215618 <400> 29Arg Fhe Aia Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gin 15 10 15 lie Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys Ala Arg 20 25 30 <210> 29 <231> 11 <212> PRT <213> Mus musculus-10-158864 - Sequence Listing.doc 201215618 <400> 29
Trp Gly Gin Gly llir Leu Val Thr Val Ser Ser 1 5 10 <21〇> 30 <211> 25 <212> PRT <213>智人 <4-00> 30Trp Gly Gin Gly llir Leu Val Thr Val Ser Ser 1 5 10 <21〇> 30 <211> 25 <212> PRT <213> Homo sapiens <4-00> 30
Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Fro Gly Gly 1 5 ]〇 15Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Fro Gly Gly 1 5 ]〇 15
Ser Leu Arg l^u Ser Cys Ala Ala Ser 20 25 <210> 31 <211> 14 <212> PRT <213>智人 <400> 31Ser Leu Arg l^u Ser Cys Ala Ala Ser 20 25 <210> 31 <211> 14 <212> PRT <213> Homo sapiens <400>
Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val GlyTrp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Gly
<210> 32 <211> 32 <212> PRT <213>智人 <400> 32<210> 32 <211> 32 <212> PRT <213> Homo sapiens <400> 32
Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin 1 5 10 15Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin 1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 33 <213> 25 <212> PRT <213>智人 <400> 33Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 33 <213> 25 <212> PRT <213> Homo sapiens <400>
Glu Val Gin Leu Val Gin Ser Gly Scr Glu Leu Lys Lys Pro Gly Ala 】 5 10 15Glu Val Gin Leu Val Gin Ser Gly Scr Glu Leu Lys Lys Pro Gly Ala 】 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 34 <21】> 32 <212> PRT <2丨3>智人 <400> 34Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 34 <21]> 32 <212> PRT <2丨3> Homo sapiens <400> 34
Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gin 15 10 15 lie Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 35 <211> 22 <212> PRT <213>小家鼠 158864-序列表.doc - 11 - 201215618 <400> 35Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gin 15 10 15 lie Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 35 <211> 22 <212> PRT <213> Mus musculus 158864 - Sequence Listing.doc - 11 - 201215618 <400> 35
Gin Leu Val Leu Thr Gin Ser Ser Ser Ala Ser Phe Ser Leu Gly Ala 15 10 15Gin Leu Val Leu Thr Gin Ser Ser Ser Ala Ser Phe Ser Leu Gly Ala 15 10 15
Ser Ala Lys Leu Thr Cys 20 <210> 36 <211> 15 <232> PRT <213>小家鼠 <400> 36Ser Ala Lys Leu Thr Cys 20 <210> 36 <211> 15 <232> PRT <213> Mus musculus <400> 36
Trp Tyr Gin Gin Gin Pro Leu Lys Pro Pro Lys Tyr Val Met Glu 15 10 15 <210> 37 <2L1> 32 <212> PRT <213>小家鼠 <400> 37Trp Tyr Gin Gin Gin Pro Leu Lys Pro Pro Lys Tyr Val Met Glu 15 10 15 <210> 37 <2L1> 32 <212> PRT <213> Mus musculus <400> 37
Gly lie Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr 1 5 10 Ϊ5Gly lie Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr 1 5 10 Ϊ5
Leu Ser lie Ser Asn lie Gin Pro Glu Asp Glu Ala He Tyr lie Cys 20 25 30 <210> 38 <211> 11 <212> PRT <213>小家鼠 <400> 38Leu Ser lie Ser Asn lie Gin Pro Glu Asp Glu Ala He Tyr lie Cys 20 25 30 <210> 38 <211> 11 <212> PRT <213> Mus musculus <400> 38
Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 1 5 10 <210> 39 <211> 22 <212> PRT <213>智人 <400> 39Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 1 5 10 <210> 39 <211> 22 <212> PRT <213> Homo sapiens <400> 39
Gin Pro Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala 15 10 15Gin Pro Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala 15 10 15
Ser Val Lys Leu Thr Cys 20 <210> 40 <211> 15 <212> PRT <213>智人 <400> 40Ser Val Lys Leu Thr Cys 20 <210> 40 <211> 15 <212> PRT <213> Homo sapiens <400> 40
Trp His Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met Lys 15 10 15 <210> 4L <211> 32 <212> PRT <213>智人 <400> 41Trp His Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met Lys 15 10 15 <210> 4L <211> 32 <212> PRT <213> Homo sapiens <400>
Gly lie Pro Asp Arg Phe Scr Gly Ser Ser Ser Gly Ala Asp Arg Tyr 12- 158864-序列表.doc 201215618 15 10Gly lie Pro Asp Arg Phe Scr Gly Ser Ser Ser Gly Ala Asp Arg Tyr 12- 158864 - Sequence Listing.doc 201215618 15 10
Leu Thr lie Ser Asn Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 42 <211> 11 <212> PRT <213>智人 <400> 42Leu Thr lie Ser Asn Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 42 <211> 11 <212> PRT <213> Homo sapiens <400>
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1 5 10 <210> 43 <211> 21 <212> PRT <213>智人 <400> 43Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 1 5 10 <210> 43 <211> 21 <212> PRT <213> Homo sapiens <400>
Ser Val Leu Thr Gin Ser Pro Ser Ala i5er Ala Ser Leu Gly Ala Ser 1 5 10 15Ser Val Leu Thr Gin Ser Pro Ser Ala i5er Ala Ser Leu Gly Ala Ser 1 5 10 15
Val Lys Leu Thr Cys 20 <210> 44 <211> 15 <212> PRT <213>智人 <400> 44Val Lys Leu Thr Cys 20 <210> 44 <211> 15 <212> PRT <213> Homo sapiens <400> 44
Trp Tyr Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met Lys 15 10 15 <210> 45 <211> 122 <212〉 PRT <213>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白重鏈多肽 <400> 45Trp Tyr Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met Lys 15 10 15 <210> 45 <211> 122 <212> PRT <213> Artificial Sequence <220><223> Artificial Sequence Description: Synthetic humanized 8G8 immunoglobulin heavy chain polypeptide <400> 45
Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGlu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gin Ala Fro Gly Gin Gly Leu Glu Trp lie 35 40 45Gly Met Asn Trp Val Arg Gin Ala Fro Gly Gin Gly Leu Glu Trp lie 35 40 45
Gly Trp lie Asn rrhr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Gly Trp lie Asn rrhr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Glv Arg Val Thr lie Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80Lys Glv Arg Val Thr lie Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80
Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 -13- 158864-序列表.doc 201215618Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 -13- 158864 - Sequence Listing.doc 201215618
Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 105 110Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 105 110
Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 46 <21i> 122 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白重鏈多肽 <4〇〇> 46Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 46 <21i> 122 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Humanized 8G8 Immunoglobulin heavy chain polypeptide <4〇〇> 46
Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 15 10 15Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Uu Glu Trp Val 35 40 45Gly Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Uu Glu Trp Val 35 40 45
Gly Trp lie Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Gly Trp lie Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Gly Arg Phe Thr Jle Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Lys Gly Arg Phe Thr Jle Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys S5 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys S5 90 95
AlaAr, Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val T,pAlaAr, Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val T,p
Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 47 <21I> 122 <212> PRT <2】3>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白重鏈多肽 <400> 47Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 47 <21I> 122 <212> PRT <2]3>Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic Human 8G8 Immunoglobulin Heavy Chain Peptide <400> 47
Glu Val Gin Leu Val Gin Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 15 10 15Glu Val Gin Leu Val Gin Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 15 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 40 45Gly Met Asn Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 40 45
Gly Trp ile Asn Tlir Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 -14- 158864-序列表,doc 201215618Gly Trp ile Asn Tlir Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 -14- 158864 - Sequence Listing, doc 201215618
Lys G!y Arg Phe Val Phe Ser Leu Asp Thr Ser Val Scr Thr Ala Tyr 65 70 75 80Lys G!y Arg Phe Val Phe Ser Leu Asp Thr Ser Val Scr Thr Ala Tyr 65 70 75 80
Leu Gin ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 1.05 110Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 1.05 110
Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 116 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白輕鏈多肽 <400> 48Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 116 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Humanized 8G8 Immunoglobulin light chain polypeptide <400> 48
Gin Pro Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Scr Leu Gly Ala 1 5 10 15Gin Pro Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Scr Leu Gly Ala 1 5 10 15
Scr Val Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr 20 25 30 lie Glu Trp His Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met 35 40 45Scr Val Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr 20 25 30 lie Glu Trp His Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met 35 40 45
Lys Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly lie Pro Asp 50 55 60Lys Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly lie Pro Asp 50 55 60
Arg Phe Scr Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr He Ser 65 70 75 80Arg Phe Scr Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr He Ser 65 70 75 80
Asn Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp 85 90 95Asn Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp 85 90 95
Thr lie Lys Glu Gin Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr lie Lys Glu Gin Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu 100 105 110
Thr Val Leu Gly 115 <210> 49 <21I> 115 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白輕鏈(突變) 多肽 <400> 49Thr Val Leu Gly 115 <210> 49 <21I> 115 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Humanized 8G8 Immunoglobulin Light Chain (Mutation ) Peptide <400> 49
Ser Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser 15 10 15Ser Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser 15 10 15
Val Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr lie 158864-序列表.doc -15- on* 201215618 20 25 30Val Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr lie 158864 - Sequence Listing. doc -15- on* 201215618 20 25 30
Glu 丁rp Tyr Gin Gin Gin Pro Gly Lys G】y Pro Arg Tyr Leu Met Lys 35 40 45Glu rp Tyr Gin Gin Gin Pro Gly Lys G】y Pro Arg Tyr Leu Met Lys 35 40 45
Leu Lys Lys Asp Gly Set His Ser Thr Gly Asp Gly He Pro Asp Arg 50 55 60Leu Lys Lys Asp Gly Set His Ser Thr Gly Asp Gly He Pro Asp Arg 50 55 60
Phe Ser Gly Set Ser Ser Gly Ala Asp Arg Tyr Leu Thr lie Ser Asn 65 70 75 80Phe Ser Gly Set Ser Ser Gly Ala Asp Arg Tyr Leu Thr lie Ser Asn 65 70 75 80
Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr 85 90 95 lie Lys Glu Gin Phe Val Tyr Val Phe Giy Gly Cty Thr Lys Leu Thr 100 105 110Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr 85 90 95 lie Lys Glu Gin Phe Val Tyr Val Phe Giy Gly Cty Thr Lys Leu Thr 100 105 110
Val Leu Gly 115 <210> 50 <21l> 122 <212> PRT <213>小家鼠 <400> 50Val Leu Gly 115 <210> 50 <21l> 122 <212> PRT <213> Mus musculus <400> 50
Gin lie His Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 15 10 15Gin lie His Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 15 10 15
Thr Va】Lys I]e Ser Cys Lys A】a Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Thr Va] Lys I]e Ser Cys Lys A] a Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45
Gly Trp He Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Gly Trp He Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80
Leu Gin lie Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys 85 90 95Leu Gin lie Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys 85 90 95
Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 105 110Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 105 110
Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5i <211> 116 <212> PRT <213>小家鼠 <400> 51Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5i <211> 116 <212> PRT <213> Mus musculus <400>
Gin Leu Val Leu Tlir Gin Scr Ser Ser Ala Ser Phe Ser Leu Gly Ala 15 10 15 •16- 158864-序列表.doc 201215618Gin Leu Val Leu Tlir Gin Scr Ser Ser Ala Ser Phe Ser Leu Gly Ala 15 10 15 •16- 158864-Sequence List.doc 201215618
Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr 20 25 30 lie GIu Trp Tyr Gin Gin Gin Pro Leu Lys Pro Pro Lys Tyr Va! Met 35 40 45Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr 20 25 30 lie GIu Trp Tyr Gin Gin Gin Pro Leu Lys Pro Pro Lys Tyr Va! Met 35 40 45
Glu Leu Lys Lys Asp Gly Scr His Ser Thr Gly Asp Gly lie Pro Asp 50 55 60Glu Leu Lys Lys Asp Gly Scr His Ser Thr Gly Asp Gly lie Pro Asp 50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ser lie Ser 65 70 75 80Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ser lie Ser 65 70 75 80
Asn lie Gin Pro Glu Asp Glu Ala lie Tyr lie Cys Gly Val Gly Asp 85 90 95Asn lie Gin Pro Glu Asp Glu Ala lie Tyr lie Cys Gly Val Gly Asp 85 90 95
Thr He Lys Glu Gin Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val 100 105 110Thr He Lys Glu Gin Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val 100 105 110
Thr Val Leu Gly 115Thr Val Leu Gly 115
<2i0> 52 <211> 117 <212> PRT <213〉智人 <400> 52<2i0> 52 <211> 117 <212> PRT <213> Homo sapiens <400> 52
Glu Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 15 10 15Glu Val 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 lie His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp lie 35 40 45Tyr lie His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp lie 35 40 45
Gly Trp He Asn Pro Gly Ser Gly Asn Thr Asn Tyr Ala Gin Lys Phe 50 55 60Gly Trp He Asn Pro Gly Ser Gly Asn Thr Asn Tyr Ala Gin Lys Phe 50 55 60
Gin Gly Arg Val Thr lie Thr Arg Asp rFhr Ser Thr Ser Thr Ala Tyr 65 70 75 80Gin Gly Arg Val Thr lie Thr Arg Asp rFhr Ser Thr Ser Thr Ala Tyr 65 70 75 80
Leu Glu Leu Ser Ser Leu Arg vSer Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Glu Leu Ser Ser Leu Arg vSer Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Thr Ala Ala Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 110Ala Arg Asp Thr Ala Ala Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 <210> 53 <2U> 117 <212> PRT <213>智人 <400> 53Val Thr Val Ser Ser 115 <210> 53 <2U> 117 <212> PRT <213> Homo sapiens <400> 53
Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 •17- 158864-序列表.doc 201215618Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 •17- 158864-Sequence List.doc 201215618
Ser Leu Atg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Leu Atg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Ala He Ser Scr Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Gly Ala He Ser Scr Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Thr Ala Ala Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu }〇〇 105 110Ala Arg Asp Thr Ala Ala Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu }〇〇 105 110
Val Thr Val Ser Ser 115Val Thr Val Ser Ser 115
<210> 54 <211> 117 <212> PRT <213>智人 <400> 54<210> 54 <211> 117 <212> PRT <213> Homo sapiens <400> 54
Glu Val Gin Leu Val Gin Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15Glu Val Gin Leu Val Gin Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 丁hr Phe Thr Ser Tyr 20 25 30Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ding hr Phe Thr Ser Tyr 20 25 30
Tyr lie His Trp Va] Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 40 45Tyr lie His Trp Va] Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 40 45
Gly Trp He Asn Pro Gly Ser Gly Asn Thr Asn Tyr Ala Gin Lys Phe 50 55 60Gly Trp He Asn Pro Gly Ser Gly Asn Thr Asn Tyr Ala Gin Lys Phe 50 55 60
Gin Gly Arg Phe Vai Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80Gin Gly Arg Phe Vai Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80
Leu Gin He Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin He Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Thr Ala Ala Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 110Ala Arg Asp Thr Ala Ala Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 110
Val Thr Val Ser Ser U5 <210> 55 <211> 112 <212> PRT <213>智人 <400> 55Val Thr Val Ser Ser U5 <210> 55 <211> 112 <212> PRT <213> Homo sapiens <400> 55
Gin Pro Val Leu Thr Gin Ser Pro Set Ala Ser Ala Ser Leu Gly Ala 15 10 15 158864-序列表.doc -18- s 201215618Gin Pro Val Leu Thr Gin Ser Pro Set Ala Ser Ala Ser Leu Gly Ala 15 10 15 158864 - Sequence Listing.doc -18- s 201215618
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gly His Ser Ser Tyr Thr 20 25 30 lie Ala Trp His Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met 35 40 45Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gly His Ser Ser Tyr Thr 20 25 30 lie Ala Trp His Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met 35 40 45
Lys Leu Asn Ser Asp Gly Ser His Ser Lys Gly Asp Gly lie Pro Asp 50 55 60Lys Leu Asn Ser Asp Gly Ser His Ser Lys Gly Asp Gly lie Pro Asp 50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr lie Ser 65 70 75 80Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr lie Ser 65 70 75 80
Asn Leu Gin Ser Glu Asp GIu Ala Asp Tyr Tyr Cys Gly Thr Trp Gly 85 90 95Asn Leu Gin Ser Glu Asp GIu Ala Asp Tyr Tyr Cys Gly Thr Trp Gly 85 90 95
Thr Gly lie Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110Thr Gly lie Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
<210> 56 <211> 345 <212> DNA <213>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白輕鏈聚核苷酸 <400> 56 tctgtgctga cccagagccc aagcgccagc gccagcctgg gcgccagcgt gaaactgacc 60 tgcaccctga gcagccagca cagcacctac accatcgaat ggiatcagca gcagccaggc 120 aaaggcccac gctacctgat gaaactgaaa aaagatggca gccacagcac cggc£atggc 180 atcccagatc gcttcagcgg cagcagcagc ggcgccgatc gctacctgac catcagcaac 240 ctgcagagcg aagatgaagc cgattactac tgcggcgtgg gcgataccat caaagaacag 300 ttcgtgtacg tgttcggcgg cggtaccaaa ctgaccgtgc tgggc 345 <210> 57 <211> 12 <212> PRT <2Π>小家鼠 <400> 57<210> 56 <211> 345 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Humanized 8G8 Immunoglobulin Light Chain Polynucleotide <400>; 56 tctgtgctga cccagagccc aagcgccagc gccagcctgg gcgccagcgt gaaactgacc 60 tgcaccctga gcagccagca cagcacctac accatcgaat ggiatcagca gcagccaggc 120 aaaggcccac gctacctgat gaaactgaaa aaagatggca gccacagcac cggc £ atggc 180 atcccagatc gcttcagcgg cagcagcagc ggcgccgatc gctacctgac catcagcaac 240 ctgcagagcg aagatgaagc cgattactac tgcggcgtgg gcgataccat caaagaacag 300 ttcgtgtacg tgttcggcgg cggtaccaaa ctgaccgtgc tgggc 345 < 210 > 57 <211> 12 <212> PRT <2Π> Mus musculus <400> 57
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly <210> 58 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 <400> 58Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Mutant Mouse Sequence <400> 58
Leu Lys Lys Asp Ala Ser His Ser Thr Gly Asp Gly 1 5 10 <210> 59 <211> 12 •19· 158864-序列表.doc 6 201215618 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 <400> 59Leu Lys Lys Asp Ala Ser His Ser Thr Gly Asp Gly 1 5 10 <210> 59 <211> 12 •19· 158864 - Sequence Listing.doc 6 201215618 <212> PRT <213>Artificial Sequence<220>;<223> Description of artificial sequence: synthetic mutant mouse sequence <400> 59
Leu Lys Lys Glu Gly Ser His Ser Thr Gly Asp Gly 1 5 10 <210> 60 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 <400> 60Leu Lys Lys Glu Gly Ser His Ser Thr Gly Asp Gly 1 5 10 <210> 60 <211> 12 <212> PRT <213>Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis Mutant mouse sequence <400> 60
Leu Lys Lys Thr Gly Ser His Ser Thr Gly Asp Gly 1 5 10Leu Lys Lys Thr Gly Ser His Ser Thr Gly Asp Gly 1 5 10
<210> 61 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 <400> 61<210> 61 <211> 12 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthetic mutant mouse sequence <400>
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Ala 1 5 10Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Ala 1 5 10
<210> 62 <213> 12 <212> PRT 人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 <400> 62<210> 62 <213> 12 <212> PRT artificial sequence <220><223> Description of artificial sequence: synthetic mutant mouse sequence <400>
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Glu Gly 1 5 10Leu Lys Lys Asp Gly Ser His Ser Thr Gly Glu Gly 1 5 10
<210> 63 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 <400> 63<210> 63 <211> 12 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthetic mutant mouse sequence <400> 63
Leu Lys Lys Asp Gly Ser His Ser Thr Gly Thr Gly 1 5 10 <210> 64 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 158864-序列表.doc •20·Leu Lys Lys Asp Gly Ser His Ser Thr Gly Thr Gly 1 5 10 <210> 64 <211> 12 <212> PRT <213>Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis Mutant mouse sequence 158864 - Sequence Listing.doc •20·
201215618 <400> 64201215618 <400> 64
Leu Lys Lys Ser Gly Ser His Ser Thr Gly Asp Gly 1 5 10 <210> 65 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列 <400> 65Leu Lys Lys Ser Gly Ser His Ser Thr Gly Asp Gly 1 5 10 <210> 65 <211> 12 <212> PRT <213>Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis Mutant mouse sequence <400> 65
Leu Lys Lys Ser Gly Ser His Ser Thr Gly Ser Gly 1 5 10 <210> 66 <211> 12 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠序列Leu Lys Lys Ser Gly Ser His Ser Thr Gly Ser Gly 1 5 10 <210> 66 <211> 12 <212> PRT <213>Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis Mutant mouse sequence
<400> 66<400> 66
Leu Lys Lys Thr Gly Ser His Ser Thr Gly Asp Ala 1 5 10 <210> 67 <211> 32 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變小鼠FR3多肽 <400> 67Leu Lys Lys Thr Gly Ser His Ser Thr Gly Asp Ala 1 5 10 <210> 67 <211> 32 <212> PRT <213>Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis Mutant mouse FR3 polypeptide <400> 67
Ala I3e Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr 15 10 15Ala I3e Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr 15 10 15
Leu Ser He Ser Asn lie Gin Pro Glu Asp Glu Ala lie Tyr lie Cys 20 25 30 <210> 68 <21l> 32 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成突變人類MFR3多肽 <400> 68Leu Ser He Ser Asn lie Gin Pro Glu Asp Glu Ala lie Tyr lie Cys 20 25 30 <210> 68 <21l> 32 <212> PRT <213> Artificial sequence <220><223> Description: Synthetic mutant human MFR3 polypeptide <400> 68
Ala He Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr 15 10 15Ala He Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr 15 10 15
Leu Thr lie Ser Asn Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 69 <211> 109 <212> PRT <213>智人 -21 · 158864·序列表.doc 201215618 <400> 69Leu Thr lie Ser Asn Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 69 <211> 109 <212> PRT <213> Homo sapiens-21 · 158864 · Sequence Listing.doc 201215618 <400> 69
Ser Tyr Glu Leu Tlir Gin Pro Pro Ser Val Ser Val Ser Pro Gly Gin 15 10 15Ser Tyr Glu Leu Tlir Gin Pro Pro Ser Val Ser Val Ser Pro Gly Gin 15 10 15
Thr Ala Arg lie Thr Cys Gin Gly Asp Ala Leu Arg Ser Tyr Tyr Ala 20 25 30Thr Ala Arg lie Thr Cys Gin Gly Asp Ala Leu Arg Ser Tyr Tyr Ala 20 25 30
Ser Trp Tyr Gin Gin Lys Fro Gly Gin Ala Pro Val Leu Val lie Tyr 35 40 45Ser Trp Tyr Gin Gin Lys Fro Gly Gin Ala Pro Val Leu Val lie Tyr 35 40 45
Lys Asp Asn Asn Arg Pro Ser Gly lie Pro Glu Arg Phe Ser Gly Ser 50 55 60Lys Asp Asn Asn Arg Pro Ser Gly lie Pro Glu Arg Phe Ser Gly Ser 50 55 60
Ser Ser Gly Asn Thr Ala Thr Leu Thr He Ser Gly Ala Gin Ala Glu 65 70 75 80Ser Ser Gly Asn Thr Ala Thr Leu Thr He Ser Gly Ala Gin Ala Glu 65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95
Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Giy 100 105Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Giy 100 105
<210> 70 <211> 117 c232> PRT <213>人類細胞巨大病毒 <400> 70<210> 70 <211> 117 c232> PRT <213> Human Cell Huge Virus <400> 70
Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala Ala Arg 15 10 15Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala Ala Arg 15 10 15
Gin Glu Glu Ala Gly Ala Gin Val Ser Val Pro Arg Ala Leu Asp Arg 20 25 30Gin Glu Glu Ala Gly Ala Gin Val Ser Val Pro Arg Ala Leu Asp Arg 20 25 30
Gin Ala Ala Leu Leu Gin lie Gin Glu Phe Met lie Thr Cys Leu Ser 35 40 45Gin Ala Ala Leu Leu Gin lie Gin Glu Phe Met lie Thr Cys Leu Ser 35 40 45
Gin Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala Val Asp 50 55 60Gin Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala Val Asp 50 55 60
Leu Ala Lys Arg Ala Leu Trp Thr Pro Asn Gin lie Thr Asp lie Thr 65 70 75 80Leu Ala Lys Arg Ala Leu Trp Thr Pro Asn Gin lie Thr Asp lie Thr 65 70 75 80
Ser Leu Val Arg Leu Val Tyr ile Leu Ser Lys Gin Asn Gin Gin His 85 90 95Ser Leu Val Arg Leu Val Tyr ile Leu Ser Lys Gin Asn Gin Gin His 85 90 95
Leu lie Pro Gin Trp Ala Leu Arg Gin Ile Ala Asp Phe Ala Leu Lys 100 105 110Leu lie Pro Gin Trp Ala Leu Arg Gin Ile Ala Asp Phe Ala Leu Lys 100 105 110
Leu His Lys Thr His 115 <210> 71 <211> 10 <212> PRT <213>智人 <400> 71Leu His Lys Thr His 115 <210> 71 <211> 10 <212> PRT <213> Homo sapiens <400> 71
Gly Phe Thr Phe Ser Pro Tyr Ser Val Phe -22·Gly Phe Thr Phe Ser Pro Tyr Ser Val Phe -22·
!58864-序列表.doc!58864-Sequence table.doc
201215618 <210> 72 <21!> 18 <2]2> PRT <213>智人 <400> 72201215618 <210> 72 <21!> 18 <2]2> PRT <213> Homo sapiens <400> 72
Ser Ser lie Asn Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 15 10 15Ser Ser lie Asn Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 15 10 15
Lys Gly <210> 73 <211> 18 <212> PRT <213>智人 <400> 73Lys Gly <210> 73 <211> 18 <212> PRT <213> Homo sapiens <400> 73
Ser Ser lie Asn Ser Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 15 10 15Ser Ser lie Asn Ser Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 15 10 15
Lys GlyLys Gly
<210> 74 <211> 18 <212> PRT <213>智人 <400> 74<210> 74 <211> 18 <212> PRT <213> Homo sapiens <400> 74
Ser Ser lie Asn Ser Asn Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 15 !0 15Ser Ser lie Asn Ser Asn Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 15 !0 15
Lys Gly <210> 75 <211> 23 <212> PRT <2】 3>智人 <400> 75Lys Gly <210> 75 <211> 23 <212> PRT <2] 3> Homo sapiens <400> 75
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 1 5 10 15Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 1 5 10 15
Tyr Tyr Tyr Gly Leu Asp Val 20 <210> 76 <211> 16 <212〉 PRT <2】3>智人 <400> 76Tyr Tyr Tyr Gly Leu Asp Val 20 <210> 76 <211> 16 <212> PRT <2]3> Homo sapiens <400> 76
Arg Ser Scr Gin Ser Leu Leu His Tlu Asn Gly Tyr Asn Tyr Leu Asp 15 10 15 <210> 77 <211> 7 <212> PRT <213>智人 <400> 77Arg Ser Scr Gin Ser Leu Leu His Tlu Asn Gly Tyr Asn Tyr Leu Asp 15 10 15 <210> 77 <211> 7 <212> PRT <213> Homo sapiens <400> 77
Leu Ala Ser Asn Arg Ala Ser 158864-序列表.doc -23- § 201215618 <210> 78 <211> 9 <212> PRT <2]3>智人 <400> 78Leu Ala Ser Asn Arg Ala Ser 158864 - Sequence Listing. doc -23- § 201215618 <210> 78 <211> 9 <212> PRT <2]3> Homo sapiens <400>
Met Gin Ala Leu Gin lie Pro Arg Thr <210> 79 <231> 25 <212> PRT <213>智人 <400> 79Met Gin Ala Leu Gin lie Pro Arg Thr <210> 79 <231> 25 <212> PRT <213> Homo sapiens <400> 79
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 人rg 8013四智80丨 0>I>2>3>Kbva 1 1 *—· 1 p <2<2<2<2<4Tr. eo LI y G1 s Ly y G, o pr va rp <210> 81 <211> 30 <212> PRT <213>智人 <400> 81Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 person rg 8013 四智80丨0>I>2>3>Kbva 1 1 *—· 1 p <2<2<2<2<4Tr. eo LI y G1 s Ly y G, o pr va rp <210> 81 <211> 30 <212> PRT <213> Homo sapiens <400>
Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe Leu Gin 1 5 10 15Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe Leu Gin 1 5 10 15
Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> 82 <2Π> Π <212> PRT <213>智人 <400> 82Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 <210> 82 <2Π> Π <212> PRT <213> Homo sapiens <400>
Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 83 <211> 23 <212> PRT <213>智人 <400> 83Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 83 <211> 23 <212> PRT <213> Homo sapiens <400> 83
Asp lie Val Met rFhr Gin Ser Pro Leu Ser Leu Ser Val Thr Pro Gly 15 10 15Asp lie Val Met rFhr Gin Ser Pro Leu Ser Leu Ser Val Thr Pro Gly 15 10 15
Glu Pro Ala Ser lie Ser Cys 20 24- 158864-序列表.doc 201215618 <210> 84 <211> 15 <212> PRT <213〉智人 <400〉 84Glu Pro Ala Ser lie Ser Cys 20 24-158864 - Sequence Listing.doc 201215618 <210> 84 <211> 15 <212> PRT <213> Homo sapiens <400> 84
Trp Tyr Val Gin Lys Pro Gly Gin Scr Pro Gin Leu Leu He Tyr 15 10 15 <230> 85 <211> 32 <212> PRT <2]3>智人 <400> g5Trp Tyr Val Gin Lys Pro Gly Gin Scr Pro Gin Leu Leu He Tyr 15 10 15 <230> 85 <211> 32 <212> PRT <2]3> Homo sapiens <400> g5
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 15 10 15
Leu Lys lie Scr Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30Leu Lys lie Scr Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30
<210> 86 <211> 10 <212> PRT <213>智人 <400> 86<210> 86 <211> 10 <212> PRT <213> Homo sapiens <400> 86
Phe Giy Gin Gly Thr Lys Val Glu lie Lys <210> 87 <211> 130 <212> PRT <2i3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類Ig重鏈多肽 <400> 87Phe Giy Gin Gly Thr Lys Val Glu lie Lys <210> 87 <211> 130 <212> PRT <2i3> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human Ig Heavy Chain Peptide <400> 87
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ΊΙιγ Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ΊΙιγ Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Lea Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Lea Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Giy Ser Leu Scr Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Giy Ser Leu Scr Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Tlu Val 115 120 125 -25- 158864-序列表.doc 201215618Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Tlu Val 115 120 125 -25- 158864 - Sequence Listing.doc 201215618
Scr Ser 130 <210> 88 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類Ig重鏈多肽 <400> 88 GIu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Fro Gly Gly 15 10 15Scr Ser 130 <210> 88 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Affinity Mature Human Ig Heavy Chain Polypeptide <400> 88 GIu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Fro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Fro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Fro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Scr Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Scr Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly ΊΉγ Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly ΊΉγ Leu Val Thr Val 115 120 125
Ser Ser 130 <210> S9 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類lg重鏈多肽 <400> 89Ser Ser 130 <210> S9 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Affinity Mature Human lg Heavy Chain Polypeptide <400> 89
Glu Glu Gin Val Leu GIu Scr Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu GIu Scr Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Ser lie Asn Ser Asn Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 158864·序列表.doc - 26 - s 201215618Scr Ser lie Asn Ser Asn Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 158864 · Sequence Listing.doc - 26 - s 201215618
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 90 <211> 112 <212> PRT <213>智人 <400> 90Ser Ser 130 <210> 90 <211> 112 <212> PRT <213> Homo sapiens <400> 90
Asp lie Val Met Thr Gin Ser Pro Leu Ser Leu Ser Val Thr Pro GlyAsp lie Val Met Thr Gin Ser Pro Leu Ser Leu Ser Val Thr Pro Gly
Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Thr 20 25 30Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Thr 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Val Gin Lys Pro Gly Gin Ser 35 40 45Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Val Gin Lys Pro Gly Gin Ser 35 40 45
Pro Gin Leu Leu lie Tyr Leu Ala Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Pro Gin Leu Leu lie Tyr Leu Ala Ser Asn Arg Ala Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 'Fhr Leu Lys lie 65 70 75 80Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 'Fhr Leu Lys lie 65 70 75 80
Ser Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ala 85 90 95Ser Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ala 85 90 95
Leu Gin lie Pro Arg Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100 105 110 <210> 91 <211> 18 <212> PRT <213>智人 <400>91Leu Gin lie Pro Arg Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100 105 110 <210> 91 <211> 18 <212> PRT <213> Homo sapiens <400>91
Ser Ser lie Asn Ser Asp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 15 10 15Ser Ser lie Asn Ser Asp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 15 10 15
Lys Gly <210> 92 <211> 130 <212> PRT <213>智人 • 27- 158864-序列表.doc 201215618 <400> 92Lys Gly <210> 92 <211> 130 <212> PRT <213> Homo sapiens • 27-158864 - Sequence Listing.doc 201215618 <400> 92
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phc Thr Phe Ser Fro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phc Thr Phe Ser Fro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Asp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Asp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Vai Trp Gly Gin Gly Thr Uu Val Thr Val Π5 120 125Tyr Tyr Tyr Gly Leu Asp Vai Trp Gly Gin Gly Thr Uu Val Thr Val Π5 120 125
Ser Ser 130 <210> 93 <211> 18 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類ig重鏈肽 <220> <221> M0DJRES <222> (4)..(4) <223> Asn或 Ser <220>Ser Ser 130 <210> 93 <211> 18 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Affinity Mature Human ig Heavy Chain Peptide <220><221> M0DJRES <222> (4)..(4) <223> Asn or Ser <220>
<221> TOJES <222> (6)..(6) <223>任何胺基酸 <220><221> TOJES <222> (6)..(6) <223> Any amino acid <220>
<221> M0D_RES <222> (8)..(8) <223>任何胺基酸 <400> 93<221> M0D_RES <222> (8)..(8) <223> Any amino acid <400>
Ser Ser lie Xaa Ser Xaa Ser Xaa Tyr Lys Tyr Tyr Ala Asp Ser Val 1 5 10 . 15Ser Ser lie Xaa Ser Xaa Ser Xaa Tyr Lys Tyr Tyr Ala Asp Ser Val 1 5 10 . 15
Lys Gly <210> 94 <211> 130 <212> PRT <213>人工序列 28- 158864-序列表.doc 201215618 <220> <223>人工序列之說明:合成親和力成熟之人類Ig重鏈多肽 <220> <221> M0D.RES <222> (52)..(52) <223> A_sn或Ser <220〉 <22]> MOD RES <222> (54).,(54) <223>任何胺基酸 <220> <221> M0DJES <222> (56)..(56) <223>任何胺基酸 <400> 94Lys Gly <210> 94 <211> 130 <212> PRT <213> Artificial sequence 28-158864 - Sequence Listing.doc 201215618 <220><223> Description of artificial sequence: synthesis of affinity mature human Ig heavy chain polypeptide <220><221> M0D.RES <222> (52)..(52) <223> A_sn or Ser <220> <22]> MOD RES <222> (54)., (54) <223> Any amino acid <220><221> M0DJES <222> (56).. (56) <223> Any amino acid <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Xaa Ser Xaa Ser Xaa Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Xaa Ser Xaa Ser Xaa Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 95 <211> 22 <212> PR丁 <213>智人 <400> 95Ser Ser 130 <210> 95 <211> 22 <212> PR Ding <213> Homo sapiens <400> 95
Leu Glu Trp Val Ser Ser lie Ser Ser Ser Ser Ser Tyr lie Tyr Tyr 15 10 15Leu Glu Trp Val Ser Ser lie Ser Ser Ser Ser Tyr lie Tyr Tyr 15 10 15
Ala Asp Ser Val Lys Gly 20Ala Asp Ser Val Lys Gly 20
<210> 96 <211> 130 <212> PRT •29- 158864-序列表.doc 201215618 <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 96<210> 96 <211> 130 <212> PRT • 29-158864 - Sequence Listing. doc 201215618 <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC Peptide <400> 96
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phc Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phc Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Ser Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Ser Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val ITir Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val ITir Val 115 120 125
Ser Ser 130 <210> 97 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 97Ser Ser 130 <210> 97 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser lie Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser lie Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Tlir lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Tlir lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp ΊΉγ Ala Val Tyr Tyr Cys 85 90 95 -30- 158864-序列表.doc 201215618Leu Gin Met Asn Ser Leu Arg Ala Glu Asp ΊΉγ Ala Val Tyr Tyr Cys 85 90 95 -30- 158864 - Sequence Listing.doc 201215618
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Scr Ser 130 <210> 98 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 98Scr Ser 130 <210> 98 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Affinity Mature Human HC Polypeptide <400>
Glu Glu G!a Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu G! A Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu 丁rp Va] 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ding rp Va] 35 40 45
Ser Ser lie Asn Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 99 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 99Ser Ser 130 <210> 99 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Giu Glu Gin Vai E.eu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Giu Glu Gin Vai E.eu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr •31 158864-序列表.doc 201215618 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr • 31 158864 - Sequence Listing.doc 201215618 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Gin Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Gin Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe ITir lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe ITir lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 100 <2il> 130 <212> PCT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 100Ser Ser 130 <210> 100 <2il> 130 <212> PCT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Phe Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Phe Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Va] Trp Gly Gin Gly Thr Leu Val Thr Vai 115 120 125Tyr Tyr Tyr Gly Leu Asp Va] Trp Gly Gin Gly Thr Leu Val Thr Vai 115 120 125
Ser Ser 130 •32- 158864-序列表.doc 201215618 <210> 101 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 1〇]Ser Ser 130 • 32-158864 - Sequence Listing. doc 201215618 <210> 101 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 1〇]
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Met Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Met Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu G3n Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu G3n Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 ]〇5 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 ]〇5 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 102 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 102Ser Ser 130 <210> 102 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly G!y Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly G!y Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Vai Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Vai Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Asn Ser Leu Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Ser Leu Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 158864-序列表.doc -33- 201215618 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 158864 - Sequence Listing.doc -33- 201215618 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Ar£ Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp ]〇〇 105 110Ala Arg Asp Ar£ Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp ]〇〇 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Scr Ser 130 <210> 103 <231> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 103Scr Ser 130 <210> 103 <231> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Gly Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Gly Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Scr Ser 130 <210> 104 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 104 158864·序列表.doc -34-Scr Ser 130 <210> 104 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of affinity matured human HC polypeptide <400> 104 158864 · Sequence Listing.doc -34-
201215618201215618
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly ] 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly ] 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Scr Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Scr Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser His Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser His Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly rFhr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly rFhr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 105 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 105Ser Ser 130 <210> 105 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Asn Ser Lys Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Ser Lys Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val -35- 158864-序列表.doc 201215618 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val -35- 158864 - Sequence Listing.doc 201215618 115 120 125
Ser Ser 】30 <210> 106 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 106Ser Ser 30 <210> 106 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400> 106
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Trp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Trp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 107 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 107Ser Ser 130 <210> 107 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu 丁rp Val 35 40 45 -36- 158864-序列表.doc 201215618Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ding rp Val 35 40 45 -36- 158864 - Sequence Listing.doc 201215618
Ser Ser lie Asn Ser Tyr Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Tyr Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Tlir Val 115 320 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Tlir Val 115 320 125
Ser Ser 130 <210> 108 <213> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 108Ser Ser 130 <210> 108 <213> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Asn Ser Val Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Ser Val Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser lie Phe 05 70 75 80Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser lie Phe 05 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp TTir Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp TTir Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 109 <211> 130 <212> PRT <213>人工序列 -37- 158864-序列表.doc 201215618 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 109Ser Ser 130 <210> 109 <211> 130 <212> PRT <213> Artificial Sequence -37-158864 - Sequence Listing.doc 201215618 <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 109
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Va] Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Va] Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Ser Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Ser Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Uu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Uu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 110 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 110Ser Ser 130 <210> 110 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Giy Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Giy Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser vSer lie Ser Ser lie Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser vSer lie Ser Ser lie Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr ile Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr ile Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 -38-Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 -38-
158864-序列表.doc s 201215618158864-Sequence List.doc s 201215618
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 111 <2il> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 111Ser Ser 130 <210> 111 <2il> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Ser Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Ser Ser Asn Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ue Scr Arg Asp Asn Ala Glu Asn Scr He Phe 65 70 75 80Lys Gly Arg Phe Thr Ue Scr Arg Asp Asn Ala Glu Asn Scr He Phe 65 70 75 80
Leu Gin Met Asn Ser Lea Arg Ala Glu Asp rrhr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Lea Arg Ala Glu Asp rrhr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 112 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 112Ser Ser 130 <210> 112 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 39· 158864-序列表.doc 201215618Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 39· 158864-Sequence List.doc 201215618
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu 丁rp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ding rp Val 35 40 45
Ser Ser lie Ser Ser Gin Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Gin Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 113 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 113Ser Ser 130 <210> 113 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Phe Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Phe Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp ΊΤίγ Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp ΊΤίγ Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Set 130 -40- 158864-序列表.doc 201215618 <210> 114 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400〉 114Ser Set 130 - 40- 158864 - Sequence Listing. doc 201215618 <210> 114 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 114
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Met Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Met Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie PheLys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 115 <211> 130 <212> PRT <213>人工序列 <220>Ser Ser 130 <210> 115 <211> 130 <212> PRT <213> artificial sequence <220>
<223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 135<223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Leu Ser Tlir Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Leu Ser Tlir Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80 158864-序列表.doc -41- 201215618Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80 158864 - Sequence Listing.doc -41- 201215618
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Va丨 Trp Gly Gin Gly Thr Leu Va丨 Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Va丨 Trp Gly Gin Gly Thr Leu Va丨 Thr Val 115 120 125
Ser Ser 130 <210> 116 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 116Ser Ser 130 <210> 116 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Aia Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Aia Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Gly Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Gly Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 117 <211> 130 <212> PRT c2】3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 117Ser Ser 130 <210> 117 <211> 130 <212> PRT c2]3>Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15 -42- 158864-序列表.doc 201215618Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15 -42- 158864 - Sequence Listing.doc 201215618
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser His Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser His Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala G]u Asp Thr Ala Val 丁yr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala G]u Asp Thr Ala Val Dyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 118 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> Π8Ser Ser 130 <210> 118 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400> Π8
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Lys Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Lys Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125 -43 - 158864-序列表.doc 201215618Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125 -43 - 158864 - Sequence Listing.doc 201215618
Ser vSer 130 <210> 119 <211> 130 <212> PRT <2I3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 119Ser vSer 130 <210> 119 <211> 130 <212> PRT <2I3> artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Trp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Trp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 】00 105 120Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 】 00 105 120
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 120 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 120Ser Ser 130 <210> 120 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly l 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly l 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Tyr Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 -44 - 158864-序列表.doc 201215618Ser Ser lie Ser Ser Tyr Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 -44 - 158864 - Sequence Listing.doc 201215618
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 121 <211> 130 <212> PRT <2i3>人工序列 <220〉Ser Ser 130 <210> 121 <211> 130 <212> PRT <2i3> artificial sequence <220>
<223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 121<223> Description of artificial sequence: Synthesis of affinity mature human HC polypeptide <400> 121
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Vai 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Tip Vai 35 40 45
Ser Ser lie Ser Ser Val Scr Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Val Scr Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Mel Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 丁yr Cys 85 90 95Leu Gin Mel Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ding yr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 Π0Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 Π0
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 122 <211> 130 <212> PRT <213>人工序列 合成親和力成熟之人類HC多肽 • 45- <220> <223>人工序列之說明 158864-序列表.doc 201215618 <40ft> 122Ser Ser 130 <210> 122 <211> 130 <212> PRT <213> Artificial sequence synthesis affinity matured human HC polypeptide • 45- <220><223> Description of artificial sequence 158864 - Sequence Listing .doc 201215618 <40ft> 122
Glu Glu Gin Val Leu Glu Ser Giy Gly Gly Leu Val Lys Pro Oly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Giy Gly Gly Leu Val Lys Pro Oly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Ser Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Ser Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Giu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Giu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp m 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp m 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 123 <211> 130 <212> PRT <2】3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 123Ser Ser 130 <210> 123 <211> 130 <212> PRT <2]3>Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400> 123
Glu Glu Gin Val Leu Giu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Giu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser lie Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser lie Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110 •46· 158864-序列表.doc 201215618Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110 •46· 158864-Sequence List.doc 201215618
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 124 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 124Ser Ser 130 <210> 124 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Scr Gly Gly Gly Leu Val Lys Fro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Scr Gly Gly Gly Leu Val Lys Fro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Asn Ser Arg 丁yr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Asn Ser Arg Dingyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Scr lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Scr lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Va】Tyr Tyr Cys 85 9Q 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Va】Tyr Tyr Cys 85 9Q 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 125 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 125Ser Ser 130 <210> 125 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400> 125
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 158864-序列表.doc -47- 201215618 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 158864 - Sequence Listing.doc -47- 201215618 35 40 45
Ser Ser He Asn Set Gin Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Set Gin Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Giu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Giu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Va] Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Va] Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210〉 126 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 126Ser Ser 130 <210> 126 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe 丁rp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 vSer Ser lie Asn Ser Phe Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Val Phe Ding rp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 vSer Ser lie Asn Ser Phe Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Hir Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Hir Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Oly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Oly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 127 <211> 130 -48· 158864·序列表.doc 201215618 <212> PRT <2i3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 127Ser Ser 130 <210> 127 <211> 130 -48·158864·Sequence List.doc 201215618 <212> PRT <2i3> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 127
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Met Ser Arg Tyr Lys Tyr Tyr Ala Asp Scr Val 50 55 60Ser Ser lie Asn Ser Met Ser Arg Tyr Lys Tyr Tyr Ala Asp Scr Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val 丁rp Gly Gin Giy Thr Leu Vai Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Ding rp Gly Gin Giy Thr Leu Vai Thr Val 115 120 125
Ser Ser 130 <210> 128 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 128Ser Ser 130 <210> 128 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser ile Asn Ser Leu Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser ile Asn Ser Leu Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys -49- 158864·序列表.doc 201215618 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys -49- 158864 · Sequence Listing.doc 201215618 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Uu Scr Asp 100 〗05 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Uu Scr Asp 100 〗 05 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 129 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 129Ser Ser 130 <210> 129 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Fro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Fro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Gly Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Gly Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Giu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Scr Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Scr Asp 100 105 110
Tyr 丁yr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val IIS 120 125Tyr Dyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val IIS 120 125
Ser Ser 130 <210> 130 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 130Ser Ser 130 <210> 130 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15 -50- 158864-序列表.doc 201215618Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15 -50- 158864 - Sequence Listing.doc 201215618
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser His Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser His Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Va! Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Va! Thr Val 115 120 125
Ser Ser 130 <210> 131 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 131Ser Ser 130 <210> 131 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Fro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Fro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Lys Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Lys Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 Π0Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 Π0
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Scr -51 - 158864-序列表.doc 201215618 130 <210> 132 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 132Ser Scr -51 - 158864 - Sequence Listing.doc 201215618 130 <210> 132 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 132
Olu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Olu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser Tie Asn Ser Trp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser Tie Asn Ser Trp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val 丁rp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Ding rp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 230 <210> 133 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 133Ser Ser 230 <210> 133 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Tyr Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 -52- 158864-序列表,doc 201215618Ser Ser lie Asn Ser Tyr Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 -52- 158864 - Sequence Listing, doc 201215618
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 1】0Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 1]0
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 134 <211> 130 <212> PRT <213>人工序列 <220>Ser Ser 130 <210> 134 <211> 130 <212> PRT <213> artificial sequence <220>
<223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 134<223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ττρ Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ττρ Val 35 40 45
Ser Ser lie Asn Ser Val Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Val Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Aia Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Aia Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <21.0> 135 <211> 130 <212> PRT <213>人工序列 <220〉 <223>人工序列之說明:合成親和力成熟之人類HC多肽 -53- 158864-序列表.doc 201215618 <400> 135Ser Ser 130 <21.0> 135 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Affinity Mature Human HC Polypeptide-53-158864- Sequence Listing.doc 201215618 <400> 135
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Ser Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Ser Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp loo 105 noAla Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp loo 105 no
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <230> 136 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 136Ser Ser 130 <230> 136 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Set Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Set Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Asn Ser lie Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Ser lie Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110 -54- 158864-序列表.doc 201215618Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110 -54- 158864 - Sequence Listing.doc 201215618
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 137 <211> 130 <212〉 PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 337Ser Ser 130 <210> 137 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ή;γ Phc Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ή; γ Phc Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Asn Ser Asn Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Ser Asn Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 SOLys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 SO
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 138 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 138Ser Ser 130 <210> 138 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 -55· 158864-序列表.doc 201215618Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 -55· 158864 - Sequence Listing.doc 201215618
Ser Ser He Asn Ser Gin Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Ser Gin Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Ixu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Ixu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 320 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 320 125
Ser Ser 130 <2I0> 139 <211> 130 <212> PRT <2]3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 139Ser Ser 130 <2I0> 139 <211> 130 <212> PRT <2]3>Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400> 139
Glu Glu Gin Val Leu Glu Ser Gly Giy Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Giy Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Phe Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Phe Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Tlir Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Tlir Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp (Jly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp (Jly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 140 <211> 130 <212> PRT <213>人工序列 -56- 158864-序列表.doc 201215618 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 140Ser Ser 130 <210> 140 <211> 130 <212> PRT <213> Artificial Sequence -56-158864 - Sequence Listing.doc 201215618 <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 140
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Met Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Met Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Scr 130 <210> 141 <211> 130 <212> PRT <2i3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 141Ser Scr 130 <210> 141 <211> 130 <212> PRT <2i3> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Ser lie Asn Ser Leu Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Scr Ser lie Asn Ser Leu Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 •57- 158864·序列表.doc 201215618Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 •57- 158864 · Sequence Listing.doc 201215618
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 142 <211> 130 <212> PRT <2I3>人工序列 <22Q> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 142Ser Ser 130 <210> 142 <211> 130 <212> PRT <2I3> artificial sequence <22Q><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Gly Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Gly Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Scr He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Scr He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 143 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 143Ser Ser 130 <210> 143 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Va] Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Va] Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 -58- 158864-序列表.doc 201215618Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 -58- 158864 - Sequence Listing.doc 201215618
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser His Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser His Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 . 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 . 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Tin Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Tin Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 144 <211> 130 <212> PRT <2i3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <4〇〇> 144Ser Ser 130 <210> 144 <211> 130 <212> PRT <2i3> artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <4〇〇> ; 144
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu 丁rp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ding rp Val 35 40 45
Ser Ser lie Asn Ser Lys Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Lys Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 1]5 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 1]5 120 125
Ser Ser 130 -59- 158864-序列表.doc 201215618 <210> 145 <213> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 145Ser Ser 130 -59- 158864 - Sequence Listing.doc 201215618 <210> 145 <213> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 145
Glu Glu Gin Vai Leu Glu Ser Gly Gly Gly Leu Val Lys Fro Gly Gly 15 10 15Glu Glu Gin Vai Leu Glu Ser Gly Gly Gly Leu Val Lys Fro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Scr Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Scr Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Trp Ser Lys 丁yr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Trp Ser Lys Ding yr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 146 <21l> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 146Ser Ser 130 <210> 146 <21l> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 Ϊ5Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 Ϊ5
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Asn Ser Tyr Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Asn Ser Tyr Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80 -60· 158864-序列表,doc 201215618Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80 -60· 158864 - Sequence Listing, doc 201215618
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 147 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 147Ser Ser 130 <210> 147 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Scr lie Asn Scr Val Ser Lys Tyr Lys Tyr Tyr Ala Asp Set Val 50 55 60Scr Scr lie Asn Scr Val Ser Lys Tyr Lys Tyr Tyr Ala Asp Set Val 50 55 60
Lys Gly Arg Phe Thr I】e Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr I】e Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser 丁yr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Dyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Scr Ser 130 <210> 148 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 148Scr Ser 130 <210> 148 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly -61 - 158864-序列表.doc 201215618 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly -61 - 158864 - Sequence Listing.doc 201215618 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Va) Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Va) Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Ser Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Ser Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp !00 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp !00 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val TTir Val 115 320 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val TTir Val 115 320 125
Ser Ser 130 <210> 149 <211> 130 <212> PRT <2I3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 149Ser Ser 130 <210> 149 <211> 130 <212> PRT <2I3> artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Ser Ser lie Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Ser Ser lie Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125 -62- 158864-序列表.doc 201215618Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125 -62- 158864 - Sequence Listing.doc 201215618
Ser Ser 130 <210> 150 <211> 330 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 150Ser Ser 130 <210> 150 <211> 330 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu GIu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu GIu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Giy Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Giy Leu Glu Trp Val 35 40 45
Ser Scr lie Ser Ser Asn Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Scr lie Ser Ser Asn Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Tht lie Ser Arg Asp Asn Ala Glu Asn Scr lie Phe 65 70 75 80Lys Gly Arg Phe Tht lie Ser Arg Asp Asn Ala Glu Asn Scr lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 205 110Ala Arg Asp Arg Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 205 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 151 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 151Ser Ser 130 <210> 151 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 】5Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 】5
Ser Leu Arg Leu Scr Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Scr Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Gin Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 158864-序列表.doc -63- 201215618 50 55 60Ser Ser lie Ser Ser Gin Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 158864 - Sequence Listing.doc -63- 201215618 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 315 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 315 120 125
Ser Ser 130 <210> 152 <211> 230 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 152Ser Ser 130 <210> 152 <211> 230 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Giy Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Giy Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Phe Ser Ar£ Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Phe Ser Ar £ Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp A$n Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp A$n Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Aia Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Aia Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 】00 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 】 00 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 153 <211> 130 <212> PRT <213>人工序列 <220> -64- 158S64_ 序列表.doc 201215618 <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 153Ser Ser 130 <210> 153 <211> 130 <212> PRT <213> Artificial Sequence <220> -64- 158S64_ Sequence Listing.doc 201215618 <223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 153
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Scr Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Scr Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe 丁rp Val Arg G]n Ala Pro Giy Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Ding rp Val Arg G]n Ala Pro Giy Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Met Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Met Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Scr Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Scr Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly ITir Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly ITir Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 154 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 154Ser Ser 130 <210> 154 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly GJy Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly GJy Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Scr Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Scr Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Scr lie Ser Ser Leu Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Scr Scr lie Ser Ser Leu Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 SOLys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 SO
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 158864-序列表.doc -65- s· 201215618 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 158864 - Sequence Listing.doc -65- s· 201215618 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <2I0> 155 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 155Ser Ser 130 <2I0> 155 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Set Gly Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Set Gly Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr AU Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr AU Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 156 <211> 130 <212> PRT <213>人工序列 <220〉 <223>人工序列之說明:合成親和力成熟之人類HC多肽 <>156Ser Ser 130 <210> 156 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 66-Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 66-
158864-序列表.doc s 201215618158864-Sequence List.doc s 201215618
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser His Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser His Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser He Phc 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser He Phc 65 70 75 80
Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phc Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phc Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Giy llir Leu Val Thr Val 115 320 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Giy llir Leu Val Thr Val 115 320 125
Ser Ser 130 <230> 157 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <4〇〇> 157Ser Ser 130 <230> 157 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <4〇〇> ; 157
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Lys Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Lys Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Scr Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Scr Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Giy Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Giy Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 158 -67- 158864·序列表.doc 201215618 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 158Ser Ser 130 <210> 158 -67- 158864 · Sequence Listing.doc 201215618 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 158
Glu Glu Gin Val Leu Glu Scr Gly Gly Gly Leu Val Lys Pro Gly Gly 】 5 10 15Glu Glu Gin Val Leu Glu Scr Gly Gly Gly Leu Val Lys Pro Gly Gly 】 5 10 15
Ser Leu Ar£ Leu Ser Cys Ala Ala Ser Gly Phc Thr Phe Ser Pro Tyr 20 25 30Ser Leu Ar£ Leu Ser Cys Ala Ala Ser Gly Phc Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Trp Ser Arg Tyr Lys Tyf Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Trp Ser Arg Tyr Lys Tyf Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 159 <211> 130 <2i2> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 159Ser Ser 130 <210> 159 <211> 130 <2i2> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Giy Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Giy Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Aia Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Aia Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Scr lie vSer Ser Tyr Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Va] 50 55 60Ser Scr lie vSer Ser Tyr Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Va] 50 55 60
Lys Gly Arg Phe Thr IJe Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80 -68- 158864-序列表.doc 201215618Lys Gly Arg Phe Thr IJe Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80 -68- 158864 - Sequence Listing.doc 201215618
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <21〇> 160 <211> 130 <212> FRT <2]3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 160Ser Ser 130 <21〇> 160 <211> 130 <212> FRT <2]3> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Affinity Mature Human HC Polypeptide<400> 160
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phc Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phc Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Val Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Val Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser Me Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser Me Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr 丁yr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Va] Thr Val 115 120 125Tyr Tyr Dyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Va] Thr Val 115 120 125
Ser Ser 130 <210> 161 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 161Ser Ser 130 <210> 161 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Giu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15 •69- 158864·序列表.doc 201215618Glu Glu Gin Val Leu Giu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15 •69- 158864 · Sequence Listing.doc 201215618
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Fhe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Fhe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Ser Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Ser Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 162 <211> 130 <212> PKT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 162Ser Ser 130 <210> 162 <211> 130 <212> PKT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Vai Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Vai Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ata Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ata Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser He Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser He Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125 -70- 158864-序列表.doc 201215618Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125 -70- 158864 - Sequence Listing.doc 201215618
Scr Ser 130 <210> 163 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 163Scr Ser 130 <210> 163 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly G!y 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly G!y 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Asn Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser ValSer Ser lie Ser Ser Asn Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val
Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 80
Leu Girt Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Girt Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 164 <211> 130 <212> FRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 164Ser Ser 130 <210> 164 <211> 130 <212> FRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Scr Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Scr Pro Tyr 20 25 30
Scr Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Scr Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Ser Ser Gin Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Ser Ser Gin Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
158864-序列表.doc -71 - 201215618158864 - Sequence Listing.doc -71 - 201215618
Lys Giy Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Giy Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Ixu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Ixu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Giy Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Giy Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Giy Leu Asp Va] Trp Giy Gin Giy Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Giy Leu Asp Va] Trp Giy Gin Giy Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 165 <211> 130 <212> PRT <213>人工序列 <220>Ser Ser 130 <210> 165 <211> 130 <212> PRT <213> artificial sequence <220>
<223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 165<223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Giy Giy Giy Leu Val Lys Pro Giy Giy 15 10 15Glu Glu Gin Val Leu Glu Ser Giy Giy Giy Leu Val Lys Pro Giy Giy 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Giy Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Giy Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Giy Lys Giy Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Giy Lys Giy Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Phe Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Phe Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Giy Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Giy Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Giy Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Giy Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Giy Leu Asp Val Trp Giy Gin Giy Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Giy Leu Asp Val Trp Giy Gin Giy Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 166 <211> 130 <212> PRT <213>人工序列 <220> <223〉人工序列之說明:合成親和力成熟之人類HC多肽 158864·序列表.doc -72-Ser Ser 130 <210> 166 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of Affinity Mature Human HC Polypeptide 158864 Sequence Listing.doc -72-
S 201215618 <400> 166S 201215618 <400> 166
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Fro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Fro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Ser lie Ser Ser Met Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Scr Ser lie Ser Ser Met Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 】30 <210> 167 <211> 130 <232> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 167Ser Ser 30 <210> 167 <211> 130 <232> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Scr lie Ser Ser Leu Ser lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Scr Scr lie Ser Ser Leu Ser lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Aig Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Aig Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110 -73- 158864-序列表.doc 201215618Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110 -73- 158864 - Sequence Listing.doc 201215618
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 168 <2Π> 130 <232> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 168Ser Ser 130 <210> 168 <2Π> 130 <232> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Giu Glu Gin Val Leu Glu Ser Gly Gly Gly I^eu Val Lys Pro Gly Gly 15 10 15Giu Glu Gin Val Leu Glu Ser Gly Gly Gly I^eu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Scr Gly Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Scr Gly Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin G]y Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin G]y Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 169 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 169Ser Ser 130 <210> 169 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe 丁rp Va】Arg Gin Ala Pro Gly Lys Gly Leu G】u Trp Vai 35 40 45 •74-Ser Val Phe Ding rp Va】Arg Gin Ala Pro Gly Lys Gly Leu G】u Trp Vai 35 40 45 •74-
158864-序列表.doc 201215618158864-SEQ ID NO.doc 201215618
Ser Ser lie Ser Ser His Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser His Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Scr Ser 130Scr Ser 130
<210> 170 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 170<210> 170 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Gla Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Gla Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Scr Scr Lys Scr Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Scr Scr Lys Scr Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Scr Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp llir Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp llir Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Uu Scr Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Uu Scr Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130Ser Ser 130
<210> 171 <211> 130 <212> PRT -75- 158864-序列表.doc 201215618 <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 171<210> 171 <211> 130 <212> PRT -75-158864 - Sequence Listing.doc 201215618 <213>Artificial Sequence<220><223> Description of Artificial Sequence: Synthesis of Mature Human HC Peptide <400> 171
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Ar^ Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Ar^ Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Trp Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Trp Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val 丁rp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Ding rp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 172 <212> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 172Ser Ser 130 <210> 172 <212> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Tyr Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Tyr Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Jle Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr Jle Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Tlir Ala Val Tyr Tyr Cys 85 90 95 -76- 158864-序列表.doc 201215618Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Tlir Ala Val Tyr Tyr Cys 85 90 95 -76- 158864 - Sequence Listing.doc 201215618
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val rrhr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val rrhr Val 115 120 125
Ser Ser 130 <230> 173 <211> 130 <212> PRT <2i3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 173Ser Ser 130 <230> 173 <211> 130 <212> PRT <2i3> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Fro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Fro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ττρ Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Ττρ Val 35 40 45
Ser Ser lie Ser Ser Val Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Val Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 174 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 174Ser Ser 130 <210> 174 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr -77- 158864-序列表.doc 201215618 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr -77- 158864 - Sequence Listing.doc 201215618 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Ser lie Asn Ser Ala Ser Tlir Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Scr Ser lie Asn Ser Ala Ser Tlir Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Scr Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 175 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 175Ser Ser 130 <210> 175 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Lea Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Lea Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Ala Ser Ήιγ Tyr Lys Tyr Tyr Ala Asp Ser Val SO 55 60Ser Ser lie Ser Ser Ala Ser Ήιγ Tyr Lys Tyr Tyr Ala Asp Ser Val SO 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr teu Val Thr Val Π5 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr teu Val Thr Val Π5 120 125
Ser Ser 130 -78- 158864-序列表.doc 201215618 <210> Ϊ76 <21I> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 176 GIu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly I 5 10 15Ser Ser 130 -78- 158864 - Sequence Listing.doc 201215618 <210> Ϊ76 <21I> 130 <212> PRT <213>Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic Affinity Mature Human HC polypeptide <400> 176 GIu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly I 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala vSer Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala vSer Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Scr Ser Ile'Asn Ser Ala Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Scr Ser Ile'Asn Ser Ala Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ne Ser Arg Asp Asn A!a Glu Asn Ser He Phe 65 70 75 80Lys Gly Arg Phe Thr Ne Ser Arg Asp Asn A!a Glu Asn Ser He Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr 丁yr Gly Leu Asp Val Trp Gly G】n G]y Thr Leu Val Thr Val 115 320 125Tyr Tyr Dyr yr Gly Leu Asp Val Trp Gly G]n G]y Thr Leu Val Thr Val 115 320 125
Ser Ser 130 <210> 177 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 177Ser Ser 130 <210> 177 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15Glu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Ala Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Ala Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe llir lie Ser Arg Asp Asn Ala Glu Asn Scr lie Phe 158864-序列表.doc - 79 - 201215618 65 70 75 80Lys Gly Arg Phe llir lie Ser Arg Asp Asn Ala Glu Asn Scr lie Phe 158864 - Sequence Listing.doc - 79 - 201215618 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr G】y Leu Asp Val Trp Gly Gin G!y Thr Leu Val 丁hr Val 115 120 125Tyr Tyr Tyr G】y Leu Asp Val Trp Gly Gin G!y Thr Leu Val Ding hr Val 115 120 125
Ser Ser 130 <210> 178 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 178Ser Ser 130 <210> 178 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Scr Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Scr Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Ala Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Ala Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Scr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Giy Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Giy Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 179 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 179 •80- 158864-序列表.doc 201215618Ser Ser 130 <210> 179 <211> 130 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthesis of affinity matured human HC polypeptide <400> 80-158864-Sequence List.doc 201215618
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Ala Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Ala Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Scr Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 180 <211> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 180Ser Ser 130 <210> 180 <211> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Set Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Set Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser ile Asn Ser Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser ile Asn Ser Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val -81 - 158864-序列表.doc 201215618 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val -81 - 158864 - Sequence Listing.doc 201215618 115 120 125
Ser Ser 130 <2i0> 181 <211> 130 <212> PRT <2]3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 181Ser Ser 130 <2i0> 181 <211> 130 <212> PRT <2]3>Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400> 181
Glu Glu Gin Va) Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly G!y 15 10 35Glu Glu Gin Va) Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly G!y 15 10 35
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Ser Ser Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Ser Ser Asp Ser Arg Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 SOLys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser He Phe 65 70 75 SO
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 noAla Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 no
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 182 <2il> 130 <212> PRT <2i3>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 182Ser Ser 130 <210> 182 <2il> 130 <212> PRT <2i3> artificial sequence <220><223> Description of artificial sequence: synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Giy 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Giy 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 -82- 158864-序列表.doc 201215618Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 -82- 158864 - Sequence Listing.doc 201215618
Ser Ser lie Asn Ser Asp Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Asp Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala GIu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala GIu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110Ala Arg Asp Arg Scr Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125
Ser Ser 130Ser Ser 130
<210> 1S3 <21!> 130 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 183<210> 1S3 <21!> 130 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of affinity matured human HC polypeptide <400>
Glu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu GIu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser He Ser Ser Asp Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser He Ser Ser Asp Ser Lys Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala GIu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala GIu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110 丁yr 丁yr Tyr Gly Leu Asp Val Trp Gly Gin G!y Thr Leu Val Thr Val 115 120 125Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Ser Leu Ser Asp 100 105 110 Dingyr Dyr Tyr Gly Leu Asp Val Trp Gly Gin G!y Thr Leu Val Thr Val 115 120 125
Ser Ser 130 <210> 184 <211) 130 <212> PRT <213>人工序列 -83- 158864-序列表.doc 201215618 <220> <223>人工序列之說明:合成親和力成熟之人類HC多肽 <400> 184Ser Ser 130 <210> 184 <211) 130 <212> PRT <213> Artificial sequence -83-158864 - Sequence Listing.doc 201215618 <220><223> Description of artificial sequence: synthetic affinity mature Human HC polypeptide <400> 184
Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15Glu Glu Gin Val Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30
Scr Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Scr Val Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser lie Asn Ser Asp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60Ser Ser lie Asn Ser Asp Ser Thr Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Glu Asn Ser lie Phe 65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Sex Leu Ser Asp 100 105 110Ala Arg Asp Arg Ser Tyr Tyr Ala Phe Ser Ser Gly Sex Leu Ser Asp 100 105 110
Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 1.15 120 125Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gin Gly Thr Leu Val Thr Val 1.15 120 125
Ser Ser 130 <210> 185 <211> 366 <212> DNA <213>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白重鏈多肽 <220> <221> (DS <222> (1)..(366) <400> 185 gag gtt cag ctg gtg cag tct ggc gcc gaa gtg aaa aaa cca ggg gcc 48 G]u Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tea gtg aaa gtg tcc tgt aaa get tct ggc tac acc tic acc aac tac 96Ser Ser 130 <210> 185 <211> 366 <212> DNA <213>Artificial sequence <220><223> Description of artificial sequence: synthetic humanized 8G8 immunoglobulin heavy chain polypeptide <220>;<221> (DS <222> (1)..(366) <400> 185 gag gtt cag ctg gtg cag tct ggc gcc gaa gtg aaa aaa cca ggg gcc 48 G]u Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tea gtg aaa gtg tcc tgt aaa get tct ggc tac acc tic acc aac tac 96
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 ggc atg aac Xgg gtg cgt cag gcc ccg ggt cag ggc ctg gaa tgg ate 144Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 ggc atg aac Xgg gtg cgt cag gcc ccg ggt cag ggc ctg gaa tgg ate 144
Gly Met Asn Trp Val Arg Gla Ala Pro Gly Gin Gly Leu Glu Trp lie 35 40 45 ggc tgg ate aac acc tac acc ggc gaa cca acc tac gcc gat gat ttc 192Gly Met Asn Trp Val Arg Gla Ala Pro Gly Gin Gly Leu Glu Trp lie 35 40 45 ggc tgg ate aac acc tac acc ggc gaa cca acc tac gcc gat gat ttc 192
Gly Trp lie Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 aaa ggc cgt gtg act ala acc cgt gac acc tcc acc age aca gcc tac 240Gly Trp lie Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 aaa ggc cgt gtg act ala acc cgt gac acc tcc acc age aca gcc tac 240
Lys 6Ty Arg VaT Thr lie Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 -84 158864-序列表.doc 201215618 eta gaa ctg age age tta aga age gag gac act gee gtc tat tat tgc 288Lys 6Ty Arg VaT Thr lie Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 -84 158864 - Sequence Listing.doc 201215618 eta gaa ctg age age tta aga age gag gac act gee gtc tat tat tgc 288
Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 geg cgt age tgg tac tac gtg age aac tac tgg lac ttc gat gtg tgg 336Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 geg cgt age tgg tac tac gtg age aac tac tgg lac ttc gat gtg tgg 336
Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 105 110 ggt caa gga acc ctg gtc acc gtc tee teg 366Ala Arg Ser Trp Tyr Tyr Val Ser Asn Tyr Trp Tyr Phe Asp Val Trp 100 105 110 ggt caa gga acc ctg gtc acc gtc tee teg 366
Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 320 <210> 186 <211> 345 <212> DNA <213>人工序列 <220> <223>人工序列之說明:合成人類化8G8免疫球蛋白輕鏈(突變)多肽Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 320 <210> 186 <211> 345 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Humanized 8G8 Immunoglobulin light chain (mutant) polypeptide
<220> <221> CDS <222> (1)..(345) <400> 186 let gtg ctg acc cag age cca age gee age gee age ctg ggc gee age Ser Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser 1 5 10 15 gtg aaa ctg acc igc acc ctg age age cag cac age acc tac acc ate Val Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr lie 20 25 30 gaa tgg tat cag cag cag cca ggc aaa ggc cca ege tac ctg atg aaa Glu Trp Tyr Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met Lys 35 40 45 ctg aaa aaa gat ggc age cac age acc ggc gat ggc ate cca gat ege Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly He Pro Asp Arg 50 55 60 ttc age ggc age age age ggc gee gat ege tac ctg acc ate age aac Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr lie Ser Asn 65 70 75 80 ctg cag age gaa gat gaa gee gat tac tac tgc ggc gtg ggc gat acc Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr 85 90 95 ate aaa gaa cag ttc gtg tac gtg tic ggc ggc ggt acc aaa ctg acc lie Lys Glu Gin Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr 100 105 110 48 96 144 192 240 gtg ctg ggc 288 336 345<220><221> CDS <222> (1)..(345) <400> 186 let gtg ctg acc cag age cca age gee age gee age ctg ggc gee age Ser Val Leu Thr Gin Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser 1 5 10 15 gtg aaa ctg acc igc acc ctg age age cag cac age acc tac acc ate Val Lys Leu Thr Cys Thr Leu Ser Ser Gin His Ser Thr Tyr Thr lie 20 25 30 gaa tgg tat Cag cag cag cca ggc aaa ggc cca ege tac ctg atg aaa Glu Trp Tyr Gin Gin Gin Pro Gly Lys Gly Pro Arg Tyr Leu Met Lys 35 40 45 ctg aaa aaa gat ggc age cac age acc ggc gat ggc ate cca gat ege Leu Lys Lys Asp Gly Ser His Ser Thr Gly Asp Gly He Pro Asp Arg 50 55 60 ttc age ggc age age ggc gee gat ege tac ctg acc ate age aac Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr lie Ser Asn 65 70 75 80 ctg cag age gaa gat gaa gee gat tac tac tgc ggc gtg ggc gat acc Leu Gin Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr 85 90 95 ate aaa gaa cag ttc gtg tac gtg tic ggc ggc ggt Acc aaa ctg acc lie Lys Glu Gin Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr 100 105 110 48 96 144 192 240 gtg ctg ggc 288 336 345
Val Leu Gly 115 <210> 187 <211> 22 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成IGHV3-21*01肽 <400〉 187Val Leu Gly 115 <210> 187 <211> 22 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: Synthesis of IGHV3-21*01 peptide <400> 187
Leu Glu Trp Val Ser Ser lie Ser Ser Ser Ser Ser Tyr lie Tyr Tyr 15 10 15 '•mi 15_8864-序列表.doc -85- 201215618 A.a Asp Ser Val Lys Gly <210> 188 <211> 22 <212> PRT <2】3>人工序列 <220> <223>人工序列之說明:合成MSL-109肽 <220> <221〉MOD RES <222> (10)..(10) <223〉入sp、$er、Thr、Asn、Gin、Phe、Met 或Leu <220> <221> MOD RES <222> (12)..(12) <223> Thr或人rg <400> 188 Leu Glu Trp Val Ser Ser lie Asn Ser Xaa Ser Xaa Tyr Lys Tyr Tyr 1 5 10 15Leu Glu Trp Val Ser Ser lie Ser Ser Ser Ser Serr Tyr Tyr 15 10 15 '•mi 15_8864-Sequence List.doc -85- 201215618 Aa Asp Ser Val Lys Gly <210> 188 <211> 22 <212> PRT <2]3>Artificial sequence <220><223> Description of artificial sequence: Synthesis of MSL-109 peptide <220><221>MOD RES <222> (10).. (10 <223> into sp, $er, Thr, Asn, Gin, Phe, Met or Leu <220><221> MOD RES <222> (12)..(12) <223> Thr or Human rg <400> 188 Leu Glu Trp Val Ser Ser lie Asn Ser Xaa Ser Xaa Tyr Lys Tyr Tyr 1 5 10 15
Ala Asp Ser Val Lys Gly 20 <210> 189 <211> 15 <212> IM <213>人工序列 <220> <223>人工序列之說明:合成寡核苷酸 <400> 189 tcgcgcccga agagg 15 <210> 190 <2Π> 17 <212> DNA <213>人工序列 <220> <223>人工序列之說明:合成寡核苷酸Ala Asp Ser Val Lys Gly 20 <210> 189 <211> 15 <212> IM <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Oligonucleotide <400> 189 tcgcgcccga agagg 15 <210> 190 <2Π> 17 <212> DNA <213> Artificial sequence <220><223> Description of artificial sequence: synthetic oligonucleotide
<400> 190 cggccggatt gtggatt 17 <210> 191 <211> 23 <212> DNA <213>人工序列 <220> <223>人工序列之說明:合成寡核苷酸 <400> 191 caccgacgag gaticcgaca acg <210> 192 23 158864-序列表.doc -86- s 201215618 <211> 28 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成肽 <400> 192<400> 190 cggccggatt gtggatt 17 <210> 191 <211> 23 <212> DNA <213> Artificial sequence <220><223> Description of artificial sequence: synthetic oligonucleotide <400> 191 caccgacgag gaticcgaca acg <210> 192 23 158864 - Sequence Listing. doc -86-s 201215618 <211> 28 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence : synthetic peptide <400> 192
Thr Ala Glu Lys Asn Asp Tyr Tyr Arg Val Pro His Tyr Trp Asp Ala 1 5 10 15Thr Ala Glu Lys Asn Asp Tyr Tyr Arg Val Pro His Tyr Trp Asp Ala 1 5 10 15
Cys Ser Arg Ala Leu Pro Asp Gin Thr Arg Tyr Lys 20 25 <210> 193 <211> 28 <2J2> PRT <2]3>人工序列 <220> <223>人工序列之說明:合成肽Cys Ser Arg Ala Leu Pro Asp Gin Thr Arg Tyr Lys 20 25 <210> 193 <211> 28 <2J2> PRT <2]3> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic peptide
<400> 193<400> 193
Cys Pro His Val Trp Met Pro Pro Gin Thr Thr Pro His Asp Trp Lys 15 10 15Cys Pro His Val Trp Met Pro Pro Gin Thr Thr Pro His Asp Trp Lys 15 10 15
Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His 20 25 <210> 194 <211> 28 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成肽 <400> 194Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His 20 25 <210> 194 <211> 28 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Peptide <400> 194
Ser Arg Ala Leu Pro Asp Gin Thr Arg Tyr Lys Tyr Val Glu Gin Leu 15 10 15Ser Arg Ala Leu Pro Asp Gin Thr Arg Tyr Lys Tyr Val Glu Gin Leu 15 10 15
Val Asp Leu Thr Leu Asn Tyr His Tyr Asp Ala Ser 20 25 <210> 195 <211> 29 <212> PRT <213:>人工序列 <220> <223>人工序列之說明:合成肽 <400> 195Val Asp Leu Thr Leu Asn Tyr His Tyr Asp Ala Ser 20 25 <210> 195 <211> 29 <212> PRT <213:> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic peptide <400> 195
His Pro His His Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser 1 5 10 15His Pro His His Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser 1 5 10 15
Ser Gly Arg Arg Asp His Ser Leu Glu Arg Leu Thr Arg 20 25 <210> 196 <211> 28 <212> PRT <213>人工序列 -87- 158864·序列表.doc 201215618 <220> <223>人工序列之說明: <400> 196 Cys Pro His Val Trp MetSer Gly Arg Arg Asp His Ser Leu Glu Arg Leu Thr Arg 20 25 <210> 196 <211> 28 <212> PRT <213> Artificial Sequence -87-158864 · Sequence Listing.doc 201215618 <220><223> Description of the artificial sequence: <400> 196 Cys Pro His Val Trp Met
Gly Ser His Thr Thr Ser 20 合成肽 Pro Pro G!n Thr Thr Pro His Asp Trp Lys 10 15 Gly Leu His Arg Pro His 25 <210> 197 <211> 28 <212> PRT <213>人工序列 <220> <223>人工序列之說明: <400〉 197 Cys Gly Leu Pro Pro GluGly Ser His Thr Thr Ser 20 Synthetic Peptide Pro Pro G!n Thr Thr Pro His Asp Trp Lys 10 15 Gly Leu His Arg Pro His 25 <210> 197 <211> 28 <212> PRT <213> Sequence <220><223> Description of the artificial sequence: <400> 197 Cys Gly Leu Pro Pro Glu
His Ser Arg Tyr Gly Pro 20 合成肽 Leu Lys Gin Thr Arg Val Asn Leu Pro Ala 10 35 Gin Ala Val Asp Ala Arg 25 > > > > > 012 3 03 11 Τ* 11 ΙΑ ΟΛ <2<2<T2<2<2<2 列 序 8 τ ^1928咫人 明說之 列 序工人 <400> 198 Val Gly Leu Asp Gin TyrHis Ser Arg Tyr Gly Pro 20 Synthetic peptide Leu Lys Gin Thr Arg Val Asn Leu Pro Ala 10 35 Gin Ala Val Asp Ala Arg 25 >>>>> 012 3 03 11 Τ* 11 ΙΑ ΟΛ <2< ;2<T2<2<2<2 column order 8 τ ^1928 咫人明说列序工<400> 198 Val Gly Leu Asp Gin Tyr
Asp Val Cys Arg Ala Lys 20 合成肽 Leu Glu Ser Val Lys Lys His Lys Arg Leu 10 15 Met Gly Tyr Met Leu Gin 25 <210> 199 <211> 25 <212> PRT <213>人工序列 <220> <223>人工序列之說明: <400> 199 Arg Gin Val Val His Asn 1 5Asp Val Cys Arg Ala Lys 20 Synthetic peptide Leu Glu Ser Val Lys Lys His Lys Arg Leu 10 15 Met Gly Tyr Met Leu Gin 25 <210> 199 <211> 25 <212> PRT <213>Artificial sequence<;220><223> Description of the artificial sequence: <400> 199 Arg Gin Val Val His Asn 1 5
Tyr Leu Glu Ala Asp Gly 20 合成肽 Lys Leu Thr Ser Cys Asn Tyr Asn Pro Leu 10 15 Arg lie Arg 25 <210> 200 <21i> 28 <212> PRT <213>人工序列 <220> <223>人工序列之說明: 合成肽 158864-序列表.doe -88- 201215618 <400> 200Tyr Leu Glu Ala Asp Gly 20 Synthetic peptide Lys Leu Thr Ser Cys Asn Tyr Asn Pro Leu 10 15 Arg lie Arg 25 <210> 200 <21i> 28 <212> PRT <213>Artificial sequence<220><223> Description of artificial sequence: synthetic peptide 158864 - Sequence Listing. doe -88 - 201215618 <400> 200
Phe Thr Glu Ala 15Phe Thr Glu Ala 15
Arg Asp Tyr Ser Val Ser Arg Gin Val Arg Leu Thr 1 5 10Arg Asp Tyr Ser Val Ser Arg Gin Val Arg Leu Thr 1 5 10
Asn Asn Gin Thr Tyr Thr Phe Cys rrhr His Pro Asn 20 25 <210> 201 <211> 27 <212> PHT <213>人工序列 <220> 人工序列之說明:合成肽 <400> 201Asn Asn Gin Thr Tyr Thr Phe Cys rrhr His Pro Asn 20 25 <210> 201 <211> 27 <212> PHT <213> Artificial Sequence <220> Description of Artificial Sequence: Synthetic Peptide <400> 201
Ser Pro Trp Phe Thr Leu Thr Ala Asn Gin Asn Pro Ser Pro Pro Trp 15 10 15Ser Pro Trp Phe Thr Leu Thr Ala Asn Gin Asn Pro Ser Pro Pro Trp 15 10 15
Ser Lys Leu Thr Tyr Pro Lys Pro His Asp Cys 20 25Ser Lys Leu Thr Tyr Pro Lys Pro His Asp Cys 20 25
<210> 202 <2il> 28 <212> PRT <213>人工序列 <220> <223>人工序列之說明:合成肽 <400> 202<210> 202 <2il> 28 <212> PRT <213> Artificial sequence <220><223> Description of artificial sequence: synthetic peptide <400> 202
Thr Ala Glu Lys Asn Asp Tyr Tyr Arg Val Pro His Tyr Trp Asp Ala 15 10 15Thr Ala Glu Lys Asn Asp Tyr Tyr Arg Val Pro His Tyr Trp Asp Ala 15 10 15
Cys Ser Arg Ala Leu Pro Asp Gin Thr Arg Tyr Lys 20 25Cys Ser Arg Ala Leu Pro Asp Gin Thr Arg Tyr Lys 20 25
<210> 203 <211> 129 <212> PRT <213>人類細胞巨大病毒 <400> 203<210> 203 <211> 129 <212> PRT <213> Human Cell Huge Virus <400> 203
Met Arg Leu Cys Arg Val Trp Leu Ser Val Cys Leu Cys Ala Va3 Val 15 10 15Met Arg Leu Cys Arg Val Trp Leu Ser Val Cys Leu Cys Ala Va3 Val 15 10 15
Leu Gly Gin Cys Gin Arg Glu Thr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30Leu Gly Gin Cys Gin Arg Glu Thr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30
Val Pro His Tyr Trp Asp Ala Cys Ser Arg Ala Leu Pro Asp Gin Thr 35 40 45Val Pro His Tyr Trp Asp Ala Cys Ser Arg Ala Leu Pro Asp Gin Thr 35 40 45
Arg Tyr Lys Tyr Val Glu Gin Leu Val Asp Leu Thr Leu Asn Tyr His 50 55 60Arg Tyr Lys Tyr Val Glu Gin Leu Val Asp Leu Thr Leu Asn Tyr His 50 55 60
Tyr Asp Ala Ser His Gly Leu Asp Asn Phe Asp Val Leu Lys Arg lie 65 70 75 80Tyr Asp Ala Ser His Gly Leu Asp Asn Phe Asp Val Leu Lys Arg lie 65 70 75 80
Asn Val Tlir Glu Val Ser Leu Leu He Ser Asp Phe Arg Arg Gin Asn 85 90 95 «hr· 158864-序列表.doc -89- 201215618Asn Val Tlir Glu Val Ser Leu Leu He Ser Asp Phe Arg Arg Gin Asn 85 90 95 «hr· 158864-Sequence List.doc -89- 201215618
Arg Arg Gly Gly Tlir Asn Lys Arg Thr Thr Phe Asn Ala Ala Gly Ser 100 105 110Arg Arg Gly Gly Tlir Asn Lys Arg Thr Thr Phe Asn Ala Ala Gly Ser 100 105 110
Leu Ala Pro His Ala Arg Ser Leu GIu Phe Ser Val Arg Leu Phe Ala 115 120 125Leu Ala Pro His Ala Arg Ser Leu GIu Phe Ser Val Arg Leu Phe Ala 115 120 125
AsnAsn
<210> 204 <221> 214 <212> PRT <213>人類細胞巨大病毒 <400> 204<210> 204 <221> 214 <212> PRT <213> Human Cell Huge Virus <400> 204
Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 15 10 15Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 15 10 15
Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Phe Thr Leu Tlir 20 25 30Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Phe Thr Leu Tlir 20 25 30
Ala Asn Gin Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Pro Lys 35 40 45Ala Asn Gin Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Pro Lys 35 40 45
Pro His Asp AJa Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60Pro His Asp AJa Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60
Pro Arg Ser Pro Ser Gin Phe Ser Gly Phe Gin Arg Val Ser Thr Gly 65 70 75 80Pro Arg Ser Pro Ser Gin Phe Ser Gly Phe Gin Arg Val Ser Thr Gly 65 70 75 80
Pro Glu Cys Arg Asn GIu Ήιγ Lcu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95Pro Glu Cys Arg Asn GIu Ήιγ Lcu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95
Gin Thr Leu Val Glu Arg Ser Ser ΊΙιγ Trp Val Lys Lys Val He Trp 100 105 110Gin Thr Leu Val Glu Arg Ser Ser ΊΙιγ Trp Val Lys Lys Val He Trp 100 105 110
Tyr Leu Ser Gly Arg Asn Gin Thr lie Leu Gin Arg Met Pro Arg Thr 115 320 125Tyr Leu Ser Gly Arg Asn Gin Thr lie Leu Gin Arg Met Pro Arg Thr 115 320 125
Ala Ser Lys Pro Ser Asp Gly Asn Val Gin lie Ser Val Glu Asp Ala 130 135 140Ala Ser Lys Pro Ser Asp Gly Asn Val Gin lie Ser Val Glu Asp Ala 130 135 140
Lys He Fhe Gly Ala His Met Val Pro Lys Gin Thr Lys Leu Leu Arg 145 150 155 160Lys He Fhe Gly Ala His Met Val Pro Lys Gin Thr Lys Leu Leu Arg 145 150 155 160
Phe Val Val Asn Asp Gly Thr Arg Tyr Gin Met Cys Val Met Lys Leu 165 170 175Phe Val Val Asn Asp Gly Thr Arg Tyr Gin Met Cys Val Met Lys Leu 165 170 175
Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gin Val 180 185 190Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gin Val 180 185 190
Arg Leu Thr Phe Thr Glu Ala Asn Asn Gin Thr Tyr Thr Phe Cys Thr 195 200 205Arg Leu Thr Phe Thr Glu Ala Asn Asn Gin Thr Tyr Thr Phe Cys Thr 195 200 205
His Pro Asn Leu lie Val 210 -90- 158864·序列表.doc 201215618His Pro Asn Leu lie Val 210 -90- 158864 · Sequence Listing.doc 201215618
<210> 205 <21l> 171 <212> PRT <213>人類細胞巨大病毒 <400> 205<210> 205 <21l> 171 <212> PRT <213> Human Cell Huge Virus <400> 205
Met Ser Pro Lys Asn Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 15 10 15Met Ser Pro Lys Asn Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 15 10 15
Leu G!y His Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30Leu G!y His Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30
Phe lie Asn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45Phe lie Asn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45
Cys Asn Arg Phe Thr Val Ala Leu Arg Cys Pro Asp Gly Glu Val Cys 50 55 60Cys Asn Arg Phe Thr Val Ala Leu Arg Cys Pro Asp Gly Glu Val Cys 50 55 60
Tyr Ser Pro Glu Lys Thr Ala Glu He Arg Gly lie Val Thr Thr Met 65 70 75 80Tyr Ser Pro Glu Lys Thr Ala Glu He Arg Gly lie Val Thr Thr Met 65 70 75 80
Thr His Ser Leu Thr Arg Gin Val Val His Asn Lys Leu Thr Ser CysThr His Ser Leu Thr Arg Gin Val Val His Asn Lys Leu Thr Ser Cys
Asn Tyr Asn Pro Leu Tyr Leu Glu Ala Asp Gly Arg lie Arg Cys Gly 100 105 110Asn Tyr Asn Pro Leu Tyr Leu Glu Ala Asp Gly Arg lie Arg Cys Gly 100 105 110
Lys Val Asn Asp Lys Ala Gin Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125Lys Val Asn Asp Lys Ala Gin Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125
Pro Tyr Arg Trp lie Asn Leu Glu Tyr Asp Lys lie Thr Arg lie Val 130 135 HOPro Tyr Arg Trp lie Asn Leu Glu Tyr Asp Lys lie Thr Arg lie Val 130 135 HO
Gly Leu Asp Gin Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160Gly Leu Asp Gin Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160
Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gin 165 170Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gin 165 170
<210> 206 <21]> 743 <212> PRT <213>人類細胞巨大病毒 <400> 206<210> 206 <21]> 743 <212> PRT <213> Human Cell Huge Virus <400> 206
Met Arg Pro Gly Leu Pro Phe Tyr Leu Thr Val Phe Ala Val Tyr Leu 15 10 15Met Arg Pro Gly Leu Pro Phe Tyr Leu Thr Val Phe Ala Val Tyr Leu 15 10 15
Leu Ser His Leu Pro Ser Gin Arg Tyr Gly Ala Asp Ala Ala Ser Glu 20 25 30Leu Ser His Leu Pro Ser Gin Arg Tyr Gly Ala Asp Ala Ala Ser Glu 20 25 30
Ala Leu Asp Pro His Ala Fhe His Leu leu Leu Asn Thr Tyr Gly Arg 35 40 45Ala Leu Asp Pro His Ala Fhe His Leu leu Leu Asn Thr Tyr Gly Arg 35 40 45
Pro lie Arg Phe Leu Arg Glu Asn Thr Thr Gin Cys Thr Tyr Asn Ser 50 55 60Pro lie Arg Phe Leu Arg Glu Asn Thr Thr Gin Cys Thr Tyr Asn Ser 50 55 60
Ser Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala He Ser Phe Asn 65 70 75 80 •91 - 158864-序列表.doc 201215618Ser Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala He Ser Phe Asn 65 70 75 80 •91 - 158864 - Sequence Listing.doc 201215618
Phe Phe Gin Ser Tyr Asn Gin Tyr Tyr Val Phe His Met Pro Arg Cys 85 90 95Phe Phe Gin Ser Tyr Asn Gin Tyr Tyr Val Phe His Met Pro Arg Cys 85 90 95
Leu Phe Ala Gly Pro Leu Ala Glu Gin Phe Leu Asn Gin Val Asp Leu 100 105 110Leu Phe Ala Gly Pro Leu Ala Glu Gin Phe Leu Asn Gin Val Asp Leu 100 105 110
Thr Glu Thr Leu Glu Arg Tyr Gin Gin Arg Leu Asn Thr Tyr Ala Leu 115 120 125Thr Glu Thr Leu Glu Arg Tyr Gin Gin Arg Leu Asn Thr Tyr Ala Leu 115 120 125
Val Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Pro Gin Gin Leu Lys 130 135 140Val Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Pro Gin Gin Leu Lys 130 135 140
Ala Gin Asp Ser Leu Gly Gin Gin Pro Thr Thr Val Pro Pro Pro lie 145 150 155 160Ala Gin Asp Ser Leu Gly Gin Gin Pro Thr Thr Val Pro Pro Pro lie 145 150 155 160
Asp Leu Ser lie Pro His Val Trp Met Pro Pro Gin Thr Tlir Pro His 165 170 175Asp Leu Ser lie Pro His Val Trp Met Pro Pro Gin Thr Tlir Pro His 165 170 175
Asp Trp Lys Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe 180 185 190Asp Trp Lys Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe 180 185 190
Asn Gin Thr Cys lie Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr 195 200 205Asn Gin Thr Cys lie Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr 195 200 205
Val Thr Pro Cys Leu His Gin Gly Phe Tyr Leu Met Asp Glu Leu Arg 210 215 220Val Thr Pro Cys Leu His Gin Gly Phe Tyr Leu Met Asp Glu Leu Arg 210 215 220
Tyr Val Lys lie Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser 225 230 235 240Tyr Val Lys lie Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser 225 230 235 240
He Asp Asp Asp Thr Pro Met Leu Leu lie Phe Gly His Leu Pro Arg 245 250 255He Asp Asp Asp Thr Pro Met Leu Leu lie Phe Gly His Leu Pro Arg 245 250 255
Val Leu Phe Lys Ala Pro Tyr Gin Arg Asp Asn Phe lie Leu Arg Gin 260 265 270Val Leu Phe Lys Ala Pro Tyr Gin Arg Asp Asn Phe lie Leu Arg Gin 260 265 270
Thr Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Thr Gin Leu Asn 275 280 285Thr Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Thr Gin Leu Asn 275 280 285
Arg His Ser Tyr Leu Lys Asp Ser Asp Phe Leu Asp Ala Ala Leu Asp 290 295 300Arg His Ser Tyr Leu Lys Asp Ser Asp Phe Leu Asp Ala Ala Leu Asp 290 295 300
Phe Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg 305 310 315 320Phe Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg 305 310 315 320
Tyr Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gin Met Leu Asp Arg 325 330 335Tyr Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gin Met Leu Asp Arg 325 330 335
Arg Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala A]a 340 345 350Arg Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala A]a 340 345 350
Ala Arg Gin Glu Glu Ala Gly Thr Glu lie Ser lie Pro Arg Ala Leu 355 360 365Ala Arg Gin Glu Glu Ala Gly Thr Glu lie Ser lie Pro Arg Ala Leu 355 360 365
Asp Arg Gin Ala Ala Leu Uu Gin He Gin Glu Phe Met lie Thr Cys -92- 158864-序列表.doc 201215618 370 375 380Asp Arg Gin Ala Ala Leu Uu Gin He Gin Glu Phe Met lie Thr Cys -92- 158864 - Sequence Listing.doc 201215618 370 375 380
Leu Ser G3n Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala 385 390 395 400Leu Ser G3n Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala 385 390 395 400
Val Asp Leu Ala Lys Arg AU Leu 丁rp Thr Pro Asp Gin lie Thr Asp 405 410 415Val Asp Leu Ala Lys Arg AU Leu Ding rp Thr Pro Asp Gin lie Thr Asp 405 410 415
He Thr Ser Leu Val Arg Leu Val Tyr lie Leu Ser Lys Gin Asn Gin 420 425 430He Thr Ser Leu Val Arg Leu Val Tyr lie Leu Ser Lys Gin Asn Gin 420 425 430
Gin His Leu lie Pro Gin Trp Ala Leu Arg Gin He Ala Asp Phe Ala 435 440 445Gin His Leu lie Pro Gin Trp Ala Leu Arg Gin He Ala Asp Phe Ala 435 440 445
Leu Gin Leu His Lys Thr His Leu Ala Ser Phe Leu Ser Ala Phe Ala 450 455 460Leu Gin Leu His Lys Thr His Leu Ala Ser Phe Leu Ser Ala Phe Ala 450 455 460
Arg Gin Glu Leu Tyr Leu Met Gly Ser Leu Val His Ser Met Leu Val 465 470 475 480Arg Gin Glu Leu Tyr Leu Met Gly Ser Leu Val His Ser Met Leu Val 465 470 475 480
His Thr Thr Glu Arg Arg Glu lie Phe He Val Glu Tiir Gly Leu Cys 485 490 495His Thr Thr Glu Arg Arg Glu lie Phe He Val Glu Tiir Gly Leu Cys 485 490 495
Ser Leu Ala Glu Leu Ser His Phe Thr Gin Leu Leu Ala His Pro His 500 505 510Ser Leu Ala Glu Leu Ser His Phe Thr Gin Leu Leu Ala His Pro His 500 505 510
His Glu Tyr Leu Ser Asp Leu 丁yr Thr Pro Cys Ser Ser Ser Gly Ar£ 515 520 525His Glu Tyr Leu Ser Asp Leu Dingyr Thr Pro Cys Ser Ser Ser Gly Ar£ 515 520 525
Arg Asp His Ser Leu Glu Arg Leu Thr Arg Leu Fhe Pro Asp Ala Thr 530 535 540Arg Asp His Ser Leu Glu Arg Leu Thr Arg Leu Fhe Pro Asp Ala Thr 530 535 540
Val Pro Ala Thr Val Pro Ala Ala Leu Ser lie Leu Ser Thr Met Gin 545 550 555 560Val Pro Ala Thr Val Pro Ala Ala Leu Ser lie Leu Ser Thr Met Gin 545 550 555 560
Pro Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly 565 570 575Pro Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly 565 570 575
Glu Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Val Val 580 585 590Glu Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Val Val 580 585 590
Thr Asn Gin Tyr Leu lie Lys Gly He Ser Tyr Pro Val Ser Tlir Thr 595 600 605Thr Asn Gin Tyr Leu lie Lys Gly He Ser Tyr Pro Val Ser Tlir Thr 595 600 605
Val Val Gly Gin Scr Leu lie He Thr G!n Thr Asp Ser Gin Ser Lys 610 615 620Val Val Gly Gin Scr Leu lie He Thr G!n Thr Asp Ser Gin Ser Lys 610 615 620
Cys Glu Leu Thr Arg Asn Met His Thr Thr His Ser lie Thr Ala Ala 625 630 635 640Cys Glu Leu Thr Arg Asn Met His Thr Thr His Ser lie Thr Ala Ala 625 630 635 640
Leu Asn [le Ser Leu Glu Asn Cys Ala Phe Cys Gin Ser Ala Leu Leu 645 650 655Leu Asn [le Ser Leu Glu Asn Cys Ala Phe Cys Gin Ser Ala Leu Leu 645 650 655
Glu Tyr Asp Asp Thr Gin Gly Val lie Asn lie Met Tyr Met His Asp 660 665 670 -93- 158864-序列表.doc 201215618Glu Tyr Asp Asp Thr Gin Gly Val lie Asn lie Met Tyr Met His Asp 660 665 670 -93- 158864 - Sequence Listing.doc 201215618
Ser Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val 675 6S0 685Ser Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val 675 6S0 685
Ser Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly ITir Val 690 695 700Ser Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly ITir Val 690 695 700
Leu Glu Val Thr Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 720Leu Glu Val Thr Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 720
Met Met Ser Val Tyr Ala Leu Ser Ala lie lie Gly lie Tyr Leu Leu 725 730 735Met Met Ser Val Tyr Ala Leu Ser Ala lie lie Gly lie Tyr Leu Leu 725 730 735
Tyr Arg Met Leu Lys Thr Cys 740Tyr Arg Met Leu Lys Thr Cys 740
<210> 207 <211> 464 <212> PRT <213>人類細胞巨大病毒 <400> 207<210> 207 <211> 464 <212> PRT <213> Human Cell Huge Virus <400>
Met Gly Arg Lys Glu Asp Met Arg Ser lie Ser Lys Leu Phe Phe lie 15 10 15 lie Ser Leu Thr Val Leu Leu Phe Ser lie lie Asn Cys Lys Val Val 20 25 30Met Gly Arg Lys Glu Asp Met Arg Ser lie Ser Lys Leu Phe Phe lie 15 10 15 lie Ser Leu Thr Val Leu Leu Phe Ser lie lie Asn Cys Lys Val Val 20 25 30
Arg Pro Pro Gly Arg Tyr Trp Leu Gly Thr Val Leu Ser Thr He Gly 35 40 45Arg Pro Pro Gly Arg Tyr Trp Leu Gly Thr Val Leu Ser Thr He Gly 35 40 45
Lys Gin Lys Leu Asp Lys Phe Lys Leu Glu lie Leu Lys Gin Leu Glu 50 55 60Lys Gin Lys Leu Asp Lys Phe Lys Leu Glu lie Leu Lys Gin Leu Glu 50 55 60
Arg Glu Pro Tyr Hu Lys Tyr Phe Asn Met Thr Arg Gin His Val Lys 65 70 75 SOArg Glu Pro Tyr Hu Lys Tyr Phe Asn Met Thr Arg Gin His Val Lys 65 70 75 SO
Asn Leu Tlir Met Asn Met Thr Gin Phe Pro Gin Tyr Tyr lie Leu Ala 85 90 95Asn Leu Tlir Met Asn Met Thr Gin Phe Pro Gin Tyr Tyr lie Leu Ala 85 90 95
Gly Pro lie Arg Asn Asp Ser He Thr Tyr Leu Trp Phe Asp Phe Tyr 100 105 110Gly Pro lie Arg Asn Asp Ser He Thr Tyr Leu Trp Phe Asp Phe Tyr 100 105 110
Ser Thr Gin Leu Arg Lys Pro Ala Lys Tyr Val Tyr Ser Gin Tyr Asn 115 120 125Ser Thr Gin Leu Arg Lys Pro Ala Lys Tyr Val Tyr Ser Gin Tyr Asn 115 120 125
His Thr Ala Lys Thr lie Thr Phe Arg Fro Pro Ser Cys Gly Tlir Val 130 135 140His Thr Ala Lys Thr lie Thr Phe Arg Fro Pro Ser Cys Gly Tlir Val 130 135 140
Pro Ser Met Thr Cys Leu Ser Glu Met Leu Asn Val Ser Lys Arg Asa 145 150 155 160Pro Ser Met Thr Cys Leu Ser Glu Met Leu Asn Val Ser Lys Arg Asa 145 150 155 160
Asp llir Gly Glu Gin Gly Cys Gly Asn Phe Thr Thr Phe Asn Pro Met 165 170 175Asp llir Gly Glu Gin Gly Cys Gly Asn Phe Thr Thr Phe Asn Pro Met 165 170 175
Phe Phe Asn Val Pro Arg Trp Asn Thr Lys Leu Tyr Val Gly Pro Thr 180 185 190Phe Phe Asn Val Pro Arg Trp Asn Thr Lys Leu Tyr Val Gly Pro Thr 180 185 190
Lys Val Asn Val Asp Ser Gin Thr lie Tyr Phe Leu Gly Leu Thr Ala -94- 158864-序列表.doc 201215618 195 200 205Lys Val Asn Val Asp Ser Gin Thr lie Tyr Phe Leu Gly Leu Thr Ala -94- 158864 - Sequence Listing.doc 201215618 195 200 205
Leu Leu Leu Arg Tyr Ala Gin Arg Asn Cys Thr His Ser Phe Tyr Leu 210 215 220Leu Leu Leu Arg Tyr Ala Gin Arg Asn Cys Thr His Ser Phe Tyr Leu 210 215 220
Val Asn Ala Met Ser Arg Asn Leu Phe Arg Val Pro Lys Tyr lie Asn 225 230 235 240Val Asn Ala Met Ser Arg Asn Leu Phe Arg Val Pro Lys Tyr lie Asn 225 230 235 240
Gly Tlir Lys Leu Lys Asn Thr Met Arg Lys Leu Lys Arg Lys Gin Ala 245 250 25SGly Tlir Lys Leu Lys Asn Thr Met Arg Lys Leu Lys Arg Lys Gin Ala 245 250 25S
Pro Val Lys Glu Gin Leu Glu Lys Lys Thr Lys Lys Ser Gin Ser Thr 260 265 270Pro Val Lys Glu Gin Leu Glu Lys Lys Thr Lys Lys Ser Gin Ser Thr 260 265 270
Thr frhr Pro Tyr Phe Ser Tyr Thr Thr Ser Thr Ala Leu Asn Val Thr 275 280 285Thr frhr Pro Tyr Phe Ser Tyr Thr Thr Ser Thr Ala Leu Asn Val Thr 275 280 285
Thr Asn Ala Thr Tyr Arg Val Thr Thr Scr Ala Lys Arg lie Pro Thr 290 295 300Thr Asn Ala Thr Tyr Arg Val Thr Thr Scr Ala Lys Arg lie Pro Thr 290 295 300
Ser Thr lie Ala Tyr Arg Pro Asp Ser Ser Phe Met Lys Ser He Met 305 310 315 320Ser Thr lie Ala Tyr Arg Pro Asp Ser Ser Phe Met Lys Ser He Met 305 310 315 320
Ala Thr Gin Leu Arg Asp Leu Ala Thr Trp Val Tyr Thr Thr Leu Arg 325 330 335Ala Thr Gin Leu Arg Asp Leu Ala Thr Trp Val Tyr Thr Thr Leu Arg 325 330 335
Tyr Arg Asn Glu Pro Phe Cys Lys Pro Asp Arg Asn Arg Thr Ala Val 340 345 350Tyr Arg Asn Glu Pro Phe Cys Lys Pro Asp Arg Asn Arg Thr Ala Val 340 345 350
Ser Glu Phe Met Lys Asn Thr His Val Leu He Arg Asn Glu Thr Pro 355 360 365Ser Glu Phe Met Lys Asn Thr His Val Leu He Arg Asn Glu Thr Pro 355 360 365
Tyr Thr lie Tyr Gly Thr Uu Asp Met Ser Ser Leu Tyr Tyr Asn Glu 370 375 380Tyr Thr lie Tyr Gly Thr Uu Asp Met Ser Ser Leu Tyr Tyr Asn Glu 370 375 380
Thr Met Ser Val Glu Asn Glu Thr Ala Ser Asp Asn Asn Glu Thr Thr 385 390 395 400Thr Met Ser Val Glu Asn Glu Thr Ala Ser Asp Asn Asn Gn Thr Thr 385 390 395 400
Pro Thr Ser Pro Ser Thr Arg Phe Gin Lys Thr Phe lie Asp Pro Leu 405 410 415Pro Thr Ser Pro Ser Thr Arg Phe Gin Lys Thr Phe lie Asp Pro Leu 405 410 415
Trp Asp Tyr Leu Asp Ser Leu Leu Phe Leu Asp Lys lie Arg Asn Phe 420 425 430Trp Asp Tyr Leu Asp Ser Leu Leu Phe Leu Asp Lys lie Arg Asn Phe 420 425 430
Ser Leu Gin Leu Pro Ala Tyr Gly Asn Leu Thr Pro Pro Glu His Arg 435 440 445Ser Leu Gin Leu Pro Ala Tyr Gly Asn Leu Thr Pro Pro Glu His Arg 435 440 445
Arg Ala Val Asn Leu Ser Thr Leu Asn Ser Leu Trp Trp Trp Leu Gin 450 455 460Arg Ala Val Asn Leu Ser Thr Leu Asn Ser Leu Trp Trp Trp Leu Gin 450 455 460
<210> 208 <211> 278 <212> PRT <213>人類細胞巨大病毒 <400> 208<210> 208 <211> 278 <212> PRT <213> Human Cell Huge Virus <400> 208
Mel Cys Arg Arg Pro Asp Cys Gly Phe Ser Phe Ser Pro Gly Pro Val 15 10 15 -95- 158864-序列表.doc 201215618Mel Cys Arg Arg Pro Asp Cys Gly Phe Ser Phe Ser Pro Gly Pro Val 15 10 15 -95- 158864 - Sequence Listing.doc 201215618
Val Leu Leu Trp Cys Cys Leu Leu Leu Pro lie Val Ser Ser Val Ala 20 25 30Val Leu Leu Trp Cys Cys Leu Leu Leu Pro lie Val Ser Ser Val Ala 20 25 30
Val Ser Val Ala Pro Thr Ala Ala Glu Lys Val Pro Ala Glu Cys Pro 35 40 45Val Ser Val Ala Pro Thr Ala Ala Glu Lys Val Pro Ala Glu Cys Pro 35 40 45
Glu Leu Thr Arg Arg Cys Leu Leu Gly Glu Val Phe Gin Gly Asp Lys 50 55 60Glu Leu Thr Arg Arg Cys Leu Leu Gly Glu Val Phe Gin Gly Asp Lys 50 55 60
Tyr Glu Ser Trp Leu Arg Pro Leu Val Asn Val Thr Gly Arg Asn Gly 65 70 75 80Tyr Glu Ser Trp Leu Arg Pro Leu Val Asn Val Thr Gly Arg Asn Gly 65 70 75 80
Pro Leu Ser Gin Leu I】e Arg Tyr Arg Pro Val Thr Pro Glu /Ua Ala 85 90 95Pro Leu Ser Gin Leu I】e Arg Tyr Arg Pro Val Thr Pro Glu /Ua Ala 85 90 95
Asn Ser Val Leu Leu Asp Asp Ala Phe Lea Asp Thr Lea Ala Leu Leu 100 105 110Asn Ser Val Leu Leu Asp Asp Ala Phe Lea Asp Thr Lea Ala Leu Leu 100 105 110
Tyr Asn Asn Pro Asp Gin Leu Arg Ala Leu Leu Thr Leu Leu Ser Ser 115 120 125Tyr Asn Asn Pro Asp Gin Leu Arg Ala Leu Leu Thr Leu Leu Ser Ser 115 120 125
Asp Thr Ala Pro Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys 130 135 140Asp Thr Ala Pro Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys 130 135 140
Gly Asp Gly Ser Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg 145 350 155 160Gly Asp Gly Ser Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg 145 350 155 160
Gly Tyr Asp Leu Thr Arg Leu Ser Tyr Gly Arg Ser lie Phe Thr Glu 165 170 175Gly Tyr Asp Leu Thr Arg Leu Ser Tyr Gly Arg Ser lie Phe Thr Glu 165 170 175
His Val Leu Gly Phe Glu Leu Val Pro Pro Ser Leu Fhe Asn Val Val ISO 185 190His Val Leu Gly Phe Glu Leu Val Pro Pro Ser Leu Fhe Asn Val Val ISO 185 190
Val Ala He Arg Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg Leu 195 200 205Val Ala He Arg Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg Leu 195 200 205
Pro Val Ser Thr Ala Ala Ala Fro GJu Gly lie Thr Leu Phe Tyr Gly 210 215 220Pro Val Ser Thr Ala Ala Ala Fro GJu Gly lie Thr Leu Phe Tyr Gly 210 215 220
Leu Tyr Asn Ala Val Lys Glu Phe Cys Leu Arg His Gin Leu Asp Pro 225 230 235 240Leu Tyr Asn Ala Val Lys Glu Phe Cys Leu Arg His Gin Leu Asp Pro 225 230 235 240
Pro Leu Leu Arg His Leu Asp Lys Tyr Tyr Ala Gly Leu Pro Pro Glu 245 250 255Pro Leu Leu Arg His Leu Asp Lys Tyr Tyr Ala Gly Leu Pro Pro Glu 245 250 255
Leu Lys Gin Thr Arg Val Asn Leu Pro Ala His Ser Arg Tyr Gly Pro 260 265 270Leu Lys Gin Thr Arg Val Asn Leu Pro Ala His Ser Arg Tyr Gly Pro 260 265 270
Gin Ala Val Asp Ala Arg 275Gin Ala Val Asp Ala Arg 275
<210> 209 <211> 472 <212> PRT <213>人類細胞巨大病毒 -96-<210> 209 <211> 472 <212> PRT <213> Human cell giant virus -96-
158864-序列表.doc s 201215618 <400> 209158864-Sequence Listing.doc s 201215618 <400> 209
Met Gly Lys Lys GIu Met lie Met Val Lys Gly lie Pro Lys lie Met 15 10 15Met Gly Lys Lys GIu Met lie Met Val Lys Gly lie Pro Lys lie Met 15 10 15
Leu Leu lie Ser lie Thr Phe Leu Leu Leu Ser Leu He Asn Cys Asn 20 25 30Leu Leu lie Ser lie Thr Phe Leu Leu Leu Ser Leu He Asn Cys Asn 20 25 30
Val Leu Val Asn Ser Arg Gly Thr Arg Arg Ser Trp Pro Tyr Thr Val 35 40 45Val Leu Val Asn Ser Arg Gly Thr Arg Arg Ser Trp Pro Tyr Thr Val 35 40 45
Leu Ser Tyr Arg Gly Lys Glu lie Leu Lys Lys Gin Lys Glu Asp lie 50 55 60Leu Ser Tyr Arg Gly Lys Glu lie Leu Lys Lys Gin Lys Glu Asp lie 50 55 60
Leu Lys Arg Leu Met Ser Thr Ser Ser Asp G]y 丁yr Arg Phe Leu Met 65 70 75 80Leu Lys Arg Leu Met Ser Thr Ser Ser Asp G]y Ding yr Arg Phe Leu Met 65 70 75 80
Tyr Pro Ser Gin Gin Lys Fhe His Ala lie Val lie Ser Met Asp Lys 85 90 95Tyr Pro Ser Gin Gin Lys Fhe His Ala lie Val lie Ser Met Asp Lys 85 90 95
Phe Pro Gin Asp Tyr lie Leu Ala Gly Pro He Arg Asn Asp Ser He 100 105 110Phe Pro Gin Asp Tyr lie Leu Ala Gly Pro He Arg Asn Asp Ser He 100 105 110
Thr His Met Trp Phe Asp Phe Tyr Ser Thr Gin Leu Arg Lys Pro Ala 115 120 125Thr His Met Trp Phe Asp Phe Tyr Ser Thr Gin Leu Arg Lys Pro Ala 115 120 125
Lys Tyr Va] Tyr Ser Glu Tyr Asn His Thr Ala His Lys He Thr Leu 130 135 140Lys Tyr Va] Tyr Ser Glu Tyr Asn His Thr Ala His Lys He Thr Leu 130 135 140
Arg Pro Pro Pro Cys Gly Thr Val Pro Ser Met Asn Cys Leu Ser Glu 145 150 155 160Arg Pro Pro Pro Cys Gly Thr Val Pro Ser Met Asn Cys Leu Ser Glu 145 150 155 160
Met Leu Asn Val Ser Lys Arg Asn Asp Thr Gly Glu Lys Gly Cys Gly 165 170 175Met Leu Asn Val Ser Lys Arg Asn Asp Thr Gly Glu Lys Gly Cys Gly 165 170 175
Asn Phe Thr Thr Phe Asn Pro Met Phe Phe Asn Val Pro Arg Trp Asn 180 185 190Asn Phe Thr Thr Phe Asn Pro Met Phe Phe Asn Val Pro Arg Trp Asn 180 185 190
Thr Lys Leu Tyr lie Gly Ser Asn Lys Val Asn Val Asp Ser Gin Thr 195 200 205 lie Tyr Phe Leu Gly Leu Thr AU Leu Leu Leu Arg Tyr Ala Gin Arg 210 215 220Thr Lys Leu Tyr lie Gly Ser Asn Lys Val Asn Val Asp Ser Gin Thr 195 200 205 lie Tyr Phe Leu Gly Leu Thr AU Leu Leu Leu Arg Tyr Ala Gin Arg 210 215 220
Asn Cys Thr Arg Ser Phe Tyr Leu Val Asn Ala Met Ser Arg Asn Leu 225 230 235 240Asn Cys Thr Arg Ser Phe Tyr Leu Val Asn Ala Met Ser Arg Asn Leu 225 230 235 240
Phe Arg Val Pro Lys Tyr lie Asn Gly Thr Lys Leu Lys Asn Thr Met 245 250 255Phe Arg Val Pro Lys Tyr lie Asn Gly Thr Lys Leu Lys Asn Thr Met 245 250 255
Arg Lys Leu Lys Arg Lys Gin Ala Leu Val Lys Glu Gin Pro Gin Lys 260 265 270Arg Lys Leu Lys Arg Lys Gin Ala Leu Val Lys Glu Gin Pro Gin Lys 260 265 270
Lys Asn Lys Lys Ser Gin Ser Thr Thr Thr Pro Tyr Leu Ser Tyr Thr 275 280 285Lys Asn Lys Lys Ser Gin Ser Thr Thr Thr Pro Tyr Leu Ser Tyr Thr 275 280 285
Thr Ser Thr Ala Phe Asn Val Thr Thr Asn Val Thr Tyr Ser Ala Thr 290 295 300 -97- 158864-序列表.doc 201215618Thr Ser Thr Ala Phe Asn Val Thr Thr Asn Val Thr Tyr Ser Ala Thr 290 295 300 -97- 158864 - Sequence Listing.doc 201215618
Ala Ala Val Thr Arg Val Ala Thr Ser Thr Thr Gly Tyr Arg Pro Asp 305 310 315 320Ala Ala Val Thr Arg Val Ala Thr Ser Thr Thr Gly Tyr Arg Pro Asp 305 310 315 320
Ser Asn Phc Met Lys Ser lie Met Ala Thr Gin Leu Arg Asp Leu Ala 325 330 335Ser Asn Phc Met Lys Ser lie Met Ala Thr Gin Leu Arg Asp Leu Ala 325 330 335
Thr Trp Val Tyr Thr Thr Leu Arg Tyr Arg Asn Glu Pro Phe Cys Lys 340 345 350Thr Trp Val Tyr Thr Thr Leu Arg Tyr Arg Asn Glu Pro Phe Cys Lys 340 345 350
Pro Asp Arg Asn Arg Thr Ala Val Scr Glu Phe Met Lys Asn Thr His 355 360 365Pro Asp Arg Asn Arg Thr Ala Val Scr Glu Phe Met Lys Asn Thr His 355 360 365
Val Leu lie Arg Asn Glu Thr Pro Tyr Thr lie Tyr Gly Thr Leu Asp 370 375 380Val Leu lie Arg Asn Glu Thr Pro Tyr Thr lie Tyr Gly Thr Leu Asp 370 375 380
Met Ser Ser Leu Tyr Tyr Asn Glu Thr Met Ser Val Glu Asn Glu llir 385 390 395 400Met Ser Ser Leu Tyr Tyr Asn Glu Thr Met Ser Val Glu Asn Glu llir 385 390 395 400
Ala Ser Asp Asn Asn Glu Thr Thr Pro Thr Ser Pro Ser ITir Arg Phe 405 410 415Ala Ser Asp Asn Asn Glu Thr Thr Pro Thr Ser Pro Ser ITir Arg Phe 405 410 415
Gin Arg ΊΤιγ Phe He Asp Pro Leu Trp Asp Tyr Leu Asp Ser Leu Leu 420 425 430Gin Arg ΊΤιγ Phe He Asp Pro Leu Trp Asp Tyr Leu Asp Ser Leu Leu 420 425 430
Phe Leu Asp Lys lie Arg Asn Phe Ser Leu Gin Leu Pro Ala Tyr Giy 435 440 445.Phe Leu Asp Lys lie Arg Asn Phe Ser Leu Gin Leu Pro Ala Tyr Giy 435 440 445.
Asn Leu Thr Pro Pro Glu His Arg Arg Ala Ala Asn Leu Ser Thr Leu 450 455 460Asn Leu Thr Pro Pro Glu His Arg Arg Ala Ala Asn Leu Ser Thr Leu 450 455 460
Asn Ser Leu Trp Trp Trp Ser Gin 465 470 -98·Asn Ser Leu Trp Trp Trp Ser Gin 465 470 -98·
158864_ 序列表.doc s158864_ Sequence Listing.doc s
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