TW201704260A - 具有組織標的功能的抗發炎分子 - Google Patents

具有組織標的功能的抗發炎分子 Download PDF

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TW201704260A
TW201704260A TW105101334A TW105101334A TW201704260A TW 201704260 A TW201704260 A TW 201704260A TW 105101334 A TW105101334 A TW 105101334A TW 105101334 A TW105101334 A TW 105101334A TW 201704260 A TW201704260 A TW 201704260A
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gly
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TWI608015B (zh
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張子文
陳柔含
朱鑫懋
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免疫功坊股份有限公司
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Abstract

本揭示內容提出了多種分子構建體，這些分子構建體具有一標的元件與一效應元件。此處亦揭示了利用所述分子構建體來治療多種疾病的方法。

Description

具有組織標的功能的抗發炎分子
本揭示內容是關於藥學領域；更明確地說，是關於多功能的分子構建體，譬如具有標的元件與效應元件以將效應元件(如治療藥物)遞送至標的部位的分子構建體。
近年來，本領域發展出多種篩檢與選擇以抗原為標的之單株抗體(monoclonal antibodies，mAbs)的方法，進而帶動許多治療用抗體的研發，為一些不久前還被認為是不治之症的疾病帶來曙光。根據治療性抗體資料庫(Therapeutic Antibody Database)的統計，有多達2,800種左右的抗體已經或即將投入人體臨床試驗研發，而由政府藥物管理機構核准可用於臨床治療的抗體則約有80種。從與抗體療效相關的大量數據中可知抗體是基於何種藥理機制而發揮療效。
以抗體作為治療藥劑時，其主要的藥理機制是以抗體來中和或誘捕造成疾病的介質；所述介質可能是存在於血液循環、間質空間(interstitial space)或淋巴結中的細胞介素或免疫成分。中和所述介質的活性可抑制造成疾病的介質和其受體之間的互動。此外，可將細胞介素的可溶性受體或受體的細胞外部分和免疫球蛋白IgG的Fc部分製成融合蛋白，此種融合蛋白可利用類似中和抗體的機制來中和上述細胞介素或免疫因子；因此目前也開發出多種融合蛋白治療劑。
另外，某些取得臨床使用許可或正在進行臨床研究的治療性抗體則是採用了與上述主要抗體機制不同的機制，來發揮其藥理作用，譬如可藉由和受體結合以阻斷這些受體和其配體之間的互動。對此類抗體藥物來說，其主要的藥理機制並非由Fc-介導的機制(如抗體依賴性細胞毒性(antibody-dependent cellular cytotoxicity，ADCC)或是補體介導的細胞溶解(complement-mediated cytolysis，CMC)等)。
另一些治療性抗體可和目標細胞上的某些表面抗原結合，並藉此在目標細胞上發揮Fc介導的功能以及其他機制。最重要的Fc介導的機制為抗體依賴性細胞毒性(ADCC)以及補體介導的細胞溶解(CMC)，這兩種機制都會將與抗體結合的目標細胞溶解。這些抗體和特定的細胞表面抗原結合後，可使得與其結合的目標細胞發生細胞凋亡。
有些抗體也可以作為載體，用來攜帶細胞毒性分子或其他治療藥劑，而抗體本身則沒有顯著的治療效果。一般來說，此類抗體可結合至目標細胞上的「腫瘤相關」抗原，但其本身不會使細胞溶解。本領域已知有多種對B淋巴瘤上的CD19與CD22專一的抗體。在過去許多年間，大半的研究著重於探究如何以這些抗體作為載體來攜帶細胞毒性藥物，包括半衰期極短的放射性核種，如90 Y、131 I以及177 Lu。某些抗體也可作為載藥微脂體的標的藥劑，上述載藥微脂體載有細胞毒性藥物，如阿黴素(doxorubicin)、紫衫醇(paclitaxel)以及兩性黴素B (amphotericin B)。近年來，抗體藥物複合體(antibody drug conjugate，ADC)的研發成長快速，這主要可歸因於具有極高細胞毒性的藥物(如奧里斯他汀(auristatin)、美登木素(maytansine)、卡奇黴素(calicheamicin)以及喜樹鹼(camptothecin)等)、以及將細胞毒性分子複合至抗體分子上的方法等的發展。這些ADC經過設計可標的至表現一或多種獨特的CD標記的血液、淋巴系統與骨髓中的擴散性(或液態)腫瘤，包括多種類型的淋巴瘤與白血病。也有某些針對固態腫瘤所開發出的ADC。此類新一代的抗體藥物複合體中，已有少數經核准可用於臨床治療，也有許多ADC正在進行臨床試驗。
然而，在第一代ADC中，細胞毒性藥物分子是透過非選擇性的方式連接到抗體中的半胱胺酸(cysteine，C)或離胺酸(lysine，K)殘基，因此會得到異質性的ADC混合物，其中每一個ADC分子所攜帶的藥物分子數可能不同。此一現象會導致安全性與有效性上的疑慮。舉例來說，美國食品暨藥物管理局(US Food and Drug Administration，FDA)核准的首例ADC—吉妥單抗奧佐米星(gemtuzumab ozogamicin)—可用以治療急性骨髓性白血病，但在上市後卻因毒性過高而在FDA的要求下退出市場。
製備具有雙重專一性的抗體的概念與方法，早在三十年前就已萌芽。近年來，隨著重組抗體工程方法的進步、以及對於更好藥物的需求驅使下，發展出多種具有不同結構構形的雙專一性抗體。
舉例來說，雙價或多價抗體可能帶有二或更多種抗原結合位。已知有多種方法可用以製備多價抗體，這些方法通常利用連接結構將三或四種抗原結合片段(antigen-binding fragment，Fab)共價連接在一起。舉例來說，已透過重組工程製備出能表現三或四個串聯(tandem)的Fab重複片段的抗體。
此外，利用合成的交聯物(crosslinker)，透過化學方法將不同的抗體或結合部位連接在一起，以製造出多價抗體的方法也是本領域公知的技術。其中一種方式是利用不同的接合物(linker)，將三、四或更多個分離的Fab片段，透過化學交聯的方式彼此接合。另一種方式是先製備一個具有多個Fab的構建體，將這些Fab組裝成一維的DNA支架(one-dimensional DNA scaffold)。這些經設計可和目標分子結合的各種多價抗體構建體彼此的尺寸、半衰期、構形彈性以及調節免疫系統的能力各不相同。
基於以上說明，已有部分研究著眼於製備效應元件數目固定的分子構建體或具有二或更多種不同功能性元件(譬如至少一標的元件與至少一效應元件)的分子構建體。然而，通常很難利用化學合成或重組技術等方法，來建立具有特定標的與效應元件組合的分子構建體。因此，本領域亟需一種新穎的分子平台，其可用以建構適用於多種疾病的各種分子。
發明內容旨在提供本揭示內容的簡化摘要，以使閱讀者對本揭示內容具備基本的理解。此發明內容並非本揭示內容的完整概述，且其用意並非在指出本發明具體實施例的重要/關鍵元件或界定本發明的範圍。發明內容的唯一目的在於對此處揭示的某些概念提供簡化的說明，以利理解後文所述的實施方式。
本揭示內容的第一態樣是關於以可結晶片段(fragment crystallizable，Fc)為基礎的分子構建體(下文稱為Fc型分子構建體)，其具有直接或間接連接到免疫球蛋白CH2-CH3區域的至少一標的元件與至少一效應元件。本發明Fc型分子構建體讓開發人員能夠進行各種標的與效應元件的組合。因此，本發明Fc型分子構建體可作為建構多價分子的平台。
根據本揭示內容某些實施方式，Fc型分子構建體包含一對IgG.Fc的CH2-CH3區段、第一對效應元件及第一對標的元件。
在某些實施方式中，本發明Fc型分子構建體可用以治療免疫疾病(特別是自體免疫疾病)或骨質疏鬆症。在本例中，第一對效應元件由兩個效應元件所組成，這兩個效應元件是對腫瘤壞死因子-α (tumor necrosis factor-α，TNF-α)、介白素-17 (interleukin-17，IL-17)、IL-17受體(IL-17R)、IL-1、IL-6、IL-6R、IL-12/IL-23、B細胞活化因子(B cell activating factor，BAFF)或細胞核因子κB受體活化因子配體(ligand of receptor activator of nuclear factor κB，RANKL)專一的抗體片段；或TNF-α或IL-1的可溶性受體。再者，第一對標的元件是由兩個標的元件所組成，這兩個標的元件是對α-蛋白聚糖(α-aggrecan)、第I型膠原蛋白(collagen I)、第II型膠原蛋白、第III型膠原蛋白、第V型膠原蛋白、第VII型膠原蛋白、第IX型膠原蛋白、第XI型膠原蛋白或骨結合素(osteonectin)專一的抗體片段。當第一對效應元件連接於成對CH2-CH3區段的N-端時，第一對標的元件則連接於成對CH2-CH3區段的C-端，反之亦然。或者是，當第一對效應元件與第一對標的元件為都是單鏈可變片段(single-chain variable fragment，scFv)的形式時，則第一對標的元件以串聯雙抗體(tandem)或雙抗體(diabody)的構形而連接於第一對效應元件的N-端，因而形成一對連接於成對CH2-CH3區段N-端的雙專一性scFv。
於一些實施方式中，該成對CH2-CH3區段衍生自人類IgG重鏈γ4或人類IgG重鏈γ1。
在某些例子中，第一對效應元件或第一對標的元件形成抗原結合片段(antigen-binding fragment，Fab)的構形(即，由VH -CH1區域以及VL -Cκ區域所組成)；此種Fab片段連接於第一與第二重鏈的N-端，而使得Fc型分子構建體形成IgG構形。在這些情形中，未採用Fab構形的該對元件則連接於成對CH2-CH3區段的C-端。
根據其他實施方式，所述的Fc型分子構建體更包含第二對效應元件，其係由兩個額外的效應元件所組成，這些額外的效應元件同樣選自上文所述的效應元件。根據多種實施方式中，第二對效應元件所選用的元件和第一對效應元件不同。在這些實施方式中，第二對效應元件連接於CH2-CH3區段的自由C-端。
或者是，本發明Fc型分子構建體更包含第二對標的元件，其所含的兩個標的元件同樣選自上文所述的標的元件。根據多種實施方式中，第二對標的元件元件所選用的元件和第一對標的元件不同。在這些實施方式中，第二對標的元件連接於CH2-CH3區段的自由C-端。
根據多種視需要的實施方式，可視需求來組合上文所述的標的元件與效應元件，進而達到所欲的療效。附隨的申請專利範圍以及下文會進一步說明用以治療免疫疾病的一些例示性的效應元件與標的元件的組合。
本揭示內容的第二態樣是關於用以治療多種疾病的方法。一般來說，上述方法涉及了對需要此一治療的個體投予治療有效量的Fc型分子構建體，所述Fc型分子構建體可以是根據本揭示內容第一態樣與相關實施方式的任一種Fc型分子構建體。
於一些實施方式中，本發明方法是關於治療免疫疾病；特別是自體免疫疾病。
根據本揭示內容某些實施方式，上述自體免疫疾病為類風濕性關節炎、牛皮癬性關節炎或關節黏連性脊椎炎。在本例中，效應元件為對TNF-α、IL-12/IL-23、IL-1、IL-17或IL-6專一的抗體片段，而標的元件可以是對第II型膠原蛋白、第IX型膠原蛋白、第XI型膠原蛋白或α-蛋白聚糖專一的抗體片段。
根據多種實施方式中，上述自體免疫疾病為牛皮癬。在本例中，效應元件為對TNF-α、IL-12/IL-23或IL-17專一的抗體片段，而標的元件是對第I型膠原蛋白或第VII型膠原蛋白專一的抗體片段。
根據一些其他實施方式，上述自體免疫疾病為全身性紅斑狼瘡(systemic lupus erythematosus，SLE)、皮膚狼瘡(cutaneous lupus)或乾燥綜合症(Sjogren’s syndrome)。在本例中，效應元件為對BAFF專一的抗體片段，而標的元件為對第I型膠原蛋白或第VII型膠原蛋白專一的抗體片段。
根據某些實施方式，上述自體免疫疾病為發炎性腸症(inflammatory bowel disease)，譬如克羅恩氏病(Crohn’s disease)或潰瘍性結腸炎(ulcerative colitis)。在本例中，效應元件為對TNF-α專一的抗體片段，而標的元件為對第III型膠原蛋白或第V型膠原蛋白專一的抗體片段。
此處所提出的方法還可以用於治療骨質疏鬆症。根據本揭示內容的實施方式，用於治療骨質疏鬆症的效應元件包含對RANKL專一的抗體片段，而標的元件則包含對第I型膠原蛋白或骨結合素專一的抗體片段。
為了使本揭示內容的敘述更加詳盡與完備，下文針對了本發明的實施態樣與具體實施例提出了說明性的描述；但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而，亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。
為了便於說明，此處整理了說明書、實施例與附隨申請專利範圍中所用的某些詞彙。除非本說明書另有定義，此處所用的科學與技術詞彙的含義與本發明所屬技術領域中具有通常知識者所理解與慣用的意義相同。
在不和上下文衝突的情形下，本說明書所用的單數名詞涵蓋該名詞的複數型；而所用的複數名詞時亦涵蓋該名詞的單數型。此外，在本說明書與申請專利範圍中，「至少一」與「一或更多」等表述方式的意義相同，兩者都代表包含了一、二、三或更多。更有甚者，在本說明書與申請專利範圍中，「A、B及C其中至少一者」、「A、B或C其中至少一者」以及「A、B和/或C其中至少一者」係指涵蓋了僅有A、僅有B、僅有C、A與B兩者、B與C兩者、與C兩者、以及A、B與C三者。
雖然用以界定本發明較廣範圍的數值範圍與參數皆是約略的數值，此處已盡可能精確地呈現具體實施例中的相關數值。然而，任何數值本質上不可避免地含有因個別測試方法所致的標準偏差。在此處，「約」通常係指實際數值在一特定數值或範圍的正負10%、5%、1%或0.5%之內。或者是，「約」一詞代表實際數值落在平均值的可接受標準誤差之內，視本發明所屬技術領域中具有通常知識者的考量而定。當可理解，除了實驗例之外，或除非另有明確的說明，此處所用的所有範圍、數量、數值與百分比(例如用以描述材料用量、時間長短、溫度、操作條件、數量比例及其他相似者)均經過「約」的修飾。因此，除非另有相反的說明，本說明書與附隨申請專利範圍所揭示的數值參數皆為約略的數值，且可視需求而更動。至少應將這些數值參數理解為所指出的有效位數與套用一般進位法所得到的數值。在此處，將數值範圍表示成由一端點至另一端點或介於二端點之間；除非另有說明，此處所述的數值範圍皆包含端點。
本揭示內容一般係關於多種分子構建體，其中每一分子構建體包含一標的元件(T)與一效應元件(E)，在本說明書中有時將這些分子構建體稱為「T-E分子」、「T-E藥物」或「T-E藥品」。
在此處，「標的元件(targeting element)」一詞係指分子構建體能夠直接或間接結合到一目標標的(如，一細胞表面上的受體或一組織中的蛋白質)的部分，因而能夠協助將本發明分子構建體遞送到目標標的。在某些例子中，標的元件可將所述分子構建體導引至鄰近目標細胞處。在其他情形中，標的元件可專一地結合到目標細胞表面上的分子；或標的元件可和第二分子專一地結合，而此一第二分子能夠專一地結合到目標細胞表面上的分子。在某些例子中，一旦標的元件與目標標的接合之後，標的元件可將本發明分子構建體內化，而使其移動到目標細胞的細胞質內。標的元件可以是細胞表面受體的抗體或配體；或者是可和上述抗體或配體結合的分子，因而能夠間接地將本發明分子構建體標的到目標部位(譬如所選定的細胞表面)。相較於沒有標的功能的治療藥物，利用本發明分子構建體能夠加強或有利於效應元件(治療藥劑)在疾病部位的局部化(localization)。上述局部化是一種程度或相對的比例，而不是讓效應元件在疾病部位絕對或完全的局部化。
根據本發明，「效應元件(effector element)」一詞係指一旦分子構建體到達目標部位後，所述分子構建體中能夠發揮生物活性(譬如，誘發免疫反應、發揮細胞毒性及與其相似者)或其他功能活性(譬如招募其他經半抗原標記的治療用分子)的部分。「效應」可指治療性或診斷性的效果。效應元件包含能夠和細胞和/或細胞外免疫調節因子結合的元件。上述效應元件包含如蛋白質、核酸、脂質、碳水化合物、醣蛋白、藥物部分(包含小分子藥與生物製劑)、化合物元素及同位素等物質，也包含上述物質的片段。
雖然在此處利用「第一」、「第二、「第三」等詞彙來描述多種元件、部件、區域和/或區段，這些元件(以及部件、區域和/或區段)不受上述修飾詞的限制。此外，除非上下文有明示的說明，使用這些序數並未隱含序列或順序。反之，這些詞彙僅是用來區分各元件。因此，亦可將下文所述的第一元件命名為第二元件，而不會悖離例示性實施方式所揭示的內容。
在此處，「連接(link)」、「耦接(couple)」以及「複合(conjugate)」等詞可互換使用，且都是用來指稱透過直接或間接的連接關係，將兩個元件連接在一起。
「多肽(polypeptide)」一詞在此係指具有至少兩個胺基酸殘基的聚合物。一般來說，多肽包含2到200個胺基酸殘基；在較佳的情形中，是2到50個殘基。在本說明書中所提及的胺基酸序列涵蓋了此種序列的L-、D-或β-胺基酸等形式。多肽亦包括胺基酸聚合物，其中有一或多個胺基酸殘基是與一天然胺基酸相應的人工化學類似物，當然也包括天然產生的胺基酸聚合物。此外，此一詞彙亦涵蓋以多肽鏈或其他方式連接的胺基酸，上述其他方式如經修飾的連接(modified linkage)，譬如以α-酯(α-ester)、β-酯(β-ester)、硫醯胺(thioamide)、磷醯胺(phosphoramide)、氨基甲酸酯(carbomate)、羥基酯(hydroxylate)鍵、以及與其相似者來取代多肽鍵。
於一些實施方式中，本發明範圍亦涵蓋了其他相較於所述序列具有保守性置換的蛋白。於多種實施方式中，有一、二、三、四或五個不同的殘基經過置換。在此處，「保守性置換(conservative substitution)」一詞是指所述胺基酸置換不會明顯地改變分子活性(譬如生物或功能活性和/或專一性)。一般來說，保守性胺基酸置換是利用另一種具有相似化學性質(如電荷或疏水性)的胺基酸來取代某一胺基酸。某些保守性置換包括「類似物置換」，亦即以非標準(如罕見或合成等)胺基酸來取代標準胺基酸，而上述非標準胺基酸和被取代的原有胺基酸之間的差異極小。可由標準胺基酸經人工修飾而在不會大幅改變原有胺基酸結構的前提下，得到所述的胺基酸類似物，譬如異構物或代謝前驅物等皆屬之。
於一些實施方式中，本案的範圍亦涵蓋和任何所述序列具有至少80%序列相似度的任何序列，上述序列相似度較佳為至少85%或90%、更佳為至少95%或98%。
此處針對多肽序列所述的「胺基酸序列相似度百分比(Percentage (%) amino acid sequence identity)」係指候選序列的胺基酸殘基與參考多肽序列的胺基酸殘基完全相同的百分比；於進行上述比對時，可將所述的候選多肽片段與所述的特定多肽片段並排，並於必要時引入間隙，以使二序列形成最高的序列相似度；在計算相似度時，保守性置換的胺基酸殘基視為不同的殘基。相關領域已有多種方法可用以進行上述並排，譬如可公開取得的軟體如BLAST、BLAST-2、ALIGN或Megalign (DNASTAR)等。本發明所屬技術領域中具有通常知識者在進行並排時，可選擇適當的參數與計算方式，以得到最佳的排列方式。在本說明書中，二多肽序列間的序列比較是採用美國國家生物科技資訊中心(Nation Center for Biotechnology Information，NCBI)所提供的蛋白質-蛋白質BLAST分析資料庫Blastp來進行。候選多肽序列A相較於參考多肽序列B的胺基酸序列相似度(在本說明書中亦稱之為多肽序列A與多肽序列B具有特定百分比(%)的胺基酸序列相似度)的計算方式如下：% 其中X是利用BLAST序列並排程式對序列A、B進行排列後所得到的相同胺基酸殘基數目(identical matches)，而Y是A、B二序列中較短者的胺基酸殘基總數。
「聚乙二醇化胺基酸(PEGylated amino acid)」一詞在此係指帶有一個胺基(amino group)與一個羧基(carboxyl group)的聚乙二醇(polyethylene glycol，PEG)鏈。一般來說，聚乙二醇化胺基酸的結構式為NH2 -(CH2 CH2 O)n -COOH。在本揭示內容中，n值介於1到20之間；較佳是介於2至12。
在此處一多肽的「端」係指位於該多肽N-或C-端點的胺基酸殘基。在描述一聚合物(譬如此處所述的聚乙二醇)的組成單元時，「端」則是指在聚合物骨架末端的部分。在本說明書與申請專利範圍中，「自由端」一詞是用來指稱並未與另一個分子形成化學鍵結的末端胺基酸殘基或構成單元。
「抗原(antigen或Ag)」一詞係指能夠導致免疫反應的分子。此種免疫反應可能涉及分泌性(secretory)、荷爾蒙性(humoral)和/或細胞級(cellular)的抗原專一反應。在本揭示內容中，「抗原」一詞係指蛋白質、多肽(包括其突變株或具有生物活性的片段)、多醣、醣蛋白、醣脂質、核酸或上述之組合。
在本說明書與申請專利範圍中，「抗體(antibody)」一詞應以廣義方式解釋，且包含完整組裝的抗體、可和抗原結合的抗體片段，譬如抗原結合片段(Fab/Fab’)、F(ab’)2 片段(具有彼此以雙硫鍵連接的兩個Fab部分)、可變片段(variable fragment，Fv)、單鏈可變片段(scFv)、雙單一性單鏈可變片段(bi-scFv)、奈米抗體(nanobodies)、單抗體(unibodies)以及雙體抗體(diabodies)。「抗體片段」包含完整抗體的一部分，較佳為包括完整抗體的抗原結合區域或可變區域。在典型的例子中，「抗體」係指實質上由免疫球蛋白基因或其片段所編碼的一或多種多肽所組成的蛋白質。習知的免疫球蛋白基因包括κ (kappa)、λ (lambda)、α (alpha)、γ (gamma)、δ (gamma)、ε (epsilon)與μ (mu)等恆定區基因(constant region gene)、以及無數的免疫球蛋白可變區基因(variable region genes)。輕鏈通常歸類為κ (kappa)或λ (lambda)。重鏈通常歸類為γ (gamma)、μ (mu)、α (alpha)、δ (gamma)或ε (epsilon)，基於這些結構，定義出了以下類型的免疫球蛋白：IgG、IgM、IgA、IgD以及IgE。典型的免疫球蛋白抗體結構是一種四聚物(tetramer)。每一個四聚物是由兩條相同的多肽鏈所組成，且每一對分別有一「輕」鏈(約25 kDa)與一「重」鏈(約50-70 kDa)。每一鏈的N-端界定了一個可變區域，包含約100至110個或更多的胺基酸，其主要負責抗原辨識。可變輕鏈(variable light chain，VL )與可變重鏈(variable heavu chain，VH )等詞就是分別指上述的輕鏈與重鏈。根據本揭示內容的實施方式，可藉由改變天然抗體或利用重組DNA方式重新合成上述抗體片段。於本揭示內容一些實施方式中，上述抗體和/或抗體片段可具有雙專一性，且可有多種不同的構形。舉例來說，雙專一性抗體可包含二個不同的抗原結合部位(可變區域)。於多種實施方式中，可利用融合瘤技術或重組DNA技術來製備雙專一性抗體。於一些實施方式中，雙專一性抗體對至少兩種不同的表位(epitope)有結合專一性。
「專一地結合(specifically bind)」一詞在此處係指抗體或其抗原結合片段能夠和抗原結合的能力，上述結合的解離常數(dissociation constant，Kd)小於約1×10− 6 M、1×10− 7 M、1×10− 8 M、1×10− 9 M、1×10− 10 M、1×10− 11 M或1×10− 12 M；或者是或額外地，相較於其對於非專一性抗原的結合親和力，上述抗體或其抗原結合片段與抗原結合的親和力為兩倍以上。
「免疫疾病(immune disorder)」一詞在此處是指涉及體液免疫(humoral immunity)缺失、細胞介導免疫(cell-mediated immunity)缺失、綜合免疫缺失(combined immunity deficiency)、不明免疫缺失(unspecified immunity deficiency)以及自體免疫疾病的相關疾病。
「治療(treatment)」一詞係指預防性(如，預防用藥)、療癒性或緩和性的處置，藉以達到所欲的藥學和/或生理學效果；而治療的行為在此包括上述預防性、療癒性或緩和性的處置。具體來說，治療在此係指對於可能患有一醫療疾患、症狀、疾病或與疾患相關的異常、或易於罹患一疾患的個體施用或投予本發明分子構建體或包含此分子構建體的藥學組合物，，以期能部分地或完全地緩和、改善、減輕特定異常和/或病症的一或多種症狀或特徵，或是延緩其發生、阻礙其進展、減輕其嚴重性和/或減低發生率。亦可對尚未表出現疾病、異常和/或病症徵兆的個體和/或出現早期徵兆的個體進行治療，以期降低發展出與該疾病、異常和/或病症相關的病理變化的風險。
在此處，「有效量(effective amount)」一詞係指本發明分子構建體的用量足以招致所欲的治療反應。藥劑的有效量不必然能夠治癒疾病或病症，但能夠延緩、阻礙或防止該疾病或病症的發生，或是可緩減與疾病或病症相關的病徵。可將治療有效量可分成一、二或更多劑，而以適當的劑型在指定期間內施用一次、二次或更多次。具體的治療有效量取決於多種因素，例如欲治療的特定狀況、個體的生理條件(如，個體體重、年齡或性別)、接受治療的個體類型、治療持續時間、併行治療(如果有的話)的本質以及所用的具體配方和化合物或其衍生物的結構。舉例來說，可將治療有效量表示成活性成分的總重量，譬如以克、毫克或微克來表示；或表示成活性成分重量相對於體重的比例，譬如表示為每公斤體重多少毫克(mg/kg)。
所述「施用(application)」與「投予(administration)」等詞在此可交替使用，其係指將本發明之分子構建體或藥學組合物提供給需要治療的個體。
「個體(subject)」或「患者(patient)」等詞在此可交互使用，且是指可接受本揭示內容之分子構建體、藥學組合物和/或方法處置的動物(包含人類)。除非另有指明，「個體」或「患者」一般包含雄性與雌性。「個體」或「患者」包含任何可因本揭示內容的處置而獲益的哺乳類動物。所述「個體」或「患者」的例示包含，但不限於：人類、大鼠、小鼠、天竺鼠、猴子、豬、山羊、牛、馬、狗、貓、鳥和禽類。在一實例中，所述患者為人類。所述哺乳類動物涵蓋哺乳動物綱的所有成員，包括人類、靈長類動物、家畜和農畜(如兔子、豬、綿羊和牛)、動物園動物或競賽用動物、寵物，以及齧齒類動物(如，小鼠和大鼠)。「非人類哺乳動物」一詞則涵蓋除了人類以外的所有哺乳動物綱成員。
本揭示內容至少部分是基於建構出了T–E藥物，此種T-E藥物可被遞送至目標細胞、目標組織或器官，且其在這些部位的比例相對高於在血液循環、淋巴系統以及其他細胞、組織和器官中的比例。當實現上述情境時，即可提升T-E藥物的療效，且其副作用與毒性的數目和嚴重性都會降低。相較於不含標的元件的藥物，以T-E分子的形式來投遞藥物時，發揮療效的效應物所用的濃度可能較低。因此，可在較低的劑量下施用治療用的效應物而不會減損其有效性，但卻能同時降低其副作用與毒性。
可因較佳藥物標的而獲益的疾病
若是可將藥物標的到疾病部位，亦即若是可使藥物在疾病部位(相對於在正常組織或器官)局部化或有較優勢的濃度，就能夠改善用以治療許多疾病的藥物，使其具備較佳的療效與安全性。下文提出一些主要的例示性疾病，若是能使得這些疾病所用藥物在疾病部位或細胞有較佳的分布比例，就可以改善這些藥物。
I 免疫疾病
可利用本發明方法而以本揭示內容之分子構建體來治療的疾病、病症和/或異常可以是免疫疾病；例如，自體免疫疾病；所述的自體免疫疾病包括，但不限於：牛皮癬、全身性紅斑狼瘡、皮膚狼瘡、乾燥綜合症、類風濕性關節炎、牛皮癬性關節炎關節黏連性脊椎炎以及發炎性腸症。
大多數的自體免疫疾病(如類風濕性關節炎、全身性紅斑狼瘡，乾燥綜合症、牛皮癬、克羅恩氏病、發炎性腸症及其他)都會影響結締組織。自體免疫疾病有多種致病機制，譬如可能是由環境、遺傳、表觀遺傳(epigenetic)等因素或其組合所造成，但不論其致病本質為何，受影響的組織都會因為長時間的發炎過程而受到損傷。本發明合理推論，可利用細胞外基質作為標的抗原，將抗發炎治療劑(如抗TNF-α、抗IL-17、抗BAFF、抗IL-6、抗IL-12/IL-23等)帶到發病的結締組織所在處。適當的標的抗原可包括許多種類的膠原蛋白、層連結蛋白(laminins)、彈性蛋白(elastins)、小纖維蛋白(fibrillins)、纖網蛋白(fibronectins)以及生腱蛋白(tenascins)。結締組織填充於人體幾乎所有部分。然而，由於在不同位置對於結構組織的結構性與功能性要求不同，這些細胞外基質成分的種類也不一樣，因此這些細胞外基質成分具有極佳的組織標的專一性。
相較於選用細胞表面抗原來作為抗發炎治療劑的標的對象，選用細胞外基質成分的優點在於在多樣的基質蛋白間類型提供了不同的選擇性，而且細胞外基質蛋白的含量非常豐富。更有甚者，由於並未使用細胞作為抗原標的，也可避免抗發炎治療劑直接與細胞結合可能帶來的危害。
I-(i) 類風濕性關節炎、牛皮癬性關節炎或關節黏連性脊椎炎
已有多種抗TNF-α抗體(譬如英夫利昔單抗(infliximab)與阿達木單抗)以及TNF-α受體和IgG.Fc融合蛋白(如依那西普(etanercept))經核准可用於類風濕性關節炎、關節黏連性脊椎炎以及其他自體免疫疾病的人體臨床試驗。介白素-1(IL-1)受體的細胞外部分—阿那白滯素(anakinra)—則經核准可用以治療類風濕性關節炎。針對IL-12以及IL-23共有的p40蛋白的抗體，譬如優特克單抗(ustekinumab)以及貝伐珠單抗(briakinumab)，則經核准可用以治療牛皮癬性關節炎或用於類風濕性關節炎的臨床試驗。抗IL-6受體的抗體(托珠單抗(tocilizumab))經核准可用於類風濕性關節炎與全身幼年性自發關節炎(systemic juvenile idiopathic arthritis)；而多種抗IL-6抗體，譬如沙里魯單抗(sarilumab)與奧洛珠單抗(olokizumab)，則正在進行治療類風濕性關節炎的臨床試驗。對IL-17專一的抗體—蘇金單抗(secukinumab)—經核准可用以治療牛皮癬，且也已用於類風濕性關節炎與關節黏連性脊椎炎的臨床試驗。
雖然上述治療藥劑比起先前所用的藥劑更能夠減緩較為嚴重的症狀，但卻會在接受治療的患者體內引發一系列的副作用。舉例來說，英夫利昔單抗會導致嚴重的血液疾病(如白血球減少症(leukopenia)以及血小板減少症(thrombocytopenia))、嚴重的感染、淋巴瘤與其他固態腫瘤、B型肝炎病毒再活化與結核病、以及其他嚴重的問題。阿那白滯素經常會造成感染，且會對腸胃道、呼吸道與造血器官造成嚴重的副作用。降低這些廣泛使用的治療藥劑的嚴重副作用，且同時保持甚至提升其療效，是非常重要的議題。
類風濕性關節炎患者的膝蓋、手指、腳趾關節與其他關節會受到影響，而關節黏連性脊椎炎患者的脊椎關節與骨盆的薦髂關節(sacroiliac joint)會受到影響。在發病的關節處，骨頭的表面還有襯接骨頭表面的關節軟骨會遭受關節中的發炎性免疫成分攻擊。關節中的關節軟骨是含有細胞外基質的平滑軟骨。軟骨沒有血管，水分佔了約60%的重量，而其他成分則是由膠原蛋白與α-蛋白聚糖(一種蛋白多醣)以及其他基質分子所組成。第II型膠原蛋白形成了軟骨中的主要纖維。蛋白聚糖是軟骨中含量第二高的成分。第XI型膠原蛋白可結合到第II型膠原蛋白原纖維(fibril)表面，以利形成原纖維網絡，且第IX型膠原蛋白與第II型膠原蛋白和第XI型膠原蛋白相關聯。軟骨有較大的表面積，而α-蛋白聚糖的結構與形狀則和羽毛相近。除了軟骨形成之外，關節處還有負責連接相鄰骨骼的韌帶，如十字韌帶以及連接肌肉和骨骼的肌腱。韌帶和肌腱是由第I、II與III型膠原蛋白還有彈性蛋白與小纖維蛋白1、2組成的纖維網絡所形成。
本發明合理推論可利用對第II型膠原蛋白、α-蛋白聚糖、第XI型膠原蛋白或第IX型膠原蛋白專一的抗體片段(如scFv)、或者是對第I型膠原蛋白彈性蛋白或小纖維蛋白1專一的抗體片段(如scFv)作為標的成分，以將TNF-α、IL-1以及IL-12/IL-23的拮抗劑遞送到疾病部位。較佳的抗第II型膠原蛋白抗體可結合至關節中的原生第II型膠原蛋白，但不會和在原纖維組裝過程中會被切除的N-端與C-端原胜肽(propeptide)結合。較佳的抗蛋白聚糖抗體可結合至整個原生的α-蛋白聚糖分子，而不會和其被切除並進入血液循環中的片段結合。藉由採用本發明分子構建體，以抗第II型膠原蛋白的scFv作為標的成分，相較於傳統的抗TNF-α、抗IL-1與抗IL12/IL-23的IgG，本發明治療劑在疾病部位的比例應該較高，而在不相關的正常組織(特別是淋巴器官)的比例則較低，故因此副作用較少。
I-(ii) 牛皮癬
大多數的牛皮癬或斑塊狀牛皮癬患者主要會在皮膚出現發炎症狀，而不會出現在其他組織與器官。牛皮癬患者最主要的症狀是皮膚某些部分的細胞出現角質化的現象。全身性地投予抗TNF-α、抗IL-12/IL-23與抗IL-17或抗IL-17受體(抗IL-17R)的單株抗體或其他抗發炎藥劑(如抗IL-6)，會導致上文所討論的多種副作用。已有許多文獻記錄了這些免疫調解抗體的嚴重副作用。
許多膜蛋白或細胞外蛋白(如絲聚合蛋白(filaggrin)、第I型膠原蛋白)在皮膚組織的表現量遠高於在其他組織的表現量，因此想要將治療劑運送到皮膚部位時，可以考慮用這些蛋白作為標的蛋白。絲聚合蛋白存在於細胞間的緊密接合處，而當抗體進到罹病組織內時，就可以接觸到這些蛋白。雖然第I型膠原蛋白也存在於骨基質以及許多身體部位中，其在皮膚真皮層中的含量極高。
患病的角質細胞所造成的發炎反應會導致牛皮癬的症狀，想要緩減此類發炎反應，不一定要讓抗發炎抗體藥物和角質細胞接觸。角質細胞位於皮膚最外層的表皮層中；血管、汗腺以及膠原纖維位於皮膚中層的真皮層中，而最裡層的下皮層主要是脂肪組織。上述三層構成了厚度約2至3釐米的人類皮膚。若利用對第I型膠原蛋白專一的scFv將抗發炎抗體運送到真皮層，抗體就可以擴散至其他層中。或者是，抗體可以將發炎性細胞介素困在上述三種皮膚層中。
亦可利用存在於真皮-上皮接面的某些蛋白作為標的，以將治療劑攜帶至皮膚。這些蛋白包括第VII型膠原蛋白、第XVII型膠原蛋白以及層連結蛋白第5、6或10型。真皮-上皮接面是將皮膚的表皮層與真皮層接合的組織區域。表皮基底層中的基底細胞透過半胞小體(hemidesmosomes)的絲狀纖維固定到基底膜。真皮的乳突層細胞藉由錨原纖維連接至基底膜，錨原纖維是由第VII型膠原蛋白所組成。第XVII型膠原蛋白是表現在角質細胞上的跨膜蛋白(亦稱為BP180)，屬於半胞小體的結構成分，半胞小體是位於真皮-上皮基底膜區域的多蛋白複合物，其可介導角質細胞與下方膜的連接。層連結蛋白存在於是基底膜中，屬於結構性的非膠原蛋白醣蛋白。在多種層連結蛋白中，第5、6與10型對位於分層上皮下方的基底層有專一性。
I-(iii) 全身性紅斑狼瘡、皮膚狼瘡或乾燥綜合症
全身性紅斑狼瘡(SLE)是一種自體免疫疾病，涉及多種自體抗原，譬如核酸、組織蛋白以及其他核蛋白。乾燥綜合症也是自體免疫疾病，患者的免疫系統攻擊外分泌腺(特別是用以產生唾液與淚液的唾腺與淚腺)造成眼睛乾燥或口乾等症狀，進而導致感染與多種其他問題。這些疾病好發於女性，特別是處於生育年齡(即，15至30歲)的女性；一般來說女性的發生率比男性高出九倍。SLE是全身性的免疫結締組織疾病，且會影響許多器官與組織。一般來說，這些組織與器官(如心臟、肺、膀胱與腎臟)具有彈性且可擴張與收縮，也都含有膠原蛋白網絡。在多種類型的SLE中，皮膚都會出現明顯的發炎症狀。
在對BAFF專一的人類單株抗體--貝利木單抗(belimumab)核准問世前，有長達50年以上的期間，都沒有任一種單一的製劑能夠治療SLE。然而，貝利木單抗對SLE的療效非常有限。此外，貝利木單抗會導致一系列副作用，包括嚴重感染的感染率較高，且治療組的死亡率也高於安慰劑組。值得注意的是，在以貝利木單抗治療乾燥綜合症的第二期臨床試驗中，其療效優於對SLE的療效。
除了BAFF之外，研究人員也一直在尋找其他的SLE治療劑。雖然目前還無法在SLE的病理過程中找出任一種主要的發炎性細胞介素，但已有許多研究觀察到，受到干擾素刺激而引發的一系列下游事件中，涉及一群基因的表現，又稱為第一型干擾素標誌(type 1 interferon signature)。已知SLE的致病機轉和類鐸受體(toll-like receptor) 7與9 (TLR7與TLR9)有關，上述TLR會誘導一群基因的標表現，這些基因與受到IFN-α活化的基因相近。
SLE的臨床試驗中使用了幾種對IFN-α專一的單株抗體，包括隆利珠單抗(rontalizumab)、西法木單抗(sifalimumab)以及阿利弗魯單抗(anifrolumab)。由於IFN-α涉及到多種生理功能，全身性地投予抗IFN-α抗體而沒有將其標的到疾病部位可能會導致嚴重的副作用。
I-(iv) 發炎性腸症
抗TNF-α抗體(如阿達木單抗)亦經許可用於治療克羅恩氏病與潰瘍性結腸炎(發炎性腸症的一種形式)。然而，如上文所述，施用抗TNF-α抗體會導致多種副作用，包括嚴重的感染性疾病以及B細胞淋巴瘤。因此，在以抗TNF-α抗體治療罹患克羅恩氏病或潰瘍性結腸炎的患者時，較理想的狀況應該是讓所投予的抗TNF-α抗體主要分布在小腸與結腸處。已知第III型與第V型膠原蛋白在小腸與大腸的結締組織中含量非常豐富。
II 骨質疏鬆症
RANKL又稱CD254，是細胞核因子κB受體活化因子(RANK)的配體；地諾單抗(denosumab)是對RANKL體專一的抗體，經核准可用於治療骨質疏鬆症。地諾單抗的研發是骨質疏鬆症治療的一大進步。然而，投予地諾單抗會導致一些常見的副作用，諸如尿道與呼吸道感染、白內障、便秘、紅疹與關節疼痛等等。因此需要將此一治療藥劑主要帶往骨頭的位置。
RANKL是一種膜蛋白，屬於腫瘤壞死因子配體家族(tumor necrosis factor ligand family)成員之一。在肺部、胸腺與淋巴結可偵測到大量的RANKL。在骨髓、胃、周邊血、脾臟、胎盤、白血球、心臟、甲狀腺與骨骼肌中的含量則較低。由於抗RANKL IgG，如地諾單抗，可作為骨質疏鬆症的治療藥劑，本發明所述的分子構建體應該會是比傳統抗RANKL IgG更為優異的治療藥劑。
由SOST基因編碼的硬化蛋白(sclerostin)也可以作為開發治療骨質疏鬆症抗體的標的。這是一種由骨細胞製造與分泌的醣蛋白，會負向調控骨母細胞骨骼生成(osteoblastic bone formation)。SOST基因的缺失或缺陷性突變會導致漸進式的骨骼增厚。另一種形式的SOST基因缺陷性突變會促進骨骼生成作用。抗硬化蛋白抗體也會促進骨骼生成作用、提升骨質密度，而使得骨骼更為強壯。布索珠單抗(blosozumab)與羅莫珠單抗(romosozumab)都是人源化抗硬化蛋白單株抗體，這兩種抗體的第一期與第二期臨床試驗顯示，抗體治療可提升骨質密度與骨骼生長作用，且會降低骨再吸收作用(bone resorption)。
有鑑於上述說明，本揭示內容提出的分子平台可用以建構本發明所述的T-E分子。下文提供了與這種以「Fc」構形為基礎之結構的相關敘述，也討論了這些分子構建體實際的應用潛力。
第一節 具有組織標的功能的抗發炎分子
以廣義的Fc構形來看，可利用免疫球蛋白抗體作為一標的或效應元件的基礎，並將與其搭配的效應或標的元件以scFv區域的形式引入其兩條γ重鏈的C-端。以傳統的「Fc」構形來看，可利用雙鏈IgG.Fc作為分子平台的基礎。每一多肽鏈可和一或兩個標的元件以及一或兩個效應元件融合，使得每一鏈共帶有二至三個元件。具有Fc構形的T-E分子總計可帶有四至六個元件(譬如scFv，生長因子或細胞介素)。在可任選的實施方式中，所述分子構建體的Fc部分亦帶有Fc介導的效應功能，如ADCC和/或補體介導的活化作用。然而在某些其他應用中，不會出現此種Fc介導的效應功能。
在設計Fc型分子構建體時，標的元件可位於N-或C-端。若效應元件透過和細胞表面元件(如CD3、CD16a、PD-1、PD-L1或CTLA-4)結合而發揮功用，應使其設於構建體的末端。若效應元件是透過和可溶性因子(如VEGF、TNF-α、IL-17或BAFF)結合並中和其功效而發揮作用，則其可位於末端的標的或效應元件和CH2-CH3之間。
在本揭示內容某些實施方式中，CH2-CH3區段(或稱CH2-CH3鏈)所攜帶的效應元件與標的元件幾乎都是由胺基酸所組成，而為了方便討論，亦將這些分子構建體稱為具有組織標的功能的抗發炎分子或抗發炎Fc型分子構建體。舉例來說，所述效應元件可以是抗體片段或可溶性受體，而標的元件也是抗體片段。本節將進一步討論Fc型分子構建體的例示性結構。
第1A圖繪示了根據本揭示內容某些實施方式的Fc型分子構建體800A。如圖所示，Fc型分子構建體800A包含兩條相同的CH2-CH3鏈810、連接於CH2-CH3鏈810的N-端的第一對效應元件E1、以及連接於CH2-CH3鏈810的C-端的第一對標的元件T1。在此一例示性的構形中，標的元件T1與效應元件E1都是抗體片段。
在某些實施方式中，CH2-CH3鏈是來自人類免疫球蛋白γ1或γ4。一般來說，當想要發揮Fc介導的功能(如抗體依賴性細胞毒性(ADCC)以及補體介導活性(發炎活化或目標細胞溶解))時，可以選擇γ1。當不想要發揮Fc介導的功能時，可以選用γ4來建構本發明Fc型分子構建體。
第1B圖所示的Fc型分子構建體800B與第1A圖的Fc型分子構建體800A結構非常相似，不同之處在於兩個效應元件E1分別連接於CH2-CH3鏈810的C-端，而兩個標的元件T1則分別連接於CH2-CH3鏈810的N-端。
根據某些實施方式，效應元件和標的元件兩者皆連接於CH2-CH3鏈的N-端。舉例來說，當效應元件與標的元件都是單鏈可變片段(scFv)時，可將效應元件與標的元件以串聯雙抗體(tandem)或雙抗體(diabody)的構形連接，因而形成連接於CH2-CH3鏈N-端的雙專一性scFv。
Fc型分子構建體800C (第1C圖)包含一Fc部分，且因此，每一CH2-CH3鏈810的N-端上接有T1-E1雙專一性scFv。
如上文所述，在某些實施方式中，抗發炎Fc型分子構建體亦可使用可溶性受體(譬如TNF-α或IL-1的可溶性受體)作為效應元件。在這些情形中，Fc型分子構建體800D (第1D圖)可帶有分別連接於CH2-CH3鏈810的N-端的兩個效應元件E1，以及分別連接於CH2-CH3鏈810的C-端的兩個標的元件T1。也可將效應元件與標的元件分別設於CH2-CH3鏈的C-端與N-端；譬如，第1E圖的Fc型分子構建體800E。
在某些例子中，第一對效應元件或第一對標的元件形成Fab構形(即，由VH -CH1區域與VL -Cκ區域組成)；此種Fab片段係連接於CH2-CH3鏈的N-端，而使得Fc型分子構建體成為IgG 構形。在這些情形中，不具備Fab構形的該對元件可連接於成對CH2-CH3區段的C-端。
舉例來說，在第1F圖所示的Fc型分子構建體800F中，兩個標的元件T1分別包含VH -CH1區域820與VL- Cκ區域825，因而形成連接於CH2-CH3鏈810的N-端的Fab構形830，而使得Fc型分子構建體800F成為IgG構形。在本例中，成對的效應元件E1是連接於成對CH2-CH3鏈810的C-端。
如上文所述，本發明Fc型分子構建體最多可帶有共六個元件。此一額外的元件可以是第二對效應元件或第二對標的元件。
根據其他實施方式，Fc型分子構建體900A (第2A圖)包含第二對標的元件T2。在這些情形中，標的元件T1與T2是以串聯雙抗體或雙抗體的構形連接，而形成雙專一性scFv並連接於CH2-CH3鏈910的N-端，而效應元件E1則連接於CH2-CH3鏈910的C-端。
根據第2B圖所例示的實施方式，Fc型分子構建體900B包含第二對效應元件E2。在這些情形中，效應元件E1與E2是以串聯雙抗體或雙抗體的構形連接，而形成雙專一性scFv並連接於CH2-CH3鏈910的N-端，而標的元件T1則連接於CH2-CH3鏈910的C-端。
在討論了抗發炎Fc型分子構建體的基本結構之後，下文進一步說明某些由特定效應元件(們)與標的元件(們)所形成的組合。
根據某些實施方式，效應元件為對TNF-α專一的scFv，且標的元件是對第II型膠原蛋白、第IX型膠原蛋白或α-蛋白聚糖專一的scFv。根據某些實施方式，二效應元件分別是對IL-17專一的scFv，而標的元件是對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。又或者是，二效應元件分別是對BAFF專一的scFv，且標的元件是對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。在某些實施方式中，每一效應元件是對TNF-α專一的scFv，而標的元件是對第III型膠原蛋白或第V型膠原蛋白專一的scFv。在某些其他實施方式中，二效應元件形成對RANKL專一的Fab，且標的元件是對第I型膠原蛋白或骨結合素專一的scFv。舉例來說，此種分子構建體可採用第1A至1C以及1F圖任一者所示的構形。
在某些實施方式中，第一對效應元件包括對TNF-α專一的scFv或對IL-17專一的scFv，而第一對標的元件包括一對第II型膠原蛋白專一的scFv或對第IX型膠原蛋白專一的scFv。在某些替代性的實施方式中，第一對效應元件包括對TNF-α專一的scFv或對IL-17專一的scFv，而第一對標的元件包括對第I型膠原蛋白專一的scFv或對第VII型膠原蛋白專一的scFv。或者是，二效應元件分別是對BAFF專一的scFv，而標的元件是對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。又或者是，二效應元件分別是對RANKL專一的scFv，而標的元件是對第I型膠原蛋白或骨結合素專一的scFv。這些分子構建體可採用第1B、1D以及1F圖任一者所示的構形。
於一些實施方式中，二效應元件為對TNF-α專一的Fab抗體，而標的元件是對第II型膠原蛋白或第IX型膠原蛋白專一的scFv。在某些實施方式中，二效應元件是對IL-17專一的Fab抗體，而標的元件是對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。或者是，二效應元件是對BAFF專一的Fab抗體，而標的元件是對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。又或者是，二效應元件是對TNF-α專一的Fab抗體，且標的元件是對第III型膠原蛋白或第V型膠原蛋白專一的scFv。
本發明的精髓在於採用特定標的與效應元件的組合或搭配。在較佳的實施方式中，所述分子構建體採用了Fc融合構形。本發明所屬技術領域具有通常知識者當可想見，亦可利用其他分子平台來連接本發明標的與效應元件的組合；上述分子平台諸如多肽、蛋白質(譬如白蛋白)、聚醣、聚乙二醇以及其他類型的聚合物，只要其能夠作為連接多個分子元件的結構基礎即可。
第二節 具有組織標的功能的抗發炎分子的用途
本揭示內容的另一態樣是關於上文第一節所述的抗發炎Fc型分子構建體的用途。
當可理解，上文第4節(i)部分中關於選擇適當標的與效應元件為的論述亦適用於本節。舉例來說，用以治療多種免疫疾病的抗發炎Fc型分子構建體可含有對第II型膠原蛋白、第XI型膠原蛋白或α-蛋白聚糖專一的抗體片段(譬如scFv、Fab及與其相似者)以作為標的元件，並含有對TNF-α或IL-17專一的抗體片段(譬如scFv、Fab及與其相似者)以作為效應元件。
根據本揭示內容多種實施方式，本發明治療方法涉及對需要治療的個體投予適當的抗發炎Fc型分子構建體。下文討論了用以治療多種免疫疾病(特別是自體免疫疾病)的抗發炎Fc型分子構建體的具體例子。
根據某些實施方式，本發明方法可用以治療類風濕性關節炎、牛皮癬性關節炎或關節黏連性脊椎炎。在這些情形中，抗發炎Fc型分子構建體的每一效應元件可以是對TNF-α、IL-12/IL-23、IL-1、IL-17或IL-6專一的抗體片段，而每一標的元件可以是對第II型膠原蛋白、第IX型膠原蛋白、第XI型膠原蛋白或α-蛋白聚糖專一的抗體片段。舉例來說，第一對效應元件的每一效應元件是對TNF-α專一的scFv，而第一對標的元件的每一標的元件則是對第II型膠原蛋白專一的抗體片段。在其他實施方式中，效應元件是對TNF-α專一的scFv，而標的元件是對第IX型膠原蛋白專一的抗體片段。或者是，效應元件是對TNF-α專一的scFv，而標的元件是對α-蛋白聚糖專一的抗體片段。根據多種實施方式，此種抗發炎Fc型分子構建體可具有上文所述的800A、800B或800C構形。
在另一種用於治療類風濕性關節炎、牛皮癬性關節炎或關節黏連性脊椎炎的抗發炎Fc型分子構建體中，兩個效應元件形成了對TNF-α專一的Fab抗體。在這些情形中，第一對標的元件的兩個標的元件都是對第II型膠原蛋白專一的scFv或對第IX型膠原蛋白專一的scFv。這些Fc型分子構建體的構形如第1F圖所示。
本發明方法亦可用於治療牛皮癬。舉例來說，抗發炎Fc型分子構建體可包含對TNF-α、IL-12/IL-23或IL-17專一的抗體片段以作為效應元件，並包含對第I型膠原蛋白或第VII型膠原蛋白專一的抗體片段以作為標的元件。根據某些實施方式，效應元件是對IL-17專一的scFv，而標的元件是對第I型膠原蛋白專一的scFv。或者是，效應元件是對IL-17專一的scFv，而標的元件是對第VII型膠原蛋白專一的scFv。這些抗發炎Fc型分子構建體的構形如上文800A、800B或800C所示。
在另一種用於治療牛皮癬的抗發炎Fc型分子構建體中，成對的效應元件形成了對IL-17專一的Fab抗體。在這些情形中，第一對標的元件的兩個標的元件都是對第I型膠原蛋白專一的scFv或對第VII型膠原蛋白專一的scFv。這些Fc型分子構建體的構形如第1F圖所示。
另一組可利用本發明抗發炎Fc型分子構建體來治療的疾病是全身性紅斑狼瘡、皮膚狼瘡或乾燥綜合症。在這些實施方式中，每一效應元件是對BAFF專一的抗體片段，而每一標的元件是對第I型膠原蛋白或第VII型膠原蛋白專一的抗體片段。這些抗發炎Fc型分子構建體的構形如第1A至1C圖所示。當可理解，成對的效應元件也可以是對BAFF專一的Fab抗體，而成對的標的元件則可以是對第I型膠原蛋白或第VII型膠原蛋白專一的scFv，其構形如第1F圖所示的分子構建體800F。
在其他實施方式中，本發明方法可用以治療發炎性腸症，如克羅恩氏病或潰瘍性結腸炎。在這些情形中，每一效應元件是對TNF-α專一的抗體片段，而每一標的元件是對第III型膠原蛋白或第V型膠原蛋白專一的抗體片段。這些Fc型分子構建體的構形如第1A圖至1C所示。當可想見，成對的效應元件也可以是對TNF-α專一的Fab抗體，而成對的標的元件則可以是對第III型膠原蛋白或第V型膠原蛋白專一的scFv，因而形成如第1F圖所示的構形。
本發明抗發炎Fc型分子構建體亦可用於治療骨質疏鬆症。舉例來說，效應元件可以是對RANKL專一的抗體片段，而標的元件則是對第I型膠原蛋白或骨結合素專一的抗體片段。當可理解，對RANKL專一的抗體片段可以是scFv，而使得Fc型分子構建體具有第1A至1C圖所示的構形；或可以形成Fab的形式，而使得Fc型分子構建體具有第1F圖所示的構形。
當可想見，上述例子僅為說明，且利用本發明發炎Fc型分子構建體並搭配其他的T-E組合來治療疾病亦屬於本揭示內容之範圍。
實驗例
實驗例 1 ：建構編碼雙鏈 IgG4.Fc 融合蛋白 ( 含有對人類第 II 型膠原蛋白專一的 scFv 與對 TNF-α 專一的 scFv) 的基因片段
可產生抗第II型膠原蛋白抗體的小鼠B細胞融合瘤II-II6B3係購自艾荷華大學Developmental Studies Hybridoma Bank。利用SuperScript III RT-PCR系統(Invitrogen, Waltham, USA)進行Poly(A)+ RNA反轉錄，並合成第一股cDNA。II-II6B3的VH 與VL 核苷酸序列與胺基酸序列尚未公開；因此，利用Ig-primer Sets (Novagen, Madison, USA)提供的DNA引子組並根據製造商的操作說明進行PCR，以決定II-II6B3可變區域的序列。對第II型膠原蛋白(CII或COL2)專一的II6B3單株抗體VH 與VL 的胺基酸序列如序列編號：3與4所示。對TNF-α專一的scFv的VL 與VH 序列即為阿達木單抗的VL 與VH 序列。
雙鏈IgG4.Fc融合蛋白分子構建體的構形如下所示。在scFv1-scFv2-CH2-CH3 (人類γ4)重組鏈中，透過可撓性鉸鏈區將兩個二scFv融合到IgG4.Fc的CH2區域N-端；上述兩個scFv一個是對人類第II型膠原蛋白專一的scFv，而另一個是對人類TNF-α專一的scFv。第一scFv (對第II型膠原蛋白專一)的方向是VL -接合物-VH ，而第二scFv (對TNF-α專一)的方向是VH -接合物-VL 。這兩個scFv中各別的VL 與VH 是透過親水性接合物(GSTSGSGKPGSGEGSTKG)而連接。兩個scFv之間則是透過可撓性接合物((GGGGS)3 )而連接。下圖所示的IgG4.Fc融合蛋白分子構建體中的重組鏈序列如序列編號：5所示。
上述雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc分子構建體的構形如下圖所示。
實驗例 2 ：重組雙鏈 (scFv α CII)-(scFv α TNF-α)-hIgG4.Fc 融合蛋白的表現與純化
在本實施例中，將編碼的基因序列置於pcDNA3表現匣中。將Expi293F細胞以每毫升2.0 × 106 個活細胞的密度播於Expi293F表現培養基中，並維持18至24小時後進行轉染，以確保進行轉染時細胞處於分裂狀態。進行轉染時，將帶有7.5×108 個細胞的255 ml培養基置於2-L艾氏燒瓶中，並加入ExpiFectamine™ 293轉染試劑。於轉染後，將轉染細胞在37°C下以定軌搖動器(125 rpm)培育16至18小時；之後在燒瓶中加入ExpiFectamine™ 293轉染強化劑1與強化劑2，並繼續培育7天。收集培養上清液，並使用蛋白A親和力層析法純化培養基中的重組雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc融合蛋白。將緩衝液置換為PBS，之後利用SDS-PAGE測定(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc蛋白的濃度並進行分析；參見第3圖；以10% SDS-PAGE來分析II-II6B3 (電泳線1)與(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc (電泳線2)。在約80 kDa的位置觀察到含Fc-融合分子構建體的主要電泳條，其大小與預期相符。
實驗例 3 ：重組雙鏈 (scFv α CII)-(scFv α TNF-α)-hIgG4.Fc 融合蛋白結合力的 ELISA 分析
透過ELISA來分析重組雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc融合蛋白對第II型膠原蛋白的結合能力，並以阿達木單抗與小鼠親本單株抗體II-II6B3作為對照。以5 μg/mL的人類第II型膠原蛋白(人類COL2)、小鼠第II型膠原蛋白(小鼠COL2)、以及雞第II型膠原蛋白(雞COL2)分別塗覆在ELISA盤上。1D11是人類抗塵蟎IgG1抗體，用作同型控制。分別利用HRP標記的山羊抗人類IgG4.Fc、山羊抗小鼠IgG.Fc以及山羊抗人類IgG1.Fc來偵測重組雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc融合蛋白、純化的抗第II型膠原蛋白抗體(II-II6B3)以及阿達木單抗。ELISA結果如第4A圖所示。
透過ELISA來分析重組雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc融合蛋白對人類TNF-α的結合能力，並以阿達木單抗與小鼠親本單株抗體II-II6B3作為對照。以1 μg/mL的人類TNF-α與1 μg/mL的人類血清白蛋白(控制組)塗覆在ELISA盤上。分別利用HRP標記的山羊抗人類IgG4.Fc、山羊抗小鼠IgG.Fc以及山羊抗人類IgG1.Fc來偵測重組雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc融合蛋白、純化的抗第II型膠原蛋白抗體(II-II6B3)以及阿達木單抗。第4B圖的ELISA結果顯示，重組雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc融合蛋白對於人類TNF-α有明顯的結合活性；HSA (人類血清白蛋白)為控制組。
實驗例 4 ：製備帶有對人類第 II 型膠原蛋白專一的 scFv 、對 TNF-α 專一的 scFv 以及對人類 IL-17 專一的 scFv 的雙鏈 IgG4.Fc 融合蛋白
scFv1-scFv2-CH2-CH3-scFv3 (人類γ4)重組鏈透過可撓性鉸鏈區將兩個二scFv融合到IgG4.Fc的CH2區域N-端；上述兩個scFv一個是對人類第II型膠原蛋白專一的scFv，而另一個是對人類TNF-α專一的scFv；另外，將第三個scFv (對IL-17專一)融合到CH3區域的C-端 。
對第II型膠原蛋白專一的scFv的VH 與VL 和II6B3單株抗體相同；對TNF-α專一的scFv的VL 與VH 和阿達木單抗相同；對IL-17專一的scFv的VH 與VL 和蘇金單抗相同。第一scFv (對第II型膠原蛋白專一)的方向是VL -接合物-VH 、第二scFv (對TNF-α專一)的方向是VH -接合物-VL 、且第三scFv (對IL-17專一)的方向是VL -接合物-VH 。這三個scFv中各別的VL 與VH 是透過親水性接合物(GSTSGSGKPGSGEGSTKG)而連接。第一和第二scFv之間則是透過可撓性接合物((GGGGS)3 )而連接。
下圖所示的IgG4.Fc融合蛋白分子構建體中的重組鏈序列如序列編號：6所示。利用上文實驗例所述的方法，在Expi293F細胞中表現所建構的基因並純化所表現的融合蛋白。利用SDS-PAGE與ELISA分析來定性所製備的新構建體。蘇金單抗(Cosentyx)係購自長庚醫院(臺北，臺灣)；人類IL-17抗體係購自Peprotech (NJ, USA)。第5A圖的SDA-PAGE結果顯示新構建體的重組鏈大小約為110 kDa，和預期大小相符。第5B圖的ELISA結果則顯示上述重組Fc-融合蛋白對人類第II型膠原蛋白、人類TNF-α與人類IL-17有結合活性。
上述雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17)分子構建體的構形如下圖所示。
實驗例 5 ：製備帶有 TNF-α 可溶性受體以及對第 II 型膠原蛋白專一的 scFv 的雙鏈 IgG1.Fc 融合蛋白
藉由融合人類TNF-α受體、IgG1.Fc、以及對第II型膠原蛋白專一的scFv，以建構(TNF-α受體)-CH2-CH3-(scFv α CII) (人類γ1)重組鏈。TNF-α受體與IgG1.Fc的受體與依那西普相同。依那西普和scFv則透過可撓性接合物((GGGGS)3 )而融合。IgG4.Fc融合蛋白分子構建體中重組鏈的序列如序列編號：7所示。
利用上文實驗例所述的方法，在Expi293F細胞中表現所建構的基因並純化所表現的融合蛋白。利用SDS-PAGE與ELISA分析來定性所製備的新構建體。依那西普(Enbrel)係購自長庚醫院(臺北，臺灣)。第6A圖的SDS-PAGE結果顯示此一新分子構建體的重組鏈大小約為100 kDa，與預期大小相符(一個依那西普分子的分子量為150 kDa，故其中一條鏈的大小約為75 kDa；scFv的大小則約為27 kDa)。第6B圖的ELISA結果顯示上述分子構建體可結合至人類TNF-α、人類第II型膠原蛋白以及小鼠第II型膠原蛋白。
上述雙鏈(可溶性TNF-α受體)-IgG1.CH2-CH3-(scFv α CII)分子構建體的構形如下圖所示。
實驗例 6 ：製備帶有抗人類 TNF-α 完整抗體以及對第 II 型膠原蛋白專一的 scFv 的雙鏈融合 蛋白
藉由融合對人類TNF-α專一的完整抗體以及對第II型膠原蛋白專一的scFv，來建構IgG-scFv (人類γ1)重組鏈(如下所示)。完整抗體的序列即阿達木單抗序列。阿達木單抗和scFv間透過可撓性接合物((GGGGS)3 )而融合。
將兩個基因插入具有複數個選殖位的pG1K表現匣中，以建構重組基因的重鏈與輕鏈。利用上文實驗例所述的方法，將表現載體轉染至Expi293F細胞中，以製備帶有完整抗人類TNF-α抗體以及對第II型膠原蛋白專一的scFv的雙鏈融合蛋白。帶有完整抗人類TNF-α抗體以及對第II型膠原蛋白專一的scFv的雙鏈融合蛋白的重鏈胺基酸序列如序列編號：8所示，其輕鏈序列如序列編號：9所示。
第7圖的SDS-PAGE結果顯示，上述延伸型IgG分子構建體重組重鏈的大小約為75 kDa，與預期大小相符。
上述C-端帶有對人類第II型膠原蛋白專一的scFv的抗人類TNF-α延伸IgG分子構建體的構形如下圖所示。
實驗例 7 ：製備帶有抗人類 IL-17 完整抗體以及對第 VII 型膠原蛋白專一的 scFv 的雙鏈融合 蛋白
藉由融合對人類IL-17專一的完整抗體以及對第VII型膠原蛋白專一的scFv (序列編號：20)，來建構IgG-scFv (人類γ1)重組鏈(如下所示)。完整抗體的序列即蘇金單抗序列。蘇金單抗和scFv間透過可撓性接合物((GGGGS)3 )而融合。
將兩個基因插入具有複數個選殖位的pG1K表現匣中，以建構重組基因的重鏈與輕鏈。利用上文實驗例所述的方法，將表現載體轉染至Expi293F細胞中，以製備帶有完整抗人類IL-17抗體以及對第VII型膠原蛋白專一的scFv的雙鏈融合蛋白。帶有完整抗人類IL-17抗體以及對第VII型膠原蛋白專一的scFv的雙鏈融合蛋白的重鏈胺基酸序列如序列編號：10所示，其輕鏈序列如序列編號：11所示。
第8A圖的SDS-PAGE結果顯示，上述延伸型IgG分子構建體重組重鏈的大小約為75 kDa，與預期大小相符。第8B圖的ELISA結果顯示，上述分子構建體可結合至人類IL17與人類第VII型膠原蛋白。對第VII型膠原蛋白的結合力相對較低可能是因為用來塗覆ELISA盤的抗原濃度較低的關係。
上述C-端帶有對人類第VII型膠原蛋白專一的scFv的抗人類IL-17延伸IgG分子構建體的構形如下圖所示。
實驗例 8 ：製備帶有對人類第 VII 型膠原蛋白專一的 scFv 以及對 BAF 專一的 FscFv 的雙鏈 IgG4.Fc 融合蛋白
利用小鼠B細胞融合瘤LH7.2 mAb的cDNA來製備抗第VII型膠原蛋白抗體，上述cDNA是倫敦大學瑪莉皇后學院英國皮膚與癌症研究所皮膚腫瘤研究室Dr. Purdie所贈。在上文的實驗例中，決定了LH7.2單株抗體的VH 與VL 序列，分別如序列編號：1與2所示。對BAFF專一的scFv的VH 與VL 序列分別和貝利木單抗的VL 與VH 的序列相同。
於建構scFv1-CH2-CH3-scFv2 (人類γ4)重組鏈(序列編號：12)時，將兩個scFv融合到IgG4.Fc的兩端；如下圖所示，其中一個scFv是對第VII型膠原蛋白專一的scFv；另一個是對BAFF專一的scFv，透過可撓性接合物((GGGGS)3 )連接到IgG4.Fc的CH3區域的C-端。第一scFv (對第VII型膠原蛋白專一)的方向是VL -接合物-VH ；而第二scFv (對BAFF專一)的方向是VH -接合物-VL 。這兩個scFv中各別的VL 與VH 是透過親水性接合物(GSTSGSGKPGSGEGSTKG)而連接。
利用上文實驗例所述的方法，在Expi293F細胞中表現所建構的基因並純化所表現的融合蛋白。利用SDS-PAGE與ELISA分析來定性所製備的新構建體。抗體貝利木單抗(Belynsta)係購自長庚醫院(臺北，臺灣)；人類抗BAFF抗體係購自GenScript (NJ, USA)。第9A圖的SDS-PAGE結果顯示此一新分子構建體的重組鏈大小約為80-90 kDa，與預期大小相符或略大於預期大小。第9B圖的ELISA結果顯示上述分子構建體可專一地結合至人類BAFF與人類第VII型膠原蛋白。
上述雙鏈Fc-融合分子構建體(scFv α CVII)-IgG4.CH2-CH3-(scFv α BAFF)的構形如下圖所示。
實驗例 9 ：製備帶有抗人類 BAFF 完整抗體與對人類第 VII 型膠原蛋白專一的 scFv 的雙鏈 IgG4.Fc 融合蛋白
藉由融合抗人類BAFF完整抗體以及對第VII型膠原蛋白專一的scFv來建構IgG-scFv (人類γ1)重組鏈。完整抗體的序列即貝利木單抗序列。貝利木單抗和scFv間透過可撓性接合物((GGGGS)3 )而融合。
將兩個基因插入具有複數個選殖位的pG1K表現匣中，以建構重組基因的重鏈與輕鏈。利用上文實驗例所述的方法，將表現載體轉染至Expi293F細胞中，以製備帶有完整抗人類BAFF抗體以及對第VII型膠原蛋白專一的scFv的雙鏈融合蛋白。
帶有完整抗人類BAFF抗體以及對第VII型膠原蛋白專一的scFv的雙鏈融合蛋白的重鏈胺基酸序列如序列編號：13所示，其輕鏈序列如序列編號：14所示。第10A圖的SDS-PAGE結果顯示此一新分子構建體的重組重鏈的大小約為80 kDa，與預期大小相符。第10B圖的ELISA結果顯示此一新穎延伸型IgG構建體可專一地結合至人類BAFF與人類第VII型膠原蛋白。
上述C-端帶有對人類第VII型膠原蛋白專一的scFv的抗人類BAFF延伸IgG分子構建體的構形如下圖所示。
實驗例 10 ：製備帶有對人類骨結合素專一的 scFv 以及對 RANKL 專一的 scFv 的雙鏈 IgG4.Fc 融合蛋白
可產生抗骨結合素(SPARC)抗體的小鼠B細胞融合瘤AON-1係購自艾荷華大學的發育研究融合瘤庫(Developmental Studies Hybridoma Bank)。利用SuperScript III RT-PCR系統(Invitrogen)進行Poly(A)+ RNA反轉錄，並合成第一股cDNA。AON-1的VH 與VL 核苷酸序列與胺基酸序列尚未公開；因此，利用Ig-primer Sets (Novagen)提供的DNA引子組並根據製造商的操作說明進行PCR，以決定AON-1可變區域的序列。對骨結合素專一的AON-1單株抗體VH 與VL 的胺基酸序列如序列編號：15與16所示。對RANKL專一的scFv的VL 與VH 序列即為地諾單抗的VL 與VH 序列。
scFv1-scFv2-CH2-CH3 (人類γ4)重組鏈透過可撓性鉸鏈區將兩個二scFv融合到IgG4.Fc的CH2區域N-端；上述兩個scFv一個是對骨結合素專一的scFv，而另一個是對RANKL專一的scFv；其序列如序列編號：17所示，結構如下圖所示。第一scFv (對骨結合素專一)的方向是VL -接合物-VH ，而第二scFv (對RANKL專一)的方向是VH -接合物-VL 。這兩個scFv中各別的VL 與VH 是透過親水性接合物(GSTSGSGKPGSGEGSTKG)而連接。兩個scFv之間則是透過可撓性接合物((GGGGS)3 )而連接。
利用上文實驗例所述的方法，在Expi293F細胞中表現所建構的基因並純化所表現的融合蛋白。利用SDS-PAGE與ELISA分析來定性所製備的新構建體。利用上文實驗例所述的方法，以ELISA分析此融合蛋白和人類骨結合素與RANKL的結合能力。利用SDS-PAGE與ELISA進行此一新穎構建體的分析。抗體地諾單抗(Prolia)係購自長庚醫院；人類RANKL與人類骨結合素(SPARC)則是購自GenScript。第11A圖的SDS-PAGE結果顯示此一新分子構建體的重組鏈大小約為80 kDa。第11B圖的ELISA結果顯示上述新穎Fc-融合構建體可專一地結合至人類SPARC與人類RANKL。
上述雙鏈(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc分子構建體的構形如下圖所示。
實驗例 11 ：製備帶有抗人類 RANKL 完整抗體與對人類骨結合素專一的 scFv 的雙鏈融合 蛋白
藉由融合抗人類RANKL完整抗體以及對人類骨結合素專一的scFv來建構IgG-scFv (人類γ1)重組鏈。完整抗體的序列即地諾單抗序列。地諾單抗和scFv間透過可撓性接合物((GGGGS)3 )而融合。
將兩個基因插入具有複數個選殖位的pG1K表現匣中，以建構重組基因的重鏈與輕鏈。利用上文實驗例所述的方法，將表現載體轉染至Expi293F細胞中，以製備帶有完整抗人類RANKL抗體以及對人類骨結合素專一的scFv的雙鏈融合蛋白。
帶有完整抗人類RANKL抗體以及對人類骨結合素專一的scFv的雙鏈融合蛋白的重鏈胺基酸序列如序列編號：18所示，其輕鏈序列如序列編號：19所示。第12A圖的SDS-PAGE結果顯示此一新分子構建體的重組重鏈的大小約為80 kDa。第12B圖的ELISA結果顯示此一新穎延伸型IgG構建體可專一地結合至人類SPARC與人類BAFF。
上述C-端帶有對人類骨結合素專一的scFv的抗人類RANKL延伸IgG分子構建體的構形如下圖所示。
實驗例 12 ：帶有對人類第 II 型膠原蛋白專一的 scFv 以及對 TNF-α 專一的 scFv 的雙鏈 IgG4.Fc 融合蛋白與關節軟骨結合的免疫組織化學分析
由中央研究院基因體研究中心的核心設施單位進行免疫組織分析，以評估所述分子構建體(即上一實驗例所述的雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc)與軟骨結合的親和力。利用二氧化碳犧牲FVB/N小鼠，並取得小鼠骨骼與軟骨樣本。取得與脛骨以及膝股骨端相連的股骨，並利用10%中性緩衝甲醛在室溫下放置48小時以固定樣本。之後，將樣本在10% EDTA (pH 7.4)中放置七天，並每天更換溶液，以將樣本去鈣。去鈣完成後，在室溫下將樣本置於10%中性緩衝甲醛中24小時以進行後固定，並在ASP6025 Tissue Processor (Leica)中，以70%乙醇在4°C下進行脫水，再進行石蠟包埋。
根據Schmitz 等人於2010年提出的方式，進行番紅(safranin O)。於進行免疫染色時，利用Leica AutoStainer XL將厚度3 μm的切片脫蠟並復水，之後根據酪胺訊號放大生物素套組(Tyramide Signal Amplification Biotin kit) (PerkinElmer)的染色流程進行染色。簡言之，加入3% H2 O2 處理15分鐘以淬滅切片中的內源性過氧化酶活性；接著進行抗原修復，加入1 mg/mL玻尿酸酶(Sigma Aldrich)在37°C下處理20分鐘，並加入20 μg/mL蛋白酶k (TOOLS)在室溫下處理10分鐘。之後利用TSA套組中的TNB緩衝液，將切片封閉。以小鼠II-II6B3抗體進行染色時，在TNB封閉之前先加入額外的小鼠IgG封閉試劑(Vector Laboratories)。初級抗II-II6B3抗體以及(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc的使用量為50 μg/mL。引入HRP時，山羊抗小鼠IgG Fc以及山羊抗人類IgG Fc (Jackson ImmunoResearch)的用量為1.6 μg/mL，使其和後續加入的生物素-酪胺反應。接著以b卵白素-HRP來標定生物素標記，並以二胺基聯苯胺(diaminobenzidine)受質(BioGenex)顯色。以蘇木素將切片對比染色，並裝載於Leica CV5030 Coverslipper上。
第13圖的圖框(A)至(C)是小鼠骺骨的免疫染色結果，進行免疫染色時，先將小鼠骺骨和單株抗體II-II6B3 (第13圖(A))、阿達木單抗(第13圖(B))或上文實驗例所製得的(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc (第13圖(C))反應後，再加入HRP-標記的山羊抗小鼠或山羊抗人類次級抗體並進行酪胺擴增。在第13圖的圖框(A)與(C)中，可在骺關節軟骨(AC)與生長板(GP)處觀察到第II型膠原蛋白。結果顯示，抗第II型膠原蛋白單株抗體II-II6B3可明顯地將AC與GP部位染色(第13圖(A))，而阿達木單抗則沒有明顯的染色效果(第13圖(B))。本案所製備的構建體亦可將將AC與GP部位染色(第13圖(C))。原始染色圖中，正向的染色結果呈棕色，此處將其轉換為灰階色彩；比例尺代表250 μm。
實驗例 13 ：帶有對第 II 型膠原蛋白專一的 scFv 、對 TNF-α 專一的 scFv 以及對 IL-17 專一的 scFv 的雙鏈 IgG1.Fc 融合蛋白與關節軟骨結合的免疫組織化學分析
利用上文實驗例所述的方式，製備組織薄切片，並以上文實驗例製備的雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17)分子構建體與對照組抗體進行染色。
第13圖的圖框(D)與(E)為小鼠骺骨經第II型膠原蛋白免疫染色的結果。以單株康體將厚度3-μm的小鼠膝股端切片染色，所用抗體分別是抗IL17a小鼠抗體(購自PeproTech, NJ, USA) (第13圖(D))以及上文製備的雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17)構建體(第13圖(E))，接著再加入HRP-標記的山羊抗小鼠或山羊抗人類次級抗體進行酪胺擴增。結果顯示。抗IL17單株抗體沒有明顯的染色效果，而本案製備的構建體則可觀察到中等程度的染色。
實驗例 14 ：帶有 TNF-α 可溶性受體以及對第 II 型膠原蛋白專一的 scFv 的雙鏈 IgG1.Fc 融合蛋白與關節軟骨結合的免疫組織化學分析
利用上文實驗例所述的方式，製備組織薄切片，並以上文實驗例製備的雙鏈(可溶性TNF-α受體)-IgG1.CH2-CH3-(scFv α CII)分子構建體與對照組抗體進行染色。
染色程序與上文實驗例相同。小鼠骺骨組織切片樣本係來自同一批次處理。結果顯示，陽性對照組II-II6B3有很明顯的染色(第14圖(A))、依那西普則沒有明顯的(第14圖(B))，而含有第II型膠原蛋白成分的AC與GP部分經過本案所製備的構建體染色後，可觀察到正向的染色結果(第14圖(C))。
實驗例 15 ：利用活體內顯影系統分析帶有對人類骨結合素專一的 scFv (SPARC) 以及對 RANKL 專一的 scFv 的重組雙鏈 IgG4.Fc 融合蛋白的生物分布
根據製造商的說明，利用Dylight 680 Antibody Labeling Kit (Thermo Scientific)來標記地諾單抗與(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc (實驗例56)。將PBS或40 μg經標記的抗體以靜脈內注射的方式注入8至10週大的BALB/c小鼠體內。在不同的時間點，利用在氧氣中的異氟烷將小鼠麻醉並以仰臥位置放置於IVIS Spectrum In Vivo Imaging System (PerkinElmer)中。使用Living Image Software V3.2在ex/em = 675/720的條件下拍攝螢光照片。
藉由觀察並比較腹部的螢光信號，來探究小鼠體內抗體的組織分並情形，以瞭解(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc的標的效果。在注射與DyLight 680複合的抗體後30分鐘、3小時與28小時，分別擷取BALB/c小鼠的螢光照片。在注射後30分鐘，抗SPARC抗體、(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc還有BoneTag滲透進入四肢的比例高於地諾單抗的比例。除了膀胱累積之外，在注射後3小時，地諾單抗、抗SPARC抗體以及(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc的分布範圍較廣，而，而BoneTag則主要限於四肢。在施用抗體後28小時，經抗SPARC抗體處處理的小鼠仍可清楚觀察到骨骼結構。
第15圖為經過螢光標記的抗體在活體內的生物分布情形。BALB/c小鼠經靜脈注射以下成分：PBS (1)、地諾單抗(2)、抗SPARC mAb (3)、(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc (4)、以及BoneTag (5)。在圖中所示時間點，利用IVIS Spectrum顯影裝置擷取影像，並以Living Image software分析。進行光譜解混(Spectral unmixing)以區隔組織自身螢光與DyLight 680信號。結果顯示，在處理後30分鐘，相較於地諾單抗，本案所製備的構建體的分布情形較貼近抗SPARC單株抗體的分布情形。隨著時間過去，分布情形會受到試劑半衰期的影響而有所不同。抗SPARC抗體與地諾單抗都是抗體，故其血清半衰期相近。
當可理解，上文實施方式的說明僅為例示，且本發明所屬技術領域具有通常知識者可對其進行各種修飾。上文的說明、實驗例與資料完整地說明了本發明例示性實施方式的結構與用途。雖然上文實施方式中揭露了本發明的具體實施例，然其並非用以限定本發明，本發明所屬技術領域中具有通常知識者，在不悖離本發明之原理與精神的情形下，當可對其進行各種更動與修飾，因此本發明之保護範圍當以附隨申請專利範圍所界定者為準。
主要元件符號如下：
800A‧‧‧Fc型分子構建體
810‧‧‧CH2-CH3鏈
T1‧‧‧標的元件
E1‧‧‧效應元件
為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂，茲將所附圖式簡單說明如下。
第1A圖至1F為根據本揭示內容多種實施方式的Fc型分子構建體的示意圖。
第2A圖與2B圖為根據本揭示內容多種實施方式的Fc型分子構建體的示意圖。
第3圖是(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc的SDS-PAGE分析結果。
第4A、4B圖分別是(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc對第II型膠原蛋白以及TNF-α的結合力的ELISA分析結果。
第5A、5B圖分別是雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17)的SDS-PAGE與ELISA分析結果。
第6A、6B圖分別是雙鏈(可溶性TNF-α受體)-IgG1.CH2-CH3-(scFv α CII)的SDS-PAGE與ELISA分析結果。
第7圖是含有完整抗人類TNF-α抗體與對第II型膠原蛋白專一的scFv的雙鏈融合蛋白的SDS-PAGE分析結果。
第8A、8B圖分別是含有完整抗人類IL-17抗體與對第VII型膠原蛋白專一的scFv的雙鏈融合蛋白的SDS-PAGE與ELISA分析結果。
第9A、9B圖分別是(scFv α CVII)-IgG4.CH2-CH3-(scFv α BAFF)的SDS-PAGE與ELISA分析結果。
第10A、10B圖分別是含有完整抗人類BAFF抗體與對第VII型膠原蛋白專一的scFv的雙鏈融合蛋白的SDS-PAGE與ELISA分析結果。
第11A、11B圖分別是雙鏈(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc分子構建體的SDS-PAGE與ELISA分析結果。
第12A、12B圖分別是含有完整抗人類RANKL抗體與對人類骨結合素專一的scFv的雙鏈融合蛋白的SDS-PAGE與ELISA分析結果。
第13圖是(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc以及雙鏈(scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17)於小鼠骺骨的免疫染色圖。
第14圖是雙鏈(可溶性TNF-α受體)-IgG1.CH2-CH3-(scFv α CII)於小鼠骺骨的免疫染色圖。
第15圖是經螢光標記的(scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc在BALB/c小鼠體內的生物分布分析結果。
根據慣常的作業方式，圖中各種特徵與元件並未依比例繪製，其繪製方式是為了以最佳的方式呈現與本發明相關的具體特徵與元件。此外，在不同圖式間，儘可能以相同或相似的元件符號來指稱相似的元件/部件。
＜110＞ 免疫功坊股份有限公司   ＜120＞ 具有組織標的功能的抗發炎分子   ＜130＞ P2913-TW   ＜140＞ TW 105101334   ＜141＞ 2015/01/18   ＜150＞ US 62/104405   ＜151＞ 2015/01/16   ＜150＞ US 62/114427   ＜151＞ 2015/02/10   ＜150＞ US 62/137737   ＜151＞ 2015/03/24   ＜160＞ 20   ＜170＞ BiSSAP 1.3   ＜210＞ 1 ＜211＞ 112 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 1F10-VH   ＜400＞ 1 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1               5                   10                  15      Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr             20                  25                  30           Ser Met His Trp Val Lys Gln Ala Pro Gly Glu Gly Leu Lys Trp Met         35                  40                  45              Gly Trp Ile Asn Thr Glu 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                  80  Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys                 85                  90                  95      Tyr Tyr Gly Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala             100                 105                 110               ＜210＞ 2 ＜211＞ 111 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 1F10-VL   ＜400＞ 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1               5                   10                  15      Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp             20                  25                  30           Gly Asp Gly Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro         35                  40                  45              Lys Leu Leu Ile Phe Ala Ala Ser Asn Leu Lys Ser Gly Ile Pro Ala     50                  55                  60                   Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65                  70                  75                  80  Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Asn                 85                  90                  95      Glu Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100                 105                 110       ＜210＞ 3 ＜211＞ 134 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ II6B3-VH   ＜400＞ 3 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1               5                   10                  15      Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe Ser Asp Tyr             20                  25                  30           Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val         35                  40                  45              Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His Tyr Ala Glu     50                  55                  60                   Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65                  70                  75                  80  Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr                 85                  90                  95      Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly Gln Gly Thr             100                 105                 110         Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro         115                 120                 125             Leu Val Pro Gly Ser Leu     130                   ＜210＞ 4 ＜211＞ 129 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ II6B3-VL   ＜400＞ 4 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1               5                   10                  15      Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser             20                  25                  30          Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35                  40                  45              Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50                  55                  60                  Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65                  70                  75                  80  Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85                  90                  95      Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100                 105                 110         Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Lys         115                 120                 125             Leu       ＜210＞ 5 ＜211＞ 741 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ (scFv anti-collagen II)-(scFv anti-TNF-alpha)-hIgG4.Fc   ＜400＞ 5 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1               5                   10                  15      Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser             20                  25                  30          Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35                  40                  45               Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50                  55                  60                  Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65                  70                  75                  80  Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85                  90                  95      Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100                 105                 110         Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser         115                 120                 125             Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln     130                 135                 140                  Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe 145                 150                 155                 160 Ser Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu                 165                 170                 175     Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His             180                 185                 190         Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser         195                 200                 205             Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr     210                 215                 220                 Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly 225                 230                 235                 240 Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Gly Gly Gly                 245                 250                 255     Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu             260                 265                 270         Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu         275                 280                 285             Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp     290                 295                 300                 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr 305                 310                 315                 320 Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe                 325                 330                 335     Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn             340                 345                 350         Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser         355                 360                 365             Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu     370                 375                 380                 Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser 385                 390                 395                 400 Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser                 405                 410                 415     Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala             420                 425                 430         Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly         435                 440                 445             Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly     450                 455                 460                 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 465                 470                 475                 480 Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln                 485                 490                 495     Arg Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu             500                 505                 510          Ile Lys Arg Ala Ser Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe         515                 520                 525             Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr     530                 535                 540                  Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 545                 550                 555                 560 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val                 565                 570                 575     Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser             580                 585                 590         Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu         595                 600                 605             Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser     610                 615                 620                 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 625                 630                 635                 640 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln                 645                 650                 655     Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala             660                 665                 670         Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr         675                 680                 685             Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu     690                 695                 700                 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 705                 710                 715                 720 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser                 725                 730                 735     Leu Ser Leu Gly Lys             740       ＜210＞ 6 ＜211＞ 1015 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白   ＜400＞ 6 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1               5                   10                  15      Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser             20                  25                  30          Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35                  40                  45              Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50                  55                  60                  Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65                  70                  75                  80  Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85                  90                  95      Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100                 105                 110         Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser         115                 120                 125             Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln     130                 135                 140                 Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe 145                 150                 155                 160 Ser Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu                 165                 170                 175     Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His             180                 185                 190         Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser         195                 200                 205             Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr     210                 215                 220                 Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly 225                 230                 235                 240 Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Gly Gly Gly                 245                 250                 255     Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu             260                 265                 270         Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu         275                 280                 285             Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp     290                 295                 300                 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr 305                 310                 315                 320 Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe                 325                 330                 335     Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn             340                 345                 350          Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser         355                 360                 365             Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu     370                 375                 380                  Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser 385                 390                 395                 400 Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser                 405                 410                 415     Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala             420                 425                 430         Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly         435                 440                 445             Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly     450                 455                 460                 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 465                 470                 475                 480 Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln                 485                 490                 495     Arg Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu             500                 505                 510         Ile Lys Arg Ala Ser Gly Gly Ser Pro Pro Cys Pro Ser Cys Pro Ala         515                 520                 525             Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro     530                 535                 540                 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 545                 550                 555                 560 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val                 565                 570                 575     Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln             580                 585                 590         Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln         595                 600                 605             Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly     610                 615                 620                 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 625                 630                 635                 640 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr                 645                 650                 655     Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser             660                 665                 670         Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr         675                 680                 685             Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr     690                 695                 700                 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 705                 710                 715                 720 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys                 725                 730                 735     Ser Leu Ser Leu Ser Leu Gly Lys Gly Thr Gly Gly Gly Gly Ser Gly             740                 745                 750         Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser         755                 760                 765             Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys     770                 775                 780                 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln 785                 790                 795                 800 Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg                 805                 810                 815      Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp             820                 825                 830         Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr         835                 840                 845              Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Cys Thr Phe Gly Gln Gly Thr     850                 855                 860                 Arg Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly 865                 870                 875                 880 Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly                 885                 890                 895     Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala             900                 905                 910         Ser Gly Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala         915                 920                 925             Pro Gly Lys Gly Leu Glu Trp Val Ala Ala Ile Asn Gln Asp Gly Ser     930                 935                 940                 Glu Lys Tyr Tyr Val Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 945                 950                 955                 960 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Val                 965                 970                 975     Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Asp Tyr Tyr Asp Ile Leu             980                 985                 990         Thr Asp Tyr Tyr Ile His Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly         995                 1000                1005            Thr Leu Val Thr Val Ser Ser     1010                1015   ＜210＞ 7 ＜211＞ 735 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白   ＜400＞ 7 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1               5                   10                  15      Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys             20                  25                  30          Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr         35                  40                  45              Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu     50                  55                  60                  Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65                  70                  75                  80  Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                 85                  90                  95      Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100                 105                 110         Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115                 120                 125             Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130                 135                 140                 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145                 150                 155                 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165                 170                 175     Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val             180                 185                 190          His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr         195                 200                 205             Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly     210                 215                 220                  Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 225                 230                 235                 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly                 245                 250                 255     Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             260                 265                 270         Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         275                 280                 285             Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     290                 295                 300                 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305                 310                 315                 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 325                 330                 335     Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             340                 345                 350         Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         355                 360                 365             Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     370                 375                 380                 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385                 390                 395                 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 405                 410                 415     Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             420                 425                 430         Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         435                 440                 445             His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     450                 455                 460                 Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 465                 470                 475                 480 Gly Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser                 485                 490                 495     Leu Gly Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val             500                 505                 510         His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly         515                 520                 525             Gln Ser Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly     530                 535                 540                 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 545                 550                 555                 560 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe                 565                 570                 575     Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu             580                 585                 590         Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu         595                 600                 605             Gly Ser Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu     610                 615                 620                  Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile 625                 630                 635                 640 Thr Phe Ser Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys                 645                 650                 655     Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala             660                 665                 670         Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp         675                 680                 685              Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu     690                 695                 700                 Asp Thr Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr 705                 710                 715                 720 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr                 725                 730                 735   ＜210＞ 8 ＜211＞ 719 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白重鏈   ＜400＞ 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1               5                   10                  15      Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr             20                  25                  30          Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35                  40                  45              Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val     50                  55                  60                   Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65                  70                  75                  80  Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85                  90                  95      Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly             100                 105                 110         Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115                 120                 125              Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130                 135                 140                 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145                 150                 155                 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165                 170                 175     Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180                 185                 190         Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195                 200                 205             Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210                 215                 220                 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225                 230                 235                 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245                 250                 255     Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260                 265                 270         Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275                 280                 285             His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290                 295                 300                 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305                 310                 315                 320 Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325                 330                 335     Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340                 345                 350         Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Arg Asn Gln Val Ser         355                 360                 365             Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370                 375                 380                 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385                 390                 395                 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405                 410                 415     Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420                 425                 430         His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435                 440                 445             Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly     450                 455                 460                 Gly Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro 465                 470                 475                 480 Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr Phe Ser                 485                 490                 495     Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu             500                 505                 510         Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His Tyr         515                 520                 525              Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys     530                 535                 540                 Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly 545                 550                 555                 560 Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly Gln                 565                 570                 575     Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Gly Ser Thr Ser             580                 585                 590         Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Val         595                 600                 605             Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln     610                 615                 620                  Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly 625                 630                 635                 640 Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys                 645                 650                 655     Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg             660                 665                 670         Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg         675                 680                 685             Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His     690                 695                 700                 Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 705                 710                 715                   ＜210＞ 9 ＜211＞ 214 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白輕鏈   ＜400＞ 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1               5                   10                  15       Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr             20                  25                  30          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35                  40                  45               Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50                  55                  60                  Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65                  70                  75                  80  Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr                 85                  90                  95      Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100                 105                 110         Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115                 120                 125             Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130                 135                 140                 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145                 150                 155                 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165                 170                 175     Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180                 185                 190         Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195                 200                 205             Phe Asn Arg Gly Glu Cys     210                   ＜210＞ 10 ＜211＞ 721 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白重鏈   ＜400＞ 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1               5                   10                  15      Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr             20                  25                  30          Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35                  40                  45              Ala Ala Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Val Gly Ser Val     50                  55                  60                  Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65                  70                  75                  80  Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys                 85                  90                  95       Val Arg Asp Tyr Tyr Asp Ile Leu Thr Asp Tyr Tyr Ile His Tyr Trp             100                 105                 110         Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala         115                 120                 125              Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser     130                 135                 140                 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145                 150                 155                 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly                 165                 170                 175     Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu             180                 185                 190         Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr         195                 200                 205             Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg     210                 215                 220                 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225                 230                 235                 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                 245                 250                 255     Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             260                 265                 270         Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr         275                 280                 285             Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     290                 295                 300                 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305                 310                 315                 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 325                 330                 335     Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln             340                 345                 350         Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met         355                 360                 365             Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro     370                 375                 380                 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385                 390                 395                 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu                 405                 410                 415     Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val             420                 425                 430         Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln         435                 440                 445             Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly     450                 455                 460                 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Met Leu Val Glu Ser Gly 465                 470                 475                 480 Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala                 485                 490                 495     Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr             500                 505                 510         Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Asp         515                 520                 525             Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg     530                 535                 540                 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Arg Arg Ser 545                 550                 555                 560 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Gly Glu Ile Gly Tyr                 565                 570                 575     Gly Ser Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             580                 585                 590          Val Ser Ala Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu         595                 600                 605             Gly Ser Thr Lys Gly Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu     610                 615                 620                  Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys 625                 630                 635                 640 Ser Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr                 645                 650                 655     Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro             660                 665                 670         Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile         675                 680                 685             Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His     690                 695                 700                 Asn Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 705                 710                 715                 720 Arg       ＜210＞ 11 ＜211＞ 215 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白輕鏈   ＜400＞ 11 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1               5                   10                  15      Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20                  25                  30          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35                  40                  45               Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50                  55                  60                  Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65                  70                  75                  80  Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                 85                  90                  95      Cys Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala             100                 105                 110         Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser         115                 120                 125             Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu     130                 135                 140                  Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145                 150                 155                 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu                 165                 170                 175     Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val             180                 185                 190         Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys         195                 200                 205             Ser Phe Asn Arg Gly Glu Cys     210                 215   ＜210＞ 12 ＜211＞ 742 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白   ＜400＞ 12 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1               5                   10                  15      Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr             20                  25                  30          Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile         35                  40                  45              Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly     50                  55                  60                  Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65                  70                  75                  80  Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr                 85                  90                  95      Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser             100                 105                 110         Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val         115                 120                 125             Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu     130                 135                 140                 Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 145                 150                 155                 160 Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr                 165                 170                 175     Ile Thr Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly             180                 185                 190          Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln         195                 200                 205             Met Ser Ser Arg Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg     210                 215                 220                  His Gly Glu Ile Gly Tyr Gly Ser Ser Ala Trp Phe Ala Tyr Trp Gly 225                 230                 235                 240 Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Gly Gly Ser Pro Pro                 245                 250                 255     Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe             260                 265                 270         Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro         275                 280                 285             Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val     290                 295                 300                 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305                 310                 315                 320 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val                 325                 330                 335     Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys             340                 345                 350         Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser         355                 360                 365             Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro     370                 375                 380                 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 385                 390                 395                 400 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly                 405                 410                 415     Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp             420                 425                 430         Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp         435                 440                 445             Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His     450                 455                 460                 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly 465                 470                 475                 480 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln                 485                 490                 495     Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Arg             500                 505                 510         Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn Ala Ile Asn         515                 520                 525             Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile     530                 535                 540                 Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe Gln Gly Arg 545                 550                 555                 560 Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser Met Glu Leu                 565                 570                 575     Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser             580                 585                 590         Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro Trp Gly Arg         595                 600                 605             Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly     610                 615                 620                  Gly Ser Gly Gly Gly Gly Ser Ala Phe Ser Ser Glu Leu Thr Gln Asp 625                 630                 635                 640 Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln                 645                 650                 655     Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro             660                 665                 670         Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser         675                 680                 685              Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser     690                 695                 700                 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 705                 710                 715                 720 Ser Ser Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly Gly Gly Thr                 725                 730                 735     Glu Leu Thr Val Leu Gly             740           ＜210＞ 13 ＜211＞ 717 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白重鏈   ＜400＞ 13 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1               5                   10                  15      Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn             20                  25                  30          Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35                  40                  45              Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe     50                  55                  60                  Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser 65                  70                  75                  80  Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85                  90                  95      Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro             100                 105                 110         Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115                 120                 125             Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly     130                 135                 140                 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145                 150                 155                 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165                 170                 175     Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180                 185                 190         Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val         195                 200                 205             Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys     210                 215                 220                 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225                 230                 235                 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                 245                 250                 255     Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val             260                 265                 270         Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val         275                 280                 285             Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser     290                 295                 300                 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305                 310                 315                 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala                 325                 330                 335     Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro             340                 345                 350         Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln         355                 360                 365             Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala     370                 375                 380                 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385                 390                 395                 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu                 405                 410                 415     Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser             420                 425                 430         Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser         435                 440                 445             Leu Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly     450                 455                 460                  Gly Gly Gly Ser Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val 465                 470                 475                 480 Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr                 485                 490                 495     Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg             500                 505                 510         Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Asp Tyr Thr Phe Tyr         515                 520                 525              Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys     530                 535                 540                 Asn Thr Leu Tyr Leu Gln Met Ser Ser Arg Arg Ser Glu Asp Thr Ala 545                 550                 555                 560 Met Tyr Tyr Cys Ala Arg His Gly Glu Ile Gly Tyr Gly Ser Ser Ala                 565                 570                 575     Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly             580                 585                 590         Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys         595                 600                 605             Gly Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro     610                 615                 620                 Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys 625                 630                 635                 640 Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu                 645                 650                 655     Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser             660                 665                 670         Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu         675                 680                 685             Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro     690                 695                 700                 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 705                 710                 715           ＜210＞ 14 ＜211＞ 214 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白輕鏈   ＜400＞ 14 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1               5                   10                  15      Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala             20                  25                  30          Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr         35                  40                  45               Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser     50                  55                  60                  Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65                  70                  75                  80  Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Asn His                 85                  90                  95      Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly Gln Pro Lys             100                 105                 110         Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln         115                 120                 125             Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly     130                 135                 140                 Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly 145                 150                 155                 160 Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala                 165                 170                 175     Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser             180                 185                 190         Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val         195                 200                 205             Ala Pro Thr Glu Cys Ser     210                   ＜210＞ 15 ＜211＞ 140 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ SPARC VH   ＜400＞ 15 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1               5                   10                  15      Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His             20                  25                  30          Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile         35                  40                  45              Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe     50                  55                  60                   Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Thr Tyr 65                  70                  75                  80  Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys                 85                  90                  95      Ala Arg Leu Ala Phe Gly Ile Ile Thr Thr Val Val Arg Tyr Phe Asp             100                 105                 110         Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr         115                 120                 125              Ala Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Leu     130                 135                 140   ＜210＞ 16 ＜211＞ 125 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ SPARC VL   ＜400＞ 16 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1               5                   10                  15      Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr             20                  25                  30          Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val         35                  40                  45              Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly     50                  55                  60                  Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65                  70                  75                  80  Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Phe                 85                  90                  95      Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala             100                 105                 110         Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Lys Leu Gly         115                 120                 125   ＜210＞ 17 ＜211＞ 751 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白   ＜400＞ 17 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1               5                   10                  15      Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr             20                  25                  30          Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val         35                  40                  45              Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly     50                  55                  60                  Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65                  70                  75                  80  Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Phe                 85                  90                  95      Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser             100                 105                 110         Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val         115                 120                 125             Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala Ser Val     130                 135                 140                 Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His Thr Ile 145                 150                 155                 160 His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Tyr                 165                 170                 175     Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly             180                 185                 190         Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Thr Tyr Met Gln         195                 200                 205             Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg     210                 215                 220                 Leu Ala Phe Gly Ile Ile Thr Thr Val Val Arg Tyr Phe Asp Val Trp 225                 230                 235                 240 Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Gly Gly                 245                 250                 255     Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln             260                 265                 270         Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg         275                 280                 285             Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser     290                 295                 300                 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile 305                 310                 315                 320 Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg                 325                 330                 335      Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met             340                 345                 350         Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp         355                 360                 365              Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp Gly Gln Gly     370                 375                 380                 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Thr Ser Gly 385                 390                 395                 400 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val                 405                 410                 415     Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala             420                 425                 430         Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg Tyr Leu Ala         435                 440                 445             Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly     450                 455                 460                 Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly 465                 470                 475                 480 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp                 485                 490                 495     Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Arg Thr Phe             500                 505                 510         Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Ser Gly Gly Ser Pro         515                 520                 525             Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val     530                 535                 540                 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 545                 550                 555                 560 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu                 565                 570                 575     Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys             580                 585                 590         Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser         595                 600                 605             Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys     610                 615                 620                 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 625                 630                 635                 640 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro                 645                 650                 655     Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu             660                 665                 670         Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn         675                 680                 685             Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser     690                 695                 700                 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 705                 710                 715                 720 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu                 725                 730                 735     His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys             740                 745                 750       ＜210＞ 18 ＜211＞ 721 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白重鏈   ＜400＞ 18 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1               5                   10                  15      Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20                  25                  30          Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35                  40                  45              Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50                  55                  60                  Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65                  70                  75                  80  Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85                  90                  95      Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp             100                 105                 110         Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115                 120                 125             Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr     130                 135                 140                 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145                 150                 155                 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165                 170                 175     Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180                 185                 190          Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn         195                 200                 205             His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser     210                 215                 220                  Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225                 230                 235                 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245                 250                 255     Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260                 265                 270         His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu         275                 280                 285             Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr     290                 295                 300                 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305                 310                 315                 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro                 325                 330                 335     Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340                 345                 350         Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val         355                 360                 365             Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370                 375                 380                 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385                 390                 395                 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr                 405                 410                 415     Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val             420                 425                 430         Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435                 440                 445             Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly     450                 455                 460                 Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 465                 470                 475                 480 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe                 485                 490                 495     Thr Asp His Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu             500                 505                 510         Glu Trp Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn         515                 520                 525             Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser     530                 535                 540                 Thr Thr Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 545                 550                 555                 560 Tyr Phe Cys Ala Arg Leu Ala Phe Gly Ile Ile Thr Thr Val Val Arg                 565                 570                 575     Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala             580                 585                 590         Lys Thr Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu         595                 600                 605             Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu     610                 615                 620                 Ser Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly 625                 630                 635                 640 Asn Ile His Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser                 645                 650                 655      Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro             660                 665                 670         Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile         675                 680                 685              Asn Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe     690                 695                 700                 Trp Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 705                 710                 715                 720 Arg       ＜210＞ 19 ＜211＞ 215 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ 融合蛋白輕鏈   ＜400＞ 19 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1               5                   10                  15      Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg             20                  25                  30          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35                  40                  45              Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50                  55                  60                  Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65                  70                  75                  80  Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                 85                  90                  95      Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala             100                 105                 110          Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser         115                 120                 125             Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu     130                 135                 140                  Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145                 150                 155                 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu                 165                 170                 175     Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val             180                 185                 190         Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys         195                 200                 205             Ser Phe Asn Arg Gly Glu Cys     210                 215   ＜210＞ 20 ＜211＞ 260 ＜212＞ PRT ＜213＞ 人工序列   ＜220＞ ＜223＞ CVII scFv   ＜400＞ 20 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1               5                   10                  15      Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr             20                  25                  30          Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile         35                  40                  45              Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly     50                  55                  60                  Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65                  70                  75                  80  Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr                 85                  90                  95      Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser             100                 105                 110         Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val         115                 120                 125             Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu     130                 135                 140                 Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 145                 150                 155                 160 Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr                 165                 170                 175     Ile Thr Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly             180                 185                 190         Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln         195                 200                 205             Met Ser Ser Arg Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg     210                 215                 220                  His Gly Glu Ile Gly Tyr Gly Ser Ser Ala Trp Phe Ala Tyr Trp Gly 225                 230                 235                 240 Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly                 245                 250                 255     Gly Gly Ser Cys             260
800A‧‧‧Fc型分子構建體
810‧‧‧CH2-CH3鏈
T1‧‧‧標的元件
E1‧‧‧效應元件

Claims (14)

  1. 一種分子構建體包含： 一對IgG.Fc的CH2-CH3區段； 一第一對效應元件，其中該效應元件為對腫瘤壞死因子-α (tumor necrosis factor-α，TNF-α)、介白素-17 (IL-17)、IL-17受體(IL-17R)、IL-1、IL-6、IL-6R、IL-12、IL-23、B細胞活化因子(B cell activating factor，BAFF)或細胞核因子κB受體活化因子配體(ligand of receptor activator of nuclear factor κB，RANKL)專一的一抗體片段、或TNF-α或IL-1的一可溶性受體；以及 一第一對標的元件，其中該標的元件為對α-蛋白聚糖、第I型膠原蛋白、第II型膠原蛋白、第III型膠原蛋白、第V型膠原蛋白、第VII型膠原蛋白、第IX型膠原蛋白、第XI型膠原蛋白或骨結合素專一的一抗體片段，其中： 當該第一對效應元件係連接於該對CH2-CH3區段的N-端，則該第一對標的元件連接於該對CH2-CH3區段的C-端，反之亦然；或 當該第一對效應元件與該第一對標的元件都是單鏈可變片段(single-chain variable fragment，scFv)的形式時，則該第一對標的元件以串聯雙抗體(tandem)或雙抗體(diabody)的構形而連接於該第一對效應元件的N-端，因而形成連接於該對CH2-CH3區段的N-端的一對雙專一性scFv。
  2. 如請求項1所述的分子構建體，其中該對CH2-CH3區段係衍生自人類γ4或γ1免疫球蛋白。
  3. 如請求項1所述的分子構建體，其中當該第一對效應元件為一抗原結合片段(antigen-binding fragment，Fab)的形式且當該第一對標的元件為scFv的形式時，反之亦然，則該Fab以及scFv分別連接於該CH2-CH3區段的N-與C-端，而使得該分子構建體形成一延伸IgG構形。
  4. 如請求項1所述的分子構建體，更包含一第二對效應元件或一第二對標的元件，其中該第二對效應或標的元件係連接於該CH2-CH3區段的自由C-端。
  5. 如請求項1所述的分子構建體，其中：該效應元件為對TNF-α專一的scFv；以及該標的元件為對第II型膠原蛋白、第IX型膠原蛋白或α-蛋白聚糖專一的scFv。
  6. 如請求項1所述的分子構建體，其中：該二效應元件為對TNF-α專一的Fab；以及該標的元件為對第II型膠原蛋白或第IX型膠原蛋白專一的scFv。
  7. 如請求項1所述的分子構建體，其中：該效應元件為對IL-17專一的scFv；以及該標的元件為對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。
  8. 如請求項1所述的分子構建體，其中：該二效應元件為對IL-17專一的Fab；以及該標的元件為對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。
  9. 如請求項1所述的分子構建體，其中：該效應元件為對BAFF專一的scFv；以及該標的元件為對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。
  10. 如請求項1所述的分子構建體，其中：該二效應元件為對BAFF專一的Fab；以及該標的元件為對第I型膠原蛋白或第VII型膠原蛋白專一的scFv。
  11. 如請求項1所述的分子構建體，其中：該效應元件為對TNF-α專一的scFv；以及該標的元件為對第III型膠原蛋白或第V型膠原蛋白專一的scFv。
  12. 如請求項1所述的分子構建體，其中：該二效應元件為對TNF-α專一的Fab；以及該標的元件為對第III型膠原蛋白或第V型膠原蛋白專一的scFv。
  13. 如請求項1所述的分子構建體，其中：該效應元件為對RANKL專一的scFv；以及該標的元件為對第I型膠原蛋白或骨結合素專一的scFv。
  14. 如請求項1所述的分子構建體，其中：該二效應元件為對RANKL專一的Fab；以及該標的元件為對第I型膠原蛋白或骨結合素專一的scFv。
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