TWI840987B - 單株抗體 - Google Patents
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- TWI840987B TWI840987B TW111138053A TW111138053A TWI840987B TW I840987 B TWI840987 B TW I840987B TW 111138053 A TW111138053 A TW 111138053A TW 111138053 A TW111138053 A TW 111138053A TW I840987 B TWI840987 B TW I840987B
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Abstract
本發明揭示一種單株抗體,其可以2.86 × 10
-10之解離常數結合至HyIL-6且顯著地抑制IL-6/IL-6R/gp130複合物形成。另外,本發明之單株抗體有效地抑制經HyIL-6刺激之信號轉導及轉錄活化因子3 (STAT3)活化及相關血管內皮生長因子(VEGF)誘導。氫氘交換質譜法(HDX-MS)之資料顯示,本發明之抗體主要結合至IL-6之位點IIIa且阻斷gp130與IL-6/IL-6R六聚體複合物之間的相互作用中之最終步驟。另外,ELISA結合分析及動力學分析之資料顯示,本發明之抗體同時與IL-6及IL-6R相互作用,然而其不單獨與IL-6R相互作用。本發明之抗體之獨特特徵為IL-6阻斷提供新的替代方案,且闡明了針對 IL-6 的更好的治療干預。
Description
本發明係關於對單株抗體之探索及表徵,一種新的治療性抗體,其對IL-6/IL-6Rα複合物的結合比IL-6具有更高的親和力,且藉由阻斷與gp130 (位點III)之相互作用而干擾形成功能齊全的受體複合物。
介白素-6 (IL-6) ,最初被認為是B細胞刺激因子2,是一種關鍵細胞因子,其在正常生理學疾病中具有多種功能。IL-6在廣泛的生理過程中介導多種生物功能,包括能量恆定、骨頭代謝、急性期反應、再生過程及神經功能。作為細胞介素網路的重要成員,IL-6在急性發炎中有著核心作用,且與先天免疫反應與後天性免疫反應相關。IL-6之過度合成與由諸如嵌合抗原受體T細胞(CAR-T)療法等免疫相關療法所引發之細胞介素釋放症候群(CRS)和包括嚴重急性呼吸道症候群冠狀病毒2 (SARS-CoV-2)在內的病毒感染密切相關。CRS為潛在危及生命之全身發炎反應,且IL-6阻斷已證實可以逆轉與新型T細胞接合療法相關之CRS及SARS-CoV-2。此外,IL-6在發炎過程中從嗜中性球向單核球募集得轉變中有著核心的作用,且失調的IL-6基本上會持續導致慢性發炎疾病及自體免疫疾病中之組織損傷的發生。靶向IL-6治療法的療效已證實了IL-6在類風濕性關節炎、全身性幼年型特發性關節炎、多關節型幼年型特發性關節炎及巨細胞動脈炎中之顯著致病作用。此外,增生性淋巴結之胚芽中心中之過度的IL-6產生會導致卡斯爾曼氏病(Castleman disease)一系列的臨床表現。在2014年,IL-6靶向療法已經批准用於多中心卡斯爾曼氏病。此等研究一起驗證了IL-6信號傳導路徑為重要分子治療目標。
IL-6目標細胞在其表面上表現不含轉導活性的低親和力受體(IL-6Rα)。IL-6與IL-6Rα結合後,再與同一細胞之第二膜蛋白gp130結合,該gp130進行二聚合且起動細胞內信號傳導。細胞內之信號轉導涉及Janus激酶(JAK)及轉錄活化因子(STAT)路徑及RAS依賴性促分裂原活化蛋白激酶(MAPK)信號傳導級聯之活化。這種所謂的經典信號傳導僅與少許表現IL-6Rα之細胞類型(肝細胞、一些上皮細胞及白血球)中相關。賦予IL-6之許多生物活性調節中的關鍵特點是標識可溶性IL-6Rα (sIL-6Rα)。sIL-6Rα與IL-6形成對抗複合物,該對抗複合物結合普遍的跨膜gp130以觸發細胞反應(IL-6反式信號傳導)。值得注意的是,IL-6反式信號傳導之致病作用已愈來愈地與許多發炎疾病、自體免疫疾病及發炎相關癌症有關。或者,已發現IL-6/IL-6Rα向鄰近細胞呈現,此稱為IL-6反式呈現,發生在樹突狀細胞(DC)與T細胞之抗原特異性相互作用的情形下。具體言之,IL-6-IL-6Rα複合物形成於DC之細胞內腔室中且隨後向細胞表面呈現,接著誘導自在CD4
+細胞上表現之gp130之信號傳導以生成高度組織破壞性表現型T輔助17細胞。此三種IL-6信號傳導模式在調節IL-6之多效性功能方面上並非功能冗餘的,此顯示不同的IL-6信號傳導模式之選擇性阻斷在疾病病理學方面有不同的結果。鑑於IL-6之複雜生物學,抑制反式信號傳導及反式呈現、同時保留IL-6經典信號傳導之恆定功能將為針對IL-6阻斷之關鍵治療策略。
針對IL-6信號傳導組裝之結晶學資料分析顯示,三元形式複合體依序且協調地組裝含有兩個IL-6、兩個IL-6Rα及兩個gp130的六聚體。IL-6為一種四α螺旋束細胞因子。IL-6首先以經由位於A螺旋及D螺旋中之位點I結合抗原表位與IL-6Rα之D3/D2域相互作用形成初始IL-6/IL-6Rα二元複合物。IL-6/IL-6Rα複合物包含與gp130細胞因子結合同源區(CHR)相互作用之複合抗原表位(位點IIa及IIb)。位點IIa係在IL-6 A及C螺旋面與gp130 CHR之間。位點IIb係在IL-6Rα D3結構域與gp130 D3結構域之間,提供了額外表面以增強整體結合親和力。功能性IL-6六聚體信號傳導複合物之後續組裝需要第三受體結合抗原表位,亦即位點III。位點IIIa具有在IL-6四螺旋束之尖端與gp130之D1結構域之間的寬闊介面。相比之下,IIIb係在gp130 D1結構域之尖端與IL-6Rα之D2結構域之間。幾種靶向IL-6受體複合物之限定抗原表位區的抑制劑展現了不同的作用模式。舉例而言,當抑制性抗體結合至IL-6位點I (司妥昔單抗(Siltuximab);西魯庫單抗(Sirukumab);克拉扎珠單抗(Clazakizumab))或IL-6Rα D3/D2 (托珠單抗(Tocilizumab);沙立蘆單抗(Sarilumab))時,其會阻斷IL-6與IL-6Rα的結合,從而阻斷對IL-6的經典信號傳導及反式信號傳導而非IL-6反式呈現。相比之下,抗IL-6抗體奧洛珠單抗(olokizumab)結合至IL-6之位點IIIa且藉由阻斷與gp130的相互作用來干擾完全作用的受體複合物。此外,奧拉西普(Olamkicept) (sgp130Fc)藉由干擾IL-6/IL-Rα複合物之位點II及位點III與gp130之結合來專門抑制IL-6反式信號傳導。在各種疾病狀態下,此等抑制劑之作用產生藥物動力學及功效差異。更重要地,關於對IL-6/IL-6Rα/gp130結構內之不同功能性抗原表位之阻斷是否可轉換成獨特的臨床效益仍很大程度上未知。
本發明之發明內容旨在提供本發明之簡化發明內容,以使得讀者對本發明有基礎的理解。本發明之發明內容並非本發明之完整綜述,且其並不意圖指出本發明之實施例之重要/關鍵要素或界定本發明之範疇。
鑑於與失調IL-6表現相關之疾病之治療之習知技術的缺陷及缺點,本發明之主要目標在於提供對人類介白素-6 (IL-6)/介白素-6受體(IL-6R)複合物具有特異性之單株抗體(在下文可稱為C14mab)。
此外,該單株抗體特異性結合至IL-6內之抗原表位,且該抗原表位包含SEQ ID NO: 1之胺基酸序列。
此外,該單株抗體特異性結合至IL-6R內之抗原表位,且該抗原表位包含SEQ ID NO: 2之胺基酸序列。
此外,該單株抗體包含輕鏈(L鏈)可變區(VL區),且該VL區包含互補決定區1 (CDR-L1),該互補決定區包含SEQ ID NO: 3之胺基酸序列。
此外,該VL區包含互補決定區2 (CDR-L2),該互補決定區包含SEQ ID NO: 4之胺基酸序列。
此外,該VL區包含互補決定區3 (CDR-L3),該互補決定區包含SEQ ID NO: 5之胺基酸序列。
此外,該單株抗體包含重鏈可變區(VH區),且該VH區包含互補決定區1 (CDR-H1),該互補決定區包含SEQ ID NO: 6之胺基酸序列。
此外,該VH區包含互補決定區2 (CDR-H2),該互補決定區包含SEQ ID NO: 7之胺基酸序列。
此外,該VH區包含互補決定區3 (CDR-H3),該互補決定區包含SEQ ID NO: 8之胺基酸序列。
此外,該VL區包含SEQ ID NO: 9之胺基酸序列。
此外,該VH區包含SEQ ID NO: 10之胺基酸序列。
本發明之另一目標在於提供包含上文所描述之單株抗體之醫藥組合物。
本發明之另一目標在於提供編碼上文所描述之單株抗體之核酸分子。
本發明之另一目標在於提供包含上文所描述之核酸分子之載體。
本發明之另一目標在於提供包含上文所描述之核酸分子或表現上文所描述之核酸分子的細胞。
本發明之另一目標在於提供用於治療IL-6/IL-6R介導之疾病的方法,其包含向需要該治療之受試者投與上文所描述之單株抗體的治療有效量。
在提及以下實施例之後,本發明所屬技術領域中具有通常知識者可易於理解本發明之基礎精神及其目的以及本發明所採用之技術手段及實施態樣。
在此章節中,本發明之內容將經由以下實例得到詳細描述。此等實例僅用於說明,且熟習此項技術者可容易想到各種修改及改變。本發明之各種實施例將在下文得到詳細描述。在本說明書及隨附專利申請案中,除非上下文清楚地指示,否則「一」及「該」亦可解釋為複數的。
靶向
IL-6/
IL-6Rα
之
單株抗體
靶向IL-6信號傳導以治療許多人類疾病已進入臨床實踐。然而,仍存在許多問題,且IL-6抑制性抗體基於其阻斷模式而具有相當不同之特點。為用於各種IL-6介導之疾病選擇具有針對IL-6/IL-6Rα/gp130信號傳導之適當的作用機制之適合的抗體仍為一項挑戰。本發明揭示C14mab特異性辨識IL-6之位點III中含有關鍵胺基酸殘基之抗原表位以組裝IL-6/IL-6Rα/gp130複合物,此與當前臨床研發之抗體奧洛珠單抗(Olokizumab)類似。C14mab與奧洛珠單抗之間的關鍵差異在於,用於IL-6/IL-6Rα複合物之C14mab之抗原表位在IL-6及IL-6Rα 中空間上分離,因此會優先結合至IL-6/IL-6Rα複合物。此類獨特特點可增加針對各種IL-6/IL-6Rα介導之疾病之特異性及潛在功效。
醫藥組合物
在各個實施例中,考慮包含本文所描述之抗體之醫藥組合物。在一些實施例中,經調配用於視投與途徑而以各種單位劑型投與之醫藥組合物。在較佳實施例中,提供與醫藥學上可接受之載劑一起調配之含有一種本文所描述之抗體或本文所描述之抗體組合的醫藥組合物或其免疫結合物。
如本文所使用,「醫藥學上可接受之載劑」包括任何及所有生理學上可相容之溶劑、分散介質、塗層、抗菌劑及抗真菌劑、等張劑及吸收延遲劑以及其類似物。較佳地,載劑適用於靜脈內、肌肉內、皮下、非經腸、經脊髓或經表皮投與(例如藉由注射或輸注)。
包含本文所描述之抗體之醫藥組合物可藉由此項技術中已知之各種方法投與。如熟習此項技術者應了解,投與途徑及/或模式將視所需結果而不同。活性化合物可與保護該化合物以免快速釋放之載劑一起製備,諸如受控釋放型調配物,包括植入劑、經皮貼片及微囊封遞送系統。可使用可生物降解、可生物相容的聚合物,諸如乙烯乙酸乙烯酯、聚酸酐、聚乙醇酸、膠原蛋白、原酸酯聚合體及聚乳酸。
生產抗體或其結合片段
在一些實施例中,抗體或其結合片段以重組方式表現,且編碼該抗體或其結合片段之核酸係由以化學方式合成之寡核苷酸組裝而成,該等寡核苷酸可參與含有編碼該抗體之序列之部分之重疊寡核苷酸的合成、彼等寡核苷酸之黏合及接合以及後續的藉由PCR進行之接合寡核苷酸之擴增。或者,編碼抗體之核酸分子視情況由適合的來源(例如抗體cDNA庫或由任何表現免疫球蛋白之組織或細胞生成之cDNA庫)藉由使用可與序列之3’端及5’端雜交之合成引子進行PCR擴增或藉由使用對特定基因序列具有特異性之寡核苷酸探針進行選殖而生成。
本發明之包含抗體之核酸的載體及其細胞
在一些實施例中,提供包含抗體之核苷酸序列之表現載體或抗體之核苷酸序列。可藉由習知技術(例如電穿孔、脂質體轉染及磷酸鈣沉澱)將載體轉移至宿主細胞,且隨後藉由習知技術培養經轉染細胞以生產抗體。在具體實施例中,抗體之表現由組成性、誘導性或組織特異性啟動子調節。
在一些實施例中,利用各種宿主-表現載體系統來表現本文所描述之抗體。該等宿主-表現系統代表藉以生產抗體之編碼序列且隨後純化之媒劑,但亦代表當經適當的核苷酸編碼序列轉型或轉染時原位表現抗體或其結合片段之細胞。此等系統包括但不限於微生物體,諸如經含有編碼抗體或其結合片段之序列之重組噬菌體DNA、質體DNA或黏質體DNA表現載體轉型的細菌;經含有編碼抗體或其結合片段之序列之重組酵母表現載體轉型的酵母;經含有編碼抗體或其結合片段之序列之重組病毒表現載體感染的昆蟲細胞系統;或具有含有衍生自哺乳動物細胞之基因體之啟動子(例如金屬硫蛋白啟動子)或衍生自哺乳動物病毒之啟動子(例如腺病毒晚期啟動子;牛痘病毒7.5K啟動子)之重組表現構築體的哺乳動物細胞系統(例如COS、CHO、BH、293、293T、3T3細胞)。
治療應用
本文亦提供用於治療患有IL-6或IL-6/IL-6R相關疾病或病症之受試者的組合物及方法。該方法包括向受試者投與治療有效量之包含本文所描述之調配物的組合物。在實施例中,該方法包括鑑別患有本文所描述之IL-6相關疾病或病症之受試者;及向受試者投與治療有效量之包含如本文所描述之調配物的組合物。
例如關於藉由投與本文所描述之組合物或調配物進行之治療,IL-6相關疾病或病症可能與IL-6表現或活性增加或升高相關。在一實施例中,IL-6相關疾病或病症之一或多種症狀與IL-6表現或活性增加或升高相關。可確定與在該疾病或該疾病之症狀發作之前的IL-6表現量相比受試者之IL-6表現增加或升高。可確定與另一不患有IL-6相關疾病或病症之受試者相比受試者之IL-6表現增加或升高。
在各個實施例中,IL-6相關疾病為「IL-6介導之發炎」或「IL-6介導之發炎性病症」,係指其中已知或懷疑IL-6促成發炎之病因學或症狀之發炎或發炎相關病症。在各個實施例中,患者患有腎病。在一些實施例中,腎病為慢性腎病(CKD)。在各個實施例中,患者患有心血管疾病。在各個實施例中,患者患有貧血。在一些實施例中,患者患有慢性疾病貧血。在一些實施例中,患者患有鐵難治性缺鐵性貧血(IRIDA)。在一些實施例中,患者患有糖尿病。在一些實施例中,患者患有肝病。在一些實施例中,患者患有骨質疏鬆症。在一些實施例中,患者患有抑鬱症。在一些實施例中,患者患有氣喘。在一些實施例中,患者患有神經發炎性病症,諸如阿茲海默氏病(Alzheimer's disease)、帕金森氏病(Parkinson's disease)、多發性硬化症及肌肉萎縮性脊髓側索硬化症(ALS)。在一些實施例中,患者患有年齡相關黃斑點退化(AMD)。在各個實施例中,患者患有癌症,諸如實體腫瘤、小細胞肺癌、非小細胞肺癌、血液癌、多發性骨髓瘤、白血病、慢性淋巴球性白血病(CLL)、慢性骨髓性白血病(CML)、淋巴瘤及霍奇金氏淋巴瘤(Hodgkin's lymphoma)。在一些實施例中,患者患有皮膚病。在一些實施例中,抗IL-6抗體調配物預防患者衰老。
實施例
以下為用於實施本發明之具體實施例之實例。該等實例僅出於說明目的而提供,且不意欲以任何方式限制本發明之範疇。已作出努力來確保關於所使用之數值(例如量、溫度等)之準確度,但當然應允許一些實驗誤差及偏差。
抗
HyIL-6
融合瘤細胞的生成且表徵
臨床使用或研發之大多數靶向IL-6軸的抗體可防止IL-6結合至IL-6Rα從而中和IL-6活性(阻斷位點I)。為了探索可靶向IL-6/IL-6Rα/gp130結構內之其他功能性抗原表位之新穎抗體,吾等使小鼠免疫接種藉由使人類IL-6及人類sIL-6Rα與13-aa連接子融合而構成的重組人類Hyper-IL-6 (HyIL-6)蛋白。後續抗體探索引起生成32種融合瘤殖株,該等殖株在初始ELISA篩檢中由HyIL-6辨識。接下來,測試該32種融合瘤殖株之上清液阻斷gp130與IL-6/IL-6Rα複合物之結合的能力。理論上,此方法可篩檢靶向除位點I以外之不同的結合位點之抗體候選物。圖1顯示使用競爭性ELISA生成數據鑑別可顯著地干擾HyIL-6與gp130結合的幾種潛在抗體候選物。選擇排名前10之候選物用於使用間接ELISA分析進一步表徵其對HyIL-6及IL-6之結合親和力。圖2顯示各種抗體候選物分別與HyIL-6及IL-6之結合的代表性結合曲線。吾等數據顯示,大多數測試候選物對HyIL-6具有優異親和力,而C40殖株與IL-6及HyIL-6顯示類似的結合特徵。
為了進一步驗證此等抗體候選物對IL-6/HyIL-6誘導之於目標細胞上之信號傳導的抑制能力,在表現低含量IL-6Rα次單元之HeLa細胞中使用免疫墨點分析酪胺酸磷酸化STAT3 (p-STAT3)。圖3(a)顯示IL-6刺激導致在HeLa細胞中較差的信號傳導反應,而HyIL-6刺激產生穩健的STAT3活化。如所預期,IL-6及HyIL-6介導之STAT3磷酸化位準均在存在含有抗體候選物之融合瘤上清液之情況下以劑量依賴性方式減少。在IL-6Rα-零位C33A細胞中HyIL-6誘導之STAT3活化之情況下獲得類似結果(圖3(b))。值得注意的是,C14殖株尤其顯示優異的針對HyIL-6介導之反式信號傳導之抑制活性(圖3(c))。因此,吾等選擇C14殖株用於進一步研發及表徵。
單株抗體
C14
之結合親和力
首先,使純化的C14單株抗體(C14mab)進行基於ELISA之用IL-6、IL-6Rα及HyIL-6進行之結合實驗。圖4a顯示C14mab的結合特性之代表圖顯示,C14mab結合至IL-6及HyIL-6,但不結合至IL-6Rα。值得注意的是,HyIL-6具有比IL-6強的C14mab結合活性(圖4(b))。ELISA分析顯示,C14mab結合經塗佈HyIL-6之EC
50值(4.296 ng/ml)是IL-6之EC
50值(EC
50= 15.32 ng/ml)的3.5倍,而HyIL-6之最大OD值亦明顯地高於IL-6。此外,儘管C14mab對人類HyIL-6展現高親和力結合,但C14mab對小鼠HyIL-6缺乏親和力,顯示C14mab不展現交叉物種反應性(圖4(c)) 。
吾等接下來測定純化的C14mab與IL-6及HyIL-6之結合動力學。代表性感測圖如圖5所示。IL-6及HyIL-6的結合親和力分別為1.13 x 10
-9及2.86 x 10
-10(表1)。實際上,C14mab與HyIL-6之結合強度為IL-6之結合強度的約四倍。純化的C14mab之結合特性與ELISA結合分析之結果一致。對於IL-6,C14mab遵循快速結合(fast-on) (6.05 x 10
5M
-1s
-1)及緩慢解離(slow-off) (6.65 x 10
-4S
-1)結合動力學,從而產生1.13 x 10
-9M之K
D。對於HyIL-6,C14mab遵循快速結合(4.98 x 10
5M
-1s
-1)及緩慢解離(1.44 x 10
-4S
-1)結合動力學,從而產生2.86 x 10
-10M之K
D。高結合速率為類似的,此意味著IL-6及HyIL-6需要極低的能量來與C14mab形成複合物。另一方面,HyIL-6之結合之解離速率比IL-6低,此意味著C14mab與HyIL-6之解離較慢,顯示複合物更穩定。
表1.IL-6及HyIL-6與C14mab結合相互作用之動力學速率常數及平衡解離常數。
複合物 | K on(M -1S -1) | K off(S -1) | K D(M) | ||
IL-6-C14mab | 6.05 ( ±0.91) x 10 5 | 6.65 ( ±1.16) x 10 -4 | 1.13 ( ±0.33) x 10 -9 | ||
HyIL-6-C14mab | 4.98 ( ±0.20) x 10 5 | 1.44 (±0.42) x 10 -4 | 2.86 ( ±0.72) x 10 -10 | ||
C14mab
之中和活性
為了驗證C14mab之中和活性,吾等藉由免疫墨點比較C14mab與托珠單抗(Tocilizumab)對IL-6介導之p-STAT3的抑制特性。圖6(a)顯示,C14mab及托珠單抗在Hela細胞中劑量依賴性抑制IL-6誘導之p-STAT3,且C14mab顯示相較於托珠單抗優異的效力。IL-6與sIL-6Rα之組合在 IL-6Rα-零位C33A 細胞中顯著地誘導p-STAT3,而單獨的IL-6治療並不如此。圖6(b)顯示, C14mab、托珠單抗及奧洛珠單抗實質上抑制C33A細胞中IL-6/sIL-6Rα組合介導之p-STAT3誘導。像位點I抑制劑一樣,托珠單抗為一種抑制游離IL-6與IL-6Rα之結合的抗IL-6Rα抗體。然而,托珠單抗無法防止由預先存在的IL-6/IL-6Rα複合物(HyIL-6)引發之STAT3活化(圖6(c))。相比之下,C14mab以劑量依賴性方式抑制HyIL-6誘導之STAT3活化,從而防止IL-6/IL-6Rα複合物與gpl30結合且起動信號傳導之能力(圖6(c)) 。
IL-6/HyIL-6信號傳導使發炎與血管生成相關。血管內皮生長因子(VEGF)之誘導係重要的STAT3信號傳導下游事件之一。因此,吾等藉由ELISA評估C14mab對HyIL-6誘導之VEGF表現之作用。圖7(a)顯示,HyIL-6實質上誘導C33A細胞中之VEGF產生,該產生可劑量依賴性地因C14mab而減少,當在C33A細胞中施用>0.1 µg/ml C14mab時完全阻斷。此外,IL-6在經由反式信號傳導生成分泌IL-17A之CD4+ T細胞(Th17細胞)中有著不可或缺的作用。圖7(b)顯示,HyIL-6支持TGF-β介導之Th17細胞之分化。與托珠單抗形成對比,C14mab及奧洛珠單抗以劑量依賴性方式明顯地抑制培養物上清液中之IL-17含量(圖7(b))。
IL-6為肝急性期蛋白之主要誘導劑。為了探究C14mab在體內消除IL-6反應之功效,吾等檢驗類澱粉蛋白A血清(SAA)含量,此為小鼠之一級急性期蛋白及發炎標記物。在Balb/c小鼠腹膜內HyIL-6注射後,SAA含量在刺激之後3小時顯著升高。與同型對照抗體相比,C14mab顯著地抑制HyIL-6引發之SAA產生(圖7(c))。圖7(d)顯示,C14mab以劑量依賴性方式消除小鼠中HyIL-6引發之SAA產生。在高於0.1 mg/kg的劑量下,吾等數據顯示,C14mab顯著地抑制小鼠之SAA表現。總而言之,吾等在體外及在體內分析證實C14mab在中和IL-6路徑中之重大功效。
C14mab
之抗原表位表徵
為了獲得C14mab結合抗原表位之分子細節,應用氫/氘交換質譜法(HDX-MS)以藉由對HyIL-6之HDX與C14mab/HyIL-6複合物之HDX進行比較來定位抗體之抗原表位。在交換反應之後,藉由胃蛋白酶消化蛋白質,且藉由液相層析法-質譜法分析所得肽。吾等分析生成30種涵蓋HyIL-6序列之80.7%之HyIL-6肽,該序列構成參與IL-6六聚體形成之HyIL-6之功能性抗原表位的89.2%。HyIL-6及C14mab/HyIL-6複合物之差異HDX結果展現於圖8(a)中。氘交換之量之減少顯示結合事件對該區域之保護。區域103-111、130-141、148-158、159-168、207-217、235-249、250-273、274-284、306-314、315-325及390-399中之多種肽在C14mab結合後展現些許HDX減少,其中總減少< 1 Da,此視為不顯著的。吾等HDX數據顯示作為潛在結合抗原表位之一個基本區(由殘基498–510構成) (圖8(a)),在C14mab結合後顯示>70%氘交換。此等殘基位於IL-6中之gp130相互作用位點IIIa,包括位點IIIa介面中之IL-6之關鍵殘基Trp157。
基於動力學分析,C14mab與HyIL-6之結合(K
D= 0.286 nM)比IL-6 (K
D=1.13 nM)更強,表示C14mab與HyIL-6之間的介面可自IL-6跨越至IL-6Rα。然而,吾等HDX-MS量測未鑑別到IL-6Rα中任何潛在的結合抗原表位,此可能歸因於實驗條件下有限的氘交換位準。為了進一步理解HyIL-6中之C14mab結合機制,吾等使用C14mab作為偵測抗體來執行間接ELISA分析。簡言之,將IL-6Rα直接固定在盤上且隨後將IL-6添加至IL-6Rα中使其結合。將未結合之IL-6洗掉,再藉由C14mab偵測IL-6/IL-6Rα複合物(圖8(b))。值得注意的是,當添加C14mab而不洗去未結合之IL-6時,ELISA讀值顯著地增加(圖8(b)),顯示C14mab改變IL-6/IL-6Rα相互作用之平衡狀態。總體而言,吾等數據顯示,C14mab辨識由IL-6及IL-6Rα形成之複合物且使其穩定,而非辨識單獨的IL-6。
C14mab
之同型分析
確定抗體之類別(例如IgG相對於IgM)及子類(例如IgG1相對於IgG2a)身份對於選擇藉以使其純化且用於其他研究中之免疫分析中的方法尤其重要。因此,吾等培養C14mab之融合瘤殖株且藉由商用同型分析套組來鑑別IgG重(H)鏈及輕(L)鏈之亞型。如圖10中所示,證實C14mab之H鏈為IgG1,而L鏈為κ。
論述
使用設計的細胞介素HyIL-6作為免疫抗原,本發明已成功地篩檢出抑制性抗體C14mab,該抗體具有不同的辨識sIL-6Rα/IL-6之融合蛋白之結合模式,且防止與gp130形成信號傳導勝任型受體複合物。在體外及體內功能分析均顯示,C14mab潛在地抑制IL-6介導之信號轉導及生物活性。ELISA結合分析及動力學分析之數據顯示C14mab同時與IL-6及IL-6Rα相互作用,表示C14mab結合表面連續地或不連續地跨越IL-6及IL-6Rα之介面。儘管C14mab結合至單獨的IL-6,但其對單獨的IL-6Rα不具有可量測親和力。值得注意的是,C14mab優先結合至sIL-6Rα/IL-6之融合蛋白而非IL-6,且提高IL-6/IL-6Rα複合物之平衡濃度,同時干擾信號傳導搭配物gp130之募集。C14mab的此特性使其成為IL-6之獨特中和抗體,從而提供不同的設計針對IL-6阻斷之特定治療劑之方式。
生物物理學及結構分析顯示,由C14mab辨識之sIL-6Rα/IL-6之抗原表位主要位於IL-6分子內。HDX-MS之數據顯示,C14mab靶向IL-6之位點IIIa,從而涵蓋胺基酸殘基W
157, 該胺基酸殘基為參與功能性人類IL-6六聚體信號傳導複合物裝配中之最後一個步驟之關鍵芳族位點III特徵殘基。本發明進一步分析差異HDX-MS及經胃蛋白酶消化之肽與胺基酸殘基W
507之間的空間分布。彼等具有HyIL-6氘含量> 25%、C14mab結合後差異> 9%及肽至W
507之距離< 30 Å之特性的肽視為抗體結合抗原表位之潛在候選物。僅一種肽
130-141滿足上文所提及之準則。因此,本發明提出由C14mab可變區之同源性模型組成之複雜模型,該可變區跨越約30 Å之距離且使用其CDR同時接合IL-6及IL-6Rα,疊加至IL-6/IL-6Rα/gp130結構上(圖9 (a))。IL-6/IL-6Rα複合物之兩個不同區域肽
498-510(SEQ ID NO:1)及肽
130-141(SEQ ID NO:2)中之C14mab結合殘基係在C14mab之重鏈及輕鏈之可變域之近似大小內(圖9(b) )。此等殘基分別位於IL-6中之gp130相互作用位點IIIa及IL-6Rα中之位點IIIb。此外,C14mab與gp130 D1結構域在空間上顯著地重疊,顯示C14mab阻斷IL-6/IL-6Rα/gp130複合物形成及後續信號轉導。
IL-6/IL-6Rα/gp130六聚體複合物之依序組裝為治療干預提供幾種替代方案。已研發出靶向IL-6信號傳導級聯中之不同步驟之抑制劑。然而,關於此等阻斷模式中哪一種為優異的及此差異是否可轉譯成臨床效益之問題仍存在。托珠單抗為第一個經FDA審批通過之用於治療類風濕性關節炎之靶向IL-6路徑之生物製劑。托珠單抗辨識mIL-6Rα及sIL-6Rα且經由對IL-6結合之競爭性阻斷來抑制IL-6信號傳導。與抗IL-6抗體相比,用抗IL-6Rα抗體治療患者會防止血清IL-6積聚及諸如傷寒、疲勞及高鈣血症之相關不利反應。儘管如此,靶向如IL-6Rα之受體而非個別細胞介素IL-6已降低特異性。已知人類IL-6Rα不僅結合至IL-6,且亦結合至人類CNTF及IL-30,顯示IL-6Rα阻斷亦可能會影響其他信號傳導路徑。同樣地,用sgp130Fc阻斷IL-6反式信號傳導之奧拉西普(Olamkicept)之實例干擾由IL-11反式信號傳導路徑引發之信號傳導。C14mab對IL-6/IL-6Rα複合物而非單獨的IL-6Rα之阻斷的特性使其能夠靶向單一信號傳導實體且不會干擾IL-6家族中之其他細胞介素之信號傳導。
對IL-6之阻斷提供最直接的IL-6抑制模式而不干擾可經由IL-6Rα傳導信號之其他細胞因子。IL-6具有三個不同的結合位點來結合至其受體次單元。臨床使用/研發之諸如司妥昔單抗(Siltuximab)、西魯庫單抗(Sirukumab)及克拉扎珠單抗(Clazakizumab)之大多數抗IL-6抗體針對位點I,以干擾IL-6與其膜結合IL-6Rα或可溶性IL-6Rα之初始結合。顯然,位點I阻斷無法區分經典信號傳導與反式信號傳導。此外,位點I阻斷不會阻斷IL-6反式呈現,該反式呈現為病原性T
H17細胞之啟動所需。值得注意的是,奧拉西普有效地抑制反式信號傳導,但不抑制反式呈現,此歸因於位阻。另一方面,可能會干擾IL-6/IL-6Rα/gp130複合物之組裝之抗IL-6RαMP16-1抑制IL-6反式呈現。在此情形下,如C14mab之非位點I阻斷理論上抑制經典信號傳導及反式信號傳導以及反式呈現,表示其有潛在的治療缺口。此外,與IL-6相比,C14mab對IL-6/IL-6Rα複合物具有結合偏好,且此類特性是否可相對地選擇性阻斷IL-6活性而非引發整體IL-6抑制仍有很大程度上未知,需要進一步地研究來證明此等概念。
材料及方法
重組蛋白
為了構築及表現具生物活性的設計細胞因子HyIL-6,由藉由編碼胺基酸序列Arg-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Val-Glu之合成DNA連接子融合之人類slL-6Rα cDNA (對應於胺基酸殘基113-323)及人類IL-6 cDNA (對應於胺基酸殘基30-212)組成的卡匣,係藉由PCR技術構築且使用限制酶NdeI及BamHI將其選殖至哺乳動物表現載體IgMu/pEF-BOS中。經轉染哺乳動物細胞中之信號肽之裂解引起具有C端6 His標籤之HyIL-6之分泌。使用經純化蛋白作為免疫原以用於單株抗體研發。另外,使用IL-6 (R&D systems)、IL-6Rα (R&D systems)、人類IL-6Rα/IL-6嵌合體(R&D systems)及小鼠IL-6/IL-6Rα複合物(R&D systems)用於ELISA、細胞功能
、體內模型及動力學分析。
融合瘤細胞株生成及抗體探索
使用HyIL-6蛋白用於Balb/c小鼠注射。用佐劑乳化HyIL-6蛋白以用於抗原啟動。兩週後,將HyIL-6蛋白與佐劑組合且乳化直至其不再分離。每週向小鼠注射且接著進行用於抗體滴定的繁殖程序。在第3至4次加強注射之後,間接ELISA測試抗體力價。進一步使用抗體力價大於1:10,000之小鼠用於生成融合瘤。融合瘤製備遵循先前研究。在三輪限制性稀釋之後,使用間接ELISA篩檢融合瘤候選物之培養物上清液。隨後,所選候選物經歷擴增用於大規模抗體生產及冷凍保存。與對照相比,在SDS-PAGE上運行IgG上清液培養物,估計上清液之IgG濃度且進行標準化調節。
競爭篩檢分析
用100 μl含10 μg/ml經純化sgp130-Fc溶液(來自Stefan Rose-John, University of Kiel, Germany之禮物)之DPBS緩衝液塗佈ELISA盤。提前製備50 μl配位體(HyIL-6,100 ng/ml)與50 μl來自所選融合瘤之上清液的混合物。添加100 μL/孔之混合物且在室溫下培育1小時。之後,依序培育初級抗sIL-6Rα小鼠單株抗體及HRP偶合之山羊抗小鼠IgG二級抗體。使用四甲基聯苯胺(TMB)溶液(0.12 mg/mL TMB及含0.04%過氧化氫之25 mM NaH
2PO
4)作為受質。5分鐘後用1 M H
2SO
4停止反應。量測在450 nm下之光學密度(OD) ,其中參考值為630 nm。
ELISA
結合分析
在4℃下用100 μl含所製備之目標蛋白之PBS緩衝液塗佈ELISA盤隔夜,並用含1% BSA之PBS阻斷一小時。在用含有0.05% Tween-20之PBS洗滌之後,在室溫下添加連續稀釋濃度之分析物(培養物上清液,抗體) 1小時。在進一步洗滌之後,使用與HRP偶合之山羊抗小鼠IgG顯示結合抗體。添加TMB受質(100 μl) 10-20分鐘,且隨後添加50 μl停止溶液(1N H
2SO
4)。直接讀取在450 nm下之吸光度。
抗體表現及純化
融合瘤在培養基(高葡萄糖DMEM、10% FBS、1% P/S)中於37℃、富含5% CO
2氛圍下培養。培養物在10-cm培養皿中達到約80%匯合度之後,由無血清培養基(高葡萄糖DMEM)置換10% FBS培養基以生產mAb。使用含有蛋白-A樹脂之管柱(GenScript)自培養物上清液純化IgG。在100 mM甘胺酸中溶離免疫球蛋白。使用十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳(SDS-PAGE)、粒徑篩析液相層析法及熱熔融分析純化的C14mab的特性。使用布拉福分析(Bradford assay)測定抗體濃度。
細胞信號轉導分析
將Hela細胞、C33A細胞(1 × 10
5個細胞/孔、1 × 10
6個細胞/孔)接種於6孔組織培養皿中並培養隔夜。在血清飢餓5小時之後,用含有IL-6 (R&D systems)、IL-6/sIL-6Rα組合(R&D Systems)或HyIL-6 (R&D systems)有或沒有抗體之預混合物刺激細胞15分鐘。各混合期之培育時間為15 分鐘。用冰冷的PBS洗滌細胞兩次,且溶解於補充有蛋白酶及磷酸酶抑制劑之RIPA緩衝液中。使用抗pSTAT3抗體及抗STAT3抗體(Cell Signaling technology)進行西方墨點法分析。在與HRP標記山羊抗兔抗體(Cell Signaling technology)或HRP標記山羊抗小鼠抗體(Cell Signaling technology)一起培育之後,藉由增強型化學發光HRP受質(Merck)偵測膜。
動力學分析
八位元組(Octet)基於用於量測蛋白質-蛋白質相互作用的生物層干涉術(BLI)。BLI分析自兩個表面反射之白色光之干擾模式:生物感測器尖端上之固定蛋白層及內部參考層。量測使用Biosensor AMC尖端(ForteBio, Pall公司)固定在生物感測器尖端表面上之C14mab與IL-6 (R&D systems)及HyIL-6 (R&D systems)複合物之間的結合。在Octet® System Octet 96 Red (ForteBio, Pall公司)上即時量測結合至生物感測器或自生物感測器解離之干擾模式以生成反應概況。藉由使用三項獨立實驗之具有全局擬合之1:1結合模型,用Octet Red分析軟體評估動力學參數。
所培養之細胞
VEGF
分析
在刺激之前,在6孔盤中之未補充MEM中培養血清飢餓C33A細胞(1x10
5個細胞/孔) 15小時。在存在或不存在抗體之情況下用重組人類HyIL-6 (50 ng/ml,R&D Systems)處理C33A細胞24小時。收集培養基用於測定VEGF含量。藉由人類VEGF DuoSet ELISA (R&D systems)量測上清液中之VEGF濃度,且一式三份地重複各量測。
鼠
TH17
細胞之活體外擴增
使用EasySep
TM小鼠CD4
+T細胞分離套組(Stemcell)自6至8週齡C57BL/6小鼠分離小鼠原生CD4
+T細胞。在補充有10%(v/v) FCS、2 mM L-麩醯胺、100 U/ml青黴素、100 μg/ml鏈黴素、1 mM丙酮酸鈉,及50 μM β-巰乙醇(全部來自Invitrogen, Carlsbad, CA)之RPMI 1640中培養細胞。在經抗CD3 (2 μg/ml,R&D Systems)及抗CD28 (10 μg/ml,BD Biosciences, San Jose, CA)塗佈之96孔盤中培養總計2.5 x 10
5個細胞/孔。向培養物補充TGF-β1 (2 ng/ml,R&D Systems)、抗IL-2 (10 μg/ml,R&D Systems)及HyIL-6 (50 ng/ml)。如所指示包括抗體。
小鼠中
HyIL-6
誘導之
SAA
之活體內中和
在測試之前,將體重為23–27 g之雄性Balb/c小鼠(Harlan Laboratories)圈養1週。在腹膜內投與1 μg/小鼠之人類HyIL-6 (R&D systems)前一小時,小鼠接受0.01、0.03、0.1 mg/kg C14mab、DPBS或0.1mg/kg同型對照抗體(Biolegend)之腹膜內投與 。將血液收集至Eppendorf管中以製備血漿及藉由小鼠血清類澱粉蛋白A DuoSet ELISA (R&D systems)評估SAA含量。
HDX-MS
在存在及不存在小鼠單株抗體之情況下,使用HDX-MS藉由經胃蛋白酶消化之片段量測目標重組蛋白中之氫-氘交換。在室溫下將重組蛋白(60
pmol)及蛋白質-抗體複合物(60
pmol: 72
pmol)以1:9比例稀釋在交換緩衝液(含99.9% D
2O之PBS,pH 7.4)中以起動HD交換。在6個時間點(0 s、30 s、180 s、600 s、1800 s、5400 s),抽吸等分試樣(10
pmol目標蛋白)並將其與預冷凍淬滅緩衝液混合(達到1.7 M胍鹽酸鹽、250 mM三(2-羧乙基)膦及0.8%甲酸之最終濃度)。將混合物立即裝載於自製胃蛋白酶管柱上用於線上消化。隨後,將經消化肽混合物裝載於逆相管柱(Zorbax 300SB-C18,0.3 x 5 mm;Agilent Technologies, Wilmington, DE, USA)上。接著 在逆相管柱(nanoEase M/Z肽BEH C18 300Å,1.7 µm,100 µm I.D. x 50 mm)上使用多步線性梯度之8%–95% HPLC緩衝液(99.5%乙腈/0.5%甲酸)分離去鹽肽10分鐘,其中流速為0.4 µl/min。使LC裝置與使用Xcalibur 2.2軟體(Thermo Fisher, San Jose, CA, USA)操作之2D線性離子阱質譜儀(Orbitrap Classic;Thermo Fisher, San Jose, CA, USA)耦合。全掃描MS在400至1,600 Da之範圍內及以在m/z 400下30,000之解析度的Orbitrap中進行。使用在m/z 445.1200下[Si(CH
3)
2O]
6H+之離子信號作為鎖定質量執行內部校準。電噴灑電壓經設定為2.4 kV,且毛細管溫度經設定為220℃。MS及MS/MS自動增益控制經設定為1,000 ms (全掃描)及200 ms (MS/MS),或對於最大累積時間或離子分別為2 x 10
6個離子(全掃描)及5 x 10
3個離子(MS/MS) 。
胜肽鑑別及
HDX
資料分析
使用Proteome Discoverer軟體(2.3版, Thermo Fisher Scientific)進行肽鑑別。使用SEQUEST搜尋引擎針對單一蛋白資料庫搜尋MS/MS光譜。為了進行肽鑑別,對於完整肽塊,准許10 ppm質量公差;而對於CID片段離子,准許0.5 Da。隨後,根據肽鑑別之高信賴度及第1名搜尋引擎過濾肽譜匹配(PSM)以確保整體錯誤發現率低於0.01。對於HDX概況分析,基於目標蛋白鑑別之LC-MS/MS結果製作肽鑑別模板。接著,將模板預裝載於安裝在MATLAB環境中之ExMS模組中。裝載HDX-MS光譜且進行分析以計算各肽之所併入之氘原子之數目,該數目隨後呈現為三項獨立實驗之氘併入之平均次數。
C14mab 之序列 C14mab VH 區段:重鏈可變區(鹼基對:363個,胺基酸:121個)
核苷酸序列(SEQ ID NO: 11) :
GAAGTGAACCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGCAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAGTTAGAACCAAAGGTTATTATCCTGTAACATTCTATGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGCCCCACTATGGGTACGGATACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA
胺基酸序列(SEQ ID NO: 10):
EVNLEESGGGLVQPGGSMQLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEVRTKGYYPVTFYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTRPHYGYGYFDVWGAGTTVTVSS
VL 區段:輕鏈可變區(鹼基對:336個,胺基酸:112個)
核苷酸序列(SEQ ID NO: 12):
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGTCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGCTAATTTTGGCATGAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGGTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAGCGG
胺基酸序列(SEQ ID NO: 9):
DIVLTQSPASLAVSLGQRATISCRASESVANFGMSFMNWFQQKPGQPPKLLIYGASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIKR
CDR1 | GFTFSDAWMD (SEQ ID NO: 6) |
CDR2 | EVRTKGYYPVTFYAESVKG (SEQ ID NO: 7) |
CDR3 | TRPHYGYGYFDV (SEQ ID NO: 8) |
CDR1 | RASESVANFGMSFMN(SEQ ID NO: 3) |
CDR2 | GASNQGS(SEQ ID NO: 4) |
CDR3 | QQSKEVPWT(SEQ ID NO: 5) |
應理解,本文所描述之實例及實施例僅用於說明目的,且將向熟習此項技術者建議根據其之各種修改或改變,且該等修改或改變將包括在本申請案之精神及範圍以及隨附申請專利範圍之範疇內。本文所引用之所有公開案、專利及專利申請案出於所有目的特此以全文引用之方式併入。
無
為了使上述之本發明及其他目標、特點、優點及實施例更加顯而易見及可理解,其圖式描述如下:
圖1說明了藉由競爭性ELISA進行融合瘤篩檢的代表性結果。將融合瘤之培養物上清液與HyIL-6混合以用於分析。顏色形成呈報為各融合瘤所觀測之吸光度(OD
450)。排名前10之抗體根據其中和HyIL-6與sgp130-Fc之結合的能力被選擇為抗體候選物。長條圖顯示針對各融合瘤所觀測之吸光度。
圖2說明了融合瘤培養物上清液之抗體與IL-6及HyIL-6的結合特徵。在用IL-6 (20 ng/孔)或HyIL-6 (60 ng/孔)塗佈微量孔盤隔夜之後,以IgG標準化融合瘤培養物上清液的連續稀釋液進行間接ELISA。融合瘤候選物C40、C14、C85、C92、C148及C155之OD
450結合曲線示於(a-f)中。數據來自三項獨立實驗。值表示平均值± SEM。
圖3說明了候選抗體在體外針對IL-6及HyIL-6的活性。在連續稀釋或不稀釋之融合瘤培養物上清液的情況下用含有呈相同莫耳比之重組人類IL-6 (20 ng/ml)或HyIL-6 (60 ng/ml)之混合物刺激Hela細胞(1 × 10
5個細胞/孔)。在連續稀釋或不稀釋之各種融合瘤培養物上清液的情況下,在IL-6/HyIL-6刺激後15 分鐘,利用西方墨點法在體外針對IL-6及HyIL-6之抗體候選物進行測定STAT3活化(p-STAT3)。
圖4說明了純化的C14mab之結合特徵。(a)用人類IL-6 (20 ng/孔)、HyIL-6 (60 ng/孔)及IL-6 Rα (40 ng/孔)塗佈微量孔盤。在室溫下添加C14mab連續稀釋液1小時。結合ELISA數據係用OD
450讀值及其相關結合百分比相對於對數抗體濃度進行製圖。數據來自三項獨立實驗。值表示平均值± SEM。(b)將數據個別地標準化至IL-6及HyIL-6之最大信號。藉由非線性回歸擬合可變斜率、四個參數劑量-反應模型,使用GraphPad Prism軟體(San Diego,CA,USA)獲得EC
50值。(c)如(a)中所描述,藉由ELISA量測小鼠HyIL-6 (60 ng/孔)與C14mab之結合。值表示三項獨立實驗之平均值± SEM。
圖5說明了C14mab之結合動力學。進行C14mab與IL-6 (a)及HyIL-6 (b)之結合的生物膜干涉術分析。簡言之,將C14mab加載於Octet抗小鼠Fc-捕捉(AMC)感測器上,並且分別在一定範圍之濃度(1.56–25 nM)之IL-6及HyIL-6上培育。藉由使用具有全局擬合之1:1結合模型評估動力學參數。各濃度之實驗曲線(實線)沿著擬合曲線(虛線)顯示。顯示三項獨立實驗之一組代表性曲線。比較線圖繪製隨時間推移之C14mab (在1.56–25 nM範圍之濃度上培育)與IL-6 (圖a)及HyIL-6 (圖b)之結合。
圖6說明了各種IL-6介導之信號傳導中C14mab之抑制概況。(a)在刺激之前使HeLa細胞(1 x 10
5個細胞/孔)血清飢餓5小時。將IL-6 (30 ng/ml)與指定濃度之C14mab或托珠單抗(Tocilizumab)預混合15分鐘。用該等混合物處理細胞15 分鐘,並藉由西方墨點法測定p-STAT3 (左圖)。藉由ImageJ軟體分析抑制數據且用p-STAT3活性百分比相對於對數抗體製圖(右圖)。(b)在刺激之前使C33A細胞(1 x 10
6個細胞/孔)血清飢餓5小時。將IL-6 (100 ng/ml)與sIL-6 Rα (200 ng/ml)混合15 分鐘,隨後再將IL-6/sIL-6R組合與指定濃度之C14mab、托珠單抗(tocilizumab)或奧洛珠單抗(olokizumab)一起培育15 分鐘。用該等混合物處理細胞15 分鐘,且藉由西方墨點法測定p-STAT3 。(c)對於HyIL-6介導之信號傳導阻斷,用HyIL-6 (10 ng/ml)及所指示之抗體刺激HeLa細胞(1 x 10
5個細胞/孔)。如(a)中所描述,藉由西方墨點法測定p-STAT3含量(左圖)且藉由Image J軟體進行分析(右圖)。圖(a)左:IL-6以及各種C14mab及托珠單抗濃度之西方墨點法分析。圖(a)右:繪製p-STAT3活性百分比相對於對數抗體(C14mab相對於托珠單抗)之折線圖。圖(b) IL-6及IL-6R以及各種濃度之C14mab、托珠單抗及奧洛珠單抗之西方墨點法分析。圖(c)左:HyIL-6以及各種濃度之C14mab及托珠單抗之西方墨點法分析。圖(c)右:繪製p-STAT3活性百分比相對於對數抗體(C14mab相對於托珠單抗)之折線圖。
圖7說明了C14mab之中和活性。(a) C14mab抑制C33A中HyIL-6誘導之VEGF表現。藉由ELISA評估在存在或不存在所指示濃度之C14mab的情況下,用HyIL-6 (50 ng/ml)處理後,進行培育之後C33A細胞培養物上清液中的VEGF蛋白表現。收集培養基用於測定VEGF含量。數據表示為平均值± SD,n = 4。*相對於陽性對照之統計學上顯著之差異;P < .05。**相對於陽性對照之統計學上顯著之差異;P < .01 (b)在存在TGF-β1 (2 ng/ml)及抗IL-2 (10 ug/ml)的情況下,用抗CD3及抗CD28刺激野生型原生CD4+ T細胞四天。在培養物中使用所指示濃度之C14mab、托珠單抗及奧洛珠單抗來阻斷HyIL-6 (50 ng/ml)介導之信號傳導。藉由ELISA (R&D Systems)測定培養物上清液中之IL-17A含量。值表示平均值± SD;n = 3。**相對於陽性對照之統計學上顯著之差異;P < .01。(c)向BALB/c小鼠注射C14mab (0.1 mg/ml)、DPBS或同型對照抗體(0.1 mg/kg)。1小時後,用重組人類HyIL-6 (1 ug)攻擊小鼠。再6小時後,收集全血。藉由使用小鼠SAA ELISA從血清中測定SAA含量。(d) C14mab劑量依賴性地減少小鼠之HyIL-6誘導之SAA含量。向BALB/c小鼠注射所指示劑量之C14mab。1小時後,用重組人類HyIL-6 (1 ug)攻擊小鼠。再6小時後,收集全血。藉由使用小鼠SAA ELISA從血清中測定SAA含量。數據表示為平均值± SD,n = 5。**相對於陽性對照之統計學上顯著之差異;P < .01。
圖8說明了HyIL-6/C14mab相互作用的表位。(a) C14mab結合時之HyIL-6的差示HDX。疊加散射圖顯示對應於在氘化30、180、600、1800及5400秒時獲得之數據的粉紅色、紫色、綠色、橙色、藍色及紅色點。與其他序列相比,C14mab結合時之序列
498LTKLQAQNQWLQD
510顯示顯著的HDX減少。(b)描繪分析程序之示意圖(左圖)。在4℃下用含sIL- 6 Rα (50 ng/孔)之PBS緩衝液塗佈微量孔盤隔夜,且用含1% BSA之PBS阻斷1小時。隨後,用含有0.05% Tween-20之PBS洗滌盤。以上;無洗滌組,在室溫下添加IL-6 (10 ng/孔) 1小時。隨後,再直接添加連續稀釋濃度之C14mab持續2小時。以下;洗滌組,在室溫下添加IL-6 (10 ng/孔) 1小時。在用含有0.05% Tween-20之PBS洗滌盤之後,再直接添加連續稀釋濃度之C14mab持續1小時。藉由ELISA測定結合C14mab。在C14mab培育之前有洗滌或無洗滌之結合結果分別以藍色及紅色示出(右圖)。顯示了3個代表性分析中之一個。
圖9說明了IL-6/IL-6R/C14mab之建議模型。(a)六聚體信號傳導複合物內之IL-6、IL-6 R及gp130之三個關鍵相互作用位點。IL-6/IL-6 R/C14mab之建議模型與IL-6/IL-6 R/gp130之三聚體結構疊加。IL-6、IL-6 R、gp130及C14mab由卡通圖表示。結合候選物為肽498–510及肽130–141。gp130結構域I的表面表示在空間上與在IL-6及IL-6 R上相互作用之C14mab重疊。(b)應用HDX-MS以藉由對HyIL-6之H/D交換與HyIL-6-C14mab複合物之H/D交換進行比較來將C14mab之抗原表位定位至HyIL-6上。IL-6R、連接子及IL-6分別為肽1-339 (SEQ ID NO: 13)、肽340-351 (SEQ ID NO: 14)及肽352-534 ((SEQ ID NO: 15)。在C14mab存在的情況下顯著地減少之候選殘基顯示為肽498-510 (SEQ ID NO: 1),而具有低減少位準之候選殘基為肽130-141 (SEQ ID NO: 2)。具有灰色背景之序列為參與IL-6與gp130 (位點III)之結合之肽序列。
圖10說明了C14mab之同型。
Claims (10)
- 一種單株抗體,其對人類介白素-6(IL-6)/介白素-6受體(IL-6R)複合物具有特異性;其中該單株抗體包含輕鏈可變區(VL區)及重鏈可變區(VH區),該VL區包含互補決定區1(CDR-L1)、互補決定區2(CDR-L2)以及互補決定區3(CDR-L3);其中該CDR-L1包含SEQ ID NO:3之胺基酸序列,該CDR-L2包含SEQ ID NO:4之胺基酸序列,而該CDR-L3包含SEQ ID NO:5之胺基酸序列;該VH區包含互補決定區1(CDR-H1)、互補決定區2(CDR-H2)以及互補決定區3(CDR-H3);其中該CDR-H1包含SEQ ID NO:6之胺基酸序列,該CDR-H2包含SEQ ID NO:7之胺基酸序列,而該CDR-H3包含SEQ ID NO:8之胺基酸序列。
- 如請求項1之單株抗體,其中該單株抗體特異性結合至IL-6內之抗原表位,且該抗原表位包含SEQ ID NO:1之胺基酸序列。
- 如請求項1之單株抗體,其中該單株抗體特異性結合至IL-6R內之抗原表位,且該抗原表位包含SEQ ID NO:2之胺基酸序列。
- 如請求項1之單株抗體,其中該VL區包含SEQ ID NO:9之胺基酸序列。
- 如請求項4之單株抗體,其中該VH區包含SEQ ID NO:10之胺基酸序列。
- 一種醫藥組合物,其包含如請求項1至5中任一項之單株抗體。
- 一種核酸分子,其編碼如請求項1至5中任一項之單株抗體。
- 一種載體,其包含如請求項7之核酸分子。
- 一種細胞,其包含如請求項7之核酸分子或表現如請求項7之核酸分子。
- 一種如請求項1之單株抗體用於製備藥物的用途,其中該藥物 係用於治療IL-6/IL-6R介導之疾病。
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Title |
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期刊 Heo et. al., " Potential therapeutic implications of IL-6/IL-6R/gp130- targeting agents in breast cancer" Oncotarget, 7(13), Impact Journals, 2016 Mar 29, p.15460-73. |
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