TW201827464A - 人程序性死亡受體pd-1的單株抗體及其片段 - Google Patents
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Abstract
本發明提供了針對人PD-1的分離的單株抗體或其抗原結合片段,包含表2中所列的重鏈CDR1、CDR2和CDR3以及表2中所列的輕鏈CDR1、CDR2和CDR3。
Description
本發明涉及生物製藥領域,具體而言,涉及針對人PD-1的單株抗體及其在疾病治療中的應用。
程序性死亡受體1(PD-1)是在激活的T細胞和B細胞上表現的免疫抑制性受體。阻斷PD-1與其配體之一PD-L1間的相互作用提高腫瘤特異性CD8+T細胞的免疫性,因此有助於免疫系統清除腫瘤細胞。非專利文獻1中詳細綜述了PD-1及相關研究。
在文獻中已確定了PD-1在癌症中的作用。業已在人的很多原發性腫瘤中發現PD-1(在腫瘤浸潤淋巴細胞上)及/或PD-L1(在腫瘤細胞上)。這樣的組織包括肺癌、肝癌、卵巢癌、子宮頸癌、皮膚癌、結腸癌、神經膠質瘤、膀胱癌、乳腺癌、腎癌、食道癌、胃癌、口腔鱗狀細胞癌、尿道上皮細胞癌和胰腺癌以及頭頸腫瘤。
阻斷PD-1/PD-L1的相互作用可導致腫瘤特異性T細胞的免疫性提高,因此,有助於由免疫系統清除腫瘤細胞。
PCT國際申請PCT/US2008/007463揭露了一種針對人程序性死亡受體PD-1的抗體。
PD-1/PD-L1路徑是開發癌症治療的抗體療法的被充分證實的靶標。抗PD-1抗體還可用於慢性病毒性感染。最近,有研究顯示PD-1 在來自HIV感染的個體的T細胞中高度表現。
【非專利文獻1】 Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway, Vassiliki A. Boussiotis, NEJM n engl j med 375;18 nejm.org November 3, 2016
本發明欲提供親和力更高,更有效果的單株抗體。
本發明提供了一種針對人PD-1的分離的單株抗體或其抗原結合片段,包含:表2中所列的重鏈CDR1、CDR2及CDR3,以及表2中所列的輕鏈CDR1、CDR2及CDR3。
在一個實施方案中,前述抗體或其抗原結合片段,其中,其係包含:(i)序列表SEQ ID NO:3所示的重鏈胺基酸序列,及序列表SEQ ID NO:4所示的輕鏈胺基酸序列;或(ii)不包含信號肽部分的序列表SEQ ID NO:3所示的重鏈胺基酸序列,及不包含信號肽部分的序列表SEQ ID NO:4所示的輕鏈胺基酸序列。
其中,SEQ ID NO:3所示的重鏈胺基酸序列的第1位-第19位(即,MNFGLSLIFLVLVLKGVLC)是信號肽部分;SEQ ID NO:4所示的輕鏈胺基酸序列的第1位-第20位(即, MEKDTLLLWVLLLWVPGSTG)是信號肽部分。
信號肽是出於方便純化等目的而加入的,並非抗體結構中必不可少的部分。該領域具有通常知識者可以理解,在本發明的抗體或抗體片段中,信號肽是任選的,也就是說,可以包含信號肽,也可以不包含信號肽。
本發明還提供了針對人PD-1的人源化的分離的單株抗體或其抗原結合片段,其包含:(1)選自序列表SEQ ID NO:13、14、15及16的重鏈胺基酸序列,或不包含信號肽部分的序列表SEQ ID NO:13、14、15及16的重鏈胺基酸序列;以及(2)選自序列表SEQ ID NO:17、18、19及20的輕鏈胺基酸序列,或不包含信號肽部分的序列表SEQ ID NO:17、18、19及20的輕鏈胺基酸序列。
其中,SEQ ID NO:13、14、15和16所示的重鏈胺基酸序列的第1位-第24位(即,MDPKGSLSWRILLFLSLAFELSYG)是信號肽部分;SEQ ID NO:17、18、19和20所示的輕鏈胺基酸序列的第1位-第20位(即,METDTLLLWVLLLWVPGSTG)是信號肽部分。
在一些實施方案中,前述抗原結合片段選自:Fab、Fab'、Fab'-SH、Fv、scFv、F(ab')2和雙抗體。
本發明提供了編碼前述抗體或其抗原結合片段的分離的多核苷酸。
在一些實施方案中,前述多核苷酸,其中,重鏈編碼序列 如序列表SEQ ID NO:1所示,輕鏈編碼序列如序列表SEQ ID NO:2所示。
在一些實施方案中,前述多核苷酸編碼人源化抗體或其抗原結合片段,並且重鏈編碼序列選自序列表SEQ ID NO:5、6、7及8,輕鏈編碼序列選自序列表SEQ ID NO:9、10、11及12。
本發明提供了包含前述分離的多核苷酸的表現載體,以及包含前述表現載體的宿主細胞。
本發明提供了藥物組合物,包含前述抗體或其抗原結合片段以及可藥用載體。
本發明提供了提高免疫細胞活性的方法,其特徵係包括使免疫細胞與前述抗體或其抗原結合片段接觸。
本發明提供了治療疾病的方法,前述方法其特徵係包括將治療有效量的本發明的抗體或其抗原結合片段給予需要治療的受試者。
在一些實施方案中,其中,前述疾病選自:癌症、感染性疾病、移植排斥、自身免疫性疾病和神經疾病。在一些實施方案中,前述方法包括對受試者聯合應用其他藥物或療法,包括化療、放射治療、靶向治療、基因治療、細胞治療、幹細胞治療。
本發明提供了前述抗體或其抗原結合片段在製備用於治療疾病的藥物中的用途,其中,前述疾病選自:癌症、感染性疾病、移植排斥、自身免疫性疾病和神經疾病。
本發明提供了調節細胞功能的方法,其特徵係包括將編碼本發明的抗體或其抗原結合片段的多核苷酸序列遞送至前述細胞中。
在一些實施方案中,其中,前述方法用於基因治療、CAR- T治療或CRISPR治療。
在一些實施方案中,利用敲除了PD-1基因的小鼠來產生抗人PD-1單株抗體,親和力更高,阻斷效果更好,細胞殺傷活力更高。試驗結果表明這種方法能夠得到高親和力的單株抗體。其親和力比目前上市的PD-1抗體親和力高出大約4-7倍,體外阻斷PD-L1/PD-1的相互作用非常有效。同時體外抑制腫瘤生長活性也有所提高。
本發明試驗結果表明藉由本發明能夠得到高親和力的單株抗體。其親和力比目前上市的PD-1抗體親和力高出大約4-7倍,體外阻斷PD-L1/PD-1的相互作用非常有效。同時體外抑制腫瘤生長活性也有所提高。
【圖1】 顯示了PD-1抗體與PD-1蛋白的結合活性。
【圖2】 顯示了PD-1抗體對PD-1蛋白與PD-L1蛋白結合的阻斷效應。
【圖3】 顯示了不同抗體競爭結合實驗結果。
【圖4】 顯示了DNA重組抗體的結合和阻斷作用。
【圖5】 顯示了人源化抗體的與PD-1蛋白的結合活性
【圖6】 顯示了人源化抗體的阻斷作用。
【圖7】 顯示了人源化母本抗體不同突變體親和力之比較。
【圖8】 顯示了人源化PD-1抗體和食蟹猴淋巴細胞PD-1蛋白結合之情形。
【圖9】 顯示了人源化PD-1抗體體外增強效應細胞殺傷腫瘤細胞之情形。
【圖10】 顯示了人源化PD-1抗體阻斷人淋巴細胞的PD-1的誘導表現。
【圖11】 顯示了自研PD-1抗體與上市PD-1抗體動力學和親和力之比較。
【圖12】 顯示了體外抑制腫瘤生長作用之比較。
“抗體”是指表現出所需生物學活性(例如抑制配體與其受體的結合或通過抑制配體誘導的受體信號轉導)的抗體的任何形式。因此,“抗體”以其最廣泛的意義來使用,並明確包括但不限於單株抗體(包括全長單株抗體)、多株抗體和多特異性抗體(例如雙特異性抗體)。
“抗體片段”和“抗原結合片段”在本文中可互換使用,意指抗體的抗原結合片段及抗體類似物,其通常包括至少部分母體抗體(parental antibody)的抗原結合區或可變區(例如一個或多個CDR)。抗體片段保留母體抗體的至少某些結合特異性。理想地,抗體片段保留至少50%、70%、80%、90%、95%或100%或更多的母體抗體對靶標的結合親和力。抗體片段實例包括但不限於:Fab、Fab'、F(ab')2和Fv片段;雙抗體;線性抗體(linear antibody);單鏈抗體分子,例如sc-Fv、單抗體(技術 來自Genmab);奈米抗體(技術來自Domantis);結構域抗體(技術來自Ablynx);和由抗體片段形成的多特異性抗體。工程改造的抗體變體綜述於Holliger和Hudson(2005)Nat.Biotechnol.23:1126-1136中。
“Fab片段”由一條輕鏈和一條重鏈的CH1及可變區組成。Fab分子的重鏈不能與另一個重鏈分子形成二硫鍵。
“Fc”區含有包含抗體的CH1和CH2結構域的兩個重鏈片段。兩個重鏈片段由兩個或多個二硫鍵並通過CH3結構域的疏水作用保持在一起。
“Fab'片段”含有一條輕鏈和包含VH結構域和CH1結構域以及CH1和CH2結構域之間區域的一條重鏈的部分,由此可在兩個Fab'片段的兩條重鏈之間形成鏈間二硫鍵以形成F(ab')2分子。
“F(ab')2片段”含有兩條輕鏈和兩條包含CH1和CH2結構域之間的恆定區的部分的重鏈,由此在兩條重鏈間形成鏈間二硫鍵。因此,F(ab')2片段由通過兩條重鏈間的二硫鍵保持在一起的兩個Fab'片段組成。
“Fv區”包含來自重鏈和輕鏈二者的可變區,但缺少恆定區。
“單鏈Fv抗體”(或“scFv抗體”)是指包含抗體的VH和VL結構域的抗體片段,其中這些結構域存在於單個多肽鏈中。一般而言,Fv多肽另外在VH和VL結構域之間包含多肽連接子,該連接子使得scFv能形成用於抗原結合的所需結構。參見國際專利申請WO88/01649。
“雙抗體”為具有兩個抗原結合位點的小抗體片段。前述 片段包含在相同的多肽鏈中與輕鏈可變結構域(VL)連接的重鏈可變結構域(VH)(VH-VL或VL-VH)。通過使用短至不能在同一鏈的兩個結構域之間配對的連接子,迫使前述結構域與另一條鏈的互補結構域配對並形成兩個抗原結合位點。
術語“嵌合”抗體指這樣的抗體以及表現出所需生物學活性的前述抗體的片段:其中部分重鏈及/或輕鏈與來源於特定物種或屬於特定抗體類別或亞類的抗體相應的序列等同或同源,同時前述鏈的其餘部分與來源於另一物種或屬於另一抗體類別或亞類的抗體相應的序列等同或同源(參見例如美國專利第4,816,567號和Morrison等,1984、Proc.Natl.Acad.Sci.USA 81:6851-6855)。
非人類(例如鼠)抗體的“人源化”形式為含有最小限度的來源於非人類免疫球蛋白序列的嵌合抗體。一般而言,人源化抗體包含至少一個且通常為兩個可變結構域的幾乎全部,其中全部或幾乎全部高度變異環(hypervariable loop)對應於非人類免疫球蛋白的高度變異環,全部或幾乎全部FR區為人免疫球蛋白序列的FR區。人源化抗體還任選包含至少部分免疫球蛋白(通常為人免疫球蛋白)恆定區(Fc)。更詳細的資料參見Jones et al,Nature 321:522-525(1986);Riechmann et al,Nature 332:323-329(1988)。
“分離的”抗體為業已被鑒定並與其天然環境組分相分離的抗體。在一些實施方案中,將前述抗體純化到以下程度:(1)超過95%重量的抗體,最理想為超過99%重量,其由Lowry法測定;(2)足以通過用旋杯式測序儀(spinning cup sequencer)獲得N-末端或內部胺基酸序列的至 少15個殘基的程度;或(3)通過在還原或非還原條件下用考馬斯藍或優選銀染色的SDS-PAGE確定為同質。
“分離的”核酸分子為被鑒定並與至少一種污染性核酸分子(通常在抗體核酸的天然來源中與其締合)分離的核酸分子。因此,分離的核酸分子與在其天然細胞中存在的核酸分子有區別。
本文所用術語“單株抗體”是指從基本上同種抗體群中獲得的抗體,即除了可能少量存在的可能的天然突變體外,構成前述群的各個抗體是一致的。單株抗體具有高度特異性,可針對單個的抗原位點。此外,與通常包括針對多個不同的決定位(表位)的多種不同抗體的常規(多株)抗體製備物相反,每種單株抗體僅針對抗原上的單個決定位。修飾語“單株”表示從基本上同種抗體群獲得的抗體的特性,不應理解為需要通過任何特定方法來製備前述抗體。本文單株抗體明確包括“嵌合”抗體。
關於抗體及其應用的更多介紹參見中國專利申請CN201510367410.4,在此通過援引將其全部內容併入本文。
本文所用術語“免疫細胞”包括具有造血的起源並在免疫反應中起作用的細胞。免疫細胞包括:淋巴細胞,例如B細胞和T細胞;自然殺手細胞;髓細胞,例如單核細胞、巨噬細胞、嗜酸性白血球、肥大細胞、嗜鹼性白血球和粒細胞。
本文所用序列“變體”是指在一個或多個胺基酸殘基處不同於所示的序列但基本保留其生物學活性的序列。例如,變體可以保留原序列至少50%,理想為至少70%,甚至90%的生物學活性。
本文所用術語“約”是指數值在由本領域一般技術人員所 測定的具體值的可接受誤差範圍內,前述數值部分取決於怎樣測量或測定(即測量體系的限度)。例如,“約”或“基本上包含”可意味著不超過20%的範圍,理想為不超過10%,更理想為不超過5%。除非另外說明,否則當具體值在本文中出現時,“約”的含義應該假定為在該具體值的可接受誤差範圍內。
當用“給予”和“治療”提及動物、人、細胞或受試者時,是指將外源性藥物、治療劑、診斷劑或組合物與動物、人、細胞或受試者接觸。“給予”和“治療”可指例如治療方法、藥動學方法、診斷方法、研究方法和實驗方法。“給予”和“治療”還意味著例如通過試劑、診斷劑、療法、聯合用藥或通過其他細胞對細胞進行體外和離體治療。
本文所使用的術語“治療有效量”指的是足以在受試者體內引起臨床醫師所期望的生物學或醫學反應的活性化合物的量。本發明抗體的“治療有效量”可由該領域具有通常知識者根據給藥途徑、受試者的體重、年齡、病情等因素而確定。例如,典型的週總劑量可以為至少0.05μg/kg體重,典型地為至少1μg/kg體重。
本發明所提供的藥物可以製成粉末、注射劑等臨床可接受的劑型。可以使用任何適當的途徑給受試者施用本發明的藥物組合物,例如可通過口服、靜脈內輸注、肌肉內注射、皮下注射、腹膜下、直腸、舌下,或經吸入、透皮等路徑給藥。
本發明的藥物製劑包含本發明PD-1抗體或其抗原結合片段以及可藥用載體。為了製備藥物組合物或無菌組合物,讓抗體或其片段與可藥用載體(或賦形劑)混合。
本發明藥物組合物還可含有其它藥劑,包括但不限於細胞毒殺劑、細胞生長抑制劑、抗血管形成藥物或抗代謝藥物、靶向腫瘤藥物、免疫刺激劑或免疫調節劑或與細胞毒殺劑、細胞生長抑制劑或其它毒性藥物綴合的抗體。也可與其它治療形式(例如手術、化療及放射治療)一起施用前述藥物組合物。
與人PD-1特異性結合的本發明抗體或抗原結合片段可用於增加、提高、刺激或上調免疫反應。本發明抗體及抗體片段尤其適用於治療罹患可通過提高T細胞介導的免疫反應來治療的疾病的受治療者。理想的受治療者包括需要提高免疫反應的人類患者。
本發明抗體或抗原結合片段可用於治療癌症(即抑制腫瘤細胞的生長或存活)。可用本發明抗體抑制其生長的理想的癌症包括通常對免疫療法有反應的癌症,但還包括迄今與免疫療法尚無關聯的癌症。用於治療的理想癌症的非限制性實例包括黑色素瘤(例如惡性轉移性黑色素瘤)、腎癌(例如透明細胞癌)、前列腺癌(例如激素難控制的前列腺腺癌)、胰腺腺癌、乳腺癌、結腸癌、肺癌(例如非小細胞肺癌)、食道癌、頭頸鱗狀細胞癌、肝癌、卵巢癌、子宮頸癌、甲狀腺癌、膠質母細胞瘤、神經膠質瘤、白血病、淋巴瘤和其它惡性腫瘤。另外,本發明包括可用本發明抗體抑制其生長的難治性或復發性癌。
本發明抗體或其抗原結合片段可單獨使用或與以下其它物質聯合使用:抗腫瘤藥或免疫原劑(例如減弱的癌細胞、腫瘤抗原(包括重組蛋白質、肽和糖類分子)、抗原呈現細胞例如用來源於腫瘤的抗原或核酸刺激的樹突細胞、免疫刺激細胞因子(例如IL-2、IFNa2、GM-CSF)和用編 碼免疫刺激細胞因子(例如但不限於GM-CSF)的基因轉染的細胞;標準癌症治療(例如化療、放射治療或手術);或其它抗體(包括但不限於針對以下物質的抗體:VEGF、EGFR、Her2/neu、VEGF受體、其它生長因子受體、CD20、CD40、CTLA-4、OX-40、4-IBB和ICOS)。
本發明抗體或其抗原結合片段還可用於防止或治療感染和感染性疾病。抗體或其抗原結合片段可單獨使用,或與疫苗聯合使用,以刺激針對病原體、毒素和自體抗原的免疫反應。抗體或其抗原結合片段可用於刺激對感染人的病毒的免疫反應,這些病毒例如但不限於人免疫缺陷病毒、甲、乙、丙型肝炎病毒、愛潑斯坦-巴爾病毒、人巨細胞病毒、人乳頭瘤病毒、皰疹病毒。抗體或其抗原結合片段可用於刺激對細菌或真菌寄生蟲及其它病原體引起的感染的免疫反應。
本發明抗體或其抗原結合片段還可用於防止或治療移植排斥、自身免疫性疾病和神經疾病。
本文中使用的術語“自身免疫性疾病”是指由自身免疫反應所引起的任何疾病,即,免疫反應針對受試者身體內的物質。
應當注意的是由於自身免疫性可以影響受試者的任何器官或組織,例如受試者的大腦、皮膚、腎臟、肺、肝臟、心臟或甲狀腺,疾病的臨床表現取決於受影響的部位。
典型的自身免疫性疾病包括但不限於類風濕性關節炎、銀屑病性關節炎、骨關節炎、斯蒂爾病(Still’s disease)、青少年關節炎、狼瘡、糖尿病、重症肌無力症、橋本甲狀腺炎、奧德甲狀腺炎、格雷夫斯病(Graves'disease)、類風濕性關節炎症候群、多發性硬化症、傳染性神經元 炎、急性播散性腦脊髓炎、阿狄森病(Addison)、視性眼陣攣-肌陣攣綜合症、強直性脊椎炎、抗磷脂抗體綜合症、再生障礙性貧血、自身免疫性肝炎、乳糜瀉、古德帕斯徹症候群、特發性血小板減少性紫癲、視神經炎、硬皮病、原發性膽汁性肝硬化、萊特爾症候群(Reiter syndrome)、高安動脈炎(Takayasu's arteritis)、顳動脈炎、溫型自身免疫性溶血性貧血、韋格納肉芽腫病、銀屑病、全身脫毛、貝赫切特病(Behcet's Disease)、慢性疲勞、家族性自主神經功能異常、子宮內膜異位、間質性膀胱炎、神經肌強直、硬皮病和外陰痛等。
本文中使用的術語"神經疾病"是指影響中樞神經系統及/或具有在中樞神經系統中的病因的疾病或病症。示例性的神經疾病的具體例子包括但不限於:帕金森病、阿爾茨海默病(Alzheimer’s disease)、普里昂疾病(prion disease)、亨廷頓病(Huntington's disease)、雷特症候群(Rett syndrome)等。
本發明抗PD-1抗體或其抗原結合片段可以與其他藥物或療法聯合應用,例如與一種或多種其它治療劑(例如細胞毒劑、放射性毒性劑或免疫抑制劑)聯合應用。前述抗體可與前述藥劑連接(作為免疫複合體),或可與治療劑分開給予,例如可在給予治療劑之前、之後或同時給予抗體。此外,本發明抗體可與化療、放射治療、靶向治療、基因治療、細胞治療、幹細胞治療等聯合應用,尤其是用於癌症的治療。
本發明抗PD-1抗體或其抗原結合片段或其編碼基因還可以被導入細胞中,以調節細胞功能或活性,或用於治療疾病。該方法包括體外應用和體內應用。該方法特別適合用於基因治療、CAR-T治療或 CRISPR治療。
本發明抗體及抗體片段的非治療性應用包括:用於流式細胞術分析、免疫組織化學和體外功能分析、西方點墨法和ELISA等。本發明抗體還可用作親和純化試劑。
前述抗體還可用於診斷測定,例如用於檢測PD-1在特定的細胞、組織或血清中的表現。為了診斷應用,通常用可檢測的部分標記(直接或間接)抗體。可利用眾多標記物,其通常分為以下類別:生物素、螢光染料、放射性核苷酸、酶、碘和生物合成標記物。
本發明抗體可用於任何已知的測定法中,例如競爭性結合測定法,直接和間接酵素免疫分析法和免疫沉澱測定法。抗體還可用於體內診斷測定。例如用放射性核素(例如111In、99Tc、4C、31I、125I、3H、32P、35S或18F)標記抗體,以便定位抗原或表現抗體的細胞。
通過參考以下實施例將更充分地理解本發明。然而,這些實施例不應該理解為限制本發明範圍。
實施例1 產生及篩選PD-1抗體
1.產生人PD-1融合蛋白
從人周邊血單核細胞(PBMC)利用MACS磁珠(MiltenyiBiotec)分離出T淋巴細胞,提取RNA(RNeasy mini kit,QIAGEN)並獲得第一鏈cDNA(SuperScript First-Strand Synthesis System,Invitrogen)並根據人PD-1基因DNA序列(NCBI Reference Sequence:NM_005018.2)通過PCR 反應獲得人PD-1分子全長序列。人PD-1融合蛋白是將PD-1分子胞外部分序列插入到一個含有小鼠免疫球蛋白G2aCH2-CH3結構域的表現質體mIgV(H Dong et al Nat Med.1999;5:1365-1369)中。利用暫態表現方法(transient expression)將質體DNA用PEI試劑(Sigma-Aldrich)轉染293T細胞產生人PD-1小鼠Ig融合蛋白(hPD-1mIg),細胞分泌上清液經IgA純化柱(HiTrap Protein A HP,GE healthcare)純化蛋白並經SDS-PAGE凝膠電泳鑒定確認。
2.構建PD-1穩定表現細胞株
為檢測和鑒定PD-1抗體,本發明構建了人PD-1蛋白穩定表現細胞株。將上述得到的PD-1全長序列插入到pcDNA3.1(-)表現質體,利用lipofactamin 2000轉染試劑(Thermo Fisher Scientific)轉染中國倉鼠卵巢成纖維細胞(CHO),經G418篩選得到穩定表現的陽性(positive)細胞株(CHO/hPD1)。
3.免疫小鼠
取6-8周齡大小的雌性Balb/c小鼠,用hPD-1mIg融合蛋白與福氏完全佐劑(CFA,Sigma)混合乳化液皮下多點接種免疫,每隻小鼠80-100μg蛋白,3周後用相同融合蛋白與不完全佐劑(IFA,Sigma)乳化液進行加強免疫,共免疫3次。每次在小鼠免疫後2周左右取血清測定抗體效價,血清效價用CHO/hPD1細胞通過流式細胞儀測定。二抗用螢光標記的羊抗鼠IgG(eBioscience)。當血清效價達到足夠高時,用80μg hPD-1mIg融合蛋白溶於PBS中,腹腔注射加強免疫一次,5天后取出脾臟進行融合。
4.製備及篩選雜交瘤
雜交瘤細胞的產生通過免疫小鼠的脾細胞與小鼠骨髓瘤細胞系(SP2/0-Ag14)在聚乙二醇(PEG1500,Roche)融合劑作用下進行融合,融合的細胞經過HAT(Sigma Aldrich)選擇培養,利用發明人實驗室建立的高通量轉染和篩選系統(S Yao et al.Immunity.2011;34(5):729-40.)鑒定培養上清,篩選得到的陽性單株抗體再經過流式細胞儀檢測確認能夠和PD-1陽性表現細胞結合。挑選出的陽性株(positive clone)細胞株經過5遍以上有限稀釋法進行次選植,確保是單一來源的陽性株。
實施例2 抗PD-1單株抗體的特性分析
單株抗體同種型(isotype)的鑒定
得到的幾株單株抗體用小鼠IgG同種型檢測套組(Mouse Immunoglobulin Isotyping Kit,BD Biosciences)進行測定,5個單株抗體重鏈均為IgG1,輕鏈均為κ鏈。
單株抗體特異性結合的評估
種屬交叉反應的鑒定顯示幾株PD-1抗體株只和人PD-1蛋白結合,與小鼠PD-1無交叉結合。
PD-1抗體與PD-1蛋白的結合反應:將發明人實驗室得到的5個PD-1抗體用流式細胞儀檢測及比較與CHO/hPD-1細胞結合活性,結果顯示這些抗體與PD-1蛋白都有較強的結合活性(圖1)。
PD-1抗體對PD-1受體蛋白和其配體PD-L1結合的阻斷作用:腫瘤細胞上表現PD-L1配體蛋白與其在淋巴細胞表現的PD-1受體結合會誘導產生免疫細胞功能抑制而導致腫瘤逃避宿主免疫系統監視作用,阻斷PD-1與PD-L1之間的相互作用是PD-1抗體抗腫瘤免疫治療的一個 重要作用機制。為檢測抗體的阻斷活性,本發明將100ng hPD-1hIg融合蛋白分別與100ng、200ng、400ng和800ng不同劑量的不同PD-1抗體混合,並設mIgG和PBS緩衝液對照組。4℃培養30分鐘,然後與PD-L1陽性表現細胞(CHO/PD-L1)在4℃作用30分鐘,用含1%小牛血清PBS緩衝液離心洗一遍,加入螢光標記的羊抗人IgG,4℃培養20分鐘,洗後用流式細胞儀檢測。結果顯示2個PD-1抗體沒有阻斷活性,其餘3個抗體都能夠以劑量依賴性阻斷PD-1與PD-L1蛋白間的結合(圖2)。
抗體競爭結合實驗:為弄清這些PD-1抗體是否識別同一個或不同抗原表位,本發明做了抗體競爭結合實驗。首先分別用10μg不同PD-1抗體與CHO/hPD-1細胞在4℃培養30分鐘,用含1%小牛血清PBS緩衝液離心洗一遍後加入0.1μg的生物素(Biotin)標記的Ab4或Ab5,4℃培養20分鐘,洗後加入螢光標記的鏈黴親和素(Streptavidin,SA;eBioscience),4℃培養20分鐘,洗後用流式細胞儀進行分析。結果顯示Ab4和Ab5相互競爭結合同一抗原表位,這兩個抗體與Ab3有部分競爭結合,與Ab1和Ab2不存在競爭結合(圖3)。
實施例3 抗PD-1抗體的序列測定和人源化
為了將PD-1抗體能夠用於臨床腫瘤治療,本發明根據上述抗體活性的測試結果選擇了Ab4進行序列測定和人源化。
單株抗體序列測定
首先利用RNA純化套組(RAeasy Mini Kit,Qiagen 74104,QIAshredder,Qiagen 79654)從雜交瘤細胞提取和純化總RNA。然後用套組(SMARter RACE cDNA Amplification Kit,Clontech)並按公司試劑操作 手冊將RNA反轉錄合成第一鏈cDNA鏈。利用5’RACE技術,用套組提供的通用引子UPM(universal primer)作為上游引子,和根據小鼠IgG1重鏈可變區和輕鏈κ鏈基因序列設計的基因特異引子GSP(genespecific primer)作為下游引子,以SMART第一鏈cDNA為模板進行PCR循環(cycle)。然後將PCR產物分別連接到T載體(Zero Blunt TOPO PCR Cloning Kit,Invitrogen K2875-J10)。選殖進行序列測定和分析,分別得到抗體的輕鏈和重鏈可變區序列,再根據重鏈可變區序列和小鼠IgG1序列設計引子,通過PCR獲得重鏈全長序列,利用VBASE2對序列進行分析,確定了該抗體輕、重鏈的胺基酸序列以及相應的CDR區域(見表2)。其DNA編碼序列參見表1。
單株抗體DNA重組序列和抗體表現
為確保選殖抗體序列的正確性,分別把抗體的輕、重鏈全長序列插入到pcDNA3.1(-)表現質體中,並通過暫態轉染293T細胞得到蛋白產物,經純化和鑒定確認蛋白產物與原抗體一樣能夠和PD-1蛋白結合(圖4.A)並能夠阻斷PD-1蛋白與PD-L1的結合(圖4.B)。
抗體序列人源化
為了使抗體能夠應用臨床病人治療,對抗體序列進行了人源化改造。首先將抗體重鏈的Fc部分換為人IgG4序列,並將IgG鉸鏈區4的CPSCP換為IgG1的CPPCP(S228P),同時把抗體輕鏈換成人的κ鏈,得到人鼠嵌合的母本抗體(Chimeric Antibody)。在確認母本抗體的功能活性後對重鏈可變區和輕鏈可變區序列進行人源化改造,將所得到的3個人源化重鏈變異體(表3序列6-8,表4序列14-16)、3個人源化輕鏈變異 體(表3序列10-12,表4序列18-20)互相組合,共得到9個不同的變異體序列(Variants 1至Variants 9)。將1個母本和9個變異體序列分別選殖到高表現載體中,用暫態轉染293細胞得到抗體上清,純化後的抗體經SDS-PAGE膠蛋白電泳和定序驗證確認正確。抗體的DNA和胺基酸序列見表3、表4。其中,Variants 1的重鏈胺基酸序列為SEQ ID NO:6,輕鏈胺基酸序列為SEQ ID NO:10;Variants 2的重鏈胺基酸序列為SEQ ID NO:6,輕鏈胺基酸序列為SEQ ID NO:11;Variants 3的重鏈胺基酸序列為SEQ ID NO:6,輕鏈胺基酸序列為SEQ ID NO:12;Variants 4的重鏈胺基酸序列為SEQ ID NO:7,輕鏈胺基酸序列為SEQ ID NO:10;Variants 5的重鏈胺基酸序列為SEQ ID NO:7,輕鏈胺基酸序列為SEQ ID NO:11;Variants 6的重鏈胺基酸序列為SEQ ID NO:7,輕鏈胺基酸序列為SEQ ID NO:12;Variants 7的重鏈胺基酸序列為SEQ ID NO:8,輕鏈胺基酸序列為SEQ ID NO:10;Variants 8的重鏈胺基酸序列為SEQ ID NO:8,輕鏈胺基酸序列為SEQ ID NO:11;Variants 9的重鏈胺基酸序列為SEQ ID NO:8,輕鏈胺基酸序列為SEQ ID NO:12。
實施例4 人源化抗體的特性和功能
人源化抗體的與PD-1蛋白之結合活性:對嵌合母本和突變抗體進行測試和比較,取CHO/hPD-1細胞懸浮於含1%小牛血清的PBS並分裝於流式管中,分別加入不同劑量的母本和突變體抗體,4℃培養30分鐘,用含1%小牛血清PBS緩衝液離心洗一遍後加入螢光標記的羊抗人IgG二抗(eBioscience),4℃培養20分鐘洗後用流式細胞儀進行分析。結果顯示所有人源化突變體均可與PD-1蛋白結合,與嵌合母本抗體比較, 部分突變抗體的結合能力更高,有的相對要低一些(圖5)。
人源化抗體對PD-1受體蛋白和其配體PD-L1結合的阻斷作用:用400ng、300ng、200ng、100ng和50ng不同PD-1抗體分別與100ng hPD-1mIg融合蛋白混合,設hIgG和PBS緩衝液對照組,4℃培養30分鐘,然後與PD-L1陽性表現細胞(CHO/PD-L1)作用30分鐘,用含1%小牛血清PBS緩衝液離心洗一遍後加入螢光標記的羊抗鼠IgG-,4℃培養20分鐘,洗後用流式細胞儀檢測。結果顯示所有人源化突變體抗體都能夠阻斷PD-1與PD-L1蛋白間的結合並呈劑量依賴性,部分突變體的阻斷作用高於母本抗體(圖6)。
人源化抗體親和力;利用Biacore儀器(Biacore T100,GE Healthcare Life Sciences)對母本抗體和突變體抗體進行了親和力的分析比較。將蛋白抗原hPD-1mIg偶聯在CM5晶片表面,檢測抗體作為分析物(流動相),檢測相互作用。結果顯示突變體抗體與母本抗體比較,結合速率(Ka)差異不大,解離速率(Kd)有2個突變體較嵌合抗體略慢,親和力(KD)母本抗體為9.89E-11M,具有較強的親和力,有2個突變體KD值與嵌合抗體接近(圖7)。
實施例5 人源化抗體其它生物功能特性
人源化PD-1抗體與食蟹猴的淋巴細胞PD-1蛋白交叉結合:為今後臨床前的毒理測試需要,本發明測試PD-1抗體能否結合猴子PD-1蛋白。取食蟹猴的周邊單核細胞,用淋巴細胞分離液分離出淋巴細胞懸浮於10%小牛血清RPMI 1640培養液裡,加入固著1μg/ml抗人CD3抗體的24孔培養板中或加入沒有固著抗體的24孔培養板中,培養液中加入 10ng/ml抗hIFNγ和50imL hIL2,5%CO2,37℃條件培養2天。收穫細胞用PBS洗1遍,加入PD-1抗體,4℃培養30分鐘,洗後再加入螢光標記的抗人IgG,4℃培養20分鐘,洗後用流式細胞儀檢測。結果顯示人源化PD-1抗體能夠和食蟹猴淋巴細胞PD-1蛋白結合(圖8)。
體外抗腫瘤作用:發明人根據PD-1抗腫瘤作用的機制設計了一個體外模型。首先利用從人周邊血淋巴細胞(PBMC)分離出的CD8+淋巴細胞,與經過放射線照射的轉染hB7-1的人黑色素瘤細胞(624 Mel/B7-1)共培養,產生能夠殺傷624 Mel瘤細胞的同種異體細胞毒性淋巴細胞(alloCD8+CTL)。然後將allo CTL效應細胞和624 Mel瘤細胞按一定比例混合後,加入PD-1抗體或對照蛋白培養5天,用0.5%結晶紫染色細胞後,用酵素免疫分析測讀儀讀取OD(450nm)值,根據瘤細胞存活計算殺傷活性,結果顯示部分人源化的PD-1突變體抗體體外情形下能夠增強效應細胞殺傷腫瘤細胞的功能(圖9,E:T=效應細胞(Effect cells):靶細胞(target cells))。
人源化PD-1抗體抑制人淋巴細胞PD-1的表現:腫瘤細胞表現PD-L1可誘導周邊的淋巴細胞表現PD-1而導致淋巴細胞活性受抑制而不能殺傷腫瘤細胞,發明人利用體外模型類比這種情況,檢測在加入PD-1抗體後能否抑制人淋巴細胞PD-1的表現從而恢復淋巴細胞的正常功能。發明人將轉染PD-L1的人黑色素瘤細胞(624 Mel/hPD-L1)與alloCD8+CTL按比例混合加入24孔培養板,並在培養液中分別加入10μg/ml不同PD-1抗體及對照蛋白,培養4天后,用螢光標記的PD-1和CD8抗體染色細胞後用流式細胞儀檢測淋巴細胞上PD-1的表現情況。結果顯示培養 液和hIgG對照組的CD8+淋巴細胞表現PD-1表現陽性,而加入母本抗體和突變抗體的各組CD8+淋巴細胞PD-1蛋白均為陰性(圖10),說明加入PD-1抗體後能抑制淋巴細胞表面的PD-1表現。
實施例6 人源化PD-1抗體與商業化抗體功能比較
發明人選擇了目前已獲批在臨床治療腫瘤病人的2個廠家的商業化PD-1抗體和本發明的抗體做基本特性的比較,包括Keytruda(penbrolizumab)(Merck & CO.,INC),100mg/4ml,Lot MO23694 Exp 2017JUL13;Opdivo(nivolumab)(Bristol-Myers Squibb),100mg/10ml,Lot AAK5824Exp 02.2018;Opdivo(nivolumab)(Bristol-Myers Squibb),40mg/4ml,Lot AAK2713 Exp10.2017。
6.1 抗體動力學和親和力比較
抗體動力學及親和力比較使用Biacore T200儀器(GE Healthcare Life Sciences),在GE公司技術工程師操作下,檢測了本發明所得PD-1抗體FMU100(即,Variant 3)與3個上市抗體對PD-1mFc蛋白的動力學及親和力的差異。將PD1-mFc蛋白分別偶聯在CM5晶片的4通道(33RU),本發明所得抗體和商品抗體作為分析物(流動相),檢測相互作用。經摸索後,將PD1抗體均稀釋到50nM作為最高濃度;1.5625,3.125,6.25,12.5,25,50nM進行濃度梯度進樣,使用Glycine pH1.7作為再生條件。測試結果顯示結合速率Ka差異不大,本發明所得抗體較商品抗體稍低;解離速率Kd,本發明所得抗體較商品抗體慢10倍;親和力KD,本發明所得抗體較商品抗體高4-7倍(圖11)。
6.2 體外抑制腫瘤生長作用比較
如前所述,將alloCD8+CTL效應細胞和624 Mel瘤細胞按一定比例混合後,加入本發明所得PD-1抗體和3個上市抗體或對照蛋白共培養5天,用0.5%結晶紫染色細胞後,用酵素免疫分析測讀儀讀取OD(450nm)值,根據瘤細胞存活計算殺傷活性,結果顯示本發明的PD-1抗體和3個上市抗體體外都能夠增強效應細胞殺傷腫瘤細胞的功能,本發明的抗體增強作用高於2個上市抗體(圖12,E:T=效應細胞(Effect cells):靶細胞(target cells))。
<110> 大有華夏生物醫藥集團有限公司
<120> 人程序性死亡受體PD-1的單株抗體及其片段
<130> T00022-P01
<140> 107102117
<141> 2018-01-19
<150> CN 201710046148.2
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<223> PD-1單株抗體DNA序列(對應於Ab4):重鏈序列
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<211> 690
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體DNA序列(對應於Ab4):輕鏈序列
<400> 2
<210> 3
<211> 466
<212> PRT
<213> Artificial Sequence
<220>
<223> PD-1單株抗體的胺基酸序列(對應於Ab4):重鏈序列
<400> 3
<210> 4
<211> 229
<212> PRT
<213> Artificial Sequence
<220>
<223> PD-1單株抗體的胺基酸序列(對應於Ab4):輕鏈序列
<400> 4
<210> 5
<211> 1425
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列: 單株抗體母本重鏈hIgG4-S228P
<400> 5
<210> 6
<211> 1425
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列:人源化重鏈變異體1
<400> 6
<210> 7
<211> 1425
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列:人源化重鏈變異體2
<400> 7
<210> 8
<211> 1425
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列:人源化重鏈變異體3
<400> 8
<210> 9
<211> 717
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列:單株抗體母本kappa輕鏈
<400> 9
<210> 10
<211> 717
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列:人源化輕鏈變異體1
<400> 10
<210> 11
<211> 717
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列:人源化輕鏈變異體2
<400> 11
<210> 12
<211> 717
<212> DNA
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體DNA序列:人源化輕鏈變異體3
<400> 12
<210> 13
<211> 474
<212> PRT
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體胺基酸序列:單株抗體母本重鏈hIgG4-S228P
<400> 13
<210> 14
<211> 474
<212> PRT
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體胺基酸序列:人源化重鏈變異體1
<400> 14
<210> 15
<211> 474
<212> PRT
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體胺基酸序列:人源化重鏈變異體2
<400> 15
<210> 16
<211> 474
<212> PRT
<213> Artificial Sequence
<220>
<223> PD-1單株抗體母本和人源化變異體胺基酸序列:人源化重鏈變異體3
<400> 16
<210> 17
<211> 238
<212> PRT
<213> Artificial Sequence
<220>
<223> 單株抗體母本kappa輕鏈
<400> 17
<210> 18
<211> 238
<212> PRT
<213> Artificial Sequence
<220>
<223> 人源化輕鏈變異體1
<400> 18
<210> 19
<211> 238
<212> PRT
<213> Artificial Sequence
<220>
<223> 人源化輕鏈變異體2
<400> 19
<210> 20
<211> 238
<212> PRT
<213> Artificial Sequence
<220>
<223> 人源化輕鏈變異體3
<400> 20
Claims (21)
- 一種針對人PD-1的分離的單株抗體或其抗原結合片段,包含:表2中所列的重鏈CDR1、CDR2及CDR3,以及表2中所列的輕鏈CDR1、CDR2及CDR3。
- 如申請專利範圍第1項所記載之單株抗體或其抗原結合片段,其中,其係包含:(i)序列表SEQ ID NO:3所示的重鏈胺基酸序列,及序列表SEQ ID NO:4所示的輕鏈胺基酸序列;或(ii)不包含信號肽部分的序列表SEQ ID NO:3所示的重鏈胺基酸序列,及不包含信號肽部分的序列表SEQ ID NO:4所示的輕鏈胺基酸序列。
- 如申請專利範圍第1項所記載之單株抗體或其抗原結合片段,其中,前述單株抗體或其抗原結合片段係被人源化,並且包含:(1)選自序列表SEQ ID NO:13、14、15及16的重鏈胺基酸序列,或不包含信號肽部分的序列表SEQ ID NO:13、14、15及16的重鏈胺基酸序列;以及(2)選自序列表SEQ ID NO:17、18、19及20的輕鏈胺基酸序列,或不包含信號肽部分的序列表SEQ ID NO:17、18、19及20的輕鏈胺基酸序列。
- 如申請專利範圍第1項所記載之單株抗體或其抗原結合片段,其中,其係包含:序列表SEQ ID NO:13所示的重鏈胺基酸序列,及序列表SEQ ID NO:17所示的輕鏈胺基酸序列。
- 如申請專利範圍第1至4項中任一項所記載之單株抗體或其抗原結合片 段,其中,前述抗原結合片段選自:Fab、Fab '、Fab '-SH、Fv、scFv、F(ab ') 2及雙抗體。
- 一多核甘酸,其係編碼申請專利範圍第1至5項中任一項之抗體或其抗原結合片段的分離的多核苷酸。
- 如申請專利範圍第6項所記載之分離的多核苷酸,其中,重鏈編碼序列如序列表SEQ ID NO:1所示,輕鏈編碼序列如序列表SEQ ID NO:2所示。
- 如申請專利範圍第6項所記載之分離的多核苷酸,該多核苷酸係編碼人源化抗體或其抗原結合片段,且其中:重鏈編碼序列選自序列表SEQ ID NO:5、6、7及8,輕鏈編碼序列選自序列表SEQ ID NO:9、10、11及12。
- 包含如申請專利範圍第6-8中任一項的分離的多核苷酸的表現載體。
- 一種宿主細胞,其特徵係包含如申請專利範圍第9的表現載體。
- 一種藥物組合物,其特徵係包含申請專利範圍第1至5項中任一項的抗體或其抗原結合片段以及可藥用載體。
- 一種提高免疫細胞活性的方法,其特徵係包括使前述免疫細胞與申請專利範圍第1至5項中任一項之抗體或其抗原結合片段接觸。
- 一種治療疾病的方法,其特徵係包括將治療有效量的申請專利範圍第1至5項中任一項的抗體或其抗原結合片段給予需要治療的受試者。
- 如申請專利範圍第13項所記載之方法,其中,前述疾病選自:癌症、感染性疾病、移植排斥、自身免疫性疾病及神經疾病。
- 如申請專利範圍第13或14項所記載之方法,其中,進一步包括對前 述受試者聯合應用其他藥物或療法。
- 如申請專利範圍第15項所記載之方法,其中,前述其他療法選自:化療、放射治療、靶向治療、基因治療、細胞治療、幹細胞治療。
- 一種申請專利範圍第1至5項中任一項之抗體或其抗原結合片段在製備用於治療疾病的藥物中的用途,其中,前述疾病選自:癌症、感染性疾病、移植排斥、自身免疫性疾病及神經疾病。
- 如申請專利範圍第17項所記載之應用,其中,前述藥物係與其他藥物或療法進行聯合應用。
- 如申請專利範圍第18項所記載之應用,其中,前述其他療法選自:化療、放射治療、靶向治療、基因治療、細胞治療、幹細胞治療。
- 一種調節細胞功能的方法,其特徵係包括將編碼申請專利範圍第1至5項中任一項之抗體或其抗原結合片段的多核苷酸序列遞送至前述細胞中。
- 如申請專利範圍第20項所記載之方法,其中,前述方法用於基因治療、CAR-T治療或CRISPR治療。
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KR20180053752A (ko) | 2015-10-02 | 2018-05-23 | 심포젠 에이/에스 | 항-pd-1 항체 및 조성물 |
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CN105566496B (zh) * | 2015-11-26 | 2019-04-02 | 大庆东竺明生物技术有限公司 | 阻断人程序性死亡因子1(pd-1)功能的单克隆抗体及其编码基因和应用 |
CN106008714B (zh) | 2016-05-24 | 2019-03-15 | 瑞阳(苏州)生物科技有限公司 | 抗人pd-1人源化单克隆抗体及其应用 |
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RU2656181C1 (ru) | 2016-07-13 | 2018-05-31 | Закрытое Акционерное Общество "Биокад" | Анти-pd-1-антитела, способ их получения и способ применения |
CN109789205B (zh) | 2016-09-14 | 2022-12-13 | 艾伯维生物制药股份有限公司 | 抗pd-1(cd279)抗体 |
CN108203464B (zh) | 2016-12-25 | 2021-07-20 | 南京金斯瑞生物科技有限公司 | 高亲和力、高特异性、多抗原识别表位的具有更高功能性的抗人pd-1抗体 |
CN110382536B (zh) | 2017-01-20 | 2023-12-19 | 大有华夏生物医药集团有限公司 | 抗pd-1抗体及其用途 |
JP7519985B2 (ja) | 2018-07-19 | 2024-07-22 | タユー ファシャ バイオテック メディカル グループ カンパニー, リミテッド | 抗pd-1抗体、投薬量、およびその使用 |
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2018
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TWI771361B (zh) | 2022-07-21 |
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CN110382536A (zh) | 2019-10-25 |
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KR102365972B1 (ko) | 2022-02-21 |
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CA3049047A1 (en) | 2018-07-26 |
CN110291109B (zh) | 2023-01-31 |
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US20240182579A1 (en) | 2024-06-06 |
KR20190085553A (ko) | 2019-07-18 |
EP3571231A1 (en) | 2019-11-27 |
AU2018209556B2 (en) | 2020-06-25 |
AU2018209556A1 (en) | 2019-06-20 |
EP3571231A4 (en) | 2020-12-02 |
JP7275030B2 (ja) | 2023-05-17 |
BR112019013238A2 (pt) | 2020-02-11 |
WO2018133837A1 (en) | 2018-07-26 |
US11560430B2 (en) | 2023-01-24 |
CN110382536B (zh) | 2023-12-19 |
KR20220025938A (ko) | 2022-03-03 |
KR102536145B1 (ko) | 2023-05-30 |
JP2020505386A (ja) | 2020-02-20 |
WO2018133837A9 (en) | 2019-02-21 |
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