TW202221045A - 抗tigit抗體或其抗原結合片段及其生產方法與應用,以及包含其的核酸、載體、細胞、藥物組合物及試劑盒 - Google Patents
抗tigit抗體或其抗原結合片段及其生產方法與應用,以及包含其的核酸、載體、細胞、藥物組合物及試劑盒 Download PDFInfo
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Abstract
本發明涉及生物醫藥領域,具體而言,是關於一種抗TIGIT抗體或其抗原結合片段及其生產方法與應用,以及包含其的核酸、載體、細胞、藥物組合物及試劑盒。該抗體或其抗原結合片段與TIGIT具有較高的親和力,且具有多方面的功能特性,可單獨或與其它試劑組合用於治療癌症、免疫及炎症相關疾病。
Description
本發明涉及生物醫藥領域,具體而言,是關於一種抗TIGIT抗體或其抗原結合片段。
TIGIT(T cell Ig and ITIM domain,也稱為WUCAM,Vstm3,VSIG9)是含ITT結構域(免疫球蛋白酪氨酸尾部基序結構域)及ITIM結構域(免疫受體酪氨酸抑制基序結構域)的T細胞和NK細胞共有的抑制性受體,屬於I型跨膜蛋白,包括IgV胞外段以及免疫球蛋白酪氨酸尾巴樣磷酸化片段。
該基因在2008年由Genentech公司的研究組發現。該研究組通過在基因組中搜索符合特定條件(①表達在免疫細胞上;②屬於I型跨膜蛋白;③胞外帶有免疫球蛋白結構域;④胞內帶有免疫調節結構域)的基因而尋找到這個基因。通過序列比對,發現這個蛋白隸屬於一個較大的PVR蛋白家族,其成員包括與TIGIT競爭配體的活化型受體CD226和CD96,以及它們的配體PVR,等等。2012年,TIGIT分子的晶體結構通過X射線衍射得到解析,研究發現免疫細胞上表達的TIGIT首先形成同一細胞上的順式同源二聚體,然後該二聚體各通過一側的一個TIGIT分子結合一個PVR分子,並且發現預先形成的同源二聚體對於TIGIT-PVR相互作用是必須的,因為如果預先將TIGIT-TIGIT結合界面的氨基酸進行突變,則TIGIT-PVR的相互作用會被破壞。
TIGIT的主要配體為CD155(Necl-5,PVR脊髓灰質炎病毒受體)以及CD112(PVRL2,Nectin-2)。其中又以與CD155的結合親和力最高(3.15nM,Kd),兩個TIGIT分子與兩個CD155分子形成四聚體「鎖鑰結構」(TIGIT的Try113與CD155的AX6G,以及CD155的Phe128和TIGIT的AX6G分別形成「鑰-鎖」)。這兩個配體同時也是CD226(DNAM-1)的配體,CD226與TIGIT競爭,刺激T細胞活性。配體與TIGIT之間的相互作用打敗了CD226,使免疫活性受到抑制,腫瘤細胞上調CD155和CD122以逃避免疫介導的破壞作用。因此,對TIGIT具有特異性的拮抗性抗體可抑制CD155和CD112誘導的T細胞反應的抑制且增強抗腫瘤免疫。
本發明的目標是獲得一種能夠特異性識別TIGIT的抗體或其抗原結合片段,其輕鏈CDR1、CDR2、CDR3選自SEQ ID NO:1~3、SEQ ID NO:4~6、SEQ ID NO:7~9、SEQ ID NO:10~12、SEQ ID NO:13~15、SEQ ID NO:16~18、SEQ ID NO:19~21組合中的至少一種;
其重鏈CDR1、CDR2、CDR3選自SEQ ID NO:22~24、SEQ ID NO:25~27、SEQ ID NO:28~30、SEQ ID NO:31~33、SEQ ID NO:34~36、SEQ ID NO:37~39、SEQ ID NO:40~42中的至少一種。
本發明還提供所述抗體或其抗原結合片段相關的核酸、載體、細胞。
本發明還涉及一種生產如上所述的抗體或其抗原結合片段的方法,包括:
在培養基中培養如上所述的細胞;以及
從培養基中或從所培養的細胞中回收如此產生的抗體。
根據本發明的再一方面,還涉及如上所述的抗體或其抗原結合片段在製備用於治療或預防感染性疾病、免疫性疾病或腫瘤的藥物中的應用。
本發明還提供試劑盒,其包含下述成分中的至少一種:
i)如上所述的抗體或其抗原結合片段,以及任選的用於承裝所述抗體或其抗原結合片段的容器;
ii)如上所述的藥物組合物,以及任選的用於承裝所述藥物組合物的容器。
上述抗體與TIGIT具有較高的親和力,且具有多方面的功能特性,可單獨或與其它試劑組合用於治療癌症、免疫及炎症相關疾病。
現將詳細地提供本發明實施方式的參考,其一個或多個實例描述於下文。提供每一實例作為解釋而非限製本發明。實際上,對本領域技術人員而言,顯而易見的是,可以對本發明進行多種修改和變化而不背離本發明的範圍或精神。例如,作為一個實施方式的部分而說明或描述的特徵可以用於另一實施方式中,來產生更進一步的實施方式。
因此,旨在本發明覆蓋落入所附權利要求的範圍及其等同範圍中的此類修改和變化。本發明的其它對象、特徵和方面公開於以下詳細描述中或從中是顯而易見的。本領域普通技術人員應理解本討論僅是示例性實施方式的描述,而非意在限製本發明更廣闊的方面。
本發明涉及一種抗體,其結合人TIGIT的抗體或其抗原結合片段,其重鏈CDR1、CDR2、CDR3選自SEQ ID NO:1~3、SEQ ID NO:4~6、SEQ ID NO:7~9、SEQ ID NO:10~12、SEQ ID NO:13~15、SEQ ID NO:16~18、SEQ ID NO:19~21組合中的至少一種;
其輕鏈CDR1、CDR2、CDR3選自SEQ ID NO:22~24、SEQ ID NO:25~27、SEQ ID NO:28~30、SEQ ID NO:31~33、SEQ ID NO:34~36、SEQ ID NO:37~39、SEQ ID NO:40~42中的至少一種。
在一些實施方式中,所述抗體或其抗原結合片段具有選自如下CDR序列組合中的任一種:
。
H-CDR1 | H-CDR2 | H-CDR3 | L-CDR1 | L-CDR2 | L-CDR3 | |
Tigit-5 | SEQ ID NO:1 | SEQ ID NO:2 | SEQ ID NO:3 | SEQ ID NO:22 | SEQ ID NO:23 | SEQ ID NO:24 |
3Tigit-16 | SEQ ID NO:4 | SEQ ID NO:5 | SEQ ID NO:6 | SEQ ID NO:25 | SEQ ID NO:26 | SEQ ID NO:27 |
3Tigit-24 | SEQ ID NO:7 | SEQ ID NO:8 | SEQ ID NO:9 | SEQ ID NO:28 | SEQ ID NO:29 | SEQ ID NO:30 |
3Tigit-29 | SEQ ID NO:10 | SEQ ID NO:11 | SEQ ID NO:12 | SEQ ID NO:31 | SEQ ID NO:32 | SEQ ID NO:33 |
14Tigit-1-1 | SEQ ID NO:13 | SEQ ID NO:14 | SEQ ID NO:15 | SEQ ID NO:34 | SEQ ID NO:35 | SEQ ID NO:36 |
14Tigit-3-2 | SEQ ID NO:16 | SEQ ID NO:17 | SEQ ID NO:18 | SEQ ID NO:37 | SEQ ID NO:38 | SEQ ID NO:39 |
3Tigit-12 | SEQ ID NO:19 | SEQ ID NO:20 | SEQ ID NO:21 | SEQ ID NO:40 | SEQ ID NO:41 | SEQ ID NO:42 |
該抗體或其抗原結合片段的一個重要優點在於其與TIGIT具有較高的親和力。
該抗體或其抗原結合片段的一個重要優點在於其具有阻斷CD112與TIGIT結合的活性;
該抗體或其抗原結合片段的一個重要優點在於其具有阻斷PVR與TIGIT結合的活性;
該抗體或其抗原結合片段的一個重要優點在於其具有活化NK92-TIGIT殺傷能力的活性;
該抗體或其抗原結合片段的一個重要優點在於其與TIGIT的結合表位是新的。
因具有上述特性,該抗體或其抗原結合片段優選可作為抗體藥物使用。
根據本發明的抗TIGIT抗體或其抗原結合片段能夠通過TIGIT / CD155相互作用的信號傳遞以抵消癌細胞對免疫細胞的抑制信號,誘導免疫應答的再激活以有效攻擊癌細胞,從而提供抗癌作用。最終,抗TIGIT抗體或其抗原結合片段可用於靶向TIGIT(一種腫瘤免疫抑製劑)的免疫抗癌療法。特別地,根據本發明的抗TIGIT抗體或其抗原結合片段降低或抑制患有癌症的受試者中TIGIT的表達或活性,並誘導T細胞或NK細胞的持續抗癌反應,從而提供治療癌症的效果。
在本發明中,「抗體或其抗原結合片段」此技術術語是結合特定抗原的蛋白,其泛指包含互補決定區(CDR區)的一切蛋白及蛋白片段。「抗體」特別指全長抗體。「全長抗體」此用語包括多克隆抗體及單克隆抗體,術語「抗原結合片段」是包含抗體CDR的一部分或全部的物質,其缺乏至少一些存在於全長鏈中的氨基酸但仍能夠特異性結合至抗原。此類片段俱生物活性,因為其結合至靶抗原,且可與其他抗原結合分子(包括完整抗體)競爭結合至給定表位。在一些實施方式中,抗原結合片段具有特異性識別並結合TIGIT的作用。在一些實施方式中,抗原結合片段是具有阻斷CD112與TIGIT結合,和/或阻斷PVR與TIGIT結合,和/或具有活化NK92-TIGIT殺傷能力功能的片段。在一個方面中,此類片段將包含單個重鍊和單個輕鏈,或其部分。所述片段可通過重組核酸技術產生,或可通過抗原結合分子(包括完整抗體)的酶裂解或化學裂解產生。
術語「互補性決定區」或「CDR」是指免疫球蛋白的重鍊和輕 鏈的高度可變區。有三種重鏈CDR和三種輕鏈CDR。此處,取決於情況,術語「CDR」和「CDRs」用於指包含一種或多種或者甚至全部的對抗體或其抗原結合片段與其識別的抗原或表位的結合親和力起作用的主要氨基酸殘基的區域。在另一具體實施方式中,CDR區或CDR是指IMGT定義的免疫球蛋白的重鍊和輕鏈的高度可變區。
在本發明中,重鏈的互補決定區用HCDR表示,輕鏈的互補決定區用LCDR表示。本領域常用的CDR標示方法包括: Kabat編號方案、Chothia和Lesk編號方案以及1997年Lefranc等人為免疫球蛋白超家族的所有蛋白質序列引入的新的標準化編號系統。 Kabat等人是第一個為免疫球蛋白可變區提出標準化編號方案的人。在他們的「免疫學蛋白質序列」(Sequences of Proteins of Immunological Interest)的彙編中,輕鏈(λ、κ)可變區和抗體重鏈的氨基酸序列,以及T細胞受體的可變區(α、β、γ、δ)對齊並編號。在過去的幾十年中,序列的積累導致了KABATMAN數據庫的創建,Kabat編號方案通常被認為是編號抗體殘基廣泛採用的標準。本發明採用Kabat註釋標準標示CDR區,但其他方法標示的CDR區也屬於本發明的保護範圍。
術語「特異性識別」、「選擇性結合」、「選擇性地結合」和「特異性地結合」或其類似表述是指抗體或其抗原結合片段對預先確定的抗原上的表位的結合。通常,抗體或其抗原結合片段以大約小於10
-6M,例如大約小於10
-7M、10
-8M、10
-9M或10
-10M或更小的親和力(KD)結合。
在本發明中,所提供的抗體或其抗原結合片段特異性識別的對象可以為多種屬來源的TIGIT,例如人、小鼠、猴(如食蟹猴(cynomolgus monkey))。
抗體或其抗原結合片段的變體也在本發明範圍內,例如各自與本發明所述的各個CDR或FR、或可變區VL和/或VH、或抗體全長的氨基酸或核苷酸序列具有至少70%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或高於99%同一性的序列。在一些情況下,抗體或其抗原結合片段的變體至少包括上述6個CDR;在一些情況下,抗體或其抗原結合片段的變體至少包括一個重鍊和一個輕鏈,而在其他情況下,變體形式含有兩個相同的輕鍊和兩個相同的重鏈(或其子部分)。在一些情況下,抗體或其抗原結合片段的變體是在本發明所提供的抗體或其抗原結合片段序列上發生保守修飾或保守置換或取代所得到的。「保守修飾」或「保守置換或取代」是指具有類似特徵(例如電荷、側鏈大小、疏水性/親水性、主鏈構象和剛性等)的其它氨基酸置換蛋白中的氨基酸,使得可頻繁進行改變而不改變蛋白的生物學活性。本領域技術人員知曉,一般而言,多肽的非必需區域中的單個氨基酸置換基本上不改變生物學活性(參見例如華生(Watson)等(1987)基因的分子生物學(Molecular Biology of the Gene),班本傑明/卡明斯出版社(The Benjamin/Cummings Pub. Co.,),第224頁,(第4版))。另外,結構或功能類似的氨基酸的置換不大可能破環生物學活性。在一些情況下,變體保留阻斷TIGIT與其配體CD112或PVR結合的能力。所屬領域技術人員將能夠使用熟知的技術確定如本文所闡明的抗原結合分子的合適變體。在某些實施方案中,所屬領域技術人員可鑑別分子的可通過靶據信對於活性而言不重要的區來改變而不破壞活性的合適區域。對於核苷酸和氨基酸序列,術語「同一性」表明當具有適當的插入或缺失的情況下最佳比對和比較時兩個核酸或兩個氨基酸序列之間的同一性程度。
在一些實施方式中,所述抗體或其抗原結合片段重鏈FR1、FR2、FR3、FR4選自SEQ ID NO:43~46、SEQ ID NO:47~50、SEQ ID NO:51~54、SEQ ID NO:55~58、SEQ ID NO:59~62、SEQ ID NO:63~66、SEQ ID NO:67~70組合中的至少一種;
其輕鏈FR1、FR2、FR3、FR4選自SEQ ID NO:71~74、SEQ ID NO:75~78、SEQ ID NO:79~82、SEQ ID NO:83~86、SEQ ID NO:87~90、SEQ ID NO:91~94、SEQ ID NO:95~98中的至少一種;
可選地,所述抗體或其抗原結合片段還包含選自如下骨架區序列組合中的至少一種:
。
H-FR1 | H-FR2 | H-FR3 | H-FR4 | L-FR1 | L-FR2 | L-FR3 | L-FR4 | |
Tigit-5 | SEQ ID NO:43 | SEQ ID NO:44 | SEQ ID NO:45 | SEQ ID NO:46 | SEQ ID NO:71 | SEQ ID NO:72 | SEQ ID NO:73 | SEQ ID NO:74 |
3Tigit-16 | SEQ ID NO:47 | SEQ ID NO:48 | SEQ ID NO:49 | SEQ ID NO:50 | SEQ ID NO:75 | SEQ ID NO:76 | SEQ ID NO:77 | SEQ ID NO:78 |
3Tigit-24 | SEQ ID NO:51 | SEQ ID NO:52 | SEQ ID NO:53 | SEQ ID NO:54 | SEQ ID NO:79 | SEQ ID NO:80 | SEQ ID NO:81 | SEQ ID NO:82 |
3Tigit-29 | SEQ ID NO:55 | SEQ ID NO:56 | SEQ ID NO:57 | SEQ ID NO:58 | SEQ ID NO:83 | SEQ ID NO:84 | SEQ ID NO:85 | SEQ ID NO:86 |
14Tigit-1-1 | SEQ ID NO:59 | SEQ ID NO:60 | SEQ ID NO:61 | SEQ ID NO:62 | SEQ ID NO:87 | SEQ ID NO:88 | SEQ ID NO:89 | SEQ ID NO:90 |
14Tigit-3-2 | SEQ ID NO:63 | SEQ ID NO:64 | SEQ ID NO:65 | SEQ ID NO:66 | SEQ ID NO:91 | SEQ ID NO:92 | SEQ ID NO:93 | SEQ ID NO:94 |
3Tigit-12 | SEQ ID NO:67 | SEQ ID NO:68 | SEQ ID NO:69 | SEQ ID NO:70 | SEQ ID NO:95 | SEQ ID NO:96 | SEQ ID NO:97 | SEQ ID NO:98 |
重鍊和輕鏈的可變區按照FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的方式組合得到。
在優選的實施方式中,重鍊和輕鏈的可變區按照上述表格中Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2、3Tigit-12行中的內容將CDR和FR一一對應進行組合。
在一些實施方式中,所述抗原結合片段為Fab、F(ab’)
2、Fd、Fv、scFv、雙特異抗體和抗體最小識別單位中的任一種,優選F(ab’)
2、Fab、scFv以及雙特異抗體中的一種。
在一些實施方式中,所述抗體具有恆定區,重鏈恆定區序列選自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任意一種的恆定區序列;輕鏈恆定區為κ或λ鏈。
在一些實施方式中,所述恆定區的種屬來源選自牛、馬、豬、綿羊、山羊、大鼠、小鼠、狗、貓、兔、駱駝、驢、鹿、貂、雞、鴨、鵝或人。
本發明還涉及核酸,其編碼如上所述的抗體或其抗原結合片段。
核酸通常是RNA或DNA,核酸分子可以是單鍊或雙鏈的,但優選是雙鏈DNA。當將核酸與另一個核酸序列置於功能關係中時,核酸是「有效連接的」。例如,如果啟動子或增強子影響編碼序列的轉錄,那麼啟動子或增強子有效地連接至所述編碼序列。當其連入載體時優選採用DNA核酸。
此外,鑑於抗體為膜蛋白,所以核酸通常帶有信號肽序列。
本發明還涉及載體,其包含如上所述的的核酸。
術語「載體(vector)」是指,可將多聚核苷酸插入其中的一種核酸運載工具。當載體能使插入的多核苷酸編碼的蛋白獲得表達時,載體稱為表達載體。載體可以通過轉化,轉導或者轉染導入宿主細胞,使其攜帶的遺傳物質元件在宿主細胞中獲得表達。載體是本領域技術人員公知的,包括但不限於:質粒;噬菌粒;柯斯質粒;人工染色體,例如酵母人工染色體(YAC)、細菌人工染色體(BAC)或P1來源的人工染色體(PAC);噬菌體如λ噬菌體或M13噬菌體及動物病毒等。可用作載體的動物病毒包括但不限於,逆轉錄酶病毒(包括慢病毒)、腺病毒、腺相關病毒、皰疹病毒(如單純皰疹病毒)、痘病毒、桿狀病毒、乳頭瘤病毒、乳頭多瘤空泡病毒(如SV40)。在一些實施方式中,本發明所述載體中包含基因工程中常用的調控元件,例如增強子、啟動子、內部核醣體進入位點(IRES)和其他表達控制元件(例如轉錄終止信號,或者多腺苷酸化信號和多聚U序列等)。
本發明還提供細胞,其包含如上所述的核酸或如上所述的載體。
本文使用的表述「細胞」、「細胞系」和「細胞培養物」可互換使用,並且所有這類名稱都包括後代。因此,單詞「轉化體」和「轉化細胞」包括原代受試細胞和由其衍生的培養物,而不考慮轉移數目。還應當理解的是,由於故意或非有意的突變,所有後代在DNA含量方面不可能精確相同。包括具有與最初轉化細胞中篩選的相同的功能或生物學活性的突變後代。在意指不同名稱的情況下,其由上下文清楚可見。
適用於表達本發明的抗原結合蛋白的宿主細胞或細胞系包括:哺乳動物細胞諸如NS0、Sp2/0、CHO、COS、HEK、成纖維細胞和骨髓瘤細胞。可以使用人細胞,因而允許分子用人糖基化模式來修飾。或者,可以採用其他真核細胞系。合適的哺乳動物宿主細胞的選擇,以及用於轉化、培養、擴增、篩选和產物產生和純化的方法,是本領域已知的。
可以證明,細菌細胞可用作宿主細胞,其適合表達本發明的重組Fab或其他實施方案。但是,由於在細菌細胞中表達的蛋白傾向於未折疊的形式或不正確地折疊的形式或非糖基化形式,必須篩選在細菌細胞中產生的任何重組Fab,以保留抗原結合能力。如果細菌細胞表達的分子以適當地折疊的形式產生,該細菌細胞將是期望的宿主,或者,在可替代的實施方案中,可以在細菌宿主中表達分子,然後隨後進行重新折疊。例如,用於表達的各種大腸桿菌菌株,是生物技術領域中眾所周知的宿主細胞。枯草芽孢桿菌、鏈黴菌屬、其他芽孢桿菌屬等的各種菌株,也可以用於該方法中。
如果需要,本領域技術人員已知的酵母細胞菌株以及昆蟲細胞,例如果蠅和鱗翅目昆蟲和病毒表達系統,也可用作宿主細胞。
根據本發明的再一方面,還涉及一種生產如上所述的抗體或其抗原結合片段的方法,包括:
在合適的培養條件下培養如上所述的細胞;以及
從培養基中或從所培養的細胞中回收如此產生的抗體或其抗原結合片段。
常見的生產抗體或其抗原結合片段的方法包括體外培養所述的細胞例如雜交瘤,並從上清中收集抗體或其抗原結合片段。
或者將所述細胞移入免疫缺陷動物(例如裸鼠)中,再從動物的血清等位置收集純化抗體或其抗原結合片段。
本發明還提供藥物組合物,其包括如上所述的抗體或其抗原結合片段,以及藥學上可接受的賦形劑、稀釋劑或載體中的一種或多種。
術語「藥學上可接受的賦形劑、稀釋劑或載體」是指在藥理學和/或生理學上與受試者和活性成分相容的賦形劑、稀釋劑或載體。
根據本發明的再一方面,本發明還涉及如上所述的抗體或其抗原結合片段在製備用於治療或預防感染性疾病、免疫性疾病或腫瘤的藥物中的應用。
本發明還涉及試劑盒,其包含下述成分中的至少一種:
i)如上所述的抗體或其抗原結合片段,以及任選的用於承裝所述抗體或其抗原結合片段的容器;
ii)如上所述的藥物組合物,以及任選的用於承裝所述藥物組合物的容器。
另外,本發明還涉及治療受試者的疾病的方法,其包括向所述受試者施用有效量的如上所述的抗體或其抗原結合片段,其任選地與另一治療劑或治療程序聯合;
所述疾病選自感染性疾病、免疫性疾病或腫瘤。
在一些實施方式中,所述受試者為動物。
在一些實施方式中,所述受試者為哺乳動物。
在一些實施方式中,所述受試者為靈長類動物。
在一些實施方式中,所述受試者為人。
術語「任選地」僅用於描述目的,而不能理解為指示或暗示相對重要性。由此,限定有「任選地」的特徵可以明示或者隱含地包括或不包括該特徵。
下面將結合實施例對本發明的實施方案進行詳細描述。
實施例 1 :抗人 TIGIT 抗體生成及製備
基於常規雜交瘤技術產生抗人TIGIT單克隆抗體,具體流程如下:
1. 抗原製備:
使用內部製備或商業化採購(AcroBiosystems, Catalog# TIT-H5253)的可溶人TIGIT融合蛋白作為抗原進行免疫。
內部製備抗原選取人TIGIT胞外段,在其C端融合小鼠mIgG2a-Fc片段,採用標準的重組蛋白表達技術在HEK293細胞中進行瞬轉表達,收集所述細胞分泌表達上清,使用蛋白A親和層析柱純化獲得目標蛋白,除菌過濾後分裝至-80°C凍存備用。
2. 小鼠免疫:
為了產生抗人TIGIT單克隆抗體,取6-10週齡的Balb/c母鼠用前面所述人TIGIT-mFc蛋白進行免疫。人TIGIT-mFc蛋白通過腹腔或皮下注入免疫小鼠,使用劑量:25µg/100µL,每週1針一般共免疫6針;或50µg/100µL,每兩週1針一般共免疫5針,具體的免疫針數與每支小鼠的實際免疫效果有關,一般免疫效果好的小鼠提前進行後續步驟,免疫效果不好的小鼠則繼續進行免疫。免疫效果通過監測小鼠血清效價判斷,一般在免疫3針後,通過ELISA方法檢測小鼠血清對抗原的結合能力(即效價)。選取最高效價的小鼠進行加強免疫,使用50µg抗原通過脾臟注入,加強免疫3天后準備融合。
3. 雜交瘤融合:
使用常規技術取小鼠脾臟並分離得到小鼠脾細胞,將小鼠瘤細胞SP2/0與免疫脾細胞按照1:10細胞數量比例混合併轉移至50ml離心管中,用RPMI1640基礎培養基洗滌一次。棄上清,將細胞混勻,緩慢加入1ml 50% PEG1500融合。融合1min後,加入15ml RPMI1640基礎培養基終止細胞融合。1000rpm離心5min,棄上清。用50ml RPMI1640篩選培養基輕輕混懸,平分於96孔板中,共10塊,50µl/孔,37°C、5% CO2細胞培養箱靜置培養。培養至第六天,更換HT培養基(含HT的RPMI1640完全培養基)兩次。
融合檢測:用0.05M碳酸緩衝液稀釋攜帶人Fc標籤的人TIGIT重組蛋白(內部製備)至終濃度為2µg/ml,按100µl/孔加入96孔ELISA檢測板中,37°C孵育2h或2-4°C包被過夜。棄上清,使用洗板液(1×PBS)清洗5次後,按200µl/孔加入封閉液(1×PBS+1%BSA),37°C封閉1h。重組融合後第7天,按100µl/孔加入細胞上清,37°C靜置孵育30min。用1×PBS洗滌3次。按100µl/孔加入HRP標記的羊抗鼠IgG(西格瑪(Sigma)A0168-1ML),37°C靜置孵育30min,用1×PBS洗滌3次後,進行顯色反應後,於酶標儀採用OD450/OD630nm讀取數據。挑選OD450nm-OD630nm ≥ 1.0的融合子,採用有限稀釋法進行克隆化,至少2次。
陽性亞克隆檢測:採用用0.05M碳酸緩衝液稀釋攜帶人Fc標籤的人TIGIT重組蛋白(內部製備)至終濃度為2µg/ml,按100µl/孔加入96孔ELISA檢測板中,37°C孵育2h或2-4°C包被過夜。棄上清,使用洗板液(1×PBS)清洗5次後,按200µl/孔加入封閉液(1×PBS+1%BSA),37°C封閉1h。按100µl/孔加入亞克隆上清液,37°C靜置孵育30min。用1×PBS洗滌3次。按100µl/孔加入HRP標記的羊抗鼠IgG(西格瑪A0168-1ML),37°C靜置孵育30min,用1×PBS洗滌3次後,進行顯色反應後,於酶標儀採用OD450/OD630nm讀取數據。所得的陽性亞克隆經體外培養進行保種和表達。
4. 鼠單抗表達及純化:
待產生抗人TIGIT抗體的克隆生長至匯合率至80%時,將細胞吹落,1000rpm離心5min後,用30ml含5%FBS的SFM完全培養基重懸細胞,並轉移至150ml SF搖瓶中,37°C,8%CO
2,120rpm培養2-3天。當細胞密度達到5×10
5/ml時,1000rpm離心5min,用50ml無血清的SFM基礎培養基重懸細胞,並轉移至250ml SF搖瓶中,37°C,8%CO
2,120rpm培養4-5天,9000rpm離心20min,收集上清進行純化。純化採用ProA親和層析。過程如下,使用AKTA avant 150層析設備,用至少5CV平衡緩衝液(10mM PBS)平衡層析柱(如MabSelectSuRe LX,GE),加載樣品至層析柱,使目標蛋白吸附在層析柱上而其他雜質穿透分離。完成上樣後使用至少5CV平衡緩衝液(10mM PBS)再次沖洗層析柱,隨後使用洗脫緩衝液(20mM NaAc,pH=3.4)洗脫目標蛋白,收集管中預先加入中和緩衝液(1M Tris,pH8.0),中和緩衝液的加入體積根據洗脫樣品的預估含量而定,一般加入10%洗脫體積量。
樣品使用Biotek - Epoch - Take-3進行濃度測定,利用A280方法檢測抗體濃度,即以消光係數(E.C.)=1.37,光程=0.05mm(Take-3板不同孔光程有細微差異,會自動校正),通過設備檢測樣品吸光值,按照蘭伯特-比爾定律(Lambert-Beer law)計算待測抗體的濃度。樣品濃度過低則需要進行超濾濃縮,使用超濾濃縮管(Amicon®超-15離心過濾裝置(Ultra-15 Centrifugal Filter Devices),30kD)按照說明書提供的通用操作方法,將樣品濃度濃縮至> 0.5mg/ml;收集濃縮端樣品,0.22um無菌針頭濾器除菌過濾(科百特,PES,0.22um,直徑13mm)後分裝凍存備用;
5. 鼠單抗亞型鑑定:
用0.05M pH9.5碳酸緩衝溶液稀釋攜帶人Fc標籤重組人TIGIT重組蛋白(內部製備),使其終濃度為2µg/mL,按100µl/孔加入96孔ELISA檢測板中,37°C孵育2h或2-4°C包被過夜。棄上清,使用洗板液(1×PBS)清洗5次後,按200µl/孔加入封閉液(1×PBS+1%BSA),37°C封閉1h。用含1%BSA的1×PBS稀釋抗體至1µg/ml,按100µl/孔加入上述獲得抗人TIGIT鼠單抗,37°C孵育30分鐘。用1×PBS洗滌3次。按100µl/孔加入HRP標記的羊抗鼠IgG(西格瑪A0168-1ML)、HRP標記的羊抗鼠IgG2a(賽默飛世爾(Thermo Fisher)M32207)、HRP標記的羊抗鼠IgG1(賽默飛世爾PA1-74421)、HRP標記的羊抗鼠IgG2b(賽默飛世爾M32407)、HRP標記的羊抗鼠IgG3(賽默飛世爾M32607)、HRP標記的羊抗鼠IgM(賽默飛世爾31440),37°C靜置孵育30min,用1×PBS洗滌3次後,進行顯色反應後,於酶標儀採用OD450/OD630nm讀取數據。
實施例 2 :抗 TIGIT 抗體結合阻斷篩選
1. 鼠單抗結合CHO-hTIGIT能力檢測:
將待檢測的重組表達全長人TIGIT的CHO工程細胞CHO-hTIGIT細胞進行細胞計數,300g離心5 min,FCM緩衝液(FCM buffer)重懸,調整細胞密度至4×10
6個/mL備用。使用FCM緩衝液將待測樣品(包括陽性對照抗體、鼠單抗)稀釋至20 µg/mL,然後使用FCM緩衝液進行3倍系列稀釋12個濃度點;使用FCM緩衝液將同型對照抗體稀釋至20 µg/mL。按照實驗設計分別96孔V型板每孔加入50µL抗體和50µL細胞(每株細胞加入到96孔V型板中的量為2×105個/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入二抗(PE羊抗鼠IgG(Goat anti-mouse IgG),1:500稀釋)至96孔V型板(按照100 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入100 µL 1×PBS重懸,上機檢測。
結果分析,R0223來源於專利WO2016106302(A1)中22G2 IgG1.1f,R0223-CH1是將R0223的Fc區替換為mIgG1的Fc區。R0300來源於專利WO2017053748(A2)中tiragolumab,R0300是將Fc區替換為mIgG1的Fc區。R0223、R0300作為篩選對照抗體,由圖1A、圖1B、圖1C結果可知,Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2對CHO-hTIGIT的結合親和力好於對照抗體。
2. 鼠單抗結合健康人PBMC能力檢測:
為了驗證我們製備的抗人TIGIT抗體能夠有效結合天然表達TIGIT能力,專門進行了一下實驗。將待檢測的健康人PBMC(外周血單核細胞)細胞進行細胞計數,300g離心5 min,FCM緩衝液重懸,調整細胞密度至4×10
6個/mL備用。使用FCM緩衝液將待測樣品(包括陽性對照抗體、鼠單抗)稀釋至20 µg/mL,然後使用FCM緩衝液進行3倍系列稀釋12個濃度點;使用FCM緩衝液將同型對照抗體稀釋至20 µg/mL。按照實驗設計分別96孔V型板每孔加入50µL抗體和50µL細胞(每株細胞加入到96孔V型板中的量為2×105個/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入二抗(PE羊抗鼠IgG,1:500稀釋)至96孔V型板(按照100 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入100 µL 1×PBS重懸,上機檢測。
結果分析,由圖2A、圖2B、圖2C結果可知,Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2對健康人PBMC的結合親和力好於對照抗體。
3. 鼠單抗阻斷CHO-hTIGIT與hPVR-hFc結合能力檢測:
為了驗證抗人TIGIT抗體能夠阻斷CHO-hTIGIT與hPVR-hFc結合能力,專門進行以下實驗。將待檢測的重組表達全長人TIGIT的CHO工程細胞CHO-hTIGIT細胞進行細胞計數,300g離心5 min,FCM緩衝液重懸,調整細胞密度至4×106個/mL備用。使用FCM緩衝液將待測樣品(包括陽性對照抗體、鼠單抗)稀釋至20 µg/mL,然後使用FCM緩衝液進行3倍系列稀釋12個濃度點;使用FCM緩衝液將配體蛋白hPVR-hFc稀釋至20 µg/mL;使用FCM緩衝液將同型對照抗體稀釋至20 µg/mL。按照實驗設計分別96孔V型板每孔加入50µL抗體和50µL細胞(每株細胞加入到96孔V型板中的量為2×10
5個/孔),冰上避光孵育30 min。加入配體蛋白至96孔V型板(按照50 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入二抗(PE抗人IgG(anti-human IgG)Fc,1:500稀釋)至96孔V型板(按照100 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入100 µL 1×PBS重懸,上機檢測。
結果分析,由圖3A、圖3B結果可知,Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-3-2能夠很好的阻斷CHO-hTIGIT與hPVR-hFc的結合,並且阻斷效果要好於對照抗體。
4. 鼠單抗阻斷CHO-hPVR與hTIGIT-hFc結合能力檢測:
為了驗證抗人TIGIT抗體能夠阻斷CHO-hPVR與hTIGIT-hFc結合能力,專門進行以下實驗。使用FCM緩衝液將待測樣品(包括陽性對照抗體、鼠單抗)稀釋至40 µg/mL,然後使用FCM緩衝液進行3倍系列稀釋12個濃度點;使用FCM緩衝液將抗原蛋白hTIGIT-hFc稀釋至0.6µg/mL;使用FCM緩衝液將同型對照抗體稀釋至40 µg/mL。加入待測抗體和抗原蛋白hTIGIT-hFc至96孔V型板(抗體和抗原分別按照50µL/孔加入)冰上避光孵育30 min。將待檢測的重組表達全長人PVR的CHO工程細胞CHO-hPVR細胞進行細胞計數,300g離心5 min,FCM緩衝液重懸,加入細胞至96孔V型板,每株細胞加入到96孔V型板中的量為2×10
5個/孔(按照50 µL/孔加入),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入二抗(PE抗人IgG(anti-human IgG)Fc,1:500稀釋)至96孔V型板(按照100 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入100 µL 1×PBS重懸,上機檢測。
結果分析,由圖4A、圖4B結果可知,Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2能夠很好的阻斷CHO-hPVR與hTIGIT-hFc的結合,並且阻斷效果要好於對照抗體。
5. 鼠單抗阻斷天然腫瘤細胞結合hTIGIT-hFc能力檢測
為了驗證抗人TIGIT抗體能夠阻斷天然腫瘤細胞表達hPVR與hTIGIT-hFc結合能力,專門進行以下實驗。將待檢測的U2-OS細胞(人骨肉瘤細胞,經檢測天然高表達PVR、CD112)進行細胞計數,300g離心5 min,FCM緩衝液重懸,調整細胞密度至4×10
6個/mL備用。使用FCM緩衝液將待測樣品(包括陽性對照抗體、鼠單抗)稀釋至20 µg/mL,然後使用FCM緩衝液進行3倍系列稀釋12個濃度點;使用FCM緩衝液將抗原蛋白hTIGIT-hFc稀釋至120µg/mL;使用FCM緩衝液將同型對照抗體稀釋至20 µg/mL。按照實驗設計分別96孔V型板每孔加入50µL抗原蛋白和50µL細胞(每株細胞加入到96孔V型板中的量為2×10
5個/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 μL FCM緩衝液洗滌一次,離心(300 g /5 min),棄上清。加入抗體至96孔V型板(按照100 µL/孔加入)冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入二抗(APC抗人IgG(anti-human IgG)Fc,1:500稀釋)至96孔V型板(按照100 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入100 µL 1×PBS重懸,上機檢測。
結果分析,由圖5A、圖5B結果可知,Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2能夠很好的阻斷腫瘤細胞與TIGIT的結合,並且阻斷效果要好於對照抗體。
6. 鼠單抗阻斷CHO-hCD112與hTIGIT-hFc結合能力檢測
為了驗證抗人TIGIT抗體能夠阻斷CHO-hCD112與hTIGIT-hFc結合能力,專門進行以下實驗。將待檢測的重組表達全長人CD112的CHO工程細胞CHO-hCD112細胞進行細胞計數,300g離心5 min,FCM緩衝液重懸,調整細胞密度至4×10
6個/mL備用。使用FCM緩衝液將待測樣品(包括陽性對照抗體、鼠單抗)稀釋至20 µg/mL,然後使用FCM緩衝液進行3倍系列稀釋12個濃度點;使用FCM緩衝液將抗原蛋白hTIGIT-hFc稀釋至120µg/mL;使用FCM緩衝液將同型對照抗體稀釋至20 µg/mL。按照實驗設計分別96孔V型板每孔加入50µL抗原蛋白和50µL細胞(每株細胞加入到96孔V型板中的量為2×10
5個/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g /5 min),棄上清。加入抗體至96孔V型板(按照100 µL/孔加入)冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入二抗(APC抗人IgG(anti-human IgG)Fc,1:500稀釋)至96孔V型板(按照100 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入100 µL 1×PBS重懸,上機檢測。
結果分析,由圖6A、圖6B結果可知,Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2能夠很好的阻斷CHO-CD112與TIGIT的結合,並且阻斷效果要好於對照抗體。
綜上,通過篩選獲得的鼠單抗可以相對更好的結合CHO-hTIGIT、健康人PBMC,並且能夠相對更好的阻斷CHO-hTIGIT、CHO-hPVR、CHO-hCD112、US-OS等細胞上TIGIT受體與配體PVR的結合(CHO-hPVR是阻斷細胞上PVR配體與受體TIGIT的結合)。
實施例 3 :抗人 TIGIT 抗體種屬交叉結合能力
鼠單抗結合CHO-cynoTIGIT能力檢測:
為了驗證抗人TIGIT抗體與猴TIGIT結合能力,專門進行以下實驗。將待檢測的重組表達全長cynoTIGIT的CHO工程細胞CHO-cynoTIGIT細胞進行細胞計數,300g離心5 min,FCM緩衝液重懸,調整細胞密度至4×10
6個/mL備用。使用FCM緩衝液將待測樣品(包括陽性對照抗體、鼠單抗)稀釋至20 µg/mL,然後使用FCM緩衝液進行3倍系列稀釋12個濃度點;使用FCM緩衝液將同型對照抗體稀釋至20 µg/mL。按照實驗設計分別96孔V型板每孔加入50µL抗體和50µL細胞(每株細胞加入到96孔V型板中的量為2×10
5個/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入二抗(PE羊抗鼠IgG(Goat anti-mouse IgG),1:500稀釋)至96孔V型板(按照100 µL/孔),冰上避光孵育30 min。離心(300 g/5 min),棄上清,加入200 µL FCM緩衝液洗滌一次,離心(300 g/5 min),棄上清。加入100 µL 1×PBS重懸,上機檢測。
結果分析,由圖7A、圖7B結果可知,Tigit-5、3Tigit-16、3Tigit-29、14Tigit-1-1能夠很好的與cynoTIGIT結合,並且結合效果要好於對照抗體。
實施例 4 :抗人 Tigit 抗體結合動力學 KD 分析
採用MD ForteBIO QKe平台進行抗hTigit抗體與hTigit的結合動力學常數分析。實驗方法如下,用平衡緩衝液(1×PBS+0.02%吐溫20(Tween 20))稀釋攜帶his標籤的人Tigit胞外區重組蛋白hTigit-his(自產)至終濃度為3µg/ml。用平衡緩衝液(1×PBS+0.02%吐溫20)2倍倍比稀釋抗hTigit抗體,起始濃度為10µg/ml,共7個濃度。待anti-Penta-HIS生物傳感器(ForteBIO,Cat.18-5122)充分水化後,先固化hTigit-his重組蛋白120秒,平衡90秒後,與抗hTigit抗體結合,其中結合時間為300秒,解離時間為600秒。整個反應在25°C,1000rpm條件下進行。最後用Octet分析軟件進行曲線擬合,獲得抗hTigit抗體的結合動力學常數KD。
如表1所示,抗人Tigit抗體與人Tigit重組蛋白的結合動力學常數均在pM級別。
[表1]
實施例 5 :抗 TIGIT 抗體活化 NK 細胞功能實驗
抗體名稱 | KD,×10 -10(M) |
R0300-CH1 | 0.631 |
Tigit-5 | 0.605 |
3Tigit-12 | 1.19 |
3Tigit-16 | 2.02 |
3Tigit-24 | 1.93 |
3Tigit-29 | 1.55 |
14Tigit-1-1 | 1.72 |
14Tigit-3-2 | 0.627 |
利用96孔實時無標記細胞分析儀對前述抗體進行體外功能篩選。大部分的細胞檢測方法採用的是傳統的終點法,僅僅給出最終結果,而且往往需要標記細胞和破壞細胞。這些方法無法得到細胞在生長時的真正狀態,也無法對細胞的生長過程做出動態的監測和分析。實時無標記細胞分析儀使用一種非侵入式的方法,記錄細胞的實時生長狀態。這種成像方法,被稱為「實時細胞內涵成像」(Live Content Imaging),擴充了實驗過程中記錄和理解細胞生長、細胞行為和細胞形態的途徑。
本實施例利用安捷倫生物RTCA SP檢測鼠單抗活化NK細胞殺傷功能的能力,鼠單抗活化NK細胞能力是通過阻斷NK細胞表面TIGIT與靶細胞表面的PVR結合而達成的。具體實驗流程如下。首先在需要做實驗的96孔板中加入50µL/孔的1640培養基,進行儀器自動校正階段;傳代培養的U2OS細胞(天然高表達PVR) 用TE緩衝液消化,消化得到的細胞懸液用1640 培養基重懸後計數:
U2OS-P4T2-1640 7.31×10
5個/mL 99.14% 16.07µM
根據所需要的細胞數量用1640培養基將細胞懸液調整至2×10
5個/mL,然後將細胞以50µL/孔的體積加入到實驗孔板中,在生物安全櫃中靜置15min後,將舖有細胞的孔板放置在儀器中,培養過夜。 20h後,U2OS緊貼細胞板底部後,開始準備抗體和NK92-hTIGIT細胞(重組表達hTIGIT)。將傳代培養的NK92-TIGIT細胞吹散成單細胞懸液,1640培養基重懸後計數:
NK92-TIGIT -1640 1.3×10
5個/mL 45.98% 12.32µM
取上述細胞懸液,將NK92-TIGIT細胞調整數量至1×10
5個/mL;同時將待檢測的鼠單抗稀釋到所需要的濃度,然後按照實驗設計將抗體和NK92-TIGIT細胞分別以50µL/孔的體積加入到對應的孔板中,空白組補上相應體積的1640培養基。將細胞培養板放置在生物安全櫃中靜置30min,同時調整儀器參數歸一化處理;繼續把培養板放入檢測卡槽內,在培養箱中繼續培養並記錄。
觀察儀器中參數細胞指數(cell index)至下臨界(bottom)值後停止實驗,分析數據,並按照下面公式計算細胞殺傷率:
細胞毒性(Cytotoxicity)=(E+T指數(index E+T) - 實驗指數(index Exp)/ E+T指數(index E+T)×100%
以時間為X軸,殺傷效率為Y軸作圖。
結果分析,由圖8A、圖8B結果可知,鼠單抗均有較好的活化NK92-hTIGIT殺傷能力,其中Tigit-5、3Tigit-12、3Tigit-16、3Tigit-24對NK92-hTIGIT殺傷功能的活化作用明顯好於對照樣品。
實施例 6 :抗 TIGIT 抗體活化 NK 細胞殺傷功能實驗
利用雙熒光法進一步檢測抗TIGIT抗體活化NK細胞殺傷功能:
靶細胞鋪板
TE消化培養的細胞U2OS計數,取實驗足量的細胞。將細胞用PBS 10mL混勻,400g/5min 離心後去上清。用2mL重懸細胞,加入2µL CFSE染色細胞37°C 15min。補加8mL 的PBS重懸細胞。400g/5min離心細胞後用10mL的PBS再次重懸細胞。400g/5min離心,棄上清,用合適的10% FBS 1640培養基重懸細胞。細胞再次計數。用培養基調整細胞數量到2×10
5/mL,以100µL的體積加入到96孔板中(中間60孔,邊緣用100µL PBS封上)。繼續培養細胞20 h。
加入效應細胞和抗體
20h後,U2OS緊貼細胞板底部後,開始準備抗體和NK92-TIGIT 細胞。將待檢測抗體和同型(isotype)hIgG1 稀釋到所需要濃度的四倍。將傳代培養的NK92-TIGIT細胞吹散成單細胞懸液,1640培養基重懸後計數。取上述細胞懸液,將NK92-TIGIT細胞調整數量至4×10
5個/mL。按照實驗設計將抗體和NK92-TIGIT細胞分別以50µL/孔的體積加入到對應的孔板中,空白組補上相應體積的1640培養基。繼續在培養箱中培養。
流式檢測
16h後取出96孔板,將細胞上清轉移到另一個96孔板中,注意一一對應,往其中加入35µL的PBS清洗板底,並將清洗液轉移到對應的96孔板中。往96孔板中每孔加入35µL的0.25% Trypsin-EDTA,至於37°C培養箱中約5min。將之前的96板中的液體一一對應的轉移到現在消化完畢的96孔板中,終止消化,輕輕吹散細胞。加入每孔10µL的PI溶液,使其終濃度為1µg/mL,輕輕混勻,立即上機檢測,檢測前的間隔時間不要超過45min。流式細胞儀分析各孔細胞。
數據分析
CFSE和PI雙陽性細胞為被殺傷細胞,CFSE單陽性細胞認為是所有靶細胞總和。因此抗體的絕對殺傷效率即為細胞毒性(Cytotoxicity)=(PI和CFSE雙陽性細胞數)/(CFSE單陽性細胞數)×100%;先計算每個加入抗體組的不同濃度的實驗孔(細胞毒性(Cytotoxicity))(%),以T+E孔為陰性對照孔;再計算相對的細胞殺傷效率=(實驗組-對照孔)/對照孔×100%。用GraphPad Prism 5軟件以抗體濃度作為橫坐標,以計算出來的相對殺傷效率(細胞毒性(Cytotoxicity))(%)的值作為縱坐標進行非線性擬合,計算EC50值。
結果分析,由圖9結果可知,Tigit-5、3Tigit-16能夠活化NK92-hTIGIT殺傷U2-OS能力,並且其活化作用要明顯好於對照樣品。
實施例 7 :抗人 Tigit 抗體競爭表位分析
1. 抗原表位研究(epitope binning)
採用MD ForteBIO QKe平台進行抗人Tigit抗體的表位競爭分析。實驗方法如下,用平衡緩衝液(1×PBS+0.02%吐溫20)稀釋攜帶his標籤的人Tigit胞外區重組蛋白hTigit-his(Acrobiosystem,Cat. TIT-H2H3,lot-1336-76GF1-F9)至終濃度為1.5µg/ml。用平衡緩衝液(1×PBS+0.02%吐溫20)稀釋抗人Tigit抗體,其終濃度為5µg/ml。待anti-Penta-HIS生物傳感器(ForteBIO,Cat.18-5122)充分水化後,先固化hTigit-his重組蛋白180秒,平衡30秒後,與第一種抗人Tigit抗體結合180秒,平衡30秒後,再與第二種抗人Tigit抗體結合其中結合時間為300秒。整個反應在25°C,1000rpm條件下進行。最後用Octet分析軟件進行抗原表位研究(epitope binning)分析。
如表2和表3所示,1株抗人Tigit抗體(9Tigit-45)與R0223表位不同。 15株抗人Tigit抗體(Tigit-5、Tigit-17、Tigit-18、Tigit-22、3Tigit-2、3Tigit-14、3Tigit-21-1、3Tigit-21-2、3Tigit-22、3Tigit-33、3Tigit-41、3Tigit-56、9Tigit-17、9Tigit-21、9Tigit-45)與R0300表位不同。
[表2]
Tigit-5 | Tigit-17 | Tigit-18 | Tigit-22 | 3Tigit-2 | 3Tigit-3 | 3Tigit-9 |
0.0443 | 0.047 | 0.0383 | 0.0353 | 0.0118 | 0.0105 | 0.0165 |
3Tigit-12 | 3Tigit-14 | 3Tigit-16 | 3Tigit-21-1 | 3Tigit-21-2 | 3Tigit-22 | 3Tigit-24 |
0.01 | 0.0126 | 0.008 | 0.0133 | 0.0206 | 0.0583 | 0.0379 |
3Tigit-29 | 3Tigit-33 | 3Tigit-41 | 3Tigit-48 | 3Tigit-49 | 3Tigit-51 | 3Tigit-52 |
-0.0018 | 0.0225/0.059 | 0.0108 | 0.0153 | 0.0413 | 0.0613 | 0.0307 |
3Tigit-56 | 6Tigit-10 | 8Tigit-4 | 8Tigit-7 | 9Tigit-17 | 9Tigit-21 | 9Tigit-30 |
0.0323 | 0.0341 | 0.026 | 0.0148 | 0.0395 | 0.0504 | 0.0537 |
9Tigit-31 | 9Tigit-45 | R0223 | ||||
0.0484 | 0.3347 | 0.0566 |
[表3]
Tigit-5 | Tigit-17 | Tigit-18 | Tigit-22 | 3Tigit-2 | 3Tigit-3 | 3Tigit-9 |
0.6679 | 0.6297 | 0.5439 | 0.6136 | 0.2683 | 0.0198 | 0.0109 |
3Tigit-12 | 3Tigit-14 | 3Tigit-16 | 3Tigit-21-1 | 3Tigit-21-2 | 3Tigit-22 | 3Tigit-24 |
0.0196 | 0.3038 | 0.0157 | 0.3001 | 0.3142 | 0.8526 | 0.0132 |
3Tigit-29 | 3Tigit-33 | 3Tigit-41 | 3Tigit-48 | 3Tigit-49 | 3Tigit-51 | 3Tigit-52 |
0.0254 | 0.9022 | 0.295 | 0.0035 | 0.032 | 0.0165 | 0.0166 |
3Tigit-56 | 6Tigit-10 | 8Tigit-4 | 8Tigit-7 | 9Tigit-17 | 9Tigit-21 | 9Tigit-30 |
0.4351 | 0.0124 | 0.015 | 0.0161 | 0.5831 | 0.6536 | 0.0238 |
9Tigit-31 | 9Tigit-45 | R0223 | R0300 | |||
0.0289 | 0.8743 | 0.0687 | 0.0316 |
2. 流式分析細胞水平的抗體表位競爭
用1×FCM緩衝液(1×FBS+3%BSA)稀釋抗Tigit抗體,其中第一種抗體包括對標抗體R0223、R0300(hIgG1亞型)、hIgG1同型對照抗體,其濃度為10µg/ml,第二種抗體包括抗人Tigit抗體、對標抗體R0223-CH1、R0300-CH1(mIgG1亞型)和mIgG1同型對照抗體,其濃度為1µg/ml。用1×FCM緩衝液重懸重組表達人Tigit的CHO穩定細胞株,調整細胞密度為2×10E6/ml,按100µl/孔分於96孔板中。250×g離心5min後,吸棄上清,加入第一種抗體100µl,冰上孵育30min後,加入第二種抗體100µl,冰上孵育30min。250×g離心5min後,吸棄上清。用1×FCM緩衝液洗滌2次後,按100µl/孔加入用1×FCM緩衝液稀釋的PE標記的羊抗鼠Fc熒光二抗(稀釋倍數為1:500)(百進生技(Biolegend),Cat. 405307),冰上孵育30min。250×g離心5min後,吸棄上清,加入1×FCM緩衝液洗滌2次後,按100µl/孔加入1×PBS重懸細胞,採用流式細胞儀(貝克曼(Beckman),cytoFLEX)進行檢測。反之,第一種抗體包括抗人Tigit抗體、對標抗體R0223-CH1、R0300-CH1(mIgG1亞型)和mIgG1同型對照抗體,其濃度為10µg/ml,第二種抗體包括對標抗體R0223、R0300(hIgG1亞型)、hIgG1同型對照抗體,其濃度為1µg/ml。熒光二抗為PE標記的羊抗人Fc熒光二抗(稀釋倍數為1:500)(百進生技,Cat. 409304)。
如圖10A、圖10B所示,結果與實施例7中的步驟1一致,1株抗人Tigit抗體與R0223表位不同。15株抗人Tigit抗體與R0300表位不同,並且R0223與R0300表位有重疊。
本發明涉及的抗體序列如下:
其中粗體字為CDR序列,非粗體字為FR序列,括號中的數字代表序列編號。
Tigit-5鼠單抗的VH序列:
QVQLQQPGAELVRPGASVKLSCKASGYSFT(43)
SYWMN ( 1 )WVKQRPGQGLEWIG(44)
MIRPSDSETRLNQMFKD ( 2 )KATLTVDKSSSTAYMQLSSPTSDDSAVYYCAG(45)
IHDYGHGAY ( 3 )WGQGTLVTVSA(46)
Tigit-5鼠單抗的VL序列:
DIQMIQSPASLSVSVGETVTITC(71)
RASENIYSNLA ( 22 )WYQQKQGKSPQLLVY(72)
AASHLPD ( 23 )GVPSRFSGSGSGTQYSLKINSLQSEDFGSYYC(73)
QHFWGTPRT ( 24 )FGGGTKLEIKR(74)
3Tigit-16鼠單抗的VH序列:
EVQLQQSGPELVKPGASVKMSCKASGFTFT(47)
DYFMD ( 4 )WVKQSRGASFEWIG(48)
RLNPNNGRTSYNQKFKG ( 5 )KATLTVDKSSSTAYMELNSLTSEDSAVYYCAR(49)
GDLGRWYFDV ( 6 )WGAGTTVTVSS(50)
3Tigit-16鼠單抗的VL序列:
QIVLTQSPAIMSASPGEKVTMTC(75)
SASSSVSYMH ( 25 )WYQQKSGTSPKRWIY(76)
DTSKLAS ( 26 )GVPTRFSGSGSGTSYSLTISSMEAEDAASYFC(77)
QQWSSNSLT ( 27 )FGAGTKLELKR(78)
3Tigit-24鼠單抗的VH序列:
QVQLQQSGAELMKPGASVKISCKATGYTFS(51)
SYWIE ( 7 )WVKQRPGHGLEWIG(52)
EILSGSGRTYFNEKFKG ( 8 )KATFTADTSSNTSYMQLSSLTSEDSAVYFCAR(53)
RGLRGPYYFDY ( 9 )WGQGTTLTVSS(54)
3Tigit-24鼠單抗的VL序列:
DIVLTQSPASLAVSLGQRATISY(79)
RASKSVSTSGYSYMH ( 28 )WNQQKPGQPPRLLIY(80)
LVSNLES ( 29 )GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC(81)
QHIRELTR ( 30 )SGGGTKLEIKR(82)
3Tigit-29鼠單抗的VH序列:
EVQLQQSGPELVKPGASVKISCKASGYTFT(55)
DYNMH ( 10 )WVKQSLGKSLEWIG(56)
YVNSFNGNTGYSQKFKS ( 11 )KVTLTVDRSSRTAYMDLRSLTSEDSAVFYCAR(57)SSRGGFAY(12)WGQGTLVTVSA(58)
3Tigit-29鼠單抗的VL序列:
QIVLTQSPAIMSASPGEKVTMTC(83)
SASSSVSYMH ( 31 )WYQQKSGTSPKRWIY(84)
DTSKLAS ( 32 )GVPARFSGSVSGTSYSLTISSMEAEDAATYYC(85)
QQWSSNSLT ( 33 )FGAGTKLELKR(86)
14Tigit-1-1鼠單抗的VH序列:
DVQLQESGPGLVKPSQSLSLTCTVTGYSIT(59)
SDYAWN ( 13 )WIRQFPGNKLEWMG(60)
YISYSGSTRSNPSLKS ( 14 )RISITRDTSKNQFFLQLNSMTAEDTATYYCGG(61)WEVRNYYAMDY(15)WGQGTSVTVSS(62)
14Tigit-1-1鼠單抗的VL序列:
DIVMTQSHKFMSTSVGDRVSITC(87)
KASQHVSTAVA ( 34 )WYQQKPGQSPKLLIY(88)
SASYRYT ( 35 )GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC(89)
QQHYNTPWT ( 36 )FGGGTKLEIKR(90)
14Tigit-3-2鼠單抗的VH序列:
EVQLQQSGAELVKPGASVKLSCTASGFNIK(63)
DTYIH ( 16 )WVKQRPDQGLEWIG(64)
GIGPANGNTKFDPKFQG ( 17 )KATITADTSSNTAYLQLSGLTSEDTAVYYCAK(65)
LLLRFYVLAY ( 18 )WGQGTSVTVSS(66)
14Tigit-3-2鼠單抗的VL序列:
DIVLTQSPASLAVSLGQRATISY(91)
RASKSVSTSGYSYMH ( 37 )WNQQKPGQPPRLLIY(92)
LVSNLES ( 38 )GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC(93)
QHIRELTR ( 39 )SGGGTKLEIKR(94)
3Tigit-12鼠單抗的VH序列:
QVQLQQSGAELVRPGTSVKMSCKAAGYTFT(67)
NYWIG ( 19 )WVKQRPGHGLEWIG(68)
DIYPGGGYTNYDEKFKG ( 20 )KATLTADTSSRTAYMQLSSLTSEDSAIYHCVN(69)
GGYGSTYGYFDV ( 21 )WGAGTTVTVSS(70)
3Tigit-12鼠單抗的VL序列:
DIVMSQSPSSLPVSVGEKVTMSC(95)
KSSQSLLFSSDQKNYLA ( 40 )WYQQKPGQSPKLLIY(96)
WASTRES ( 41 )GVPDRLTGSGSGTDFTLTISSVKAEDLAVYYC(97)
QQYHKYPFT ( 42 )FGGGTKLEIKR(98)
以上所述實施例的各技術特徵可以進行任意的組合,為使描述簡潔,未對上述實施例中的各個技術特徵所有可能的組合都進行描述,然而,只要這些技術特徵的組合不存在矛盾,都應當認為是本說明書記載的範圍。
以上所述實施例僅表達了本發明的幾種實施方式,其描述較為具體和詳細,但並不能因此而理解為對發明專利範圍的限制。應當指出的是,對於本領域的普通技術人員來說,在不脫離本發明構思的前提下,還可以做出若干變形和改進,這些都屬於本發明的保護範圍。因此,本發明專利的保護範圍應以所附發明申請專利範圍為準。
無
為了更清楚地說明本發明具體實施方式或現有技術中的技術方案,下面將對具體實施方式或現有技術描述中所需要使用的附圖作簡單地介紹,顯而易見地,下面描述中的附圖是本發明的一些實施方式,對於本領域普通技術人員來講,在不付出創造性勞動的前提下,還可以根據這些附圖獲得其他的附圖。
圖1A、圖1B、圖1C為本發明一個實施例中通過流式細胞術(FACS)驗證Tigit-5、3Tigit-12、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2等對CHO-hTIGIT的結合親和力;
圖2A、圖2B、圖2C為本發明一個實施例中通過流式細胞術(FACS)驗證Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2對健康人PBMC的結合親和力;
圖3A、圖3B為本發明一個實施例中通過流式細胞術(FACS)驗證Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-3-2對CHO-hTIGIT與hPVR-hFc結合的阻斷效果;
圖4A、圖4B為本發明一個實施例中通過流式細胞術(FACS)驗證Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2對CHO-hPVR與hTIGIT-hFc結合的阻斷效果;
圖5A、圖5B為本發明一個實施例中通過流式細胞術(FACS)驗證Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2對腫瘤細胞與TIGIT結合的阻斷效果;
圖6A、圖6B為本發明一個實施例中通過流式細胞術(FACS)驗證Tigit-5、3Tigit-16、3Tigit-24、3Tigit-29、14Tigit-1-1、14Tigit-3-2對CHO-CD112與TIGIT結合的阻斷效果;
圖7A、圖7B為本發明一個實施例中通過流式細胞術(FACS)驗證Tigit-5、3Tigit-16、3Tigit-29、14Tigit-1-1與cynoTIGIT結合能力;
圖8A、圖8B為本發明一個實施例中Tigit-5、3Tigit-12、3Tigit-16、3Tigit-24對NK92-hTIGIT殺傷功能的活化作用驗證;
圖9為本發明一個實施例中Tigit-5、3Tigit-16活化NK92-hTIGIT殺傷U2-OS能力驗證;
圖10A本發明一個實施例中驗證不同抗體與R0223表位是否相同;
圖10B本發明一個實施例中驗證不同抗體與R0300表位是否相同。
<![CDATA[<110> 廣東菲鵬制藥股份有限公司]]> <![CDATA[<120> 抗TIGIT抗體或其抗原結合片段及其生產方法與應用,以及包含其的核酸、載體、細胞、藥物組合物及試劑盒]]> <![CDATA[<160> 98 ]]> <![CDATA[<170> PatentIn version 3.3]]> <![CDATA[<210> 1]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 1]]> Ser Tyr Trp Met Asn 1 5 <![CDATA[<210> 2]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 2]]> Met Ile Arg Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Met Phe Lys 1 5 10 15 Asp <![CDATA[<210> 3]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 3]]> Ile His Asp Tyr Gly His Gly Ala Tyr 1 5 <![CDATA[<210> 4]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 4]]> Asp Tyr Phe Met Asp 1 5 <![CDATA[<210> 5]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 5]]> Arg Leu Asn Pro Asn Asn Gly Arg Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <![CDATA[<210> 6]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 6]]> Gly Asp Leu Gly Arg Trp Tyr Phe Asp Val 1 5 10 <![CDATA[<210> 7]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 7]]> Ser Tyr Trp Ile Glu 1 5 <![CDATA[<210> 8]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 8]]> Glu Ile Leu Ser Gly Ser Gly Arg Thr Tyr Phe Asn Glu Lys Phe Lys 1 5 10 15 Gly <![CDATA[<210> 9]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 9]]> Arg Gly Leu Arg Gly Pro Tyr Tyr Phe Asp Tyr 1 5 10 <![CDATA[<210> 10]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 10]]> Asp Tyr Asn Met His 1 5 <![CDATA[<210> 11]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 11]]> Tyr Val Asn Ser Phe Asn Gly Asn Thr Gly Tyr Ser Gln Lys Phe Lys 1 5 10 15 Ser <![CDATA[<210> 12]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 12]]> Ser Ser Arg Gly Gly Phe Ala Tyr 1 5 <![CDATA[<210> 13]]> <![CDATA[<211> 6]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 13]]> Ser Asp Tyr Ala Trp Asn 1 5 <![CDATA[<210> 14]]> <![CDATA[<211> 16]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 14]]> Tyr Ile Ser Tyr Ser Gly Ser Thr Arg Ser Asn Pro Ser Leu Lys Ser 1 5 10 15 <![CDATA[<210> 15]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 15]]> Trp Glu Val Arg Asn Tyr Tyr Ala Met Asp Tyr 1 5 10 <![CDATA[<210> 16]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 16]]> Asp Thr Tyr Ile His 1 5 <![CDATA[<210> 17]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 17]]> Gly Ile Gly Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe Gln 1 5 10 15 Gly <![CDATA[<210> 18]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 18]]> Leu Leu Leu Arg Phe Tyr Val Leu Ala Tyr 1 5 10 <![CDATA[<210> 19]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 19]]> Asn Tyr Trp Ile Gly 1 5 <![CDATA[<210> 20]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 20]]> Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asp Glu Lys Phe Lys 1 5 10 15 Gly <![CDATA[<210> 21]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 21]]> Gly Gly Tyr Gly Ser Thr Tyr Gly Tyr Phe Asp Val 1 5 10 <![CDATA[<210> 22]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 22]]> Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <![CDATA[<210> 23]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 23]]> Ala Ala Ser His Leu Pro Asp 1 5 <![CDATA[<210> 24]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 24]]> Gln His Phe Trp Gly Thr Pro Arg Thr 1 5 <![CDATA[<210> 25]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 25]]> Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <![CDATA[<210> 26]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 26]]> Asp Thr Ser Lys Leu Ala Ser 1 5 <![CDATA[<210> 27]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 27]]> Gln Gln Trp Ser Ser Asn Ser Leu Thr 1 5 <![CDATA[<210> 28]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 28]]> Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <![CDATA[<210> 29]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 29]]> Leu Val Ser Asn Leu Glu Ser 1 5 <![CDATA[<210> 30]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 30]]> Gln His Ile Arg Glu Leu Thr Arg 1 5 <![CDATA[<210> 31]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 31]]> Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <![CDATA[<210> 32]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 32]]> Asp Thr Ser Lys Leu Ala Ser 1 5 <![CDATA[<210> 33]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 33]]> Gln Gln Trp Ser Ser Asn Ser Leu Thr 1 5 <![CDATA[<210> 34]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 34]]> Lys Ala Ser Gln His Val Ser Thr Ala Val Ala 1 5 10 <![CDATA[<210> 35]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 35]]> Ser Ala Ser Tyr Arg Tyr Thr 1 5 <![CDATA[<210> 36]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 36]]> Gln Gln His Tyr Asn Thr Pro Trp Thr 1 5 <![CDATA[<210> 37]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 37]]> Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <![CDATA[<210> 38]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 38]]> Leu Val Ser Asn Leu Glu Ser 1 5 <![CDATA[<210> 39]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 39]]> Gln His Ile Arg Glu Leu Thr Arg 1 5 <![CDATA[<210> 40]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 40]]> Lys Ser Ser Gln Ser Leu Leu Phe Ser Ser Asp Gln Lys Asn Tyr Leu 1 5 10 15 Ala <![CDATA[<210> 41]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 41]]> Trp Ala Ser Thr Arg Glu Ser 1 5 <![CDATA[<210> 42]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 42]]> Gln Gln Tyr His Lys Tyr Pro Phe Thr 1 5 <![CDATA[<210> 43]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 43]]> Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30 <![CDATA[<210> 44]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 44]]> Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 <![CDATA[<210> 45]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 45]]> Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Pro Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys Ala Gly 20 25 30 <![CDATA[<210> 46]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 46]]> Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10 <![CDATA[<210> 47]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 47]]> Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr 20 25 30 <![CDATA[<210> 48]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 48]]> Trp Val Lys Gln Ser Arg Gly Ala Ser Phe Glu Trp Ile Gly 1 5 10 <![CDATA[<210> 49]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 49]]> Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <![CDATA[<210> 50]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 50]]> Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <![CDATA[<210> 51]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 51]]> Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser 20 25 30 <![CDATA[<210> 52]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 52]]> Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly 1 5 10 <![CDATA[<210> 53]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 53]]> Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ser Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 20 25 30 <![CDATA[<210> 54]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 54]]> Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <![CDATA[<210> 55]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 55]]> Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <![CDATA[<210> 56]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 56]]> Trp Val Lys Gln Ser Leu Gly Lys Ser Leu Glu Trp Ile Gly 1 5 10 <![CDATA[<210> 57]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 57]]> Lys Val Thr Leu Thr Val Asp Arg Ser Ser Arg Thr Ala Tyr Met Asp 1 5 10 15 Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys Ala Arg 20 25 30 <![CDATA[<210> 58]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 58]]> Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10 <![CDATA[<210> 59]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 59]]> Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 20 25 30 <![CDATA[<210> 60]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 60]]> Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly 1 5 10 <![CDATA[<210> 61]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 61]]> Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln 1 5 10 15 Leu Asn Ser Met Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Gly Gly 20 25 30 <![CDATA[<210> 62]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 62]]> Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <![CDATA[<210> 63]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 63]]> Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys 20 25 30 <![CDATA[<210> 64]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 64]]> Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile Gly 1 5 10 <![CDATA[<210> 65]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 65]]> Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln 1 5 10 15 Leu Ser Gly Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 20 25 30 <![CDATA[<210> 66]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 66]]> Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <![CDATA[<210> 67]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 67]]> Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Thr 20 25 30 <![CDATA[<210> 68]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 68]]> Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly 1 5 10 <![CDATA[<210> 69]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 69]]> Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Arg Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr His Cys Val Asn 20 25 30 <![CDATA[<210> 70]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 70]]> Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <![CDATA[<210> 71]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 71]]> Asp Ile Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys 20 <![CDATA[<210> 72]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 72]]> Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr 1 5 10 15 <![CDATA[<210> 73]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 73]]> Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser 1 5 10 15 Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys 20 25 30 <![CDATA[<210> 74]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 74]]> Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 <![CDATA[<210> 75]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 75]]> Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys 20 <![CDATA[<210> 76]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 76]]> Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 1 5 10 15 <![CDATA[<210> 77]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 77]]> Gly Val Pro Thr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Ser Tyr Phe Cys 20 25 30 <![CDATA[<210> 78]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 78]]> Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 1 5 10 <![CDATA[<210> 79]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 79]]> 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 Tyr 20 <![CDATA[<210> 80]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 80]]> Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr 1 5 10 15 <![CDATA[<210> 81]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 81]]> Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <![CDATA[<210> 82]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 82]]> Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 <![CDATA[<210> 83]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 83]]> Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys 20 <![CDATA[<210> 84]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 84]]> Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 1 5 10 15 <![CDATA[<210> 85]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 85]]> Gly Val Pro Ala Arg Phe Ser Gly Ser Val Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <![CDATA[<210> 86]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 86]]> Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 1 5 10 <![CDATA[<210> 87]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 87]]> Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys 20 <![CDATA[<210> 88]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 88]]> Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 1 5 10 15 <![CDATA[<210> 89]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 89]]> Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys 20 25 30 <![CDATA[<210> 90]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 90]]> Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 <![CDATA[<210> 91]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 91]]> 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 Tyr 20 <![CDATA[<210> 92]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 92]]> Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr 1 5 10 15 <![CDATA[<210> 93]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 93]]> Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <![CDATA[<210> 94]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 94]]> Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 <![CDATA[<210> 95]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 95]]> Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys 20 <![CDATA[<210> 96]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 96]]> Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 1 5 10 15 <![CDATA[<210> 97]]> <![CDATA[<211> 32]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 97]]> Gly Val Pro Asp Arg Leu Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys 20 25 30 <![CDATA[<210> 98]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<400> 98]]> Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10
Claims (10)
- 一種能夠特異性識別TIGIT的抗體或其抗原結合片段,其重鏈CDR1、CDR2、CDR3選自SEQ ID NO:1~3、SEQ ID NO:4~6、SEQ ID NO:7~9、SEQ ID NO:10~12、SEQ ID NO:13~15、SEQ ID NO:16~18、SEQ ID NO:19~21組合中的至少一種; 其輕鏈CDR1、CDR2、CDR3選自SEQ ID NO:22~24、SEQ ID NO:25~27、SEQ ID NO:28~30、SEQ ID NO:31~33、SEQ ID NO:34~36、SEQ ID NO:37~39、SEQ ID NO:40~42中的至少一種; 可選地,所述抗體或其抗原結合片段具有選自如下CDR序列組合中的至少一種:
H-CDR1 H-CDR2 H-CDR3 L-CDR1 L-CDR2 L-CDR3 Tigit-5 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 3Tigit-16 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27 3Tigit-24 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30 3Tigit-29 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33 14Tigit-1-1 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:34 SEQ ID NO:35 SEQ ID NO:36 14Tigit-3-2 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 3Tigit-12 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42 - 如請求項1所述能夠特異性識別TIGIT的抗體或其抗原結合片段,其重鏈FR1、FR2、FR3、FR4選自SEQ ID NO:43~46、SEQ ID NO:47~50、SEQ ID NO:51~54、SEQ ID NO:55~58、SEQ ID NO:59~62、SEQ ID NO:63~66、SEQ ID NO:67~70組合中的至少一種; 其輕鏈FR1、FR2、FR3、FR4選自SEQ ID NO:71~74、SEQ ID NO:75~78、SEQ ID NO:79~82、SEQ ID NO:83~86、SEQ ID NO:87~90、SEQ ID NO:91~94、SEQ ID NO:95~98中的至少一種; 可選地,所述抗體或其抗原結合片段還包含選自如下骨架區序列組合中的至少一種:
H-FR1 H-FR2 H-FR3 H-FR4 L-FR1 L-FR2 L-FR3 L-FR4 Tigit-5 SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45 SEQ ID NO:46 SEQ ID NO:71 SEQ ID NO:72 SEQ ID NO:73 SEQ ID NO:74 3Tigit-16 SEQ ID NO:47 SEQ ID NO:48 SEQ ID NO:49 SEQ ID NO:50 SEQ ID NO:75 SEQ ID NO:76 SEQ ID NO:77 SEQ ID NO:78 3Tigit-24 SEQ ID NO:51 SEQ ID NO:52 SEQ ID NO:53 SEQ ID NO:54 SEQ ID NO:79 SEQ ID NO:80 SEQ ID NO:81 SEQ ID NO:82 3Tigit-29 SEQ ID NO:55 SEQ ID NO:56 SEQ ID NO:57 SEQ ID NO:58 SEQ ID NO:83 SEQ ID NO:84 SEQ ID NO:85 SEQ ID NO:86 14Tigit-1-1 SEQ ID NO:59 SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:87 SEQ ID NO:88 SEQ ID NO:89 SEQ ID NO:90 14Tigit-3-2 SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65 SEQ ID NO:66 SEQ ID NO:91 SEQ ID NO:92 SEQ ID NO:93 SEQ ID NO:94 3Tigit-12 SEQ ID NO:67 SEQ ID NO:68 SEQ ID NO:69 SEQ ID NO:70 SEQ ID NO:95 SEQ ID NO:96 SEQ ID NO:97 SEQ ID NO:98 - 如請求項1或2所述的能夠特異性識別TIGIT的抗體或其抗原結合片段,其中所述抗原結合片段為F(ab’) 2、Fab、scFv以及雙特異抗體中的一種; 可選地,所述抗體具有恆定區,重鏈恆定區序列選自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任意一種的恆定區序列;輕鏈恆定區為κ或λ鏈; 可選地,所述恆定區的種屬來源選自牛、馬、豬、綿羊、山羊、大鼠、小鼠、狗、貓、兔、駱駝、驢、鹿、貂、雞、鴨、鵝或人。
- 一種核酸,其編碼如請求項1至3中任一項所述的抗體或其抗原結合片段。
- 一種載體,其包含如請求項4所述的核酸。
- 一種細胞,其包含如請求項4所述的核酸或如請求項5所述的載體。
- 一種生產如請求項1至3中任一項所述的抗體或其抗原結合片段的方法,包括: 在培養基中培養如請求項6所述的細胞;以及 從培養基中或從所培養的細胞中回收如此產生的抗體或其抗原結合片斷。
- 一種藥物組合物,其包括如請求項1至3中任一項所述的抗體或其抗原結合片段,以及藥學上可接受的賦形劑、稀釋劑或載體中的一種或多種。
- 一種如請求項1至3中任一項所述的抗體或其抗原結合片段在製備用於治療或預防感染性疾病、免疫性疾病或腫瘤的藥物中的應用。
- 一種試劑盒,其包含下述成分中的至少一種: i)如請求項1至3中任一項所述的抗體或其抗原結合片段,以及任選的用於承裝所述抗體或其抗原結合片段的容器; ii)如請求項8所述的藥物組合物,以及任選的用於承裝所述藥物組合物的容器。
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CN116003606B (zh) * | 2023-01-03 | 2023-06-20 | 上海百英生物科技股份有限公司 | 一种tigit纳米抗体及其制备方法与应用 |
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MA40662B1 (fr) * | 2014-12-23 | 2020-12-31 | Bristol Myers Squibb Co | Anticorps contre tigit |
CN115925931A (zh) * | 2015-08-14 | 2023-04-07 | 默沙东公司 | 抗tigit抗体 |
JP6861418B2 (ja) * | 2015-09-02 | 2021-04-28 | イッサム リサーチ デベロップメント カンパニー オブ ザ ヘブリュー ユニバーシティー オブ エルサレム リミテッド | ヒトt細胞免疫グロブリン及びitimドメイン(tigit)に特異的な抗体 |
KR20200087283A (ko) | 2015-09-25 | 2020-07-20 | 제넨테크, 인크. | 항-tigit 항체 및 이의 이용 방법 |
EP3689909A4 (en) * | 2017-09-29 | 2021-12-29 | Jiangsu Hengrui Medicine Co., Ltd. | Tigit antibody, antigen-binding fragment thereof, and medical use thereof |
TW202400654A (zh) * | 2017-12-30 | 2024-01-01 | 英屬開曼群島商百濟神州有限公司 | 抗tigit抗體及其作為治療和診斷的用途 |
SG11202005595YA (en) * | 2018-01-15 | 2020-07-29 | Nanjing Legend Biotech Co Ltd | Antibodies and variants thereof against tigit |
US10973853B2 (en) * | 2018-02-06 | 2021-04-13 | I-Mab Biopharma Us Limited | Antibodies to T cell immunoreceptor with IG and ITIM domains (TIGIT) and uses thereof |
JP2021532058A (ja) * | 2018-07-25 | 2021-11-25 | イノベント バイオロジックス (スウツォウ) カンパニー,リミテッド | 抗tigit抗体及びその使用 |
CN109384846B (zh) * | 2018-09-25 | 2020-03-03 | 合肥瑞达免疫药物研究所有限公司 | 能够结合tigit的抗体或其抗原结合片段及用途 |
CN111748580B (zh) * | 2019-03-28 | 2022-09-13 | 百奥泰生物制药股份有限公司 | 一种检测免疫检查点抗体活性的方法 |
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