CN110938146B - Tim-3单域抗体及其应用 - Google Patents
Tim-3单域抗体及其应用 Download PDFInfo
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- CN110938146B CN110938146B CN201911146557.5A CN201911146557A CN110938146B CN 110938146 B CN110938146 B CN 110938146B CN 201911146557 A CN201911146557 A CN 201911146557A CN 110938146 B CN110938146 B CN 110938146B
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- antigen binding
- binding fragment
- domain antibody
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Abstract
本发明提供了能够与免疫检查点分子TIM‑3特异结合的单域抗体或其抗原结合片段、编码所述单域抗体或抗原结合片段的多核苷酸、基于上述单域抗体或抗原结合片段、多核苷酸的用途。本发明所提供的单域抗体或其抗原结合片段是由抗体重链可变区(VH)的框架区(frame region)和抗原结合互补决定区(CDR)构成，框架区和CDR区可以按照不同的方式组合。本发明所提供的单域抗体或抗原结合片段能够阻断Galectin‑9和TIM‑3的结合，提高T细胞的活性。本发明所提供的单域抗体或其抗原结合片段可以用于肿瘤、免疫性疾病、感染性疾病等疾病的诊断、治疗、辅助治疗，也可以用于检测生物样本中是否存在TIM‑3蛋白及其含量。
Description
技术领域
本发明属于生物技术领域。本发明涉及新的抗人TIM-3蛋白的单域抗体及其应用。
背景技术
肿瘤是严重威胁人类健康的疾病之一。近年来以嵌合抗原受体T细胞(CAR-T)和免疫检查点抑制剂型抗体为代表的肿瘤免疫治疗技术为肿瘤的治疗带来了革命性的进步。免疫检查点是表达在免疫细胞(T细胞、单核/巨噬细胞)表面的一类调节蛋白，肿瘤细胞表面高表达的抑制性配体与免疫检查点蛋白结合后可以抑制免疫细胞的活性，帮助肿瘤细胞逃避免疫系统的监视。靶向免疫检查点的单克隆抗体可以阻断肿瘤细胞对免疫细胞的抑制作用，强化免疫细胞的抗肿瘤活性。目前已经有靶向免疫检查点CTLA-4和PD-1的单抗药物被FDA批准，还有更多的免疫检查点被陆续鉴定，这些新的免疫检查点是抗体药物研发的重要靶点，TIM-3(T cell immunoglobulin domain and mucin domain-3)就是其中的一个代表。
2001年，McIntire在小鼠的11号染色体上发现了一个新的基因家族，结构上含有免疫球蛋白V区和粘蛋白区，将其命名为T细胞免疫球蛋白黏蛋白分子(T cellimmunoglobulin-and mucin-domain-containing molecules,TIM)，它属于T细胞表面跨膜蛋白家族。该家族在小鼠中有8个成员，而在人类中只有3个(TIM-1、TIM-3、TIM-4)，位于第5号染色体上(Mcintire et al 2001)。TIM-3由信号肽、特征性的免疫球蛋白V区(IgV)结构域、粘蛋白样区、跨膜区和含有磷酸化位点的胞内尾巴区五个domain构成。TIM-3选择性表达于CD4+辅助性T细胞(Th1)表面与CD8+T细胞毒性T细(TC)上。除了其在T细胞上的表达，TIM-3在调节性T细胞(Treg)和其他先天性免疫细胞(如DCs,NK细胞以及单核细胞等)上都能检测到。
TIM-3最重要的功能是参与Th1型免疫负性调节，能够特异性从Th2细胞中识别Th1。CD4+或CD8+效应T细胞单独具有显著抗肿瘤活性，而TIM-3对于CD4+和CD8+效应T细胞是一个关键的靶标(See et al 2015)。TIM-3+CD4+T细胞积聚在人体肿瘤组织内并产生较低水平的Th1型细胞因子,并且，TIM-3+CD4+T细胞能显著抑制自体产生的CD8+T细胞(Yan et al2013)。阻断TIM-3可以拯救肿瘤浸润性T细胞的功能，从而增强宿主的抗肿瘤能力。目前，TIM-3已经被认为是肿瘤免疫治疗的一个重要靶标，许多文献和制药公司已经开展了靶向TIM-3的免疫治疗研究，有些已经进入临床I期测试。另外，在肝癌抗肿瘤免疫中可以通过至少两种不同通路起到负调节作用，如果同时阻断TIM-3和PD-1可以作为肿瘤免疫治疗的一个靶点，从而更好地恢复T细胞免疫功能。
TIM-3的配体是Galectin-9(beta-galactoside lectin protein)，属于半乳凝素家族成员，高表达于淋巴瘤、肺癌、乳腺癌、肝癌等多种肿瘤类型中。Galectin-9可通过与Th1、Th17细胞上的TIM-3结合，诱导上述T效应细胞的凋亡或抑制其分化，下调免疫反应，诱导免疫耐受(Suk et al 1999,Sanchez-Fueyo et al 2003,Tsuboi et al 2007)。Galectin-9在上述免疫耐受诱导中的作用，被认为是一个自身免疫性疾病的干预靶点。靶向TIM-3的单克隆抗体能阻断TIM-3与Galectin-9的结合。传统的单克隆抗体有VH和VL两个抗原结合域，分子量比较大，而小型化的VH单域抗体(domain antibody)具有多方面的独特优势，比如更强的组织渗透性(tissue penetration)，更容易改型和用以构建多特异性抗体等。在抗体药物研发中最具吸引力的单域抗体是骆驼来源的VHH抗体(也称纳米抗体)、人源和兔源的VH domain。现有技术下，人们展开了TIM-3单域抗体的研究，例如中国CN201611041970.1公开了一种针对人TIM-3的全人源单域抗体，CN201780044843.3公开了多特异性抗原结合蛋白，其中所述第一表位来自PD-1、PD-L1、PD-L2、CTLA-4、B7-H3、TIM-3、LAG-3、VISTA、ICOS、4-1BB、OX40、GITR和CD40免疫检查点分子。然而由于单域抗体的亲和力相对于传统的单抗有所减弱。现有技术下对单域抗体的研究均没有考虑TIM-3单域抗体与Galectin-9的竞争结合能力。目前尚无成功阻断Galectin-9和TIM-3结合的TIM-3单域抗体的报道。
发明内容
本发明以免疫检查点分子TIM-3为靶点，采用重组TIM-3蛋白免疫家兔和噬菌体展示相结合的技术，制备靶向TIM-3的VH单域抗体。
本发明具体技术方案如下：
TIM-3单域抗体或抗原结合片段，具有如下任一特征：
a)所述单域抗体或抗原结合片段的氨基酸序列选自包含SEQ ID NO:1-5任一项所述的序列；或者，
b)所述单域抗体或者抗原结合片段的CDR序列的1个、2个或3个包含SEQ ID NO:6-16任一项所述的序列；或者，
c)所述单域抗体或者抗原结合片段的CDR序列的1个、2个或3个进一步包含一个或多个氨基酸残基的替代、插入、缺失，但仍然保持与所述单域抗体竞争相同的抗原表位的特性；或者，
d)所述单域抗体或者抗原结合片段的CDR序列的1个、2个或3个与SEQ ID NO:6-16有至少80％序列同源性，但仍然保持与所述单域抗体竞争相同的抗原表位的特性。
本发明所提供的TIM-3单域抗体或抗原结合片段是由由抗体重链可变区(VH)的框架区(frame region)和3个抗原结合互补决定区(CDR)构成，框架区和CDR区可以按照不同的方式组合，但仍然保持与所述单域抗体竞争相同的抗原表位的特性。框架区部分或全部可以是人源的(如SEQ ID NO.1-3任一序列中的框架区)，也可以是兔源的(如SEQ ID NO.4-5序列中的框架区)。CDR区(CDR1、CDR2、CDR3)的1个、2个或3个选自SEQ ID NO:6-16任一项所述的序列。本申请中一个具体的示例，TIM-3单域抗体或抗原结合片段的氨基酸序列如SEQ ID NO:1-5所示。
一个优选的方案，本发明所述TIM-3单域抗体或抗原结合片段的重链可变区CDR1选自包含SEQ ID NO:6、9、10、11、14任一项所示的氨基酸序列；重链可变区CDR2选自包含SEQ ID NO:7、12、15任一项所示的氨基酸序列；重链可变区CDR3选自包含SEQ ID NO:8、13、16任一项所示的氨基酸序列。
优选的，所述TIM-3单域抗体或抗原结合片段的氨基酸序列选自如下组合：CDR1选自包含SEQ ID NO:6、9、10任一项所示的氨基酸序列；重链可变区CDR2选自包含如SEQ IDNO:7所示的氨基酸序列；重链可变区CDR3选自包含SEQ ID NO:8所示的氨基酸序列；或者，
CDR1选自包含SEQ ID NO:11或14所示的氨基酸序列；重链可变区CDR2选自包含如SEQ ID NO:12或15所示的氨基酸序列；重链可变区CDR3选自包含SEQ ID NO:13或16所示的氨基酸序列；或者，
CDR1选自包含SEQ ID NO:11所示的氨基酸序列；重链可变区CDR2选自包含如SEQID NO:12所示的氨基酸序列；重链可变区CDR3选自包含SEQ ID NO:13所示的氨基酸序列。
或者，
CDR1选自包含SEQ ID NO:14所示的氨基酸序列；重链可变区CDR2选自包含如SEQID NO:15所示的氨基酸序列；重链可变区CDR3选自包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，本发明所述的单域抗体或抗原结合片段可以用来构建双特异性抗体或融合蛋白。双特异抗体包含两个抗原结合片段，一个可以是TIM-3抗原，另外一个可以是肿瘤细胞抗原。
在某些实施方式中，本发明所述的单域抗体或抗原结合片段、双特异性抗体或融合蛋白可以与效应分子偶联。优选的，所述效应分子选自荧光标记、放射性标记、亲和素、生物素、酶、毒素或化疗剂中的一种或几种。
本发明另一目的在于公开本发明所述的单域抗体或抗原结合片段、双特异性抗体或融合蛋白、免疫偶联物、核酸分子在制备免疫检查点抑制剂中的应用。所述免疫检查点抑制剂为诊断或治疗自身免疫疾病、慢性病毒感染或肿瘤的药物。所述的肿瘤为非霍奇金淋巴瘤、慢性淋巴细胞白血病、霍奇金氏病、实体瘤或转移瘤。
本发明所述的TIM-3单域抗体、编码基因、表达TIM-3单域抗体的载体、表达框或细胞、双特异性抗体、融合蛋白或免疫偶联物可以单独成药，也可以与其它具有抗肿瘤作用或者辅助治疗作用的活性物质形成药物组合物。药物组合物可以包含本发明所述的单域抗体或或抗原结合片段、双特异性抗体或融合蛋白、免疫偶联物或者核酸分子中的一种或几种。本发明所述的药物或药物组合物可以与GITR、CD137、PD-1、PD-L1、OX40、CTLA4、TIGIT、LAG3、CD47、SIRPa免疫检查点抑制剂中的一种或几种形成药物组合物。所述的药物或药物组合物还进一步包括药学上可接受的载体、赋形剂和/或稀释剂。
本申请还提供了编码所述的单域抗体或抗原结合片段、双特异性抗体或融合蛋白、免疫偶联物的核酸分子。具体的示例中，核酸分子序列如SEQ ID NO:17-21所示。
本发明所述的药物或药物组合物还可以包括激活的或未激活的T细胞、经过遗传修饰的T细胞、经过体外诱导、激活与培养的人淋巴细胞。
本发明还提供了一种宿主细胞，携带多核苷酸分子并能编码本发明所述的单域抗体或抗原结合片段、双特异性抗体和融合蛋白、免疫偶联物或核酸分子。
本发明还提供了一种检测试剂、诊断试剂或诊断试剂盒，本发明所述的单域抗体或抗原结合片段、双特异性抗体或融合蛋白、免疫偶联物、核酸分子检测生物样本中是否存在TIM-3蛋白和/或TIM3蛋白的含量。
本发明的优势：
TIM-3是T细胞表面的一种免疫检查点分子，其抑制性配体Galectin-9与TIM-3的结合导致T细胞的功能耗竭和最终凋亡，从而抑制T细胞的抗肿瘤活性。本发明提供的单域抗体不仅能够阻断免疫检查点分子TIM-3与其抑制性配体的结合，增强T细胞的抗肿瘤活性，而且由于本发明提供的单域抗体属于新一代的微型化抗体片段，具有更好的肿瘤组织渗透性，是更为理想的肿瘤药物候选物。
附图说明
图1本发明所述TIM-3单域抗体与TIM-3重组蛋白的亲和力曲线图。
图2本发明所述TIM-3单域抗体阻断Galectin-9与TIM-3的结合曲线图。
图3CAR-T联合本发明所述TIM-3单域抗体对移植瘤小鼠的治疗活性结果。
发明详述
本申请的以下描述只为说明本申请的多种实施方式。因此，此处讨论的具体修改方式不应理解为对申请范围的限制。本领域的技术人员在不偏离本申请范围的情况下即可很容易地得出多种等同方式，变化和修改，应理解这样的等同实施方式包含在本发明范围内。在本申请中引用的所有文献，包含公开出版物、专利和专利申请都通过引用的方式全文并入。
I.缩写
BSA,牛血清白蛋白
CDR,互补决定区
ELISA,酶联免疫吸附测定
FACS，荧光激活细胞分选
CAR-T，嵌合抗原受体T细胞
II.术语和方法
除非另有说明，技术术语以生物化学与分子生物学、免疫学领域常规用法使用。为了便于阅读本发明内容的各个实施方案，提供了以下具体术语的解释。
抗体：包含至少轻链或重链免疫球蛋白可变区的抗原结合蛋白，识别并结合抗原如TIM-3或其片段的表位。一个天然的完整抗体包含两条重(H)链和两条轻(L)链。哺乳动物的重链可分为α、δ、ε、γ和μ，每条重链由一可变区(VH)和第一、第二、第三恒定区(分别为CH1、CH2、CH3)组成；哺乳动物的轻链可分为λ或κ，每条轻链由一可变区(VL)和一恒定区组成。抗体包括完整的免疫球蛋白和变体以及本领域熟知的抗体的一个部分，例如单结构域抗体(也称单域抗体，如VH结构域抗体)、Fab片段、Fab’片段、F(ab)’2片段、单链Fv片段(也称scFv)和二硫键稳定化的Fv蛋白(dsFv)。轻链和重链的可变区决定抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)(轻链的CDR包含LCDR1、LCDR2、LCDR3，重链的CDR包含HCDR1、HCDR2、HCDR3)。本发明中公开的抗体和抗原结合片段的CDR边界可通过Kabat，IMGT，AbM，Chothia或Al-Lazikani命名法命名或识别。(Al-Lazikani,B.，Chothia,C.，Lesk,A.M.，J.Mol.Biol.，273(4),927(1997)；Chothia,C.等，J Mol Biol.Dec 5；186(3):651-63(1985)；Chothia,C和Lesk,A.M.，J.Mol.Biol.，196,901(1987)；Chothia,C.等，Nature.Dec 21-28；342(6252):877-83(1989)；Kabat E.A.等，National Institutes ofHealth，马里兰州Bethesda(1991)；Marie-Paule Lefranc等,Developmental andComparative Immunology,27:55-77(2003)；Marie-Paule Lefranc等,ImmunomeResearch,1(3),(2005)；Marie-Paule Lefranc,Molecular Biology of B cells(第2版),第26章,481-514,(2015))。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。重链和轻链的恒定区与抗原结合无关，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或异构体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。
抗原结合片段：指由含有一个或多个CDR的抗体部分或者任何其他结合抗原但不具有完整抗体结构的抗体片段所形成的一种抗体片段。抗原结合片段的例子包含，但不限于，如双功能抗体(diabody)、Fab、Fab'、F(ab')2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、二硫键稳定的双功能抗体(ds diabody)、单链抗体分子(scFv)、scFv二聚体(双价的双功能抗体)、双价单链抗体(BsFv)、骆驼化单域抗体(camelizedsingle domainantibody)、纳米抗体、域抗体和双价域抗体。抗原结合片段可以与母体抗体结合相同的抗原表位。
单域抗体：指仅有抗体的重链可变区或抗体的轻链可变区构成的抗体片段。
双功能抗体(diabody)或dAb：包含带有两个抗原结合位点的小抗体片段，其中该片段含有在同一条多肽链上相连的VH域和VL域(VH-VL或VH-VL)(请参见，Holliger P.等，Proc Natl Acad Sci USA.Jul 15；90(14):6444-8(1993)；EP404097；WO93/11161)。两个域之间衔接物很短，使同一条链上的两个域不能互相配对，从而迫使两个域与另一条链的互补域配对，形成两个抗体结合位点。在某些实施方式中，“scFv二聚体”是双价双功能抗体或双价ScFv(BsFv)，包含VH-VL与另一个VH-VL部分的二聚化(由多肽连接子连接)，使得一个部分的VH与另一个部分的VL协作形成两个结合位点，这两个结合位点可靶向相同抗原(或抗原表位)。
本申请所述的术语“同源”和“同源的”可以互换使用，指当最佳比对时核酸序列(或其互补链)或氨基酸序列具有与另一条序列至少80％(如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的同一性。
当“百分比序列同一性”用于氨基酸序列(或核酸序列)时，是指在进行序列比对，并且必要时引入间隔使相同氨基酸(或核酸)数目达到最多后，在候选序列中，与参比序列相同的氨基酸(或核酸)残基占所述候选序列的氨基酸(或核酸)残基的百分比。所述氨基酸残基的保守替代可以认为或可以不认为是相同残基。可以通过本领域公开的工具，例如BLASTN,BLASTp(美国国家生物技术信息中心网站(NCBI)，也可参见，Altschul S.F.等，J.Mol.Biol.，215:403–410(1990)；Stephen F.等，Nucleic Acids Res.，25:3389–3402(1997))、ClustalW2(欧洲生物信息研究所网站，可参见，Higgins D.G.等，MethodsinEnzymology，266:383-402(1996)；Larkin M.A.等，Bioinformatics(Oxford、England)，23(21):2947-8(2007))和ALIGN或Megalign(DNASTAR)软件，对序列进行比对以确定氨基酸(或核酸)序列的百分比序列同一性。本领域技术人员可以使用所述工具的默认参数或根据比对的需要适当调整参数，例如通过挑选合适的算法。
在本申请中当“保守替代”用于氨基酸序列时，是指将一个氨基酸残基用另一个具有相似理化性质的侧链的氨基酸残基替代。例如，可以在疏水侧链氨基酸残基间(例如Met、Ala、Val、Leu和Ile)、中性亲水侧链残基间(例如Cys、Ser、Thr、Asn和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)或方向侧链残基间(例如Trp、Tyr和Phe)进行保守替代。本领域已知保守替代通常不会引起蛋白构象结构的显著变化，因此能够保留蛋白质的生物活性。
本申请中使用的“表位”是指抗原分子中与抗体结合的那部分氨基酸或原子基团。如果两种抗体表现出对抗原的竞争性结合，则可能结合抗原上的相同或密切相关的表位。例如，如果抗体或其抗原结合片段阻断参比抗体与抗原至少85％或至少90％或至少95％的结合，那么所述抗体或其抗原结合片段可以被认为与所述参比抗体结合相同/密切相关的表位。
本申请中的“TIM-3”来源于任何脊椎动物来源，包含哺乳物动，如灵长类(例如人、猴)和啮齿类(例如小鼠和大鼠)。人TIM-3的示例性序列包含人TIM-3蛋白(NCBI登录号GI:18182535)。TIM-3的示例性序列包含Mus musculus(小鼠)TIM-3蛋白(NCBI登录号GI:18182531)、Rattus norvegicus(大鼠)TIM-3蛋白(NCBI登录号GI:39725405)和Macacafascicularis(猴)TIM-3蛋白(NCBI登录号GI:355750365)。
本申请中的术语“TIM-3”旨在涵盖任意形式的TIM-3，例如1)天然未经处理的TIM-3分子、“全长”TIM-3链或TIM-3天然存在的变体(包含例如剪接变体或等位基因变体)；2)TIM-3由在细胞中的处理而产生的任何形式；或3)通过重组方法产生的TIM-3亚单位的全长、片段(例如截短的形式、胞外/跨膜域)或修饰的形式(例如突变形式、糖基化/聚乙二醇化的形式、组氨酸标签/免疫荧光融合的形式)。
本发明中“宿主细胞”是指导入外源多核苷酸和/或载体的细胞，包括但不限于大肠杆菌细胞，酵母细胞，哺乳动物细胞如CHO细胞、NSO细胞、PER.C6细胞、HEK293细胞等。
表1列出了本发明提供的单域抗体的氨基酸序列。
表2列出了本发明提供的单域抗体的CDR区氨基酸序列。
表3提供了本发明所述单域抗体的编码核酸序列。
表1单域抗体的氨基酸序列
抗体的框架区比CDR更加高度保守并形成一个支架支撑CDR环，不同种属来源的抗体的框架区可以被替换，比如兔抗体和小鼠抗体的框架区可以被替换为人源抗体的框架区，换言之，SEQ ID NO.4和SEQ ID NO.5抗体中的框架区可以被替换为人源抗体的框架区，而基本上保持与TIM-3特异性结合的亲和力。
表2单域抗体的CDR区氨基酸序列
已知CDR负责抗原结合，然而已发现单域抗体的3个CDR并不都是不可缺少或不可改变的。换言之，可以替换或改变或修饰SEQ ID NO:6-16中的一个或多个CDR，而基本上保持与TIM-3特异性结合的亲和力。
表3单域抗体的编码核酸序列
在某些实施方式中，所述分离的多核苷酸包含一个或多个如SEQ ID NO:17-21所示的核酸序列，和/或与之具有至少80％(例如至少85％、88％、90％、92％、93％、94％、95％、96％、97％、98％或99％)的序列同一性的同源序列，和/或仅具有简并替代的变体，并且编码如本申请所述的示例性抗体。编码所述单克隆抗体的DNA可以通过常规的方法分离和测序(例如可以使用寡核苷酸探针，该探针可特异性与编码所述抗体的重链和轻链的基因结合)。所述编码DNA也可以通过合成的方法获得。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不局限于实施例范围。在本发明中使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。下面结合具体实施例并参照数据进一步详细描述本发明，应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
实施例1噬菌体展示的TIM-3单域抗体库的构建及筛选
以TIM-3重组蛋白(购自于北京义翘神州科技有限公司)对新西兰大白兔进行免疫，取脾脏，提取总RNA，经反转录，扩增抗体可变区基因，将抗体基因扩增片段连接噬菌体载体，经纯化，连接产物电转化入TG1感受态细胞，制备TIM-3单域抗体的文库，库容是6×109。同时将合成的人VH库(参考Thomas Tiller,et al.A fully synthetic human Fabantibody library based on fixed VH/VL framework pairings with favorablebiophysical properties.mAbs 5:3,445–470；May/June 2013)一起连接噬菌体载体，经纯化，连接产物电转化入TG1感受态细胞。采用常规的文库筛选方法，以重组TIM-3蛋白对文库进行4轮筛选，最后获得编码TIM-3单域抗体的克隆，经过基因测序鉴定后，获得5个单域抗体，分别编号为TIM-3-CX101，TIM-3-CX106，TIM-3-CX119，TIM-3-CX123，TIM-3-CX153。抗体基因的亚克隆与表达载体的构建按照标准的分子生物技术进行操作。
实施例2单域抗体的原核表达与纯化
将实施例1获得的单域抗体基因亚克隆至原核表达载体pET28,导入表达菌株BL21，从生长过夜的氨苄平皿上挑取单个菌落，通过在LB培养基中加入IPTG诱导，30℃诱导8小时。采用超声波破壁后，离心收集上清液，并用His镍柱纯化获得单域抗体TIM-3-CX101(SEQ ID NO:1)，TIM-3-CX106(SEQ ID NO:2)，TIM-3-CX119(SEQ ID NO:3)，TIM-3-CX123(SEQ ID NO:4)，TIM-3-CX153，(SEQ ID NO:5)。
实施例3 ELISA测定TIM-3单域抗体的亲和力
TIM-3重组蛋白与实施例2制得的五种TIM-3单域抗体的ELISA亲和力分析，TIM-3-CX101、TIM-3-CX106、TIM-3-CX123、TIM-3-CX119、TIM-3-CX153-HisFlag为实验组，以Galectin-9-rFc为阳性对照，TIM-3以5μg/mL包被，用生物素标记TIM-3单域抗体蛋白和Galectin-9-rFc蛋白，以首孔200μg/mL，1:2进行梯度稀释，使用HRP-Streptavidin检测，酶标仪在450nm波长下读取OD值，并用Prism软件拟合出亲和力曲线图，以OD值作为纵坐标，实验组和对照组的蛋白浓度作为横坐标，通过Prism软件计算出结合常数。结果如图1所示，结果显示抗体的多样性导致亲和力存在差异：TIM-3-CX153与TIM-3重组蛋白结合能力最强，其它单域抗体的亲和力均低于Galectin-9-rFc重组蛋白。这些单域抗体的亲和力Kd值在278-8740nM之间，属于正常范围，亲和力最高的是TIM-3-CX153，略低于Galectin-9对TIM-3的亲和力。
实施例4 ELISA分析TIM-3单域抗体对TIM-3/Galectin-9互作的中和活性
采用竞争结合ELISA的方法，检测TIM-3单抗阻断生物素化Galectin-9与固定化TIM-3的结合。TIM-3(5μg/mL)包被ELISA平板，分别加入TIM-3-CX101、TIM-3-CX106、TIM-3-CX123、TIM-3-CX119、TIM-3-CX153孵育30min后，加入5μg/mL生物素化的Galectin-9-rFc，用HRP-Streptavidin检测。酶标仪在450nm波长下读取OD值，并通过Prism软件拟合出竞争曲线，以OD值作为纵坐标，实验组蛋白浓度作为横坐标。TIM-3-CX153和TIM-3-CX123可以竞争配体Galectin-9与TIM-3的结合，两者在1-200μg/mL的范围呈现浓度梯度依赖性的阻断效应，具有免疫检查点抑制活性。TIM-3-CX101、TIM-3-CX106、TIM-3-CX119和配体Galectin-9之间没有竞争或者竞争很弱。
实施例5 CAR-T联合TIM-3单抗的体内抗肿瘤活性
CAR-T是一种前沿的肿瘤免疫治疗技术，但是由于CAR-T细胞会上调表达TIM-3等免疫检查点分子，CAR-T细胞的活性受到抑制。TIM-3单域抗体可以促进CAR-T细胞的抗肿瘤活性。活性测试可以采用移植瘤模型。对裸鼠接种H9肿瘤细胞，待成瘤后对其分组进行实验，对照组尾静脉注射PBS，单独CAR-T组腹腔注射5million CAR-T细胞，联合组腹腔注射5million CAR-T细胞+TIM-3-CX153单抗(尾静脉注射，每两天一次，剂量5mg/kg)。结果如图3所示，图中箭头代表CAR-T细胞+TIM-3-CX153单域抗体组每次注射药物的时间(CAR-T只在day7注射一次)。结果表明，相比较Control组，CAR-T组和CAR-T+TIM-3-CX153单域抗体组肿瘤生长大大减缓，CAR-T+TIM-3-CX153单域抗体联合治疗组比CAR-T单独治疗组显示出显著性差异，具有更好的抑瘤活性。
序列表
<110> 华中农业大学
<120> TIM-3单域抗体及其应用
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<170> SIPOSequenceListing 1.0
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<211> 116
<212> PRT
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Thr Val Ser Ser
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<213> 人工序列(Artificial Sequence)
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Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
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Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
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Arg Val Ser Phe Asp Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
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Thr Val Ser Ser
115
<210> 4
<211> 118
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Gln Lys Gln Leu Met Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly
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Gly Ala Ile Ser Ser Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys
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Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Arg Ile
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Gly Ala Ser Ser Asp Gly Ile Tyr Tyr Asn Leu Trp Gly Gln Gly Thr
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Leu Val Thr Val Ser Ser
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<210> 5
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<213> 人工序列(Artificial Sequence)
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Gln Ser Met Glu Glu Ser Gly Gly Arg Leu Val Lys Pro Asp Glu Thr
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Ile Ile Gly Thr Thr Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
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Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr
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Ala Asp Gly Val Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val
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Ser Ser
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<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
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Glu Trp Thr Lys Gln Val Pro Glu Met Ser
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<210> 7
<211> 16
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<213> 人工序列(Artificial Sequence)
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Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
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<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
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Val Ser Phe Asp Arg Phe Asp Tyr
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<210> 9
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<213> 人工序列(Artificial Sequence)
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Glu Trp Thr Arg Ala Val Pro Glu Met Ser
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<210> 10
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<213> 人工序列(Artificial Sequence)
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Glu Trp Thr Arg Val Val Pro Glu Met Ser
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<210> 11
<211> 10
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<213> 人工序列(Artificial Sequence)
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Gly Ile Asp Leu Ser Ser Tyr Ala Met Gly
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<213> 人工序列(Artificial Sequence)
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Ala Ile Ser Ser Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
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Glu Gly Ala Ser Ser Asp Gly Ile Tyr Tyr Asn Leu
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<213> 人工序列(Artificial Sequence)
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Gly Phe Ser Leu Ser Ser Tyr Ser Met Ile
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<211> 16
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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Tyr Tyr Ala Asp Gly Val Phe Asp Leu
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60
tcctgtgcag cctctgagtg gactaagcag gttccggaga tgagctgggt ccgccaggct 120
ccagggaagg gcctagagtg gattgggagt atctatcata gtgggagcac ctactacaac 180
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caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60
tcctgtgcag cctctgagtg gacgcgtgct gttccggaga tgagctgggt ccgccaggct 120
ccagggaagg gcctagagtg gattgggagt atctatcata gtgggagcac ctactacaac 180
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caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60
tcctgtgcag cctctgagtg gacgcgtgtt gttccggaga tgagctgggt ccgccaggct 120
ccagggaagg gcctagagtg gattgggagt atctatcata gtgggagcac ctactacaac 180
ccgtccctca agagtcgagt caccatctcc agagacaatt ccaagaacac gctgtatctg 240
caaatgaaca ccctgagagc cgaggacaca gccacgtatt actgtgcgag agtttcgttt 300
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<210> 20
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
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cagaagcagc tgatggagtc cgggggtcgc ctggtcacgc ctggaggatc cctgacactc 60
acctgcacag tctctggaat cgacctcagt agctatgcaa tgggctgggt ccgccaggct 120
ccagggaagg ggctggaatg gatcggagcc attagtagta gtggtaacac atactacgcg 180
agctgggcga aaggccgatt caccatctcc aaaacctcga ccacggtgga tctgaggatc 240
accagtccga caaccgagga cacggccacc tatttctgtg ccagagaggg tgctagtagt 300
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<210> 21
<211> 342
<212> DNA
<213> 人工序列(Artificial Sequence)
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cagtcgatgg aggagtccgg gggtcgcctt gtcaagcctg acgaaaccct gacactcacc 60
tgcaccgtct ctggattctc cctcagtagc tattcaatga tctgggtccg ccaggctcca 120
gggaaggggc tggaatggat cggaatcatt ggtactactg gtaacacata ctacgcgagc 180
tgggcaaaag gccgattcac catctccaag acatcgacca cggtggatct gaaaatgacc 240
agtctgacaa gcgaggacac ggccacctat ttctgtgcca gatattatgc tgatggtgtc 300
tttgacttgt ggggccaagg caccctggtc accgtctctt ca 342
Claims (13)
1.TIM-3单域抗体或抗原结合片段，其特征在于：
所述单域抗体或者抗原结合片段的CDR1为SEQ ID NO: 14所述的序列，CDR2为SEQ IDNO: 15所述的序列，CDR3为SEQ ID NO: 16所述的序列。
2.根据权利要求1所述的TIM-3单域抗体或抗原结合片段，其特征在于所述单域抗体或抗原结合片段的氨基酸序列为SEQ ID NO: 5所述的序列。
3.根据权利要求1或2所述的TIM-3单域抗体或抗原结合片段，其特征在于所述单域抗体或者抗原结合片段框架区的部分或全部为人源抗体的框架区，但仍然保持与所述单域抗体竞争相同的抗原表位的特性。
4.双特异性抗体或融合蛋白，其特征在于所述双特异性抗体或融合蛋白含有权利要求1-3任一项所述的单域抗体或抗原结合片段。
5.免疫偶联物，其特征在于所述免疫偶联物含有权利要求1-3任一项所述的单域抗体或抗原结合片段或含有权利要求4所述的双特异性抗体或融合蛋白。
6.如权利要求5所述的免疫偶联物，其特征在于所述免疫偶联物的效应分子选自荧光标记，放射性标记，亲和素，生物素，酶、毒素或化疗剂中的一种或几种。
7.一种核酸分子，其特征在于所述核酸分子能够编码如权利要求1-3任一项所述的单域抗体或抗原结合片段，或权利要求4所述的双特异性抗体或融合蛋白。
8.权利要求1-3任一项所述的单域抗体或抗原结合片段、权利要求4所述的双特异性抗体或融合蛋白、或权利要求5或6所述的免疫偶联物、或者权利要求7所述的核酸分子在制备免疫检查点抑制剂中的应用。
9.一种药物组合物，其特征在于所述药物组合物包含权利要求1-3任一项所述的单域抗体或抗原结合片段、权利要求4所述的双特异性抗体或融合蛋白、权利要求5或6所述的免疫偶联物或权利要求7所述的核酸分子中的一种或几种。
10.如权利要求9所述的药物组合物，其特征在于所述药物组合物还包括GITR、CD137、PD-1、PD-L1、OX40、CTLA4、TIGIT、LAG3、CD47、SIRPa免疫检查点抑制剂中的一种或几种。
11.如权利要求9所述的药物组合物，其特征在于所述药物组合物包括激活的或未激活的T细胞、经过遗传修饰的T细胞、经过体外诱导、激活与培养的人淋巴细胞。
12.宿主细胞，其特征在于所述宿主细胞携带多核苷酸分子并能编码权利要求1-3任一项所述的单域抗体或抗原结合片段、权利要求4所述的双特异性抗体或融合蛋白、权利要求5或6所述的免疫偶联物或权利要求7所述的核酸分子。
13.检测试剂、诊断试剂或诊断试剂盒，其特征在于包括权利要求1-3任一项所述的单域抗体或抗原结合片段、权利要求4所述的双特异性抗体和融合蛋白、权利要求5或6所述的免疫偶联物或权利要求7所述的核酸分子，检测生物样本中是否存在TIM-3蛋白和/或TIM3蛋白的含量。
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