CN118221816A - Lag3单克隆抗体及其应用 - Google Patents
Lag3单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明提供了多个能高亲和力地识别淋巴细胞激活基因3(LAG3)的单克隆抗体。所述抗体不仅亲和力高,而且能够阻断LAG3蛋白和其配体MHC‑II或FGL1的结合。本发明所提供的中和性单克隆抗体不仅具有良好的体外阻断活性,而且具有非常强的热稳定性,能够在体外促进PBMC细胞增殖和细胞因子释放,增强双特异性抗体和CAR‑T的肿瘤杀伤活性。
Description
技术领域
本发明属于生物技术领域,具体涉及识别LAG3的高亲和力单克隆抗体及其应用。
背景技术
淋巴细胞激活基因3(LAG3,CD223)是免疫球蛋白超家族的一员,是T细胞表面的一种重要的负调控因子。1990年LAG3在激活的NK细胞和T细胞上被发现,它包含4个胞外IgSF结构域和1个跨膜结构域以及1个胞内结构域,它在D4结构域和跨膜域中间可以通过蛋白酶ADAM10和ADAM17切割变为分泌型sLAG3。此外LAG3在DC细胞,B细胞,肿瘤浸润淋巴细胞,NK细胞,调节性T细胞,耗竭的CD8+T细胞等细胞上都检测到有表达。
在基因结构上LAG3同MHC-II有着大约20%的同源性,同时相比CD4蛋白,LAG3同MHC-II有着更高的亲和力。但是与CD4不同的是CD4主要表达在细胞表面,而LAG3却有很大一部分在胞内,当抗原刺激后LAG3迅速的转移到细胞表面行使功能。T细表面的LAG3分子上调表达引起了T细胞增殖的下调,由于LAG3同MHC-II分子亲和力相比CD4更高,同时通过抗体阻断LAG3同MHC-II相互作用可以显著的增强T细胞活性,因此过去普遍认为LAG3下调T细胞活性是通过和MHC-II分子相互作用。但是近年来发现一些针对LAG3同时不阻断其与MHC-II结合的抗体仍有一定的抗肿瘤活性,因此LAG3可能存在其他的配体来行使功能其中就包括Galectin-3,LSECtin,FGL1等。2019年《Cell》上的一篇文章表明FGL1可能也是的LAG3的主要抑制性配体(Fibrinogen-like Protein 1Is a Major Immune Inhibitory Ligandof LAG3,Wang J,et al,Cell,2019;176:334-347)。通过制备抗体阻断LAG3-MHC-II或LAG3-FGL1的相互作用,是肿瘤免疫治疗过程中的一种新策略。
发明内容
本发明的目的在于提供高亲和力识别LAG3的单克隆抗体。所提供的抗体包括抗体片段(例如单链可变区片段(scFv))和效应分子(例如毒素蛋白)的免疫缀合物。还提供了包括特异性结合LAG3的抗体的组合物、编码这些抗体的核酸分子、包含所述核酸分子的表达载体以及表达所述核酸分子的分离的宿主细胞。
本发明具体技术方案如下:
一种淋巴细胞激活基因3的单克隆抗体或其抗原结合片段,包括重链可变区和轻链可变区,所述单克隆抗体或其抗原结合片段包括SEQ ID NO:1所示的重链可变区CDR1、SEQ ID NO:2所示的重链可变区CDR2、SEQ ID NO:3所示的重链可变区CDR3,SEQ ID NO:4所示的轻链可变区CDR1、SEQ ID NO:5所示的轻链可变区CDR2、SEQ ID NO:6所示的轻链可变区CDR3。
人LAG3有三种已知的亚型(亚型1-3)。LAG3的三种亚型的核酸和氨基酸序列是已知的,包括GenBank登录号:NM_002286.6和NP_002277.4(亚型1);NM_001414176.1和NP_001401105.1(亚型2);NM_001414177.14和NP_001401106.1(亚型3)。本发明所述的抗体可结合所述三种人LAG3亚型的一种或多种,或其保守变体。
具体的,本发明所述的LAG3的抗体或其抗原结合片段为:
(1)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:7所示,轻链可变区氨基酸序列如SEQ ID NO:8所示;
或者,(2)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:9所示,轻链可变区氨基酸序列如SEQ ID NO:10所示。
或者,(3)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:11所示,轻链可变区氨基酸序列如SEQ ID NO:12所示。
或者,(4)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:13所示,轻链可变区氨基酸序列如SEQ ID NO:14所示。
或者,(5)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:15所示,轻链可变区氨基酸序列如SEQ ID NO:16所示。
本发明所述抗体是单链抗体、双链抗体、单克隆抗体或嵌合抗体。
本发明所述的单克隆抗体可以为任何同种型。可以为例如IgM或IgG抗体,例如IgG1或IgG2。可特异性结合LAG3的抗体的种类可根据已知的方法彼此转换(例如,IgG可转换为IgM)。种类转换还可用于使一种IgG亚类转换成另一亚类,例如从IgG1转换成IgG2。
本发明所述的抗体可以为:
(1)Fab,包含抗体分子的单价抗原结合片段的片段,其可以通过用木瓜蛋白酶消化完整抗体以产生完整轻链和一条重链的一部分而产生;
(2)Fab',可以通过用胃蛋白酶处理完整抗体、之后进行还原以产生完整轻链和重链的一部分而获得的抗体分子片段;每个抗体分子得到两个Fab'片段;
(3)(Fab')2,可以通过将完整的抗体用胃蛋白酶处理、但之后不进行还原而得到的抗体片段;F(ab')2是两个Fab'片段通过两个二硫键连接在一起的二聚体;
(4)Fv,含有表达为2条链的轻链可变区和重链可变区的基因程片段;
(5)单链抗体(例如scFv),含有轻链可变区和重链可变区并通过合适的多肽接头将其连接为遗传上融合的单链分子的基因工程分子;
(6)单链抗体的二聚体(scFv2),定义为scFv的二聚体(还被称为“微型抗体”);
(7)VH单结构域抗体,由重链可变区组成的抗体片段。
本领域技术人员会了解,可以制备抗体的保守变体。可以在所述VH和/或VL区进行氨基酸置换(例如1个、2个、3个、4个或5个氨基酸置换),置换后的VH和VL仍然保留结合LAG3的能力,或者对LAG3的结合能力更强。功能类似的氨基酸的保守置换是本领域普通技术人员所熟知的。以下六组是被认为是互为保守置换的氨基酸的实例:
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T);
2)天冬氨酸(D)、谷氨酸(E);
3)天冬酰胺(N)、谷氨酰胺(Q);
4)精氨酸(R)、赖氨酸(K);
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V);
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本发明另一目的在于提供一种重组蛋白,所述的重组蛋白包括本发明所述的抗体或其抗原结合片段和协助表达和/或纯化的标签序列。所述的标签序列包括但不限于6xHis标签。
本发明另一目的在于公开本发明所述的单克隆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、慢性病再感染或肿瘤的约物。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。
本发明所述可检测的标记物是可以被同位素分析仪、酶标仪、生物发光检测仪、化学发光检测仪、电化学发光检测仪、荧光分析仪器检测,或裸眼可视化的物质,这些物质包括但不限于放射性同位素(如3H、14C、15N、35S、90Y、、99Tc、111In、125I、131I)、可用于检测的酶类(如辣根过氧化物酶、β-半乳糖苷酶、碱性磷酸酶、葡萄糖氧化酶等)、荧光蛋白(如绿色荧光蛋白(GFP)、黄色荧光蛋白(YFP)、别藻蓝蛋白APC、藻红蛋白PE)、生物发光标记物(如萤光素酶)、荧光化合物(如荧光素、异硫氰酸荧光素、罗丹明、5-二甲基氨基-1-萘磺酰氯、藻红蛋白、荧光染料Cy3、Cy5、稀土无机发光材料、量子点等)、生物素、磁性试剂(例如钆)、电化学发光试剂(如三联吡啶钌)、胶体金。
效应分子可使用本领域技术人员已知的任何方式连接至本发明所述的抗体。例如,所述抗体可以被功能性地连接(通过化学偶联、基因融合、非共价缔合或其它)到一种或多种其他分子实体。根据效应分子的化学结构,连接所述效应分子和抗体的方法有所不同。多肽一般含有多个官能团;例如羧酸(COOH)、游离氨基(-NH2)或巯基(-SH),其可用于与抗体上合适的官能团发生反应以与所述效应分子结合。或者,可将所述抗体衍生化以暴露或连接另外的反应性官能团。所述衍生化可包括连接任意的多种已知接头分子。所述接头可以是用于接合所述抗体与效应分子的任何分子。所述接头能够与所述抗体和效应分子形成共价键。合适的接头对于本领域技术人员是熟知的,包括但不限于直链或支链的碳接头、杂环碳接头或肽接头。在所述抗体和效应分子是多肽的情况下,所述接头可以通过其侧基(例如通过半胱氨酸的二硫键)接合到组成氨基酸或者接合至末端氨基酸的α碳氨基和羧基。通常,衍生化所述抗体或其部分使得与靶抗原的结合不受所述衍生化或标记的不利影响。
在某些情况中,当所述免疫缀合物已经到达其靶位点时,需要从所述抗体释放所述效应分子。因此,在这些情况中,免疫缀合物会包含所述靶位点附近可切割的键。切割所述接头以将所述效应分子从所述抗体释放可以由酶活性引起,或由所述免疫缀合物在所述靶细胞内或靶位点附近所处的条件引起。
本发明另一目的在于提供一种多核苷酸,编码本发明所述的抗体,重组蛋白或者免疫缀合物。
本发明另一目的在于提供一种载体,含有本发明所述的多核苷酸。所述的载体包括:细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。
本发明另一目的在于提供一种药物组合物,包括本发明所述的抗体或其抗原结合片段、重组蛋白、双特异性抗体、免疫缀合物、多核苷酸、载体或遗传工程化的宿主细胞中的一种或几种。所述药物组合物还包括药学上可接受的载体。所述抗体、重组蛋白、双特异性抗体、免疫缀合物、多核苷酸、载体或遗传工程化的宿主细胞可溶于水性载体,例如缓冲盐水等。还可以含有接近生理条件所需要的可药用辅料,例如pH调节剂和缓冲试剂等,乙酸钠、氯化钠、氯化钾、氯化钙或乳酸钠等。
本发明所述的药物或药物组合物可以与GITR、CD137、PD-1、PD-Ll、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。
本发明另一目的在于提供本发明所述的抗体或其抗原结合片段、重组蛋白、免疫缀合物、多核苷酸、载体或遗传工程化的宿主细胞在制备自身免疫疾病、病毒感染或癌症的治疗药物或诊断试剂中的应用。
所述癌症为肝癌、胃癌、结直肠癌、肺癌、卵巢癌,或表达LAG3的任何其他类型的癌症。
本发明公开的单克隆抗体或其抗原结合片段还可用于制备嵌合抗原受体(CAR;也称为嵌合T细胞受体、人造T细胞受体或嵌合免疫受体)或双特异性抗体。
本发明所述的药物或约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检测试剂、诊断试剂或诊断试剂盒包含本发明所述的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、核酸分子或载体中的一种或几种,利用其检测生物样本中是否存在LAG3蛋白和/或检测LAG3蛋白的含量。
本发明具体实施例中公开了高亲和力的LAG3单克隆抗体。本发明还公开了该抗体体外活性以及联合hYP7-OKT3双特性抗体(T细胞型双特异性抗体在肝癌和小细胞肺癌中的抗肿瘤活性研究,陈鑫,2020,华中农业大学博士学位论文)治疗肝癌的效果。所述的抗体和组合物可以用于治疗LAG3表达阳性的肿瘤。
本发明所提供的抗体和组合物可用于多种目的,例如用于肿瘤的分子诊断。所述样品可以为任何样品,包括但不限于来自活组织检查、尸体解剖和病理标本的组织。生物样品还包括组织的切片,例如为组织学目的获取的冷冻切片。生物样品还包括体液,例如血液、血清、血浆、痰、脊髓液或尿。生物样品一般获自哺乳动物,包括人,非人灵长类,小鼠等。
本发明优点:
本发明所提供的单克隆抗体不仅亲和力高(Kd值均在nM和pM级,甚至更高),而且热稳定性好。本发明所提供的单克隆抗体能够以高活性阻断LAG3与其配体MHC-II或FGL1的相互作用。本发明所提供的单克隆抗体与双特异性抗体或PD1抗体联用可以明显增强单药抗肿瘤活性。
附图说明
图1.本发明所述LAG3单克隆抗体M234 scFv-HF与LAG3ECD-hFc亲和力测定。
图2.本发明所述LAG3单克隆抗体M234 scFv-HF与LAG3D1D2-hFc亲和力测定。
图3.本发明所述M234 IgG与Bio-LAG3D1D2-hFc亲和力测定。
图4.本发明所述M234 IgG结合激活的PBMC细胞。
图5.Bio-LAG3D1D2-hFc与FGL1-hFc亲和力。
图6.本发明所述M234 IgG阻断BioLAG3D1D2-hFc与FGL1-hFc结合的能力。
图7.M234 IgG阻断BioLAG3D1D2-hFc与Raji细胞表面MHC-II互作。
图8.M234 IgG抑制Huh-7肿瘤细胞在NOD-SCID小鼠体内的生长。
图9.M234 IgG治疗Huh7荷瘤小鼠后的体重变化。
图10.人源化M234单克隆抗体的亲和力检测。
具体实施方式
本发明公开描述了结合LAG3的单克隆抗体的制备和鉴定。具体的实施方案公开了靶向LAG3单克隆抗体的分离和表征。
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。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。
缩写
CDR互补决定区
CTL细胞毒性T淋巴细胞
ELISA酶联免疫吸附测定
FACS荧光激活细胞分选
GPC3磷脂酰肌醇蛋白聚糖3
BLI 生物膜干涉技术
HCC 肝细胞癌
hFc人Fc
Ig免疫球蛋白
mAb单克隆抗体
PE藻红蛋白
实施例1本发明所述LAG3的单克隆抗体的制备
本实施例描述了针对肿瘤相关LAG3的高亲和力单克隆抗体的制备。
使用293F细胞表达、纯化LAG3ECD和LAG3D1D2融合hFc的截短蛋白(截短信息参考NP_002277.4)。以LAG3ECD-hFc和LAG3D1D2-hFc蛋白免疫6周龄小鼠,每次免疫50μg,每次间隔3周,免疫2次之后采集小鼠的脾脏,提取脾脏中的总RNA,逆转录为cDNA,作为建立噬菌体抗体文库的模板,并构建小鼠抗体噬菌体文库(库容量4.6×109)。利用LAG3ECD-hFc蛋白作为抗原,对噬菌体文库进行3次淘选后,随机挑取单个克隆进行测序,获得高亲和力LAG3单克隆抗体M234。
抗体M234互补决定区(CDR区)以及可变区氨基酸序列如表1和2所示:
表1.LAG3 M234单克隆抗体的CDR氨基酸序列(根据Kabat和IMGT)
表2.LAG3M234单克隆抗体的可变区氨基酸序列
实施例2本发明所述LAG3的单克隆抗体M234 scFv-HF对LAG3蛋白亲和力
使用HB2151原核表达实施例1的单克隆抗体scFv-HF,表达产物用Ni-NTA层析柱(GE healthcare)进行纯化。利用ELISA法对抗体亲和力进行检测。
首先将LAG3ECD-hFc蛋白或LAG3D1D2-hFc蛋白分别包被在ELISA板底,随后分别加入上述制得的M234 scFv-HF,首孔200nM,1:3梯度稀释,与之孵育,使用HRP标记的山羊抗FLAG抗体作为二抗进行检测。使用TMB显色液显色后用多功能酶标仪检测吸光度,最后使用GraphPad Prism分析结果。结果如图1和图2所示,M234与LAG3ECD-hFc和LAG3D1D2-hFc亲和力分别为0.2nM和0.14nM。
实施例3本发明所述M234 IgG对LAG3蛋白的亲和力
将实施例1得到的抗体M234重链可变区连接IgG1型抗体的重链恒定区,轻链可变区连接轻链恒定区后分别构建到有两启动子的IgG表达载体上,使用293F细胞进行表达,表达产物用ProteinA层析柱(GE healthcare)进行纯化。利用ELISA法对抗体亲和力进行检测。
首先将M234 IgG蛋白分别包被在ELISA板底,同时使用NHS-Biotin(生工生物)冰上标记LAG3D1D2-hFc蛋白,得到生物素标记的Bio-LAG3D1D2。随后将生物素标记的BioLAG3D1D2-hFc以首孔200nM 1:3梯度稀释,与之孵育,使用HRP标记的链霉亲和素作为二抗进行检测。使用TMB显色液显色后用多功能酶标仪检测吸光度,最后使用GraphPad Prism分析结果。结果如图3所示,BioLAG3D1D2-hFc与M234单克隆抗体亲和力为0.45nM。
实施例4M234 IgG稳定性的检测
Tm值是定量描述蛋白的热稳定性的参数,是成药性评价中最为常用的指标之一。Tm值越高意味着蛋白构象越稳定。利用Nano Temper公司的Prometheus NT.48测定实施例1制得的抗体的稳定性:将抗体浓度稀释成500μg/ml,然后上样,1℃/min的速度,从25℃上升到95℃。最后得出抗体的Tm值。结果如表3所示,抗体M234的Tm1为61.67℃,Tm2为87.73℃,聚集温度为89.12℃,M234的热稳定性和聚集性均强于已上市的LAG3单克隆抗体Relatlimab。
表3本发明所述单克隆抗体的Tm值
实施例4FACS检测本发明所述M234 IgG与LAG3阳性细胞系的结合
将PBMC细胞用CD3Ab/CD28Ab和IL-2激活后使用1640培养基(Invitrogen,Carlsbad,CA)添加10%胎牛血清(HyClone,Logan,UT)、1%L-谷氨酰胺和1%青霉素-链霉素(Invitrogen,Carlsbad,CA)进行培养。收获细胞后,将实施例3制得的抗体与激活的PBMC细胞进行结合,加入PE标记的羊抗人Fc二抗,检测结合至细胞表面上的抗体。结果如图4所示,实施例3制得的M234与PBMC细胞亲和力为0.10nM。
实施例5本发明所述M234 IgG阻断LAG3与FGL1的结合
首先将FGL1包被在ELISA板底,同时使用NHS-Biotin(生工生物)冰上标记LAG3D1D2-hFc蛋白,得到生物素标记的Bio-LAG3D1D2。Bio-LAG3D1D2以1:2的稀释度从1000nM开始稀释,加入ELISA板底后37℃孵育30min,使用HRP标记的链霉亲和素作为二抗进行检测。使用TMB显色液显色后用多功能酶标仪检测吸光度,最后使用GraphPad Prism分析结果。结果显示LAG3-FGL1亲和力如图5所示。
抗体阻断实验首先将FGL1包被ELISA板底,然后首孔200nM 1:3梯度稀释分别将实施例3制得的M234 IgG与100nM BioLAG3D1D2共孵育30min后加入ELISA板中,用HRP标记的链霉亲和素作为二抗进行检测。使用TMB显色液显色后用多功能酶标仪检测吸光度,最后使用GraphPad Prism分析结果。抗体阻断活性如图6所示,结果显示抗体M234具有LAG3-FGL1阻断活性,IC50为15nM。
实施例6本发明所述M234 IgG阻断LAG3与MHC-II的结合
Raji细胞天然高表达MHC-II,阻断实验使用Raji细胞。使用1640培养基(Invitrogen,Carlsbad,CA)添加10%胎牛血清(HyClone,Logan,UT)、1%L-谷氨酰胺和1%青霉素-链霉素(Invitrogen,Carlsbad,CA)进行Raji细胞的培养。10μg/ml LAG3ECD-hFc蛋白与梯度稀释的实施例3制得的抗体M234 IgG共孵育30min,然后加入收集到的Raji细胞中冰上孵育30min。最后加入PE标记的羊抗人二抗,检测结合至细胞表面上的抗体。结果如图7所示,抗体M234具有LAG3-MHC-II阻断活性,其阻断IC50为6.2nM。
实施例7M234 IgG在小鼠体内抑制肿瘤的生长
选择肝癌细胞系Huh-7作为抗体抑瘤活性的测试细胞。Huh-7细胞使用DMEM培养基(Invitrogen,Carlsbad,CA)添加10%胎牛血清(HyClone,Logan,UT)、1%L-谷氨酰胺和1%青霉素-链霉素(Invitrogen,Carlsbad,CA)进行培养。细胞消化后每只小鼠注射5×106个细胞,待到成瘤后,每只小鼠注射5×106个PBMC细胞,以0.2mg/kg hYP7-OKT3双特异抗体(抗体制备过程参考陈鑫博士的博士学位论文(T细胞型双特异性抗体在肝癌和小细胞肺癌中的抗肿瘤活性研究,华中农业大学,2020))联合10mg/kg实施例3制备的LAG3单克隆抗体进行免疫治疗。小鼠肿瘤生长曲线如图8和表4所示,体重变化如图9所示。结果显示本发明所述的M234 IgG单克隆抗体联合双特异性抗体可以显著增强抗肿瘤活性。
表4小鼠肿瘤体积
实施例8M234的人源化及人源化M234 IgG的亲和力检测
将单克隆抗体M234使用人源抗体Germline骨架替换的方法进行人源化改造,得到人源化Hu1M234的序列,对人源化M234的重链进行回复突变得到Hu2M234,对人源化M234的轻链回复突变得到Hu3M234,轻链和重链同时回复突变得到Hu4M234,人源化序列如表5所示。
将人源化M234的重链可变区连接人源IgG1型抗体的重链恒定区并在Fc区后添加Flag标签,将人源化M234的轻链可变区连接人源抗体κ轻链恒定区,然后分别克隆到IgG表达载体上,构建IgG表达质粒,并使用293F细胞进行表达,表达产物用ProteinA层析柱(GEhealthcare)进行纯化。利用ELISA方法对HuM234 IgG的亲和力进行检测。
表5人源化M234的VH和VL序列
首先将LAG3ECD-hFc蛋白包被在ELISA板底,随后加入人源化的M234 IgG-Flag抗体,首孔200nM,其余各孔浓度按照1:3梯度稀释,与包被的LAG3ECD蛋白孵育,然后使用HRP标记的FLAG抗体进行检测,使用TMB显色液显色后用多功能酶标仪检测吸光度,最后使用GraphPad Prism分析实验结果。结果如图10所示,人源化的HuM234 IgG亲和力分别为0.96nM,0.52nM,0.59nM,0.16nM,其中Hu4M234亲和力与原始M234抗体亲和力基本一致。
Claims (17)
1.靶向人淋巴细胞激活基因3的单克隆抗体或其抗原结合片段,包括重链可变区和轻链可变区,其特征在于:
所述单克隆抗体或其抗原结合片段包括SEQ ID NO:1所示的重链可变区CDR1、SEQ IDNO:2所示的重链可变区CDR2、SEQ ID NO:3所示的重链可变区CDR3,SEQ ID NO:4所示的轻链可变区CDR1、SEQ ID NO:5所示的轻链可变区CDR2、SEQ ID NO:6所示的轻链可变区CDR3。
2.根据权利要求1所述的单克隆抗体或其抗原结合片段,其特征在于:
(1)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:7所示,轻链可变区氨基酸序列如SEQ ID NO:8所示;
或者,(2)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:9所示,轻链可变区氨基酸序列如SEQ ID NO:10所示。
或者,(3)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:11所示,轻链可变区氨基酸序列如SEQ ID NO:12所示。
或者,(4)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:13所示,轻链可变区氨基酸序列如SEQ ID NO:14所示。
或者,(5)所述抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:15所示,轻链可变区氨基酸序列如SEQ ID NO:16所示。
3.根据权利要求1所述的单克隆抗体或其抗原结合片段,其特征在于所述抗体是单链抗体、双链抗体、嵌合抗体或人源化抗体。
4.根据权利要求1所述的单克隆抗体或其抗原结合片段,其特征在于所述抗体是VH单结构域抗体、Fab片段、Fab'片段、F(ab)'2片段、单链可变片段scFv或二硫键稳定的可变片段dsFv。
5.一种重组蛋白,其特征在于,所述的重组蛋白包括权利要求1-4任一项所述的单克隆抗体或其抗原结合片段和协助表达和/或纯化的标签序列。
6.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1-4任一项所述的单克隆抗体或其抗原结合片段或者含有权利要求5所述的重组蛋白。
7.免疫偶联物,其特征在于所述免疫偶联物含有权利要求1-4任一项所述的单克隆抗体或其抗原结合片段、权利要求5所述的重组蛋白或含有权利要求6所述的双特异性抗体或融合蛋白。
8.根据权利要求7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签放射性标记, 亲和素, 生物素, 酶、毒素或化疗剂中的一种或几种。
9.一种多核苷酸, 其特征在于其编码根据权利要求1-4任一项所述的抗体或其抗原结合片段,或权利要求5所述的重组蛋白, 或权利要求6所述的双特异性抗体或融合蛋白, 或者权利要求7或8所述的免疫缀合物。
10.一种载体, 其特征在于含有权利要求9所述的多核苷酸。
11.权利要求1-4任一项所述的单克隆抗体或其抗原结合片段、权利要求5所述的重组蛋白、权利要求6所述的双特异性抗体或融合蛋白、权利要求7或8所述的免疫偶联物、权利要求9所述的多核苷酸或者权利要求10所述的载体在制备免疫检查点抑制剂中的应用。
12.根据权利要求11所述的应用, 其特征在于所述免疫检查点抑制剂为诊断或治疗免疫性疾病、慢性病毒感染或肿瘤的试剂或药物。
13.一种药物组合物, 其特征在于所述约物组合物包含权利要求1 - 4所述的单克隆抗体或其抗原结合片段、权利要求5所述的重组蛋白、权利要求6所述的双特异性抗体或融合蛋白、权利要求7或8所述的免疫偶联物、权利要求9所述的多核苷酸或权利要求10所述的载体中的一种或几种。
14.根据权利要求13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、TI GIT、LAG3、CD47、SIRPa免疫检查点抑制剂中的一种或几种。
15.根据权利要求13所述的药物组合物, 其特征在于所述药物组合物还包括激活的或未激活的T细胞、经过遗传修饰的T细胞或者经过体外诱导、激活与培养的人淋巴细胞中的一种或几种。
16.宿主细胞, 其特征在于所述宿主细胞携带权利要求9所述的多核苷酸或权利要求10所述的载体。
17.LAG-3蛋白检测试剂、诊断试剂或诊断试剂盒, 其特征在于包含权利要求1-4所述的单克隆抗体或其抗原结合片段、权利要求5或6所述的双特异性抗体和融合蛋白、权利要求7或8所述的免疫偶联物、权利要求9所述的核酸分子或权利要求10所述的载体中的一种或几种。
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