CN113234152B - 程序性死亡受体-配体1(pd-l1)特异性结合多肽及应用 - Google Patents
程序性死亡受体-配体1(pd-l1)特异性结合多肽及应用 Download PDFInfo
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Abstract
本发明涉及针对程序性死亡受体‑配体1(programmed cell death‑Ligand 1，PD‑L1)特异性结合多肽及其应用，属于生物技术领域。其中，所述多肽由葡萄球菌蛋白A(SPA)免疫球蛋白结合区域通过突变1‑20个氨基酸残基获得(多肽序列见SEQ ID：2‑5)，能够特异性结合PD‑L1并阻断PD‑1/PD‑L1相互作用。本发明通过四轮富集从全合成亲和小体多肽分子库淘选获得PD‑L1特异性结合多肽编码序列，并利用原核表达系统对获得的多肽进行了大量制备，该多肽与PD‑L1特异结合，与BSA蛋白无明显亲和，可用于PD‑L1检测。所述多肽同时具有PD‑1/PD‑L1相互作用阻断能力，其对PD‑1/PD‑L1相互作用阻断能力随浓度增加而增强。
Description
技术领域
本发明属于生物技术领域，具体涉及特异性结合程序性死亡受体-配体1(programmed cell death-Ligand 1，PD-L1)的多肽、或其变体、或其功能性片段，以及所述多肽、或其变体、或功能性片段的应用。
背景技术
免疫检查点(immune checkpoint)是免疫系统中通过共信号(共刺激或共抑制)分子调控T细胞免疫应答强度的信号通路，在正常生理条件下对维持自身免疫耐受至关重要。程序性死亡蛋白1(programmed cell death 1，PD-1)是一个重要免疫细胞活化负调控分子，可通过与其配体结合而发挥免疫抑制作用。PD-1主要表达于激活的效应T细胞、调节性T细胞、B细胞、单核细胞、和天然杀伤T细胞表面，不表达于静息T细胞，但可在T细胞活化的数小时内检测到(Keir ME，Butte MJ，et al.PD-1 and its ligands in tolerance andimmunity.Annual Review of Immunology，2008，26：677-704.)。PD-1有两个配体，即PD-L1与PD-L2。其中，PD-L1广泛表达于T细胞、B细胞、单核细胞、巨噬细胞、多种肿瘤细胞等。PD-1和PD-L1的结合抑制了CD8+细胞毒性淋巴细胞(CTL)的增殖、存活和效应功能，并促进肿瘤浸润性淋巴细胞(TIL)的凋亡(Kim JW，Nam KH，et al.Prognostic implications ofimmunosuppressive protein expression in tumors as well as immune cellinfiltration within the tumor microenvironment in gastric cancer.GastricCancer，2016，19(1)：42-52)。许多肿瘤细胞系及肿瘤细胞通过高表达PD-L1，削弱机体肿瘤免疫应答，从而导致肿瘤免疫逃逸发生。
近年来，以PD-1/PD-L1免疫检查点为靶点的免疫检查点阻断治疗在肿瘤免疫治疗领域取得了极大的成功，其中PD-1/PD-L1单克隆抗体抑制剂在肿瘤临床治疗中疗效显著(Liu B，Song Y，et al.Recent development in clinical applications of PD-1 andPD-L1 antibodies for cancer immunotherapy.Journal of Hematology&Oncology，2017，10(1)：174)，包括观察到肿瘤明显缩小、使肿瘤患者体内产生了持久的免疫应答等，但是作为一种四多肽链大蛋白(分子量为～150kDa)，单抗药物存在着与其复杂分子结构相关的固有缺点：①结构复杂，制备较为困难，生产成本增加，导致治疗费用过高，广泛应用受限。②稳定性不好，对外界条件高度敏感，一些常见的外界干扰都会导致抗体活性丧失，需要特殊的运输和储存条件保证其活性。③分子较大，穿透肿瘤组织能力差，不能有效运抵肿瘤组织内部，肿瘤位点药物浓度很低，对实体瘤治疗效果欠佳。
亲和小体(affibody)分子是一类新型人工合成的功能上类似于抗体的亲和性配体，由葡萄球菌蛋白A(Staphylococcal protein A，SPA)的免疫球蛋白结合区域B段衍化而来(Nilsson B，Moks T et al.A synthetic IgG-binding domain based onstaphylococcal protein A.Protein Engineering，1987，1(2)：107-113)。Affibody分子为单链结构，由58个氨基酸残基组成，相对分子质量约为6.5kDa，结构中含有3个α-螺旋，其受体结合部位包含13个氨基酸残基，分别是第一条α-螺旋的Q9(谷氨酰胺)、Q10(谷氨酰胺)、N11(天冬酰胺)、F13(苯丙氨酸)、Y14(酪氨酸)、L17(亮氨酸)、H18(组氨酸)和第二条α-螺旋的E24(谷氨酸)、E25(谷氨酸)、R27(精氨酸)、N28(天冬酰胺)、Q32(谷氨酰胺)、K35(赖氨酸)，将这13个氨基酸残基进行饱和突变，所形成的突变体就构成了affibody的分子库，不同的affibody分子分别对不同靶分子具有特异性结合活性和高度亲和力(Nord K，Nilsson J et al.A combinatorial library of an alpha-helical bacterialreceptor domain.Protein Engineering，1995，8(6)：601-608)。多种重要蛋白质的特异结合affibody分子已被成功筛选到，包括胰岛素、纤维蛋白(fibrinogen)、转铁蛋白(TRF)、肿瘤坏死因子α(TNF-α)、人血清白蛋白、白细胞介素-8(IL-8)、gpl20、CD28、IgA、IgE、IgM、人表皮生长因子受体2(HER2)和表皮生长因子受体(EGFR)等。相比于抗体，亲和小体affibody具有相对分子量小、稳定性高、应用灵活、易制备等优势，在生物科学的许多领域中得到广泛应用。因此，开发PD-L1特异性结合亲和小体多肽，将为研发PD-1/PD-L1免疫检查点多肽类阻断药物及肿瘤PD-L1非入侵性精准检测奠定重要的物质基础和必要的数据支撑。
发明内容
本发明利用全合成亲和小体多肽噬菌体展示技术平台，通过四轮富集淘选出一系列PD-L1特异性结合多肽，获得多肽序列为SEQ ID No：2-5。利用原核大肠杆菌表达系统制备PD-L1特异性结合多肽，通过ELISA结合实验验证淘选到的亲和小体多肽与PD-L1的结合能力及结合特异性。结果表明，所获得的4个亲和小体多肽均可与PD-L1特异性结合，且可有效阻断PD-1/PD-L1相互作用，可用于抑制PD-1/PD-L1免疫抑制信号通路而使免疫增强或PD-L1的检测。
为实现上述目的，本发明所采用的技术方案如下：
针对PD-L1特异性结合的多肽序列为由葡萄球菌蛋白A(SPA)免疫球蛋白结合区域通过突变1-20个氨基酸残基获得，多肽序列如SEQ ID No：2-5，具有PD-L1高特异亲和活性。
所述的PD-L1特异性结合多肽序列，包括所述的多肽、或其变体、或其功能性片段，及以该多肽、或其变体、或其功能片段为核心，任何对该多肽序列所进行的调整及修饰；修饰材料包括但不限制在特定蛋白质、酶、荧光蛋白、荧光发光基团、生物素、放射相关基团、纳米材料。
本发明还提供了所述PD-L1特异性结合多肽、或其变体、或其功能性片段的制备方法。在不实质性影响PD-L1亲和特性的前提下，本领域技术人员可以对本发明的序列替换、添加和/或缺失一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述多肽或其功能性片段之序列的变体。它们都被视为包括在本发明保护的范围内。
所述PD-L1特异性结合多肽序列用于PD-L1的检测，包括但不局限于活体、组织水品、细胞和亚细胞水平的分子影像学检测。
一种所述的多肽在PD-L1检测中的应用。
一种所述的多肽在抑制PD-1/PD-L1免疫抑制信号通路而使免疫增强中的应用。
本发明的优点和积极效果：
1、本发明基于噬菌体展示技术，通过四轮富集从全合成亲和小体多肽分子库淘选出了PD-L1特异性结合多肽，该多肽特异亲和力好，且相比于单克隆抗体具有制备成本低、稳定性好等显著优点。
2、本发明所得到的PD-L1特异结合多肽或其变体、或其功能性片段作为活体、组织水品、细胞和亚细胞水平的分子影像学检测中PD-L1的特异性识别元件，也可作为PD-1/PD-L1免疫抑制信号通路阻断剂增强机体免疫。
附图说明
图1为噬菌体展示筛选特异性结合PD-L1的多肽过程中每一轮富集倍数。
图2为筛选到的阳性噬菌体克隆噬菌体ELISA结果，BSA为阴性对照。
图3为特异性结合PD-L1的多肽纯化结果。
图4为特异性结合PD-L1多肽在体外与PD-L1亲和力及特异性验证结果。
图5为特异性结合PD-L1多肽阻断PD-1/PD-L1相互作用结果。
图6为不同浓度特异性结合PD-L1多肽阻断PD-1/PD-L1相互作用结果。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的保护范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1
PD-L1特异结合多肽的淘选与鉴定。
1、突变体构建及单克隆挑选
针对葡萄球菌蛋白A的免疫球蛋白结合区域多肽第9、10、11、13、14、17、18、24、25、27、28、32和35位氨基酸残基设计以NNK为密码子的简并引物，利用简并引物通过over-lapPCR得到亲和小体多肽文库的编码基因。将多肽突变体编码基因克隆至噬菌体展示载体，重组质粒电转化至TG1宿主细胞获得库容为5×107的亲和小体多肽突变体文库。利用辅助噬菌体将亲和小体多肽突变体进行展示，进而得到多肽突变体噬菌体展示文库。
以PD-L1胞外域蛋白(购自Sino Biological，货号为10084-H08H)为靶标，加入亲和小体噬菌体展示文库，经过四轮富集淘选，第四轮捕获的噬菌体较第一轮富集了200倍，收集四轮富集得到的噬菌体上清液，通过间接ELISA实验针对每一轮噬菌体对PD-L1的亲和力进行验证，结果见附图1，噬菌体上清液与PD-L1亲和力逐轮增加。
2、噬菌体ELISA检测多肽与PD-L1的结合能力
经过四轮淘选，挑取单克隆，接种至1mL 2×YTAG培养基(1.6g胰蛋白胨，1g酵母提取物，0.5g氯化钠定容至100mL，高压灭菌；再加入终浓度为2％葡萄糖，100μg/mL氨苄青霉素)中，37℃，180rpm过夜培养；将培养物按照1∶50接种至5mL 2×YTAG培养基中，37℃，180rpm培养至对数期；按照1∶20加入辅助噬菌体M13KO7(购自New England Biolabs，货号)，37℃静置30min进行感染，然后37℃，180rpm震荡培养30min；取1mL细菌噬菌体培养物，7000rpm，离心10min，弃上清；用5mL 2×YTAK培养基(1.6g胰蛋白胨，1g酵母提取物，0.5g氯化钠定容至100mL，高压灭菌；再加入终浓度为100μg/mL氨苄青霉素和50μg/mL卡那霉素)重悬菌体，30℃，185rpm，过夜培养。将培养物10000rpm，4℃，离心10min，收集上清即为展示多肽突变体的噬菌体单克隆。
用pH 9.6的碳酸盐buffer稀释PD-L1和BSA(阴性对照)至5μg/mL，包被96孔酶标板(购自Nunc，货号)，4℃过夜；用3％脱脂奶粉-PBS封闭，37℃ 2h；加入收集的噬菌体单克隆上清，室温孵育2h；加入1∶2000稀释的HRP标记的抗M13抗体(购自义翘神州，货号11973-MM05T-H)，室温孵育2h；加入TMB底物显色10min；加入2M H2SO4终止反应，在酶标仪读取OD450。ELISA过程中，每一步都要用PBST(PBS+0.5％吐温-20)洗板4次。对于ELISA结果，选择阳性值/阴性值＞2.1的克隆为阳性克隆，一共选择了20株阳性克隆，各阳性克隆的具体数据结果见附图2。
3、DNA测序
将优良阳性噬菌体克隆进行DNA测序，其中个别克隆为相同序列，分别将不同序列命名为ZPD-L1 A2，ZPD-L1 D3、ZPD-L1 E3、ZPD-L1 GA2、ZPD-L1 GA2、ZPD-L1 GE2、ZPD-L1 P2、ZPD-L1 P10、ZPD-L1 P3、ZPD-L1 P9，根据DNA测序结果及密码子表可获得氨基酸序列。
实施例2
PD-L1特异结合多肽的表达与纯化。
从实施例1中鉴定出来的10种特异结合PD-L1的Affibody多肽作为进一步研究的对象，利用原核表达系统进行多肽的大量表达。将10种Affibody编码基因克隆至表达载体选择pET-21b(实验室保存)，通过双酶切组合DNA测序技术鉴定重组表达载体是否构建成功。表达宿主选择E.coli BL21(DE3)(本实验室保存)，将测序正确的重组表达载体转化E.coli BL21(DE3)，挑取单克隆至5mL LB/A培养基(1g胰蛋白胨，0.5g酵母提取物，1g氯化钠定容至100mL，高压灭菌；再加入终浓度100μg/mL氨苄青霉素)中，37℃，180rpm过夜培养；将培养物按照1∶50接种至200mL LB/A培养基中，37℃，180rpm培养至对数期，加入终浓度0.1mM IPTG，20℃，180rpm过夜诱导。将培养物于4℃，4400rpm离心收集菌体；用20mL预冷的PBS(8g NaCl，Na2HPO4·12H2O，0.2g KCl，0.24gNaH2PO4·2H2O)重悬菌体，超声破碎(超3s，停10s)20min后，12000rpm离心15min，收集上清。
Affibody多肽的纯化选择Ni-TED柱(购自生工生物工程股份有限公司，货号C600803)。首先用10个柱体积的磷酸盐缓冲液平衡Ni-TED柱；收集的蛋白破碎上清经0.45μm滤膜过滤后进行上样，流穿4次，流穿液保留进行SDS-PAGE分析；用含20mM咪唑的磷酸盐缓冲液(20个柱体积)清洗柱子，洗掉杂蛋白；准备含50mM、100mM、250mM、400mM咪唑的磷酸盐缓冲液，分别用2个柱体积洗脱目的蛋白。将不同浓度咪唑收集到的蛋白样品进行SDS-PAGE分析；根据SDS-PAGE结果，选择纯度高的蛋白，用磷酸盐缓冲液进行透析，透析后的蛋白进行SDS-PAGE分析，结果见附图3，10种亲和小体多肽均纯化成功，其中ZPD-L1 J1，ZPD-L1 J2为阳性对照(实验室现存PD-L1特异亲和Affibody)。
实施例3
PD-L1特异性结合多肽结合特性分析。
在本实验中，以实施例2中表达纯化的8种PD-L1特异结合Affibody多肽作为进一步研究对象，利用ELISA方法检测其与PD-L1的亲和特性，同时选择BSA为无关抗原阴性对照，具体操作入下：
用pH 9.6的碳酸盐buffer稀释PD-L1/BSA至10μg/mL，包被96孔酶标板，4℃过夜；用3％脱脂奶粉-PBS封闭，37℃2h；加入带有HA标签的ZPD-L1 A2，ZPD-L1 D3、ZPD-L1 E3、ZPD-L1 GA2、ZPD-L1 GA2、ZPD-L1 GE2、ZPD-L1 P2、ZPD-L1 P10、ZPD-L1 P9重组蛋白，37℃孵育2h；加入稀释的HA抗体(购自生工生物工程股份有限公司，货号D191044)，37℃孵育2h；加入稀释的HRP标记的兔抗小鼠二抗(购自生工生物工程股份有限公司，货号D110098)，37℃孵育45min；加入TMB底物显色10min；加入2M H2SO4终止反应，在酶标仪读取OD450。ELISA过程中，每一步都要用PBST(PBS+0.5％吐温-20)洗板4次。结果见附图4。可以看出，8种Affibody均具备PD-L1的特异结合能力。
实施例4
PD-L1特异性结合Affibody多肽对PD-1/PD-L1相互作用阻断特性分析。
1.PD-L1特异性结合Affibody多肽对PD-1/PD-L1相互作用阻断能力鉴定
在本实验中，以实施例3中具有PD-L1特异性结合能力的8种PD-L1特异结合Affibody多肽作为进一步研究对象，利用竞争ELISA方法检测其对PD-1/PD-L1相互作用的阻断，具体操作入下：
96孔板中预包被100μL(1μg/mL)PD-1-Fc-His(购自Sino Biological，货号为10377-H03H)/Fc-His(实验室自制)蛋白，4℃过夜包被；以5％脱脂奶粉-PBST封闭，37℃2h；加入PD-L1-His-biotin(购自Sino Biological，货号为10084-H08H-B)和PD-L1特异性结合Affibody多肽(0.5μg)，37℃ 2h；加入HRP-链霉亲和素37℃ 45min。加入TMB底物显色10min；加入2M H2SO4终止反应，在酶标仪读取OD450。ELISA过程中，每一步都要用PBST(PBS+0.5％吐温-20)洗板4次。结果见附图5。ZPD-L1 P9，ZPD-L1 D3、ZPD-L1 E3、ZPD-L1 P2具有良好的PD-1/PD-L1相互作用阻断能力。
2.PD-L1特异性结合Affibody多肽对PD-1/PD-L1相互作用阻断浓度依赖性鉴定
在本实验中，以ZPD-L1 P9，ZPD-L1 D3、ZPD-L1 E3、ZPD-L1 P2多肽作为进一步研究对象，利用竞争ELISA方法检测不同浓度Affibody多肽对PD-1/PD-L1相互作用的阻断，具体操作入下：
96孔板中预包被100μL(1μg/mL)PD-1-Fc-His/Fc-His蛋白，4℃过夜包被；以5％脱脂奶粉-PBST封闭，37℃ 2h；加入PD-L1-His-biotin和PD-L1特异性结合Affibody多肽(加入量分别为0.5、1、2、4、8μg)，37℃ 2h；加入HRP-链霉亲和素37℃ 45min。加入TMB底物显色10min；加入2M H2SO4终止反应，在酶标仪读取OD450。ELISA过程中，每一步都要用PBST(PBS+0.5％吐温-20)洗板4次。结果见附图6。ZPD-L1 P9，ZPD-L1 D3、ZPD-L1 E3、ZPD-L1 P2对PD-1/PD-L1相互作用阻断能力随浓度增加而增强。
Claims (2)
1.一种特异性结合程序性死亡受体-配体1(programmed cell death-Ligand 1，PD-L1)胞外结构域的多肽或其功能性片段，所述特异性结合PD-L1胞外结构域的多肽由葡萄球菌蛋白A(staphylococcal protein A，SPA)免疫球蛋白结合区域通过突变13个氨基酸残基获得，其氨基酸序列如SEQ ID NO：4所示。
2.应用权利要求1所述多肽在制备PD-L1胞外结构域检测试剂或药物中的用途。
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