CN113476619B - 一种18f标记纳米抗体探针及其制备方法和应用 - Google Patents
一种18f标记纳米抗体探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种18F标记纳米抗体探针及其制备方法和应用,涉及核医学和分子影像领域,该探针的靶向载体包括纳米抗体MM01、纳米抗体MM02或纳米抗体MM02的衍生物,其制备方法为:合成小分子化合物前体RJDJ01;制备18F标记前体18F[F]‑RJDJ01;通过点击化学反应制备氟‑18(18F)标记纳米抗体探针;该探针为用于检测多发性骨髓瘤CD38特异性纳米抗体。本发明通过创制CD38特异性新型纳米抗体探针18F[F]‑MM01,实现了对CD38表达的无创可视化,无创检测多发性骨髓瘤。该探针具有制备工艺简单、成本低廉、特异性高、稳定性高、显像周期短、辐射剂量低、易于临床转化等优点。
Description
技术领域
本发明涉及核医学和分子影像领域,尤其涉及一种18F标记纳米抗体探针及其制备方法和应用。
背景技术
1993年比利时科学家Hamers等人在《Nature》杂志中首次报道在羊驼外周血液中存在一种天然缺失轻链的抗体(Nature.1993;363(6428):446-8.),这种具有特殊结构域的抗体即为重链抗体(Heavy-chain antibodies,HCAbs)。通过分子生物学手段,克隆重链抗体的可变区即可得到只有重链可变区的抗原结合片段,即为纳米抗体(VHH, VariableDomain of Heavy Chain of Heavy Chain Antibody)。VHH晶体宽为2.5nm,长 4nm,分子量只有15KDa,因此也被称为纳米抗体(Ablynx公司注册商品名)。纳米抗体是目前已知的可结合目标抗原的最小抗体单位,具有亲和力高、分子量小、制备成本低廉(既可以运用大肠杆菌表达,也可以运用酵母、中国仓鼠卵巢细胞等真核表达体系表达)、易于临床转化和推广应用的优点。
纳米抗体是近年来构筑分子影像探针的热门靶向载体(Theranostics. 2014;4(4):386-98.)。目前,多种短半衰期核素已用于标记纳米抗体,制备纳米抗体分子影像探针。锝-99m(99mTc;T1/2=6.02h)标记靶向程序性死亡配体1(PD-L1) 的纳米抗体探针已成功转化至临床,用于非小细胞肺癌患者的无创诊断(J Nucl Med. 2019;60(9):1213-1220.);镓-68(68Ga;T1/2=1.1h)标记靶向人类表皮生长因子受体 (HER2)的纳米抗体探针也已成功转化至临床,用于乳腺癌的无创诊断(J Nucl Med. 2016;57(1):27-33.)。以上实例说明放射性核素标记纳米抗体探针极具临床转化应用前景,可用于人类恶性肿瘤的早期无创诊断、关键致病靶点的可视化、单克隆抗体(mAb) 治疗患者的筛选以及单克隆抗体治疗后疗效评价。但是,99mTc属于单光子发射放射性核素,所标记纳米抗体探针的显像性能欠佳;68Ga一般需要通过锗镓发生器或配置有固体靶的医用回旋加速器制备,制备价格昂贵,且半衰期较短,所标记纳米抗体探针不适宜运输及推广应用。氟-18(18F;T1/2=109.8min h)是临床正电子发射断层显像(PET)最长使用的放射性核素,其正电子发射率高达97%,正电子射程为0.5mm,不发射伽马射线,是创制PET显像探针的最佳核素之一。但是,因为放射化学产率 (RCY)较低、标记条件苛刻(需要高温、有机溶剂)等原因的限制,18F并没有广泛用于纳米抗体的核素标记(Chem Rev.2020;120(8):3787-3851.)。
多发性骨髓瘤(MM)是一种B细胞来源的血液***恶性肿瘤,目前临床尚无有效的诊疗措施。CD38是多发性骨髓瘤特异性的生物标志物。靶向CD38的单克隆抗体达雷妥尤单克隆抗体注射液(daratumumab)在欧美和我国均已获批临床,用于新发或复发难治性多发性骨髓瘤的治疗。诸多因素介导达雷妥尤单抗的治疗疗效,其中最主要的因素是CD38蛋白表达水平。目前,临床判断多发性骨髓瘤细胞CD38表达水平严重依赖于骨髓穿刺物的流式细胞学检测。但是,该方法创伤性较大、且可重复性较差。因此,临床亟需一种可用于无创可视化CD38表达水平、早期精准诊断多发性骨髓瘤的分子影像学方法。
有机融合了PET显像的高灵敏度和抗体高亲和力的免疫PET(immuno-positronemission tomography,immunoPET)显像是一种新型的分子影像模式。免疫PET显像探针的制备主要基于放射性核素随机或定点标记的单克隆抗体、抗体片段或纳米抗体。其中,18F标记的纳米抗体探针最具临床应用前景。目前,多种前体已用于18F标记生物大分子。[18F]-对氟苯甲醛([18F]FBA)是最常用的18F标记前体; N-Succinimidyl-4-[18F]-fluorobenzoate([18F]SFB)也是较常用的18F标记前体,该前体可与抗体赖氨酸(Lys)氨基反应形成稳定的酰胺键; N-[2-(4-[18F]-Fluorobenzamido)-ethyl]maleimide([18F]FBEM)是一种具有硫醇反应活性的标记前体,可用于纳米抗体的定点标记,但其合成步骤繁琐、放射化学产率较低; 2,3,5,6-tetrafluorophenyl6-[18F]-fluoronicotinate([18F]TFPFN)虽然标记纳米抗体的条件较温和(37-40℃,15min,pH 8.5-9.0),但放射化学产率只有5%左右,严重限制其应用。此外,其他18F标记方法常需乙腈等有机溶剂,且需要在酸性环境(pH=2.0 -2.5)中反应。但抗体在极端环境中易变性或失活,因此常用标记方法不适用于纳米抗体的标记。临床亟需一种标记条件温和、可重复性好、放射化学产率高的18F标记纳米抗体方法,以实现靶点特异性纳米抗体的室温、高效18F标记。
在MM诊断方面,目前评估CD38表达水平的方式主要是穿刺骨髓液的流式细胞学检测或免疫组织化学染色(IHC)。但是骨髓穿刺不仅创伤大、可重复性差,并且取样误差、肿瘤异质性等因素,往往导致活检穿刺结果呈现假阴性或假阳性。
临床亟需一种可用于无创可视化CD38表达水平、早期精准检测多发性骨髓瘤抗体的分子影像学方法,需要一种价格低廉、辐射剂量低、显像周期短、且更易临床转化应用的纳米抗体探针,需要一种标记条件温和、可重复性好、放射化学产率高的18F 标记纳米抗体方法,以实现靶点特异性纳米抗体的室温、高效18F标记。
因此,本领域的技术人员致力于开发一种价格低廉、辐射剂量低、显像周期短、且更易临床转化应用的CD38特异性纳米抗体探针18F[F]-MM01,实现CD38的无创可视化和MM靶点特异性无创诊断。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是本发明提供一种运用自动化合成模块18F高效标记纳米抗体的方法;通过该技术方法创新,制备一种价格低廉、辐射剂量低、显像周期短、且更易临床转化应用的CD38特异性纳米抗体探针18F[F]-MM01,实现CD38的无创可视化和MM靶点特异性无创诊断。
为实现上述目的,本发明提供了一种18F标记纳米抗体探针,其靶向载体包括纳米抗体MM01、纳米抗体MM02或纳米抗体MM02的衍生物。
进一步地,上述纳米抗体MM02的衍生物包括定点PEG修饰的MM02纳米抗体的衍生物。
本发明还提供一种18F标记纳米抗体探针的制备方法,包括以下步骤:
步骤1、合成小分子化合物前体RJDJ01;
步骤2、18F标记步骤1得到的小分子化合物前体RJDJ01,制备用于纳米抗体快速点击化学标记的18F标记前体,即18F[F]-RJDJ01;
步骤3、通过点击化学反应制备18F标记纳米抗体探针;点击化学反应的反应物包括点击化学底物1和点击化学底物2,点击化学底物1为步骤2得到的18F[F]-RJDJ01,点击化学底物2为纳米抗体,点击化学反应的产物为18F标记纳米抗体探针。
进一步地,上述步骤1中小分子化合物前体RJDJ01的化学结构式为:
进一步地,上述步骤1中小分子化合物前体RJDJ01的化学合成路径为:
首先经三缩四乙二醇与对甲苯磺酰氯和氢氧化钾反应,制备粗产品;将所述粗产品溶解于二氯甲烷中,加入3-羟基-2-硝基吡啶和氯化钠反应后,柱层析分离得到2-(2-(2-(2-((2-硝基吡啶-3-基)氧基)乙氧基)乙氧基)乙氧基)4-甲基苯磺酸乙酯;将2-(2-(2-(2-((2-硝基吡啶-3-基)氧基)乙氧基)乙氧基)乙氧基)4-甲基苯磺酸乙酯与叠氮化钠反应,经二氯甲烷萃取后,层析得到3-(2-(2-(2-(2-叠氮基乙氧基)乙氧基)乙氧基)乙氧基)-2-硝基吡啶,即小分子化合物前体RJDJ01。
进一步地,上述步骤2中前体18F[F]-RJDJ01的化学结构式为:
进一步地,上述步骤3中通过点击化学反应制备18F标记纳米抗体探针包括18F 随机标记纳米抗体制备方式或者18F定点标记纳米抗体制备方式。
进一步地,18F随机标记纳米抗体制备方式中点击化学底物2为DBCO随机偶连的CD38、BCMA、TROP-2、HER2、EGFR、VEGFR、HER2、CD47、CD146、ICAM-1、Nectin-4、CAIX、GPC3、GPA33、Claudin18.2、GD2、MM01或PD-L1特异性纳米抗体。
进一步地,18F定点标记纳米抗体制备方式中点击化学底物2为DBCO定点偶连的CD38、BCMA、TROP-2、HER2、EGFR、VEGFR、HER2、CD47、CD146、ICAM-1、 Nectin-4、CAIX、GPC3、GPA33、Claudin18.2、GD2、MM01、MM02或PD-L1特异性纳米抗体;其中,MM02通过连接子GGGGSCGSGSGSLLQS中的半胱氨酸 (Cysteine,Cys,C)与二苯基环辛炔-马来酰亚胺(DBCO-Mal,CAS#:1395786-30-7, MeloPEG)定点反应,制备DBCO-MM02;或者通过微生物谷氨酰胺转氨酶(mTGase) 介导的酶促反应,实现二苯基环辛炔-胺(DBCO-Amine,CAS#:1255942-06-3, MeloPEG)与MM01或MM02连接子GGGGSCGSGSGSLLQS中的谷氨酰胺 (Glutamine,Gln,Q)定点反应,从而制备DBCO-MM01或DBCO-MM02;或者通过微生物谷氨酰胺转氨酶(mTGase)介导二苯基环辛炔-聚乙二醇-胺 (DBCO-PEG-Amine,MeloPEG)与MM01或MM02连接子GGGGSCGSGSGSLLQS 中的谷氨酰胺(Glutamine,Gln,Q)定点反应,制备DBCO-PEG-MM01或DBCO-PEG-MM02。
进一步地,一种CD38特异性纳米抗体MM01,其具有如序列表中的SEQ ID No.1 所示的氨基酸序列,如序列表中的SEQ ID No.2所示的基因序列。
进一步地,一种CD38特异性纳米抗体MM02,其具有如序列表中的SEQ ID No.3 所示的氨基酸序列,如序列表中的SEQ ID No.4所示的基因序列。
进一步地,经随机标记所制备探针18F[F]-MM01为人CD38特异性纳米抗体探针。
进一步地,PEG可为不同的分子量,即100Da,200Da,500Da,1KDa,5KDa, 10KDa或20KDa。
本发明还提供一种18F标记纳米抗体探针的应用方法,其应用于检测多发性骨髓瘤的CD38特异性纳米抗体。
在本发明的较佳实施方式中,详细说明合成RJDJ01的路径;
在本发明的另一较佳实施方式中,详细说明点击化学反应底物1即18F[F]-RJDJ01的自动化制备过程;
在本发明的另一较佳实施方式中,详细说明制备点击化学反应底物 DBCO-MM01,经点击化学反应制备新型CD38特异性纳米抗体探针18F[F]-MM01;
在本发明的另一较佳实施方式中,详细说明18F[F]-MM01质量控制方法;
在本发明的另一较佳实施方式中,详细说明18F[F]-MM01免疫PET显像检测播散型多发性骨髓瘤;
在本发明的另一较佳实施方式中,详细说明mTGase酶促反应介导纳米抗体定点PEG修饰。
目前,多发性骨髓瘤的诊断所做的检查项目包括:血液检查、尿液检查、骨髓检查和影像学检查等。其中骨髓穿刺检查创伤较大、可重复性较差,且对异质性多发性骨髓瘤的检出率较低。现有的分子影像学检查手段,例如X线平片、计算机断层扫描 (CT)和核磁共振(MRI)等,均是反应疾病的结构性变化,不能反应疾病的功能或分子层面的变化,因此对早期多发性骨髓瘤的检出率较低,且缺乏特异性。氟代脱氧葡萄糖(18F-FDG)PET/CT检查也已用于多发性骨髓瘤的诊断,但仍面面临灵敏度低、特异性低等诸多缺点。CD38是多发性骨髓瘤最特异性的标志物之一,靶向CD38的单克隆抗体,例如达雷妥尤单抗,已在美国和中国获批,用于多发性骨髓瘤的临床治疗 (Blood.2018;131(1):13-29.)。研发CD38特异性分子影像探针将有望实现多发性骨髓的早期精准诊断,达雷妥尤单抗治疗患者的有效筛选,及治疗前后CD38表达的无创动态评估。
目前国内外尚无CD38特异性分子影像探针,基于探针68Ga[Ga]-NOTA-MMO1 的免疫PET显像取得了较好的检测效果,但缺点表现为:68Ga需要经锗镓发生器制备,因此探针68Ga[Ga]-NOTA-MMO1制备成本较高;68Ga半衰期(T1/2=1.1h),致使探针的应用范围受限。
本发明通过创制CD38特异性新型纳米抗体探针18F[F]-MM01,实现了对CD38 表达的无创可视化,进一步实现了多发性骨髓瘤的无创诊断。新型探针18F[F]-MM01 具有制备工艺简单、成本低廉、特异性高、稳定性高、显像周期短、辐射剂量低、易于临床转化等优点,具有以下有益的技术效果:
1、本发明提供的一种18F快速高效标记纳米抗体的新方法,该方法通过点击化学介导的“两步法”有效避开了18F标记前体化合物过程中高温和有机溶剂等因素对纳米抗体活性和结构的影响,降低其制备成本。
2、制备了18F标记CD38特异性纳米抗体探针18F[F]-MM01,实现了多发性骨髓瘤的快递、精准检出。氟-18(18F;T1/2=109.8min h)半衰期比68Ga长,其标记的探针应用范围广泛。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例1的RJDJ01化学合成路径示意图;
图2是本发明的一个较佳实施例1的用高效液相色谱仪(Agilent 1260 HPLC and6120 MSD)检测产物的色谱图及质谱检测器(MSD)总离子色谱图(TIC);
图3是本发明的一个较佳实施例1的用质谱法检测产物的质谱图;
图4是本发明的一个较佳实施例1的用核磁共振(NMR)法检测产物的结果图;
图5是本发明的另一个较佳实施例3的18F随机标记纳米抗体示意图;
图6是本发明的另一个较佳实施例5的18F[F]-MM01免疫PET/CT检测原位多发性骨髓瘤的显像图;
图7是本发明的另一个较佳实施例5的达雷妥尤单克隆抗体(daratumumab)封闭后18F[F]-MM01免疫PET/CT显像图;
图8是本发明的另一个较佳实施例6的18F定点标记纳米抗体示意图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
实施例1合成RJDJ01
如图1所示的合成路径自主合成RJDJ01:
1.精确称量1g三缩四乙二醇(约5.1mmol),将其溶解于15mL的二氯甲烷中,而后将2g(约10.5mmol)对甲苯磺酰氯和0.59g氢氧化钾(约10.5mmol)缓慢加入到上述混合液中室温反应过夜。经过水洗后分液,得到2g粗产品(收率约为78%)。
2.上述粗产品溶解于二氯甲烷中,而后加入0.56g(约4mmol)3-羟基-2-硝基吡啶和96mg氯化钠(约4mmol),室温反应过夜后,柱层析分离得到1g(约2.1mmol) 的2-(2-(2-(2-((2-硝基吡啶-3-基)氧基)乙氧基)乙氧基)乙氧基)4-甲基苯磺酸乙酯,收率约为51%。
3.940mg(约2mmol)的2-(2-(2-(2-((2-硝基吡啶-3-基)氧基)乙氧基)乙氧基) 乙氧基)4-甲基苯磺酸乙酯溶解于10mL二氯甲烷中,然后将130mg(约2mmol)的叠氮化钠的2mL水溶液加入到上述反应液中,室温反应5小时后,二氯甲烷萃取,并柱层析得到产物477mg的3-(2-(2-(2-(2-叠氮基乙氧基)乙氧基)乙氧基)乙氧基)-2-硝基吡啶,即本专利述及的RJDJ01,收率约为70%。
将上述产物使用液相色谱法、质谱法和核磁共振的检测方法进行验证,其中,液相色谱法使用Agilent 1260 HPLC配置紫外检测器,检测结果如图2,上图是在波长为220nm,产物的色谱图,保留时间为2.711min,下图为质谱法质谱检测器6120MSD 的总离子色谱图(TIC),保留时间为2.724min;图3为质谱法检测的质谱图,在341.9m/z处有最强峰;图4为核磁共振(NMR)的检测结果图用以表征RJDJ01,以上结果都表明产物具有RJDJ01的正确的分子量及化学结构。
实施例2点击化学反应底物1 18F[F]-RJDJ01的自动化制备
具体实验方案如下:称取2–2.5mg RJDJ01,溶解于1mL二甲基亚砜(DMSO);用预平衡的阴离子交换柱转移18F-fluoride(33.49–39.39GBq),用溶解于1.4mL 乙腈的碳酸钾(3mg)和 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane(15mg)作为流动相淋洗阴离子交换柱;将溶剂蒸干后,加入溶解于1mL DMSO的RJDJ01;将反应体系置于120℃加热14分钟;加3mL灭菌注射用水至反应体系,转移反应体系至C18 柱子,设置流动相为含32%乙腈的0.1%三氟乙酸(trifluoroacetic acid),设定流速为 4.6mL/min,经HPLC纯化、收集18F-RJDJ01至梨形瓶;加10mL灭菌注射用水至梨形瓶稀释18F[F]-RJDJ01,并转移C18柱子,用3mL乙醇萃取18F[F]-RJDJ01,加热至100℃蒸干乙醇,再加100uL磷酸盐缓冲液(PBS)重悬18F[F]-RJDJ01。
实施例3制备点击化学反应底物DBCO-MM01,经点击化学反应制备新型CD38 特异性纳米抗体探针18F[F]-MM01
反应原理如图5 18F随机标记纳米抗体示意图所示,制备点击化学反应底物 DBCO-MM01,经点击化学反应制备新型CD38特异性纳米抗体探针18F[F]-MM01。
DBCO-NHS ester(CAS#:1353016-71-3,MeloPEG)随机偶连MM01,制备点击化学反应底物DBCO-MM01。具体步骤如下:将1mg CD38特异性纳米抗体MM01 溶于磷酸盐缓冲液(PBS),体积约1mL,加80–100μL 0.1M碳酸钠(Na2CO3) 缓冲液将纳米抗体溶液pH调至9.0–10。以DBCO-NHS ester/MM01摩尔比为10:1 的比例,将新鲜溶于二甲基亚砜(DMSO)的DBCO-NHS ester加至纳米抗体溶液,即670nM/0.27mg。将反应体系置于室温反应2小时,然后以PBS作为流动相,用预平衡的PD-10脱盐柱(GE Healthcare)纯化经DBCO随机偶连的纳米抗体,收集 DBCO-MM01;再用截断值为10KDa的超滤管(Merck Millipore)浓缩纳米抗体样本,最后用NanoDrop测定DBCO-MM01浓度,将DBCO-MM01置于4℃冰箱备用。
经点击化学反应制备新型CD38特异性纳米抗体探针18F[F]-MM01的具体实验方案如下:取DBCO-MM01 332uL(320ug),加20mL 18F[F]-MM01至DBCO-MM01 纳米抗体溶液;将上述反应体系置于恒温振荡器,45℃反应45分钟;结束后以PBS 作为流动相,再次用预平衡的PD-10脱盐柱分离未反应18F-RJDJ01,纯化收集终产物18F[F]-MM01。
实施例4 18F[F]-MM01质量控制
吸取2μL 18F[F]-MM01在硅胶板上点样,用0.1M柠檬酸钠溶液(pH=5) 作为流动相,用放射性薄层色谱仪(Radio-TLC,Eckert&Ziegler Radiopharma Inc) 测定探针的放射化学纯度(Radiochemical purity,RCP);再用高效液相色谱(HPLC, Agilent)进一步测定所制备纳米抗体探针的完整度、RCP及免疫反应性。
实施例5 18F[F]-MM01免疫PET显像检测播散型多发性骨髓瘤
包括以下步骤:本研究所涉及的小动物PET/CT显像采集均使用IRIS小动物 PET/CT扫描仪(Inviscan Imaging Systems)完成,每只荷瘤NCG小鼠经尾静脉注射3.7–7.4MBq18F[F]-MM01(每组3只),在注射后的1小时用混有氧气的异氟烷(浓度为2%)麻醉荷瘤裸鼠,并将进入深度麻醉状态的裸鼠以仰卧位姿势置于 PET/CT扫描床上,续惯采集PET和CT图像,用IRIS***自带软件完成图像重建,如图6所示,CD38特异性纳米抗体探针18F[F]-MM01在肿瘤组织有较高的摄取,显像结果显示双侧股骨(1、2)、胫骨(3、4)有明显的显像剂摄取,18F[F]-MM01免疫PET显像可精准检测播散型多发性骨髓瘤,包括双侧股骨和胫骨等处的病灶。用 OsiriX Lite图像处理工作站(Pixmeo SARL)在重建后的PET图像上勾画肿瘤、心脏、及主要组织脏器(肝脏、脾脏、肺脏、肾脏、胰腺、肌肉)等感兴趣区,以%ID/g (percent ofinjected dose per gram)为单位计算肿瘤组织及重要组织器官的放射性摄取值,在主要***(肾脏)和代谢(肝脏)组织也要较高的非特异性摄取;显像结束后,取肿瘤组织及主要的组织器官做体外生物分布实验,CD38特异性纳米抗体探针18F[F]-MM01同样有较高的摄取。
CD38特异性免疫PET显像探针18F[F]-MM01特异性检测多发性骨髓瘤,达雷妥尤单克隆抗体可有效封闭骨骼组织对18F-MM01的摄取,基于达雷妥尤单抗可有效降低多发性骨髓瘤累及骨骼组织显像剂摄取,将上述实验中每只荷瘤小鼠提前24小时注射1mg达雷妥尤单抗,然后按照上述步骤每只荷瘤NCG小鼠经尾静脉注射3.7 –7.4MBq 18F[F]-MM01(每组3只),在注射后的1小时用混有氧气的异氟烷(浓度为2%)麻醉荷瘤裸鼠,并将进入深度麻醉状态的裸鼠以仰卧位姿势置于PET/CT扫描床上,续惯采集PET和CT图像,用IRIS***自带软件完成图像重建,则结果如图 7所示,达雷妥尤单克隆抗体(daratumumab)封闭后18F[F]-MM01免疫PET/CT显像,显像结果显示经达雷妥尤单克隆抗体封闭后,双侧股骨(1、2)、胫骨(3、4)的显像剂摄取明显降低,说明达雷妥尤单克隆抗体有效封闭了18F[F]-MM01在MM.1S肿瘤细胞上的结合位点,证明探针18F[F]-MM01具有高度特异性,且其抗原结合位点与daratumumab的抗原结合位点重合。达雷妥尤单抗封闭后双侧肱骨、双侧肩胛骨、胸骨、椎体、双侧髂骨及双侧股骨等部位18F[F]-MM01摄取显著降低,说明18F[F]-MM01探针对人CD38具有高度特异性、且18F[F]-MM01探针和达雷妥尤单抗具有相同的抗原结合位点。
实施例6mTGase酶促反应介导纳米抗体定点PEG修饰
反应原理如图818F定点标记纳米抗体示意图所示。具体实施路线如下:将3mg C 端带有LLQS标签的MM01、1mg PEG-NH2(5KDa)或2mg PEG-NH2(10KDa) 及1mg mTGase溶解于1mLPBS溶液,将反应体系置于恒温振荡器在室温反应1 小时;用截断值为10KDa的超滤管(Merck Millipore)浓缩样本体积至300μL;用配有SuperdexTM75Increase柱子的pure蛋白纯化仪(Cytiva,formerly GE Healthcare Life Science)纯化、收集经不同分子量PEG定点修饰的MM01衍生物。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
序列表
<110> 上海交通大学医学院附属仁济医院
<120> 一种18F标记纳米抗体探针及其制备方法和应用
<130> 2020
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 145
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met His His His His His His Asp Val Gln Leu Gln Glu Ser Gly Gly
1 5 10 15
Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Thr Gly Ser
20 25 30
Gly Arg Thr Phe Arg Asn Tyr Pro Met Ala Trp Phe Arg Gln Ala Pro
35 40 45
Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Thr Trp Val Gly Ala Ser
50 55 60
Thr Leu Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
65 70 75 80
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
85 90 95
Asp Thr Ala Val Tyr Ser Cys Ala Ala Gly Arg Gly Ile Val Ala Gly
100 105 110
Arg Ile Pro Ala Glu Tyr Ala Asp Trp Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Leu Gln
130 135 140
Ser
145
<210> 2
<211> 450
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
catatgcacc atcatcatca tcacgacgtc caactgcaag aatcgggcgg cggtctggtc 60
caagcgggcg gttccctgcg tctgtcatgc accggcagcg gtcgtacgtt tcgcaactat 120
ccgatggcat ggttccgtca ggctccgggc aaagaacgcg aatttgtggc gggcattacc 180
tgggttggtg ccagtacgct gtacgcagat tttgctaaag gtcgtttcac catctcccgc 240
gacaacgcga aaaatacggt ttatctgcag atgaatagcc tgaaaccgga agataccgca 300
gtctactctt gtgccgcggg tcgtggtatt gttgccggtc gtatcccggc cgaatatgca 360
gactggggcc aaggtacgca ggtgacggtt tcttctggtg gtggcggctc tggtggtggc 420
ggttctctgc tgcaaagtta atgaaagctt 450
<210> 3
<211> 147
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met His His His His His His Asp Val Gln Leu Gln Glu Ser Gly Gly
1 5 10 15
Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Thr Gly Ser
20 25 30
Gly Arg Thr Phe Arg Asn Tyr Pro Met Ala Trp Phe Arg Gln Ala Pro
35 40 45
Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Thr Trp Val Gly Ala Ser
50 55 60
Thr Leu Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
65 70 75 80
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
85 90 95
Asp Thr Ala Val Tyr Ser Cys Ala Ala Gly Arg Gly Ile Val Ala Gly
100 105 110
Arg Ile Pro Ala Glu Tyr Ala Asp Trp Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser Gly Gly Gly Gly Ser Cys Gly Ser Gly Ser Gly Ser Leu
130 135 140
Leu Gln Ser
145
<210> 4
<211> 456
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
catatgcatc atcatcatca tcacgacgtc caactgcaag aatctggcgg cggtctggtt 60
caagcgggcg gtagcctgcg tctgtcatgt accggcagcg gtcgtacgtt tcgcaactat 120
ccgatggcat ggttccgtca ggctccgggc aaagaacgcg aatttgtggc gggcattacc 180
tgggttggtg ccagtacgct gtacgcagat tttgctaaag gtcgtttcac catctcccgc 240
gacaacgcga aaaatacggt ttatctgcaa atgaatagcc tgaaaccgga agataccgca 300
gtctactctt gtgccgcggg tcgtggtatt gttgccggtc gtattccggc cgaatatgca 360
gactggggtc agggtacgca agtcacggtc tcttcaggcg gtggcggttc gtgtggctcg 420
ggctcgggct ctctgctgca atcgtaatga aagctt 456
Claims (2)
1.一种针对多发性骨髓瘤的18F标记纳米抗体探针的制备方法,其特征在于,所述方法包括以下步骤:
步骤1、合成小分子化合物前体RJDJ01;
步骤2、18F标记步骤1得到的小分子化合物前体RJDJ01,制备用于纳米抗体快速点击化学标记的18F标记前体,即18F[F]-RJDJ01;
步骤3、通过点击化学反应制备所述18F标记纳米抗体探针;所述点击化学反应的反应物包括点击化学底物1和点击化学底物2,所述点击化学底物1为步骤2得到的所述18F[F]-RJDJ01,所述点击化学底物2为所述纳米抗体,所述点击化学反应的产物为所述18F标记纳米抗体探针;
所述步骤1中所述小分子化合物前体RJDJ01的化学结构式为:
所述步骤1中所述小分子化合物前体RJDJ01的化学合成路径为:首先经三缩四乙二醇与对甲苯磺酰氯和氢氧化钾反应,制备粗产品;将所述粗产品溶解于二氯甲烷中,加入3-羟基-2-硝基吡啶和氯化钠反应后,柱层析分离得到2-(2-(2-(2-((2-硝基吡啶-3-基)氧基)乙氧基)乙氧基)乙氧基)4-甲基苯磺酸乙酯;将所述2-(2-(2-(2-((2-硝基吡啶-3-基)氧基)乙氧基)乙氧基)乙氧基)4-甲基苯磺酸乙酯与叠氮化钠反应,经二氯甲烷萃取后,层析得到3-(2-(2-(2-(2-叠氮基乙氧基)乙氧基)乙氧基)乙氧基)-2-硝基吡啶,即所述小分子化合物前体RJDJ01;
所述步骤2中所述前体18F[F]-RJDJ01的化学结构式为:
所述步骤3中所述通过点击化学反应制备18F标记纳米抗体探针包括18F随机标记纳米抗体制备方式或者18F定点标记纳米抗体制备方式;
所述18F随机标记纳米抗体制备方式中所述点击化学底物2为DBCO随机偶连的MM01特异性纳米抗体;
所述18F定点标记纳米抗体制备方式中所述点击化学底物2为所述DBCO定点偶连的所述MM01特异性纳米抗体;其中,所述定点偶连的方式为通过微生物谷氨酰胺转氨酶(mTGase)介导的酶促反应,实现二苯基环辛炔-胺与所述MM01连接子GGGGSCGSGSGSLLQS中的谷氨酰胺定点反应,从而制备DBCO-MM01;或者通过微生物谷氨酰胺转氨酶(mTGase)介导二苯基环辛炔-聚乙二醇-胺(DBCO-PEG-Amine,MeloPEG)与所述MM01连接子GGGGSCGSGSGSLLQS中的谷氨酰胺定点反应,制备DBCO-PEG-MM01。
2.如权利要求1所述的18F标记纳米抗体探针的制备方法,其特征在于,所述探针应用于检测多发性骨髓瘤的CD38特异性纳米抗体。
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