CN114848853B - 一种靶向cd19抗体的多肽分子探针及其应用 - Google Patents
一种靶向cd19抗体的多肽分子探针及其应用 Download PDFInfo
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Abstract
本发明公开了一种靶向CD19抗体的多肽分子探针及其应用，所述多肽分子探针，具有以下化学结构的化合物中的一种或几种，或它们在药学上可接受的盐，P1‑Rn，P2‑Rn，P3‑Rn，P4‑Rn，P5‑Rn，P6‑Rn。本发明开发的靶向CD19抗体的分子探针能够示踪细胞疗法体内输运过程、肿瘤靶向能力、体内增殖和持续性，有助于全过程监测细胞疗法的有效性及安全性，在疗效评估、治疗方案指导方面有着重要的临床价值。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种靶向CD19抗体的多肽分子探针及其应用。
背景技术
复发和难治性急性淋巴细胞白血病(ALL)仍然是一种难以治疗的疾病，尽管前期治疗取得了进展，并且改善了新生ALL的生存率，但20多年来看到的结果改善甚微。过继性转移表达嵌合抗原受体(CAR)的T细胞已成为一种强大的靶向免疫疗法，在高度难治性人群中显示出惊人的反应。据报道，在复发和难治性ALL的儿童和成人中，使用针对B细胞特异性抗原CD19的CAR修饰T 细胞治疗，完全缓解率高达90％。尽管针对CD19的CAR修饰T细胞疗法已取得巨大的成功，其仍然面临着许多的挑战，比如体内输运过程、肿瘤靶向能力、体内增殖和持续性等信息都未知，体内活性也难以控制。
因此开发靶向CD19抗体的分子探针对示踪细胞疗法体内输运过程、肿瘤靶向能力、体内增殖和持续性等信息至关重要。有助于全过程监测细胞疗法的有效性及安全性，在疗效评估、治疗方案指导方面有着重要的临床价值。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。
作为本发明其中一个方面，本发明提供一种靶向CD19抗体的多肽分子探针。
为解决上述技术问题，本发明提供了如下技术方案：一种靶向CD19抗体的多肽分子探针，其中：所述多肽分子探针，具有以下化学结构的化合物中的一种或几种，或它们在药学上可接受的盐，
P1-Rn，P2-Rn，P3-Rn，P4-Rn，P5-Rn，P6-Rn；
其中，P1、P2、P3、P4、P5、P6，其氨基酸序列分别为SEQ ID No.1、SEQ ID No.2、SEQID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6；
R包括由连接基团、螯合剂、金属核素、靶向多肽或单抗、小分子抑制剂、小分子药物、荧光分子、纳米颗粒中的一种或几种；n代表R的数量，n＝0-10。
作为本发明所述的靶向CD19抗体的多肽分子探针的一种优选方案：所述 CD19抗体，包括IgG、Fab、scFv、scFv-Fc、diabody、minibody、nanobody、 triabody、VH、scFab、dAb中一种或几种的抗体及其片段结构，或其表达在细胞中的融合蛋白。
作为本发明所述的靶向CD19抗体的多肽分子探针的一种优选方案：所述连接基团包括PEGn或Glyn或ACPn。
作为本发明所述的靶向CD19抗体的多肽分子探针的一种优选方案：所述金属螯合物包括DOTA、NOTA、NOTP、PCTA、NODA、DFO、DTPA、DATA、TRAP、 THP、HBED、DEDPA、NETA、AATZA、Hynic中的一种或几种。
作为本发明所述的靶向CD19抗体的多肽分子探针的一种优选方案：所述金属核素，包括Ga-68、F-18、Cu-64、Zr-89、Y-86、Mn-52、I-124、Tc-99m、 Ga-67、In-111、Tl-201、Y-90、Re-188、Lu-177、Gd-159、I-125和I-131 中的一种或几种。
作为本发明所述的靶向CD19抗体的多肽分子探针的一种优选方案：所述多肽分子探针，能够用于制备PET、SPECT、CT、MR、US、光学成像的产品。
作为本发明所述的靶向CD19抗体的多肽分子探针的一种优选方案：所述 CD19抗体，包括单克隆抗体。
作为本发明所述的靶向CD19抗体的多肽分子探针的一种优选方案：所述多肽分子探针，包括GCGLKNRSSE-FITC、RWNVSDLGGLGCGLKNRSSE-FITC、 RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga、GCGLKNRSSE-FITC-NOTA-68Ga、 RWNVSDLGGLGCGLKNRSSE-NOTA-68Ga、GCGLKNRSSE-NOTA-68Ga。
作为本发明的另一个方面，本发明提供所述的多肽分子探针在制备肿瘤诊断产品和/或细胞治疗监测产品中的用途。
作为所述的多肽分子探针在制备肿瘤诊断产品和/或细胞治疗监测产品中的用途：所述多肽分子用于制备细胞治疗的体内外示踪试剂、试剂盒。
本发明的有益效果：本发明开发的靶向CD19抗体的分子探针能够示踪细胞疗法体内输运过程、肿瘤靶向能力、体内增殖和持续性，有助于全过程监测细胞疗法的有效性及安全性，在疗效评估、治疗方案指导方面有着重要的临床价值。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：
图1为为实施例1中所示的GCGLKNRSSE-FITC多肽分子探针的化学结构式。
图2为实施例1中所示GCGLKNRSSE-FITC多肽分子探针的质谱图。
图3为实施例2中所示多肽分子探针结合表达抗CD19的CART细胞的流式分析结果。
图4为实施例3中所示RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga多肽分子探针的化学结构式。
图5为实施例3中所示RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga多肽分子探针的放射性高效液相分析结果。
图6为实施例4中所示RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga多肽分子探针的PET成像结果。
图7为实施例5中所示RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga多肽分子探针的PET成像在肿瘤诊断中的应用。
图8为多肽分子探针特异性比较。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
本发明筛选出的分子探针的序列：
实施例1：GCGLKNRSSE-FITC的制备
1.GCGLKNRSSE多肽通过固相多肽合成法进行合成。2.多肽GCGLKNRSSE通过ACP连接基团进行N端修饰再与FITC进行反应制备得到GCGLKNRSSE-FITC，其化学结构式如图1所示。如图2所示，其分子量为1552.68。
实施例2：多肽分子探针的体外靶向能力
FITC修饰的多肽分子探针SPSGKLMSPK-FITC， RWNVSDLGGLGCGLKNRSSE-FITC，KDRPEIWEGE-FITC，GCGLKNRSSEGPSSPSGKLM-FITC，RWNVSDLGGLGCGLKNRSSEGPSSPSGKLM-FITC的制备参考实施例1。取表达 antiCD19 scFv的CART细胞(上海优卡迪生物医药科技有限公司提供)，用细胞计数仪各计数取10^6个细胞于6孔板中，加入含50微克不同的多肽分子探针的细胞培养基溶液1mL。共同孵育1小时。将细胞吸出置于离心管中1000rpm 离心5分钟，弃上清液，加入新鲜培养基重悬，将此离心重悬操作重复一次。将此细胞悬液用BD流式细胞仪进行分析。如图3所示，表达antiCD19 scFv的CART细胞与空白对照细胞相比，SEQ No.2,3，5，6的阳性率明显增加，从 0提高到最高4％以上，其中SEQ No.2,3阳性率和荧光强度均最高，SEQ ID No.3 阳性率达到4.78％，说明GCGLKNRSSE-FITC、RWNVSDLGGLGCGLKNRSSE-FITC分子探针对antiCD19 scFv有较好的体外靶向能力。
实施例3：RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga的制备
1.RWNVSDLGGLGCGLKNRSSE-FITC的制备方法与GCGLKNRSSE-FITC的制备方法相同；
2.将RWNVSDLGGLGCGLKNRSSE-FITC与NOTA-MAL反应6小时,并通过半制备型高效液相色谱进行纯化，可制得RWNVSDLGGLGCGLKNRSSE-FITC-NOTA；
3.68Ga标记：将RWNVSDLGGLGCGLKNRSSE-FITC-NOTA溶解到醋酸钠缓冲液(0.5mL，pH6.8)中，加入2mL 68GaCl3的0.05M盐酸溶液，混合物在100 摄氏度下加热反应10分钟，通过C18固相萃取小柱进行纯化，1mL 50％乙醇淋洗，并通过无菌滤膜进入产品瓶，加入4mL生理盐水进行稀释得到 RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga。其化学结构式见图4。
通过radio-HPLC测定放化纯。如图5所示，放化纯接近90％。
实施例4：RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga在细胞示踪中的应用
将5000个细胞数的表达antiCD19 scFv的CART细胞用50微升基质胶混匀后用注射器注射到6-8周龄的昆明鼠肩头，半小时后，尾静脉注射实施例3 中的分子探针RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga，放射剂量为200μCi。在分子探针注射30分钟和60分钟后对小鼠进行PET成像。从图6可以看出，该分子探针对表达antiCD19 scFv的CART细胞的成像特异性高，跟对侧未注射细胞的位置摄取值明显提高，摄取值达到2％，并且在30分钟和60分钟的时间区间内，成像效果维持较好的水平。
实施例5：RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga在制备肿瘤诊断产品中的应用
6-8周龄的NSG小鼠尾静脉注射10^6Raji-Luc细胞，构建小鼠淋巴瘤原位模型，一周后通过腹腔注射荧光素底物并IVIS成像确认小鼠建模成功。尾静脉注射10^7细胞数量的表达antiCD19 scFv的CART细胞，在细胞治疗7天后对小鼠同时通过腹腔注射荧光素底物并IVIS成像，然后尾静脉注射实施例3 中的分子探针RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga，并在探针注射1小时后进行PET成像。从图7可以看出，IVIS成像展示的是负荷肿瘤的病灶，主要集中在外周血中，并有腿部骨髓转移。PET成像结果表明，分子探针RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga成像位置与负荷肿瘤的病灶重叠，主要集中在外周血中，并在腿部骨髓转移灶同样有高摄取，该结果表明该分子探针在肿瘤诊断中具有较高的特异性，可以对原位病灶和转移病灶进行高特异性成像。
实施例6：多肽分子探针特异性比较
利用SEQ ID No.2,3,5,6多肽制备分子探针GCGLKNRSSE-NOTA-68Ga，RWNVSDLGGLGCGLKNRSSE-NOTA-68Ga，GCGLKNRSSEGPSSPSPSGKLM-NOTA-68Ga，RWNVSDLGGLGCGLKNRSSEGPSSPSGKLM-NOTA-68Ga，方法同实施例3。将5000个细胞数的表达antiCD19 scFv的CART细胞用50微升基质胶混匀后用注射器注射到6-8周龄的昆明鼠肩头，半小时后，对小鼠分别尾静脉注射200μCi放射剂量的4种分子探针,1小时后进行PET成像。从图8可以看出，SEQ INo.3对应的分子探针RWNVSDLGGLGCGLKNRSSE-NOTA-68Ga具有最高的摄取值，摄取值高于 2％ID/g；GCGLKNRSSEGPSSPSPSGKLM-NOTA-68Ga和RWNVSDLGGLGCGLKNRSSEGPS SPSGKLM-NOTA-68Ga探针摄取值约1％，从图像上也可以直观看出， RWNVSDLGGLGCGLKNRSSE-NOTA-68Ga具有最佳对比度,GCGLKNRSSE-NOTA-68Ga次之。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 江苏省原子医学研究所
<120> 一种靶向CD19抗体的多肽分子探针及其应用
<140> 202110075078X
<141> 2021-01-20
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Claims (3)
1.一种靶向CD19抗体的多肽分子探针，其特征在于：所述多肽分子探针为RWNVSDLGGLGCGLKNRSSE-FITC-NOTA-68Ga；所述CD19抗体为scFv-Fc。
2.权利要求1所述的多肽分子探针在制备肿瘤诊断产品和/或细胞治疗监测产品中的用途。
3.根据权利要求2所述的多肽分子探针在制备肿瘤诊断产品和/或细胞治疗监测产品中的用途，其特征在于：所述多肽分子探针用于制备细胞治疗的体内外示踪试剂、试剂盒。
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| CN110028588A (zh) * | 2018-01-11 | 2019-07-19 | 上海细胞治疗研究院 | 抗原-Fc融合蛋白及其检测阳性CAR-T细胞的应用 |
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