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大多数哺乳动物体内的单克隆抗体由2条重链和2条轻链组成,分子量约150 kDa。1993年,比利时科学家Hamers-Casterman等[1]在鲨鱼和骆驼中发现了没有轻链、只有重链的免疫球蛋白(immunoglobulin,Ig)G,并基于此IgG进一步开发了单域抗体(single domain antibody,sdAb),也称之为纳米抗体(nanobody)[2]。纳米抗体相较于单克隆抗体,具有分子量小(约15 kDa)、溶解性好、稳定性高、组织穿透能力强且可快速从血液中清除并经肾脏排泄等诸多优点。此外,纳米抗体可通过生物工程技术在细菌、酵母或哺乳动物细胞中大规模生产,制备成本较低,从而有效促进其临床应用。纳米抗体作为构筑分子影像探针的新型靶向分子,经放射性核素标记后,具有短期内获取高质量图像、对疾病进行全面评估及指导个体化精准治疗等优势 [3]。迄今为止,已经涌现出多种针对不同分子靶标的纳米抗体分子影像探针(简称纳米抗体探针)[4],并在肿瘤早期靶点特异性诊断方面发挥了重要作用。我们旨在综述纳米抗体探针领域的最新研究进展,并对该领域的未来发展作相应的展望。
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纳米抗体可应用于多种分子影像学成像技术,如PET/CT、SPECT/CT、光学成像和超声成像等。目前纳米抗体探针的主要靶点包括肿瘤膜抗原及肿瘤微环境中的靶点等,主要包括但不限于表皮生长因子受体(epidermal growth factor receptor,EGFR)、人表皮生长因子受体(human epidermal growth factor receptor,HER)2、血管内皮生长因子受体(VEGFR)、细胞分化抗原(cell differentiation,CD)、程序性细胞死亡蛋白-1(PD-1)、程序性细胞死亡配体-1(programmed cell death ligand 1,PD-L1)、淋巴细胞激活基因-3(lymphocyte activation gene-3,LAG-3)等。表1中列举了部分已应用于临床试验的纳米抗体探针。
靶点 探针名称 临床分期 显像方式 年份 研究者[文献]或临床试验号 HER2 68Ga-HER2-Nanobody Ⅰ期 PET/CT 2016 Keyaerts[5] 68Ga-NOTA-Anti-HER2 VHH1 Ⅱ期 PET/CT 2019 NCT03331601 131I-SGMIB-Anti-HER2 VHH1 Ⅰ期 SPECT/CT 2016 NCT02683083 99mTc-MIRC208 招募中 SPECT/CT 2020 NCT04591652 MMR 68Ga-NOTA-Anti-MMR-VHH2 Ⅰ/Ⅱa期 PET/CT 2019 NCT04168528 PD-L1 99mTc-NM01 Ⅰ期 SPECT/CT 2019 Xing[6] 注:HER2为人表皮生长因子受体2;MMR为巨噬细胞甘露糖受体;PD-L1为程序性细胞死亡配体1;NOTA为1,4,7-三氮杂环壬烷-1,4,7-三乙酸;VHH为重链抗体可变区;PET为正电子发射断层显像术;CT为计算机体层摄影术;SPECT为单光子发射计算机体层摄影术;NCT为国家的临床试验 表 1 临床级别的纳米抗体分子影像探针及其靶点
Table 1. Clinical-grade nanobody-based molecular imaging probes and their targets
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使用放射性核素68Ga、18F等标记纳米抗体,可制备短半衰期PET/CT分子影像探针。目前,PET/CT显像纳米抗体探针的靶点主要包括HER2和CD38等。
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HER2是乳腺癌经典的生物标志物之一。抗HER2治疗可显著延长HER2阳性乳腺癌患者的生存期。基于纳米抗体的免疫PET显像(immunoPET)有望实现HER2异质性表达的无创可视化。Vaidyanathan等[7]构建了靶向HER2的纳米抗体探针18F-RL-I-5F7,证明了该探针可有效评估体内HER2的表达情况。2016年Keyaerts等[5]制备了68Ga-HER2-Nanobody,并在一项纳入了20例乳腺癌患者的临床试验中评估了该探针的诊断效能,结果显示,与周围正常组织相比,HER2阳性乳腺癌病灶中的示踪剂摄取明显增高,这充分证实了68Ga-HER2-Nanobody是一种极具临床应用价值的探针。目前该团队正在开展另一项临床试验,研究该探针对于乳腺癌脑转移灶的检出能力(NCT03331601)。
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CD38是一种在多发性骨髓瘤(multiple myeloma,MM)、非霍奇金淋巴瘤(NHL)及多种实体瘤细胞中显著高表达的跨膜蛋白。靶向CD38的抗体达雷木单抗(daratumumab)已获批临床,用于MM的治疗。最近,我们团队开发了首个靶向CD38的纳米抗体探针68Ga[Ga]-NOTA-Nb1053(NOTA: 1,4,7-三氮杂环壬烷-1,4,7-三乙酸,1,4,7-triacyclononane-1,4,7-tracetic acid),研究结果显示,该探针可特异性可视化皮下及原位骨髓瘤模型中浆细胞表面CD38的表达水平,进而实现MM的无创、靶点特异性精准诊断[8]。该探针的临床转化应用将有望为MM的早期诊断、靶向治疗患者的筛选及治疗后疗效评价提供全新的路径。关于该探针的临床转化前景,可见最新的述评[9]。
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TAM是肿瘤微环境的重要组成部分,具有多种促肿瘤活性。在肿瘤缺氧部位,具有较强促血管生成作用的TAM高度表达巨噬细胞甘露糖受体(macrophage mannose receptor,MMR)[10]。Blykers等[11]制备了纳米抗体探针18F-anti-MMR sdAb,基于该探针的免疫PET显像成功实现了肿瘤基质中促癌巨噬细胞亚群的无创可视化。Xavier等[12]利用68Ga标记的靶向MMR的纳米抗体探针进一步实现了其临床转化。目前一项临床实验(NCT04168528)的研究者正在研究68Ga-NOTA-Anti-MMR-VHH2(VHH: 重链抗体可变区,variable domains of heavy chain-only antibodies)免疫PET显像在乳腺癌、头颈部恶性肿瘤或黑色素瘤中的诊断价值。
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靶向免疫检查点的免疫治疗正在逐渐改变肿瘤治疗的现状,PD-L1是经典的免疫检查点之一[13]。然而,病灶内PD-L1是否表达及表达程度将直接影响免疫治疗的效果。Nature Medicine杂志刊登的一项临床试验初步报道了PD-L1特异性单克隆抗体免疫PET显像探针可视化PD-L1异质性表达、预测PD-L1特异性免疫治疗疗效的价值[14]。近期, 国内研究者构建了一种靶向PD-L1的纳米抗体探针68Ga-NOTA-Nb109,该探针可以利用PET/CT显像无创显示肿瘤PD-L1的表达情况并且及时准确评估免疫检查点靶向治疗的效果[15-16]。
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ECM对于促进肿瘤细胞的发生发展、增殖及迁移等具有重要的影响[17]。纤维连接蛋白(fibronectin)是一种糖蛋白,是构成肿瘤细胞外基质和新生血管的主要成分。Jailkhani等[18]从靶向ECM蛋白的纳米抗体文库中筛选出了一种靶向纤维连接蛋白剪接体EIIIB的纳米抗体NJB2,通过64Cu标记构建了64Cu-labeled-NJB2,最后通过免疫PET显像证明了该探针在检测肿瘤原发灶、肿瘤转移灶、纤维化病变及以ECM沉积为特征的疾病中具有良好的特异度和灵敏度。
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肿瘤浸润的CD8阳性(CD8+)T淋巴细胞在抗肿瘤免疫应答中起着重要作用。目前多项临床试验正在评估靶向CD8+ T淋巴细胞分子探针(89Zr-Df-IAB22M2C)的临床应用价值[19-21]。Rashidian等[22]及我们团队[23]分别构建了靶向CD8+ T细胞的纳米抗体探针89Zr-PEGylated-VHH-X118及68Ga-NOTA-SNA006a,上述探针在CD8+ T细胞的可视化及免疫治疗疗效的监测方面展现出了一定的价值,其临床应用价值有待进一步研究。
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除了上述的靶点之外,还有多种针对其他不同靶点的纳米抗体探针处于临床前研究阶段,如前列腺特异性膜抗原(PSMA)[24-25]、MM细胞的表面标志物M蛋白[26]和癌胚抗原(CEA)[27]等。
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使用发射γ射线的放射性同位素(如99Tcm、111In等)标记纳米抗体可进行SPECT/CT显像。EGFR是酪氨酸激酶受体家族的跨膜蛋白,其过度表达与多种恶性肿瘤的总体生存率和复发率相关。7D12是第1个被应用于分子影像的靶向EGFR的纳米抗体[28]。Renard等[29]合成了111In-labeled DTPA-IRDye700DX-7D12 ,该探针可进行SPECT/CT及荧光双模态显像,准确可视化肿瘤细胞EGFR的表达情况,有望筛选可从EGFR靶向治疗中获益的患者。赵晋华团队首次将99Tcm标记的抗PD-L1纳米抗体(NM-01)探针应用于非小细胞肺癌患者的SPECT/CT显像,研究结果显示,99Tcm-NM-01 SPECT/CT显像安全无不良反应,且在注射后2 h显示出良好的T、NT,肿瘤的摄取水平与组织活检所示PD-L1的表达情况具有良好的一致性[6]。近期,Lecocq 等[30]报道了一种靶向免疫检查点LAG-3的纳米抗体探针99Tcm-labeled nanobody 3132,利用SPECT/CT显像可以无创监测肿瘤浸润淋巴细胞(TILs)表面LAG-3的动态演变,从而有望预测肿瘤患者的免疫治疗效果及远期预后。
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活体光学成像包括生物发光技术和荧光技术。生物发光技术是采用荧光素酶基因(luciferase)转染细胞,培养出能稳定表达荧光素酶的细胞株,进而通过光学成像进行生物发光;而荧光技术则采用荧光报告基团标记相应靶点进行显像。近期,Nix等[31] 通过细胞表面蛋白质组学揭示了CD72是预后不良的KMT2A/MLL1(赖氨酸甲基轻移酶2A基因易位的混合谱系白血病,Lysine methyltransferase 2A translocations of the mixed lineage leukemia)重组急性B淋巴细胞白血病的潜在靶标,并构建了靶向CD72的纳米抗体NbD4,制备了基于NbD4主动靶向的嵌合抗原受体T细胞NbD4-CAR-T,进一步通过生物发光显像技术无创评估了NbD4-CAR-T对于KMT2A/MLL1重组急性B淋巴细胞白血病的治疗效果。其研究结果显示, CD72-CAR-T可延长CD19-CAR-T治疗失败的KMT2A/MLL1 重组急性B淋巴细胞白血病患者的生存期;此外,将靶向HER2的纳米抗体11A4与近红外荧光团IRDye 800CW偶联后可特异性地定位于HER2 阳性异种移植小鼠的肿瘤部位。因此,利用近红外荧光成像进行图像引导手术,可精准切除HER2 阳性肿瘤[32]。
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超声分子成像是一种新颖的肿瘤诊断方法,将携带靶向特异性的抗体或配体的超声造影剂注射入体内,通过血液循环聚集于肿瘤部位,进而进行超声分子成像。碳酸酐酶Ⅸ(CAⅨ)在包括肾细胞肾癌在内的多种恶性实体瘤细胞膜高度表达,是构建分子影像探针的良好靶标。Zhu等[33]构建了一种带有CAⅨ多肽的靶向纳米气泡,体内外研究结果显示,该气泡具有良好的穿透能力和特异性,可进行多种肿瘤的超声分子成像。
纳米抗体分子影像探针的研究进展
Reserch progress of molecular imaging with radiolabeled nanobodies
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摘要: 纳米抗体凭借其优越的靶向性、稳定性、溶解度及组织穿透能力,已成为分子影像领域中的热门靶向分子。多种基于纳米抗体的分子影像探针在临床前研究和临床试验中均已展现出良好的诊断效能。笔者主要介绍纳米抗体分子影像探针在肿瘤中的最新研究进展,并进一步讨论其临床转化面临的挑战和应对策略。
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关键词:
- 分子探针 /
- 纳米抗体 /
- 放射免疫显像 /
- 正电子发射断层显像术 /
- 肿瘤诊断
Abstract: Nanobodies have become popular targeting molecules in the field of molecular imaging due to their excellent targeting ability, superior stability, good solubility, and enhanced tissue penetration ability. A variety of nanobody-based molecular imaging probes have shown great promise for noninvasive diagnosis of human malignancies in preclinical and clinical settings. This review mainly introduces the latest research progress on nanobody-based molecular imaging probes in tumors, and further discusses the challenges and strategies for future clinical translation.-
Key words:
- Molecular probes /
- Nanobody /
- Radioimmunodetection /
- Positron-emission tomography /
- Tumor diagnostics
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表 1 临床级别的纳米抗体分子影像探针及其靶点
Table 1. Clinical-grade nanobody-based molecular imaging probes and their targets
靶点 探针名称 临床分期 显像方式 年份 研究者[文献]或临床试验号 HER2 68Ga-HER2-Nanobody Ⅰ期 PET/CT 2016 Keyaerts[5] 68Ga-NOTA-Anti-HER2 VHH1 Ⅱ期 PET/CT 2019 NCT03331601 131I-SGMIB-Anti-HER2 VHH1 Ⅰ期 SPECT/CT 2016 NCT02683083 99mTc-MIRC208 招募中 SPECT/CT 2020 NCT04591652 MMR 68Ga-NOTA-Anti-MMR-VHH2 Ⅰ/Ⅱa期 PET/CT 2019 NCT04168528 PD-L1 99mTc-NM01 Ⅰ期 SPECT/CT 2019 Xing[6] 注:HER2为人表皮生长因子受体2;MMR为巨噬细胞甘露糖受体;PD-L1为程序性细胞死亡配体1;NOTA为1,4,7-三氮杂环壬烷-1,4,7-三乙酸;VHH为重链抗体可变区;PET为正电子发射断层显像术;CT为计算机体层摄影术;SPECT为单光子发射计算机体层摄影术;NCT为国家的临床试验 -
[1] Hamers-Casterman C, Atarhouch T, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature, 1993, 363(6428): 446−448. DOI: 10.1038/363446a0. [2] Muyldermans S. Nanobodies: natural single-domain antibodies[J]. Annu Rev Biochem, 2013, 82: 775−797. DOI: 10.1146/annurev-biochem-063011-092449. [3] Bao GF, Tang M, Zhao J, et al. Nanobody: a promising toolkit for molecular imaging and disease therapy[J/OL]. EJNMMI Res, 2021, 11(1): 6[2021-10-10]. https://ejnmmires.springeropen.com/articles/10.1186/s13550-021-00750-5. DOI: 10.1186/s13550-021-00750-5. [4] Verhaar ER, Woodham AW, Ploegh HL. Nanobodies in cancer[J]. Semin Immunol, 2021, 52: 101425. DOI: 10.1016/j.smim.2020.101425. [5] Keyaerts M, Xavier C, Heemskerk J, et al. Phase Ⅰ study of 68Ga-HER2-nanobody for PET/CT assessment of HER2 expression in breast carcinoma[J]. J Nucl Med, 2016, 57(1): 27−33. DOI: 10.2967/jnumed.115.162024. [6] Xing Y, Chand G, Liu CC, et al. Early phase Ⅰ study of a 99mTc-labeled anti-programmed death ligand-1 (PD-L1) single-domain antibody in SPECT/CT assessment of PD-L1 expression in non-small cell lung cancer[J]. J Nucl Med, 2019, 60(9): 1213−1220. DOI: 10.2967/jnumed.118.224170. [7] Vaidyanathan G, McDougald D, Choi J, et al. Preclinical evaluation of 18F-labeled anti-HER2 nanobody conjugates for imaging HER2 receptor expression by immuno-PET[J]. J Nucl Med, 2016, 57(6): 967−973. DOI: 10.2967/jnumed.115.171306. [8] Wang C, Chen YM, Hou YN, et al. ImmunoPET imaging of multiple myeloma with [68Ga]Ga-NOTA-Nb1053[J]. Eur J Nucl Med Mol Imaging, 2021, 48(9): 2749−2760. DOI: 10.1007/s00259-021-05218-1. [9] Shi SX, Goel S, Lan XL, et al. ImmunoPET of CD38 with a radiolabeled nanobody: promising for clinical translation[J]. Eur J Nucl Med Mol Imaging, 2021, 48(9): 2683−2686. DOI: 10.1007/s00259-021-05329-9. [10] Movahedi K, Schoonooghe S, Laoui D, et al. Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages[J]. Cancer Res, 2012, 72(16): 4165−4177. DOI: 10.1158/0008-5472.CAN-11-2994. [11] Blykers A, Schoonooghe S, Xavier C, et al. PET imaging of macrophage mannose receptor-expressing macrophages in tumor stroma using 18F-radiolabeled camelid single-domain antibody fragments[J]. J Nucl Med, 2015, 56(8): 1265−1271. DOI: 10.2967/jnumed.115.156828. [12] Xavier C, Blykers A, Laoui D, et al. Clinical translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT imaging of protumorigenic macrophages[J]. Mol Imaging Biol, 2019, 21(5): 898−906. DOI: 10.1007/s11307-018-01302-5. [13] Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer[J]. N Engl J Med, 2012, 366(26): 2443−2454. DOI: 10.1056/NEJMoa1200690. [14] Bensch F, Van Der Veen EL, Lub-De Hooge MN, et al. 89Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer[J]. Nat Med, 2018, 24(12): 1852−1858. DOI: 10.1038/s41591-018-0255-8. [15] Liu QZ, Jiang L, Li K, et al. Immuno-PET imaging of 68Ga-labeled nanobody Nb109 for dynamic monitoring the PD-L1 expression in cancers[J]. Cancer Immunol Immunother, 2021, 70(6): 1721−1733. DOI: 10.1007/s00262-020-02818-y. [16] Lv GC, Sun XR, Qiu L, et al. PET imaging of tumor PD-L1 expression with a highly specific nonblocking single-domain antibody[J]. J Nucl Med, 2020, 61(1): 117−122. DOI: 10.2967/jnumed.119.226712. [17] Cox TR. The matrix in cancer[J]. Nat Rev Cancer, 2021, 21(4): 217−238. DOI: 10.1038/s41568-020-00329-7. [18] Jailkhani N, Ingram JR, Rashidian M, et al. Noninvasive imaging of tumor progression, metastasis, and fibrosis using a nanobody targeting the extracellular matrix[J]. Proc Natl Acad Sci U S A, 2019, 116(28): 14181−14190. DOI: 10.1073/pnas.1817442116. [19] Pandit-Taskar N, Postow MA, Hellmann MD, et al. First-in-humans imaging with 89Zr-Df-IAB22M2C Anti-CD8 minibody in patients with solid malignancies: preliminary pharmacokinetics, biodistribution, and lesion targeting[J]. J Nucl Med, 2020, 61(4): 512−519. DOI: 10.2967/jnumed.119.229781. [20] Farwell MD, Gamache RF, Babazada H, et al. CD8-targeted PET imaging of tumor-infiltrating T cells in patients with cancer: a phase Ⅰ first-in-humans study of 89Zr-Df-IAB22M2C, a radiolabeled anti-CD8 minibody[J]. J Nucl Med, 2022, 63(5): 720−726. DOI: 10.2967/jnumed.121.262485. [21] Griessinger CM, Olafsen T, Mascioni A, et al. The PET-tracer 89Zr-Df-IAB22M2C enables monitoring of intratumoral CD8 T-cell infiltrates in tumor-bearing humanized mice after T-cell bispecific antibody treatment[J]. Cancer Res, 2020, 80(13): 2903−2913. DOI: 10.1158/0008-5472.Can-19-3269. [22] Rashidian M, Ingram JR, Dougan M, et al. Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells[J]. J Exp Med, 2017, 214(8): 2243−2255. DOI: 10.1084/jem.20161950. [23] Zhao HT, Wang C, Yang YL, et al. ImmunoPET imaging of human CD8+ T cells with novel 68Ga-labeled nanobody companion diagnostic agents[J/OL]. J Nanobiotechnology, 2021, 19(1): 42[2021-10-10]. https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-021-00785-9. DOI: 10.1186/s12951-021-00785-9. [24] Rosenfeld L, Sananes A, Zur Y, et al. Nanobodies targeting prostate-specific membrane antigen for the imaging and therapy of prostate cancer[J]. J Med Chem, 2020, 63(14): 7601−7615. DOI: 10.1021/acs.jmedchem.0c00418. [25] Chatalic KLS, Veldhoven-Zweistra J, Bolkestein M, et al. A novel ¹¹¹In-labeled anti-prostate-specific membrane antigen nanobody for targeted SPECT/CT imaging of prostate cancer[J]. J Nucl Med, 2015, 56(7): 1094−1099. DOI: 10.2967/jnumed.115.156729. [26] Lemaire M, D'Huyvetter M, Lahoutte T, et al. Imaging and radioimmunotherapy of multiple myeloma with anti-idiotypic nanobodies[J]. Leukemia, 2014, 28(2): 444−447. DOI: 10.1038/leu.2013.292. [27] Wang H, Meng AM, Li SH, et al. A nanobody targeting carcinoembryonic antigen as a promising molecular probe for non-small cell lung cancer[J]. Mol Med Rep, 2017, 16(1): 625−630. DOI: 10.3892/mmr.2017.6677. [28] Vosjan MJWD, Perk LR, Roovers RC, et al. Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET[J]. Eur J Nucl Med Mol Imaging, 2011, 38(4): 753−763. DOI: 10.1007/s00259-010-1700-1. [29] Renard E, Camps EC, Canovas C, et al. Site-specific dual-labeling of a VHH with a chelator and a photosensitizer for nuclear imaging and targeted photodynamic therapy of EGFR-positive tumors[J/OL]. Cancers (Basel), 2021, 13(3): 428[2021-10-10]. https://www.mdpi.com/2072-6694/13/3/428. DOI: 10.3390/cancers13030428. [30] Lecocq Q, Awad RM, De Vlaeminck Y, et al. Single-domain antibody nuclear imaging allows noninvasive quantification of LAG-3 expression by tumor-infiltrating leukocytes and predicts response of immune checkpoint blockade[J]. J Nucl Med, 2021, 62(11): 1638−1644. DOI: 10.2967/jnumed.120.258871. [31] Nix MA, Mandal K, Geng HM, et al. Surface proteomics reveals CD72 as a target for in vitro-evolved nanobody-based CAR-T cells in KMT2A/MLL1-rearranged B-ALL[J]. Cancer Discov, 2021, 11(8): 2032−2049. DOI: 10.1158/2159-8290.CD-20-0242. [32] Kijanka M, Warnders FJ, El Khattabi M, et al. Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery[J]. Eur J Nucl Med Mol Imaging, 2013, 40(11): 1718−1729. DOI: 10.1007/s00259-013-2471-2. [33] Zhu LH, Guo YL, Wang LF, et al. Construction of ultrasonic nanobubbles carrying CAIX polypeptides to target carcinoma cells derived from various organs[J/OL]. J Nanobiotechnology, 2017, 15(1): 63[2021-10-10]. https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-017-0307-0. DOI: 10.1186/s12951-017-0307-0. [34] Rashidian M, Wang L, Edens JG, et al. Enzyme-mediated modification of single-domain antibodies for imaging modalities with different characteristics[J]. Angew Chem Int Ed Engl, 2016, 55(2): 528−533. DOI: 10.1002/anie.201507596. [35] Ehlerding EB, Sun LY, Lan XL, et al. Dual-targeted molecular imaging of cancer[J]. J Nucl Med, 2018, 59(3): 390−395. DOI: 10.2967/jnumed.117.199877. [36] Sikic BI, Lakhani N, Patnaik A, et al. First-in-human, first-in-class phase Ⅰ trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers[J]. J Clin Oncol, 2019, 37(12): 946−953. DOI: 10.1200/JCO.18.02018. [37] Ma LL, Zhu M, Gai JW, et al. Preclinical development of a novel CD47 nanobody with less toxicity and enhanced anti-cancer therapeutic potential[J/OL]. J Nanobiotechnology, 2020, 18(1): 12[2021-10-10]. https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-020-0571-2. DOI: 10.1186/s12951-020-0571-2. [38] Feng YT, Zhou ZY, McDougald D, et al. Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent[J]. Nucl Med Biol, 2021, 92: 171−183. DOI: 10.1016/j.nucmedbio.2020.05.002. [39] Akizawa H, Uehara T, Arano Y. Renal uptake and metabolism of radiopharmaceuticals derived from peptides and proteins[J]. Adv Drug Deliv Rev, 2008, 60(12): 1319−1328. DOI: 10.1016/j.addr.2008.04.005. [40] Zhou ZY, Meshaw R, Zalutsky MR, et al. Site-specific and residualizing linker for 18F labeling with enhanced renal clearance: application to an Anti-HER2 single-domain antibody fragment[J]. J Nucl Med, 2021, 62(11): 1624−1630. DOI: 10.2967/jnumed.120.261446. [41] Zhou ZY, Devoogdt N, Zalutsky MR, et al. An efficient method for labeling single domain antibody fragments with 18F using tetrazine- trans-cyclooctene ligation and a renal brush border enzyme-cleavable linker[J]. Bioconjug Chem, 2018, 29(12): 4090−4103. DOI: 10.1021/acs.bioconjchem.8b00699. [42] Hong HF, Zhou ZF, Zhou K, et al. Site-specific C-terminal dinitrophenylation to reconstitute the antibody Fc functions for nanobodies[J]. Chem Sci, 2019, 10(40): 9331−9338. DOI: 10.1039/c9sc03840j. [43] Xenaki KT, Dorrestijn B, Muns JA, et al. Homogeneous tumor targeting with a single dose of HER2-targeted albumin-binding domain-fused nanobody-drug conjugates results in long-lasting tumor remission in mice[J/OL]. Theranostics, 2021, 11(11): 5525−5538[2021-10-10]. https://www.thno.org/v11p5525.htm. DOI: 10.7150/thno.57510. [44] Lee W, Bobba KN, Kim JY, et al. A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images[J]. J Mater Chem B, 2021, 9(13): 2993−2997. DOI: 10.1039/d0tb02911d. [45] Peplau E, De Rose F, Reder S, et al. Development of a chimeric antigen-binding fragment directed against human galectin-3 and validation as an immuno-positron emission tomography tracer for the sensitive in vivo imaging of thyroid cancer[J]. Thyroid, 2020, 30(9): 1314−1326. DOI: 10.1089/thy.2019.0670. [46] Wei WJ, Rosenkrans ZT, Liu JJ, et al. ImmunoPET: concept, design, and applications[J]. Chem Rev, 2020, 120(8): 3787−3851. DOI: 10.1021/acs.chemrev.9b00738. [47] Panikar SS, Banu N, Haramati J, et al. Nanobodies as efficient drug-carriers: Progress and trends in chemotherapy[J]. J Control Release, 2021, 334: 389−412. DOI: 10.1016/j.jconrel.2021.05.004. [48] D'Huyvetter M, De Vos J, Caveliers V, et al. Phase Ⅰ trial of 131I-GMIB-Anti-HER2-VHH1, a new promising candidate for HER2-targeted radionuclide therapy in breast cancer patients[J]. J Nucl Med, 2021, 62(8): 1097−1105. DOI: 10.2967/jnumed.120.255679.