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Abstract：
CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety-specific conjugation with deferoxamine-maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects.
摘要：
CD73是一种细胞表面的胞外酶，可水解细胞外磷酸腺苷转化为腺苷，这反过来又可以促进对免疫检查点阻断治疗的抵抗力。因此,可以通过靶向肿瘤CD73来改善免疫应答,并且这种可能性强调需要非侵入性地评估肿瘤CD73水平。在这项研究中,我们开发了一种半胱氨酸位点特异性89Zr标记的抗CD73（89Zr-CD73）IgG免疫PET技术，该技术可以成像活体中肿瘤CD73的表达。抗CD73IgG用三（2-羧乙基）膦还原，与去铁胺-马来酰亚胺进行巯基部分特异性缀合，并用89Zr放射性标记。CT26小鼠结肠癌细胞，经工程化以组成性过表达CD73的CT26/CD73细胞和4T1.2小鼠乳腺癌细胞进行细胞结合测定和蛋白质印迹。携带肿瘤的Balb/c裸鼠接受了89Zr-CD73IgGPET成像和生物分布研究。与低表达CT26细胞相比，89Zr-CD73IgG与过表达CT26/CD73细胞的结合高20倍，中等表达的4T1.2细胞显示CT26/CD73细胞的摄取为38.9±1.51%。过量的未标记抗体显著抑制了摄取。CT26和CT26/CD73细胞混合物中CD73含量成比例增加与89Zr-CD73IgG摄取的线性增加有关。89Zr-CD73IgGPET/CT在CT26/CD73肿瘤中显示出明显的积累，与CT26肿瘤相比具有更大的摄取（3.13±1.70％ID/gvs.在第8天1.27±0.31%ID/g；P=0.04）。与89Zr-CD73IgG相比，89Zr-同种型IgG的低CT26/CD73肿瘤与血液比率进一步支持了特异性（0.48±0.08vs.2.68±0.52在4天和0.53±0.07vs.第8天4.81±1.02；两者均P<0.001）。免疫印迹和免疫组织化学证实了CD73在CT26/CD73肿瘤中的强表达和在CT26肿瘤中的低表达。4T1.2肿瘤小鼠在8天时也显示出清晰的89Zr-CD73IgG积累（3.75±0.70％ID/g），与89Zr同种型IgG相比，具有较高的肿瘤与血液比率（4.91±1.74vs.1.20±0.28；P<0.005）。89Zr-CD73IgG特异性靶向CD73在体外高表达癌细胞和体内肿瘤。因此，89Zr-CD73IgG免疫PET可用于活体肿瘤中CD73表达的非侵入性监测。
