Solomon Viswas Raja, Barreto Kris, Bernhard Wendy, Alizadeh Elahe, Causey Patrick, Perron Randy, Gendron Denise, Alam Md Kausar, Carr Adriana, Geyer C Ronald, Fonge Humphrey
Department of Medical Imaging, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada.
Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada.
Cancers (Basel). 2020 Nov 20;12(11):3449. doi: 10.3390/cancers12113449.
To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc ( = 3) at 48 h post injection. The EC of Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly ( = 0.0017) prolonged the survival of mice (64 days) compared to Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly ( > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.
为了开发成像和治疗剂，抗体通常使用赖氨酸的伯胺（NH）或半胱氨酸的巯基（SH）随机偶联至螯合剂/放射性同位素或药物。与NH或SH基团的随机偶联可能需要极端条件，且可能影响靶点识别/结合，因此必须进行测试。在本研究中，使用∆N-SpyCatcher/SpyTag与不同的螯合剂和放射性金属对尼妥珠单抗进行位点特异性标记。尼妥珠单抗是一种耐受性良好、皮肤毒性低的抗表皮生长因子受体（EGFR）抗体。首先，使用三（2-羧乙基）膦（TCEP）还原∆N-SpyCatcher，随后与去铁胺-马来酰亚胺（DFO-mal）偶联，得到反应性∆N-SpyCatcher-DFO。使∆N-SpyCatcher-DFO与尼妥珠单抗-SpyTag反应，获得稳定的尼妥珠单抗-SpyTag-∆N-SpyCatcher-DFO。用锆进行放射性标记，并将该偶联物用于EGFR阳性的MDA-MB-468异种移植瘤的体内微型正电子发射断层扫描（microPET）成像。同样，将∆N-SpyCatcher与一种十八元大环螯合剂巨环帕-马来酰亚胺偶联，并用于用225锕（Ac）对尼妥珠单抗-SpyTag进行放射性标记，以用于体内放射治疗研究。使用生物层干涉术、流式细胞术、放射性配体结合试验、高效液相色谱法和生物分析仪对所有构建体进行表征。MicroPET/CT成像显示，注射后48小时，Zr-尼妥珠单抗-SpyTag-∆N-SpyCatcher在肿瘤中的摄取良好，为6.0±0.6%IA/cc（n = 3）。Ac-尼妥珠单抗-SpyTag-∆N-SpyCatcher和Ac-对照-IgG-SpyTag-∆N-SpyCatcher对EGFR阳性细胞系（MDA-MB-468）的半数有效浓度（EC）分别为3.7±3.3 Bq/mL（0.04±0.03 nM）和18.5±4.4 Bq/mL（0.2±0.04 nM）。在携带MDA-MB-468 EGFR阳性异种移植瘤的小鼠中，与Ac-对照IgG（28.5天）、尼妥珠单抗（28.5天）或磷酸盐缓冲液（PBS）处理的小鼠（30天）相比，Ac-尼妥珠单抗-SpyTag-∆N-SpyCatcher显著（p = 0.0017）延长了小鼠的生存期（64天）。结果表明，与非特异性偶联方法相比，使用SpyTag/∆N-SpyCatcher对尼妥珠单抗进行偶联和标记不会显著（p>0.05）改变尼妥珠单抗的受体结合。Ac-尼妥珠单抗-SpyTag-∆N-SpyCatcher在体外和EGFR阳性三阴性乳腺癌异种移植瘤模型中均有效。
