PO.ET08.02 · 实验与分子治疗
用于诊疗一体化应用的钙网蛋白结合物的从头蛋白质设计
De novo protein design of calreticulin binders for theranostic applications
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：钙网蛋白（CALR）是一种内质网（ER）蛋白，在部分内质网应激期间可易位至肿瘤细胞表面。表面CALR在正常细胞上基本不存在，但在多种处于应激状态的肿瘤细胞上上调，使其成为一个具有潜在普遍性的肿瘤特异性靶点，并可作为泛肿瘤生物标志物加以应用。用诊疗一体化制剂靶向表面CALR，可为肿瘤可视化及治疗反应监测提供一种新策略。我们假设，从头蛋白质设计能够生成一种新型CALR特异性诊疗一体化制剂。
方法：我们采用从头蛋白质设计平台BindCraft生成针对CALR的小分子（<10kDa）高亲和力结合物。一条流线化的计算机模拟到体外的筛选流程能够在两周内分离出CALR结合物。结合亲和力与特异性通过生物膜层干涉技术（BLI）和流式细胞术进行验证。在流式细胞术中，用已知的表面CALR诱导剂多柔比星处理人胰腺癌细胞。细胞与两种CALR结合物孵育30分钟，随后用荧光抗体染色。在基质胶栓（Matrigel plug）模型中，携带CALR栓与对照栓的小鼠被注射100 μCi的[64Cu]Cu-NOTA-结合物，并通过动态PET成像1小时，随后进行CT。
结果：针对CALR的BLI鉴定出两种高亲和力结合物d4和d6，其计算得到的解离常数分别为83.2 nM（kon = 512790 M-1s-1，koff = 0.04265 s-1）和31.2 nM（kon = 551797 M-1s-1，koff = 0.01723 s-1）。流式细胞术显示，两种CALR结合物d4和d6在多柔比星处理的癌细胞上相比溶媒对照分别检测到表面CALR增加1.6倍和2.1倍。PET成像显示，[64Cu]Cu-NOTA-d6在CALR栓中的摄取在注射后早至20分钟即显著高于对照，并在整个成像期间持续滞留。注射后1小时，CALR栓的SUVmean为（0.882 ± 0.270），而对照栓为（0.344 ± 0.048）（p = 0.0272）。[64Cu]Cu-NOTA-d6经肾清除，相比血池在非靶器官中摄取不显著，平均血半衰期为4.2分钟。
结论：本研究证明了从头蛋白质设计快速生成适用于诊疗一体化应用的高亲和力CALR靶向结合物的可行性。进一步的体内研究将评估d6在临床前癌症模型中的肿瘤特异性摄取和长期滞留，以支持其作为诊疗一体化制剂的转化开发。
查看英文原文 English abstract
Background: Calreticulin (CALR) is an endoplasmic reticulum (ER) protein that can translocate to tumor cell surfaces during partial ER stress. Surface CALR is largely absent on normal cells yet upregulated on many types of tumor cells undergoing stress, making it a tumor-specific target with potential ubiquity and application as a tumor-agnostic biomarker. Targeting surface CALR with theranostic agents could offer a novel strategy to visualize tumors and monitor treatment response. We hypothesized that de novo protein design could generate a novel CALR-specific theranostic agent.
Methods: We employed the de novo protein design platform BindCraft to generate small (<10kDa) high-affinity binders against CALR. A streamlined in silico to in vitro screening pipeline enabled isolation of CALR binders within two weeks. Binding affinity and specificity were validated by biolayer interferometry (BLI) and flow cytometry. For flow cytometry, human pancreatic cancer cells were treated with doxorubicin, a known surface CALR inducer. Cells were incubated with two CALR binders for 30 minutes, followed by staining with fluorescent antibody. In a Matrigel plug model, mice bearing CALR and control plugs were injected with 100 μCi of [ 64 Cu]Cu-NOTA-binder and imaged by dynamic PET for 1 hour followed by CT.
Results: BLI against CALR identified two high affinity binders, d4 and d6, with calculated dissociation constants of 83.2 nM ( k on = 512790 M -1 s -1 , k off = 0.04265 s -1 ) and 31.2 nM ( k on = 551797 M -1 s -1 , k off = 0.01723 s -1 ), respectively. Flow cytometry showed both CALR binders, d4 and d6, detected increases in surface CALR of 1.6-fold and 2.1-fold, respectively, on doxorubicin-treated cancer cells compared to vehicle. PET imaging revealed significantly higher uptake of [ 64 Cu]Cu-NOTA-d6 in CALR plugs compared to controls as early as 20 minutes post-injection, with sustained retention throughout the imaging period. At 1 hr post-injection, SUV mean of the CALR plug was (0.882 ± 0.270) compared to (0.344 ± 0.048) for the control plug ( p = 0.0272). [ 64 Cu]Cu-NOTA-d6 was cleared renally with insignificant uptake in non-target organs compared to blood pool and an average blood ½-life of 4.2 minutes.
Conclusions: This study demonstrates the feasibility of de novo protein design to rapidly generate high-affinity CALR-targeted binders suitable for theranostic applications. Further in vivo studies will evaluate tumor-specific uptake and long-term retention of d6 in preclinical cancer models to support its translational development as a theranostic agent.
利益披露 Disclosure
L. Rathbun, None..
Y. Golivi, None..
R. Guenter, None..
J. Rose, None..
B. Larimer, None.