Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA)
Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA)
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DOI：
10.1093/protein/gzv065
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发表时间：
2016-03-01
影响因子：
2.4
通讯作者：
Skerra, Arne
中科院分区：
文献类型：
--
作者：
Barinka, Cyril;Ptacek, Jakub;Skerra, Arne
尽管前列腺癌（PCa）是迄今为止男性中最常诊断的肿瘤，但目前相应的诊断和治疗方式的疗效有限。 Anticalins 包含一类基于非免疫球蛋白支架的新型结合蛋白，可以对其进行工程改造以特异性地靶向感兴趣的分子靶标。在这里，我们报告了识别人前列腺特异性膜抗原（PSMA）的Anticalins的选择和表征，PSMA是一种膜束缚金属肽酶，构成PCa以及一般实体恶性肿瘤成像和治疗的疾病相关靶点。我们使用基于人脂质运载蛋白 2 (Lcn2) 支架的随机脂质运载蛋白文库以及噬菌体展示和 ELISA 筛选来选择 PSMA 特异性变体。来自最初淘选的五种 Anticalin 候选物在大肠杆菌中表达为可溶性单体蛋白，揭示了对 PSMA 的亲和力低至低纳摩尔范围。最有希望的候选物的结合特性通过应用易错PCR的亲和力成熟得到进一步改善，然后通过噬菌体展示以及更严格条件下的细菌表面展示进行选择。在 BIAcore 测量中，最佳 Anticalin 的解离常数被确定为类似于 500 pM，与第一代候选药物相比，解离速率显着提高。最后，免疫荧光显微镜揭示了 PSMA 阳性肿瘤细胞系的特异性染色，而流式细胞术分析证实了所选 Anticalins 检测活细胞上 PSMA 的能力。总而言之，本研究产生的 Anticalins 为生物医学应用（包括 PCa 或实体瘤新血管系统的体内成像）提供了基于抗体的 PSMA 结合剂的可行替代方案。
Although prostate carcinoma (PCa) is by far the most commonly diagnosed neoplasia in men, corresponding diagnostic and therapeutic modalities have limited efficacy at present. Anticalins comprise a novel class of binding proteins based on a non-immunoglobulin scaffold that can be engineered to specifically address molecular targets of interest. Here we report the selection and characterization of Anticalins that recognize human prostate-specific membrane antigen (PSMA), a membrane-tethered metallopeptidase constituting a disease-related target for imaging and therapy of PCa as well as solid malignancies in general. We used a randomized lipocalin library based on the human lipocalin 2 (Lcn2) scaffold together with phage display and ELISA screening to select PSMA-specific variants. Five Anticalin candidates from the original panning were expressed in Escherichia coli as soluble monomeric proteins, revealing affinities toward PSMA down to the low nanomolar range. Binding characteristics of the most promising candidate were further improved via affinity maturation by applying error-prone PCR followed by selection via phage display as well as bacterial surface display under more stringent conditions. In BIAcore measurements, the dissociation constant of the best Anticalin was determined as similar to 500 pM, with a substantially improved dissociation rate compared with the first-generation candidate. Finally, immunofluorescence microscopy revealed specific staining of PSMA-positive tumor cell lines while flow cytometric analysis confirmed the ability of the selected Anticalins to detect PSMA on live cells. Taken together, Anticalins resulting from this study offer a viable alternative to antibody-based PSMA binders for biomedical applications, including in vivo imaging of PCa or neovasculature of solid tumors.