噬菌体展示筛选鉴定高亲和力 TAG-72 结合肽。

Identification of a high affinity TAG-72 binding peptide by phage display selection.

机构信息

University of Massachusetts Medical School, Worcester, Massachusetts, USA.

出版信息

Cancer Biol Ther. 2011 Jan 1;11(1):22-31. doi: 10.4161/cbt.11.1.13797.

DOI:10.4161/cbt.11.1.13797

PMID:20980835

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3047098/

Abstract

PURPOSE

Phage display was used to select novel peptides that specifically bind the TAG-72 antigen and with properties suitable for imaging TAG-72 positive cancers.

RESULTS

After three rounds of selection against TAG-72 and using two different elution conditions including a long elution, the consensus sequences FRERCDKHPQKCTKFL and DPRHCQKRVLPCPAWL were expressed on phages G3-15 and T3-15 respectively. ELISA, fluorescence-activated cell sorting analysis and fluorescence microscopy provided evidence that both phages specifically bound TAG-72 in vitro. Both peptides are stable in 37oC serum. By a cell binding competition assay, the IC50 for T3-15 was measured as 0.29 nM and therefore 36-fold higher affinity than G3-15 at 10.32 nM. The biodistribution in mice carrying LS-174T tumors in one thigh were similar for both 99mTc-peptides at 30 min, but at 90 min the 99mTc-T3-15 peptide accumulated almost three times higher in the tumor. The SPECT/CT images were consistent with the biodistribution results.

PROCEDURES

The f88-4/Cys6 phage library and two different elution conditions were used to identify two new higher affinity binding peptides for the TAG-72 antigen. One, was a single brief elution with pH 2.2 glycine buffer, and the second began with the glycine elution but was followed with a longer elution with Tris buffered saline (TBS) at pH 7.4. The phages that bound TAG-72 were evaluated by fluorescence-activated cell sorting analysis using TAG-72 positive LS-174T cells and confirmed by immunofluorescence imaging. The consensus peptides displayed on the selected phages were synthesized and conjugated with NHS-MAG3 for radiolabeling with 99mTc. The IC50 for TAG-72 binding was evaluated by cell binding competition in vitro while binding affinity was evaluated in vivo by necropsy and SPECT/CT imaging in a tumor mouse model.

CONCLUSION

We have identified a peptide with a sub nanomolar inhibition constant for the TAG-72 antigen that may have application in cancer imaging.

摘要

目的

利用噬菌体展示技术筛选能够特异性结合 TAG-72 抗原且具有适用于成像 TAG-72 阳性癌症的特性的新型肽。

结果

经过三轮针对 TAG-72 的筛选，并使用包括长洗脱在内的两种不同洗脱条件，共识序列 FRERCDKHPQKCTKFL 和 DPRHCQKRVLPCPAWL 分别在噬菌体 G3-15 和 T3-15 上表达。ELISA、荧光激活细胞分选分析和荧光显微镜检查提供了证据表明，这两种噬菌体都能在体外特异性结合 TAG-72。这两种肽在 37°C 血清中都很稳定。通过细胞结合竞争测定，T3-15 的 IC50 为 0.29 nM，因此比 G3-15 的 10.32 nM高 36 倍。在携带 LS-174T 肿瘤的小鼠中，两种 99mTc-肽在 30 分钟时的生物分布相似，但在 90 分钟时，99mTc-T3-15 肽在肿瘤中的积累量几乎高出三倍。SPECT/CT 图像与生物分布结果一致。

程序

使用 f88-4/Cys6 噬菌体文库和两种不同的洗脱条件，鉴定出两种针对 TAG-72 抗原的新型高亲和力结合肽。一种是 pH 2.2 甘氨酸缓冲液的单一短暂洗脱，另一种是从甘氨酸洗脱开始，但随后用 pH 7.4 的 Tris 缓冲盐水（TBS）进行更长时间的洗脱。通过用 TAG-72 阳性 LS-174T 细胞进行荧光激活细胞分选分析评估与 TAG-72 结合的噬菌体，并通过免疫荧光成像进行确认。在选定的噬菌体上展示的共识肽被合成并与 NHS-MAG3 缀合，用于用 99mTc 标记。通过体外细胞结合竞争评估 TAG-72 结合的 IC50，通过肿瘤小鼠模型的尸检和 SPECT/CT 成像评估体内结合亲和力。