Alon-Zchut Gal, Zalk Ran, Huynh Truc T, Zalutsky Michael R, Weizmann Yossi, Zarivach Raz, Papo Niv
Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Ilse Katz Institute for Nanoscale Science & Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Int J Biol Macromol. 2025 Aug;320(Pt 1):145693. doi: 10.1016/j.ijbiomac.2025.145693. Epub 2025 Jul 1.
Prostate-specific membrane antigen (PSMA), overexpressed in prostate cancer, is a promising target for diagnostics and therapy. However, the monoclonal antibodies in current use for PSMA targeting and inhibition have suboptimal activities due to their poor tissue and cell penetration and slow normal tissue clearance. Potentially superior alternatives are nanobodies (NBs), the single-chain variable domains of heavy-chain antibodies derived from camelids. The advantages of NBs include small size (~15 kDa), ability to bind hidden epitopes, and rapid clearance. In contrast to most known PSMA inhibitors, which bind to the same catalytic site in PMSA, NBs can bind to different PSMA epitopes, facilitating heterovalent binding strategies that could enhance their therapeutic and diagnostic potential. The objective of this study was to map these binding epitopes and hence to acquire an atomic-resolution understanding of NB-PMSA binding by investigating the structural interactions between PSMA and three NBs (NB7, NB8, and NB37). Using cryo-electron microscopy to generate high-resolution structures of NB-PSMA complexes, we found that NB7 had the highest affinity for PSMA due to a larger interface and to stabilizing interactions, including salt bridges and π-π stacking. Notably, we also found that NB7 and NB8 can bind simultaneously to different PSMA epitopes without interfering with the function of PSMA (which is still not completely known), opening the way for the development of theranostic applications for prostate cancer treatment and imaging. Importantly, NB7 binds specifically to human PSMA but not to murine PSMA, due to key amino acid differences responsible for its species specificity.
前列腺特异性膜抗原（PSMA）在前列腺癌中过表达，是诊断和治疗的一个有前景的靶点。然而，目前用于靶向和抑制PSMA的单克隆抗体由于其组织和细胞穿透性差以及正常组织清除缓慢，活性并不理想。潜在的更优替代品是纳米抗体（NBs），它是源自骆驼科动物重链抗体的单链可变结构域。纳米抗体的优势包括尺寸小（约15 kDa）、能够结合隐蔽表位以及清除迅速。与大多数已知的PSMA抑制剂不同，后者结合PMSA中的同一催化位点，纳米抗体可以结合不同的PSMA表位，有助于采用异价结合策略，从而增强其治疗和诊断潜力。本研究的目的是绘制这些结合表位图谱，从而通过研究PSMA与三种纳米抗体（NB7、NB8和NB37）之间的结构相互作用，获得纳米抗体 - PMSA结合的原子分辨率认识。利用冷冻电子显微镜生成纳米抗体 - PSMA复合物的高分辨率结构，我们发现NB7对PSMA具有最高亲和力，这归因于更大的界面以及稳定相互作用，包括盐桥和π-π堆积。值得注意的是，我们还发现NB7和NB8可以同时结合不同的PSMA表位而不干扰PSMA的功能（其功能仍不完全清楚），这为开发用于前列腺癌治疗和成像的治疗诊断应用开辟了道路。重要的是，由于关键氨基酸差异导致其具有物种特异性，NB7特异性结合人类PSMA而不结合小鼠PSMA。
