Department of Pharmaceutical Chemistry, University of California San Francisco, CA 94158, United States of America.
Molecular Biophysics and Integrated Bioimaging, Environmental Genomics and Systems Biology, and Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Biochim Biophys Acta Proteins Proteom. 2021 Feb;1869(2):140562. doi: 10.1016/j.bbapap.2020.140562. Epub 2020 Nov 19.
Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.
U33 是一种重组 Fab 抑制剂，可与 uPA 结合，其亲和力成熟用于提高与亲本 Fab 相比的亲和力和抑制效果。对 U33 的六个 CDR 环进行精氨酸扫描，以确定初始结合决定因素，因为 uPA 在其主要底物结合口袋中更喜欢精氨酸。选择两个 CDR 环来创建 U33 的工程亲和力成熟文库，该文库围绕 ArgL91（CDR L3）和 ArgH52（CDR H2）进行多样化。在严格条件下对随机化的 U33 文库进行生物淘选，得到了 8 个具有改善结合特性的 Fab。其中一种最有效的抑制剂 AB2 的 IC50 与 U33 相比降低了 13 倍，这主要是由于其离解速率降低所致。为了确定界面残基的贡献，这些残基在界面形成时可能会发生结构重排，我们使用了 X 射线足迹法和质谱法（XFMS）。有 4 个残基的溶剂可及性明显降低，它们的聚类表明 AB2 靶向活性位点，并与相邻口袋中的残基结合，该口袋是人类 uPA 所特有的。AB2 与 uPA 结合的 2.9 Å 分辨率晶体结构显示出一种结合模式，其中 CDR L1 环插入活性位点裂隙，并作为抑制的决定因素。这种结合模式的选择性决定因素与先前针对 uPA 相关丝氨酸蛋白酶、MTSP-1、HGFA 和 FXIa 的抑制性 Fab 不同。CDR H2 和 L3 环有助于界面形成，并提供关键盐桥以重塑 uPA 活性位点周围的环，从而提供特异性，并进一步证明抗体可以成为蛋白酶的有效和选择性抑制剂。
