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.

与亲本 Fab 相比，uPA 的重组 Fab 抑制剂 U33 的亲和力成熟用于提高亲和力和抑制效果。对 U33 的六个 CDR 环进行精氨酸扫描以确定初始结合决定簇，因为 uPA 在其主要底物结合口袋中更喜欢精氨酸。选择了两个 CDR 环来创建 U33 的工程亲和力成熟库，该库在 ArgL91 (CDR L3) 和 ArgH52 (CDR H2) 周围多样化。在严格条件下对随机 U33 文库进行生物淘选产生了 8 个具有改进结合特性的 Fab。与 U33 相比，最有效的抑制剂之一 AB2 的 IC50 降低了 13 倍，这主要是由于其关闭率的降低。为了确定在界面形成时可能经历结构重排的界面残基的贡献，我们使用了 X 射线足迹和质谱 (XFMS)。四个残基的溶剂可及性显着降低，它们的聚集表明 AB2 靶向活性位点，并且还与人类 uPA 独有的相邻口袋中的残基结合。AB2 与 uPA 结合的 2.9 Å 分辨率晶体结构显示了一种结合模式，其中 CDR L1 环插入到活性位点裂缝中并作为抑制的决定因素。这种结合模式的选择性决定因素不同于先前鉴定的针对 uPA 相关丝氨酸蛋白酶、MTSP-1、HGFA 和 FXIa 的抑制性 Fab。