Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity

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...

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Published in:	Biochimica et biophysica acta. Proteins and proteomics Vol. 1869; no. 2; p. 140562
Main Authors:	Sevillano, N., Bohn, M.F., Zimanyi, M., Chen, Y., Petzold, C., Gupta, S., Ralston, C.Y., Craik, C.S.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-02-2021 Elsevier
Subjects:	Affinity maturation Amino Acid Sequence - genetics Humans Protease inhibitor Quinuclidines - chemistry Quinuclidines - pharmacology Recombinant antibody Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - ultrastructure Serine Endopeptidases - chemistry Serine Endopeptidases - ultrastructure Serine Proteases - chemistry Serine Proteases - genetics Serine Proteinase Inhibitors - chemistry Serine Proteinase Inhibitors - pharmacology uPA Urokinase-Type Plasminogen Activator - antagonists & inhibitors Urokinase-Type Plasminogen Activator - chemistry Urokinase-Type Plasminogen Activator - genetics Affinity maturation Recombinant antibody Protease inhibitor uPA
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Summary:	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. •A novel mode of inhibition of a human hydrolase by a human antibody.•Atomic level resolution of the determinants of potency and specificity of an anti-proteolytic antibody.•Affinity maturation of an anti-proteolytic antibody increases potency.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 USDOE Sevillano, N: Conceptualization, Writing Zimanyi, M: Methodology, Writing Gupta, S: Methodology Co corresponding authors Bohn, M.-F: Conceptualization, Writing Chen Y: Methodology Credit author statement Petzold C: Methodology Craik C.S: Conceptualization, Writing Co first authors Ralston, C.Y: Methodology
ISSN:	1570-9639 1878-1454
DOI:	10.1016/j.bbapap.2020.140562