Extended Data Fig. 1: Additional biophysical and structural characterization of humanized anti-QSOX1 antibodies.

a, Dot-blot analysis of 15 CUMAb designs. 12 designs exhibit comparable expression levels to the AbLIFT18 design of the anti-QSOX1 antibody¹⁴. CUMAb designs are labelled according to heavy chain V-gene subgroup and then by light chain subgroup according to IMGT. If there are two chains from the same subgroup, they are labelled as ‘a’ or ‘b’ arbitrarily. ‘Mock’ refers to a control transfection where no DNA was added. ‘Prev’ refers to a construct from a prior version of CUMAb. b, Functional screen of 15 CUMAb designs, labelled as in A. Presence of an upper band, corresponding to MBP-ZG16 modified with PEG-mal 5000, indicates remaining unoxidized MBP-ZG16 as a result of inhibited QSOX1 activity. As evident from the ‘no QSOX1’ lane, the MBP-ZG16 used in the experiment was not fully reduced, but the reduced population was sufficient to distinguish between the mock-transfected sample, which lacks antibody, and the antibody-containing samples, which all show some PEG-mal-modified species indicative of QSOX1 inhibition. Designs marked with * were further analyzed. c, Titration of relative QSOX1 activity at different antibody concentrations for mαQSOX1 and four CUMAb designs (aggregate data shown in main Fig. 2B). Points represent the average of two technical repeats and errors bars represent the standard deviation. d, Thermal denaturation of mαQSOX1 and four CUMAb designs using nano differential scanning fluorimetry. Shown is the average of two technical repeats. e, Crystal structure of oxidoreductase fragment of human QSOX1 in complex with parental mouse antibody (QSOX1 in dark gray, parental mouse antibody Fv in light gray, PDB entry 4IJ3) aligned with crystal structure of oxidoreductase fragment of human QSOX1 in complex with hαQSOX1.4 (QSOX1 in purple, hαQSOX1.4 Fv in magenta, PDB entry 8AON. Structures are nearly identical (0.7 Å) despite 51 Fv mutations.

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