Biophysical characterization of leads. (A) Binding of Fab fragments to HLA-DQ2.5:DQ2.5-glia-α1a by SPR. Fabs were ranked based on off-rates. The parent clone 107 is shown in blue (n = 2). (B) Melting temperatures (Tm) of the parent clone 107 (blue) and the affinity matured Fab fragments. Error bars illustrate mean ± SD of 3–7 replicates. Statistical analysis was performed by unpaired two-tailed t-test, ^****P < 0.0001. (C) Representative sensorgrams of 4.7C and combination variant 4.7Cplus (n ≥ 2). Data were fitted to a 1:1 Langmuir binding model (dotted gray lines). (D) Table detailing position of mutations, library origin and Kd value of the individual clones. FR, framework; NA, not applicable. (E) The locations of the mutations present in the combination mutant 4.7Cplus are illustrated as spheres. VH contains the following mutations: CDR H1 Asn 31A Ser and CDR H3 Ser 96 Arg, Ser 97 Thr and Ser 98 Thr. VL contains the following mutations: FR1 Ile 2 Val, FR3 Ile 53 Val and CDR L3 Asp 90 Asn. The Fv model is based on mAb 107. (F) The candidate antibodies were reformatted to full-length hIgG1 (0.5 μg/mL) and binding to a panel of related soluble peptide:HLA-DQ2.5:gliadin complexes or controls was analysed by ELISA. Error bars illustrate mean ± SD of duplicates (n = 3).