Fig. 4: Biochemical characterization of combinatorially assembled scFvs with six designed CDRs.

a, Multiple sequence alignment of six scFvs that bind to TcdB. scFvs 1–5 originate from the same structural cluster, whereas scFv6 originates from a distinct cluster. b,c, AlphaFold3 predictions of scFv5 (b) and scFv6 (c) in complex with TcdB. scFv5 and scFv6 are predicted to bind to a similar but not identical epitope. The predicted orientation of scFv6 relative to TcdB is rotated compared with scFv5. d, Affinity of scFv5 to TcdB was 460 nM by SPR. e, Computational prediction of the scFv5–TcdB interface for VH (variable heavy-chain fragment; left) and VL (variable light-chain fragment; right). f, scFv5, when expressed as a full-length IgG1, shows a binding affinity of 380 nM to TcdB by SPR. g, Affinity of scFv6 to TcdB was 72 nM by SPR. h, Computational prediction of the scFv6–TcdB interface for VH (left) and VL (right). i, scFv6, when expressed as a full-length IgG1, shows a binding affinity of 68 nM to TcdB by SPR. j, scFv5 competes with Frizzled-7 and does not compete with CSPG4, indicating on-target binding. scFv5 was conjugated to a CM5 chip and TcdB RBD was flowed over at 50 nM either alone or mixed with 1 μM of Frizzled-7, CSPG4 or scFv5. k,l, SPR comparative analysis of B1.2.1 binding to C*07:02–PHOX2B versus C*07:02–PHOX2B(R6A). scFv was immobilized and then on-target and off-target binding was measured across an eight-step, twofold titration with an upper concentration of 5 μM. Steady-state kinetic analysis (k) and raw SPR trace (l) of on-target and off-target binding indicate specific binding to the intended target. m, AlphaFold3 predictions of HLA-C*07:02 with peptide PHOX2B (left) and PHOX2B(R6A) (right). R6 of PHOX2B is predicted to be solvent exposed. n, AlphaFold3 prediction of scFv B1.2.1 in complex with C*07:02–PHOX2B (left). Predicted polar contacts with R6 of the PHOX2B peptide (right), mediated by CDRH3, CDRL1 and CDRL2, are also shown. Figure was created using BioRender (http://biorender.com).