Fig 1.
(A) Homology model of AB1 antibody with VH (blue) and VL (green) domains, annotated with the model-predicted accuracy on the CDR H3 at ≤2.5 Å. (B) Structure of muCCL20 showing human and murine sequence differences (grey). (C) VH (blue) and VL (green) sequences of anti-muCCL20 antibody AB1.
Table 1.
Details of the five highest-ranked, by RDOCK score, rigid-body docking poses.
Fig 2.
(A) Sequence alignment of human and mouse CCL20 with vMIP-II; identical residues are highlighted in green. (B) Binding specificity of AB1 antibody to murine (blue line) but not human (red line) CCL20. AB1 also blocked the interaction between muCCL20 and its receptor muCCR6 (black line). (C) Structural alignment of muCCL20 (white) with the CXCR4 (grey) and vMIP-II (black) co-crystal structure, 4RWS. (D) Top five highest-ranked antibody-muCCL20 docked poses, 1904 (red), 1704 (orange), 1843 (yellow), 1334 (green), and 1644 (blue), (E) aligned to vMIP-II in the surrogate receptor ligand complex.
Fig 3.
Predicted intermolecular interactions between antibody and antigen for each of the five candidate poses.
Fig 4.
In silico alanine scanning mutagenesis was completed for all five poses.
Poses 1904, 1704, 1843, 1334 and 1644 are shown in red, orange, yellow, green and blue respectively. The graph shows relative ΔΔEbinding changes upon alanine mutation for the five candidate docked poses across the IMGT-defined CDR regions.
Table 2.
Relative ΔΔEbinding values for each proposed variant across the five docked conformations.
IMGT numbering. Stabilizing mutations have negative ΔΔEbinding values; destabilizing mutations have positive ΔΔEbinding values.
Fig 5.
ELISA binding assay results representing the 23 alanine variants in comparison with WT AB1.
The WT AB1 mAb is shown in black. Nine variants that retained binding are represented in green. As labelled in the inset, 11 variants (variants 4, 6, 8, 9, 11, 12, 15, 17, 21, 22, 23) were found to be non-binding and 3 variants had reduced binding (variants 5, 6, 17). The mutations in these variants are denoted in the inset.
Table 3.
Panel of 20 single-mutation variants selected for affinity maturation of the anti-muCCL20 monoclonal antibody.
Mutations 2, 10, 14 and 16 were selected from Discovery Studio; 1, 3, 4, 5, 7, 8, 11, 12, 13, 17, 18 and 19 from Schrödinger; 5, 6, 9 and 15 from Rosetta and the neutral control mutation is 20.
Fig 6.
Re-docked protein complex of muCCL20 (crystal structure in yellow) and anti-muCCL20 antibody AB1 (homology model Fv, heavy chain in blue and light chain in green).
Interfacial residues (5.0 Å) are shown in sticks representation.
Fig 7.
(A) ELISA revealed improved antigen binding with VH clone 1 and VL clone 16, as well as the combination clone with both variable domains. (B) Fab fragments of C1, C16, and C1-16 showed improved KD values for muCCL20 when compared with parental AB1. (C) A cell-based β-arrestin assay demonstrated that the affinity-matured variants led to improved CCR6 receptor blocking activity when compared with the parental AB1 clone.
Fig 8.
Comparison of minimised docked structure between AB1 and muCCL20 (VH, blue; VL, green; antigen, yellow) with (A) H:R28 and (B) L:Y35 FEP+ output structures (VH, light blue; VL, light green; muCCL20, purple). (C–J) Local structural environment of VH (C–F) and VL (G–J) mutation sites (red) in the docked conformation (C, G), MM conformations (D, H), FEP+ native (E, I) and FEP+ mutant conformations (E, H).