Fig. 5
From: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
Epitope mapping of crossreactive antibodies. a Binding of SA129 (red), SA138 (blue), SA157 (gray), Ab275 (green), and Ab276 (orange) to a defined panel of hCXCL1 alanine mutants was assessed by flow cytometry. Obtained median values were normalized to the display median fluorescence intensities of each single yeast surface displayed mutant (binding/display). Normalized values represent the means of at least three independent experiments. Mutations that do not significantly affect binding (0.75‒1.0) are shown in white, while mutations that weakly (0.5‒0.75), moderately (0.25‒0.5), or strongly (0.0‒0.25) disrupt binding are shown respectively in light, intermediate, and dark colors. b The identified contact residues of hCXCL1 (PDB ID: 1MGS) to each antibody as defined by epitope mapping are shown in red (SA129), blue (SA138), gray (SA157*), green (Ab275), and orange (Ab276). The color intensity correlates with the strength of the interaction, with weak and strong interactions shown as light and dark colors, respectively. c Sequence alignment of various CXC chemokine proteins. Positions of conserved solvent-exposed residues that appear to be involved in the interaction with SA129 (red), SA138 (blue), SA157* (gray), Ab275 (green), and Ab276 (orange) based on residues identified using hCXCL1 alanine mutants are shown. Amino-acid sequences have been listed based on binding affinity (KD), with the tightest CXC chemokine protein at the top and the weakest at the bottom. Upper case N and C letters indicate the N- and C-terminus of the amino-acid sequence, respectively. Regions including residues that are not involved in binding are not reported for space reasons. The regions denoting the ELR-motif, N-loop, 30s-loop, 40s-loop, and 50s-loop that are known to be crucial for the binding of ELR+ CXC chemokines to the cognate CXCR2 receptor are indicated at the bottom. Residues have been highlighted according to the strength of interaction determined using soluble antibodies against hCXCL1 alanine mutants, as shown in panel a
