Fig. 3

Frequency and distribution of mutations in crossreactive antibodies. a Box-and-whiskers graph comparing the total number (left y-axis) and frequencies (right y-axis) of mutated residues detected in CK1-, CK2-, and CK4-derived clones before (light gray) and after (dark gray) the first (I) and second (II) process of selection, respectively. “Inp” indicates sequenced clones picked from the random yeast-display antibody library before selection (input). “Out” indicates sequenced clones after selection (output). “n” indicates samples size. The middle line within each box represents the median, and the lower and upper boundaries of the box indicate the 25th (Q1) and 75th (Q3) percentiles. Whiskers represent the 1.5× interquartile range (IQR = Q3–Q1) extending beyond box. Statistical comparisons were made between each group using one-way analysis of variance (ANOVA), followed by Tukey’s test to calculate P-values: *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001. ns: non-significant. Homology model and frequencies of enriched mutations of engineered b CK138 and c CK157 antibodies. Left, the V_L and V_H backbones are represented as ribbons (light gray). Mutations acquired during the selection process are depicted as spheres at the Cα positions. Mutated amino acids belonging to CDR loops of CK138 and CK157 are colored in dark blue and dark red, respectively. Diversified amino acids belonging to FWR regions of CK138 and CK157 are colored in light blue and light red, respectively. Right, columns graph reporting the mutation frequency in CDR (dark gray) and FWR (light gray) regions. Only amino-acid mutations of CK1 and CK2 lineages that showed at least 20% frequency and were enriched through two iterative processes of selection are reported. Wild type and mutated amino acids are listed at the top and bottom, respectively. d Fluorescence binding signal of CK1- (left) and CK2- (right) derived clones bearing highly frequent mutations within the CDR-H3 and FWRs, respectively, that were reverted to the wild-type amino acids. ELR⁺ CXC chemokines and the corresponding binding/display values (y-axis) are indicated as differently colored filled circles and represent the means of at least three independent experiments