Figure 3: Validation for C05 variants binding against H1 HA and H3 HA.
(a) Supernatant of 293T cells transfected with plasmids encoding the indicated C05 Fab variants was used to estimate the affinity of C05 Fab variants against immobilized SI06 HA (H1) and A/Perth/16/2009 (Perth09) HA (H3). The relative Kd of WT C05 Fab was set as 1, which is indicated by the green line. For visualizing purpose, the relative Kd is capped at 100. Variants that had an H3-specific binding profile and cited in the main text are boxed. (b) The effect of changing position 100d from Ala to Ser on binding kinetics was investigated. Four pairs of C05 variants, in which variants within each pair were related by an Ala to Ser substitution at position 100d, were included in our validation experiment (a). We computed the fold change in kon and koff by comparing the binding kinetics in the variant that carried a Ser at position 4 against the variant that carried an Ala at position 4. For visualization, for a given pair of variants (i→j), the fold change in kon was calculated as on rate of j divided by on rate of i, whereas fold change in koff was calculated as off rate of i divided by off rate of j, such that a positive value at the log scale indicates a positive effect in binding. (c) The occurrence frequencies of all observed variants in the sequencing data (a total of 848,630) in round 3 post-selection library and round 1 post-selection library are compared and shown as a scatterplot. Variants with >0.4% occurrence frequency in H1 round 3 post-selection library and <0.003% in H3 round 3 post-selection library were classified as ‘H1 specialists’ (shaded in red in the scatterplot). Variants with >0.3% occurrence frequency in H3 round 3 post-selection library and <0.001% in H1 round 3 post-selection library were classified as ‘H3 specialists’ (shaded in lilac in the scatterplot).
