https://orcid.org/0000-0002-3888-9541
Figures & data
Figure 1. Deselection approaches do not prevent positive charge enrichment
(a) Schematic illustrating two differing selection strategies used to select mutagenic libraries. (b) Selection outputs (Round 1–4) from separate strategies were induced to express scFv and inhibitory activity was assessed via periprep HTRF. (c) Sanger sequencing of clones with equivalent or improved ΔF over parental revealed the percentage of the screening output represented by 9 dominant clones. (d) Sequences of the nine dominant clones, net VL CDR charge and BIAcore affinity measurements (e) AC-SINS and DNA binding scores for the dominant clones.
Figure 2. NGS of R3 selection outputs
Next-generation sequencing was performed on R3 outputs of the following selection branches: No deselection, in-solution DNA deselection and in-solution DT40 membrane deselection. (a) A slope of unity was generated with the frequency of shared clones between the non-deselected and deselected samples. (b) Overlap histograms highlighting the net CDR charge of unshared clones between non-deselected and deselected samples.
Table 1. Frequency of dominant clones within NGS dataset
Figure 3. Structurally guided interrogation of NGS datasets
(a) The structure of hIL-21 R interacting with the parental lead molecule MJ4-2 (b) Overlay of the structure of hIL-21 R in complex with both MJ4-2 Fab and IL-21. (c) Top down view of the MJ4-2 binding pocket highlighting the electrostatic potential and mutations within the binding pocket predicted to improve biophysical properties. (d) AC-SINs and DNA score for clones identified as high-frequency NGS clones, clones identified through traditional screening methods and clones rationally designed from the NGS data set or (e) Clones predicted via linear regression to have improved or poor biophysical properties.
Figure 4. Design, selection and screening of a computationally designed rational library
(a) Amino acid diversity introduced across all 6 CDRs within the rational library design. Sequence and biophysical scores of clones identified from selection and screening of a (b) rationally designed library or (c) application of linear regression models to the rational library NGS outputs. (d) Overlay of BIAcore trace of C2 parental and mAb5-Rational.
Supplemental material