Table 1. CB-EGF.1 mAb-driven selection of the original residues from libraries of EGF mutated variants.
| Targeted segment | Original residue | Unselected phage-displayed mAb- negative EGF variants | Selected mAb-positive EGF variants |
|---|---|---|---|
| P7-Y13 | P7 |
20% Pro other: ACFGHIKLMNQRSTVY |
45.2% Pro (> 2x) other: DGHLMNRSTW |
| L8 |
11.4% Leu other: ADGMNPQSTVWY |
26.1% Leu (> 2x) 64.1% hydrophobic: ILMV other: AFHKPRTWY |
|
| S9 |
5.7% Ser other: ADEGHIKLMPRTVY |
31.0% Ser (> 5x) other: AFHILNPQRTVY |
|
| H10 |
5.7% His other: ADEGIKLNPQRTVWY |
11.9% His (> 2x) other: AFILNPQSTVW |
|
| D11 |
No Asp other: EFGHKLMNPQRSTVWY |
88.1% Asp (> 88x) 95.2% negatively charged: DE Other: ST |
|
| G12 |
2.9% Gly other: ADFHMLPQRSTV |
61.9% Gly (> 21x) other: ADNPS |
|
| Y13 |
2.9% Tyr other: ACDEGHIKLMNPQRSTVW |
45.2% Tyr (> 15x) 83.3% aromatic: FWY other: AHLS |
|
| L15-V19 | L15 |
8.3% Leu other: AEFHIKMPQSTV |
6.9% Leu other: AEGMNPQRSTVW |
| H16 |
4.2% His other: ADGLMNPQRSTVW |
17.2% His (> 4x) other: ADEGLMNPQRSTY |
|
| D17 |
No Asp other: AGHIKLMPQRSTVWY |
3.4% Asp (> 3x) other: ACEGHKLNPQRSVW |
|
| G18 |
No Gly other: AEHILNPQR |
31% Gly (> 31x) other: AHQRS |
|
| V19 |
4.2% Val other: ACEHKLNPRTY |
6.9% Val other: ADEGHLMNPQRSTWY |
Residues within the EGF P7-Y13 and L15-V19 segments were replaced by random aa mixtures in two separate libraries. Samples of non-selected CB-EGF.1-negative EGF variants and positive variants selected on CB-EGF.1 mAb were sequenced. The frequencies (%) of each original residue in both groups of variants are shown. The relative increase in the frequency of a given residue after selection is indicated between parentheses. The combined prevalence (%) of residues with shared physicochemical properties at some positions is represented. Other amino acids found at each position in both groups of variants are also shown. Residues that contribute to epitope formation are shaded in gray, whereas those classified as major functional contributors are highlighted in dark gray.