Table 3.
Engineered binder sequences
| Name | Target | 23 | 24 | 25 | 26 | 27 | 28 | — | 29 | 30 | 31 | 52 | 53 | 54 | 55 | 56 | 77 | 78 | 79 | 80 | 81 | 82 | 83 | 84 | 85 | 86 | Framework | Kd (nM) | Tm (°C) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | — | D | A | P | A | V | T | — | V | R | Y | G | S | K | S | T | G | R | G | D | S | P | A | S | S | K | — | — | 85.7 |
| E4.2.1 | EGFR | Y | G | F | S | L | — | — | A | S | S | R | S | P | W | F | S | N | D | F | S | N | R | Y | S | G | — | 30±3 | 56.2 |
| E6.2.6 | EGFR | F | D | Y | A | — | — | — | V | T | Y | G | W | I | S | T | D | N | S | H | W | P | F | R | S | T | I90T | 0.26±0.13 | 65.7 |
| E6.2.10 | EGFR | Y | L | R | D | P | R | Y | V | D | Y | W | Y | L | P | E | Y | D | G | Y | R | E | S | T | P | L | — | 0.96±0.11 | — |
| EI1.4.1 | EGFR | Y | G | P | F | Y | Y | V | A | H | S | R | S | P | W | F | S | K | C | Y | D | G | — | — | S | V | — | 0.85±0.50 | 52.8 |
| EI2.4.6 | EGFR | Y | H | P | F | Y | Y | V | A | H | S | R | S | P | W | F | D | S | N | G | — | — | — | — | S | H | — | 2.9±0.3 | 63.9 |
| EI3.4.2 | EGFR | Y | G | S | S | Y | — | — | A | S | Y | R | S | P | W | F | P | S | G | I | — | — | — | — | S | A | T58I | 9.5±3.5 | — |
| EI3.4.3 | EGFR | L | H | H | R | S | D | — | V | R | S | G | S | R | S | L | W | G | S | Y | C | C | S | N | E47K | 0.25±0.05 | 72.8 | ||
| EI4.4.2 | EGFR | Y | F | R | D | P | R | Y | V | D | Y | W | Y | L | P | E | G | D | D | Q | N | A | — | — | G | L | V45A | 1.4±0.2 | 70.2 |
| IIa8.2.6 | Fcγlla | C | T | H | L | H | — | — | W | D | Y | A | L | C | P | G | V | G | G | D | — | — | — | — | D | W | R6G, T35F, V72A, I88S, K98E | 850 | — |
| IIIa6.2.6 | FcγlIIa | D | M | P | F | — | — | — | S | D | S | G | T | D | S | L | S | S | G | S | N | — | — | — | S | Y | A12V, S21N, T35A | 530 | — |
| hA2.2.1 | hA33 | Y | C | P | D | G | C | H | S | Y | Y | R | S | I | S | S | F | R | W | P | — | — | — | — | S | F | — | — | — |
| hA2.2.2 | hA33 | N | T | Y | F | S | F | — | L | Y | Y | S | S | L | H | T | G | T | W | P | — | — | — | — | S | Y | — | — | — |
| hA3.2.1 | hA33 | S | Y | S | S | Y | N | S | W | D | S | N | S | D | C | I | R | D | C | D | F | Y | — | — | S | Y | Y32F | — | — |
| hA3.2.2 | hA33 | Y | Y | H | L | R | G | — | L | D | S | R | S | Y | S | T | V | N | D | Y | I | — | — | — | S | Y | S21G Q46K T49A | — | — |
| mA3.2.1 | mA33 | S | S | S | L | Y | N | — | S | A | Y | V | W | D | C | T | P | N | Y | S | F | — | — | — | S | L | Y32F | — | — |
| mA3.2.2 | mA33 | C | C | L | F | F | — | — | S | G | Y | G | L | V | Y | W | D | N | V | G | — | — | — | — | S | N | I90V | — | — |
| mA3.2.3 | mA33 | S | F | P | C | V | — | — | S | S | S | G | D | T | T | S | S | T | C | Y | P | — | — | — | S | Y | — | — | — |
| mA3.2.4 | mA33 | S | C | P | I | C | P | R | A | T | S | A | T | — | S | S | D | Q | G | Y | D | D | — | — | S | A | I34V | — | — |
| mA3.2.5 | mA33 | Q | C | H | Y | Y | Y | — | A | Q | S | S | S | K | S | T | Y | N | W | F | L | D | S | V | S | I | A12V | — | — |
| Alb3.2.1 | hAlb | G | A | P | A | C | — | — | A | A | Y | G | S | G | T | S | S | R | Y | Y | Y | C | — | — | S | E | — | — | — |
| mI2.2.1 | mlgG | C | C | S | D | N | C | — | S | N | S | R | S | C | F | M | D | S | N | G | — | — | — | — | P | H | V72A | 4.1±0.7 | — |
“Name” is the name of each clone. “Target” is the cognate protein bound by the Fn3 clone. 23–31,52–56, and 77–86 refer to the amino acid positions diversified in the naive library. “Framework” refers to amino acid mutations outside of the diversified loops. A dash indicates no amino acid. Underline indicates wild-type amino acid.