Table 1.
Summary of mutations, equilibrium dissociation constants, and scFv yields for isolated clones
| scFv clone | Rounds of mutagenesis and screening | VH CDR and framework mutationsa | VL CDR and framework mutationsa | Kdb (nM) | Purified scFv yieldc (mg scFv/L culture) | ||
|---|---|---|---|---|---|---|---|
| CDR (#) | FR (#) | CDR (#) | FR (#) | ||||
| wt scFv13 | n/a | n/a | n/a | n/a | n/a | 200 ± 87 | 1.1 |
|
| |||||||
| scFv13.R4 | 4 | G10S (1) | G51D (2) | - | 128 ± 49 | 5.8 | |
| S52aG (2) | V48I (2) | ||||||
| S55R (2) | K75T (3) | ||||||
| A93V (3) | |||||||
|
| |||||||
| 1–4 | 1 | S55R (2) | - | - | - | nd | 3.2 |
|
| |||||||
| 2-1 | 2 | S55R (2) | - | S27aC (1) | S72F (3) | 91.5 ± 6.8 | 1.1 |
| G51D (2) | |||||||
|
| |||||||
| 2–3 | 2 | S55R (2) | L11P (1) | K42R (2) | - | nd | 3.1 |
| V48I (2) | G51D (2) | ||||||
| A93T (3) | V97D (3) | ||||||
a
Kabat numbering is used for VH and VL amino acids; see Fig. S3 for complete sequences. Value in parentheses refers to CDR or framework number. nd = not determined.
b
Equilibrium dissociation constants in the solution phase were determined by ELISA according to the protocol described by Martineau [ref. 41].
c
Yields of purified scFvs from a representative experiment.