Criteria for assigning residues to B-cell epitopes
| No. . | Substitution . | A . | AB . | B . | BC . | C . | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ESH4 . | 3E6 . | I54 . | I109 . | BO2C11 . | 1B5 . | 3D12 . | 3G6 . | 2-77 . | 2-117 . | ESH8 . | ||
| 1 | E2181A | QFD | FD | FD | ||||||||
| 2 | D2187A | NB | QFD | QFD | ||||||||
| 3 | Y2195A | FD | ||||||||||
| 4 | F2196A | FD | FD | FD | FD | |||||||
| 5 | T2197A | FD | FD | |||||||||
| 6 | N2198A | FD | FD | FD | FD | |||||||
| 7 | M2199A | FD | FD | |||||||||
| 8 | F2200A | QFD | FD | QFD | QFD | |||||||
| 9 | T2202A | FD | FD | FD | FD | |||||||
| 10 | S2206A | FD | FD | |||||||||
| 11 | K2207A | NB | FD | FD | ||||||||
| 12 | H2211A | FD | FD | FD | ||||||||
| 13 | L2212A | QFD | QFD | QFD | ||||||||
| 14 | Q2213A | NB | NB | NB | ||||||||
| 15 | R2215A | FD | ||||||||||
| 16 | R2220A | FD | NB | NB | QFD | NB | QFD | FD | FD | |||
| 17 | Q2222A | NB | ||||||||||
| 18 | N2224A | FD | ||||||||||
| 19 | N2225A | FD | QFD | QFD | QFD | |||||||
| 20 | E2228A | FD | FD | FD | FD | |||||||
| 21 | K2239A | FD | FD | |||||||||
| 22 | K2249A | FD | ||||||||||
| 23 | S2250A | FD | FD | |||||||||
| 24 | L2251A | FD | FD | |||||||||
| 25 | L2252A | FD | FD | FD | ||||||||
| 26 | T2253A | FD | FD | |||||||||
| 27 | S2254A | FD | FD | FD | ||||||||
| 28 | H2269A | FD | FD | |||||||||
| 29 | Q2270A | FD | FD | FD | ||||||||
| 30 | T2272A | FD | FD | |||||||||
| 31 | L2273A | FD | FD | FD | FD | |||||||
| 32 | V2280A | FD | FD | |||||||||
| 33 | V2282A | FD | FD | |||||||||
| 34 | R2307Q | QFD | NB | FD | ||||||||
| 35 | H2309A | QFD | QFD | QFD | QFD | QFD | ||||||
| 36 | Q2311A | FD | ||||||||||
| 37 | H2315A | FD | FD | |||||||||
| 38 | Q2316A | FD | FD | FD | ||||||||
| No. . | Substitution . | A . | AB . | B . | BC . | C . | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ESH4 . | 3E6 . | I54 . | I109 . | BO2C11 . | 1B5 . | 3D12 . | 3G6 . | 2-77 . | 2-117 . | ESH8 . | ||
| 1 | E2181A | QFD | FD | FD | ||||||||
| 2 | D2187A | NB | QFD | QFD | ||||||||
| 3 | Y2195A | FD | ||||||||||
| 4 | F2196A | FD | FD | FD | FD | |||||||
| 5 | T2197A | FD | FD | |||||||||
| 6 | N2198A | FD | FD | FD | FD | |||||||
| 7 | M2199A | FD | FD | |||||||||
| 8 | F2200A | QFD | FD | QFD | QFD | |||||||
| 9 | T2202A | FD | FD | FD | FD | |||||||
| 10 | S2206A | FD | FD | |||||||||
| 11 | K2207A | NB | FD | FD | ||||||||
| 12 | H2211A | FD | FD | FD | ||||||||
| 13 | L2212A | QFD | QFD | QFD | ||||||||
| 14 | Q2213A | NB | NB | NB | ||||||||
| 15 | R2215A | FD | ||||||||||
| 16 | R2220A | FD | NB | NB | QFD | NB | QFD | FD | FD | |||
| 17 | Q2222A | NB | ||||||||||
| 18 | N2224A | FD | ||||||||||
| 19 | N2225A | FD | QFD | QFD | QFD | |||||||
| 20 | E2228A | FD | FD | FD | FD | |||||||
| 21 | K2239A | FD | FD | |||||||||
| 22 | K2249A | FD | ||||||||||
| 23 | S2250A | FD | FD | |||||||||
| 24 | L2251A | FD | FD | |||||||||
| 25 | L2252A | FD | FD | FD | ||||||||
| 26 | T2253A | FD | FD | |||||||||
| 27 | S2254A | FD | FD | FD | ||||||||
| 28 | H2269A | FD | FD | |||||||||
| 29 | Q2270A | FD | FD | FD | ||||||||
| 30 | T2272A | FD | FD | |||||||||
| 31 | L2273A | FD | FD | FD | FD | |||||||
| 32 | V2280A | FD | FD | |||||||||
| 33 | V2282A | FD | FD | |||||||||
| 34 | R2307Q | QFD | NB | FD | ||||||||
| 35 | H2309A | QFD | QFD | QFD | QFD | QFD | ||||||
| 36 | Q2311A | FD | ||||||||||
| 37 | H2315A | FD | FD | |||||||||
| 38 | Q2316A | FD | FD | FD | ||||||||
FD, fast dissociation.
The kd(mutein) was >2.0× the kd for WT-FVIII-C2 binding to this mAb. In these cases, the kd could be estimated (by visual inspection) as more than 2.0 times the kd for WT-FVIII-C2, but the quality of the sensorgram was insufficient to fit the data to theoretical binding curves, as required to determine accurate kinetic constants for these interactions. NB, nonbinding. These amino acid substitutions completely abrogated binding to the antibody. Boldface indicate alanine substitutions that resulted in kd(mutein) > 2.0× the kd for WT-FVIII-C2 binding to this mAb, but these residues were not conformationally contiguous with the other surface-exposed candidate residues identified using the same kinetic criterion. Therefore, these outlier residues were not assigned to the corresponding epitopes. These substitutions likely had a localized effect on the stability/conformation of the protein, rather than disrupting specific interactions between the native FVIII side chains and the mAb. Twenty-two of the 60 FVIII-C2 muteins tested did not show altered binding kinetics to any of the mAbs (supplemental Table 2), and therefore they were not assigned to any of the functional B-cell epitopes.