Table 2. Effects of individual mutations on recognition of mouse IL-2 by JES6–1A12 mAb.
| Targeted mIL-2 residue | Effect of replacements | ||
|---|---|---|---|
| Tolerated | Partially tolerated | Non-tolerated | |
| Met 33 | F, I, L*, P, R | - | - |
| Gln 36 | C, L, M, T | P** | - |
| Glu 37 | G, H, I, L, M*, T, V | C | P** |
| Ser 40 | A, E, F, L, N*, R, T, V, Y | - | C, P** |
| Glu 43 | L, N*, S | G, I, W, Y | C, P, R |
| Asn 44 | Q | G, T | A, D, F, K, L, P, R, V, W |
| Tyr 45 | D, E, H, K, L, M | - | P |
| Arg 46 | K* | V | A, D, G, I, L, P, Q, S, T, Y |
| Asn 47 | C, P, Q, R, T, Y | - | - |
| Leu 48 | C, P*, R, S, W, Y | - | - |
| Leu 50 | A, M, P, T | - | - |
| Pro 51 | A, H, T* | - | - |
| Lys 90 | H, L, P, R, W | - | - |
| Gln 93 | A, E, G, H*, K, L, T | - | - |
Individual positions within the mIL-2 gene were randomized through Kunkel mutagenesis. Phages displaying diverse mIL-2-derived mutated variants were rescued at a 96-well scale. ELISA screening of phage-containing supernatants was used to evaluate their recognition by anti-mIL-2 mAbs. Protein sequences of the variants were deduced after sequencing phagemid-inserted genes. Mutated variants showing relative reactivities below 50% with JES6-1A12 mAb (compared with wt phage-displayed mIL-2) were considered to display non-tolerated replacements. Those substitutions resulting in reactivity levels between 50–75% and above 75% were classified as partially tolerated and tolerated, respectively. Residues shown to have an individual contribution to JES6-1A12 epitope formation are contained in the box. *Replacements by the equivalent residues in human IL-2. **Substitutions that also resulted in total or partial reactivity loss toward JES6-5H4 or S4B6 mAbs.