Table 2.

The evaluation results of variants derived from AIA22

	ΔΔG (kcal/mol) **		Relative Affinity***		Relative Neutralizing Activity****
Variants*IFN-α:	1b	2b	1b	2b	1b	2b	5	6	8	14	17	21
L-S34A	−0.3	0.24	0.4	0.78	0.88	1.24	–	–	3.85	5.22	–	–
H-Y32A	−0.305	−0.115	0.07	0.88	–	1.15	3.42	>10	–	8.9	0.83	–
H-Y32G	−0.045	−0.17	0.44	2.52	1.03	1.12	0.73	–	–	0.99	1.07	–
H-A33G	−8.53	0.04	0.06	0.76	0.33	0.91	–	>10	>10	2.53	2.22	>10
H-Y96W	−0.02	0	0.13	2.15	0.87	1.05	–	>10	–	2.23	0.51	1.67
H-Y99V	−13.44	−55.425	0.56	0.87	0.48	5.86	7.71	–	–	–	–	–
L-S34A+H-A33G	−1.925	0.52	0.18	1.01	–	4.34	–	>10	>10	7.48	>20	–
L-S34A+H-Y96W	−0.735	0.395	0.27	1.58	0.49	1.85	–	>10	6.02	>20	1.25	–
H-32A96W	−0.65	−0.075	0.32	7.33	–	0.91	3.15	˜7.28	–	–	2.36	–
H-32G96W	−0.36	−0.18	0.22	3.56	1.09	0.88	2.37	2.89	–	7.98	0.93	–
H-33G96W	−5.68	−0.14	0.21	1.08	0.13	1.35	–	1.22	1.05	0.87	2.15	2.23
L-S34A+H-32A96W	−1.065	0.105	0.29	2.02	–	2.77	–	1.58	–	–	–	–
L-S34A+H-32G96W	−0.05	−0.14	0.21	1.15	0.15	0.38	0.19	2.11	1.01	8.53	0.62	˜0.01
L-S34A+H-33G96W	−0.06	0.23	0.19	2.39	0.13	1.71	–	11.34	7.59	7.33	1.98	–
H-32G96W99V	0.37	0.66	0.86	0.99	0.22	2.15	9.63	–	–	–	–	–

^*The “+” symbol represents the combination of light and heavy chain. For multi-site mutation of the same chain, the initial AA is not shown in the name.

^**ΔΔG (= ΔG_Variants − ΔG_AIA22) was estimated by FoldX based on the AIA22/IFN-α complex model. ΔG represents the binding free energy between antibody (AIA22 or variants) and corresponding IFN-α subtype.

^***The value of KD_Variants/KD_AIA22 (K_D was derived from the same ELISA assay with Table S3).

^****The value of EC50_Variants/EC50_AIA22 (EC50 was derived from proliferation assay of Daudi cell).

The horizontal short line (–) indicates the value was not determined.