TABLE 1.

Mutants designed using PDA and SPA calculations

Position	29	30	38	41	64	66	75	mAb bindinga	αLI domain tetramer bindingb	%OccPDAc	%OccSPAc
								% of WT		%
WT	K	L	P	E	M	Y	T	100	100
%OccPDAc	19	2	2	7	20	12	5
%OccSPAc	0	0	92	0	0	0	9
1					K			76 ± 15	0 ± 5	1	51
2					Q			88 ± 8	19 ± 10	0	0
3					L			80 ± 6	154 ± 12	73	32
4							V	80 ± 5	242 ± 12	3	0
5							I	92 ± 7	187 ± 20	17	2
6							F	75 ± 5	114 ± 8	0	0
7			D					93 ± 6	55 ± 3	5	0
8				K				74 ± 10	116 ± 10	5	0
9	Q							75 ± 5	115 ± 5	8	49
10		W				A		1 ± 2	1 ± 2	0, 0	54, 54 (54)
11		A			L	W		0 ± 1	3 ± 7	0, 73, 0	9, 32, 5 (2)
12		A				W	I	0 ± 1	312 ± 9	0, 0, 17	9, 5, 2 (1)
13		F				V		−1 ± 1	317 ± 9	0, 0	31, 0 (0)

^aICAM-1 expression was determined with CBR IC1/11 mAb, which recognizes a conformation-dependent epitope in domain 3 of ICAM-1. % mAb binding = (mutant mAb MFI − background mAb MFI)/(wild-type mAb MFI − background mAb MFI) × 100.

^b% tetramer binding = (mutant tetramer MFI − background tetramer MFI)/(wild-type tetramer MFI − background tetramer MFI) × 100.

^c% occupancies from PDA and SPA calculations are provided for the WT (under the WT residue) and variants (across from each variant). % occupancy represents the % occurrence of the given amino acid in the set of 1000 output sequences from the Monte Carlo (PDA) or 300 output sequences from the Genetic Algorithm (SPA). For double and triple variants (10–13), the % occupancy is presented for each substitution consecutively separated by a comma, with the % occupancy of the combined variant provided in parentheses.