Table 1.

ICOSL vIgD-Fc exhibit increased affinities against ICOS, CD28, and CTLA4.

	ICOS		CD28		CTLA-4
Sample ID	KD	Fold imp.	KD	Fold imp.	KD	Fold imp.
WT ICOSL	883.4 pM		13.88 nM		77.12 nM
H160	331.5 pM	2.7	524.7 pM	26.5	677.6 pM	113.8
H180	337.9 pM	2.6	1.163 nM	11.9	1.522 nM	50.7
H183	769.1 pM	1.1	782.9 pM	17.7	832.8 pM	92.6
H184	381.7 pM	2.3	435.9 pM	31.8	646.8 pM	119.2
H2227	1.294 nM	0.7	896 pM	15.5	1.466 nM	52.6
H2229	491.8 pM	1.8	447.2 pM	31.0	644.9 pM	119.6
H2231	372.7 pM	2.4	400.5 pM	34.7	591.7 pM	130.3
H2236	471.6 pM	1.9	389.9 pM	35.6	575.9 pM	133.9
H2237	420.3 pM	2.1	293.4 pM	47.3	535.6 pM	144.0
H2239	368.6 pM	2.4	367.9 pM	37.7	719.4 pM	107.2
H2241	336.8 pM	2.6	1.042 nM	13.3	2.518 nM	30.6
H3256	542.8 pM	1.6	502.6 pM	27.6	1.059 nM	72.8
H3269	362.2 pM	2.4	553 pM	25.1	647.9 pM	119.0
H3305	477 pM	1.9	562.9 pM	24.7	1.066 nM	72.3
H3310	477.1 pM	1.9	365.9 pM	37.9	864.3 pM	89.2
H3318	339.8 pM	2.6	968.7 pM	14.3	1.122 nM	68.7
H3321	371.3 pM	2.4	1.947 nM	7.1	2.812 nM	27.4
H3322	310.6 pM	2.8	909.8 pM	15.3	1.2 nM	64.3

Protein-protein interactions between the receptors and ICOSL variants were assessed using ForteBio Octet binding assays. Human recombinant ICOS-Fc CD28-Fc, or CTLA4-Fc fusion proteins were loaded individually onto anti-human capture sensors (ForteBio Octet AHC) and wild type human ICOSL-Fc fusion protein or ICOSL vIgD-Fc were bound to the receptors in four-point titrations (100, 50, 25, and 12.5 nM). Each titration was globally fit with a 1:1 model using the ForteBio Analysis software to calculate the association and dissociation rates of each protein (data not shown). The dissociation constant (K_D) was then calculated and compared to wild type ICOSL to determine a fold improvement value.