Table 5.

Evaluation and Selection of anti-RGMa/anti-TfR1 DVD-Igs. Diverse anti-RGMa/anti-TfR1 DVD-Ig proteins incorporating different domain orientations, linkers and paired variable domains were generated using recombinant methods and % monomer was determined by SEC. They were screened using a cell-based mTfR1 binding assay and RGMa BMP potency assay for functional activity. DVD-Ig concentrations in mouse serum and brain at 24 hour post intravenous injection at the indicated doses were determined using MSD-ECL assay. Anti-TfR1(AB405)-SL-anti-RGMa DVD-Ig and anti-RGMa-GS-anti-TfR1(AB403) showed favorable brain penetration and target potency. Data was expressed as means ± SD, n = 4 mice per group.

Ab/DVD name	% Monomer	mTFR binding EC50 (nM)	BMP Assay IC50 (nM)	Serum conc. (nM)	Brain conc. (nM)
Anti-RGMa-LS-anti-TfR1(AB402)**	99.3	127.4	0.21	1,184 +/− 279	10.7 +/− 1.3
Anti-RGMa-SL- anti-TfR1(AB402)**	90.5	2.1	0.37	819 +/− 206	6.5 +/− 0.9
Anti-RGMa-LL-anti-TfR1(AB402)**	91.8	1.7	1.78	534 +/− 101	6.3 +/− 0.9
Anti-RGMa-SS-anti-TfR1(AB402)*	96.4	30.9	0.44	670 +/− 108	8.0 +/− 1.0
Anti-RGMa-GS- anti-TfR1(AB402)*	91.4	5.4	0.01	187 +/− 40	10.0 +/− 1.2
Anti-RGMa-LS- anti-TfR1(AB403)*	99.1	25.7	<0.03	481 +/− 74	18.5 +/− 5.1
Anti-RGMa-SS- anti-TfR1(AB403)*	98.2	71.0	1.18	487 +/− 92	9.3 +/− 1.1
Anti-RGMa-GS- anti-TfR1(AB403)**	98.8	4.3	<0.03	397 +/− 99	14.8 +/− 0.7
Anti-TfR1(AB405)-SL-anti-RGMa**	95.2	4.4	0.73	643 +/− 72	13.1 +/− 2.8
Anti-TfR1(AB405)-LS-anti-RGMa*	99.2	3.9	No inhibition	462 +/− 32	18.4 +/− 1.8
Anti-TfR1(AB405)-GS-anti-RGMa*	90.1	28.1	3.12	612 +/− 221	19.2 +/− 1.9
DVD-Ig control***	97.9	NA	NA	2,287+/−188	2.5+/−0.6
anti-RGMa hFc****	100%	NA	0.21	4,512 +/− 77	5.9 +/− 2.1

^*10mpk;

^**20mpk,

^***30mpk,

^****40mpk.