Antibodies targeting ADAM17 reverse neurite outgrowth inhibition by myelin-associated inhibitors

(A) Purification of the 5-mer neuron-myelin signaling complex for ELISA-based studies. The 5-mer complex was formed in the presence of GT1b-Na as described in Fig 1. Briefly, bead-bound Lingo-1-Fc was used to pull-down NgR1, p75, and MAG in the presence of GT1b-Na. The beads were gently washed, and the bound proteins were eluted with low pH (100 mM glycine-HCl, pH 3.5). After elution, the pH was raised to 7 and the complex was analyzed on SDS–PAGE. (B, C) ELISA-based assay to gauge the binding of the 5-mer neuron-myelin complex (B) or p75 (C) to immobilized ADAM17 or ADAM10 (ECD or D+C protein constructs). (A) Briefly, wells were coated with ADAM17 ECD (E406A) or ADAM17 D+C or ADAM10 ECD (E384) or ADAM10 D+C, and varying concentrations of the 5-mer complex (isolated as described in panel (A)) or p75 were added to the wells and incubated for 1 h at RT. The bound p75 was detected by mouse mAb specific to the ectodomain of human p75. To detect the bound 5-mer complex, mouse mAb to the MAG (human) ectodomain was used. Data (n = 3) was recorded at 450 nm.

(A) Binding of ADAM17 ECD (E406A) or ADAM17 D+C constructs to the neuronal receptor/co-receptor/p75 complex in the presence of GT1b-Na. Protein A-Sepharose pull-down assays were used to detect the binding of the ADAM17 ECD (active-site mutant E406A) (4 μg) or ADAM17 D+C (8 μg). The neuronal complex comprising Lingo-1-Fc, NgR1, and p75 was pre-formed in the presence of GT1b and used as a “bait.” The “prey” ADAM17 protein constructs were added, incubated, washed twice, and analyzed by SDS–PAGE. (B) Binding of ADAM17 ECD and ADAM17 D+C to the 5-mer neuron-myelin complex. Here, the pre-formed 5-mer complex was used as a “bait” to pull-down ADAM17 ECD or ADAM17 D+C. (C) Binding of ADAM17 D+C to isolated p75. The binding of ADAM17 D+C to Fc-tagged p75 was gauged using protein A-Sepharose beads with p75-Fc (4 μg) immobilized on the beads. The candidate ADAM17 D+C (8 μg) protein was added, washed, and bound proteins were detected by SDS–PAGE. (D) Binding of p75 to ADAM17 ECD. Because ADAM17 ECD and p75-Fc show identical mobility on SDS–PAGE (reducing conditions), we performed the experiment using untagged p75 (10 μg) as “prey” and ADAM17 ECD (E406A)-Fc (3 μg) immobilized on protein A-Sepharose beads.

(A) Effect of the mAbs on the binding of the neuron-myelin complex to immobilized ADAM17 ECD (E406A). As before (Fig 2B and C), wells were coated with ADAM17 ECD (E406A), overnight at 4°C. As indicated in the figure, varying concentrations of the three mAbs (D8P1C1, D5P2A11, and C12) were added to the wells and the 5-mer complex bound to ADAM17 was detected by mouse mAb to MAG (human) ectodomain. Data (n = 3) were recorded at 450 nm. (B) Effect of the C12 mAb on the binding of the neuron-myelin complex to immobilized ADAM17 D+C. The wells were coated with ADAM17 D+C and the assay was executed as described in panel (A).

(A) Endogenous expression of Lingo-1, p75, and NgR1 in the NG108-15 cell line before and after differentiation. The NG108-15 cells were differentiated with 1 mM dibutyl cAMP, which resulted in enhanced expressions of NgR1, Lingo-1, and p75. The cells were harvested, lysed, and subjected to Western blots analysis. For NogoR1, we detected minor higher molecular weight bands. This could be because of the association of membranous glycolipids, such as gangliosides, with NogoR1 in cell lysates of NG105-15, that slow the migration of NogoR1 on SDS–PAGE, or may be because of differently glycosylated species. The main 60 kD band represents the NgR1 receptor ectodomain. (B) PMA induced shedding of p75 in the presence or absence of the myelin inhibitor MAG. Briefly, 1 × 10⁵ NG108-15 cells were differentiated by dibutyl cAMP (1 mM) for 3–4 d in six-well cell-culture plates (Nunc). The cells were transferred to conditioned media without FBS and allowed to grow for 24 h. PMA (25 ng/ml) was added to the media and incubated for either 15′ or 30′. Finally, the myelin inhibitor MAG (20 μg/ml) was added followed by an additional 30′ incubation. The culture supernatants (media and secreted proteins) were then harvested, concentrated, and subjected to Western Blot analysis using the p75 specific mAb 8J2. Likewise, the cells were lysed and blotted for the loading control, GAPDH. The results show that PMA augments the shedding of p75 in the presence of the exogenously added myelin inhibitor, MAG. (C) The ADAM17 inhibitor TAPI-1 causes significant inhibition of p75 shedding in absence of PMA. Differentiated NG108-15 cells, in absence of PMA, were incubated for 30′ with 1 mM TAPI-1 (30) before the addition of MAG. MAG was then added, followed by another 30′ of incubation. The supernatants were harvested and processed as before.

(A) Inhibition by the D8P1C1 and D5P2A11 mAbs. (B) Inhibition by the C12 mAb. Small-molecule inhibitors, including Batimastat (MMP inhibitor), TAPI-1 (ADAM17 and MMP inhibitor), and GI254023X (ADAM10 inhibitor), as well as 1H5 (inhibitory anti-ADAM10 mAb), were included in the assays for comparison. The NG108-15 cells were differentiated and incubated with mAbs or small-molecule inhibitors for 30’. This was followed by the addition of 25 ng/ml of PMA (30′ incubation). Finally, MAG (20 μg/ml) was added, incubated for 30′, and the supernatants were processed as before. The results show that the anti-ADAM17 mAbs, which target either the protease or the D+C domain region of ADAM17 (Fig 1A), abrogate p75 shedding at 20 μg/ml. (C) The percent inhibition of p75 shedding by the anti-ADAM17 mAbs was quantitated using the software Image J and GAPDH as the loading control. The bar graph demonstrates mean percent inhibition for each concentration of the inhibitors and the whiskers indicate ± SEM, n = 2. (D) Table showing the inhibitory potencies of the small-molecule inhibitors and the anti-ADAM mAbs. The percent inhibition of p75 shedding by the alpha secretase inhibitors (anti-ADAM10 or ADAM17 mAbs or small-molecule inhibitors) was quantitated using Image J software as follows: [1-{cleaved p75 in presence of myelin inhibitor MAG + inhibitors, normalized with respect to the loading control GAPDH}/{cleaved p75 in presence of MAG only, normalized with respect to GAPDH}]X100 (the amount of cleaved protein is estimated by the optical intensity of the corresponding SDS–PAGE band). GAPDH was used as a loading control (5). This table shows the average percent inhibition ± SEM for all inhibitors and controls (concentration as indicated in parentheses). Percent inhibition for all inhibitors and controls was analyzed using descriptive statistics. Statistical analyses were performed using IBM SPSS version 30 and GraphPad Prism version 10.