A Neutralizing Anti-Nogo66 Receptor Monoclonal Antibody Reverses Inhibition of Neurite Outgrowth by Central Nervous System Myelin

Sylvia A. Rabacchi,Betty H. Zheng,Dane Worley,Eugene Choi,Blake Pepinsky,Laura Silvian,Dinah W.Y. Sah,Weixing Yang,Wenjun Mo,Jessica Schauer,Weiwei Li,Stephen M. Strittmatter,Colleen Mullen,Betty P. Liu,Adrienna Jirik,Ernie Chang,Daniel H.S. Lee,Nancy J. Benedetti,Lee Walus

doi:10.1074/jbc.m401803200

Sylvia A. Rabacchi, Betty H. Zheng + Show 17 more

Open Access

https://doi.org/10.1074/jbc.m401803200

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Abstract

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

Highlights

Growth-inhibitory molecules present in the CNS1 myelin contribute, at least in part, to the inability of mammalian central nervous system (CNS) axons to regenerate upon injury [1]
Comparison of IC50 values of 7E11 and sNgR310-Fc in inhibiting MAG-Fc, alkaline phosphatase (AP)-OMgp, and AP-Nogo binding to sNgR344-Fc monoclonal antibody (mAb) 7E11 Recognizes Rat and Human NgR1—7E11 mAb was generated using a synthetic peptide derived from amino acid residues 110 –125 in the rat NgR1 sequence (Fig. 1A)
We have generated a neutralizing anti-NgR1 antibody, 7E11, to test the hypothesis that reagents targeting NgR1 would antagonize the inhibitory effects of CNS myelin

Summary

EXPERIMENTAL PROCEDURES

Soluble NgR310-Fc (sNgR310-Fc) and sNgR344-Fc—The generation of sNgR310-Fc was described by Li et al. Soluble NgR344-Fc (sNgR344Fc) was generated, except that the cDNA encoding amino acid residues 27–344 of rat NgR1 was fused in-frame to the rat IgG1 hinge and Fc was contained in the expression plasmid vector PV90 [27]. FACS Analysis—COS-7 cells expressing rat NgR1 or parental COS-7 cells were incubated at 4 °C for 30 min with 7E11 at various concentrations in PBS containing 4% normal goat serum, 2 mM MgCl2, and 2 mM CaCl2. Cells were washed and incubated with R-phycoerythrinconjugated affinity purified F(abЈ) fragment goat anti-mouse IgG Fc(␥) specific secondary antibody (1:200, Jackson ImmunoResearch Laboratory) in PBS. At the end of the incubation, the cells were washed twice with PBS, resuspended in 200 ␮l of PBS containing 1% fetal bovine serum, and subjected to FACS analysis. The protein ligands, 30 nM alkaline phosphatase (AP)-Nogo nM AP-OMgp, or 33 nM rat MAG-human Fc (R & D Systems) was added to wells containing serial dilutions of 7E11 or sNgR310-Fc in Hank’s balanced salt solution containing 0.1% ovalbumin, and incubated at 25 °C for 18 h.

A Neutralizing Murine Monoclonal Anti-Nogo66 Receptor Antibody

RESULTS

DISCUSSION