Human LINGO1 Stable Cell Line - CHOK1

LINGO-1 is a central nervous system (CNS) membrane protein that inhibits axonal myelination. Preclinical studies have shown that blocking LINGO-1 function can lead to CNS repair in animal models of demyelination. The anti-LINGO-1 antibody Li81 blocks LINGO-1 function and exhibits potent remyelinating activity in animal models and is currently in Phase II clinical trials as a potential treatment for patients with relapsing forms of multiple sclerosis. Li81 has a unique property in that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity determining region and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after binding to the primary binding site. Simultaneous binding of both sites results in the formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment and higher order complexes with the intact Li81 antibody. To elucidate the role of the secondary binding site, the researchers designed a series of Li81 variant constructs that eliminated the secondary binding site while retaining the classical binding site. These Li81 mutants retained high-affinity binding to LINGO-1 but reduced Li81-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity observed in OPC-dorsal root ganglion neuron co-cultures. These mutations also attenuated antibody-induced LINGO-1 internalization in cultured cortical neurons, OPCs, and cells overexpressing LINGO-1. These studies suggest that binding of both LINGO-1 binding sites of Li81 is critical for the antibody's potent functional activity.

To assess the impact of the secondary binding site on LINGO-1 internalization, the researchers performed a series of fluorescence-activated cell sorting (FACS) studies (Figure 1). Treatment of CHO cells expressing LINGO-1 with Li81 for 23 hours at 37℃ resulted in a substantial decrease in the mean fluorescence intensity (MFI) of the fluorescently labeled anti-human detection antibody, indicating a decrease in surface levels of LINGO-1 (Figure 1a). In contrast, when CHO-LINGO-1 cells were treated with CN1373 and CN1375, surface levels of LINGO-1 were only slightly reduced. Treatment with an isotype control mAb had no effect on surface LINGO-1. Similarly, treatment of mouse hematopoietic Ba/F3 cells expressing LINGO-1 with Li81 for 3 hours resulted in an approximately 40% decrease in surface LINGO-1 levels at 37℃compared to 4℃, whereas treatment with CN1373 resulted in only a 15% decrease (Figure 1b).

Figure 1. (a) CHO cells expressing full-length LINGO-1 were treated with 1 and 3 µg/mL of 5C8 isotype control antibody (Ctl), Li81, or CN1373 at 37℃ for 23 hours and analyzed by FACS, stained with anti-human IgG. (b) Ba/F3 cells expressing full-length LINGO-1 were treated with serial dilutions of 5C8, Li81, or CN1373 for 3 hours at 4℃ or 37℃ and analyzed by FACS. (Hanf K J M, et al., 2020)