Effects of Fc glycosylation on the activity of WNT mimetic agonistic antibodies

Abstract

Monoclonal antibodies have been explored in a broad range of applications including receptor agonism. Given the importance of receptor conformation in signaling, the agonistic activity of antibodies that engage these receptors are influenced by many parameters. Tetravalent bispecific antibodies that target the frizzled and lipoprotein receptor-related protein receptors and subsequently activate WNT ("Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Int") signaling have been constructed. Because WNT activation stimulates stem cell proliferation and tissue regeneration, immune effector functions should be eliminated from therapeutic antibodies targeting this pathway. Here, we report an unexpected effect of Fc glycosylation on the agonistic activity of WNT mimetic antibodies. Our findings underscore the importance of antibody format, geometry and epitope in agonistic antibody design, and highlight the need to establish appropriate early discovery screening strategies to identify hits for further optimization.

Keywords: FZD; LRP; WNT; WNT mimetic; agonist; bispecific antibody; glycosylation; surrogate.

Figure 1

N297 mutations disrupt WNT mimetic activity. (A) Diagram of the WNT mimetic molecule FA-L6. (B) Mutation of N297 to G significantly disrupts the activity of FA-L6 WNT mimetic. (C) Mutations of N297 significantly disrupt the activity of FA-L6 WNT mimetic, whereas the LALAPG mutation does not. (D) Activity of FA-L6 Fc mutations tested in a mouse small intestinal organoid growth assay. Representative images of mouse small intestinal organoids treated with Basal media, Basal media +1 μM IWP2 or with Basal media +1 μM IWP2 plus 1 nM FA-L6 WT, 10 nM FA-L6 WT, 1 nM FA-L6 LALAPG, 10 nM FA-L6 LALAPG, 1 nM FA-L6 N297A, 10 nM FA-L6 N297A, 1 nM FA-L6 N297G and 10 nM FA-L6 N297G. Bars: 100 μm. (E–G) The removal of the N297G Fc domain by IdeS (E) rescues the molecule’s STF activity (F). (G) SDS-PAGE of IdeS digested FA-L6 N297G. (H and I) Removal of the Fc domain from FA-L6 WT does not affect the molecule’s activity. (H) STF activity of untreated and IdeS treated FA-L6 WT. (I) SDS-PAGE of IdeS digested FA-L6 WT.

Figure 2

Glycan removal at N297 through PNGase F treatment disrupts the activity of L6 appended WNT mimetics. PNGase F treatment significantly decreases the activity of FA-L6 (A) but not FA-L5 (B) or FA-36 (C). PNGase F treatment significantly decreases the activity of FB-L6 (D) but not FB-L5 (E) or FB-36 (F). (G–I) Molecules with L6 appended to different locations on FA show a significant loss of STF activity after PNGase F treatment.

Figure 3

The biophysical property comparison of FA-L6 N297 mutants to FA-L6 WT molecule. (A) The hydrodynamic diameter of FA-L6 N297 mutants (FA-L6 N297G and FA-L6 N297A) are smaller than FA-L6 WT. FA-L5 WT and N297 mutants (FA-L5 N297G and FA-L5 N297A) showed no significant hydrodynamic diameter difference. ^**P < 0.01. (one-way ANOVA). (B) Both sets of N297 mutants showed decreased thermal stability (Tm) compared to their WT molecules. (C–F) Binding of FA-L6 and its N297 mutants to LRP6E3E4 or FZD₈ CRD. The monovalent (C, D) and bivalent (E, F) binding affinities of FA-L6 WT, FA-L6 N297A and FA-L6 N297G to LRP6E3E4 or FZD₈ CRD, measured with an Octet Red 96 system. Dotted lines indicate simulated fits.