Pan-filovirus activity of an IGF2-fused monoclonal antibody: Impairment by IGF1R cross-engagement and rescue via IGF2 Y27L mutation.

BACKGROUND

Filoviruses (Ebola and Marburg) cause severe high-mortality infectious diseases. Neutralizing antibodies represent promising antiviral candidates, but broadly reactive antibodies against multiple filoviruses are rare. Antibody-ligand fusion strategies that combine conserved glycoprotein-targeting antibodies with IGF2R ligands can mediate endosomal antibody delivery. Previously, we showed that an NPC2-fused AF-03 (an antibody to the GP1 core region) exhibited pan-filovirus neutralization. However, this NPC2 fusion faced quality control challenges and its application is limited by heterogeneous glycosylation, batch inconsistency, and poor CMC feasibility.

METHODS

Human IGF2 or its Y27L variant was fused to the N-terminus of the AF-03 light chain via a peptide linker. Cellular attachment was measured by flow cytometry and ELISA. Internalization was assessed by flow cytometry, western blotting and confocal imaging. Neutralizing activity against pseudotyped filoviruses was determined using a luciferase reporter system in vitro.

RESULTS

The IGF2-fused antibody (termed AF03-IL) exhibited broad-spectrum neutralizing activity against multiple pseudotyped filovirus in vitro. However, its potency, including internalization and neutralization, was much weaker than that of the NPC2-fused antibody (termed AF03-NL). The reduced activity was caused by IGF2 binding to IGF1R on the cell surface. To overcome this limitation, we introduced a single point mutation (Y27L) into the IGF2 domain. This new construct, named AF03-ILm1, showed superior internalization and neutralization activity compared to the parental AF03-IL.

CONCLUSIONS

The anti-filovirus activity of IGF2-fused AF-03 is impaired by cross-engagement with IGF1R that is rescued by Y27L introduction. The work also might have implications into the design of IGF2-based protein degradation molecules.