Biparatopic targeting of IL-23 enables dual-epitope engagement and enhanced neutralization potency

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Abstract
Biparatopic antibodies (bpAbs) have two non-overlapping epitopes on a single antigen, offering a distinct therapeutic potential, yet their application to soluble cytokines remains underexplored. Herewithin, we engineered and characterized biparatopic antibodies targeting human interleukin-23 (IL-23) by combining the clinically validated variable regions of ustekinumab and guselkumab into 1 + 1 IgG-like and 2 + 2 Fab-IgG extended biparatopic formats. Constructs were generated using heterodimer promoting CH3 mutations and redox repair assembly of half‑antibodies, expressed in HEK-2936E cells, and benchmarked against parental monospecific antibodies. An orthogonal analytical workflow, comprising sodium dodecyl sulfate polyacrylamide gel electrophoresis, liquid chromatography-mass spectrometry, analytical size-exclusion chromatography, surface plasmon resonance (SPR), and mass photometry was used to define critical quality attributes and characterize biparatopic target engagement. SPR kinetic analysis demonstrated picomolar affinities across constructs and revealed avidity‑driven enhancements in apparent affinity. A bespoke SPR dual‑engagement bridging assay confirmed simultaneous IL-23A and IL-12B binding, validating obligate biparatopic engagement. Mass photometry independently corroborated in trans immune complex formation and revealed stoichiometry-dependent multimerisation across the bpAb formats explored. Functional neutralization was measured using an IL-23 luciferase reporter assay and supported by a bespoke, two-parameter mechanistic model, which accurately predicted observed EC50 shifts. Biparatopic formats exhibited equivalent potency relative to equimolar antibody mixtures, establishing a generalizable framework for engineering and modeling soluble cytokine‑targeting biparatopic therapeutics.
Funding Information
  • Biotechnology and Biological Sciences Research Council (BB/Y003268/1)
  • GlaxoSmithKline

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