Catherine Y Liu, Cory L Ahonen, Michael E Brown, Ling Zhou, Martin Welin, Eric M Krauland, Robert Pejchal, Paul F Widboom, Michael B Battles 

mAbs, 15(1). DOI: 10.1080/19420862.2023.2189974

March 29, 2023

CD3-targeting bispecific antibodies are a cornerstone of modern T cell engager (TCE) therapies, in which T cell activation is mediated by cross-linking the T cell receptor (TCR)–CD3 complex. CD3 serves as the signaling component, requiring precise engagement to trigger T cell stimulation. However, many TCEs exhibit high polyreactivity, resulting in poor pharmacokinetics and undesirable off-target interactions, largely due to the nature of their binding mechanism and a tendency for self-association. This study, conducted by Adimab scientists, addresses a key industry challenge: decoupling CD3 affinity from polyreactivity to enable safer, more effective bispecific antibodies through structure-guided design.

Key hypotheses and objectives

Most clinical CD3 antibodies, whilst exhibiting the desired cross-reactivity to cynomolgus monkey CD3, bind to a highly electronegative N-terminal epitope on CD3ε, leading to high polyreactivity and poor developability. These electrostatic interactions, as indicated by a high isoelectric point (pI), at the antibody-antigen interface drive both strong CD3 binding and polyreactivity, making affinity tuning challenging. The team hypothesized that structure-guided charge balancing could reduce the electrostatic liabilities, improving developability for bispecific applications, while preserving strong CD3 binding.

Approach and techniques

1. Structural analysis: The crystal structure of a novel high-affinity CD3 antibody (ADI-26906) bound to a CD3ε peptide was solved at 1.9Å resolution, revealing a key pyroglutamate-modified residue important for recognition and cynomolgus cross-reactivity. This represents the first publicly available structure of a CD3ε–antibody complex that includes the extreme N-terminus.
2. Antibody engineering: The Adimab yeast-based platform was used to introduce targeted acidic amino acid substitutions at solvent-exposed sites. This counteracted excessive electrostatic charge without disrupting CD3 binding, focusing substitutions in solvent-exposed CDR loops with minimal antigen contact to balance charge while maintaining the native binding mode.
3. Biophysical characterization: Engineered variants were rigorously tested using: 
   • Surface plasmon resonance (SPR) and biolayer interferometry (BLI) to measure CD3 affinity.
   • Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) to assess self-interaction and developability.
   • Polyspecificity reagent (PSR) binding assays to quantify polyreactivity.
4. Functional testing: The optimized antibodies were reformatted as CD20 × CD3 and HER2 × CD3 bispecifics, demonstrating antigen-dependent T-cell activation and tumor cell killing in vitro, with reduced pro-inflammatory cytokine release compared to existing CD3 binders.

Major findings and impact

• Correlation between affinity and polyreactivity broken: The novel engineered CD3 antibodies retained high affinity and cross-reactivity with cynomolgus monkey CD3 while displaying significantly lower polyreactivity, and improvement over clinical comparators. These results demonstrate that optimizing surface charge can uncouple affinity from polyreactivity.
• Broader affinity range with improved pharmacokinetics: By fine tuning CD3 binding strength without increasing off-target interactions, these antibodies support greater flexibility in bispecific design.
• Potential for safer, more effective T cell engagers: The findings provide a roadmap for optimizing CD3-targeting bispecific antibodies with improved safety, stability, and manufacturability, enhancing half-life and increasing bioavailability. In addition, by lowering Fv charge density, these engineered antibodies may mitigate pharmacokinetic and cytokine-release liabilities associated with highly charged CD3 binders.

This work highlights Adimab’s capabilities in antibody engineering to overcome long-standing challenges in the field. Read the full study to learn how structure-based insights can transform TCE therapeutics.