Profiling the Biophysical Developability Properties of Common IgG1 Fc Effector Silencing Variants

Robert Pejchal, Anthony B. Cooper, Michael E. Brown, Maximiliano Vásquez, Eric M. Krauland 

Antibodies 12(3), 54. DOI: 10.3390/antib12030054 

Overview  

Therapeutic antibodies are a significant biologic modality used for the treatment of a wide range of diseases. Beyond their ability to bind specific targets via heavy and light chain variable fragments (Fvs), they can also recruit immune effector mechanisms through their constant region (Fc). Interaction of the Fc with Fcγ receptors (FcγR) on immune cells triggers effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). In addition, when the Fc region engages the complement system, the C1 cascade leads to complement-dependent cytotoxicity (CDC). While these functions are beneficial in some therapeutic antibody applications, they are undesirable in others, such as T cell engager (TCE) bispecific antibodies, where unintended immune activation can cause serious adverse effects. 

Eliminating these effector functions, a strategy known as “Fc silencing,” is achieved through mutations in the lower hinge and the second constant domain (CH2) of IgG1. These mutations disrupt FcγR binding while preserving interaction with the FcRn, which is important for antibody recycling and half-life, and with Protein A (ProA), which is essential for purification during manufacturing. Numerous silencing mutation sets have been described in the literature and are present in clinical stage or approved antibodies, but comprehensive biophysical profiling of these variants has been lacking. This knowledge gap is clinically important because mutations that compromise antibody stability, particularly under the conditions used during manufacturing, can create development liabilities that may not become apparent until late in the pipeline. 

Key hypotheses and objectives 

Not all commonly used IgG1 Fc silencing mutations maintain equivalent biophysical developability. Some may introduce stability liabilities, particularly under the acidic conditions routinely encountered during manufacturing, and these differences are not captured by functional binding assays alone.  

An ideal silent IgG1 Fc would possess the following properties: no binding to activating FcγR and C1q, retained binding to FcRn and Protein A (ProA), and biophysical properties equivalent to wild-type IgG1.  

The objectives of this study were to: 

• Produce and characterize a comprehensive panel of IgG1 Fc effector silencing variants. 

• Evaluate the impact of individual and combined mutations on FcγR binding, FcRn binding, stability, aggregation propensity and detection of complement activation. 

• Identify optimal mutation sets that balance effector silencing with retention of wild-type-like stability. 

Approach and techniques 

A panel of representative Fc silencing mutations from the literature and clinical molecules was introduced into an anti-CD3 IgG1 backbone. Variants were generated first as single lower hinge substitutions, including charge-flip variants, and then as combinations pairing these hinge mutations with a range of CH2 substitutions derived from both IgG1 and IgG4 sequences. 

Each variant was characterized in depth using a comprehensive suite of assays. Binding to the three activating FcγRs, FcRn, C1q, and ProA was measured by surface plasmon resonance (SPR) and biolayer interferometry (BLI). FcγR engagement was also assessed by flow cytometry on monocytic cells under both standard and highly avid pre-complexed conditions, the latter providing a more sensitive test of residual binding. Complement activation was measured using a bead-based C1q/C3 assay. Biophysical developability was assessed using polyspecificity reagent (PSR) binding, affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS), hydrophobic interaction chromatography (HIC), size-exclusion chromatography (SEC), differential scanning fluorescence (DSF) for thermal stability (Tm) measurements, and SEC following exposure to pH 3.5 to measure acid-induced aggregation. 

Key findings  

Lower hinge substitutions: Lower hinge mutations alone reduced FcγR binding by approximately one order of magnitude, comparable to the level of wild-type (WT) IgG2, but did not fully abolish binding when tested under high-avidity conditions. Their biophysical properties were largely similar to WT IgG1, with a notable exception of L235K-containing variants, which showed slightly elevated Fc Tm but were more susceptible to acid-induced aggregation.  

Lower hinge and CH2 variants: Adding CH2 mutations to hinge variants produced more variable outcomes. A subset of CH2 mutations most effectively reduced FcγRI binding, with LALE (L234A/L235E) combinations showing slightly more reduction than LALA (L234A/L235A). Critically, this same subset fully eliminated binding under high-avidity cell-based conditions. FcRn binding was preserved across almost all variants, with reduced FcRn affinity only observed for L309A, as previously reported. ProA binding was preserved across the entire panel. Complement activation, as assessed by C1q/C3 bead assay, was most effectively reduced by an overlapping but distinct subset of CH2 mutations. 

The most consequential differences across the panel were observed for thermal stability and acid-induced aggregation. D265A, L328A, and P331S in combination with LALA and LALE produced the largest reductions in CH2 melting temperature. D265A showed particularly marked increases in aggregation under pH 3.5 stress. Generally, variants that produced a marked reduction in CH2 Tm also showed increased propensity to form aggerates under acidic conditions. 

Key experimental outcomes include: 

• Optimal combinations defined: Four mutation sets stood out as combining effective Fc effector silencing with WT-like biophysical stability: LALA-S267K, LALE-S267K, LALA-P329A, and LALE-P329G. These performed comparably to the well-established LALA-P329G in all functional assays while maintaining acceptable developability profiles. 

• Stability liabilities identified: Some commonly used CH2 mutations, particularly D265A and P331S, effectively reduced FcγR binding but significantly compromised stability, promoting aggregation under acidic manufacturing conditions. 

• Practical selection guidance: This comprehensive dataset enables informed selection of constant region variants during antibody development, particularly for TCE and other bispecific formats. 

Implications for therapeutic antibody development 

This study provides a practical reference for selecting Fc effector silencing variants during antibody development. For the growing pipeline of TCE bispecifics and other formats requiring Fc silencing, the choice of mutation set has direct consequences for both pharmacology and manufacturability. The identification of variants prone to aggregation under low pH conditions, which mirror the viral inactivation steps routinely used in biomanufacturing, underscores the importance of early biophysical screening that goes beyond binding assays. Therapeutic antibody developers relying on D265A- or P331S-containing constructs, both of which are present in molecules in clinical development, should be aware of potential stability risks that may not be apparent under standard assay conditions. The newly characterized combinations LALA-P329A and LALE-P329G offer effective alternatives to LALA-P329G under the conditions of the assays tested here, expanding the toolkit available to antibody engineers and providing options that may better suit specific development programs.