Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

Abstract

The widespread use of monoclonal antibodies for therapeutic applications has led to intense interest in optimizing several of their natural properties (affinity, specificity, stability, solubility and effector functions) as well as introducing non-natural activities (bispecificity and cytotoxicity mediated by conjugated drugs). A common challenge during antibody optimization is that improvements in one property (e.g., affinity) can lead to deficits in other properties (e.g., stability). Here we review recent advances in understanding trade-offs between different antibody properties, including affinity, specificity, stability and solubility. We also review new approaches for co-optimizing multiple antibody properties and discuss how these methods can be used to rapidly and systematically generate antibodies for a wide range of applications.

Keywords: CDR; Fab; Fc; aggregation; developability; mAb.

Figure 1

Overview of the key properties of monoclonal antibodies. The lines connecting different antibody properties highlight their interdependence and that optimization of any one property can lead to defects in other properties.

Figure 2

Efficient affinity maturation of antibody variable (V_H) domains requires co-selection of stabilizing mutations that compensate for the destabilizing effects of affinity-enhancing mutations. (A) Structural model of a human variable domain specific for the Alzheimer’s Aβ42 peptide that was co-evolved for enhanced affinity and stability using directed evolution methods. The evolved antibody domain acquired 12 mutations in the framework and complementarity-determining regions (CDRs). (B) Analysis of the contribution of each acquired mutation to affinity and stability. Single reversion mutations that reduce the equilibrium association constant (K_A) indicate mutations that are beneficial for affinity, while those that increase K_A correspond to mutations that are detrimental to affinity. Likewise, single reversion mutations that reduce the apparent melting temperature (T_m^*) indicate mutations that are beneficial for stability, while those that increase T_m^* correspond to mutations that are detrimental to stability. Several of the key affinity mutations (R50, R62 and N72) are strongly destabilizing and are compensated for by two key stabilizing mutations that also enhance antibody affinity (K45 and K98). The solid lines are the values of K_A (~1 × 10⁻⁷ M⁻¹) and T_m^* (66 °C) for the evolved V_H domain with 12 mutations. The CDRs are defined using Kabat numbering, and CDR4 is defined as residues 71–78. The figure is adapted from Reference [13].

Figure 3

Mutational analysis of the contributions of antibody (V_HH) mutations accumulated during affinity maturation to affinity and specificity. Single reversion mutations were generated for two affinity-matured variable domains specific for α-synuclein to determine the contribution of each acquired mutation to affinity and specificity. Values of the equilibrium association constant (K_A) were measured using fluorescence polarization. Normalized specificity was measured as the binding of the parent antibody to milk proteins divided by that for the reversion mutant (parent/reversion mutant). Reductions in affinity or specificity for the reversion mutants indicate that the original mutations acquired during affinity maturation contribute positively to either property. The figure is adapted from Reference [31].

Figure 4

Affinity maturation of an anti-nerve growth factor antibody leads to the accumulation of mutations that result in non-specific interactions and fast antibody clearance. (A) Structural model of the variable regions of the affinity-matured antibody. The residues responsible for low specificity (W30 and F31 in heavy chain CDR1 and L56 in heavy chain CDR2) are highlighted. (B) Size-exclusion chromatography reveals that the affinity-matured antibody interacts with the column matrix and displays abnormally long elution times relative to the parental antibody. (C) Antibody clearance rates from rats (dose of 0.3 mg/kg) reveals that the affinity-matured antibody is cleared much faster than the parental antibody. The figure is redrawn and adapted from Reference [44].

Figure 5

Mutations in an anti-IL-13 mAb display trade-offs between antibody affinity and solubility. Key residues that mediate high affinity while compromising solubility are three consecutive aromatic and histidine residues (Phe-Trp-His) in heavy chain CDR3. The most effective combination of mutations that increase antibody solubility while maintaining affinity are a single mutation that introduces an N-linked glycosylation site in heavy chain CDR2 and multiple mutations in the variable light (V_L) domain that increase the overall net (positive) charge. The data are from Reference [51].