The immunogenicity of an anti-EGFR single domain antibody (VHH) is enhanced by misfolded amorphous aggregation but not by heat-induced aggregation

Abstract

Amorphous aggregates of therapeutic proteins can provoke an unwanted immune response (anti-drug antibodies; ADAs), but counter-examples have led to some controversy. Amorphous aggregates can possess unique biophysical and biochemical attributes depending on both the way they are generated and the protein's biophysical/biochemical properties. Here, we examine the immunogenicity of an anti-EGFR single domain antibody (V_HH) in four types of amorphous aggregates: two heat-aggregated V_HH incubated at 65 °C (V_HH-65) and 95 °C (V_HH-95), a misfolded V_HH isolated from the insoluble fraction of the E. coli lysate (V_HH-Ins), and a low solubility misfolded V_HH produced by miss-shuffling the SS bonds of the native V_HH (V_HH-Mis). Biophysical and biochemical measurements indicated that V_HH was indeed natively folded, monomeric, and β-sheeted; that V_HH-65 was partially unfolded and formed aggregates with a Z-average (Zave) of 771 nm; whereas V_HH-95 was unfolded and formed aggregates of 1722 nm; and that both V_HH-Ins and V_HH-Mis were misfolded with non-native intermolecular SS bonds and formed aggregates with a Zave of 1846 nm and 1951 nm, respectively. The IgG level generated in Jcl:ICR mice determined by ELISA showed that the native V_HH was barely immunogenic, V_HH-95 was not immunogenic, while V_HH-65 was mildly immunogenic. By contrast, the misfolded aggregates, V_HH-Ins and V_HH-Mis, having a Zave and an aggregation propensity similar to that of V_HH-95, were highly immunogenic. These findings indicate the critical role of the biochemical and biophysical attributes of the amorphous aggregates in generating an immune response against a protein, rather than just their sizes.

Keywords: Amorphous aggregation; Anti-drug antibodies; Disulfide bond; Immunogenicity; Protein aggregation; Protein misfolding; Protein solubility; SS bond; Single domain antibody.