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

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 (VHH) in four types of amorphous aggregates: two heat-aggregated VHH incubated at 65 °C (VHH-65) and 95 °C (VHH-95), a misfolded VHH isolated from the insoluble fraction of the E. coli lysate (VHH-Ins), and a low solubility misfolded VHH produced by miss-shuffling the SS bonds of the native VHH (VHH-Mis). Biophysical and biochemical measurements indicated that VHH was indeed natively folded, monomeric, and β-sheeted; that VHH-65 was partially unfolded and formed aggregates with a Z-average (Zave) of 771 nm; whereas VHH-95 was unfolded and formed aggregates of 1722 nm; and that both VHH-Ins and VHH-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 VHH was barely immunogenic, VHH-95 was not immunogenic, while VHH-65 was mildly immunogenic. By contrast, the misfolded aggregates, VHH-Ins and VHH-Mis, having a Zave and an aggregation propensity similar to that of VHH-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.