Nanometer-Sized Aggregates Generated Using Short Solubility Controlling Peptide Tags Do Increase the In Vivo Immunogenicity of a Nonimmunogenic Protein

• Published in: Molecular pharmaceutics Vol. 17; no. 5; pp. 1629 - 1637
• Main Authors: Rahman, Nafsoon, Islam, Mohammad Monirul, Unzai, Satoru, Miura, Shiho, Kuroda, Yutaka
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.05.2020
• Subjects: antibody titer; SCP-tag (solubility controlling peptide tag); soluble aggregates; oligomers; hydrodynamic radius; ELISA
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/acs.molpharmaceut.0c00071

Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2–10 μm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (R h) of 3–4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice’s sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.