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

Abstract

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.

A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

“…Data analysis was performed using the continuous distribution c (s) analysis module in the SEDFIT program [28]. As a positive control, we used BPTI‐C5I, which was previously reported to produce subvisible oligomers [15,18].…”

“…In previous reports, we showed that short solubility controlling peptide tags (SCP‐tags) [11–13] attached at the C terminus of a host protein could be used to induce the formation of subvisible amorphous aggregates and thereby increase the immunogenicity of non‐immunogenic proteins [14,15]. In particular, a hydrophobic isoleucine tag attached to the C terminus of two model proteins, BPTI‐19A (58 residues; MW: 5.98 kDa) and DEN3ED3 (103 residues; MW 11.46 kDa), increased their immunogenicity by oligomerizing the proteins into small nanometer‐scale aggregates, and the immune response was long‐lasting with a T‐cell‐dependent activation of the B cells [16,17].…”

“…In particular, a hydrophobic isoleucine tag attached to the C terminus of two model proteins, BPTI‐19A (58 residues; MW: 5.98 kDa) and DEN3ED3 (103 residues; MW 11.46 kDa), increased their immunogenicity by oligomerizing the proteins into small nanometer‐scale aggregates, and the immune response was long‐lasting with a T‐cell‐dependent activation of the B cells [16,17]. A major advantage of the SCP‐tags is their ability to produce subvisible aggregates in a highly reproducible and reliable way [18,19], which in turn provides good control of their effect on immunogenicity [15].…”

Systematic mutational analysis of epitope-grafted ED3’s immunogenicity reveals a DENV3-DENV4 bi-serospecific ED3 mutant

“…Finally, after thorough washing with PBS-Tween, coloring was performed with 100µl/well of OPD substrate (0.4mg/mL in 50mM citrate buffer, pH4.5 supplemented with 4mM H 2 O 2 ) for 20 minutes followed by absorbance measurement at 450nm using a Multiskan Ascent (Thermofisher) microplate reader. Antibody titers were calculated from power fitting of absorbance versus reciprocal of serum dilution using a cutoff of 0.1 (OD 450nm ) above the background values [45].…”

Toward Drug-Like Multispecific Antibodies by Design

“…The challenges associated with assembly and purification of multispecific antibodies may also result in an increased risk of immunogenicity. Moreover, critical quality attributes like aggregation, particle size, and polyspecificity are also thought to increase the likelihood of immunogenicity [ 88 , 89 , 239 , 242 , 243 , 244 ]. Given the unique immunogenic liabilities of multispecific antibodies and their increasing numbers in clinical trials, new tools for the analysis and reduction of immunogenic liabilities will be of increasing importance.…”