Editor’s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response

Abstract

Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood. In the investigations described herein, the immunogenicity in mice of a humanized single chain variable antibody fragment (scFv) purified after expression in Escherichia coli has been examined. Reproducible scFv aggregates were obtained within the subvisible particle size range (mean diameter 2 µm) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1 mg/ml of aggregated or monomeric scFv induced similar IgG and IgG1 antibody responses. In contrast, aggregate preparations stimulated significantly higher levels of anti-scFv IgG2a antibody than did the monomer. In comparative studies, aggregates of ovalbumin (OVA) within the subvisible particle size range were prepared by stir stress, and their immunogenicity compared with that of monomeric OVA in mice. Aggregated and monomeric OVA induced similar anti-OVA IgG and IgG1 antibody responses, whereas IgG2a antibody levels were significantly higher in aggregate-immunized mice. Furthermore, cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were consistent with aggregated preparations inducing a T helper (Th) 1-type response. Aggregated proteins within the subvisible range were therefore shown to induce a preferential Th1 type response, whereas monomeric proteins elicited a selective Th2 response. These data indicate that protein aggregation can impact on both the vigor and quality of immune responses.

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

“…Additionally, aggregation alone significantly enhanced the anti‐scFv IgM response (*** P < 0·001). This effect of aggregation on IgM antibody levels has previously been reported 22. The addition of DnaK enhanced the IgG2a anti‐scFv response in aggregated preparations (** P < 0·01), but was without effect on IgG1 or IgM antibody production.…”

“…This was expected as we have previously shown that subvisible‐sized aggregates of scFv promote a Th1‐skewed response 22. It is possible that the Th1 skewing is an effect of differential antigen processing and presentation, and this effect is also consistent with the hypothesis that the repetitive epitopes formed by aggregation can mimic characteristics of microbes and viruses 28.…”

“…We examined the effects of DnaK on the immunogenicity of aggregated forms of two different proteins. One, an scFv, was selected because we have characterized its immunological responses previously and it displays a relatively vigorous baseline immunogenicity 22. We also studied mouse albumin, as an exemplar protein for in vivo immunogenicity assessment as it would be treated as ‘self’ by the BALB/c mice.…”

“…Cell pellets were resuspended, sonicated and centrifuged at 28 700 g for 30 min. The scFv was purified from supernatants using DEAE–Sepharose anion exchange chromatography, followed by Protein A affinity chromatography, through binding the VH region, and size exclusion chromatography 22. Protein concentrations were determined by measuring the absorbance at 280 nm using an extinction coefficient of 58 580/M/cm.…”

“…Plates were washed between incubations with 0·05% Tween‐20 in PBS. Plates were incubated with substrate o ‐phenylenediamine and urea hydrogen peroxide for 15 min and reactions were stopped with 0·5 m citric acid 22. Absorbance was read at 450 nm using an automated reader (ELx800; BioTek Instruments, Inc., Winooski, VT), using gen 5 1.10 software (BioTek Instruments).…”