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.

Keywords: OVA.; biotherapeutic; immunogenicity; protein aggregation; scFv.

FIG. 1

Characterization of immune responses to scFv: comparisons of monomer and heat stressed aggregates. scFv at 1 mg/ml in PBS pH 7 was subjected to heat treatment for 25 min at 40 °C. A, The mean particle diameter was measured by DLS before (i) and after (ii) the 40 °C incubation. B, Mice were immunized by ip injection with 250 µl of 1 mg/ml monomer or heat aggregated scFv on days 0 and 7 and serum isolated on day 14 (2 independent experiments; n = 5 and n = 3 per group). Doubling dilutions of serum samples from scFv monomer (Mono) and aggregate (Agg) immunized animals and negative control naïve serum samples were analyzed against both scFv substrate proteins (vs monomer [M] and vs aggregated protein [A]) by ELISA for IgG, IgG1, IgG2a, and IgM anti-scFv antibody content. (i) Data are displayed as OD450 nm (±SEM) for each reciprocal serum dilution (32–8192 for IgG antibodies; 128–13 1072 for IgM antibodies) (ii) Data are displayed with respect to antibody titer (log2) calculated as the lowest serum dilution at which 3x the ELISA substrate blank OD450 nm reading was reached. Individual titers are displayed with overall mean and SEM. Statistical significance of differences in antibody detection between all sera groups against each substrate was calculated using a 1-way ANOVA (*P < .05).

FIG. 2

Antibody response to scFv: influence of dose and aggregation status. Mice were immunized by ip injection with 250 µl of 1 (n = 5) or 0.1 mg/ml (n = 3) monomer or heat aggregated scFv on days 0, 7 and 14 and serum isolated on day 21. Doubling dilutions of serum samples (starting dilution 1 in 32) from scFv monomer (Mono) and aggregate (Agg) immunized animals and negative control NMS samples were analyzed against scFv substrate proteins (versus immunizing protein only results are shown) by ELISA for IgG (A), IgG1 (B), IgG2a (C), and IgM (D) anti-scFv antibody content. Data are displayed with respect to antibody titer (log2), calculated as the lowest serum dilution at which 3× the ELISA substrate blank OD450nm reading was reached. Individual titers are displayed with overall mean and SEM. Statistical significance of differences in antibody binding between all treatment groups against substrate were calculated using a 1-way ANOVA (*P < .05).

FIG. 3

Antibody responses to scFv monomer, heat stressed and stir stressed aggregates. ScFv at 1 mg/ml in PBS pH 7 was subjected to heat treatment for 25 min at 40 °C for thermal stress. To induce stir stress samples were stirred for 6 h at room temperature. A, The mean particle diameter was measured by DLS using the Malvern zetasizer before and after the 40 °C incubation or stir stress. B, Mice (n = 6) were immunized by ip injection with 250 µl of 1 mg/ml monomer or heat (Heat Agg) or stir aggregated (Stir Agg) scFv on days 0, 7, and 14 and serum isolated on day 21. Doubling dilutions of serum samples (starting dilution 1 in 32 for IgG and 1 in 128 for IgM) from immunized animals and negative control NMS samples were analyzed against scFv substrate proteins (vs immunizing protein only results are shown) by ELISA for IgG, IgG1, IgG2a, and IgM anti-scFv antibody content. Data are displayed with respect to antibody titer (log2), calculated as the lowest serum dilution at which 3× the ELISA substrate blank OD450 nm reading was reached. Individual titers (open and closed symbols) are displayed for 2 independent experiments (n = 3 per group), with overall mean and SEM. Statistical significance of differences in antibody detection between all treatment groups against substrate were calculated using a 1-way ANOVA (*P < .05).

FIG. 4

Splenocyte culture ³H-thymidine incorporation following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend), or naïve* mice (n = 3 per group) were cultured alone and in co-culture with BMDC and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv or with media alone. Cells were pulsed with ³H-thymidine 24 h before harvesting at 72 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Group mean proliferation measured as DPM is shown ±SEM. Statistical significance of differences were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.

FIG. 5

Splenocyte culture IFN-γ and IL-13 cytokine secretion following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend) or naïve* mice (n = 3 per group) were cultured alone (splenocyte only) and in co-culture with BMDC (w/BMDC) and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv or media alone. Each culture condition was performed for a single aliquot of splenocytes derived from each individual animal. Supernatants were harvested at 144 h and analyzed for the presence of IFNγ and IL-13 by cytokine specific ELISA. Data are shown as group mean ± SEM. Statistical significance of differences were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.

FIG. 6

Characterization of immune responses to OVA: comparisons of monomer and stir stressed aggregates. 1 mg/ml OVA in PBS pH 7 was subjected to stir stress for 24–28 h at room temperature. A, The mean particle diameter was measured by DLS using the Malvern zetasizer before and after stir stress. B, Mice (n = 3 per group) were immunized by ip injection with 250 µl of 1 mg/ml (n = 3) monomer or stir aggregated OVA on days 0, 7, and 14 and serum isolated on day 21. Doubling dilutions of serum samples (starting dilution 1 in 64) from OVA monomer (Mono) and aggregate (Agg) immunized animals and negative control NMS samples were analyzed against OVA substrate proteins (vs monomer [M] and vs aggregated protein [A]) by ELISA for IgG, IgG1, IgG2a, and IgM anti-OVA antibody content. (i) Data are displayed as OD450 nm ±SEM for each reciprocal serum dilution (ranging from 64 to 66532).) (ii) Data are displayed with respect to antibody titer (log2) calculated as the lowest serum dilution at which 3× the ELISA substrate blank OD450 nm reading was reached. Individual titers are displayed with overall mean and SEM (vs immunizing protein only results are shown). Statistical significance of differences in antibody detection between all treatment groups against substrate were calculated using a 1-way ANOVA (*P < .05).

FIG. 7

Splenocyte culture ³H-Thymidine incorporation following ex vivo OVA challenge. Splenocytes from naïve* (A) and monomer (B) or aggregate (B) immunized mice (as described in Figure 6 legend; n = 3 per group) were co-cultured with BMDC and challenged with 100 µg/ml monomeric OVA (Mono), stir aggregated OVA (Agg) or media alone. Cells were pulsed with ³H-thymidine 24 h before harvesting at 72 and 144 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Proliferation measured as DPM is shown ± SEM (2 bars represent 72 and 144 h time points). Statistical significance of differences in proliferation between all groups were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001, ****p P < .0001). * Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.

FIG. 8

Splenocyte culture IFN-γ, IL-4, and IL-13 cytokine secretion following ex vivo OVA challenge. Splenocytes from naïve* and monomer (Mono) or aggregate (Agg) immunized mice (as described in Figure 6 legend; n = 3 per group) were cultured alone (splenocyte only) or in co-culture with BMDC (w/BMDC) and challenged with 100 µg/ml monomer (Mono), aggregate (Agg), or media alone. Each culture condition was performed for a single aliquot of splenocytes derived from each individual animal. Supernatants were harvested at 144 h and analyzed for the presence of A, IFNγ; B, IL-4, and C, IL-13 by cytokine specific ELISA. Data are shown as group mean ±SEM. Statistical significance of differences were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001, **** P < .0001). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.