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

View Article: PubMed Central - PubMed

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.

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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).
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kfw121-F1: 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).

Mentions: To produce aggregates by heat stress, 1 mg/ml scFv in PBS was incubated at 40 °C for 25 min and DLS was employed to observe changes in mean particle diameter. A reproducible increase in size from 7 nm to 1000–3000 nm was observed (Fig. 1A). Aggregate and monomer preparations were also shown to be stable by DLS following 2 freeze/thaw cycles. Initial experiments with the scFv monomer demonstrated that a concentration of 0.1 mg/ml resulted in a weak or undetectable IgG antibody response in some animals. A robust, detectable response was required in these experiments for comparison to the aggregated protein (data not shown). A 1 mg/ml dose was found to provide a consistent antibody response, so for all further experiments this concentration was used.FIG. 1


Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response
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).
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kfw121-F1: 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).
Mentions: To produce aggregates by heat stress, 1 mg/ml scFv in PBS was incubated at 40 °C for 25 min and DLS was employed to observe changes in mean particle diameter. A reproducible increase in size from 7 nm to 1000–3000 nm was observed (Fig. 1A). Aggregate and monomer preparations were also shown to be stable by DLS following 2 freeze/thaw cycles. Initial experiments with the scFv monomer demonstrated that a concentration of 0.1 mg/ml resulted in a weak or undetectable IgG antibody response in some animals. A robust, detectable response was required in these experiments for comparison to the aggregated protein (data not shown). A 1 mg/ml dose was found to provide a consistent antibody response, so for all further experiments this concentration was used.FIG. 1

View Article: PubMed Central - PubMed

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 &micro;m) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1&thinsp;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.

No MeSH data available.


Related in: MedlinePlus