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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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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).
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kfw121-F3: 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).

Mentions: The aggregates studied thus far were formed using thermal stress, and were found to consistently induce higher titer IgG2a antibody responses than did the monomeric protein. Stir stress was employed to determine whether scFv aggregates within a similar size range could be formed using a mechanical stress method. The mean protein particle diameter of aggregates was analyzed by DLS (Fig. 3A), from which it was apparent that aggregates produced using stir stress were similar to heat stressed aggregates. Stir stressed aggregates also had a mean particle diameter of 1000–3000 nm and were stable following 2 freeze/thaw cycles. Anti-scFv antibody production patterns were identical, irrespective of whether stir aggregate or monomer was used as a substrate in the ELISA (data not shown). Monomer and aggregate protein preparations formed using heat or stir stress were administered via ip injection to mice on day 0, 7 and 14 (2 independent experiments; n = 3 in each) and sera isolated on day 21.FIG. 3


Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response
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).
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kfw121-F3: 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).
Mentions: The aggregates studied thus far were formed using thermal stress, and were found to consistently induce higher titer IgG2a antibody responses than did the monomeric protein. Stir stress was employed to determine whether scFv aggregates within a similar size range could be formed using a mechanical stress method. The mean protein particle diameter of aggregates was analyzed by DLS (Fig. 3A), from which it was apparent that aggregates produced using stir stress were similar to heat stressed aggregates. Stir stressed aggregates also had a mean particle diameter of 1000–3000 nm and were stable following 2 freeze/thaw cycles. Anti-scFv antibody production patterns were identical, irrespective of whether stir aggregate or monomer was used as a substrate in the ELISA (data not shown). Monomer and aggregate protein preparations formed using heat or stir stress were administered via ip injection to mice on day 0, 7 and 14 (2 independent experiments; n = 3 in each) and sera isolated on day 21.FIG. 3

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