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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.

No MeSH data available.


Related in: MedlinePlus

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).
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kfw121-F2: 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).

Mentions: In subsequent experiments, the ability of aggregated scFv to induce IgG2a antibody skewing was confirmed. Mice (n = 3-5) were immunized with monomer or heat stressed aggregate at 1 or 0.1 mg/ml using a more vigorous dosing regimen with an additional immunization (day 14), and termination on day 21 (Fig. 2).FIG. 2


Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response
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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036615&req=5

kfw121-F2: 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).
Mentions: In subsequent experiments, the ability of aggregated scFv to induce IgG2a antibody skewing was confirmed. Mice (n = 3-5) were immunized with monomer or heat stressed aggregate at 1 or 0.1 mg/ml using a more vigorous dosing regimen with an additional immunization (day 14), and termination on day 21 (Fig. 2).FIG. 2

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