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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.


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).
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kfw121-F6: 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).

Mentions: To determine whether aggregation and Th1 skewing was a feature solely of scFv protein or a more general property of protein aggregation per se, OVA was used as an alternative immunogenic protein. Stir stress was employed to aggregate OVA within the subvisible size range and DLS used for analysis. The mean diameters of OVA aggregates as a percentage of volume were: 80% approximately 0.8 µm, 10% approximately 0.1 µm, and 10% approximately 5 µm (Fig. 6A). Although these aggregates were less homogenous than observed with the scFv preparation, the mean diameter of the main population was similar to that of the scFv aggregates (1 µm). Monomer and aggregate protein preparations were administered via ip injection to mice on days 0, 7, and 14. On day 21, spleens and sera were isolated.FIG. 6


Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response
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).
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kfw121-F6: 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).
Mentions: To determine whether aggregation and Th1 skewing was a feature solely of scFv protein or a more general property of protein aggregation per se, OVA was used as an alternative immunogenic protein. Stir stress was employed to aggregate OVA within the subvisible size range and DLS used for analysis. The mean diameters of OVA aggregates as a percentage of volume were: 80% approximately 0.8 µm, 10% approximately 0.1 µm, and 10% approximately 5 µm (Fig. 6A). Although these aggregates were less homogenous than observed with the scFv preparation, the mean diameter of the main population was similar to that of the scFv aggregates (1 µm). Monomer and aggregate protein preparations were administered via ip injection to mice on days 0, 7, and 14. On day 21, spleens and sera were isolated.FIG. 6

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.