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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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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.
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kfw121-F8: 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.

Mentions: Antigen-induced cytokine production by cultured splenocytes was also measured (Fig. 8). Splenocytes secreted significantly more IFN-γ when stimulated with aggregate compared with monomer or medium alone (Fig. 8A). In the presence of BMDC there was an increased level of IFNγ secretion in response to monomeric protein. IL-4 and IL-13 secretion was also increased by provision of BMDC (Figs. 8B and C): here, monomer and aggregate treated wells secreted significantly more IL-4 and IL-13 compared with medium alone controls. Stimulation with monomer challenge induced significantly higher Ievels of IL-13 production (**P < .01) compared with aggregate challenge in co-cultures from aggregate immunized mice. Naïve splenocytes did not secrete significantly more cytokine in response to OVA treatment in culture when compared with the medium alone control. These data are indicative of a Th1 skewed response against the aggregated OVA preparation, and a Th2 skewed response against the monomeric preparation.FIG. 8


Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response
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.
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kfw121-F8: 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.
Mentions: Antigen-induced cytokine production by cultured splenocytes was also measured (Fig. 8). Splenocytes secreted significantly more IFN-γ when stimulated with aggregate compared with monomer or medium alone (Fig. 8A). In the presence of BMDC there was an increased level of IFNγ secretion in response to monomeric protein. IL-4 and IL-13 secretion was also increased by provision of BMDC (Figs. 8B and C): here, monomer and aggregate treated wells secreted significantly more IL-4 and IL-13 compared with medium alone controls. Stimulation with monomer challenge induced significantly higher Ievels of IL-13 production (**P < .01) compared with aggregate challenge in co-cultures from aggregate immunized mice. Naïve splenocytes did not secrete significantly more cytokine in response to OVA treatment in culture when compared with the medium alone control. These data are indicative of a Th1 skewed response against the aggregated OVA preparation, and a Th2 skewed response against the monomeric preparation.FIG. 8

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