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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 and IL-13 cytokine secretion following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend) or naïve* mice (n = 3 per group) were cultured alone (splenocyte only) and in co-culture with BMDC (w/BMDC) and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv 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 IFNγ and 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). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
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kfw121-F5: Splenocyte culture IFN-γ and IL-13 cytokine secretion following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend) or naïve* mice (n = 3 per group) were cultured alone (splenocyte only) and in co-culture with BMDC (w/BMDC) and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv 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 IFNγ and 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). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.

Mentions: To characterize further the Th1/Th2 response, culture supernatants in parallel experiments were analyzed for the presence of IFN-γ (a Th1 signature cytokine), IL-4 and IL-13 (Th2 cytokines) (Fig. 5). Secretion of IFN-γ was generally highest under splenocyte-BMDC co-culture conditions. Immunization with heat and stir aggregated scFv resulted in an increased level of IFNγ secretion from splenocyte-BMDC co-cultures in response to stimulation with scFv compared with medium alone. Monomer immunized mouse cultures did not respond to the scFv, by proliferation or by cytokine production, therefore aggregation of the immunizing material resulted in primed splenocytes that were capable of being re-stimulated by antigen in culture. Again, baseline levels of naïve cells were somewhat higher than those of immunized mice, but there was no evidence of antigen specific stimulation of these cells. Again, the nature of the scFv used for antigen challenge in culture failed to influence cytokine production levels. A similar pattern was observed for IL-13 secretion, in so far as BMDC co-culture was required for optimal production, and splenocytes derived from aggregate immunized mice displayed higher levels of cytokine production than did the monomer immunized counterparts. However, significantly higher IL-13 secretion compared with the medium control was only observed in the stir aggregate immunized co-culture group. No change in IFNγ or IL-13 production was observed with naïve splenocyte cultures. IL-4 secretion was measured in addition to IL-13 and IFN-γ; however, levels were below the limit of detection (15.6 pg/ml; data not shown).FIG. 5


Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response
Splenocyte culture IFN-γ and IL-13 cytokine secretion following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend) or naïve* mice (n = 3 per group) were cultured alone (splenocyte only) and in co-culture with BMDC (w/BMDC) and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv 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 IFNγ and 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). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
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kfw121-F5: Splenocyte culture IFN-γ and IL-13 cytokine secretion following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend) or naïve* mice (n = 3 per group) were cultured alone (splenocyte only) and in co-culture with BMDC (w/BMDC) and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv 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 IFNγ and 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). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
Mentions: To characterize further the Th1/Th2 response, culture supernatants in parallel experiments were analyzed for the presence of IFN-γ (a Th1 signature cytokine), IL-4 and IL-13 (Th2 cytokines) (Fig. 5). Secretion of IFN-γ was generally highest under splenocyte-BMDC co-culture conditions. Immunization with heat and stir aggregated scFv resulted in an increased level of IFNγ secretion from splenocyte-BMDC co-cultures in response to stimulation with scFv compared with medium alone. Monomer immunized mouse cultures did not respond to the scFv, by proliferation or by cytokine production, therefore aggregation of the immunizing material resulted in primed splenocytes that were capable of being re-stimulated by antigen in culture. Again, baseline levels of naïve cells were somewhat higher than those of immunized mice, but there was no evidence of antigen specific stimulation of these cells. Again, the nature of the scFv used for antigen challenge in culture failed to influence cytokine production levels. A similar pattern was observed for IL-13 secretion, in so far as BMDC co-culture was required for optimal production, and splenocytes derived from aggregate immunized mice displayed higher levels of cytokine production than did the monomer immunized counterparts. However, significantly higher IL-13 secretion compared with the medium control was only observed in the stir aggregate immunized co-culture group. No change in IFNγ or IL-13 production was observed with naïve splenocyte cultures. IL-4 secretion was measured in addition to IL-13 and IFN-γ; however, levels were below the limit of detection (15.6 pg/ml; data not shown).FIG. 5

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