Limits...
Trace levels of innate immune response modulating impurities (IIRMIs) synergize to break tolerance to therapeutic proteins.

Verthelyi D, Wang V - PLoS ONE (2010)

Bottom Line: However, immune responses to these products are frequent and can seriously impact their safety and efficacy.Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1(st) dose and developed long-lasting anemia following subsequent doses.Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins.

View Article: PubMed Central - PubMed

Affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America. daniela.verthelyi@fda.hhs.gov

ABSTRACT
Therapeutic proteins such as monoclonal antibodies, replacement enzymes and toxins have significantly improved the therapeutic options for multiple diseases, including cancer and inflammatory diseases as well as enzyme deficiencies and inborn errors of metabolism. However, immune responses to these products are frequent and can seriously impact their safety and efficacy. Of the many factors that can impact protein immunogenicity, this study focuses on the role of innate immune response modulating impurities (IIRMIs) that could be present despite product purification and whether these impurities can synergize to facilitate an immunogenic response to therapeutic proteins. Using lipopolysaccharide (LPS) and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFNγ, TNFα and IL-6 expression. In vivo, trace levels of these impurities synergized to increase antigen-specific IgG antibodies to ovalbumin. Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1(st) dose and developed long-lasting anemia following subsequent doses. This suggests that the presence of IIRMIs facilitated a breach in tolerance to the endogenous mouse erythropoietin. Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins.

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Related in: MedlinePlus

IFNγ and IL-6 expression in cells stimulated with trace levels of LPS and CpG ODN.Splenocytes were cultured in the presence of increasing concentrations of LPS and CpG ODN alone or in combination. a. The expression of mRNA was monitored for 24 hr and is shown as fold-increase over time 0. b. Protein expression in culture supernatants was determined at 48 hr. LPS concentrations are expressed in ng/ml. Statistical significance (* p<0.05; ***p<0.001): differences between treatment groups are noted as horizontal stars signal significant increase relative to the corresponding concentration of CpG ODN, or as vertical stars, which show significant increase of dual stimulation over the corresponding concentration of each individual stimulant (CpG ODN or LPS) (ANOVA, p<0.001).
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pone-0015252-g003: IFNγ and IL-6 expression in cells stimulated with trace levels of LPS and CpG ODN.Splenocytes were cultured in the presence of increasing concentrations of LPS and CpG ODN alone or in combination. a. The expression of mRNA was monitored for 24 hr and is shown as fold-increase over time 0. b. Protein expression in culture supernatants was determined at 48 hr. LPS concentrations are expressed in ng/ml. Statistical significance (* p<0.05; ***p<0.001): differences between treatment groups are noted as horizontal stars signal significant increase relative to the corresponding concentration of CpG ODN, or as vertical stars, which show significant increase of dual stimulation over the corresponding concentration of each individual stimulant (CpG ODN or LPS) (ANOVA, p<0.001).

Mentions: To assess whether the presence of low levels of both IIRMIs together would further increase immune activation, splenocytes were stimulated with the lowest levels of LPS (10ng/ml) and CpG ODN (20 nM) that induced a reproducible response in vitro. The breadth of the activation was determined using TaqMan low density DNA arrays (TLDA) for 92 immune-related genes (supplementary Table S1). As shown in figure 2, the addition of both stimuli together triggered a significant increase in mRNA for TBx21, IFNγ and IFNγ- inducible proteins, CXCL10 and CXCL11, as well as interleukin 6 (IL-6), granzyme B, nitric oxide synthase 2 (nos2) and cycloxygenase 2 (cox2) suggesting that the presence of these IIRMIs fosters a pro-inflammatory/type I response. In contrast, while individually LPS and CpG ODN induced small increases in IL-4, IL-5 and IL-10 mRNA expression, no synergy was evident. The increased mRNA expression was sustained for at least 24 hours and resulted in dose-dependent increases in IFNγ and IL-6 protein concentration in culture supernatants, as shown in figure 3a. In the presence of sub-stimulatory levels of CpG ODN (12.5 nM), concentrations of LPS (as low as 1ng/ml or 0.1EU) significantly increased IFNγ and IL-6 production.


Trace levels of innate immune response modulating impurities (IIRMIs) synergize to break tolerance to therapeutic proteins.

Verthelyi D, Wang V - PLoS ONE (2010)

IFNγ and IL-6 expression in cells stimulated with trace levels of LPS and CpG ODN.Splenocytes were cultured in the presence of increasing concentrations of LPS and CpG ODN alone or in combination. a. The expression of mRNA was monitored for 24 hr and is shown as fold-increase over time 0. b. Protein expression in culture supernatants was determined at 48 hr. LPS concentrations are expressed in ng/ml. Statistical significance (* p<0.05; ***p<0.001): differences between treatment groups are noted as horizontal stars signal significant increase relative to the corresponding concentration of CpG ODN, or as vertical stars, which show significant increase of dual stimulation over the corresponding concentration of each individual stimulant (CpG ODN or LPS) (ANOVA, p<0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008684&req=5

pone-0015252-g003: IFNγ and IL-6 expression in cells stimulated with trace levels of LPS and CpG ODN.Splenocytes were cultured in the presence of increasing concentrations of LPS and CpG ODN alone or in combination. a. The expression of mRNA was monitored for 24 hr and is shown as fold-increase over time 0. b. Protein expression in culture supernatants was determined at 48 hr. LPS concentrations are expressed in ng/ml. Statistical significance (* p<0.05; ***p<0.001): differences between treatment groups are noted as horizontal stars signal significant increase relative to the corresponding concentration of CpG ODN, or as vertical stars, which show significant increase of dual stimulation over the corresponding concentration of each individual stimulant (CpG ODN or LPS) (ANOVA, p<0.001).
Mentions: To assess whether the presence of low levels of both IIRMIs together would further increase immune activation, splenocytes were stimulated with the lowest levels of LPS (10ng/ml) and CpG ODN (20 nM) that induced a reproducible response in vitro. The breadth of the activation was determined using TaqMan low density DNA arrays (TLDA) for 92 immune-related genes (supplementary Table S1). As shown in figure 2, the addition of both stimuli together triggered a significant increase in mRNA for TBx21, IFNγ and IFNγ- inducible proteins, CXCL10 and CXCL11, as well as interleukin 6 (IL-6), granzyme B, nitric oxide synthase 2 (nos2) and cycloxygenase 2 (cox2) suggesting that the presence of these IIRMIs fosters a pro-inflammatory/type I response. In contrast, while individually LPS and CpG ODN induced small increases in IL-4, IL-5 and IL-10 mRNA expression, no synergy was evident. The increased mRNA expression was sustained for at least 24 hours and resulted in dose-dependent increases in IFNγ and IL-6 protein concentration in culture supernatants, as shown in figure 3a. In the presence of sub-stimulatory levels of CpG ODN (12.5 nM), concentrations of LPS (as low as 1ng/ml or 0.1EU) significantly increased IFNγ and IL-6 production.

Bottom Line: However, immune responses to these products are frequent and can seriously impact their safety and efficacy.Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1(st) dose and developed long-lasting anemia following subsequent doses.Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins.

View Article: PubMed Central - PubMed

Affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America. daniela.verthelyi@fda.hhs.gov

ABSTRACT
Therapeutic proteins such as monoclonal antibodies, replacement enzymes and toxins have significantly improved the therapeutic options for multiple diseases, including cancer and inflammatory diseases as well as enzyme deficiencies and inborn errors of metabolism. However, immune responses to these products are frequent and can seriously impact their safety and efficacy. Of the many factors that can impact protein immunogenicity, this study focuses on the role of innate immune response modulating impurities (IIRMIs) that could be present despite product purification and whether these impurities can synergize to facilitate an immunogenic response to therapeutic proteins. Using lipopolysaccharide (LPS) and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFNγ, TNFα and IL-6 expression. In vivo, trace levels of these impurities synergized to increase antigen-specific IgG antibodies to ovalbumin. Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1(st) dose and developed long-lasting anemia following subsequent doses. This suggests that the presence of IIRMIs facilitated a breach in tolerance to the endogenous mouse erythropoietin. Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins.

Show MeSH
Related in: MedlinePlus