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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

Trace levels of IIRMIs increase protein immunogencity in vivo.Balb/c mice (3–6 week old; 5 mice/group) were immunized and boosted i.p. with human ovalbumin alone (5µg/mouse) or mixed with the stated amounts of LPS and/or CpG ODN. Mice that received saline were used as controls. Mice were tail-bled before the immunization and weekly thereafter. Antibodies (IgG) to ovalbumin assessed by ELISA. Statistical analysis: ANOVA. * = p<0.05, ** p<0.005, NS: Not significant.
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pone-0015252-g005: Trace levels of IIRMIs increase protein immunogencity in vivo.Balb/c mice (3–6 week old; 5 mice/group) were immunized and boosted i.p. with human ovalbumin alone (5µg/mouse) or mixed with the stated amounts of LPS and/or CpG ODN. Mice that received saline were used as controls. Mice were tail-bled before the immunization and weekly thereafter. Antibodies (IgG) to ovalbumin assessed by ELISA. Statistical analysis: ANOVA. * = p<0.05, ** p<0.005, NS: Not significant.

Mentions: Numerous studies have demonstrated that TLR 4 and TLR 9 agonists in optimal amounts (10–100 µg for LPS and 25–100 µg for CpG ODN) significantly increased the efficacy of vaccines in murine models [17], [34]–[36]. To determine whether trace amounts of TLR agonists were sufficient to increase the immunogenicity of a foreign protein in rodents, Balb/c mice were immunized with LPS-free ovalbumin (5 µg/mouse, subcutaneously) alone or together with LPS (10 ng–1µg/animal) or CpG ODN (500 ng–5µg/animal) individually, or the combination. As shown in figure 5, administration of ovalbumin alone or together with trace or suboptimal amounts of a single TLR-agonist induced very low levels of IgG antibodies. In contrast, mice that received the same dose of ovalbumin together with 10 ng of LPS plus 500 ng of CpG ODN had significantly increased (p<0.05) IgG antibody responses to ovalbumin 3 weeks post treatment. The same group showed significantly higher antibody titers following re-exposure 4 weeks after priming (p<0.005). Indeed, the antibody levels in these mice were significantly higher than those immunized with ovalbumin together with either 5 µg of CpG ODN or 1 µg of LPS (p<0.05).


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

Verthelyi D, Wang V - PLoS ONE (2010)

Trace levels of IIRMIs increase protein immunogencity in vivo.Balb/c mice (3–6 week old; 5 mice/group) were immunized and boosted i.p. with human ovalbumin alone (5µg/mouse) or mixed with the stated amounts of LPS and/or CpG ODN. Mice that received saline were used as controls. Mice were tail-bled before the immunization and weekly thereafter. Antibodies (IgG) to ovalbumin assessed by ELISA. Statistical analysis: ANOVA. * = p<0.05, ** p<0.005, NS: Not significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008684&req=5

pone-0015252-g005: Trace levels of IIRMIs increase protein immunogencity in vivo.Balb/c mice (3–6 week old; 5 mice/group) were immunized and boosted i.p. with human ovalbumin alone (5µg/mouse) or mixed with the stated amounts of LPS and/or CpG ODN. Mice that received saline were used as controls. Mice were tail-bled before the immunization and weekly thereafter. Antibodies (IgG) to ovalbumin assessed by ELISA. Statistical analysis: ANOVA. * = p<0.05, ** p<0.005, NS: Not significant.
Mentions: Numerous studies have demonstrated that TLR 4 and TLR 9 agonists in optimal amounts (10–100 µg for LPS and 25–100 µg for CpG ODN) significantly increased the efficacy of vaccines in murine models [17], [34]–[36]. To determine whether trace amounts of TLR agonists were sufficient to increase the immunogenicity of a foreign protein in rodents, Balb/c mice were immunized with LPS-free ovalbumin (5 µg/mouse, subcutaneously) alone or together with LPS (10 ng–1µg/animal) or CpG ODN (500 ng–5µg/animal) individually, or the combination. As shown in figure 5, administration of ovalbumin alone or together with trace or suboptimal amounts of a single TLR-agonist induced very low levels of IgG antibodies. In contrast, mice that received the same dose of ovalbumin together with 10 ng of LPS plus 500 ng of CpG ODN had significantly increased (p<0.05) IgG antibody responses to ovalbumin 3 weeks post treatment. The same group showed significantly higher antibody titers following re-exposure 4 weeks after priming (p<0.005). Indeed, the antibody levels in these mice were significantly higher than those immunized with ovalbumin together with either 5 µg of CpG ODN or 1 µg of LPS (p<0.05).

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