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.

Show MeSH

Related in: MedlinePlus

Impurities can synergize to break tolerance to “self” proteins.Two month-old female C57BL/6 mice (n = 5/group) received rhuEPO (5 µg i.p. on day 0, 14 and 62) alone or together CpG ODN and/or LPS as shown (black arrows), and then bled (50 µl) every 7 or 14 days. Mice that received saline served as controls. Results are expressed as the mean (±SD) hematocrit for each individual mouse (Mean ± SEM). The gray box shows the mean +3SD of the hematocrit of untreated mice over the time of the experiment. Those mice were bled with the same schedule as the treated groups. Statistical analysis: SAS mixed model with repeated measures where HCT = treat+day+treat*day+treat*day2+treat*day3. P values<0.05 were considered significant. * p<0.05; *** p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3008684&req=5

pone-0015252-g006: Impurities can synergize to break tolerance to “self” proteins.Two month-old female C57BL/6 mice (n = 5/group) received rhuEPO (5 µg i.p. on day 0, 14 and 62) alone or together CpG ODN and/or LPS as shown (black arrows), and then bled (50 µl) every 7 or 14 days. Mice that received saline served as controls. Results are expressed as the mean (±SD) hematocrit for each individual mouse (Mean ± SEM). The gray box shows the mean +3SD of the hematocrit of untreated mice over the time of the experiment. Those mice were bled with the same schedule as the treated groups. Statistical analysis: SAS mixed model with repeated measures where HCT = treat+day+treat*day+treat*day2+treat*day3. P values<0.05 were considered significant. * p<0.05; *** p<0.01.

Mentions: Tolerance to low-abundance self proteins in sera is incomplete and may be overcome when these proteins are presented in the context of adequate adjuvants [37]–[39]. Using a model established by Ryan et al. [40], we determined whether the addition of low levels of LPS and/or CpG DNA was sufficient to induce a breach in tolerance and the induction of a neutralizing response to erythropoietin. Balb/c mice were treated with recombinant Human Erythropoietin (rhuEPO) alone or together with low levels of LPS and/or CpG ODN on days 0, 14 and 62, followed by weekly hematocrit measurements. As shown in figure 6, the hematocrit of untreated mice remains constant over time (51±3%). Despite having only 80% homology with mouse [41], rhuEPO was active in mice, eliciting a reproducible increase in the hematocrit 1 week after each treatment (15.5, 13.9 and 13.6% increase over baseline after the 1st, 2nd and 3rd dose respectively). Addition of low levels of CpG ODN (50 or 500 ng), LPS (100 pg) or the combination (50 ng CpG ODN +100pg LPS) did not modify the response. In mice treated with rhuEPO together with LPS (10ng) there was evidence of reduced hematocrit following the 2nd inoculation. In contrast, mice treated with rhuEPO in combination with 500 ng of CpG ODN plus 10 ng of LPS showed reduced response to rhuEPO following one treatment as evidenced by increases in hematocrit of 23, 7 and 1.5% over baseline after the 1st, 2nd and 3rd inoculations respectively, followed by a pronounced reduction in hematocrit (2%, 27% and 28% respectively relative to baseline; p<0.001). The reduction in the hematocrit lasted for over 30 days following the 2nd inoculation. This data shows that the rhuEPO augments the hematocrit of mice for about 7 days. The prolonged anemia observed in mice treated with rhuEPO plus LPS and CpG ODN suggests that the inoculations led to a break in tolerance to the endogenous (mouse) EPO, which is needed to maintain the hematocrit stable. Together these data suggests that in mice, the presence of low levels of impurities that can trigger TLR 4 and 9 are may foster a break in tolerance to an essential endogenous non-redundant growth factor.


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

Verthelyi D, Wang V - PLoS ONE (2010)

Impurities can synergize to break tolerance to “self” proteins.Two month-old female C57BL/6 mice (n = 5/group) received rhuEPO (5 µg i.p. on day 0, 14 and 62) alone or together CpG ODN and/or LPS as shown (black arrows), and then bled (50 µl) every 7 or 14 days. Mice that received saline served as controls. Results are expressed as the mean (±SD) hematocrit for each individual mouse (Mean ± SEM). The gray box shows the mean +3SD of the hematocrit of untreated mice over the time of the experiment. Those mice were bled with the same schedule as the treated groups. Statistical analysis: SAS mixed model with repeated measures where HCT = treat+day+treat*day+treat*day2+treat*day3. P values<0.05 were considered significant. * p<0.05; *** p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015252-g006: Impurities can synergize to break tolerance to “self” proteins.Two month-old female C57BL/6 mice (n = 5/group) received rhuEPO (5 µg i.p. on day 0, 14 and 62) alone or together CpG ODN and/or LPS as shown (black arrows), and then bled (50 µl) every 7 or 14 days. Mice that received saline served as controls. Results are expressed as the mean (±SD) hematocrit for each individual mouse (Mean ± SEM). The gray box shows the mean +3SD of the hematocrit of untreated mice over the time of the experiment. Those mice were bled with the same schedule as the treated groups. Statistical analysis: SAS mixed model with repeated measures where HCT = treat+day+treat*day+treat*day2+treat*day3. P values<0.05 were considered significant. * p<0.05; *** p<0.01.
Mentions: Tolerance to low-abundance self proteins in sera is incomplete and may be overcome when these proteins are presented in the context of adequate adjuvants [37]–[39]. Using a model established by Ryan et al. [40], we determined whether the addition of low levels of LPS and/or CpG DNA was sufficient to induce a breach in tolerance and the induction of a neutralizing response to erythropoietin. Balb/c mice were treated with recombinant Human Erythropoietin (rhuEPO) alone or together with low levels of LPS and/or CpG ODN on days 0, 14 and 62, followed by weekly hematocrit measurements. As shown in figure 6, the hematocrit of untreated mice remains constant over time (51±3%). Despite having only 80% homology with mouse [41], rhuEPO was active in mice, eliciting a reproducible increase in the hematocrit 1 week after each treatment (15.5, 13.9 and 13.6% increase over baseline after the 1st, 2nd and 3rd dose respectively). Addition of low levels of CpG ODN (50 or 500 ng), LPS (100 pg) or the combination (50 ng CpG ODN +100pg LPS) did not modify the response. In mice treated with rhuEPO together with LPS (10ng) there was evidence of reduced hematocrit following the 2nd inoculation. In contrast, mice treated with rhuEPO in combination with 500 ng of CpG ODN plus 10 ng of LPS showed reduced response to rhuEPO following one treatment as evidenced by increases in hematocrit of 23, 7 and 1.5% over baseline after the 1st, 2nd and 3rd inoculations respectively, followed by a pronounced reduction in hematocrit (2%, 27% and 28% respectively relative to baseline; p<0.001). The reduction in the hematocrit lasted for over 30 days following the 2nd inoculation. This data shows that the rhuEPO augments the hematocrit of mice for about 7 days. The prolonged anemia observed in mice treated with rhuEPO plus LPS and CpG ODN suggests that the inoculations led to a break in tolerance to the endogenous (mouse) EPO, which is needed to maintain the hematocrit stable. Together these data suggests that in mice, the presence of low levels of impurities that can trigger TLR 4 and 9 are may foster a break in tolerance to an essential endogenous non-redundant growth factor.

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