An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide.

Claire K Holley,Edward Cedrone,Barry W Neun,Duncan Donohue,Daniela Verthelyi,Eric S Pang,Marina A Dobrovolskaia

doi:10.3390/molecules26247461

Abstract

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

Highlights

Repeated administration of therapeutic drug products was shown to trigger unwanted immune responses and the production of antibodies capable of neutralizing both the therapeutic protein and its endogenous counterparts [1,2,3]
One such factor is the presence of innate immune response modulating impurities (IIRMIs) that might be inadvertently introduced during product manufacturing [4,5]
While it is nearly impossible to predict the immunogenicity of a specific biotherapeutic without directly assessing the related immune responses in vivo [3], the presence of IIRMIs contributing to the immunogenicity via priming the immune cells could be identified using in vitro methods detecting innate immunostimulatory responses, including the production of inflammatory cytokines (e.g., IL-1, IFNs, IL-8, TNFα, etc.) and activation of the complement system

Summary

Introduction

Repeated administration of therapeutic drug products was shown to trigger unwanted immune responses and the production of antibodies capable of neutralizing both the therapeutic protein and its endogenous counterparts [1,2,3]. Antibodies to recombinant biotechnology therapeutics come in a variety of isotypes (e.g., IgM vs IgG vs IgE), allotypes (e.g., reflecting genetic differences between IgG of biologically unrelated individuals), idiotypes (e.g., reflecting binding to specific epitopes within antibody variable sites), and may lead to different functional consequences for the host (e.g., binding, PK-altering, neutralizing, hypersensitivity- or anaphylaxis-triggering, and cross-reactive neutralizing). Such anti-drug antibodies (ADA) may lead to severe and, when not timely and properly treated, potentially lethal clinical consequences, loss of treatment efficacy, and the formation of autoimmunity [4,5,6,7,8]. There is an urgent need in understanding the applicability to, and performance of, in vitro assays in detecting IIRMIs presence in drug products

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Conclusion