Abstract
Therapeutic proteins can induce immune responses that affect their safety and efficacy. Product aggregates and innate immune response modulating impurities (IIRMI) are risk factors of product immunogenicity. In this study, we use Intravenous Immunoglobulin (IVIG), Avastin, and Human Serum Albumin (HSA) to explore whether increased aggregates activate innate immune cells or modify the response to IIRMI. We show that increased aggregates (shaken or stirred) in IVIG and Avastin, but not HSA, induced activation of MAPKs (pp38, pERK and pJNK) and transcription of immune-related genes including IL8, IL6, IL1β, CSF1, CCL2, CCL7, CCL3, CCL24, CXCL2, IRAK1, EGR2, CEBPβ, PPARg and TNFSF15 in human PBMC. The immunomodulatory effect was primarily mediated by FcγR, but not by TLR. Interestingly, increased aggregates in IVIG or Avastin magnified innate immune responses to TLR2/4 agonists, but diminished responses to TLR3/9 agonists. This study shows that IIRMI and aggregates can modify the activity of immune cells potentially modifying the milieu where the products are delivered highlighting the complex interplay of different impurities on product immunogenicity risk. Further, we show that aggregates could modify the sensitivity of PBMC-based assays designed to detect IIRMI. Understanding and managing immunogenicity risk is a critical component of product development and regulation.
Highlights
Therapeutic proteins and peptides, whether recombinant, synthetic, or naturally derived have the potential to induce an immune response in the host that impacts on the safety and efficacy of the product
We show that aggregates of Intravenous Immunoglobulin (IVIG), which is a complex mixture of human immunoglobulins, or Avastin, a monoclonal antibody targeting VEGF-A, can modulate the response to innate immune response modulating impurities (IIRMI) magnifying the induction of IL8, IL1β, and IL6 in response to TLR2 or TLR4 agonists
We sought to characterize better the pro-inflammatory response induced by aggregates and determine whether their presence could modify the response to trace levels of IIRMI, increasing the immunogenicity risk of a therapeutic protein
Summary
Therapeutic proteins and peptides, whether recombinant, synthetic, or naturally derived have the potential to induce an immune response in the host that impacts on the safety and efficacy of the product. Product denaturation and aggregation were linked to the emergence of neutralizing antibodies in a clinical trial for erythropoietin[10] These clinical observations are supported by several studies in mice showing that aggregated proteins are more immunogenic than monomers[4,6]. Aggregates of monoclonal antibodies were shown to crosslink Fcγ receptors (FcγR) on the surface of B cells, dendritic cells, macrophages and neutrophils embedded in the tissues with greater affinity than monomers[27] and trigger the activation of innate effector cells. This enhances antigen presentation and maturation of dendritic cells (DCs)[28]. Recent reports suggest that Human PBMC, human monocytes and THP-1 cells could interact with Toll-like receptor (TLR) 2 and TLR4 to produce cytokines and chemokines when stimulated in-vitro with aggregated proteins[29,30]
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