Aggregation of Human Recombinant Monoclonal Antibodies Influences the Capacity of Dendritic Cells to Stimulate Adaptive T-Cell Responses In Vitro

Abstract

Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses – peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated Peptide Proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells.The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of subvisible proteinaceous particles correlated very well with the pronounced increase in the peptide number and clusters presented in the context of class II HLA-DR molecules, suggesting a major involvement of a mass-action mechanism of altering the presentation.

Modulating the immune system through nanotechnology

“…Although nowadays the use of humanized or fully-human biodrugs has greatly contributed to reduce this risk, immunogenicity associated to the aggregation of biodrug molecules and other factors is still a major concern for the optimum exploitation of these modern drugs [296,297]. The association between biodrug molecules upon injection has been thought to be a natural way to enhance antigen processing and presentation in the cells [298,299]. On the other hand, the presence of impurities could also be part of the problem.…”

Therapeutic outcomes, assessments, risk factors and mitigation efforts of immunogenicity of therapeutic protein products

“…Product properties that affect immunogenicity include molecular structures, post-translational modifications, impurities, immunomodulatory properties and aggregation [7,9,57]. Among these, aggregation of TPP carries particular concern, as it occurs under some conditions with many TPP and several studies have identified associations between aggregates and increased propensity for immunogenicity [51,[81][82][83][84][85][86]. Compared with non-aggregated forms, higher immunogenicity in mice has been observed for aggregates generated under different stress conditions for various TPPs, such as human interferon alpha2b [87][88][89], human mAbs [90][91][92], human epoetin alfa [83], human Factor VIII [93,94], human interferon beta [95], and murine growth hormone [96].…”

“…Compared with non-aggregated forms, higher immunogenicity in mice has been observed for aggregates generated under different stress conditions for various TPPs, such as human interferon alpha2b [87][88][89], human mAbs [90][91][92], human epoetin alfa [83], human Factor VIII [93,94], human interferon beta [95], and murine growth hormone [96]. The detailed mechanisms of aggregate-induced immunogenicity are still unknown, but it has been observed that aggregation enhances antigen uptake, increases the total amount of peptides associated with MHC II molecules, and increases danger signals to maturate dendritic cells and activate T cells [52,86,91,97,98]. Therefore, aggregates could contribute to increased immunogenicity through enhancing antigen processing and presentation in the T cell-dependent pathway.…”

T-cell assays confirm immunogenicity of tungsten-induced erythropoietin aggregates associated with pure red cell aplasia

“…15 Specific high-affinity neutralizing IgG1 and IgG4 antibody responses to rhEPO indicate T-cell-dependent isotype switching. 2,16 Because in vitro T-cell assays have been successfully used to evaluate the mechanism of immune responses to biotherapeutics, [5][6][7][8][9] vaccines, 17,18 and self-antigens in vitro, 19,20 we investigated in vitro T-cell responses to experimentally heat-induced rhEPO aggregates, and tungsteninduced rhEPO aggregates in clinical lots associated with rhEPOneutralizing antibodies and PRCA. Furthermore, we studied ex vivo T-cell recall responses of patients treated with rhEPO without PRCA, and of patient P1 who developed PRCA after treatment with a clinical batch with elevated levels of tungsten and aggregates.…”