Evaluation of in vitro Assays to Assess the Modulation of Dendritic Cells Functions by Therapeutic Antibodies and Aggregates

Abstract

Therapeutic antibodies have the potential to induce immunogenicity leading to the development of anti-drug antibodies (ADA) that consequently may result in reduced serum drug concentrations, a loss of efficacy or potential hypersensitivity reactions. Among other factors, aggregated antibodies have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are the most efficient antigen-presenting cell population and are crucial for the initiation of T cell responses and the subsequent generation of an adaptive immune response. This work focuses on the development of predictive in vitro assays that can monitor DC maturation, in order to determine whether drug products have direct DC stimulatory capabilities. To this end, four independent laboratories aligned a common protocol to differentiate human monocyte-derived DC (moDC) that were treated with either native or aggregated preparations of infliximab, natalizumab, adalimumab, or rituximab. These drug products were subjected to different forms of physical stress, heat and shear, resulting in aggregation and the formation of subvisible particles. Each partner developed and optimized assays to monitor diverse end-points of moDC maturation: measuring the upregulation of DC activation markers via flow cytometry, analyzing cytokine, and chemokine production via mRNA and protein quantification and identifying cell signaling pathways via quantification of protein phosphorylation. These study results indicated that infliximab, with the highest propensity to form aggregates when heat-stressed, induced a marked activation of moDC as measured by an increase in CD83 and CD86 surface expression, IL-1β, IL-6, IL-8, IL-12, TNFα, CCL3, and CCL4 transcript upregulation and release of respective proteins, and phosphorylation of the intracellular signaling proteins Syk, ERK1/2, and Akt. In contrast, natalizumab, which does not aggregate under these stress conditions, induced no DC activation in any assay system, whereas adalimumab or rituximab aggregates induced only slight parameter variation. Importantly, the data generated in the different assay systems by each partner site correlated and supported the use of these assays to monitor drug-intrinsic propensities to drive maturation of DC. This moDC assay is also a valuable tool as an in vitro model to assess the intracellular mechanisms that drive DC activation by aggregated therapeutic proteins.

Fcγ Receptor Activation by Human Monoclonal Antibody Aggregates

“…9,10 Some in vitro studies using human peripheral blood mononuclear cells (hPBMCs) have suggested that mAb aggregates induce inflammatory responses and innate immune responses, which may increase the risk for immunogenicity. [17][18][19][20] It has been reported that highly aggregated mAbs, but not the monomeric form, can enhance in vitro immune responses, and this activation has been shown to be inhibited by blocking toll-like receptor (TLR) 2, TLR4, Fc gamma receptors (FcgRs), and the complement system. 18 A recent study has suggested that aggregates of intravenous immunoglobulin and bevacizumab, but not human serum albumin, activated innate immune cells, primarily via FcgRs.…”

Optimization of aPlasmodium falciparumcircumsporozoite protein repeat vaccine using the tobacco mosaic virus platform

“…Many of these human CSP mAbs are now being considered as tools for malaria control by passive transfer. Although mAbs may confer short-term protection, cost-benefit analyses may reveal hurdles in the mass administration of mAbs to infants and pregnant women (32,33). Structural data emerging from mAbs has, however, revealed vulnerabilities on parasite antigens that can now be utilized to design the next-generation malaria vaccines that focus the host response to a narrow range of protective epitopes, recapitulating the protective responses achieved by passive transfer of mAbs (34,35).…”

Growth Hormone Aggregates Activation of Human Dendritic Cells Is Controlled by Rac1 and PI3 Kinase Signaling Pathways

“…Furthermore, we recently showed that ERK phosphorylation, but not p38 or JNK, was induced in moDC in response to infliximab aggregates. 12 Previous reports also demonstrated activation of MAPKs and NF-kB subunit p65 in moDC in response to danger signals such as lipopolysaccharide and contact sensitizers, such as nickel. 14,16,19 We were then interested in the upstream signaling pathways that participated in DC response to hGH aggregates.…”

“…The choice to focus on CXCL10 was made for 2 main reasons: first, it is an important chemokine playing a central role in T-helper cell polarization, and second, we described its production in response to hGH aggregates but not with therapeutic antibody aggregates. 12 To study intracellular mechanisms regulated in aggregatesstimulated moDC, we first showed that aggregates increased phosphorylation of p38 MAPK, ERK, JNK, as well as NF-kB subunit p65 binding. This observation seems in agreement with a recent study by Polumuri et al 18 also showing the implication of intravenous immunoglobulin and bevacizumab aggregates (shaken or stirred) in the activation of MAPKs in human peripheral blood mononuclear cells.…”