Antigen-presenting cell internalization is key for understanding and evaluating therapeutic antibodies' immunogenicity

Abstract

Therapeutic antibodies have revolutionized the treatment of many diseases. However, their safety and efficacy are often altered by their immunogenicity, as many patients frequently develop anti-drug antibodies. Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system. DCs initiate the immunogenic adaptive immune response by internalizing therapeutic antibodies using different pathways and receptors, leading to antigen presentation to T-cells. Recently, studies have shown that the uptake of antibodies by immune cells could contribute to their immunogenicity. This review will present in detail the different DC internalization mechanisms and then discuss the impact of therapeutic antibodies' properties and aggregation on their uptake by DCs and, therefore, their immunogenicity. We will also highlight cellular models and strategies used to evaluate antibodies' internalization. Addressing the uptake of antibodies by DCs could help to predict the risk of immunogenicity and to develop mitigation strategies.

Keywords: dendritic cells; immunogenicity; immunoglobulin; internalization; therapeutic antibodies.

Figure 1

Summary of endocytic pathways implicated in therapeutic antibodies internalization by dendritic cells. Top: Schematic representation of major endocytic routes used by dendritic cells to internalize IgG or immune complexes, including clathrin-mediated endocytosis, macropinocytosis, and phagocytosis. Bottom: Tabular summary comparing the key features of each pathway, including the mechanism of internalization, vesicle size, and receptors involved. *includes target-mediated endocytosis. CLR, C-type lectine receptor; FcR, Fc receptor; FcRn, neonatal Fc receptor;IgG, immunoglobulin G. Created using BioRender.com.

Figure 2

Antibodies N-glycans and their recognition by dendritic cells receptors. ADCC, antibody dependent cell-mediated cytotoxicity; FcγR, Fc gamma receptors; MR, mannose receptors. Monoclonal antibodies (mAbs) glycosylation modifies their functional properties and impacts their recognition by dendritic cell (DC) receptors through carbohydrate residues. Sialic acid is primarily recognized by Siglecs and DC-SIGN on the surface of DCs. In contrast, fucosylation of mAbs reduces their interaction with FcγRIII, leading to decreased antibody-dependent cellular cytotoxicity (ADCC). However, mannosylation of mAbs enhances ADCC activity and is associated with increased clearance. Mannose-sensitive receptors, such as CD205, CD206, and DC-SIGN, are capable of recognizing fucose and mannose residues. Created using BioRender.com.

Figure 3

An evidence-based strategy for assessing the immunogenicity risk of therapeutic protein candidates, including an internalization assessment test. DC, dendritic cell; MAPPS, MHC-associated peptide proteomics; MHC, major histocompatibility complex, The immunogenicity of therapeutic antibodies is evaluated using complementary approaches. In silico evaluation, based on a database of therapeutic antibodies with known immunogenic risks, predicts MHC-II binding, identifies aggregation-prone regions (APRs), and assesses epitope-based risks and implicated alleles. This approach can guide antibody sequence optimization by selecting variants with lower MHC-II affinity, reduced APRs, and fewer T cell epitopes, while maintaining activity. The next step involves in vitro evaluation of the cellular mechanisms involved in T cell response initiation. Assessing the internalization and processing of antibodies by monocyte-derived dendritic cells (moDCs)—two key steps in this immune response—is important for quantifying antibody uptake and comparing it to a reference antibody with known immunogenicity. This is complemented by identifying internalization pathways and receptors using pharmacological inhibitors or receptor-overexpressing models, as antibody entry depends on properties such as glycosylation and charge. Optimization involves modifying the antibody’s physicochemical properties, such as testing different monoclonal antibodies (mAbs) with the same Fab but varying Fc portions (e.g., glycosylated versus non-glycosylated). The MAPPS assay complements internalization evaluation by identifying peptides presented on MHC-II molecules to CD4 T cells. Additionally, dendritic cell (DC) activation testing is crucial for assessing the biological activity of impurities or large aggregates. T cell assays encompass multiple stages of the immune response: internalization, antigen presentation, peptide recognition, and T cell activation. These assays either identify the presence of a T cell repertoire in response to an antibody, which is a prerequisite for T cell initiation, or assess T cell proliferation in response to the antibody. Based on these results, antibody sequence optimization may be considered to eliminate impurities and reduce immunogenicity by removing, masking, or reducing T cell epitopes. Creating using BioRender.com.