Anti-brolucizumab immune response as one prerequisite for rare retinal vasculitis/retinal vascular occlusion adverse events.

In October 2019, Novartis launched brolucizumab, a single-chain variable fragment molecule targeting vascular endothelial growth factor A, for the treatment of neovascular age-related macular degeneration. In 2020, rare cases of retinal vasculitis and/or retinal vascular occlusion (RV/RO) were reported, often during the first few months after treatment initiation, consistent with a possible immunologic pathobiology. This finding was inconsistent with preclinical studies in cynomolgus monkeys that demonstrated no drug-related intraocular inflammation, or RV/RO, despite the presence of preexisting and treatment-emergent antidrug antibodies (ADAs) in some animals. In this study, the immune response against brolucizumab in humans was assessed using samples from clinical trials and clinical practice. In the brolucizumab-naïve population, anti-brolucizumab ADA responses were detected before any treatment, which was supported by the finding that healthy donors can harbor brolucizumab-specific B cells. This suggested prior exposure of the immune system to proteins with structural similarity. Experiments on samples showed that naïve and brolucizumab-treated ADA-positive patients developed a class-switched, high-affinity immune response, with several linear epitopes being recognized by ADAs. Only patients with RV/RO showed a meaningful T cell response upon recall with brolucizumab. Further studies in cynomolgus monkeys preimmunized against brolucizumab with adjuvant followed by intravitreal brolucizumab challenge demonstrated that high ADA titers were required to generate ocular inflammation and vasculitis/vascular thrombosis, comparable to RV/RO in humans. Immunogenicity therefore seems to be a prerequisite to develop RV/RO. However, because only 2.1% of patients with ADA develop RV/RO, additional factors must play a role in the development of RV/RO.

T cell epitope mapping of secukinumab and ixekizumab in healthy donors.

Secukinumab, a human monoclonal antibody that selectively neutralizes IL-17A, has consistently shown low anti-drug antibody responses in patients with psoriasis, psoriatic arthritis, and ankylosing spondylitis. Secukinumab has also shown lower in vitro immunogenicity potential compared with other monoclonal antibodies used to treat psoriasis and psoriatic arthritis, and a significantly lower in vitro T cell precursor frequency compared with ixekizumab, which targets the same antigen. Here, secukinumab and ixekizumab were further examined regarding their specific T cell epitopes. Secukinumab- or ixekizumab-specific CD4 T cell lines were generated from 31 healthy, treatment-naïve donors via 28-day co-culture with mature monocyte-derived dendritic cells exposed to either antibody. Consistent with previous data, the frequency of preexisting T cells to secukinumab was significantly lower as compared with ixekizumab. Only two T cell lines from two different donors could be derived for secukinumab, but no specific T cell epitope was identified. In contrast, 32 T cell lines from eight donors were obtained for ixekizumab. For 11 of these T cell lines, the specific T cell epitopes could be identified and confirmed by major histocompatibility complex-associated peptide proteomics as being naturally presented peptides. All identified T cell epitopes cluster in four main regions that are overlapping with the complementarity-determining regions HCDR3, LCDR1, LCDR2 and LCDR3. Interestingly, ixekizumab CDRs contain amino acids that are not found in any of the germline family members. These amino acids may be associated with the higher number of T cell epitopes identified for ixekizumab light chain and may contribute to the increased in vitro immunogenicity potential observed for ixekizumab vs. secukinumab.

Aggregation of human recombinant monoclonal antibodies influences the capacity of dendritic cells to stimulate adaptive T-cell responses in vitro.

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.