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Development and qualification of a dendritic cell internalization assay contributing to the immunogenicity risk evaluation of biotherapeutics

ABSTRACT: The appended raw files, csv files and other documents were deposited into the public domain in support for the publication "Development and characterization of a dendritic cell internalization assay contributing to the immunogenicity risk evaluation of biotherapeutics" by Michel Siegel, Aman Padamsey, Anna-Lena Bolender, Patrick Hargreaves, Axel Ducret, Johannes Fraidling, Katharina Hartmann, Cary M. Looney, Olivier Rohr, Tim Hickling, Thomas Kraft, Celine Marban-Doran. The abstract reads as follows: Assessing immunogenicity risks during the development of biotherapeutics is crucial. Given the complexity of immunogenicity - influenced by myriad biological, immunological, and patient-specific factors - Individual risk assessments are poorly predictive, necessitating a holistic approach to immunogenicity risk assessment. Anti-drug antibody production starts with the internalization of drugs by antigen presenting cells such as dendritic cells, which present drug-derived peptides to CD4+ T cells as peptide-MHC-II complexes. Assessing dendritic cell function is common in preclinical immunogenicity risk assessments, including presentation of potential T cell epitopes using the MAPPs assay. However, other aspects of dendritic cell biology are often overlooked. To better understand the dendritic cell contribution to immunogenicity, we developed two flow cytometry-based assays: the dendritic cell internalization assay and the dendritic cell activation assay. Our assay addresses two issues with existing methods for measuring internalization into antigen presenting cells; lack of specificity for the cellular compartment of internalization or the recycling of internalized antibodies, and the increased risk of aggregation, when using a large payload for the detection of antibodies, that would lead to activation of irrelevant scavenger receptors in the context of dendritic cell internalization. We also developed a DC activation assay, improving on various aspects of its relevance for immunogenicity risk assessment compared to previously published protocols. Additionally, we identified DC-SIGN (CD209) and CD80 as crucial for understanding dendritic cell activation mechanisms leading to immunogenicity. To evaluate the performance of these two assays, we used a set of marketed therapeutic antibodies. The dendritic cell internalization assay revealed differences in internalization rates for marketed therapeutic antibodies, even those targeting the same antigen (e.g. PCSK9-targeting antibodies: bococizumab, evolocumab and alirocumab or TNF-targeting antibodies: adalimumab and infliximab), potentially accounting for differential immunogenicity liabilities. The DC activation assay also showed different patterns of DC activation between bococizumab, which rapidly accumulates within the lysosomes, and the other PCSK9-targeting antibodies (evolocumab and alirocumab), giving new insights into specific immunogenicity profiles. Overall, this study provides valuable information for future risk assessments of therapeutic antibodies in development. doi: 10.3389/fimmu.2024.1406804 The data deposited here were used to generate the supplementary figure 3.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Axel Ducret

PROVIDER: MSV000095578 | MassIVE | Mon Aug 12 11:51:00 BST 2024

SECONDARY ACCESSION(S): PXD054823

REPOSITORIES: MassIVE

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Project description:The appended raw files, csv files and other documents were deposited into the public domain in support for the publication "Internalization of therapeutic antibodies into Dendritic cells as a risk factor for immunogenicity" by Michel Siegel, Anna-Lena Bolender, Axel Ducret, Johannes Fraidling, Katharina Hartman, Cary M Looney, Olivier Rohr, Timothy Hickling, Martin Lechmann, Celine Marban-Doran and Thomas Kraft. The abstract reads as follows: "Immunogenicity, defined as the unwanted immune response triggered by the administration of therapeutic antibodies, leads to the production of anti-drug antibodies and is a significant risk factor for the development of therapeutic antibodies. Immunogenicity is a multifactorial phenomenon and risk factors influencing each and every step of the immune response may have major overall consequences. Previously, it has been observed that biophysical properties such as positive charge patches affect the biodistribution of therapeutic antibodies, pharmacokinetic properties like nonspecific clearance by altering their uptake into endothelial cells. The internalization of therapeutic antibodies into dendritic cells plays a crucial role in immunogenicity. Cellular internalization is mainly facilitated by nonspecific endocytosis or fluid phase pinocytosis. It involves the cell engulfing surrounding fluid, leading to the internalization of its contents. The biophysical properties of therapeutic antibodies, such as positive charge patches, can influence this process as described for endothelial cells. We therefore hypothesize that internalization of therapeutic antibodies into dendritic cells is influenced by their biophysical properties. Investigating the link between charge patches and dendritic cells internalization using tool antibodies with large positive or negative charge patches, we observed that antibodies with large positive charge patches showed higher lysosomal accumulation and epitope presentation, an important risk factor for immunogenicity. To understand the subsequent impact on T cell activation, we inserted a CD4+ T cell epitope from ovalbumin (a model antigen with well-characterized T cell epitopes) into the same tool compounds. This insertion led to no significant changes in dendritic cell internalization or T cell epitope presentation compared to wild-type antibodies, allowing for a direct comparison of the impact of internalization T cell epitope presentation on T cell activation. However, the insertion of the ovalbumin CD4+ T cell epitope into the tool antibodies increased the occurrence of a specific T cell response, coupled with enhanced internalization and epitope presentation. This increased activation is a key risk factor in immunogenicity, emphasizing the need for careful consideration during the development of biotherapeutics. In conclusion, positive charge patches-mediated internalization and presentation significantly influence the immunogenicity of therapeutic antibodies." doi: 10.3389/fimmu.2024.1406643 The data deposited here supports the generation of Fig. 2 and 4 of this manuscript.

Project description:The appended raw files, csv files and other documents were deposited in the public domain in support for the publication "Expanding the MAPPs assay to accommodate MHC-II pan re-ceptors for improved predictability of potential T cell epitopes" by Katharina Hartman, Guido Steiner, Michel Siegel, Cary M. Looney, Timothy P. Hickling, Katharine Bray-French, Sebastian Springer, Celine Marban-Doran and Axel Ducret. The abstract is as follows: A critical step in the immunogenicity cascade is attributed to human leukocyte antigen (HLA) II presentation triggering T cell immune responses. The liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based major histocompatibility complex (MHC) II-associated peptide proteomics (MAPPs) assay is implemented during preclinical risk assessments to identify bio-therapeutic-derived T cell epitopes. Although studies indicate HLA-DP and HLA-DQ alleles are linked to immunogenicity, most MAPPs studies are restricted to HLA-DR as the dominant HLA II genotype due to lack of well-characterized immunoprecipitating antibodies. Herein we ad-dress this issue by testing various commercially-available clones of MHC-II pan (CR3/43, WR18, and Tu39), HLA-DP (B7/21), and HLA-DQ (SPV-L3 and 1a3) antibodies in the MAPPs assay, and characterizing identified peptides according to binding specificity. Our results reveal that HLA II receptor-precipitating reagents with similar reported specificities differ based on clonality and that MHC-II pan antibodies do not entirely exhibit pan-specific tendencies. Since no individual antibody clone is able to recover the complete HLA II peptide repertoire, we recommend a mixed strategy of clones L243, WR18, and SPV-L3 in a single immunoprecipitation step for more robust compound-specific peptide detection. Ultimately, our optimized MAPPs strategy im-proves the predictability and additional identification of T cell epitopes in immunogenicity risk assessments. The dataset is divided in two sections, one supporting the figures 1-4, the other one supporting the figure 5-6. The collective data has aslo be used to generate the supplementary tables S1-S9.

Project description:Recombinant adenovirus vectors (rAds) are being investigated as vaccine delivery vehicles in pre-clinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. Here, we compared the infectivity and cell stimulatory capacity of rAds of serotype 5 (rAd5), 28 (rAd28) and 35 (rAd35) in association with their immunogenicity. We found that rAd28 and rAd35 infected, and led to the in vitro maturation and activation of both human and mouse dendritic cells (DCs) more efficiently than did rAd5. In stark contrast to rAd5, rAd28 and rAd35 induced production of interferon-alpha (IFNM-NM-1) and stimulated interferon-related intracellular pathways. However, the in vivo immunogenicity of rAd28 and rAd35 was significantly lower than that of rAd5. Deletion of IFNM-NM-1 signaling during vaccination with rAd28 and rAd35 vectors increased the magnitude of the insert-specific T-cell response to levels induced by vaccination with rAd5 vector. The negative impact of IFNM-NM-1 signaling on the magnitude of the T cell response could be overcome by increasing the vaccine dose, which was also associated with greater polyfunctionality and a more favorable long-term memory phenotype of the CD8 T cell response in the presence of IFNM-NM-1 signaling. Taken together, our results demonstrate that rAd-induced IFNM-NM-1 production has multiple effects on T cell immunogenicity, the understanding of which should be considered in the design of rAd vaccine vectors. FACSAria-purified DC populations were incubated with rAd5, rAd28, rAd35, TLR7/8-ligand, or unexposed, for 24 hours. RNA samples from incubated myeloid DCs (mDCs; n=5 for Ad5, Ad35 and mock; n=4 for Ad28 and TLR7/8-ligand) and plasmacytoid DCs (pDCs; n=5 for Ad28; n=4 for mock; n=3 for Ad35 and TLR7/8-ligand; n=2 for Ad5) were prepared using the Illumina BeadStation assay and hybridized to Illumina RefSeq-8 V3 BeadChips.

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Development and qualification of a dendritic cell internalization assay contributing to the immunogenicity risk evaluation of biotherapeutics

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