Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics

Siegel, Michel; Steiner, Guido; Franssen, Linnea C.; Carratu, Francesca; Herron, James N.; Hartman, Katharina; Looney, Cary M.; Ducret, Axel; Bray‐French, Katharine; Rohr, Olivier; Hickling, Timothy P.; Smith, Noel; Marban-Doran, Céline

doi:10.3390/pharmaceutics14122672

Cited by 13 publications

(12 citation statements)

References 27 publications

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“…Both in vitro assays are complementary and aim to identify potential T cell epitopes and the propensity for biotherapeutics to activate CD4+ T cells, respectively. 58,59 In the DC/CD4+ Tcell restimulation assay, the technical control KLH showed high IFN-γ release, indicative of CD4+ T cell activation, and the negative benchmark bevacizumab was associated with background levels of IFN-γ release. In addition, no significant differences were observed for any of the Q-tag-modified variants in comparison to the unconjugated anti-HER2 antibody.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Based on the in silico prediction of T cell epitopes in combination with high conjugation fidelity, a panel of three selected Q-tag sequences (IRQRQ, RWRQR, and YRYRQ), incorporated at position HC297 but also at position HC446 for IRQRQ and YRYRQ, was then tested in the MAPPs and the DC/CD4+ T-cell restimulation assays (Figure b,c). Both in vitro assays are complementary and aim to identify potential T cell epitopes and the propensity for biotherapeutics to activate CD4+ T cells, respectively. , In the DC/CD4+ T-cell restimulation assay, the technical control KLH showed high IFN-γ release, indicative of CD4+ T cell activation, and the negative benchmark bevacizumab was associated with background levels of IFN-γ release. In addition, no significant differences were observed for any of the Q-tag-modified variants in comparison to the unconjugated anti-HER2 antibody.…”

Section: Resultsmentioning

confidence: 99%

“…The Epibase DC/CD4 restimulation assay was performed by Lonza Biologics (Saffron Walden, UK) as described. 83 In short, monocytes were isolated from frozen PBMC samples by magnetic bead selection using CD14 microbeads (Miltenyi Biotec # 130-050-201, on an Auto-MACS Pro system, Miltenyi Biotec, Bergisch Gladbach, Germany) and differentiated into immature DC (iDC) using 1000 IU/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1000 IU/mL of IL-4 in a serum-free medium (CellGenix # 20805-0500, Sartorius, Goëttingen, Germany), supplemented with 0.05 mg/mL Gentamicin (Lonza # 17-518L, Basel, Switzerland) for 5 days at 37 °C and 5% CO2. iDC were then harvested, washed, and loaded with each test protein individually for 4 h at 37 °C and 5% CO2.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…MAPPs (MHC-II Associated Peptide ProteomicS). The HLA-DR-restricted MHC-II peptidome was investigated using the MAPPs assay essentially according to Steiner et al 58 (please refer to the added publication Steiner et al 58 ) with some modifications. In short, monocyte-derived dendritic (moDCs) cells were challenged with the test protein at 50 μg/mL in the presence of 1 μg/mL lipopolysaccharide (LPS) from Salmonella abortus equiv (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) for 24 h. Mature DCs were harvested, washed with PBS, and the cell pellets frozen at −80 °C.…”

Section: ■ Conclusionmentioning

confidence: 99%

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Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties

Sela,

Mansø,

Siegel

et al. 2023

Bioconjugate Chem.

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Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration via intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood–brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising in vitro activity and in vivo pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Conclusionmentioning

confidence: 99%

Section: ■ Conclusionmentioning

confidence: 99%

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Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties

Sela,

Mansø,

Siegel

et al. 2023

Bioconjugate Chem.

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“…Immunogenicity of the nanobodies was determined via a dendritic cell/T cell restimulation assay and was outsourced to Lonza (Basel, Switzerland). The assay was performed using PBMCs of 30 pre-HLAtyped healthy donors as described previously [17].…”

Section: Dendritic Cell/t Cell Restimulation Experimentsmentioning

confidence: 99%

Specific Imaging of CD8+ T-Cell Dynamics with a Nanobody Radiotracer against Human CD8β

De Groof,

Lauwers,

De Pauw

et al. 2024

Preprint

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Purpose While immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8+ T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8+ T-cell dynamics. Methods Nanobodies targeting human CD8β were generated via llama immunizations and subsequent biopanning. The lead anti-human CD8β nanobody was characterized in vitro on binding, specificity, stability and toxicity. The lead nanobody was labelled with 99mTc and 68Ga for non-invasive imaging of human T-cell lymphomas and CD8+ T cells in human CD8 transgenic mice and non-human primates via SPECT or PET/CT. Repeated imaging of CD8+ T cells in MC38 tumor-bearing mice was performed to visualize CD8+ T-cell dynamics. Results The nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8+ T cells were non-invasively visualized via SPECT and PET imaging in naïve and tumor-bearing mice and in naïve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8+ T cells and/or faster in vivo pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8+ T-cell dynamics already at early timepoints. Conclusion Overall, this study describes the development of a more specific human CD8+ T-cell-targeting immunotracer, allowing follow up of immunotherapy responses via non-invasive imaging of human CD8+ T-cell dynamics.

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Reducing Immunogenicity by Design: Approaches to Minimize Immunogenicity of Monoclonal Antibodies

Harris,

Cohen

2024

BioDrugs

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Monoclonal antibodies (mAbs) have transformed therapeutic strategies for various diseases. Their high specificity to target antigens makes them ideal therapeutic agents for certain diseases. However, a challenge to their application in clinical practice is their potential risk to induce unwanted immune response, termed immunogenicity. This challenge drives the continued efforts to deimmunize these protein therapeutics while maintaining their pharmacokinetic properties and therapeutic efficacy. Because mAbs hold a central position in therapeutic strategies against an array of diseases, the importance of conducting comprehensive immunogenicity risk assessment during the drug development process cannot be overstated. Such assessment necessitates the employment of in silico, in vitro, and in vivo strategies to evaluate the immunogenicity risk of mAbs. Understanding the intricacies of the mechanisms that drive mAb immunogenicity is crucial to improving their therapeutic efficacy and safety and developing the most effective strategies to determine and mitigate their immunogenic risk. This review highlights recent advances in immunogenicity prediction strategies, with a focus on protein engineering strategies used throughout development to reduce immunogenicity.

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Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics

Cited by 13 publications

References 27 publications

Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties

Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties

Specific Imaging of CD8+ T-Cell Dynamics with a Nanobody Radiotracer against Human CD8β

Reducing Immunogenicity by Design: Approaches to Minimize Immunogenicity of Monoclonal Antibodies

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