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A major impediment to successful use of therapeutic protein drugs is their ability to induce anti-drug antibodies (ADA) that can alter treatment efficacy and safety in a significant number of patients. To this aim, in silico, in vitro, and in vivo tools have been developed to assess sequence and other liabilities contributing to ADA development at different stages of the immune response. However, variability exists between similar assays developed by different investigators due to the complexity of assays, a degree of uncertainty about the underlying science, and their intended use. The impact of protocol variations on the outcome of the assays, i.e., on the immunogenicity risk assigned to a given drug candidate, cannot always be precisely assessed. Here, the Non-Clinical Immunogenicity Risk Assessment working group of the European Immunogenicity Platform (EIP) reviews currently used assays and protocols and discusses feasibility and next steps toward harmonization and standardization.

New immuno-oncology mAbs or agents with other structures have revolutionized treatment options for several malignancies in the past few years, and more are currently being evaluated in the clinic. The development of new therapeutics comes with a series of challenges and questions, of which one is the risk for unwanted immunogenicity which can lead to decreased efficacy and safety concerns. Today, both in silico and in vitro preclinical tools are available to identify early on therapeutic candidates with a high immunogenicity risk potential. Additionally, certain tools can be used to mitigate the immunogenicity potential, and thus improve and accelerate therapeutic drug development and reduce the number of clinical failures. Often used as a first step is an in silico T cell epitope prediction algorithm such as NetMHCIIpan which can be used to assess and compare the immunogenic potential of the lead candidates and guide de-immunization strategies. Further monitoring of the immunogenic risk can be performed using different in vitro assays: a peptide screen assay, to enable the exclusion of immunogenic peptides and the inclusion of low risk sequences; a dendritic cell activation assay to assess immunogenicity signals for the whole product; the MAPPs or MHC Associated Peptide Proteomics, assay to follow uptake, processing and presenting of biotherapeutics by dendritic cells and identify specific regions of concern, and; in vitro T cell proliferation and activation assays to determine and rank the immunogenic risk of the test proteins. The compilation of the different datasets and translation of the results into a comprehensive risk management plan, allows selection of the best candidates to move forward into humans, deimmunization of test candidates and identification and discontinuation of the high-risk candidates as early as possible. Citation Format: Amin Osmani, Sofie Pattijn, Jana Schockaert, Aurélie Mazy, Chloé Ackaert. Immunogenicity risk assessment and mitigation tools [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 701.

Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration.

UNLABELLED: Human epidermal growth factor receptor 2 (HER2) status is one of the major tumor characteristics in breast cancer to guide therapy. Anti-HER2 treatment has clear survival advantages in HER2-positive breast carcinoma patients. Heterogeneity in HER2 expression between primary tumor and metastasis has repeatedly been described, resulting in the need to reassess HER2 status during the disease course. To avoid repeated biopsy with potential bias due to tumor heterogeneity, Nanobodies directed against HER2 have been developed as probes for molecular imaging. Nanobodies, which are derived from unique heavy-chain-only antibodies, are the smallest antigen-binding antibody fragments and have ideal characteristics for PET imaging. The primary aims were assessment of safety, biodistribution, and dosimetry. The secondary aim was to investigate tumor-targeting potential. METHODS: In total, 20 women with primary or metastatic breast carcinoma (score of 2+ or 3+ on HER2 immunohistochemical assessment) were included. Anti-HER2-Nanobody was labeled with (68)Ga via a NOTA derivative. Administered activities were 53-174 MBq (average, 107 MBq). PET/CT scans for dosimetry assessment were obtained at 10, 60, and 90 min after administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor-targeting potential was assessed in primary and metastatic lesions. RESULTS: No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h after injection. Uptake was seen mainly in the kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall, with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above the background level was demonstrated in most identified sites of disease. Primary lesions were more variable in tracer accumulation. CONCLUSION: (68)Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in the kidneys, liver, and intestines but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in HER2-positive metastases is high, compared with normal surrounding tissues, and warrants further assessment in a phase II trial.

Background We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins. Objective The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy. Design BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood. Results A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation. Conclusion Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO(-)), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O₃/NO₂). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO(-), and Y83 and Y158 for O₃/NO₂. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O₃/NO₂ was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein.