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.
Hypoxia
is present in most solid tumors and is clinically correlated
with increased metastasis and poor patient survival. While studies
have demonstrated the role of hypoxia and hypoxia-regulated proteins
in cancer progression, no attempts have been made to identify hypoxia-regulated
proteins using quantitative proteomics combined with MALDI-mass spectrometry
imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome
study and are the first to investigate changes in situ using tumor
samples. In vitro quantitative mass spectrometry analysis of the hypoxic
proteome was performed on breast cancer cells using stable isotope
labeling with amino acids in cell culture (SILAC). MS analyses were
performed on laser-capture microdissected samples isolated from normoxic
and hypoxic regions from tumors derived from the same cells used in
vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated
protein localization within tumor sections. Here we identified more
than 100 proteins, both novel and previously reported, that were associated
with hypoxia. Several proteins were localized in hypoxic regions,
as identified by MALDI-MSI. Visualization and data extrapolation methods
for the in vitro SILAC data were also developed, and computational
mapping of MALDI-MSI data to IHC results was applied for data validation.
The results and limitations of the methodologies described are discussed.
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