Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins.

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T-cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD-1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN-γ, granzyme B, IL-2, and TNF-α, demonstrating successful reinvigoration. The monovalent DARPin® protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD-1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD-1 blockade by tumor-derived T-cells; however, the absolute amounts were significantly lower compared to the release from ascites-derived T-cells. Our results demonstrate that PD-1 blockade can only partially reinvigorate functionally suppressed T-cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof-of-concept for the potential use of DARPin® proteins as eligible alternative scaffold proteins to block PD-1.

Rescued Chondrogenesis of Mesenchymal Stem Cells under Interleukin 1 Challenge by Foamyviral Interleukin 1 Receptor Antagonist Gene Transfer.

Background: Mesenchymal stem cells (MSCs) and their chondrogenic differentiation have been extensively investigated in vitro as MSCs provide an attractive source besides chondrocytes for cartilage repair therapies. Here we established prototype foamyviral vectors (FVV) that are derived from apathogenic parent viruses and are characterized by a broad host range and a favorable integration pattern into the cellular genome. As the inflammatory cytokine interleukin 1 beta (IL1ß) is frequently present in diseased joints, the protective effects of FVV expressing the human interleukin 1 receptor antagonist protein (IL1RA) were studied in an established in vitro model (aggregate culture system) of chondrogenesis in the presence of IL1ß. Materials and Methods: We generated different recombinant FVVs encoding enhanced green fluorescent protein (EGFP) or IL1RA and examined their transduction efficiencies and transgene expression profiles using different cell lines and human primary MSCs derived from bone marrow-aspirates. Transgene expression was evaluated by fluorescence microscopy (EGFP), flow cytometry (EGFP), and ELISA (IL1RA). For evaluation of the functionality of the IL1RA transgene to block the inhibitory effects of IL1ß on chondrogenesis of primary MSCs and an immortalized MSC cell line (TERT4 cells), the cells were maintained following transduction as aggregate cultures in standard chondrogenic media in the presence or absence of IL1ß. After 3 weeks of culture, pellets were harvested and analyzed by histology and immunohistochemistry for chondrogenic phenotypes. Results: The different FVV efficiently transduced cell lines as well as primary MSCs, thereby reaching high transgene expression levels in 6-well plates with levels of around 100 ng/ml IL1RA. MSC aggregate cultures which were maintained in chondrogenic media without IL1ß supplementation revealed a chondrogenic phenotype by means of strong positive staining for collagen type II and matrix proteoglycan (Alcian blue). Addition of IL1ß was inhibitory to chondrogenesis in untreated control pellets. In contrast, foamyviral mediated IL1RA expression rescued the chondrogenesis in pellets cultured in the presence of IL1ß. Transduced MSC pellets reached thereby very high IL1RA transgene expression levels with a peak of 1087 ng/ml after day 7, followed by a decrease to 194 ng/ml after day 21, while IL1RA concentrations of controls were permanently below 200 pg/ml. Conclusion: Our results indicate that FVV are capable of efficient gene transfer to MSCs, while reaching IL1RA transgene expression levels, that were able to efficiently block the impacts of IL1ß in vitro. FVV merit further investigation as a means to study the potential as a gene transfer tool for MSC based therapies for cartilage repair.

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.

A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans.

The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which has so far received less attention in the scientific world with respect to the evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. Cytokine secretion into plasma or cell culture supernatants after 24h incubation with the test mAbs (anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release) was detected by a multiplex assay based on electrochemiluminescent excitation. We provide proof that this whole blood assay is a suitable new method for hazard identification of safety-relevant cytokine release in the clinic based on its ability to detect the typical cytokine signatures found in humans for the tested mAbs and on a markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™ - a clear advantage over the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine responses to the individual mAbs, in the concentration-response relationships and the prominent cytokine signatures for individual mAbs in the two formats reflect diverging mechanisms of cytokine release and different levels of dependency on high density coating even for two anti-CD28 super-agonistic antibodies. These results clearly show that one generic approach to assessment of cytokine release using in vitro assays is not sufficient, but rather the choice of the method, i.e. applying the whole blood assay or the PBMC assay needs to be well considered depending on the target characteristics and the mechanistic features of the therapeutic mAbs being evaluated.

Heparan sulfate is an attachment factor for foamy virus entry.

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.