Intact mass analysis reveals the novel O-linked glycosylation on the stalk region of PD-1 protein.

Programmed cell death protein 1 (PD-1) is a key receptor in the immune checkpoint pathway and has emerged to be a promising target for cancer therapy. PD-1 consists of an intracellular domain followed by a transmembrane domain that is connected to the extracellular domain by the stalk region. Although the PD-1 structure has been studied for more than two decades, the posttranslational modification of this protein has been incompletely characterized. In this study, we identified the previously undescribed modification sites of O-linked glycan on the stalk region of PD-1 protein using O-protease digestion coupling with intact mass analysis. The result indicates that T153, S157, S159, and T168 are modified by sialylated mucin-type O-glycan with core 1- and core 2-based structures. This study provides both information on potential novel modification sites on the PD-1 protein and an attractive method for identifying O-linked glycosylation using a specific enzyme and intact mass analysis.

Generation of bispecific antibodies by structure-guided redesign of IgG constant regions.

Bispecific antibodies (BsAbs) form an exciting class of bio-therapeutics owing to their multispecificity. Although numerous formats have been developed, generation of hetero-tetrameric IgG1-like BsAbs having acceptable safety and pharmacokinetics profiles from a single cell culture system remains challenging due to the heterogeneous pairing between the four chains. Herein, we employed a structure-guided approach to engineer mutations in the constant domain interfaces (CH1-CL and CH3-CH3) of heavy and κ light chains to prevent heavy-light mispairing in the antigen binding fragment (Fab) region and heavy-heavy homodimerization in the Fc region. Transient co-transfection of mammalian cells with heavy and light chains of pre-existing antibodies carrying the engineered constant domains generates BsAbs with percentage purity ranging from 78% to 85%. The engineered BsAbs demonstrate simultaneous binding of both antigens, while retaining the thermal stability, Fc-mediated effector properties and FcRn binding properties of the parental antibodies. Importantly, since the variable domains were not modified, the mutations may enable BsAb formation from antibodies belonging to different germline origins and isotypes. The rationally designed mutations reported in this work could serve as a starting point for generating optimized solutions required for large scale production.

Modified recombinant human erythropoietin with potentially reduced immunogenicity.

Recombinant human erythropoietin (rHuEPO) is a biopharmaceutical drug given to patients who have a low hemoglobin related to chronic kidney disease, cancer or anemia. However, some patients repeatedly receiving rHuEPO develop anti-rHuEPO neutralizing antibodies leading to the development of pure red cell aplasia (PRCA). The immunogenic antibody response activated by rHuEPO is believed to be triggered by T-cells recognizing EPO epitopes bound to MHC molecules displayed on the cell surface of APCs. Previous studies have reported an association between the development of anti-rHuEpo-associated PRCA and the HLA-DRB1*09 gene, which is reported to be entrenched in the Thai population. In this study, we used computational design to screen for immunogenic hotspots recognized by HLA-DRB1*09, and predicted seventeen mutants having anywhere between one through four mutations that reduce affinity for the allele, without disrupting the structural integrity and bioactivity. Five out of seventeen mutants were less immunogenic in vitro while retaining similar or slightly reduced bioactivity than rHuEPO. These engineered proteins could be the potential candidates to treat patients who are rHuEpo-dependent and express the HLA-DRB1*09 allele.

Prediction of the binding interface between monoclonal antibody m102.4 and Nipah attachment glycoprotein using structure-guided alanine scanning and computational docking.

Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen-antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.

Comparative secretome analysis of cholangiocarcinoma cell line in three-dimensional culture.

Cholangiocarcinoma (CCA) is a lethal malignancy which occurs with relatively high incidence in Thailand. This cancer is often difficult to diagnose and associated with high mortality. The secretome, containing the secreted proteins from cells, are potentially useful as biomarkers of cancers. Since three-dimensional (3D) cell culture may mimic growth characteristics and microenvironment of solid tumors in vivo better than monolayer culture, we have developed culture of CCA in natural collagen-based scaffold, to enable analysis of the secretome by 2DE. Our results indicated that CCA growth in 3D environment alters cell shape significantly and enhances extracellular matrix deposition. Interestingly, more secreted proteins were detected from 3D culture compared to monolayer culture. Secretome analysis using 2DE coupled with LC-MS/MS demonstrated 10 secreted proteins uniquely found in 3D culture. Moreover, 25 proteins were enriched in 3D culture compared to monolayer culture, including 14-3-3 σ, triosephosphate isomerase, phosphoglycerate mutase 1, α-enolase, and L-plastin. Immunoblotting was used to confirm the presence of L-plastin in conditioned media of CCA and of hepatocellular carcinoma (HCC) cell lines. The results revealed that L-plastin, an actin bundling protein, was uniquely expressed only in the CCA cell line and could be a promising biomarker for differential diagnosis of CCA compared to HCC.

Proteomic analysis reveals important role of 14-3-3σ in anoikis resistance of cholangiocarcinoma cells.

Cholangiocarcinoma (CCA), a high-prevalence cancer in Thailand, is highly metastatic and has high mortality rates. Anoikis resistance or the ability of cells to survive after detachment from extracellular matrix is a necessary property of metastatic cancer. Here, we report differential protein expression of an anoikis-resistant CCA cell line culture, under attachment conditions compared to nonattachment conditions, studied using 2DE coupled with protein identification by LC-MS/MS. Our data reveal the deregulation of proteins involved in stress response, cytoskeleton rearrangement, proapoptosis, cell proliferation, and glycolysis. Interestingly, 14-3-3σ (14-3-3 sigma) protein was intensely upregulated in detached CCA cells. Real-time RT-PCR analysis confirmed that only the sigma isotype was the most abundant transcript among 14-3-3 genes in CCA cells. Furthermore, silencing 14-3-3σ expression by small interfering RNA in CCA cells resulted in significantly increased percentage of cell death in detached culture. Our findings provide the first evidence showing that 14-3-3σ protein plays a crucial role in anoikis resistance of CCA cells. Therefore, 14-3-3σ might be a potential target in CCA therapy.