A novel bispecific antibody as an immunotherapeutic agent in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains one of the most common and highly fatal malignancies in humans worldwide with increasing prevalence and limited therapeutic options. For many decades, many researchers have attempted to find effective curative methods for HCC and great strides have been made. GPC3 is overexpressed in HCC, but not in normal liver, making it a rational immunotherapeutic target for HCC. GC33, a humanized mAb directed against GPC3, is a safe and well-tolerated therapy choice for patients with HCC, which tested in a phase I trial in advanced HCC patients. Phase II trials of GC33 to evaluate its efficacy and safety in advanced or metastatic HCC, showed no significant differences in overall survival and progression-free survival compared with the placebo. Retrospective analysis indicates that high drug exposure and high CD16 expression may contribute to the clinical efficacy of GC33. Chugai Pharmaceutical has restarted its Phase I trial of GC33, continuing to explore its clinical value targeting GPC3 in solid tumors. To enhance the antitumor potency of GC33, we designed a GPC3/CD16A bispecific antibody (QDEB). In this study, we obtained QDEB at high purity and assessed its effectiveness in the therapy of HCC compared with GC33. In vitro cytotoxicity assays and in vivo experiments demonstrated that QDEB could enhance anti-tumor efficacy compared with GC33. CD16A activation and increased cytokines release were associated with higher anti-tumor activity. In conclusion, this bispecific antibody may possibly help develop new therapeutic strategies for HCC and develop new treatment options in the future.

Mendelian Randomization Analyses of Chronic Immune-Mediated Diseases, Circulating Inflammatory Biomarkers, and Cytokines in Relation to Liver Cancer.

Liver cancer is closely linked to chronic inflammation. While observational studies have reported positive associations between extrahepatic immune-mediated diseases and systemic inflammatory biomarkers and liver cancer, the genetic association between these inflammatory traits and liver cancer remains elusive and merits further investigation. We conducted a two-sample Mendelian randomization (MR) analysis, using inflammatory traits as exposures and liver cancer as the outcome. The genetic summary data of both exposures and outcome were retrieved from previous genome-wide association studies (GWAS). Four MR methods, including inverse-variance-weighted (IVW), MR-Egger regression, weighted-median, and weighted-mode methods, were employed to examine the genetic association between inflammatory traits and liver cancer. Nine extrahepatic immune-mediated diseases, seven circulating inflammatory biomarkers, and 187 inflammatory cytokines were analyzed in this study. The IVW method suggested that none of the nine immune-mediated diseases were associated with the risk of liver cancer, with odds ratios of 1.08 (95% CI 0.87-1.35) for asthma, 0.98 (95% CI 0.91-1.06) for rheumatoid arthritis, 1.01 (95% CI 0.96-1.07) for type 1 diabetes, 1.01 (95% CI 0.98-1.03) for psoriasis, 0.98 (95% CI 0.89-1.08) for Crohn's disease, 1.02 (95% CI 0.91-1.13) for ulcerative colitis, 0.91 (95% CI 0.74-1.11) for celiac disease, 0.93 (95% CI 0.84-1.05) for multiple sclerosis, and 1.05 (95% CI 0.97-1.13) for systemic lupus erythematosus. Similarly, no significant association was found between circulating inflammatory biomarkers and cytokines and liver cancer after correcting for multiple testing. The findings were consistent across all four MR methods used in this study. Our findings do not support a genetic association between extrahepatic inflammatory traits and liver cancer. However, larger-scale GWAS summary data and more genetic instruments are needed to confirm these findings.

LncRNA model predicts liver cancer drug resistance and validate in vitro experiments.

Introduction: Hepatocellular carcinoma (HCC) patients may benefit from chemotherapy, but drug resistance is an important obstacle to favorable prognoses. Overcoming drug resistance is an urgent problem to be solved. Methods: Differential expression analysis was used to identify long non-coding RNAs (LncRNAs) that differed in chemotherapy-sensitive and chemotherapy-resistant patients. Machine learning algorithms including random forest (RF), lasso regression (LR), and support vector machines (SVMs) were used to identify important chemotherapy-related LncRNAs. A back propagation (BP) network was then used to validate the predictive capacity of important LncRNAs. The molecular functions of hub LncRNAs were investigated via qRT-PCR and cell proliferation assay. Molecular-docking technique was used to explore candidate drug of targets of hub LncRNA in the model. Results: A total of 125 differentially expressed LncRNAs between sensitive and resistant patients. Seventeen important LncRNAs were identified via RF, and seven factors were identified via LR. With respect to SVM, the top 15 LncRNAs of AvgRank were selected. Five merge chemotherapy-related LncRNAs were used to predict chemotherapy resistance with high accuracy. CAHM was a hub LncRNA of model and expression high in sorafenib resistance cell lines. In addition, the results of CCK8 showed that the sensitivity of HepG2-sorafenib cells to sorafenib was significantly lower than that of HepG2; and the sensitivity of HepG2-sorafenib cells transfected with sh-CAHM was significantly higher than that of Sorafenib. In the non-transfection group, the results of clone formation experiments showed that the number of clones formed by HepG2-sorafenib cells treated with sorafenib was significantly more than that of HepG2; after HepG2-sorafenib cells were transfected with sh-CAHM, the number of clones formed by Sorafenib treatment was significantly higher than that of HepG2 cells. The number was significantly less than that of HepG2-s + sh-NC group. Molecular Docking results indicate that Moschus was candidate drug for target protein of CAHM. Conclusion: Five chemotherapy-related LncRNAs could predict drug resistance in HCC with high accuracy, and the hub LncRNA CAHM has potential as a new biomarker for HCC chemotherapy resistance.

Serum level of calpains product as a novel biomarker of acute lung injury following cardiopulmonary bypass.

Objective: The aim of this study was to test the hypothesis whether serum level of calpains could become a meaningful biomarker for diagnosis of acute lung injury (ALI) in clinical after cardiac surgery using cardiopulmonary bypass (CPB) technology. Methods and results: Seventy consecutive adults underwent cardiac surgery with CPB were included in this prospective study. Based on the American-European Consensus Criteria (AECC), these patients were divided into ALI (n = 20, 28.57%) and non-ALI (n = 50, 71.43%) groups. Serum level of calpains in terms of calpains' activity which was expressed as relative fluorescence unit (RFU) per microliter and measured at beginning of CPB (baseline), 1 h during CPB, end of CPB as well as 1, 12, and 24 h after CPB. Difference of serum level of calpains between two groups first appeared at the end of CPB and remained different at subsequent test points. Univariate and multivariate logistic regression analysis indicated that serum level of calpains 1 h after CPB was an independent predictor for postoperative ALI (OR 1.011, 95% CI 1.001, 1.021, p = 0.033) and correlated with a lower PaO2/FiO2 ratio in the first 2 days (The first day: r = -0.389, p < 0.001 and the second day: r = -0.320, p = 0.007) as well as longer mechanical ventilation time (r = 0.440, p < 0.001), intensive care unit (ICU) length of stay (LOS) (r = 0.419, p < 0.001) and hospital LOS (r = 0.297, p = 0.013). Conclusion: Elevated serum level of calpains correlate with impaired lung function and poor clinical outcomes, indicating serum level of calpains could act as a potential biomarker for postoperative ALI following CPB in adults. Clinical trial registration: [https://clinicaltrials.gov/show/NCT05610475], identifier [NCT05610475].

Proteome-wide Tyrosine Phosphorylation Analysis Reveals Dysregulated Signaling Pathways in Ovarian Tumors.

The recent accomplishment of comprehensive proteogenomic analysis of high-grade serous ovarian carcinoma (HGSOC) tissues reveals cancer associated molecular alterations were not limited to variations among DNA, and mRNA/protein expression, but are a result of complex reprogramming of signaling pathways/networks mediated by the protein and post-translational modification (PTM) interactomes. A systematic, multiplexed approach interrogating enzyme-substrate relationships in the context of PTMs is fundamental in understanding the dynamics of these pathways, regulation of cellular processes, and their roles in disease processes. Here, as part of Clinical Proteomic Tumor Analysis Consortium (CPTAC) project, we established a multiplexed PTM assay (tyrosine phosphorylation, and lysine acetylation, ubiquitylation and SUMOylation) method to identify protein probes' PTMs on the human proteome array. Further, we focused on the tyrosine phosphorylation and identified 19 kinases are potentially responsible for the dysregulated signaling pathways observed in HGSOC. Additionally, elevated kinase activity was observed when 14 ovarian cancer cell lines or tumor tissues were subjected to test the autophosphorylation status of PTK2 (pY397) and PTK2B (pY402) as a proxy for kinase activity. Taken together, this report demonstrates that PTM signatures based on lysate reactions on human proteome array is a powerful, unbiased approach to identify dysregulated PTM pathways in tumors.

Methylated cis-regulatory elements mediate KLF4-dependent gene transactivation and cell migration.

Altered DNA methylation status is associated with human diseases and cancer; however, the underlying molecular mechanisms remain elusive. We previously identified many human transcription factors, including Krüppel-like factor 4 (KLF4), as sequence-specific DNA methylation readers that preferentially recognize methylated CpG (mCpG), here we report the biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. We show that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. In-depth mechanistic studies reveal that recruitment of KLF4 to the methylated cis-regulatory elements of these genes result in chromatin remodeling and transcription activation. Our study demonstrates a new paradigm of DNA methylation-mediated gene activation and chromatin remodeling, and provides a general framework to dissect the biological functions of DNA methylation readers and effectors.

Lin28A Binds Active Promoters and Recruits Tet1 to Regulate Gene Expression.

Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems.

Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant.

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.

Peptide aptamer microarrays: bridging the bio-detector interface.

In the near future, personalised medicine and phase-0 trials will require that clinical practitioners move from the "one biomarker per disease" paradigm to the use of molecular signatures of disease for diagnosis and the prediction of a patient's response to treatment. These signatures will be composed of biomarkers specific to the disease, and will include over-expression of normal protein from a gene that does not carry a mutation; loss of expression of an essential protein; expression of a protein from a mutant gene; and metabolites whose levels are altered in disease. Surrogates for protein expression, such as alterations in the messenger RNA that encode for them, have already proved their value. The next challenge, then, in clinical biosensing is to enable the multiplexed detection of protein biomarkers, and perhaps the multiplexed detection of mixed biomarkers (metabolites, RNA and proteins) all in a single test. Given the plethora of available antibodies specific for biomarkers, why is this not already happening? We believe that the limitation lies in the nature of the antibody molecule itself, and especially the fact that antibodies have evolved to function in solution, while most diagnostic tests take place at a surface. We have accordingly turned to the design of alternative antibodies, and have identified a protein that appears to be unusually stable on surfaces. The new, non-antibody, scaffold protein is derived from human Stefin A, a natural inhibitor of the cathepsin family of proteases. We have engineered this protein so that it lacks natural binding partners, and introduced a series of new binding surfaces through randomisation or directed replacement of the surfaces used by Stefin A to bind to cathepsins. Our new probes show exquisite specificity and binding affinities comparable to antibodies, and can be used to probe biology in intact cells. More importantly, together and in collaboration with other groups in Chemistry or Engineering Departments, we have shown that these designer proteins can be used in optical detection of labelled target proteins from whole cell lysates in a highly multiplexed microarray format, as well as in label-free detection of unlabelled proteins using surface plasmon resonance, QCM, microcantilevers and using electrochemical assays on gold electrodes. We believe that the combination of optimised surface chemistry, robust and combinatorial designer biological probes and novel, robust and sensitive detection technologies will enable, in the near future, the introduction of multiplexed biomarker detection in the clinical setting, most likely in cancer where multiple biomarkers are known, but probes are still lacking.

Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.

Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

A protein chip approach for high-throughput antigen identification and characterization.

Proteomics research in humans and other eukaryotes demands a large number of high-quality mAbs. Here, we report a new approach to produce high-quality mAbs against human liver proteins using a combined force of high-throughput mAb production and protein microarrays. After immunizing mice with live cells from human livers, we isolated 54 hybridomas with binding activities to human cells and identified the corresponding antigens for five mAbs via screening on a protein microarray of 1058 unique human liver proteins. Finally, we demonstrated that using the five mAbs we could characterize the expression profiles of their corresponding antigens by using tissue microarrays. Among them, we discovered that eIF1A expressed only in normal liver tissues, not in hepatocellular carcinoma in humans.