Development an engineered human enzyme for anti-vasculature immunotherapy

The tumour vasculature is an attractive target for cancer therapy because tumour growth and spread depend on these structures for oxygen and nutrient supply. Anti-vasculature therapy aims to cause rapid and selective shutdown of the established tumour vasculature, leading to secondary tumour cell death. In this study, we propose to employ an Antibody Guided Enzyme Nitrile Therapy (AGENT) approach to selectively target extra-domain B fibronectin (B-FN), which is abundantly expressed in tumour vasculature. A recombinant fusion protein, composed of the humanised BC-1 single chain fragment (huBC1 scFv) fused with a re-engineered human cytosolic β–glucosidase (rehCβG) enzyme, was investigated to target this antigen and generate cytotoxic cyanide molecules specifically within tumour sites. Naturally-occurring human β-glucosidase (hCβG) cannot hydrolyse the cyanogenic glycoside linamarin, to any significant level, but was chosen as a suitable candidate for site-directed mutagenesis due to its homology with a native cyanogenic plant enzyme, linamarase. Both huBC-1 scFv and hCβG protein were expressed in Escherichia coli and purified by affinity and size exclusion chromatography. In vitro characterisation of the huBC-1 scFv showed that it retained antigen-binding specificity of the parental antibody, Immunoglobulin-G HuBC-1 towards the same antigen. Kinetic analysis of the wild type and a panel of mutant hCβGs showed that their catalytic activity was retained. Interestingly, one of the mutants, V168A hCβG, was shown to have increased cyanogenic activity towards linamarin. The Km was improved from 2.23 ± 0.03 mM to 0.79 ± 0.02 mM and the kcat from 0.21 ± 0.01 min-1 to 5.49 ± 0.01 min-1. Following this discovery, construction and expression of the fusion protein, scFv-rehCβG (wild-type and V168A) was carried out using different expression systems to compare the best strategy in constructing the fusion protein. The huBC-1 scFv-hCβG fusion proteins were expressed cytosolically but as insoluble aggregates in inclusion bodies. Recovery of the fusion protein was made through refolding of the protein. Using the dilution method, the protein was solubilised and refolded, however it was functionally inactive. Another refolding method via dialysis experienced loss of the fusion protein. Final production of a cyanogenic scFv-hCβG requires further investigation by optimising the protein refolding protocol which could lead to further studies of AGENT

A gain of function p53 gene mutant promotes growth suppression in human liver cancer cells

Primary liver cancer is one of the most common cancer in the world with highest cancer mortality rate. The most common type of primary liver cancer is hepatocellular carcinoma (HCC). There are many risk factors for liver cancer and currently available treatments for HCC are largely inadequate. Gene mutation and dysfunction of p53 are common and is recognized as an important molecular event in hepatocarcinogenesis. Therefore, replacement of the aberrant p53 gene is an attractive approach in the treatment of HCC providing an alternative treatment for primary HCC. In this study, we assessed whether the transfection with wild-type p53 gene is able to restore the pro-apoptotic effects and evaluate the feasibility of gene therapy in fixing a faulty p53 molecule. We established a non-viral cationic lipid-based p53 gene delivery into two human HCC cell lines namely HLF and PLC/PRF/5 cells. Both cell lines have mutations in the p53 gene. We compared the results with the normal liver cell line, WRL68, that constitutively expresses the wild-type p53 gene. In this study, the introduction of wild-type p53 gene into HLF and PLC/PRF/5 cells resulted in an increased of p53 gene expression, protein expression and cells growth inhibition shown in MTS reduction cell viability assay, FITC-Annexin V and PI apoptosis assay, western blot and caspase activity assay. In summary, the study provides a promising therapeutic approach for p53 gene delivery into HCC patients. The p53 gene delivery can be instituted together with chemotherapy as a combination treatment to induce apoptosis

The HLA DPB1*02:01:02 and DQB1*05:02:01 alleles as possible risk factors for colorectal carcinoma in the Malaysian population

Many studies have shown that the immune response highly depends on the inheritance of specific HLA genes in promoting the generation of T cells for the elimination of pathogens. Loss or alteration of HLA antigen expression in tumor cells has been observed in a variety of human malignancies leading to immune escape or immune resistance. We investigated whether the inheritance of certain alleles of HLA class II genes confers susceptibility or resistance towards the development of colorectal carcinoma (CRC). Molecular typing of HLA DRB1, DQB1 and DPB1 alleles in 42 patients diagnosed with CRC and 50 ethnically matched healthy controls using the PCR-sequence based typing (PCR-SBT) was conducted. The HLA DPB1*02:01:02 was significantly higher in CRC patients (38.1%, p=0.0189) compared to healthy controls (16%). Also, HLA DQB1*05:02:01 was present in 28.6% of CRC patients but only 10% of healthy controls (p=0.0278). The odds ratios for HLA DPB1*02:01:02 and HLA DQB1*05:02:01were 3.23 and 3.60, respectively. There were no significant association observed for the DRB1 allele with CRC. Our study suggests that the HLA DPB1*02:01:02 and HLA DQB1*05:02:01 alleles may confer a higher risk for CRC

Volatolomics combined terahertz time - domain spectral analyses of colon cancer in vitro

Terahertz time-domain spectroscopy (THz-TDS) offers a great advantage for the analysis of biological samples. In this study, we combined THz-TDS with volatolomics analysis and analyzed 2 colon cell lines via in vitro settings. The release of volatile organic compounds (VOCs) was measured from the normal colon (CCD112CoN) and cancer colon (COLO320DM) cell lines which were grown in sealed flasks. Data validation were carried out with principal component analysis (PCA) and partial least square (PLS) scores while the chemometric analyses were performed using Camo Unscrambler X software. In-depth THz-TDS spectral analysis of the cancer colon (COLO320DM) cell line shows significant traces of benzamide gas when validated using gas chromatography-mass selective detection (GC-MSD) system. This preliminary data shows the potential use of identification and quantification of benzamide compound in the cancer colon cells and this could provide useful insight towards cancer drug design and therapy

Molecular Signatures of Human Regulatory T Cells in Colorectal Cancer and Polyps

Regulatory T cells (Tregs), a subset of CD4+ or CD8+ T cells, play a pivotal role in regulating immune homeostasis. An increase in Tregs was reported in many tumors to be associated with immune suppression and evasion in cancer patients. Despite the importance of Tregs, the molecular signatures that contributed to their pathophysiological relevance remain poorly understood and controversial. In this study, we explored the gene expression profiles in Tregs derived from patients with colorectal cancer [colorectal carcinoma (CRC), n = 15], colorectal polyps (P, n = 15), and in healthy volunteers (N, n = 15). Tregs were analyzed using CD4+CD25+CD127lowFoxP3+ antibody markers. Gene expression profiling analysis leads to the identification of 61 and 66 immune-related genes in Tregs derived from CRC and P patients, respectively, but not in N-derived Treg samples. Of these, 30 genes were differentially expressed both in CRC- and P-derived Tregs when compared to N-derived Tregs. Most of the identified genes were involved in cytokine/chemokine mediators of inflammation, chemokine receptor, lymphocyte activation, and T cell receptor (TCR) signaling pathways. This study highlights some of the molecular signatures that may affect Tregs’ expansion and possible suppression of function in cancer development. Our findings may provide a better understanding of the immunomodulatory nature of Tregs and could, therefore, open up new avenues in immunotherapy