Relation of hypoxia inducible factor 1α and 2α in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival

Hypoxia inducible factors HIF1α and HIF2α are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1α and 2α protein levels, as a consequence of a redox-sensitive stabilization. The HIFαs enter the nucleus, heterodimerize with the HIF1β protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1α and HIF2α expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2α expression with HIF1α expression showed a significant association (P < 0.0001, r = 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF (P < 0.004), PD-ECGF (P < 0.003) and bFGF (P < 0.04) was noted. HIF1α correlated with the expression of bek-bFGF receptor expression (P = 0.01), while HIF2α was associated with intense VEGF/KDR-activated vascularization (P = 0.002). HIF2α protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD (P = 0.006). Analysis of overall survival showed that HIF2α expression was related to poor outcome (P = 0.008), even in the group of patients with low MVD (P = 0.009). HIF1α expression was marginally associated with poor prognosis (P = 0.08). In multivariate analysis HIF2α expression was an independent prognostic indicator (P = 0.006, t-ratio 2.7). We conclude that HIF1α and HIF2α overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting the HIF pathway may prove of importance in the treatment of NSCLC. © 2001 Cancer Research Campaignhttp://www.bjcancer.co

Ki-67 expression and patients survival in lung cancer: systematic review of the literature with meta-analysis

Among new biological markers that could become useful prognostic factors for lung carcinoma, Ki-67 is a nuclear protein involved in cell proliferation regulation. Some studies have suggested an association between Ki-67 and poor survival in lung cancer patients. In order to clarify this point, we have performed a systematic review of the literature, using the methodology already described by our Group, the European Lung Cancer Working Party. In total, 37 studies, including 3983 patients, were found to be eligible. In total, 49% of the patients were considered as having a tumour positive for the expression of Ki-67 according to the authors cutoff. In all, 29 of the studies dealt with non-small-cell lung carcinoma (NSCLC), one with small-cell carcinoma (SCLC), two with carcinoid tumours and five with any histology. In terms of survival results, Ki-67 was a bad prognosis factor for survival in 15 studies while it was not in 22. As there was no statistical difference in quality scores between the significant and nonsignificant studies evaluable for the meta-analysis, we were allowed to aggregate the survival results. The combined hazard ratio for NSCLC, calculated using a random-effects model was 1.56 (95% CI: 1.30-1.87), showing a worse survival when Ki-67 expression is increased. In conclusion, our meta-analysis shows that the expression of Ki-67 is a factor of poor prognosis for survival in NSCLC.Journal ArticleMeta-AnalysisResearch Support, Non-U.S. Gov'tSCOPUS: re.jinfo:eu-repo/semantics/publishe

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli

Abstract With increased accessibility and tissue penetration, smaller antibody formats such as antibody fragments (Fab) and single chain variable fragments (scFv) show potential as effective and low-cost choices to full-length antibodies. These formats derived from the modular architecture of antibodies could prove to be game changers for certain therapeutic and diagnostic applications. Microbial hosts have shown tremendous promise as production hosts for antibody fragment formats. However, low target protein yields coupled with the complexity of protein folding result in production limitations. Here, we report an alternative antibody fragment format ‘FabH3’ designed to overcome some key bottlenecks associated with the folding and production of Fabs. The FabH3 molecule is based on the Fab format with the constant domains replaced by engineered immunoglobulin G1 (IgG1) CH3 domains capable of heterodimerization based on the electrostatic steering approach. We show that this alternative antibody fragment format can be efficiently produced in the cytoplasm of E. coli using the catalyzed disulfide-bond formation system (CyDisCo) in a natively folded state with higher soluble yields than its Fab counterpart and a comparable binding affinity against the target antigen

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli

Abstract With increased accessibility and tissue penetration, smaller antibody formats such as antibody fragments (Fab) and single chain variable fragments (scFv) show potential as effective and low-cost choices to full-length antibodies. These formats derived from the modular architecture of antibodies could prove to be game changers for certain therapeutic and diagnostic applications. Microbial hosts have shown tremendous promise as production hosts for antibody fragment formats. However, low target protein yields coupled with the complexity of protein folding result in production limitations. Here, we report an alternative antibody fragment format ‘FabH3’ designed to overcome some key bottlenecks associated with the folding and production of Fabs. The FabH3 molecule is based on the Fab format with the constant domains replaced by engineered immunoglobulin G1 (IgG₁) CH3 domains capable of heterodimerization based on the electrostatic steering approach. We show that this alternative antibody fragment format can be efficiently produced in the cytoplasm of E. coli using the catalyzed disulfide-bond formation system (CyDisCo) in a natively folded state with higher soluble yields than its Fab counterpart and a comparable binding affinity against the target antigen

Efficient production of Fc fusion proteins in the cytoplasm of Escherichia coli:dissecting and mitigating redox heterogeneity

Abstract Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG₁-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG₁ CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability

Production of neutralizing antibody fragment variants in the cytoplasm of E. coli for rapid screening:SARS-CoV-2 a case study

Abstract Global health challenges such as the coronavirus pandemic warrant the urgent need for a system that allows efficient production of diagnostic and therapeutic interventions. Antibody treatments against SARS-CoV-2 were developed with an unprecedented pace and this enormous progress was achieved mainly through recombinant protein production technologies combined with expeditious screening approaches. A heterologous protein production system that allows efficient soluble production of therapeutic antibody candidates against rapidly evolving variants of deadly pathogens is an important step in preparedness towards future pandemic challenges. Here, we report cost and time-effective soluble production of SARS-CoV-2 receptor binding domain (RBD) variants as well as an array of neutralizing antibody fragments (Fabs) based on Casirivimab and Imdevimab using the CyDisCo system in the cytoplasm of E. coli. We also report variants of the two Fabs with higher binding affinity against SARS-CoV-2 RBD and suggest this cytoplasmic production of disulfide containing antigens and antibodies can be broadly applied towards addressing future global public health threats

So you want to express your protein in Escherichia coli?

Abstract Recombinant proteins have been extensively employed as therapeutics for the treatment of various critical and life-threatening diseases and as industrial enzymes in high-value industrial processes. Advances in genetic engineering and synthetic biology have broadened the horizon of heterologous protein production using multiple expression platforms. Selection of a suitable expression system depends on a variety of factors ranging from the physicochemical properties of the target protein to economic considerations. For more than 40 years, Escherichia coli has been an established organism of choice for protein production. This review aims to provide a stepwise approach for any researcher embarking on the journey of recombinant protein production in E. coli. We present an overview of the challenges associated with heterologous protein expression, fundamental considerations connected to the protein of interest (POI) and designing expression constructs, as well as insights into recently developed technologies that have contributed to this ever-growing field