Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Impact of host cell line choice on glycan profile.

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.

Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload.

UNLABELLED: To identify polymorphisms associated with variability of iron overload severity in HFE-associated hemochromatosis, we performed exome sequencing of DNA from 35 male HFE C282Y homozygotes with either markedly increased iron stores (n = 22; cases) or with normal or mildly increased iron stores (n = 13; controls). The 35 participants, residents of the United States, Canada, and Australia, reported no or light alcohol consumption. Sequencing data included 82,068 single-nucleotide variants, and 10,337 genes were tested for a difference between cases and controls. A variant in the GNPAT gene showed the most significant association with severe iron overload (P = 3 × 10(-6) ; P = 0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of twenty-two participants with severe iron overload had glyceronephosphate O-acyltransferase (GNPAT) polymorphism p.D519G (rs11558492; 15 heterozygotes, one homozygote). No control participant had this polymorphism. To examine functional consequences of GNPAT deficiency, we performed small interfering RNA-based knockdown of GNPAT in the human liver-derived cell line, HepG2/C3A. This knockdown resulted in a >17-fold decrease in expression of the messenger RNA encoding the iron-regulatory hormone, hepcidin. CONCLUSION: GNPAT p.D519G is associated with a high-iron phenotype in HFE C282Y homozygotes and may participate in hepcidin regulation.

Bidirectional Ventricular Tachycardia in Acute Multivessel Myocardial Infarction.

Bidirectional ventricular tachycardia (BVT) is a rare and unusual ventricular dysrhythmia that is characterized by a beat-to-beat alternation of the QRS axis. This can sometimes manifest as alternating left and right bundle branch blocks. To the best of our knowledge, there are two previous cases of BVT in the setting of type I myocardial infarction. Our case would be the third and showed a subtle change in the anterior-posterior axis that can be seen in lead V2. The coronary angiography of our patient demonstrated severe multivessel coronary artery disease with complete total occlusion of the proximal dominant right coronary artery, 100% in-stent restenosis of the ostial left circumflex, 40% stenosis of left main, and 90% stenosis of mid left anterior descending artery (LAD). The BVT resolved after two amiodarone boluses followed by a drip. We attempted to transition to oral mexiletine, however, the patient was unable to tolerate the medication due to intractable nausea and vomiting. The patient subsequently underwent high risk coronary artery bypass graft surgery with no further episodes of BVT following revascularization and was discharged after six weeks of hospitalization. Although rare, type I myocardial infarction is an important differential diagnosis of BVT.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

A human expression system based on HEK293 for the stable production of recombinant erythropoietin.

Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

Endofin, a novel BMP-SMAD regulator of the iron-regulatory hormone, hepcidin.

BMP-SMAD signalling plays a crucial role in numerous biological processes including embryonic development and iron homeostasis. Dysregulation of the iron-regulatory hormone hepcidin is associated with many clinical iron-related disorders. We hypothesised that molecules which mediate BMP-SMAD signalling play important roles in the regulation of iron homeostasis and variants in these proteins may be potential genetic modifiers of iron-related diseases. We examined the role of endofin, a SMAD anchor, and show that knockdown of endofin in liver cells inhibits basal and BMP-induced hepcidin expression along with other BMP-regulated genes, ID1 and SMAD7. We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling. Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression. In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway, and the regulation of hepcidin expression and iron homeostasis. This study further defines the molecular network involved in iron regulation and provides potential targets for the treatment of iron-related disorders.

Mitotic Golgi vesiculation involves mechanisms independent of Ser25 phosphorylation of GM130.

During mitosis, the Golgi undergoes two sequential fragmentation steps to break from ribbon to individual stacks, then from stacks to vesicles. While the mechanism that regulates the first step has been studied, it remains obscure how the second vesiculation step is regulated. It has been suggested that Cdk1-dependent phosphorylation of the cis-Golgi matrix protein GM130 regulates the second step. Here we have tested if phorphorylation of GM130 by Cdk1 is required for Golgi vesiculation and mitotic progression. Inhibition of Cdk1 activity caused a failure of Golgi vesiculation and defects in chromosome congression/segregation. Expression of non-phosphorylatable mutant of GM130 (GM130S25A) in cells depleted of endogenous GM130 caused no apparent defects in Golgi vesiculation and mitotic progression. Similarly, no apparent defects in Golgi vesiculation and mitotic progression were observed when GM130S25A was expressed in GM130-deficient CHO cells. Our observations suggest that while Cdk1 based phosphorylation is essential for mitotic Golgi vesiculation, mammalian cells could possess redundant, S25 phosphorylation of GM130 independent pathways that ensure Golgi vesiculation and mitotic progression.