The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.

Targeting GD2-Positive Tumor Cells by Pegylated scFv Fragment-Drug Conjugates Carrying Maytansinoids DM1 and DM4.

Oligomerization of antibody fragments via modification with polyethylene glycol (pegylation) may alter their function and properties, leading to a multivalent interaction of the resulting constructs with the target antigen. In a recent study, we generated pegylated monomers and multimers of scFv fragments of GD2-specific antibodies using maleimide-thiol chemistry. Multimerization enhanced the antigen-binding properties and demonstrated a more efficient tumor uptake in a syngeneic GD2-positive mouse cancer model compared to monomeric antibody fragments, thereby providing a rationale for improving the therapeutic characteristics of GD2-specific antibody fragments. In this work, we obtained pegylated conjugates of scFv fragments of GD2-specific antibodies with maytansinoids DM1 or DM4 using tetravalent PEG-maleimide (PEG4). The protein products from the two-stage thiol-maleimide reaction resolved by gel electrophoresis indicated that pegylated scFv fragments constituted the predominant part of the protein bands, and most of the scFv formed pegylated monomers and dimers. The conjugates retained the ability to bind ganglioside GD2 comparable to that of the parental scFv fragment and to specifically interact with GD2-positive cells. Both induced significant inhibitory effects in the GD2-positive B78-D14 cell line, in contrast to the GD2-negative B16 cell line. The decrease in the B78-D14 cell viability when treated with scFv-PEG4-DM4 was more prominent than that for scFv-PEG4-DM1, and was characterized by a twofold lower half-maximal inhibitory concentration (IC50). Unlike the parental scFv fragment, the product of scFv and PEG4 conjugation (scFv-PEG4), consisting predominantly of pegylated scFv multimers and monomers, induced direct cell death in the GD2-positive B78-D14 cells. However, the potency of scFv-PEG4 was low in the selected concentration range, thus demonstrating that the cytotoxic effect of DM1 and DM4 within the antibody fragment-drug conjugates was primary. The suggested approach may contribute to development of novel configurations of antibody fragment-drug conjugates for cancer treatment.

Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells.

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

Therapeutic efficacy of antibody-drug conjugates targeting GD2-positive tumors.

BACKGROUND: Both ganglioside GD2-targeted immunotherapy and antibody-drug conjugates (ADCs) have demonstrated clinical success as solid tumor therapies in recent years, yet no research has been carried out to develop anti-GD2 ADCs against solid tumors. This is the first study to analyze cytotoxic activity of clinically relevant anti-GD2 ADCs in a wide panel of cell lines with varying GD2 expression and their effects in mouse models of GD2-positive solid cancer. METHODS: Anti-GD2 ADCs were generated based on the GD2-specific antibody ch14.18 approved for the treatment of neuroblastoma and commonly used drugs monomethyl auristatin E (MMAE) or F (MMAF), conjugated via a cleavable linker by thiol-maleimide chemistry. The antibody was produced in a mammalian expression system, and its specific binding to GD2 was analyzed. Antigen-binding properties and biodistribution of the ADCs in mice were studied in comparison with the parent antibody. Cytotoxic effects of the ADCs were evaluated in a wide panel of GD2-positive and GD2-negative tumor cell lines of neuroblastoma, glioma, sarcoma, melanoma, and breast cancer. Their antitumor effects were studied in the B78-D14 melanoma and EL-4 lymphoma syngeneic mouse models. RESULTS: The ch14.18-MMAE and ch14.18-MMAF ADCs retained antigen-binding properties of the parent antibody. Direct dependence of the cytotoxic effect on the level of GD2 expression was observed in cell lines of different origin for both ADCs, with IC50 below 1 nM for the cells with high GD2 expression and no cytotoxic effect for GD2-negative cells. Within the analyzed cell lines, ch14.18-MMAF was more effective in the cells overexpressing GD2, while ch14.18-MMAE had more prominent activity in the cells expressing low GD2 levels. The ADCs had a similar biodistribution profile in the B78-D14 melanoma model compared with the parent antibody, reaching 7.7% ID/g in the tumor at 48 hours postinjection. The average tumor size in groups treated with ch14.18-MMAE or ch14.18-MMAF was 2.6 times and 3.8 times smaller, respectively, compared with the control group. Antitumor effects of the anti-GD2 ADCs were also confirmed in the EL-4 lymphoma model. CONCLUSION: These findings validate the potential of ADCs targeting ganglioside GD2 in treating multiple GD2-expressing solid tumors.

Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF.

While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5' leader of POLG messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5' of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5' leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

CT-IGFBP-4 as a novel prognostic biomarker in acute heart failure.

AIMS: Insulin-like growth factor binding protein-4 (IGFBP-4) fragments have been shown to predict the risk of major adverse cardiovascular events, including segment-elevation myocardial infarction, in patients with acute coronary syndrome. We evaluated the prognostic value of the carboxy-terminal fragment of IGFBP-4 (CT-IGFBP-4) for all-cause mortality in emergency room patients with acute heart failure (AHF). METHODS AND RESULTS: CT-IGFBP-4, N-terminal pro brain natriuretic peptide (NT-proBNP), and C-reactive protein (CRP) were measured at admission from the lithium-heparin plasma of 156 patients with AHF. All-cause mortality was recorded for 1 year. Receiver operator characteristic (ROC) curves, Kaplan-Meier, and Cox proportional hazard ratio analyses were performed to evaluate the prognostic value of the various clinical variables, CT-IGFBP-4, NT-proBNP, CRP, and their combinations. During 1 year of follow-up, 52 (33.3%) patients died. CT-IGFBP-4 only weakly correlated with NT-proBNP (Pearson correlation coefficient r = 0.16, P = 0.044) and did not correlate with CRP (r = 0.08, P = 0.35), emphasizing the different nature of these biomarkers. The receiver operator characteristic area under the curve (ROC AUC) of CT-IGFBP-4 for the prediction of all-cause mortality (0.727) was significantly higher than that of NT-proBNP (0.680, P = 0.045) and CRP (0.669, P = 0.016). The combination of CT-IGFBP-4, NT-proBNP, and CRP predicted mortality significantly better (ROC AUC = 0.788) than any of the biomarkers alone (P < 0.01 for all). The addition of CT-IGFBP-4 to a clinical prediction model that included age, gender, systolic blood pressure, creatinine, and sodium levels, as well as the history of previous heart failure, coronary artery disease, and hypertension significantly improved the mortality risk prediction (ROC AUC 0.774 vs. 0.699, P = 0.025). Cox hazard analysis indicated that elevated CT-IGFBP-4 was independently associated with 1 year mortality (hazard ratio 3.26, P = 0.0008) after adjustment for age, gender, history of previous heart failure, coronary artery disease, hypertension, chronic kidney failure, history of diabetes, heart rate, haemoglobin, plasma sodium, NT-proBNP, CRP, cystatin C, and elevated cardiac troponin I or T. Patients with increased levels of either two or three of the biomarkers CT-IGFBP-4, NT-proBNP, and CRP had significantly higher mortality risk (adjusted hazard ratio 10.04, P < 0.0001) than patients with increased levels of one or none of the biomarkers. CONCLUSIONS: CT-IGFBP-4 was independently associated with all-cause mortality in patients with AHF. Compared with single biomarkers, the combination of CT-IGFBP-4, NT-proBNP, and CRP improved the prediction of all-cause mortality in patients with AHF.

Optimized dual assay for the transgenes selection and screening in CHO cell line development for recombinant protein production.

OBJECTIVE: To develop a simple robust methodology of screening multiple CHO cell clones secreting recombinant proteins to assess their specific productivity. RESULTS: We developed a dual assay based on immunoassay measurements of a recombinant protein expression combined with staining of viable cells with resazurin. Following this approach, colonies can be simultaneously assessed for cell growth rate and for production of a recombinant protein. Combination of these two assays enables to estimate productivity of a recombinant protein per cell from the very early stages of a cell line development process (CLD) and exclude poor producers from further steps. Comparison of the dual assay with a standard CLD protocol followed by only analysis of protein expression level showed at least 10-20% increase in the amount of clones that can be included into pool of high-producers at early stages. This shortens duration of a typical CLD scheme from 23 to 19 weeks. CONCLUSIONS: Our method: (i) allows to include into workflow clones that demonstrate slow growth during single cell cloning but producing high amounts of a target protein, which otherwise would be lost in standard protocols of cells screening; (ii) can be applied for testing of DNA vectors for transfection and protein production; (iii) can be used for monitoring the heterogeneity of cell population and analysis of stable pools productivity.

Glycosylated and non-glycosylated NT-IGFBP-4 in circulation of acute coronary syndrome patients.

BACKGROUND: N-terminal and C-terminal proteolytic fragments of IGF binding protein 4 (NT-IGFBP-4 and CT-IGFBP-4) were recently shown to predict adverse cardiac events in acute coronary syndrome (ACS) patients. NT-IGFBP-4 and CT-IGFBP-4 are products of the pregnancy-associated plasma protein-A (PAPP-A)-mediated cleavage of IGFBP-4. It has been demonstrated that circulating IGFBP-4 is partially glycosylated in its N-terminal region, although the influence of this glycosylation on PAPP-A-mediated proteolysis and the ratio of glycosylated/non-glycosylated IGFBP-4 fragments in human blood remain unrevealed. The aims of this study were to investigate i) the presence of glycosylated NT-IGFBP-4 in the circulation, ii) the influence of the glycosylation of IGFBP-4 on its susceptibility to PAPP-A-mediated cleavage, and iii) the influence of glycosylation on NT-IGFBP-4 immunodetection. METHODS: Affinity purification was used for the extraction of IGFBP-4 and NT-IGFBP-4 from plasma samples. Purified proteins were quantified by Western blotting and specific sandwich immunoassays, while molecular masses were determined using mass spectrometry. RESULTS: Glycosylated NT-IGFBP-4 was identified in the blood of ACS patients. The fraction of glycosylated NT-IGFBP-4 in individual plasma samples was 9.8%-23.5% of the total levels of NT-IGFBP-4. PAPP-A-mediated proteolysis of glycosylated IGFBP-4 was 3-4 times less efficient (p < 0.001) than proteolysis of non-glycosylated protein. A sandwich fluoroimmunoassay that was designed for quantitative NT-IGFBP-4 measurements recognized both protein forms with the same efficiency. CONCLUSIONS: Although glycosylation suppresses PAPP-A-mediated IGFBP-4 cleavage, a considerable amount of glycosylated NT-IGFBP-4 is present in blood. Glycosylation does not influence NT-IGFBP-4 measurements using a specific sandwich immunoassay.

Cell-type-restricted anti-cytokine therapy: TNF inhibition from one pathogenic source.

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)--a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.

Jedi--a novel transmembrane protein expressed in early hematopoietic cells.

Self-renewal and differentiation of hematopoietic stem and progenitor cells are defined by the ensembles of genes expressed by these cells. Here we report identification of a novel gene named Jedi, which is expressed predominantly in short- and long-term repopulating stem cells when compared to more mature bone marrow progenitors. Jedi mRNA encodes a transmembrane protein that contains multiple EGF-like repeats. Jedi and two earlier reported proteins, MEGF10 and MEGF11, share a substantial homology and are likely to represent a novel protein family. Studies of the potential role of Jedi in hematopoietic regulation demonstrated that the retrovirally mediated expression of Jedi in bone marrow cells decreased the number of myeloid progenitors in in vitro clonogenic assays. In addition, expression of Jedi in NIH 3T3 fibroblasts resulted in a decreased number of late and early myeloid progenitors in the non-adherent co-cultured bone marrow cells. Jedi shares a number of structural features with the Jagged/Serrate/Delta family of Notch ligands, and our experiments indicate that the extracellular domain of Jedi, similar to the corresponding domain of Jagged1, inhibits Notch signaling. On the basis of obtained results, we suggest that Jedi is involved in the fine regulation of the early stages of hematopoietic differentiation, presumably through the Notch signaling pathway.