Optimization of Culture Media and Feeding Strategy for High Titer Production of an Adenoviral Vector in HEK 293 Fed-Batch Culture.

Adenoviruses are efficient and safe vectors for delivering target antigens and adenovirus-based vaccines have been used against a wide variety of pathogens, including tuberculosis and COVID-19. Cost-effective and scalable biomanufacturing processes are critical for the commercialization of adenovirus-vectored vaccines. Adenoviral vectors are commonly produced through the infection of batch cultures at low cell density cultures, mostly because infections at high cell densities result in reduced cell-specific virus productivity and does not improve volumetric productivity.

Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus-1.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1).

Repressing expression of difficult-to-express recombinant proteins during the selection process increases productivity of CHO stable pools.

More than half of licensed therapeutic recombinant proteins (r-proteins) are manufactured using constitutively-expressing, stably-transfected Chinese hamster ovary (CHO) clones. While constitutive CHO expression systems have proven their efficacy for the manufacturing of monoclonal antibodies, many next-generation therapeutics such as cytokines and bispecific antibodies as well as biological targets such as ectodomains of transmembrane receptors remain intrinsically challenging to produce. Herein, we exploited a cumate-inducible CHO platform allowing reduced expression of various classes of r-proteins during selection of stable pools.

The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese Hamster ovary cells: retrospective analysis and future directions.

Chinese hamster ovary (CHO) cell-based platforms are the most widely used for the biomanufacturing of complex therapeutic proteins, such as monoclonal antibodies (mAbs). The development of high-producing clones that are stable and amenable to large-scale cultures is essential to advance a molecule toward clinical evaluation. Nevertheless, the generation of such clones generally relies on random integration of an expression plasmid encoding the therapeutic protein gene into the host genome.

Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells.

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production.

Putative chemopreventive molecules can increase Nrf2-regulated cell defense in some human cancer cell lines, resulting in resistance to common cytotoxic therapies.

Nrf2 is a key transcription factor, which induces a cytoprotective gene array. Nrf2 is regulated at the posttranslational level through proteasomal degradation through an interaction with the adapter protein Keap1. High levels of Nrf2, resulting from a loss of function mutation in Keap1, were reported in chemoresistant non-small cell lung cancer.

Cul3 overexpression depletes Nrf2 in breast cancer and is associated with sensitivity to carcinogens, to oxidative stress, and to chemotherapy.

Nrf2 is the key transcription factor for cytoprotective gene programs. Nrf2 is normally maintained at very low concentrations by proteasomal degradation, through its interaction with the adapter protein Keap1 and the Cul3 E3 ligase. Increased Nrf2 concentration resulting from loss of function Keap1 mutations has been described in chemoresistant non-small cell lung cancer.

The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

Background: The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms.

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells.

Background: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity.

The bioactivity and receptor affinity of recombinant tagged EGF designed for tissue engineering applications is defined by the nature and position of the tags.

For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure.

A sensitive flow cytometry-based nucleotide excision repair assay unexpectedly reveals that mitogen-activated protein kinase signaling does not regulate the removal of UV-induced DNA damage in human cells.

In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest.

Novel use of the fluorescent dye 5-(and-6)-chloromethyl SNARF-1 acetate for the measurement of intracellular glutathione in leukemic cells and primary lymphocytes.

Glutathione (GSH) plays an important role in protecting cells against injury, particularly during oxidative stress. Alterations in GSH metabolism are becoming the focus of attention in many diseases such as cancer, neurodegeneration, and AIDS. As such, a rapid assessment of GSH levels in a clinical setting is of increasing importance.

XRCC3 depletion induces spontaneous DNA breaks and p53-dependent cell death.

In vertebrate cells, Xrcc3 initiates the repair of exogenous induced-DNA breaks during S and G(2)/M phases of the cell cycle by homologous recombination. However, much less is known of the role of Xrcc3 in the response to spontaneous DNA breaks. Using a siRNA approach, we show that depletion of XRCC3 inhibits the proliferation of MCF7 breast cancer cells.

Chlorambucil cytotoxicity in malignant B lymphocytes is synergistically increased by 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase.

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways.

FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency.

Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis.

Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan.

Repression of DNA repair mechanisms in IRF-4-expressing and HTLV-I-infected T lymphocytes.

Human T cell leukemia virus (HTLV) is the causative agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes in which interferon regulatory factor-4 (IRF-4) becomes constitutively expressed, concomitant with major alterations in host gene expression. When constitutively expressed in uninfected T lymphocytes, IRF-4 caused reduced expression of critical DNA repair genes, including Rad51, XRCC1, Ung1, RPA, and proliferative cell nuclear antigen (PCNA), a transcriptional phenotype with striking similarities to the profile observed in HTLV-infected T lymphocytes. Concomitant with the inhibition of gene expression and defects in the DNA repair pathways, increased sensitivity of T lymphocytes to various genotoxic stresses that challenged all major DNA repair pathways were detected.

Cdc25A-inhibitory properties and antineoplastic activity of bisperoxovanadium analogues.

Bisperoxovanadium (bpV) compounds are irreversible protein tyrosine phosphatase (PTP) inhibitors with a spectrum of activity distinct from that of vanadium salts. We studied the efficacy of a panel of bpVs as antineoplastic agents in vitro and in vivo with a view to investigating phosphatases as potential antineoplastic targets. The Cdc25A dual-specificity phosphatase is an oncoprotein required for progression through G(1)-S.

The initiation of UV-induced G(1) arrest in human cells is independent of the p53/p21/pRb pathway but can be attenuated through expression of the HPV E7 oncoprotein.

It is well established that the initiation of G(1) arrest in cultured cells exposed to ionizing radiation (IR) is fully dependent upon the p53/p21waf1/pRb signaling cascade. However, the extent to which this pathway regulates G(1) arrest following exposure to UV is less clear. Here we demonstrate that primary human fibroblasts from either skin or lung, in which p53 has been functionally inactivated through expression of the human papillomavirus E6 oncoprotein, each undergoes a prolonged G(1) arrest upon UV irradiation.