Explore the vast range of titles available.

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

The popularity of rAAV vectors in gene therapy are placing a burden on current production systems. To improve the accessibility of these life changing treatments, increases in production yields and a reduction in the cost-of-goods are needed. Transient transfection is the most common way to introduce rAAV-encoding plasmids to producer cells but it suffers from significant drawbacks such as low and inconsistent yields as well as high cost due to its need for plasmid DNA. This study aims to address the low yield of transient transfection-based rAAV production through advanced methods in process characterization. Adherent and suspension cultures of a HEK293T cell line were triple-transfected for rAAV9 production using polyethylenimine (PEI). Samples were taken at various times post-transfection for analysis with bulk and single-cell transcriptomics. It was revealed that 46% of the cells lacked transcripts of genes from at least one plasmid, indicating that a significant proportion of the cells did not have the genes necessary for rAAV9 production. Among the remaining 54% of the cells expressing genes from all three plasmids, only 8% showed high plasmid gene expression. Flow cytometric analysis of intracellular rAAV9 confirmed these results by showing that only ~ 3% of cells contained assembled rAAV9 capsids. Titre analysis by qPCR of the supernatant and lysate of the producer cells indicated an average culture performance of 10 13 vg/L. Analysis of the single-cell transcriptomic data showed that a significant proportion of cells that had high plasmid gene expression were in the S-phase. Trajectory inference highlighted that genes involved in the G2-M phase transition, immune response, and protein unfolding were differentially expressed at the branch point between high and low plasmid expression. This study reveals a significant bottleneck in the transient transfection-based production of rAAV. With less than 5% of cells producing rAAV, significant improvements in titres can be achieved if this fraction can be increased. Moreover, regulation of the cell-cycle, inhibition of the immune response, and alleviating protein misfolding all potentially offer the key to enabling these life changing treatments to reach a wider audience.

Background There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results An n -butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n -butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin–Benson–Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden–Meyerhof–Parnas and a reduced butanol ATP demand. Conclusion These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8 + (10-15 fold) and CD4 + T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients. Current applications of CD40-directed cancer immunotherapy rely on the release of endogenous tumor antigen for T cell priming and activation. Here the authors develop a bispecific CD40 agonistic antibody for modular delivery of antigenic peptides combined with CD40 stimulation and find that this improves T cell expansion and anti-tumor response in mice.’

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1–10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1–10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1–10 in analyses. The pre-maturated GFP 1–10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1–10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1–10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.

An efficient pipeline for mapping antibody epitopes is presented. Combining bacterial surface display of peptide libraries, flow cytometric sorting, and pyrosequencing, the approach is amenable to a high-throughput format and should find future application in whole-proteome studies. We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens. Using a new method for rapid, residue-level epitope determination combining flow cytometry and surface displayed alanine substitution variants the authors determined the epitopes of three new antibodies binding the SARS-CoV-2 Spike protein.