Preclinical safety and efficacy of a therapeutic antibody that targets SARS-CoV-2 at the sotrovimab face but is escaped by Omicron.

The recurrent emerging of novel viral variants of concern (VOCs) with evasion of preexisting antibody immunity upholds severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case numbers and maintains a persistent demand for updated therapies. We selected the patient-derived antibody CV38-142 based on its potency and breadth against the VOCs Alpha, Beta, Gamma, and Delta for preclinical development into a therapeutic. CV38-142 showed in vivo efficacy in a Syrian hamster VOC infection model after post-exposure and therapeutic application and revealed a favorable safety profile in a human protein library screen and tissue cross-reactivity study. Although CV38-142 targets the same viral surface as sotrovimab, which maintains activity against Omicron, CV38-142 did not neutralize the Omicron lineages BA.1 and BA.2. These results highlight the contingencies of developing antibody therapeutics in the context of antigenic drift and reinforce the need to develop broadly neutralizing variant-proof antibodies against SARS-CoV-2.

A switchable secrete-and-capture system enables efficient selection of Pichia pastoris clones producing high yields of Fab fragments.

Pichia pastoris (syn. Komagataella phaffii) represents a commonly used expression system in the biotech industry. High clonal variation of transformants, however, typically results in a broad range of specific productivities for secreted proteins. To isolate rare clones with exceedingly high product titers, an extensive number of clones need to be screened. In contrast to high-throughput screenings of P. pastoris clones in microtiter plates, secrete-and-capture methodologies have the potential to efficiently isolate high-producer clones among millions of cells through fluorescence-activated cell sorting (FACS). Here, we describe a novel approach for the non-covalent binding of fragment antigen-binding (Fab) proteins to the cell surface for the isolation of high-producing clones. Eight different single-chain variable fragment (scFv)-based capture matrices specific for the constant part of the Fabs were fused to the Saccharomyces cerevisiae alpha-agglutinin (SAG1) anchor protein for surface display in P. pastoris. By encoding the capture matrix on an episomal plasmid harboring inherently unstable autonomously replicating sequences (ARS), this secrete-and-capture system offers a switchable scFv display. Efficient plasmid clearance upon removal of selective pressure enabled the direct use of isolated clones for subsequent Fab production. Flow-sorted clones (n = 276) displaying high amounts of Fabs showed a significant increase in median Fab titers detected in the cell-free supernatant (CFS) compared to unsorted clones (n = 276) when cells were cultivated in microtiter plates (factor in the range of ∼21-49). Fab titers of clones exhibiting the highest product titer observed for each of the two approaches were increased by up to 8-fold for the sorted clone. Improved Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask cultivation of selected candidates (factor in the range of ∼2-3). Hence, the developed display-based selection method proved to be a valuable tool for efficient clone screening in the early stages of our bioprocess development.

Design of a novel switchable antibody display system in Pichia pastoris.

Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomyces cerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichia pastoris (Komagataella phaffii). Establishment of surface display in P. pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments (DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P. pastoris formaldehyde dehydrogenase (FLD1) promoter, S. cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyces lactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S. cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. KEY POINTS: • First report of antibody display in P. pastoris using episomal plasmids. • Identification of genetic elements conferring highest levels of antibody display. • Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.

Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential.

Bacterial non-specific nucleases are ubiquitously distributed and involved in numerous intra- and extracellular processes. Although all nucleases share the basic chemistry for the hydrolysis of phosphodiester bonds in nucleic acid molecules, the catalysis comprises diverse modes of action, which offers great potential for versatile biotechnological applications. A major criterium for their differentiation is substrate specificity. Specific endonucleases are widely used as restriction enzymes in molecular biology approaches, whereas the main applications of non-specific nucleases (NSNs) are the removal of nucleic acids from crude extracts in industrial downstream processing and the prevention of cell clumping in microfabricated channels. In nature, the predominant role of NSNs is the acquisition of nutrient sources such as nucleotides and phosphates. The number of extensively characterized NSNs and available structures is limited. Moreover, their applicability is mostly challenged by the presence of metal chelators that impede the hydrolysis of nucleic acids in a metal ion-dependent manner. However, a few metal ion-independent NSNs that tolerate the presence of metal chelators have been characterized in recent years with none being commercially available to date. The classification and biotechnological potential of bacterial NSNs with a special focus on metal ion-independent nucleases are presented and discussed.Key Points • Bacterial phospholipases (PLD-family) exhibit nucleolytic activity. • Bacterial nucleases of the PLD-family are metal ion-independent. • NSNs can be used in downstream processing approaches.

Characterization of Single Amino Acid Variations in an EDTA-Tolerating Non-specific Nuclease from the Ice-Nucleating Bacterium Pseudomonas syringae.

Non-specific nuclease (NSN) can be applied in industrial downstream processing to remove nucleic acids from crude protein extracts or in cell-sorting systems to degrade nucleic acids derived from lysed cells. PsNuc from the ice-nucleating bacterium Pseudomonas syringae has the ability to decompose double- and single-stranded DNA in linear or circular form and RNA. It is not affected by the presence of metal-ion chelators such as EDTA and tolerates several protease inhibitors and reducing agents. A multiple sequence alignment of PsNuc with closely related enzymes (97-99% identity on the protein level) within the family Pseudomonaceae revealed the presence of only six amino acid residues that are variable in putative NSN from different members of the genus Pseudomonas. Single amino acid variants were produced in recombinant form in Escherichia coli, purified, and characterized. They showed similar activity compared to PsNuc, but a single variant even displayed an improved performance with an activity of > 20,000 U/mg at 35 °C, while amino acid residues S148 and V161 were found to be essential for enzymatic functionality. These results suggest that homologous nucleases from Pseudomonaceae display high activity levels in a metal-ion-independent manner and are therefore of interest for applications in biotechnology.

Comparative analysis of two non-specific nucleases of the phospholipase D family from the plant pathogen competitor bacterium Pantoea agglomerans.

Bacterial non-specific nucleases of the phospholipase D family are widely distributed among the members of the Enterobacteriaceae. Each genome mainly contains a single copy of a gene encoding a phospholipase D family protein. However, two distantly related isozymes (< 40% identity at the protein level) were identified by BLAST-analyses in the plant pathogenic competitor enterobacterium Pantoea agglomerans. The two nucleases PaNuc-1 and PaNuc-2 were produced in Escherichia coli. Identical gene constructs and expression conditions resulted in the production of PaNuc-1 in soluble form, while PaNuc-2 remained insoluble in inclusion bodies. PaNuc-2 was refolded and both proteins were purified by a combination of affinity and ion exchange chromatography. Proteolytic removal of the HIS-tag allowed the characterization of pure and mature tag-less proteins. Enzymatic properties of both isozymes revealed that they are non-specific nucleases, displaying activities against RNA, single- and double-stranded genomic DNA as well as circular plasmids. However, their biochemical activity profiles were clearly different, with PaNuc-1 being optimally active at 70 °C and pH 7.0, while PaNuc-2 was most active at 45 °C and pH 7.0. The enzymes retained > 90% nuclease activity at EDTA concentrations of 4 mM (PaNuc-2) and 20 mM (PaNuc-1), respectively. Different enzymatic properties suggest that the roles of PaNuc-1 and PaNuc-2 differ in the cell and might be the result of functional diversification after an ancient gene duplication event took place. The fact that both enzymes could be easily produced in recombinant form and their tolerance against metal ion chelators in combination with a broad substrate promiscuity might pave the way to versatile biotechnological applications.

A non-specific nucleolytic enzyme and its application potential in EDTA-containing buffer solutions.

OBJECTIVES: Metal-ion independent non-specific nucleases are of high potential for applications in EDTA-containing bioprocessing workflows. RESULTS: A novel extracellular non-specific nuclease EcNuc from the enterobacterium Escherichia coli has been identified. The recombinant gene was expressed and the protein was purified. Maximum activity of the enzyme was detected at 41.7 °C and at an acidic pH of 5.8. EcNuc tolerates EDTA in the reaction buffer at concentrations of up to 20 mM and the activity is not impaired by high concentrations of mono- and divalent metal ions in the absence of EDTA. The viscosity of crude protein extracts after cell lysis in EDTA-containing buffers is reduced when supplemented with EcNuc. CONCLUSION: Proof-of-concept has been demonstrated that a metal-ion independent non-specific nuclease can be applied for removal of nucleic acids in EDTA-containing buffers for the subsequent purification of proteins from crude extracts.

Solubilization and renaturation of biologically active human bone morphogenetic protein-4 from inclusion bodies.

Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli (E. coli) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).

Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 105 U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.