Generation of Endotoxin-Specific Monoclonal Antibodies by Phage and Yeast Display for Capturing Endotoxin.

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules.

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.

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

DLX1008 (brolucizumab), a single-chain anti-VEGF-A antibody fragment with low picomolar affinity, leads to tumor involution in an in vivo model of Kaposi Sarcoma.

Kaposi Sarcoma (KS) is among the most angiogenic cancers in humans and an AIDS-defining condition. KS-associated herpesvirus (KSHV) is necessary for KS development, as is vascular endothelial growth factor (VEGF-A). DLX1008 is a novel anti-VEGF-A antibody single-chain variable fragment (scFv) with low picomolar affinity for VEGF-A. In vivo imaging techniques were used to establish the efficacy of DLX1008 and to establish the mechanism of action; this included non-invasive imaging by ultrasound and optical fluorescence, verified by post-mortem histochemistry. The results showed that DLX1008 was efficacious in a KS mouse model. The NSG mouse xenografts suffered massive internal necrosis or involution, consistent with a lack of blood supply. We found that imaging by ultrasound was superior to external caliper measurements in the validation of the angiogenesis inhibitor DLX1008. Further development of DLX1008 against VEGF-dependent sarcomas is warranted.

Antitumor Activity of DLX1008, an Anti-VEGFA Antibody Fragment with Low Picomolar Affinity, in Human Glioma Models.

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of glioblastoma. Based on the response rate and improved progression-free survival, although not on overall survival, the 149-kDa anti-VEGF-A IgG antibody bevacizumab (Avastin) has been approved in the United States and Japan for recurrent glioblastoma and in Japan for newly diagnosed glioblastoma; however, it is not approved in the EU. Here we characterize the biologic activity of DLX1008, a 26-kDa anti-VEGF-A single-chain antibody fragment that shows 30-fold stronger affinity to human VEGF-A than bevacizumab. The small molecular size of DLX1008 is predicted to result in improved target coverage over bevacizumab. DLX1008 showed superiority to bevacizumab in the inhibition of VEGF-A binding to VEGF receptor (VEGFR) 1 in enzyme-linked immunosorbent assay by a factor of around 10 and comparable efficacy for the inhibition of VEGF-A-stimulated VEGFR2 dimerization. In a tube-formation assay with human cerebral microvascular endothelial cells, DLX1008 was at least as active as bevacizumab. In vivo, DLX1008 delayed growth in a mouse subcutaneous U87 xenograft model (P = 0.0021) and improved survival in a mouse orthotopic U87 xenograft model (P = 0.00026). Given the exceptionally high affinity and small molecular size of DLX1008, these data warrant further clinical development of DLX1008 as an antiangiogenic agent in glioblastoma.

Noninvasive measurement of cell volume changes by negative staining.

To maintain the intracellular concentration of ions and small molecules on osmotic challenges, nature has developed highly sophisticated transport systems for regulating water and ion content. An ideal measurement technique for volume changes of cells during osmotic challenges has to fulfil two requirements: it has to be osmotically inert, and it should allow online monitoring of cell volume changes. Here, a simple fluorescence microscopy-based approach is presented. Using fluorescein as a negative stain, it is possible to monitor cell volume changes without affecting the functionality of cell membranes and cell osmolarity. Measurement of Madine-Darby canine kidney (MDCK) cells after hypo- and hyperosmotic challenges reveals the main advantages of this approach: besides providing precise and reproducible quantitative data on reversible cell volume changes, the viability of the cells can be assessed directly by the appearance of stain in the cytoplasm. This becomes evident especially after hypo-osmotic challenge of glutaraldehyde-treated cells, which become leaky after fixation, followed by a massive volume change. This new approach represents a very sensitive measurement technique for cell volume changes resulting from water or ion flux, and thus seems to be an ideal tool for studying cell volume regulatory processes.