Optimizing expression of Nanobody® molecules in Pichia pastoris through co-expression of auxiliary proteins under methanol and methanol-free conditions.

BACKGROUND: Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi (Pichia pastoris) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers. RESULTS: Here we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system. CONCLUSION: Our findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris. The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.

GlycoVHH: optimal sites for introducing N-glycans on the camelid VHH antibody scaffold and use for macrophage delivery.

As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man5GlcNAc2-glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man5GlcNAc2 N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-based targeting approach for lung macrophage endolysosomal system delivery. The set of optimal artificial VHH N-glycosylation sites identified in this study can serve as a blueprint for targeted glyco-engineering of other VHHs, enabling site-specific functionalization through the rapidly expanding toolbox of synthetic glycobiology.

Knockout of RSN1, TVP18 or CSC1-2 causes perturbation of Golgi cisternae in Pichia pastoris.

The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5 GlcNAc2 modified with a Glcα-1,2-Manß-1,2-Manß-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5 GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

Yeast-secreted, dried and food-admixed monomeric IgA prevents gastrointestinal infection in a piglet model.

Oral antibodies that interfere with gastrointestinal targets and can be manufactured at scale are needed. Here we show that a single-gene-encoded monomeric immunoglobulin A (IgA)-like antibody, composed of camelid variable single domain antibodies (VHH) fused to IgA Fc (mVHH-IgA), prevents infection by enterotoxigenic Escherichia coli (F4-ETEC) in piglets. The mVHH-IgA can be produced in soybean seeds or secreted from the yeast Pichia pastoris, freeze- or spray-dried and orally delivered within food.

A simple colorimetric assay for measuring fructosamine 3 kinase activity.

BACKGROUND: Fructosamine 3 kinase (FN3K) is a deglycating enzyme, which may play a key role in reducing diabetes-induced organ damage by removing bound glucose from glycated proteins. We wanted to develop a simple colorimetric method for assaying FN3K activity in human body fluids. METHODS: Glycated bovine serum albumin (BSA) was obtained by glycation with a 10% glucose solution at 37 °C. After 72 h, glycated BSA was dialyzed against phosphate buffered saline (0.1 mol/L, pH 7.4). The dialyzed solution (containing ±1000 µmol/L fructosamine) was used as an FN3K substrate. In the assay, 300 µL of substrate was incubated with 50 µL of serum and 100 µL of MgCl2 (0.7 mmol/L)/ATP (3.2 mmol/L). The fructosamine concentration was determined at the start and after incubation (120 min, 25 °C). The decrease in fructosamine concentration over time is a measure for the FN3K activity (1 U corresponding to 1 µmol/min). Concomitantly, the FN3K SNP rs1056534 and the ferroportin SNP rs1156350 were genotyped. RESULTS: Within-assay CV was 6.0%. Reference values for FN3K activity in serum were 14.2±1.6 U/L (n=143). Reference values for FN3K were neither age- nor sex-dependent. The various FN3K SNP rs1056534 genotypes showed no significant differences in serum FN3K activity. In diabetics (n=191), values (14.0±2.2 U/L) were comparable to those of the controls. FN3K activity in erythrocytes was significantly higher (170.3±7.6 U/L). The intra-erythrocytic FN3K activity makes the results prone to hemolysis. FN3K activity depended on the ferroportin Q248H genotypes, with the highest value for the wild type genotype. Neither transferrin saturation nor ferritin were confounders for the FN3K activity. FN3K activity was significantly (p<0.0001) correlated with HbA1c values, although the correlation between FN3K and HbA1c was weak. CONCLUSIONS: The simple colorimetric method allows determining FN3K activity in human serum. The assay may be useful for studying the impact of deglycation processes in diabetes mellitus.

Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology.

Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.

Comparison of VHH-Fc antibody production in Arabidopsis thaliana, Nicotiana benthamiana and Pichia pastoris.

VHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH-Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH-Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH-Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N-glycans were expected for KDEL-tagged VHH-Fcs, several VHH-Fcs with an intact KDEL-tag carried complex-type N-glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH-Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA.

Engineering yeast for producing human glycoproteins: where are we now?

Yeast has advanced as an alternative for mammalian cell culture for the production of recombinant therapeutic glycoproteins. Engineered yeast strains not only allow to mimic the human N-glycosylation pathway but also specific types of human O-glycosylation. This is of great value for therapeutic protein production and indispensable to determine the structure-function relationships of glycans on recombinant proteins. However, as the technology matures, some limitations have come up that may hamper biomedical applications and must be considered to exploit the full potential of the unprecedented glycan homogeneity obtained on relevant biopharmaceuticals. In this special report, we focus on the recent developments in N- and O-glycosylation engineering in yeasts of industrial importance, to produce recombinant therapeutics with customized glycans.

Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor α-induced necroptosis.

Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis.

Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c(+) MHC-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.