Juliana Parsons

Production of biologically active recombinant human factor H in Physcomitrella

The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma.

Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants

Protein therapeutics represent one of the most increasing areas in the pharmaceutical industry. Plants gain acceptance as attractive alternatives for high-quality and economical protein production. However, as the majority of biopharmaceuticals are glycoproteins, plant-specific N-glycosylation has to be taken into consideration. In Physcomitrella patens (moss), glyco-engineering is an applicable tool, and the removal of immunogenic core xylose and fucose residues was realized before. Here, we present the identification of the enzymes that are responsible for terminal glycosylation (α1,4 fucosylation and β1,3 galactosylation) on complex-type N-glycans in moss. The terminal trisaccharide consisting of α1,4 fucose and β1,3 galactose linked to N-acetylglucosamine forms the so-called Lewis A epitope. This epitope is rare on moss wild-type proteins, but was shown to be enriched on complex-type N-glycans of moss-produced recombinant human erythropoietin, while unknown from the native human protein. Via gene targeting of moss galactosyltransferase and fucosyltransferase genes, we identified the gene responsible for terminal glycosylation and were able to completely abolish the formation of Lewis A residues on the recombinant biopharmaceutical.

Moss‐made pharmaceuticals: from bench to bedside

Summary Over the past two decades, the moss Physcomitrella patens has been developed from scratch to a model species in basic research and in biotechnology. A fully sequenced genome, outstanding possibilities for precise genome‐engineering via homologous recombination (knockout moss), a certified GMP production in moss bioreactors, successful upscaling to 500 L wave reactors, excellent homogeneity of protein glycosylation, remarkable batch‐to‐batch stability and a safe cryopreservation for master cell banking are some of the key features of the moss system. Several human proteins are being produced in this system as potential biopharmaceuticals. Among the products are tumour‐directed monoclonal antibodies with enhanced antibody‐dependent cytotoxicity (ADCC), vascular endothelial growth factor (VEGF), complement factor H (FH), keratinocyte growth factor (FGF7/KGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), asialo‐erythropoietin (asialo‐EPO, AEPO), alpha‐galactosidase (aGal) and beta‐glucocerebrosidase (GBA). Further, an Env‐derived multi‐epitope HIV protein as a candidate vaccine was produced, and first steps for a metabolic engineering of P. patens have been made. Some of the recombinant biopharmaceuticals from moss bioreactors are not only similar to those produced in mammalian systems such as CHO cells, but are of superior quality (biobetters). The first moss‐made pharmaceutical, aGal to treat Morbus Fabry, is in clinical trials.

Functional cross‐kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries

Plants and mammals are separated by a huge evolutionary distance. Consequently, biotechnology and genetics have traditionally been divided into green and red. Here, we provide comprehensive evidence that key components of the mammalian transcription, translation and secretion machineries are functional in the model plant Physcomitrella patens. Cross-kingdom compatibility of different expression modalities originally designed for mammalian cells, such as native and synthetic promoters and polyadenylation sites, viral and cellular internal ribosome entry sites, secretion signal peptides and secreted product proteins, and synthetic transactivators and transrepressors, was established. This mammalian expression portfolio enabled constitutive, conditional and autoregulated expression of different product genes in a multicistronic expression format, optionally adjusted by various trigger molecules, such as butyrolactones, macrolide antibiotics and ethanol. Capitalizing on a cross-kingdom-compatible expression platform, we pioneered a prototype biopharmaceutical manufacturing scenario using microencapsulated transgenic P. patens protoplasts cultivated in a Wave Bioreactor. Vascular endothelial growth factor 121 (VEGF(121)) titres matched those typically achieved by standard protonema populations grown in stirred-tank bioreactors. The full compatibility of mammalian expression systems in P. patens further promotes the use of moss as a cost-effective alternative for the manufacture of complex biopharmaceuticals, and as a valuable host system to advance synthetic biology in plants.

A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin

Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

Glyco-engineering for biopharmaceutical production in moss bioreactors

The production of recombinant biopharmaceuticals (pharmaceutical proteins) is a strongly growing area in the pharmaceutical industry. While most products to date are produced in mammalian cell cultures, namely Chinese hamster ovary cells, plant-based production systems gained increasing acceptance over the last years. Different plant systems have been established which are suitable for standardization and precise control of cultivation conditions, thus meeting the criteria for pharmaceutical production. The majority of biopharmaceuticals comprise glycoproteins. Therefore, differences in protein glycosylation between humans and plants have to be taken into account and plant-specific glycosylation has to be eliminated to avoid adverse effects on quality, safety, and efficacy of the products. The basal land plant Physcomitrella patens (moss) has been employed for the recombinant production of high-value therapeutic target proteins (e.g., Vascular Endothelial Growth Factor, Complement Factor H, monoclonal antibodies, Erythropoietin). Being genetically excellently characterized and exceptionally amenable for precise gene targeting via homologous recombination, essential steps for the optimization of moss as a bioreactor for the production of recombinant proteins have been undertaken. Here, we discuss the glyco-engineering approaches to avoid non-human N- and O-glycosylation on target proteins produced in moss bioreactors.

An Env-derived multi-epitope HIV chimeric protein produced in the moss Physcomitrella patens is immunogenic in mice

Key messageThe first report on the recombinant production of a candidate vaccine in the moss system.AbstractThe need for economical and efficient platforms for vaccine production demands the exploration of emerging host organisms. In this study, the production of an antigenic protein is reported employing the moss Physcomitrella patens as an expression host. A multi-epitope protein from the Human Immunodeficiency Virus (HIV) based on epitopes from gp120 and gp41 was designed as a candidate subunit vaccine and named poly-HIV. Transgenic moss plants were generated carrying the corresponding poly-HIV transgene under a novel moss promoter and subsequently seven positive lines were confirmed by PCR. The poly-HIV protein accumulated up to 3.7xa0µgxa0g−1 fresh weight in protonema cultures. Antigenic and immunogenic properties of the moss-produced recombinant poly-HIV are evidenced by Western blots and by mice immunization assays. The elicitation of specific antibodies in mice was observed, reflecting the immunogenic potential of this moss-derived HIV antigen. This is the first report on the production of a potential vaccine in the moss system and opens the avenue for glycoengineering approaches for the production of HIV human-like glycosylated antigens as well as other vaccine prototypes under GMP conditions in moss bioreactors.

The MFHR1 Fusion Protein Is a Novel Synthetic Multitarget Complement Inhibitor with Therapeutic Potential

The complement system is essential for host defense, but uncontrolled complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of complement factor H (FH) with the C5 convertase/C5b-9 inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH-/- mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong inhibitory capacity of MFHR1, which offers a novel basis for complement therapeutics.

Moss-Produced, Glycosylation-Optimized Human Factor H for Therapeutic Application in Complement Disorders.

Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (Physcomitrella patens). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial P. patens culture after a multistep purification process. This glycosylation-optimized factor H showed full in vitro complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.