Yoseph Shaaltiel

Taliglucerase alfa: an enzyme replacement therapy using plant cell expression technology.

Gaucher disease (GD) is a rare, genetic lysosomal storage disorder caused by functional defects of acid β-glucosidase that results in multiple organ dysfunction. Glycosylation of recombinant acid human β-glucosidase and exposure of terminal mannose residues are critical to the success of enzyme replacement therapy (ERT) for the treatment of visceral and hematologic manifestations in GD. Three commercially available ERT products for treatment of GD type 1 (GD1) include imiglucerase, velaglucerase alfa, and taliglucerase alfa. Imiglucerase and velaglucerase alfa are produced in different mammalian cell systems and require production glycosylation modifications to expose terminal α-mannose residues, which are needed for mannose receptor-mediated uptake by target macrophages. Such modifications add to production costs. Taliglucerase alfa is a plant cell-expressed acid β-glucosidase approved in the United States and other countries for ERT in adults with GD1. A plant-based expression system, using carrot root cell cultures, was developed for production of taliglucerase alfa and does not require additional processing for postproduction glycosidic modifications. Clinical trials have demonstrated that taliglucerase alfa is efficacious, with a well-established safety profile in adult, ERT-naïve patients with symptomatic GD1, and for such patients previously treated with imiglucerase. These included significant improvements in organomegaly and hematologic parameters as early as 6months, and maintenance of achieved therapeutic values in previously treated patients. Ongoing clinical trials will further characterize the long-term efficacy and safety of taliglucerase alfa in more diverse patient populations, and may help to guide clinical decisions for achieving optimal outcomes for patients with GD1.

Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems

The glycosylation of recombinant β-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize β-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different cell systems and undergo various post-translational or post-production glycosylation modifications to expose their mannose residues. This is the first study in which the glycosylation profiles of the three enzymes are compared, using the same methodology and the effect on functionality and cellular uptake is evaluated. While the major differences in glycosylation profiles reside in the variation of terminal residues and mannose chain length, the enzymatic activity and stability are not affected by these differences. Furthermore, the cellular uptake and in-cell stability in rat and human macrophages are similar. Finally, in vivo studies to evaluate the uptake into target organs also show similar results for all three enzymes. These results indicate that the variations of glycosylation between the three regulatory-approved β-glucocerebrosidase enzymes have no effect on their function or distribution.

Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience.

Protalix Biotherapeutics develops recombinant human proteins and produces them in plant cell culture. Taliglucerase alfa has been the first biotherapeutic expressed in plant cells to be approved by regulatory authorities around the world. Other therapeutic proteins are being developed and are currently at various stages of the pipeline. This review summarizes the major milestones reached by Protalix Biotherapeutics to enable the development of these biotherapeutics, including platform establishment, cell line selection, manufacturing process and good manufacturing practice principles to consider for the process. Examples of the various products currently being developed are also presented.

Characterization of a chemically modified plant cell culture expressed human α-Galactosidase-A enzyme for treatment of Fabry disease

Fabry disease is an X-linked recessive disorder caused by the loss of function of the lysosomal enzyme α-Galactosidase-A. Although two enzyme replacement therapies (ERTs) are commercially available, they may not effectively reverse some of the Fabry pathology. PRX-102 is a novel enzyme for the therapy of Fabry disease expressed in a BY2 Tobacco cell culture. PRX-102 is chemically modified, resulting in a cross-linked homo-dimer. We have characterized the in-vitro and in-vivo properties of PRX-102 and compared the results with the two commercially produced α-Galactosidase-A enzymes. Results show that PRX-102 has prolonged in-vitro stability in plasma, after 1h incubation it retains 30% activity compared with complete inactivation of the commercial enzymes. Under lysosomal-like conditions PRX-102 maintains over 80% activity following 10 days of incubation, while commercial enzymes become inactive after 2days. Pharmacokinetic profile of PRX-102 measured in male Fabry mice shows a 10 fold increase in t1/2 in mice (581min) compared to approved drugs. The enzyme has significantly different kinetic parameters to the alternative ERTs available (p-value<0.05, one way ANOVA), although these differences do not indicate any significant biochemical variations. PRX-102 is uptaken to primary human Fabry fibroblasts. The repeat administration of the enzyme to Fabry mice caused significant reduction (p-value<0.05) of Gb3 in various tissues (the measured residual content was 64% in kidney, liver was cleaned, 23% in heart, 5.7% in skin and 16.2% in spleen). PRX-102 has a relatively simple glycosylation pattern, characteristic to plants, having mainly tri-mannose structures with the addition of either α(1-3)-linked fucose or β(1-2)-linked xylose, or both, in addition to various high mannose structures, while agalsidase beta has a mixture of sialylated glycans in addition to high mannose structures. This study concludes that PRX-102 is equivalent in functionality to the current ERTs available, with superior stability and prolonged circulatory half-life. Therefore we propose that PRX-102 is a promising alternative for treatment of Fabry disease.

Plant specific N -glycans do not have proven adverse effects in humans

VOLUME 34 NUMBER 7 JULY 2016 NATURE BIOTECHNOLOGY After tracking cells with tTt (Fig. 2b), normalized NanogVENUS signal is quantified and manually curated with qTfy (Fig. 2c). Visualization of molecular dynamics through ‘heat trees’ from qTfy (Fig. 2d) demonstrates both the reported NanogVENUS heterogeneity25, including cells with low (black) and high expression (white), and the underlying single-cell expression dynamics (Supplementary Video 3). Our tTt and qTfy software enables continuous long-term quantification of cellular behavior and molecular properties over long periods of time at the level of individual cells (Fig. 2 and Supplementary Video 3) in a user-friendly way and without being limited to specific cell or image types. By making these tools available to the scientific community, we hope to support future studies of single-cell dynamics.

Establishment of a tobacco BY2 cell line devoid of plant specific xylose and fucose as a platform for the production of biotherapeutic proteins.

Summary Plant‐produced glycoproteins contain N‐linked glycans with plant‐specific residues of β(1,2)‐xylose and core α(1,3)‐fucose, which do not exist in mammalian‐derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant‐expressed proteins. We knocked out the β(1,2)‐xylosyltranferase (XylT) and the α(1,3)‐fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked‐out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N‐linked glycans lacking β(1,2)‐xylose and/or α(1,3)‐fucose. The knocked‐out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild‐type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco‐engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.

An oral administration of a recombinant anti-TNF fusion protein is biologically active in the gut promoting regulatory T cells: Results of a phase I clinical trial using a novel oral anti-TNF alpha-based therapy ☆

BACKGROUND An orally administered BY-2 plant cell-expressed recombinant anti-TNF fusion protein (PRX-106) consists of the soluble form of the human TNF receptor (TNFR) fused to the Fc component of a human IgG1 domain. Aim This study aim at determining the safety and the immune modulatory effect of an oral administration of PRX-106 in humans. METHODS Three different doses (2, 8 or 16mg/day) of PRX-106 were orally administered for five consecutive days in 14 healthy volunteered participants. Subjects were followed for safety parameters and for an effect on T lymphocytes subsets and cytokine levels. RESULTS An oral administration of PRX-106 was safe and well tolerated. The PK study showed that PRX106 is not absorbed. No effect on white blood cells and lymphocytes counts were noted. A dose dependent effect was noted on systemic lymphocytes. The oral administration of all three dosages was associated with an increase in CD4+CD25+ and CD8+CD25+ subset of suppressor lymphocytes. A marked increase in CD4+CD25+FoxP3 regulatory T cells was noted in the 8mg treated group. In addition, NKT regulatory cells, CD3+CD69+ and CD4+CD62 lymphocyte subsets increased with treatment. No changes in serum TNF alpha were observed. CONCLUSION An oral administration of the non-absorbable recombinant anti-TNF fusion protein, PRX-106, is safe, not associated with immune suppression, while inducing a favorable anti-inflammatory immune modulation. The PRX-106 may provide a safe orally administered effective anti-TNF alpha-based immune therapy for inflammatory bowel diseases and non-alcoholic steatohepatitis, as well as other autoimmune, TNF-mediated diseases.

A plant cell-expressed recombinant anti-TNF fusion protein is biologically active in the gut and alleviates immune-mediated hepatitis and colitis

The orally administered BY-2 plant cell-expressed recombinant anti-TNF fusion protein (PRX-106) (n=6) consists of the soluble form of the human TNF receptor (TNFR) fused to the Fc component of a human antibody IgG1 domain. AIM To evaluate the immune modulatory effect of the oral administration of plant cells expressing PRX-106. METHODS Mice treated with Concanavalin A (ConA) to induce immune hepatitis was orally treated with cells expressing PRX-106 containing 0.5 or 5μg PRX 106. In the colitis model, TNBS-colitis was induced in mice followed by the oral administration of plant cells expressing PRX-106. The immune modulatory effect was determined through follow-up to assess the clinical effect, histology, and serum cytokine levels and by FACS analysis for lymphocyte subsets. RESULTS The oral administration of BY-2 cells expressing PRX-106 alleviated immune-mediated liver injury. Serum AST and ALT levels decreased and were comparable to those of mice that had received high-dose steroids. The beneficial effect was also observed as a marked decrease in hepatic necrosis. In the colitis model, the oral administration of BY-2 plant cells expressing PRX-106 alleviated weight loss associated with immune-mediated colitis and improved bowel histology. A reduction in I-IkB-alpha phosphorylation in treated mice was also observed. These effects were associated with a significant alteration in the distribution of CD4+CD25+FOXP3+ cells. CONCLUSIONS Plant cells expressing recombinant anti-TNF fusion protein show biological activity when orally administered, exerting an immune modulatory effect through the alleviation of immune-mediated hepatitis and immune-mediated colitis.

Development and Analytical Characterization of Pegunigalsidase Alfa, a Chemically Cross-Linked Plant Recombinant Human α-Galactosidase-A for Treatment of Fabry Disease

The current treatment of Fabry disease by enzyme replacement therapy with commercially available recombinant human α-Galactosidase A shows a continuous deterioration of the disease patients. Human recombinant α-Galactosidase A is a homodimer with noncovalently bound subunits and is expressed in the ProCellEx plant cell-based protein expression platform to produce pegunigalsidase alfa. The effect of covalent bonding between two α-Galactosidase A subunits by PEG-based cross-linkers of various lengths was evaluated in this study. The results show that cross-linking by a bifunctional PEG polymer of 2000 Da produces a more stable protein with improved pharmacokinetic and biodistribution properties. The chemical modification did not influence the tertiary protein structure but led to an increased thermal stability and showed partial masking of immune epitopes. The developed pegunigalsidase alfa is currently tested in phase III clinical trials and has a potential to show superior efficacy versus the currently used enzyme replacement therapies in the treatment of Fabry disease patients.

Oral administration of a non-absorbable plant cell-expressed recombinant anti-TNF fusion protein induces immunomodulatory effects and alleviates nonalcoholic steatohepatitis

AIM To evaluate the immunomodulatory effect of oral administration of PRX-106 in the high-fat diet model. METHODS For 22 wk, C57BL/6 HFD-fed mice received daily oral treatments with BY-2 cells expressing recombinant anti-tumor necrosis factor alpha fusion protein (PRX-106). Mice were followed for serum liver enzyme and triglyceride levels, liver histology and intrahepatic and systemic FACS. RESULTS The orally administered non-absorbable PRX-106 was biologically active. Altered distribution of CD4+CD25+FoxP3+ between the liver and spleen and an increase in the intrasplenic-to-intrahepatic CD4+CD25+FoxP3+ ratio and a decrease in the intrasplenic-to-intrahepatic CD8+CD25+FoxP3+ ratio were observed. An increase in intrahepatic NKT cells and a decrease in the intrasplenic-to-intrahepatic NKT ratio were noted. Assessment of the CD4-to-CD8 ratios showed sequestration of CD8+ lymphocytes in the liver. These effects were associated with a decrease in serum triglyceride levels, decrease in the aspartate aminotransferase levels, serum glucose levels, and HOMA-IR score. A decrease in hepatic triglycerides content was observed in the high dose-treated mice. CONCLUSION Orally administered PRX-106 shows biological activity and exerts an immunomodulatory effect, alleviating liver damage. The data suggest that PRX-106 may provide an oral immunotherapy for nonalcoholic steatohepatitis.