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Papers by Yoseph Shaaltiel

Zeitschrift Fur Naturforschung C a Journal of Biosciences, 1990

Plant Biotechnology Journal, 2015

Protalix Biotherapeutics develops recombinant human proteins and produces them in plant cell cult... more 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.

Toxicology and Applied Pharmacology, 2015

PRX-105 is a plant-derived recombinant version of the human &... more PRX-105 is a plant-derived recombinant version of the human 'read-through' acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50nmol/kg PRX-105, 2minutes before being exposed to 1.33×LD50 and 1.5×LD50 toxin and 10minutes after exposure to 1.5×LD50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (T½) in mice was 994 (±173) minutes. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The T½ in humans was substantially longer than in mice (average 26.7hours). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation.

Plant biotechnology journal, 2015

Gaucher's disease (GD), a lysosomal storage disorder caused by mutations in the gene encoding... more Gaucher's disease (GD), a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy (ERT) using recombinant GCD that is administered intravenously every 2 weeks. However, intravenous administration includes discomfort or pain and might cause local and systemic infections that may lead to low patient compliance. An orally administered drug has the potential to alleviate these problems. In this study, we describe the potential use of plant cells as a vehicle for the oral delivery of recombinant human GCD (prGCD) expressed in carrot cells. The in vitro results demonstrate that the plant cells protect the recombinant protein in the gastric fluids and may enable absorption into the blood. Feeding experiments, with rat and pig as model animals, using carrot cells containing prGCD, show that active recombinant prGCD was found in the digestive tract and blood system and reached both, liver and spleen,...

PLoS ONE, 2009

Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocere... more Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing.

Molecular Genetics and Metabolism, 2015

Fabry disease is an X-linked recessive disorder caused by the loss of function of the lysosomal e... more 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.

Theoretical and Applied Genetics, 1988

The genetics of paraquat-resistance in Conyza bonariensis was studied. Reciprocal crosses were pr... more The genetics of paraquat-resistance in Conyza bonariensis was studied. Reciprocal crosses were prepared between resistant and sensitive individuals. The enzymes of the pathway that detoxifies superoxide to innocuous oxygen species involved in resistance were evaluated in the F1 and F2 generations. All F1 plants were as resistant as the resistant parent, irrespective of parental sex, demonstrating dominance and excluding maternal

Plant Biotechnology Journal, 2007

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocer... more Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme ® ) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme ® . In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro , which is a requirement for the production of Cerezyme ® . prGCD also displays a level of biological activity similar to that of Cerezyme ® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.

Organic & Biomolecular Chemistry, 2011

Cyclodextrin-based host-guest chemistry has been exploited to facilitate co-crystallization of re... more Cyclodextrin-based host-guest chemistry has been exploited to facilitate co-crystallization of recombinant human acid b-glucosidase (b-glucocerebrosidase, GlcCerase) with amphiphilic bicyclic nojirimycin analogues of the sp 2 -iminosugar type. Attempts to co-crystallize GlcCerase with 5-N,6-O-[N¢-(n-octyl)iminomethylidene]nojirimycin (NOI-NJ) or with 5-N,6-S-[N¢-(n-octyl)iminomethylidene]-6-thionojirimycin (6S-NOI-NJ), two potent inhibitors of the enzyme with promising pharmacological chaperone activity for several Gaucher disease-associated mutations, were unsuccessful probably due to the formation of aggregates that increase the heterogeneity of the sample and affect nucleation and growth of crystals. Cyclomaltoheptaose (b-cyclodextrin, bCD) efficiently captures NOI-NJ and 6S-NOI-NJ in aqueous media to form inclusion complexes in which the lipophilic tail is accommodated in the hydrophobic cavity of the cyclooligosaccharide. The dissociation constant of the complex of the amphiphilic sp 2 -iminosugars with bCD is two orders of magnitude higher than that of the corresponding complex with GlcCerase, allowing the efficient transfer of the inhibitor from the bCD cavity to the GlcCerase active site. Enzyme-inhibitor complexes suitable for X-ray analysis were thus grown in the presence of bCD. In contrast to what was previously observed for the complex of GlcCerase with the more basic derivative, 6-amino-6-deoxy-5-N,6-N-[N¢-(n-octyl)iminomethylidene]nojirimycin (6N-NOI-NJ), the b-anomers of both NOI-NJ and 6S-NOI-NJ were seen in the active site, even though the a-anomer was exclusively detected both in aqueous solution and in the corresponding bCD:sp 2 -iminosugar complexes. Our results further suggest that cyclodextrin derivatives might serve as suitable delivery systems of amphiphilic glycosidase inhibitors in a biomedical context. † Electronic supplementary information (ESI) available: Experimental procedures for K i and tensiometry determinations, titration plots showing the least-square fitted curves, Lineweaver-Burk plots and NMR spectra of NOI-NJ and 6S-NOI-NJ in the absence and in the presence of bcyclodextrin. See

Molecular Genetics and Metabolism, 2009

Molecular Genetics and Metabolism, 2010

Molecular Genetics and Metabolism, 2014

Zeitschrift Fur Naturforschung C a Journal of Biosciences, 1990

Plant Biotechnology Journal, 2015

Protalix Biotherapeutics develops recombinant human proteins and produces them in plant cell cult... more 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.

Toxicology and Applied Pharmacology, 2015

PRX-105 is a plant-derived recombinant version of the human &... more PRX-105 is a plant-derived recombinant version of the human 'read-through' acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50nmol/kg PRX-105, 2minutes before being exposed to 1.33×LD50 and 1.5×LD50 toxin and 10minutes after exposure to 1.5×LD50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (T½) in mice was 994 (±173) minutes. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The T½ in humans was substantially longer than in mice (average 26.7hours). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation.

Plant biotechnology journal, 2015

Gaucher's disease (GD), a lysosomal storage disorder caused by mutations in the gene encoding... more Gaucher's disease (GD), a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy (ERT) using recombinant GCD that is administered intravenously every 2 weeks. However, intravenous administration includes discomfort or pain and might cause local and systemic infections that may lead to low patient compliance. An orally administered drug has the potential to alleviate these problems. In this study, we describe the potential use of plant cells as a vehicle for the oral delivery of recombinant human GCD (prGCD) expressed in carrot cells. The in vitro results demonstrate that the plant cells protect the recombinant protein in the gastric fluids and may enable absorption into the blood. Feeding experiments, with rat and pig as model animals, using carrot cells containing prGCD, show that active recombinant prGCD was found in the digestive tract and blood system and reached both, liver and spleen,...

PLoS ONE, 2009

Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocere... more Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing.

Molecular Genetics and Metabolism, 2015

Fabry disease is an X-linked recessive disorder caused by the loss of function of the lysosomal e... more 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.

Theoretical and Applied Genetics, 1988

The genetics of paraquat-resistance in Conyza bonariensis was studied. Reciprocal crosses were pr... more The genetics of paraquat-resistance in Conyza bonariensis was studied. Reciprocal crosses were prepared between resistant and sensitive individuals. The enzymes of the pathway that detoxifies superoxide to innocuous oxygen species involved in resistance were evaluated in the F1 and F2 generations. All F1 plants were as resistant as the resistant parent, irrespective of parental sex, demonstrating dominance and excluding maternal

Plant Biotechnology Journal, 2007

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocer... more Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme ® ) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme ® . In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro , which is a requirement for the production of Cerezyme ® . prGCD also displays a level of biological activity similar to that of Cerezyme ® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.

Organic & Biomolecular Chemistry, 2011

Cyclodextrin-based host-guest chemistry has been exploited to facilitate co-crystallization of re... more Cyclodextrin-based host-guest chemistry has been exploited to facilitate co-crystallization of recombinant human acid b-glucosidase (b-glucocerebrosidase, GlcCerase) with amphiphilic bicyclic nojirimycin analogues of the sp 2 -iminosugar type. Attempts to co-crystallize GlcCerase with 5-N,6-O-[N¢-(n-octyl)iminomethylidene]nojirimycin (NOI-NJ) or with 5-N,6-S-[N¢-(n-octyl)iminomethylidene]-6-thionojirimycin (6S-NOI-NJ), two potent inhibitors of the enzyme with promising pharmacological chaperone activity for several Gaucher disease-associated mutations, were unsuccessful probably due to the formation of aggregates that increase the heterogeneity of the sample and affect nucleation and growth of crystals. Cyclomaltoheptaose (b-cyclodextrin, bCD) efficiently captures NOI-NJ and 6S-NOI-NJ in aqueous media to form inclusion complexes in which the lipophilic tail is accommodated in the hydrophobic cavity of the cyclooligosaccharide. The dissociation constant of the complex of the amphiphilic sp 2 -iminosugars with bCD is two orders of magnitude higher than that of the corresponding complex with GlcCerase, allowing the efficient transfer of the inhibitor from the bCD cavity to the GlcCerase active site. Enzyme-inhibitor complexes suitable for X-ray analysis were thus grown in the presence of bCD. In contrast to what was previously observed for the complex of GlcCerase with the more basic derivative, 6-amino-6-deoxy-5-N,6-N-[N¢-(n-octyl)iminomethylidene]nojirimycin (6N-NOI-NJ), the b-anomers of both NOI-NJ and 6S-NOI-NJ were seen in the active site, even though the a-anomer was exclusively detected both in aqueous solution and in the corresponding bCD:sp 2 -iminosugar complexes. Our results further suggest that cyclodextrin derivatives might serve as suitable delivery systems of amphiphilic glycosidase inhibitors in a biomedical context. † Electronic supplementary information (ESI) available: Experimental procedures for K i and tensiometry determinations, titration plots showing the least-square fitted curves, Lineweaver-Burk plots and NMR spectra of NOI-NJ and 6S-NOI-NJ in the absence and in the presence of bcyclodextrin. See

Molecular Genetics and Metabolism, 2009

Molecular Genetics and Metabolism, 2010

Molecular Genetics and Metabolism, 2014