Glycozym Aps

Other

Glycozym USA Inc.

Glycozm Aps

Eric Bennett

Henrik Clausen

Rudi Steffensen

Zhang Yang

Peter Ulvskov

Helle Hassan

Damian Paul Drew

Celso Albuquerque Reis

Emma Adhiambo Arigi

Jochen G&auml;tgens

Detlef Eisenkr&auml;tzer

Steven B Levery

Frau Helle Hassan

Brent Larsen Petersen

This invention discloses the development of a novel platform for recombinant production of bioactive glycoproteins and cancer specific vaccines in plants. Plants and plant cell cultures have been humanized with respect to human mucin-type protein O-glycosylation. A panel of plant cell factories for production of recombinant glycoproteins with designed human O-glycosylation, including an improved cancer vaccine candidate, has been developed. The platform provides basis for i) production of an essentially unlimited array of O-glycosylated human glycoprotein therapeutics, such as human interferon &#x3b1;2B and podoplanin, and ii) for further engineering of additional cancer specific O-glycans on glycoproteins of therapeutical value. Currently, mammalian cells are required for human O-glycosylation, but plants offer a unique cell platform for engineering O-glycosylation since they do not perform human type O-glycosylation. Introduction of O-glycosylation into plant cells requires i) that wild-type plant cells do not modify the target peptide substrates and ii) that the appropriate enzymes and substrates are introduced into of plant cells such that O-glycosylation in the secretory pathway proceed and the glycosylated peptide substrates are preferentially exported to the exterior of the cell or accumulated in the cell. In this invention i) the integrity of transiently and stably expressed 'mucin' type target peptides in plants cells has been determined and ii) mucin-type O-glycosylation has been established in plants by transient and stable introduction of aC4-epimerase, the human polypeptide GalNAc-transferases T2 and T4 (GalNAc-T2 and T4) and various human target peptides or proteins. In the present invention GalNAc-T2 and -T4 have been used to produce a Tn cancer glycoform of MUC1.

Methods for glyco-engineering plant cells for controlled human O-glycosylation

A novel gene defining a novel enzyme UDP-galactose: &#x3b2;-D-galactose-R 4-&#x3b1;-D-galactosyltransferase, termed &#x3b1;4Gal-T1, with unique enzymatic properties is disclosed. The invention provides isolated DNA molecules and DNA constructs encoding &#x3b1;4Gal-T1 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting &#x3b1;4Gal-T1 activity, as well as cloning and expression vectors including such DNA, host cells comprising DNA encoding &#x3b1;4Gal-T1, and recombinant methods for providing &#x3b1;4Gal-T1. The enzyme &#x3b1;4Gal-T1 and &#x3b1;4Gal-active derivatives thereof are disclosed. Further, the invention discloses methods of obtaining &#x3b1;1, 4galactosyl glycosylated glycosphingolipids by use of an enzymatically active &#x3b1;4Gal-T1 protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active &#x3b1;4Gal-T1 protein as an expression system for recombinant production of such glycosphingolipids. Also a method for the identification of DNA sequence variations in the &#x3b1;4Gal-T1-coding exon by PCR, and detecting the presence of DNA sequence variation, are disclosed.

UDP-galactose:&#x3b2;-D-galactose-R 4-&#x3b1;-D-galactosyltransferase, &#x3b1;4gal-T1

UDP-galactose: &#x3b2;-D-galactose-R 4-&#x3b1;-D-galactosyltransferase, &#x3b1;4Gal-T1

A novel gene defining a novel enzyme in the UDP-D-galactose: b-N-acetyl-glucosamine &#x3b2;-1,4-galactosyltransferase family, termed &#x3b2;4Gal-T2, with unique enzymatic properties is disclosed. The enzymatic activity of &#x3b2;4Gal-T2 is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding &#x3b2;4Gal-T2 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting &#x3b2;4Gal-T2 activity, as well as cloning and expression vectors including such DNA, cells transfected with the vectors, and recombinant methods for providing &#x3b2;4Gal-T2. The enzyme &#x3b2;4Gal-T2 and &#x3b2;4Gal-T2-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of &#x3b2;4Gal-T2. Further, the invention discloses methods of obtaining &#x3b2;-1,4-galactosyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymically active &#x3b2;4Gal-T2 protein or fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active &#x3b2;4Gal-T2 protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification of DNA sequence variations in the &#x3b2;4Gal-T2 gene by isolating DNA from a patient, amplifying &#x3b2;4Gal-T2-coding exons by PCR, and detecting the presence of DNA sequence variation, are disclosed.

UDP-galactose: &#x3b2;-N-acetyl-glucosamine &#x3b2;-1,4-galactosyltransferase, &#x3b2;4Gal-T2

Attachment of O-glycans to proteins is controlled by a large family of homologous polypeptide GalNAc-transferases. Polypeptide GalNAc-transferases contain a C-terminal sequence with similarity to lectins. This invention discloses that the putative lectin domains of GalNAc-transferase isoforms, GalNAc-T4, -T7, -T2, and -T3, are functional and recognize carbohydrates, glycopeptides, and peptides and discloses the lectin domains of GalNAc-T1-T16. These lectin domains have different binding specificities and modulate the functions of GalNAc-transferase isoforms differently. Novel methods for identification of inhibitors or modulators of binding activities mediated by lectin domains of polypeptide GalNAc-transferases are disclosed. Direct binding activity of GalNAc-transferase lectins has been demonstrated for the first time and methods to measure lectin mediated binding of isolated lectins or enzymes with lectin domains are disclosed. The present invention specifically discloses a novel selective inhibitor of polypeptide GalNAc-transferase lectin domains, which provides a major advancement in that this inhibitor and related inhibitors sharing common characteristics of activity bind lectin domains without serving as acceptor substrate for glycosyltransferases involved in synthesis of O-glycans. This inhibitor is represented by the &#x3b2;-anomeric configuration of GalNAc-benzyl, GalNAc&#x3b2;-benzyl. Methods for inhibiting intracellular transport, cell surface expression, and secretion of mucins and O-glycosylated glycoproteins without affecting O-glycosylation processing are disclosed using the novel selective inhibitor identified.

Methods of modulating functions of polypeptide GalNAc-transferases and of screening test substances to find agents herefor, pharmaceutical compositions comprising such agents and the use of such agents for preparing medicaments

A novel gene defining a novel enzyme UDP-Galactose: &#x3b2;-D-Galactose-R 4-&#x3b1;-D-galactosyltransferase, termed &#x3b1;4Gal-T1, with unique enzymatic properties is disclosed. The invention discloses isolated DNA molecules and DNA constructs encoding &#x3b1;4Gal-T1 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting &#x3b1;4Gal-T1 activity, as well as cloning and expression vectors including such DNA, cells transfected with vectors, and recombinant methods for providing &#x3b1;4Gal-T1. The enzyme &#x3b1;4Gal-T1 and &#x3b1;4Gal-active derivatives thereof are disclosed. Further, the invention discloses methods of obtaining &#x3b1;1, 4galactosyl glycosylated glycosphingolipids by use of an enzymatically active &#x3b1;4Gal-T1 protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active &#x3b1;4Gal-T1 protein as an expression system for recombinant production of such glycosphingolipids. Also a method for the identification of DNA sequence variations in the &#x3b1;4Gal-T1-coding exon by PCR, and detecting the presence of DNA sequence variation, are disclosed.

UDP-galactose: &#x3b2;-D-galactose-R 4-&#x3b1;-D-galac-tosyltransferase, &#x3b1;4Gal-T1

A novel gene defining a novel enzyme in the UDP-D-galactose: &bgr;-N-acetyl-glucosamine &bgr;-1,4-galactosyltransferase family, termed &bgr;4Gal-T2, with unique enzymatic properties is disclosed. The enzymatic activity of &bgr;4Gal-T2 is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding &bgr;4Gal-T2 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting &bgr;4Gal-T2 activity, as well as cloning and expression vectors including such DNA, cells transfected with the vectors, and recombinant methods for providing &bgr;4Gal-T2. The enzyme &bgr;4Gal-T2 and &bgr;4Gal-T2-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of &bgr;4Gal-T2. Further, the invention discloses methods of obtaining &bgr;-1,4-galactosyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymically active &bgr;4Gal-T2 protein or fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active &bgr;4Gal-T2 protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification of DNA sequence variations in the &bgr;4Gal-T2 gene by isolating DNA from a patient, amplifying &bgr;4Gal-T2-coding exons by PCR, and detecting the presence of DNA sequence variation, are disclosed.

UDP-galactose: &bgr;-N-acetyl-glucosamine &bgr;-1,4-galactosyl-transferase, &bgr;4Gal-T2

The use of human polypeptide N-acetylgalactosaminytransferase T-4 (GalNAc-T4) to glycosylate substrates is disclosed. The action of GalNAc-T4 can complement the activities of GalNAc-T1, -T2 and -T3. The glycosylated substrates are useful in preparation of vaccines and anti-inflammatory agents. A method of producing the soluble form of the enzyme is also disclosed.

Growing community of inventors

Lyngby, Denmark

Eric Bennett