Engineered and fully-functional customized glycoproteins
Inventors
Faridmoayer, Amirreza • Mally, Manuela • FOLLADOR, Rainer
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11913051-B2
Publication Date
2024-02-27
Expiration Date
Abstract
Described herein are compositions and methods of producing glycosylated proteins in vitro and in vivo. The methods include using host cells to produce glycosylated proteins. Also described herein are glycosylated proteins produced using such methods and uses thereof.
Core Innovation
The invention provides engineered Leishmania host cells that comprise recombinant nucleic acids encoding a target protein and a heterologous glycosyltransferase. The heterologous glycosyltransferase is an N-acetyl glucosamine transferase that is not from Leishmania and/or a galactosyltransferase that is not from Leishmania, and includes a signal sequence capable of targeting the heterologous glycosyltransferase to the secretory pathway and a retention sequence capable of retaining the heterologous glycosyltransferase in the Golgi apparatus.
The invention further describes signal and retention elements derived from Leishmania tarentolae, including CTS and GRIP variants, for localization and retention in Golgi sub-compartments. The host cells are engineered for altered N-glycosylation to build defined glycan structures on a paucimannose base and to generate glycoprotein populations with high site occupancy and high proportions of defined glycoforms.
Defined glycan structures include glycan classes such as G0, G0-Gn, G1-Gn, and G2, together with defined terminal sialylation forms including α2,3 and α2,6 sialylation. The document also describes heterologous sialyltransferases, references to CMP-sialic acid biosynthetic pathway proteins, and glycosylated protein compositions characterized by humanized N-glycans on therapeutic glycoproteins.
Claims Coverage
The consolidated claim coverage identifies one main independent claim and dependent refinements. Two core inventive features are present in the independent claim, with dependent claims adding further glycosyltransferases, trafficking elements, sequence constraints, and pathway modifications.
Leishmania host cell expressing target protein and non-Leishmania glycosyltransferase
A Leishmania host cell comprising a recombinant nucleic acid encoding a target protein and a recombinant nucleic acid encoding a heterologous glycosyltransferase, wherein the heterologous glycosyltransferase is an N-acetyl glucosamine transferase that is not from Leishmania and/or a galactosyltransferase that is not from Leishmania.
Secretory pathway targeting and Golgi retention by signal and retention sequences
The heterologous glycosyltransferase comprises a signal sequence capable of targeting the heterologous glycosyltransferase to the secretory pathway of the Leishmania host cell and a retention sequence capable of retaining the heterologous glycosyltransferase in the Golgi apparatus.
Heterologous sialyltransferase with secretory targeting and Golgi retention
A Leishmania host cell wherein the recombinant nucleic acid encodes a non-Leishmania heterologous sialyltransferase equipped with a signal sequence that targets it to the secretory pathway and a retention sequence that retains it in the Golgi apparatus.
Specific heterologous glycosyltransferase identities
The host cell wherein the N-acetyl glucosamine transferases are GnT-I and GnT-II, and the galactosyltransferase is B4GALT1.
Trafficking element derived from Leishmania tarentolae
The host cell in which the signal sequence and/or retention sequence is derived from Leishmania tarentolae.
Sequence-identity constrained CTS sequence
A host cell defined such that its CTS sequence is at least about 70% to 100% identical to one of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or includes a functionally active fragment.
Engineered N-glycan biosynthesis pathway via endogenous enzyme modification
A host cell in which one or more endogenous enzymes involved in the N-glycan biosynthesis pathway have been deleted, mutated, and/or functionally inactivated.
The claims focus on engineered Leishmania host cells that produce glycosylated therapeutic glycoproteins by expressing a target protein together with non-Leishmania heterologous N-acetyl glucosamine transferase and/or galactosyltransferase, with secretory-pathway targeting and Golgi retention. The dependent claim refinements further include heterologous sialyltransferases, specific enzyme identities, trafficking-element derivation from Leishmania tarentolae, CTS sequence-identity constraints, and modification of endogenous N-glycan biosynthesis enzymes.
Stated Advantages
Reduced glycoheterogeneity.
Improved PK/effector function.
Consistent potency.
Safer, more consistent therapeutics.
Produces glycosylated target proteins with defined N-glycan populations including core paucimannose (G0-Gn), biantennary (G2), and higher sialylated variants including α2,3- and α2,6-linked sialic acid variants.
Enables production of homogeneous paucimannose and engineered glycoforms in engineered strains.
Supports downstream therapeutic formulation using pharmaceutically acceptable carriers.
Documented Applications
Producing humanized N-glycans on therapeutic glycoproteins, including therapeutic antibodies (e.g., rituximab/anti-CD20).
Producing humanized N-glycans on therapeutic glycoproteins, including rhEPO.
Therapeutic use in pharmaceutical compositions formulated with pharmaceutically acceptable carriers.
Example application involving rituximab expression and N-glycan profiling.
Interested in licensing this patent?