Methods for preparation of fucose-linked site specific conjugates of proteins with toxins, adjuvants, detection labels and pharmacokinetic half life extenders
Inventors
Von Horsten, Hans Henning • Sandig, Volker • Jordan, Ingo • Winkler, Karsten
Assignees
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Publication Number
US-9765158-B2
Publication Date
2017-09-19
Expiration Date
Abstract
The present invention relates to eukaryotic cells for producing molecules having an atypical fucose analog on their glycomoieties and/or amino acids. It also relates to methods for producing molecules having an atypical fucose analog on their glycomoieties and/or amino acids and to molecules obtainable by said methods. It further relates to methods for producing conjugates comprising molecules having an atypical fucose analog on their glycomoieties and/or amino acids and pharmaceutical active compounds and to conjugates obtainable by said methods. In addition, the present invention relates to specific conjugates.
Core Innovation
An engineered eukaryotic cell is configured for producing a molecule comprising a fucose analogue, where the GDP-L-fucose synthesis pathway originating from GDP-D-mannose is blocked due to the presence of at least one enzyme which uses GDP-6-deoxy-D-lyxo-4-hexulose as a substrate but does not catalyze the reaction which converts GDP-6-deoxy-D-lyxo-4-hexulose into GDP-L-fucose. The cell further comprises a GDP-L-fucose analogue that is a substrate for a fucosyltransferase and enters the cell from extracellular medium. In this way, incorporation of the atypical fucose analogue into defined glycan and/or amino-acid sites is enabled by preventing competitive natural fucose incorporation.
The engineered cell limits competing fucosylation by blocking or reducing enzymatically active GDP-mannose dehydratase, GDP-fucose synthetase, and/or alpha-1,3-fucosyltransferase. The pathway block is implemented using an enzyme which uses GDP-6-deoxy-D-lyxo-4-hexulose as a substrate, including embodiments where the enzyme is selected from GDP-6-deoxy-D-lyxo-4-hexulose substrate enzymes such as RMD, Per, GTS, and variants including GDP-4-keto-6-deoxymannose-3-dehydratase and optionally a combination with GDP-L-colitose synthase.
The disclosure further provides conjugates in which the fucose analogue positions a pharmaceutically active compound through specified glycan-linked and/or amino-acid-linked structures. The conjugate structures include NG-cF*-Y_o-C and F*-Y_p-C, where cF* is a core fucose analogue and Y is a spacer unit with o or p being an integer of 0 or 1. Pharmaceutically active compound C can be a cytotoxic drug or a pharmacokinetic half-life extender, and the EGF-like repeats have independently from each other an amino acid sequence according to SEQ ID NO: 10 or variants at least 90% identical to SEQ ID NO: 10.
Claims Coverage
The document contains four independent claims: one directed to a eukaryotic cell, and three directed to defined conjugates. Across these claims, there are two main inventive feature groups: blocking the GDP-L-fucose synthesis pathway from GDP-D-mannose while supplying a GDP-L-fucose analogue that serves as substrate for fucosyltransferase, and defining conjugate structures that attach a pharmaceutically active compound to defined glycan and/or amino-acid motifs via a fucose analogue and spacer unit.
Blocking GDP-L-fucose synthesis while supplying a GDP-L-fucose analogue for fucosyltransferase substrate uptake
A eukaryotic cell for producing a molecule comprising a fucose analogue, wherein the GDP-L-fucose synthesis pathway originating from GDP-D-mannose is blocked due to the presence of at least one enzyme which uses GDP-6-deoxy-D-lyxo-4-hexulose as a substrate, wherein the enzyme does not catalyze the reaction which converts GDP-6-deoxy-D-lyxo-4-hexulose into GDP-L-fucose, and wherein the cell comprises a GDP-L-fucose analogue that is a substrate for a fucosyltransferase and enters the cell from extracellular medium.
Core fucose analogue on N-glycosylation site linked to pharmaceutically active compound as NG-cF*-Y_o-C
A conjugate comprising a protein or polypeptide comprising one or more structures —NG-cF*—Y_o—C, wherein each is attached to an N-glycosylation site comprised in said protein or polypeptide, where NG is an N-linked glycomoiety, cF* is a core fucose analogue, Y is a spacer unit with o being an integer of 0 or 1, and C is a pharmaceutically active compound, wherein the pharmaceutically active compound is a cytotoxic drug or a pharmacokinetic half-life extender.
Fucose analogue attached to serine and/or threonine in EGF-like repeats linked to pharmaceutically active compound as F*-Y_p-C
A conjugate comprising a protein or polypeptide comprising one or more EGF-like repeats comprising a serine and/or threonine residue to which a structure —F*—Y_p—C is attached, wherein F* is a fucose analogue moiety directly linked to said serine and/or threonine residue, Y is a spacer unit with p being an integer of 0 or 1, C is a pharmaceutically active compound, and wherein the EGF-like repeats have independently from each other an amino acid sequence according to SEQ ID NO: 10 or are a variant thereof which is at least 90% identical to the amino acid sequence according to SEQ ID NO: 10.
N-glycosylation site incorporation of core fucose analogue linked to pharmaceutically active compound on antibody heavy chain as NG-cF*-Y_o-C
A conjugate comprising a polypeptide comprising one or more structures —NG-cF*—Y_o—C, wherein each is attached to an N-glycosylation site comprised in said polypeptide, where NG is an N-linked glycomoiety, cF* is a core fucose analogue, Y is a spacer unit with o being an integer of 0 or 1, and C is a pharmaceutically active compound, wherein the polypeptide is an antibody heavy chain (H).
Overall, the claim set covers an engineered eukaryotic cell that blocks the GDP-L-fucose synthesis pathway originating from GDP-D-mannose while providing a GDP-L-fucose analogue that serves as a substrate for a fucosyltransferase entering from extracellular medium, and it covers defined conjugate structures where the fucose analogue, including a core fucose analogue, is positioned on N-glycosylation sites and/or within EGF-like repeats to attach a pharmaceutically active compound that is a cytotoxic drug or a pharmacokinetic half-life extender.
Stated Advantages
Homogenous, stable, site-directed coupling.
High yields.
Minor impact on thermal stability.
Enhanced ADCC control by fucose-analogue positioning.
Defined conjugate structures including NG–cF*–Y_o–C and F*–Y_p–C.
Documented Applications
Therapeutic/pharmaceutical composition use of conjugates comprising cytotoxic drugs or pharmacokinetic half-life extenders attached via fucose-analogue positioned on protein glycosylation or EGF-like repeats.
Diagnosis/vaccine/labeling contexts for fucose-analogue-containing molecules and conjugates.
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