Glycan-based antibody-drug conjugates
AssigneeMerck Sharp & Dohme Corp.
InventorsRobert Davidson, Bing Gong
Life of this patent
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Abstract
Genetically engineered antibodies containing non-native N-glycosylated sites, preparation of the antibodies in yeast and fungi, site-specific conjugation of drugs to these antibodies, and methods of treatment utilizing these antibodies are described herein.
Claims15
An engineered IgG antibody or fragment thereof comprising one to ten mutations (or a pair of mutations) in the heavy chain constant domain which generate one to ten non-native N-glycosylation sites, the mutations being selected from the group consisting of S134N, G161T, G161S, N203T, N203S, V363T, V363S, Q438N, S176N, A118N, S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T, Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S, Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T, and G194N/Q196S, according to EU numbering.
An engineered IgG antibody or fragment thereof comprising one to two mutations in the heavy chain framework domain which generate one to two non-native N-glycosylation sites, the mutations being selected from the group consisting of Q105N and S113N, according to the Kabat numbering system.
A method of preparing a conjugated N-glycosylated IgG antibody or fragment thereof containing one to ten non-native N-glycosylation sites in the heavy chain constant domain, the method comprising:
- (a) transforming a yeast or filamentous fungus host cell genetically engineered to produce N-glycans comprising terminal galactose residues of the structure Gal(1-4)GlcNAc(1-4)Man3GlcNAc2 or the structure Gal(1-2)GlcNAc(1-2)Man5GlcNAc2 with a nucleic acid encoding an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain comprises one to ten amino acid mutations (or pairs of mutations), and wherein the one to ten amino acid mutations generate at least one N-glycosylation site in the IgG heavy chain constant domain;
- b) culturing the transformed host cell under conditions that allow the expression of the IgG heavy chain constant domain comprising terminal galactose residues;
- (c) contacting the expressed IgG heavy chain constant domain with a reagent that oxidizes the terminal galactose residues;
- and
- (d) conjugating a drug to the oxidized moiety of the terminal galactose residues.
A method of preparing a conjugated N-glycosylated IgG antibody or fragment thereof containing one to ten non-native N-glycosylation sites in the heavy chain constant domain is provided, the method comprising:
- (a) transforming a yeast or filamentous fungus host cell genetically engineered to produce N-glycans comprising terminal sialic acid residues (NANA(1-4)Gal(1-4)GlcNAc(1-4)Man3GlcNAc2) with a nucleic acid encoding an IgG heavy chain contain domain, wherein the IgG heavy chain comprises one to ten amino acid mutations (or pairs of mutations), and wherein the one to ten amino acid mutation generates at least one non-native N-glycosylation site in the IgG heavy chain constant domain;
- (b) culturing the transformed yeast host cell under conditions that allow the expression of the IgG heavy chain constant domain comprising terminal sialic acid residues,
- (c) contacting the expressed IgG heavy chain constant domain with neuraminidase to remove the terminal sialic acid residues to form N-glycosylated heavy chain constant domain comprising terminal galactose residues;
- (d) contacting the expressed glycosylated heavy chain constant domain comprising terminal galactose residues of step (c), with a reagent that oxidizes the terminal galactose residues;
- (e) conjugating a drug to the oxidized moiety of the terminal galactose residues.
Dependencies are inferred from the claim text.