Site-specific glycoengineering of targeting moieties
The current disclosure provides binding polypeptides (e.g., antibodies), and targeting moiety conjugates thereof, comprising a site-specifically engineered glycan linkage within native or engineered glycans of the binding polypeptide. The current disclosure also provides nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.
1 . A method of making a PEGylated binding polypeptide comprising at least one modified glycan, wherein the at least one modified glycan comprises at least one moiety of Formula (IV):
the method comprising reacting an effector moiety of Formula (I):
NH 2 -Q-CON-X Formula (I),
with a precursor binding polypeptide comprising an oxidized glycan
wherein:
A) Q is NH or O;
B) CON is a connector moiety;
C) X is a moiety comprising a PEG moiety;
D) Gal is a galactose moiety; and
E) Sia is a sialic acid moiety,
wherein the binding polypeptide is an antibody, an antigen binding fragment thereof, or an immunoadhesin,
wherein the PEGylated binding polypeptide comprising at least one oxidized glycan is prepared by reacting a binding polypeptide comprising at least one glycan with a periodate oxidase or galactose oxidase in the presence of a salt comprising a copper metal ion to generate the PEGylated binding polypeptide comprising at least one oxidized glycan.
2 . The method of claim 1 , wherein the binding polypeptide is internalized by a cell.
3 . The method of claim 1 , wherein the oxidized glycan is reacted with at least one effector moiety represented by Formula IX or Formula XI:
wherein p has a value of 1 to 32; or
wherein p has a value of 1 to 32.
4 . The method of claim 1 , wherein the at least one modified glycan comprises at least one moiety selected from the following structural formulae:
wherein p has a value of 1 to 32; or
wherein p has a value of 1 to 32.
5 . The method of claim 1 , wherein the at least one modified glycan comprises at least one moiety selected from the following structural formulae:
wherein p has a value of 1 to 32; or
wherein p has a value of 1 to 32.
6 . The method of claim 1 , wherein the targeting moiety comprises PEG and is represented by Formula X or Formula XII:
7 . The method of claim 1 , wherein the at least one modified glycan comprises at least one moiety selected from the following structural formulae:
8 . The method of claim 1 , wherein the at least one modified glycan comprises at least one moiety selected from the following structural formulae:
9 . The method of claim 1 , wherein one or more of the following conditions are met:
a) the PEG moiety comprises mono-PEG, bi-PEG, or tri-PEG;
b) the PEG moiety comprises 3 to 3.5 PEG.
10 . The method of claim 1 , wherein the targeting moiety further comprises Dolastatin 10 (Dol10).
11 . The method of claim 1 , wherein the antibody, antigen-binding fragment thereof, or immunoadhesin comprises 1, 2, 3, 4, or more binding sites responsible for selectively binding to a target antigen of interest.
12 . The method of claim 11 , wherein at least one binding site comprises a ligand binding site of a receptor, or a receptor binding site of a ligand, optionally wherein the ligand is selected from the group consisting of a polysaccharide, a sugar molecule, a carbohydrate.
13 . The method of claim 1 , wherein the antibody, or antigen-binding fragment thereof, comprises a single chain variable region (ScFv) sequence, or the antibody, or antigen-binding fragment thereof, comprises one binding site comprising an antibody variable domain.
14 . The method of claim 1 , wherein the antibody, or antigen binding fragment thereof, is bispecific, optionally wherein the bispecific antibody, or antigen-binding fragment thereof, is a cross-over dual variable domain IgG (CODV-IgG) bispecific antibody or antigen-binding fragment thereof, or a trispecific antibody, or antigen-binding fragment thereof.
15 . The method of claim 1 , wherein the antibody, or antigen-binding fragment thereof, comprises a constant domain, and wherein:
(a) the modified glycan is N-linked to the antibody, or antigen-binding fragment thereof, via an asparagine residue at amino acid position 297 of a Fc domain, according to EU numbering,
(b) the modified glycan is N-linked to the antibody, or antigen-binding fragment thereof, via an asparagine residue at amino acid position 298 of a Fc domain, according to EU numbering, or
(c) the modified glycan is N-linked to the to the antibody, or antigen-binding fragment thereof, via an asparagine residue at amino acid position 114 of a CHI domain, according to Kabat numbering; or
(d) the Fc domain is human.
16 . A method of making a PEGylated binding polypeptide, wherein the method comprises:
(a) reacting a binding polypeptide comprising at least one glycan with a periodate oxidase or galactose oxidase in the presence of a salt comprising a copper metal ion to generate a binding polypeptide comprising at least one oxidized glycan, and
(b) conjugating the oxidized glycan to at least one amino-oxy or hydrazine containing PEG moiety, thereby generating the PEGylated binding polypeptide, wherein the binding polypeptide is an antibody, an antigen binding fragment thereof, or an immunoadhesin.
17 . The method of claim 16 , wherein the binding polypeptide is reacted with a periodate oxidase.
18 . The method of claim 17 , wherein the periodate oxidase is sodium periodate.
19 . The method of claim 18 , wherein the salt is copper acetate.
20 . The method of claim 19 , wherein the salt comprising the copper metal ion is present at a concentration of at least 0.1 mM.
21 . The method of claim 17 , wherein the binding polypeptide is an antibody or antigen binding fragment thereof.
22 . The method of claim 17 , wherein the glycan comprises a terminal sialic acid moiety.
23 . The method of claim 16 , wherein the binding polypeptide is reacted with a galactose oxidase.
24 . The method of claim 23 , wherein the salt is copper acetate.
25 . The method of claim 24 , wherein the salt comprising the copper metal ion is present at a concentration of at least 0.1 mM.
26 . The method of claim 23 , wherein the binding polypeptide is an antibody or antigen binding fragment thereof.
27 . The method of claim 23 , wherein the glycan comprises a terminal galactose moiety.