Modified FC polypeptides, FC conjugates, and methods of use thereof
View Patent ↗The present disclosure provides modified Fc domains having one or more attachment moieties for attaching a heterologous functional moiety; and methods of generating the modified Fc domains. The present disclosure provides Fc conjugates; and methods of making the conjugates. The Fc conjugates are useful in various methods, which are also provided.
1. A modified Fc polypeptide, wherein the modified Fc polypeptide is described by the following structure:
wherein:
L 1 is a linker;
X is O or NH;
R′ and R″ are each independently H, an alkyl or an aryl;
R is one or more groups, independently selected from hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a halogen, an amino, an acyl, an acyloxy, an amido and nitro;
R 1 is the sidechain of an amino acid, H, an alkyl or an aryl; and
Z 1 is an Fc polypeptide.
2. The modified Fc polypeptide of claim 1 , wherein:
i) Z 1 includes an N-terminal sequence Xaa 2 -Xaa 3 , wherein Xaa 2 and Xaa 3 are independently amino acid residues and at least one of Xaa 2 and Xaa 3 is a basic amino acid residue; and
and R 1 is the amino acid sidechain of alanine; or
ii) Z 1 includes an N-terminal sequence Xaa 2 -Xaa 3 , wherein Xaa 2 and Xaa 3 are independently amino acid residues, and at least one of Xaa 2 and Xaa 3 is an acidic amino acid residue; or
iii) Z 1 includes an N-terminal sequence Xaa 2 -Xaa 3 , wherein Xaa 2 and Xaa 3 are independently amino acid residues; and
R 1 is the amino acid sidechain of glutamic acid or aspartic acid.
3. The modified Fc polypeptide of claim 1 , wherein:
a) L 1 is —(CH 2 ) m —NHCO—CH 2 — or —(CH 2 ) m —CONH—CH 2 —, and X is O or NH, wherein m is 1 to 6; or
b) L 1 is —(CH 2 ) m —NHCO— or —(CH 2 ) m —CONH—, and X is NH, wherein m is 1 to 6.
4. The modified Fc polypeptide of claim 1 , wherein the modified Fc polypeptide comprises an antigen-binding domain.
5. A method of conjugating a heterologous functional moiety to the modified Fc polypeptide of claim 1 , the method comprising:
contacting the modified Fc polypeptide of claim 1 with a heterologous functional moiety comprising an aminophenol, a 2-methoxyphenol, an aniline, an azidophenol, or a phenylene diamine group under conditions sufficient to produce a Fc conjugate via oxidative coupling.
6. The method of claim 5 , wherein the Fc conjugate is described by one of the following structures:
wherein:
L 1 and L 5 are optional linkers;
Z 2 is a heterologous functional moiety;
X is O or NH;
R 1 is the sidechain of an amino acid, H, an alkyl or an aryl; and
Z 1 is an Fc polypeptide.
7. The method of claim 6 , wherein:
a) Z 1 includes N-terminal lysine-threonine residues, and R 1 is the amino acid sidechain of alanine; or
b) R 1 is the amino acid sidechain of glutamic acid or aspartic acid.
8. An Fc conjugate comprising:
a) a modified Fc polypeptide of claim 1 ; and
b) a covalently linked heterologous functional moiety.
9. The Fc conjugate of claim 8 , wherein the heterologous functional moiety is a polypeptide, a nucleic acid, a peptoid, an aptamer, an oligosaccharide, a nanoparticle, a small molecule drug, a ligand, a polymer, a liposome, or a drug delivery vehicle.
10. The Fc conjugate of claim 8 , wherein the Fc conjugate is described by one of the following structures:
wherein:
L 1 and L 5 are optional linkers;
Z 2 is a heterologous functional moiety;
X is O or NH;
R 1 is the sidechain of an amino acid, H, an alkyl or an aryl; and
Z 1 is an Fc polypeptide.
11. The Fc conjugate of claim 8 , wherein the modified Fc polypeptide comprises an antigen-binding domain.
12. An antibody comprising:
a) the Fc conjugate of claim 8 ; and
b) an antigen-binding region.
13. The modified Fc polypeptide of claim 1 , wherein R is H or hydroxyl and R′ and R″ are each H.
14. A method of preparing a modified Fc polypeptide, the method comprising:
contacting a parent Fc polypeptide with a transamination reagent to convert the terminal amino group of the parent Fc polypeptide to a ketone or aldehyde group and produce a modified Fc polypeptide; and
contacting the modified Fc polypeptide with a bifunctional moiety described by the formula:
wherein
R is one or more optional aryl substituents;
R′ and R″ are each independently H, an alkyl or an aryl;
L 6 is an optional linker; and
Y 1 is —CONHNH 2 , —O—NH 2 , or —NHNH 2 , under conditions sufficient to crosslink the a ketone or aldehyde group to the bifunctional moiety.
15. The method of claim 14 , wherein the parent Fc polypeptide is mutated to change the N-terminal amino acid of the parent Fc polypeptide to produce a mutated parent Fc polypeptide, and wherein the mutated parent Fc polypeptide is contacted with the transamination reagent to produce the modified Fc polypeptide.
16. The method of claim 15 , wherein:
a) the mutated Fc polypeptide comprises an N-terminal alanine or an N-terminal glutamic acid; or
b) the mutated Fc polypeptide comprises N-terminal alanine-lysine-threonine residues.
17. The method of claim 14 , wherein the transamination reagent is pyridoxal 5′-phosphate or N-methylpyridinium-4-carboxaldehyde.