Recombinant immunoglobulin heavy chains comprising a sortase conjugation loop and conjugates thereof
The disclosure describes a recombinant immunoglobulin heavy chain comprising a sortase conjugation loop, methods for conjugating and/or labeling such recombinant immunoglobulin heavy chains by use of the enzyme sortase and to the conjugates/labeled products obtained via the method.
1. A recombinant immunoglobulin heavy chain comprising a VH-domain, a CH1 domain, a hinge region down to the C-terminal most cysteine of the middle hinge region, a sortase conjugation loop, a CH2 domain and a CH3 domain,
wherein the sortase conjugation loop comprises:
a) at least 16 amino acids located between the C-terminal most cysteine of the middle hinge region and the VFX1FPP (SEQ ID NO: 2; wherein X1 is L or I) consensus sequence at the N-terminus of an immunoglobulin heavy chain CH2-domain,
b) a sortase recognition motif of the amino acid sequence LPX1TX2 (SEQ ID NO: 01), wherein X1 can be any amino acid residue and X2 is G or A,
c) N-terminal to the sequence of (b) at least two amino acids, and
d) C-terminal to the sequence of (b) at least 6 amino acids, and
wherein the amino acids C- and N-terminal to the sequence of (b) are not cysteine.
2. The recombinant immunoglobulin heavy chain of claim 1 , wherein said sortase conjugation loop comprises at least 9 amino acids C-terminal to the sequence LPX1TX2 (SEQ ID NO: 01, wherein X1 can be any amino acid residue and X2 is G or A).
3. The recombinant immunoglobulin heavy chain of claim 1 , wherein said sortase conjugation loop comprises at least 5 amino acids N-terminal to the sequence LPX1TX2 (SEQ ID NO: 01, wherein X1 can be any amino acid residue and X2 is G or A).
4. The recombinant immunoglobulin heavy chain of claim 1 , wherein said sortase conjugation loop comprises at least 8 amino acids N-terminal to the sequence LPX1TX2 (SEQ ID NO: 01, wherein X1 can be any amino acid residue and X2 is G or A).
5. The recombinant immunoglobulin heavy chain according to claim 1 , wherein said sortase conjugation loop comprises at least 5 amino acids N-terminal to the sequence LPX1TX2 (SEQ ID NO: 01, wherein X1 can be any amino acid residue and X2 is G or A), and at least 9 amino acids C-terminal to said sequence.
6. The recombinant immunoglobulin heavy chain according to claim 1 , wherein said sortase conjugation loop comprises at least 8 amino acids N-terminal to the sequence LPX1TX2 (SEQ ID NO: 01, wherein X1 can be any amino acid residue and X2 is G or A), and at least 9 amino acids C-terminal to said sequence.
7. The recombinant immunoglobulin heavy chain according to claim 1 , wherein said sortase conjugation loop is at most 50 amino acids long.
8. An antibody comprising at least two recombinant immunoglobulin heavy chains according to claim 1 .
9. An IgG class antibody having two recombinant immunoglobulin heavy chains according to claim 1 .
10. A method for producing a conjugate of a sortase cleavage fragment of a recombinant immunoglobulin heavy chain and a sortase nucleophile comprising incubating
i) the antibody according to claim 7 ,
ii) a compound comprising a sortase nucleophile, and
iii) a polypeptide with sortase activity, thereby cleaving the recombinant antibody heavy chain after the threonine in LPX1TX2 (SEQ ID NO: 01, wherein X1 can be any amino acid residue and X2 is G or A) and conjugating the N-terminus of the sortase nucleophile to said threonine.
11. The method according to claim 10 , wherein the sortase nucleophile comprises a tag of interest and has the formula (Gly) n -tag, wherein n is at least 2.
12. The method according to claim 10 , wherein the sortase nucleophile comprises a tag of interest and has the formula (Gly) n -tag and wherein n is selected from 2, 3, 4, 5 or 6.
13. The method according to claim 10 , wherein the sortase nucleophile comprises a tag of interest and has the formula (Gly) n -tag, wherein n is at most 30.