Method for selection and production of multi-specific targeting entities containing at least an antibody fc-region and an antigen binding peptide and uses thereof
Herein are reported antibody Fc-region conjugates useful for the treatment of disease and methods for producing the same comprising incubating under conditions that allow for sortase mediated transpeptidation: (i) a dimeric antibody heavy chain Fc region polypeptide comprising the amino acid sequence GGCPX4C, wherein X4 is either S or P (SEQ ID NO: 08) at its N terminus (ii) an antigen binding polypeptide comprising within its 20 C-terminal amino acid residues the amino acid sequence GnSLPX1TG, wherein X1 is any amino acid residue, with n=1, 2, or 3 (SEQ ID NO:2); and (iii) a Sortase A enzyme; and thereby producing the antibody Fc-region conjugate, wherein the antibody Fc-region conjugate comprises two polypeptide chains, and at least one polypeptide chain comprises the amino acid sequence GnSLPX1TGGCPX4C, wherein n=1, 2 or 3, X1 is any amino acid residue, and X4 is either S or P (SEQ ID NO 9).
1 . A method of producing an antibody Fc-region conjugate comprising an antibody heavy chain Fc-region polypeptide and an antigen binding polypeptide, comprising incubating under conditions for sortase-mediated transpeptidation:
i) a Fc-region comprising two antibody heavy chain Fc-region polypeptides, wherein one or both of the two antibody heavy chain Fc-region polypeptides comprise the amino acid sequence GGCPX4C, wherein X4 is either S or P (SEQ ID NO: 08), at its N-terminus;
ii) an antigen binding polypeptide comprising within its 20 C-terminal amino acid residues the amino acid sequence G n SLPX1TG, wherein X1 is any naturally occurring amino acid residue, with n=1, 2, or 3 (SEQ ID NO: 02); and
iii) a Sortase A enzyme;
wherein the Sortase A enzyme performs a transpeptidation reaction that connects the heavy chain Fc-region polypeptide comprising the amino acid sequence GGCPX4C, wherein X4 is either S or P (SEQ ID NO: 08) with the antigen binding polypeptide comprising within its 20 C-terminal amino acid residues the amino acid sequence G n SLPX1TG;
and thereby producing the antibody Fc-region conjugate, wherein the antibody Fc-region conjugate comprises two or more polypeptide chains, and at least one polypeptide chain of the two or more polypeptide chains comprises one of the antibody heavy chain Fc-region polypeptides, the antigen binding polypeptide and an amino acid sequence G n SLPX1TGGCPX4C, wherein n=1, 2 or 3, X1 is any naturally occurring amino acid residue, and X4 is either S or P (SEQ ID NO: 9).
2 . The method of claim 1 , wherein the antigen binding polypeptide is selected from the group consisting of a scFv polypeptide, a darpin polypeptide, a dsFv polypeptide, a Fab polypeptide, a Fab′ polypeptide, a Fab′-SH polypeptide, a F(ab′)2 polypeptide, a diabody polypeptide, a linear antibody polypeptide, and a scFab polypeptide.
3 . The method of claim 1 , wherein the antigen binding polypeptide comprises within its 20 C-terminal amino acid residues the amino acid sequence GSLPX1TGGSGS, wherein X1 is any naturally occurring amino acid residue (SEQ ID NO: 03).
4 . The method of claim 1 , wherein the antigen binding polypeptide comprises within its 20 C-terminal amino acid residues the amino acid sequence GGGSLPX1TGGSGS, wherein X1 is any naturally occurring amino acid residue (SEQ ID NO: 04).
5 . The method of claim 1 , wherein the antigen binding polypeptide comprises within its 20 C-terminal amino acid residues the amino acid sequence X2GSLPX1TGGSGS wherein X1 is any naturally occurring amino acid residue and X2 is any naturally occurring amino acid residue except G (SEQ ID NO: 05).
6 . The method of claim 1 , wherein the antigen binding polypeptide comprises within its 20 C-terminal amino acid residues the amino acid sequence G n SLPX1TGGSGSX3, wherein X1 is any naturally occurring amino acid residue, n=1, 2 or 3 and X3 is an amino acid sequence tag (SEQ ID NO: 06).
7 . The method of claim 1 , wherein the antigen binding polypeptide comprises within its 20 C-terminal amino acid residues the amino acid sequence X2GSLPX1TGGSGSX3, wherein X1 is any naturally occurring amino acid residue, X2 is any naturally occurring amino acid residue except G, and X3 is an amino acid sequence tag (SEQ ID NO: 07).
8 . The method of claim 1 , wherein the one or both of the two antibody heavy chain Fc-region polypeptides comprises at its N-terminus, the amino acid sequence GGCPPC, wherein X4 is P (SEQ ID NO: 08).
9 . The method of claim 1 , wherein the antibody Fc-region is of the human IgG1 subclass.
10 . The method of claim 1 , wherein one or both of the two antibody heavy chain Fc-region polypeptides comprises the amino acid sequence X2GSLPX1TGGCPX4C, wherein X1 is any naturally occurring amino acid residue, X4 is S or P, and X2 is any naturally occurring amino acid residue except G (SEQ ID NO: 10).
11 . The method of claim 1 , wherein the antibody Fc-region conjugate is a one-armed antibody.
12 . The method of claim 1 , wherein the antigen binding polypeptide is a Fab polypeptide.
13 . The method of claim 1 , wherein
(i) the antibody heavy chain Fc-region polypeptide is produced by culturing a first host cell comprising nucleic acid(s) encoding the two antibody heavy chain Fc-region polypeptides under conditions for expression of the two antibody heavy chain Fc-region polypeptides in a first culture medium; and
(ii) the antigen binding polypeptide is produced by culturing a second host cell comprising nucleic acid(s) encoding an antigen binding fragment comprising the antigen binding polypeptide under conditions for expression of the antigen binding fragment in a second culture medium.
14 . The method of claim 13 , further comprising recovering the two antibody heavy chain Fc-region polypeptides from the first host cell or the first culture medium and/or recovering the antigen binding polypeptide from the second host cell or the second culture medium prior to the incubation with Sortase A.
15 . The method of claim 1 , wherein one or both of the two antibody heavy chain Fc-region polypeptides comprises a P329G mutation, and at least one mutation selected from the group consisting of S228P, E233P, L234A, L235A, L235E, N297A, N297D, P329G, and P331S, wherein the numbering is according to EU index of Kabat.