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

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 GnSLPX1TG, 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 GnSLPX1TG;
• 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 GnSLPX1TGGCPX4C, 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).