Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof

1. A method for producing a bispecific antibody comprising the steps of:

(a) incubating under a condition that allows a sortase-mediated transpeptidation reaction

(i) an antibody Fab fragment or a scFv antibody that specifically binds to a first epitope or antigen, comprising within its 20 C-terminal amino acid residues the amino acid sequence GnSLPX1TG (SEQ ID NO:2, wherein X1 can be any amino acid residue, with n=1, 2, or 3), wherein the amino acid sequence GnSLPX1TG is located C-terminal to the variable domain of the antibody Fab fragment or scFv, and

(ii) a one-armed antibody fragment comprising a full length antibody heavy chain, a full length antibody light chain, and an antibody heavy chain Fc-region polypeptide, wherein the one-armed antibody fragment specifically binds to a second epitope or antigen,

wherein the full length antibody heavy chain and the full length antibody light chain are cognate antibody chains complementary to each other and the pair of variable domains (VH and VL) thereof forms an antigen binding site,

wherein the full length antibody heavy chain and the antibody heavy chain Fc-region polypeptide are covalently linked to each other via one or more disulfide bonds forming an antibody hinge region, and

wherein the antibody heavy chain Fc-region polypeptide has the amino acid sequence GGCPX4C (SEQ ID NO: 08, wherein X4 is either S or P), at its N-terminus

and

(iii) a Sortase A enzyme,

and

(b) producing the bispecific antibody.

2. The method of claim 1 , wherein the Fc-region comprises a mutation of the naturally occurring amino acid residue at position 329 and at least one further mutation of at least one amino acid residue selected from the group consisting of amino acid residues at position 228, 233, 234, 235, 236, 237, 297, 318, 320, 322 and 331 to a different residue, wherein the residues in the Fc-region are numbered according to the EU index of Kabat.

3. The method of claim 1 , wherein the antibody Fab fragment or scFv antibody is an antibody Fab fragment.

4. A bispecific antibody obtained by a method according to claim 1 .

5. A pharmaceutical formulation comprising a bispecific antibody produced by the method of claim 1 .

6. A method for producing a bispecific antibody comprising:

(i) determining the cell surface makers present in a cell-containing sample and selecting thereof at least a first cell surface marker or its ligand and a second cell surface marker or its ligand,

(ii) incubating under a condition that allows a sortase-mediated transpeptidation reaction

(a) an antibody Fab fragment or a scFv antibody comprising within the 20 C-terminal amino acid residues the amino acid sequence GnSLPX1TG (SEQ ID NO:02, wherein X1 can be any amino acid residue with n=1, 2, or 3), wherein the amino acid sequence GnSLPX1TG is located C-terminal to the variable domain of the antibody Fab fragment or scFv, and wherein the Fab fragment or scFv antibody specifically binds to the first cell surface marker or its ligand and

(b) a one-armed antibody fragment comprising a full length antibody heavy chain, a full length antibody light chain, and an antibody heavy chain Fc-region polypeptide, wherein the full length antibody heavy chain and the full length antibody light chain are cognate antibody chains complementary to each other and the pair of variable domains (VH and VL) thereof forms an antigen binding site that specifically binds to the second cell surface marker or its ligand,

wherein the full length antibody heavy chain and the antibody heavy chain Fc-region polypeptide are covalently linked to each other via one or more disulfide bonds forming an antibody hinge region, and wherein the antibody heavy chain Fc-region polypeptide has the amino acid sequence GGCPX4C (SEQ ID NO: 08, wherein X4 is either S or P) at its N-terminus, and

(c) a Sortase A enzyme,

and

(iii) producing the bispecific antibody.

7. A method for determining a combination of antigen binding sites comprising:

(i) incubating under a condition that allows a sortase-mediated transpeptidation reaction

(a) each member of a first multitude of antibody Fab fragments or scFv antibody fragments wherein each member comprises within its 20 C-terminal amino acid residues the amino acid sequence GnSLPX1TG (SEQ ID NO: 02), wherein X1 can be any amino acid residue with n=1, 2, or 3, wherein the amino acid sequence GnSLPX1TG is located C-terminal to the variable domain of the antibody Fab fragment or scFv, and

wherein the Fab fragment or scFv antibody specifically binds to a first epitope or antigen, with

(b) each member of a multitude of one-armed antibody fragments comprising a full length antibody heavy chain, a full length antibody light chain, and an antibody heavy chain Fc-region polypeptide, wherein the full length antibody heavy chain and the full length antibody light chain are cognate antibody chains complementary to each other and the pair of variable domains (VH and VL) thereof forms an antigen binding site that specifically binds to a second epitope or antigen,

wherein the full length antibody heavy chain and the antibody heavy chain Fc-region polypeptide are covalently linked to each other via one or more disulfide bonds forming an antibody hinge region, and wherein the antibody heavy chain Fc-region polypeptide has the amino acid sequence GGCPX4C (SEQ ID NO: 08), wherein X4 is either S or P at its N-terminus, and

(c) a Sortase A enzyme,

wherein a multitude of bispecific antibodies is prepared,

(ii) determining the binding specificity, or selectivity, or affinity, or effector function, or in vivo half-life of the multitude of bispecific antibodies,

(iii) choosing the bispecific antibody with suitable binding specificity, or selectivity, or affinity, or effector function, or in vivo half-life and

(iv) determining a combination of antigen binding sites.

8. The method of claim 7 , wherein the antibody Fab fragment or scFv antibody is an antibody Fab fragment.