METHOD FOR THE PRODUCTION AND SELECTION OF MOLECULES COMPRISING AT LEAST TWO DIFFERENT ENTITIES AND USES THEREOF
First Claim
1. A method for producing a polypeptide comprising at least two polypeptide domains comprising the step ofcultivating a cell comprisinga) a nucleic acid encoding a soluble S. aureus sortase A with a C-terminal endoplasmic reticulum retention signal,b) a nucleic acid encoding a first polypeptide domain comprising at its C-terminus a sortase motif followed by an endoplasmic reticulum retention signal, andc) a nucleic acid encoding a second polypeptide domain comprising at its N-terminus at least a diglycine,wherein the cell secretes the sortase A conjugate of the first polypeptide domain and the second polypeptide domain,thereby producing a polypeptide comprising at least two polypeptide domains.
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Abstract
Herein is reported a method for producing a polypeptide comprising at least two polypeptide domains comprising the step of cultivating a cell comprising (a) a nucleic acid encoding a soluble S. aureus sortase A with a C-terminal endoplasmic reticulum retention signal, (b) a nucleic acid encoding a first polypeptide domain comprising at its C-terminus a sortase motif followed by an endoplasmic reticulum retention signal, and (c) a nucleic acid encoding a second polypeptide domain comprising at its N-terminus at least a diglycine, whereby the cell secretes the sortase A conjugate of the first polypeptide domain and the second polypeptide domain, thereby producing a polypeptide comprising at least two polypeptide domains.
20 Citations
21 Claims
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1. A method for producing a polypeptide comprising at least two polypeptide domains comprising the step of
cultivating a cell comprising a) a nucleic acid encoding a soluble S. aureus sortase A with a C-terminal endoplasmic reticulum retention signal, b) a nucleic acid encoding a first polypeptide domain comprising at its C-terminus a sortase motif followed by an endoplasmic reticulum retention signal, and c) a nucleic acid encoding a second polypeptide domain comprising at its N-terminus at least a diglycine, wherein the cell secretes the sortase A conjugate of the first polypeptide domain and the second polypeptide domain, thereby producing a polypeptide comprising at least two polypeptide domains.
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2. A method for producing a multispecific binder comprising at least two binding entities comprising the step of
cultivating a cell comprising a) a nucleic acid encoding a soluble S. aureus sortase A with a C-terminal endoplasmic reticulum retention signal, b) a nucleic acid encoding a first binding entity comprising at its C-terminus a sortase motif followed by an endoplasmic reticulum retention signal, and c) a nucleic acid encoding a second binding entity comprising at its N-terminus at least a diglycine, wherein the cell secretes the sortase A conjugate of the first binding entity and the second binding entity, and wherein the first binding entity specifically binds to a first antigen or target and the second binding entity specifically binds to a second antigen or target, thereby producing a multispecific binder comprising at least two binding entities.
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13. A method for producing a bispecific antibody comprising the following steps
(i) determining the cell surface makers present in a cell containing sample and selecting thereof at least a first surface marker and a second surface marker, (ii) transfecting a cell with (a) a nucleic acid encoding an antibody Fab fragment, or an antibody scFab, or a scFv antibody comprising within the 20 C-terminal amino acid residues the amino acid sequence LPXTG (SEQ ID NO: - 01, wherein X can be any amino acid residue) followed by an endoplasmic reticulum retention signal KDEL (SEQ ID NO;
02), wherein the Fab fragment or scFv antibody specifically binds to the first surface marker or its ligand, (b) a nucleic acid encoding an 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 surface marker or its ligand, and 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 an oligoglycine Gm (m=2, or 3, or 4, or
5) SEQ ID NO;
53) amino acid sequence at its N-terminus, and (c) a nucleic acid encoding a soluble S. aureus sortase A with a C-terminal endoplasmic reticulum retention signal,and thereby producing the bispecific antibody. - View Dependent Claims (15, 17, 18, 20, 21)
- 01, wherein X can be any amino acid residue) followed by an endoplasmic reticulum retention signal KDEL (SEQ ID NO;
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14. A method for determining a combination of antigen binding sites comprising the following steps
(i) determining the binding specificity and/or selectivity and/or affinity and/or effector function and/or in vivo half-life of a multitude of bispecific antibodies prepared by combining (a) each member of a first multitude of antibody Fab fragments or scFv antibody fragments wherein each member comprises within the 20 C-terminal amino acid residues the amino acid sequence LPXTG (SEQ ID NO: - 01, wherein X can be any amino acid residue) followed by an endoplasmic reticulum retention signal KDEL (SEQ ID NO;
02), and 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 an oligoglycine Gm (m=2, or 3, or 4, or
5) SEQ ID NO;
53) amino acid sequence at its N-terminus, via a S. aureus sortase A-mediated intracellular enzymatic coupling reaction,and (ii) choosing the bispecific antibody with suitable binding specificity and/or selectivity and/or affinity and/or effector function and/or in vivo half-life and thereby determining a combination of antigen binding sites.
- 01, wherein X can be any amino acid residue) followed by an endoplasmic reticulum retention signal KDEL (SEQ ID NO;
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16. A bispecific antibody comprising the amino acid sequence LPXTG (SEQ ID NO:
- 01, wherein X can be any amino acid residue) in one of its heavy chains.
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19. (canceled)
Specification