Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof
Herein is reported a method for producing an antibody Fc-region conjugate comprising as first component an antibody Fc-region and as second component at least one binding entity that specifically binds to a target using a transpeptidase for enzymatic conjugation of the antibody Fc-region to at least one binding entity.
1. A method for producing an antibody Fc-region conjugate, which comprises as a first component an antibody Fc-region and as a second component a Fab that specifically binds to a target, wherein the method comprises incubating:
(i) a Fab fragment comprising the amino acid sequence KSCGSLPETGGSGSHHHHHH (SEQ ID NO: 76) within its 20 C-terminal amino acid residues, and an antibody Fc-region, comprising two heavy chain fragments each comprising an N-terminus, which comprises at one of its N-termini the amino acid sequence GGCPPC (SEQ ID NO: 17), with the enzyme Sortase A; or
(ii) a Fab fragment comprising the amino acid sequence KSCGGGSLPETGGSGSHHHHHH (SEQ ID NO: 75), and an antibody Fc-region, comprising two heavy chain fragments each comprising an N-terminus, which comprises at one of its N-termini the amino acid sequence GGCPPC (SEQ ID NO: 17), with the enzyme Sortase A,
thereby producing the antibody Fc-region conjugate.
2. The method of claim 1 , further comprising, prior to incubating the antibody Fc-region and the Fab fragment:
(a) culturing a first host cell comprising a nucleic acid encoding the antibody Fc-region component of the conjugate under conditions suitable for expression of the antibody Fc-region; and
(b) culturing a second host cell comprising a nucleic acid encoding the Fab fragment under conditions suitable for expression of the Fab fragment.
3. The method of claim 1 , wherein the Fc-region comprises, at both N-termini, the amino acid sequence GGCPPC (SEQ ID NO: 17).