Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format

A method for making an Fc-containing protein comprising an Fc region that is heterodimeric with respect to Protein A binding, comprising:

• (a) introducing into a mammalian cell a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a first polypeptide that comprises a first functional CH3 domain of a human IgG1 or a human IgG4 constant region, wherein the first functional CH3 domain binds to Protein A;
• and the second nucleic acid sequence encodes a second polypeptide that comprises a second functional CH3 domain of a human IgG1 or a human IgG4 constant region, wherein the second functional CH3 domain comprises a modification that reduces or eliminates binding of the second functional CH3 domain to Protein A, wherein the modification that reduces or eliminates binding of the second functional CH3 domain to Protein A comprises a 95R modification, a 96F modification, or both a 95R and a 96F modification;
• (b) allowing the cell to express an Fc-containing protein comprising the first and second polypeptides;
• and
• (c) isolating the Fc-containing protein based on the ability of the Fc-containing protein to bind Protein A.

A method for making a bispecific antibody, comprising:

• (a) culturing a cell containing:
• (i) a first nucleic acid sequence encoding a first immunoglobulin heavy chain comprising, from N-terminus to C-terminus, a first variable domain that selectively binds a first epitope, an immunoglobulin constant region that comprises a first CH3 domain of a human IgG selected from IgG1, IgG2 and IgG4, wherein the first CH3 domain binds Protein A;
• (ii) a second nucleic acid sequence encoding a second human immunoglobulin heavy chain comprising, from N-terminus to C-terminus, a second variable domain that selectively binds a second epitope, an immunoglobulin constant region that comprises a second CH3 domain of a human IgG selected from IgG1, IgG2 and IgG4, wherein the second CH3 domain comprises a modification that reduces or eliminates binding of the second CH3 domain to Protein A, wherein the modification that reduces or eliminates binding of the second functional CH3 domain to Protein A comprises a 95R modification, a 96F modification, or both a 95R and a 96F modification;
• and
• (iii) a third nucleic acid sequence encoding an immunoglobulin light chain that pairs with the first and the second immunoglobulin heavy chains;
• under conditions that allow the cell to express the first and the second immunoglobulin heavy chains and the immunoglobulin light chain to generate the bispecific antibody, wherein the bispecific antibody is heterodimeric with respect to Protein A binding;
• and
• (b) isolating the bispecific antibody from the cell using Protein A.