Recombinant cell surface capture proteins

A method of detecting and isolating cells that produce high levels of a bispecific antibody, comprising:

• (a) transfecting cells with a nucleic acid that encodes a cell surface capture protein (CSCP), which is a fusion protein comprising a recombinant antigen-binding protein and a membrane anchor domain, wherein the transfected cells express the bispecific antibody, wherein the bispecific antibody comprises multiple subunits and a first site on the bispecific antibody resides on a first subunit, and a second site on the bispecific antibody resides on a second subunit, and wherein (i) the CSCP binds to the first site on the bispecific antibody to form a CSCP-bispecific antibody complex inside the transfected cells, (ii) the CSCP-bispecific antibody complex is transported through the transfected cells, and (iii) then displayed on the surface of the transfected cells;
• (b) detecting a cell of (a) that expresses the CSCP in high yield;
• (c) isolating and culturing the cell that expresses the CSCP in high yield;
• (d) detecting the bispecific antibody on the surface of the isolated and cultured cell of step (c) with a detection molecule (DM), comprising a labeled recombinant antigen-binding domain, wherein the detection molecule binds to the second site on the bispecific antibody;
• and
• (e) isolating the cell detected in step (d) that bears the detected bispecific antibody on its surface;
• wherein the first site on the first subunit of the bispecific antibody comprises:
• (i) a CH3 domain having a histidine residue at position 95 (95H) according to the IMGT exon numbering system, and a tyrosine residue at position 96 (96Y) according to the IMGT exon numbering system (Fc);
• and
• wherein the second site on the second subunit of the bispecific antibody comprises:
• (ii) a CH3 domain containing an arginine residue at position 95 (95R) according to the IMGT exon numbering system and a phenylalanine residue at position 96 (96F) according to the IMGT exon numbering system (Fc*);
• wherein the bispecific antibody comprises both the Fc and the Fc*;
• wherein the recombinant antigen-binding protein specifically binds the Fc;
• wherein the DM specifically binds to the Fc*.

A method of high expression of a bispecific antibody of interest, comprising:

• (a) transfecting a mammalian cell with (i) a nucleic acid encoding the bispecific antibody of interest and (ii) a nucleic acid encoding a cell surface capture molecule that comprises a recombinant antigen-binding protein and a membrane anchor;
• wherein the bispecific antibody of interest comprises multiple subunits, and a first site on the bispecific antibody of interest resides on a first subunit, and a second site on the bispecific antibody of interest resides on a second subunit;
• (b) culturing the mammalian cell under conditions in which the bispecific antibody of interest and cell surface capture molecule are expressed, and a bispecific antibody of interest-cell surface capture molecule complex is formed and displayed on the cell surface of the mammalian cell;
• (c) contacting the mammalian cell with a detection molecule (DM) comprising a labeled recombinant antigen-binding domain, wherein the detection molecule (DM) binds to the second site on the bispecific antibody of interest;
• and
• (d) detecting and isolating the mammalian cell that displays on the cell surface the binding of the detection molecule (DM) with the bispecific antibody of interest-cell surface capture molecule complex;
• wherein the first site on the first subunit of the bispecific antibody of interest comprise:
• (i) a CH3 domain having a histidine residue at position 95 (95H) according to the IMGT exon numbering system, and a tyrosine residue at position 96 (96Y) according to the IMGT exon numbering system (Fc);
• and
• wherein the second site on the second subunit of the bispecific antibody of interest comprises:
• (ii) a CH3 domain containing an arginine residue at position 95 (95R) according to the IMGT exon numbering system and a phenylalanine residue at position 96 (96F) according to the IMGT exon numbering system (Fc*);
• wherein the bispecific antibody comprises both the Fc and the Fc*;
• wherein the recombinant antigen-binding protein specifically binds the Fc;
• wherein the detection molecule (DM) specifically binds to the Fc*.