Recombinant Cell Surface Capture Proteins

Abstract

Recombinant cell surface capture proteins and detection molecules that are useful for isolating and detecting cells that produce a secreted heterodimeric protein of interest (POI) that has an immunoglobulin CH3 domain and/or substituted CH3 domain are provided. Recombinant cell surface capture proteins and detection molecules that isolate and detect bispecific antibodies are also provided. The invention also provides recombinant antigen-binding proteins that are capable of recognizing and binding to proteins of interest that contain a CH3 domain and/or a modified CH3 domain, such as a CH3 domain with or without amino acid substitutions at H95 and Y96 (IMGT).

1 - 56 . (canceled) 
     
     
         57 . A method of detecting or isolating a cell that stably expresses a heterodimeric protein comprising the steps of:
 (a) expressing in a host cell a cell surface capture protein (CSCP) comprising a recombinant antigen-binding protein, and a heterodimeric protein, wherein the heterodimeric protein comprises multiple subunits and a first site on the heterodimeric protein resides on a first subunit, and a second site on the heterodimeric protein resides on a second subunit and, wherein (i) the CSCP binds to the first site on the heterodimeric protein to form a CSCP-heterodimeric protein complex inside the host cell, (ii) the CSCP-heterodimeric protein complex is transported through the host cell, and (iii) then displayed on the surface of the host cell;   (b) contacting the host cell with a detection molecule, wherein the detection molecule binds to the second site on the heterodimeric protein; and   (c) selecting the host cell which binds the detection molecule.   
     
     
         58 . The method of  claim 57 , wherein:
 (a) the heterodimeric protein comprises an antibody;   (b) the first subunit of the heterodimeric protein comprises a heavy chain domain comprising a wild type CH3 domain; or   (c) the first subunit of the heterodimeric protein comprises a heavy chain comprising a CH3 domain having a histidine residue at position 95 according to the IMGT exon numbering system and a tyrosine residue at position 96 according to the IMGT exon numbering system.   
     
     
         59 . The method of  claim 57  further comprising a step of contacting the cell with a blocking molecule, wherein the blocking molecule binds to the CSCP that is not bound to the heterodimeric protein, but does not bind to the CSCP-heterodimeric protein complex. 
     
     
         60 . The method of  claim 59 , wherein the blocking molecule is a non-human IgG or a human Fc molecule. 
     
     
         61 . The method of  claim 57 , wherein the cell is detected and selected by fluorescence activated cell sorting. 
     
     
         62 . The method of  claim 57 , wherein the detection molecule comprises a recombinant antigen-binding protein that specifically binds a substituted CH3 polypeptide comprising one or more amino acid substitutions selected from the group consisting of (a) 95R, and (b) 95R and 96F according to the IMGT exon numbering system, or (a′) 435R, and (b′) 435R and 436F according to the EU numbering system. 
     
     
         63 . The method of  claim 57 , wherein the CSCP comprises a recombinant antigen-binding protein that binds a human IgG1-Fc domain, a human IgG2-Fc domain, or a human IgG4-Fc domain. 
     
     
         64 . The method of  claim 57 , wherein the recombinant antigen-binding protein binds a polypeptide comprising an amino acid sequence of SEQ ID NO:26. 
     
     
         65 . The method of  claim 57 , wherein the recombinant antigen-binding protein comprises a heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:27, an HCDR-2 having the amino acid sequence of SEQ ID NO:28, an HCDR-3 having the amino acid sequence of SEQ ID NO:29, a light chain CDR-1 (LCDR-1) having the amino acid sequence of SEQ ID NO:30, and an LCDR-2 having the amino acid sequence of SEQ ID NO:31. 
     
     
         66 . The method of  claim 57 , wherein the recombinant antigen-binding protein comprises Protein A or a functional fragment of Protein A. 
     
     
         67 . The method of  claim 57 , wherein the recombinant antigen-binding protein binds the heterodimeric protein with a KD of less than about 40 nM as measured in a surface plasmon resonance assay. 
     
     
         68 . The method of  claim 58 , wherein the CSCP comprises an ScFv fusion protein comprising (a) a heavy chain variable domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:15, (b) a light chain variable domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:16, and (c) a membrane anchor domain. 
     
     
         69 . The method of  claim 68 , wherein the membrane anchor domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:17 or SEQ ID NO:21. 
     
     
         70 . The method of  claim 57 , wherein the CSCP is an ScFv fusion protein comprising the amino acid sequence of SEQ ID NO: 19. 
     
     
         71 . The method of  claim 57 , wherein the detection molecule comprises a recombinant antigen-binding protein comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:15, and a light chain variable domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:16. 
     
     
         72 . The method of  claim 57 , wherein:
 (a) the second subunit of the heterodimeric protein comprises a heavy chain comprising a CH3 domain that comprises an arginine residue at position 95 according to the IMGT exon numbering system and a phenylalanine residue at position 96 according to the IMGT exon numbering system; and   (b) the detection molecule (DM) comprises a labeled recombinant antigen-binding protein that binds a human IgG1-Fc domain, a human IgG2-Fc domain, or a human IgG4-Fc domain wherein the Fc domain comprises an arginine residue at position 95 according to the IMGT exon numbering system and a phenylalanine residue at position 96 according to the IMGT exon numbering system.   
     
     
         73 . The method of  claim 57 , wherein the detection molecule comprises a recombinant antigen-binding protein that specifically binds a substituted CH3 polypeptide comprising one or more amino acid substitutions selected from the group consisting of (a) 95R, and (b) 95R and 96F according to the IMGT exon numbering system, or (a′) 435R, and (b′) 435R and 436F according to the EU numbering system wherein:
 (a) the recombinant antigen-binding protein comprises an antibody or ScFv that binds a polypeptide comprising an amino acid sequence of SEQ ID NO:42; 
 (b) the recombinant antigen-binding protein comprises an antibody or ScFv that binds a polypeptide comprising an amino acid sequence of SEQ ID NO:42 with a KD of less than about 60 nM as measured in a surface plasmon resonance assay; or 
 (c) the recombinant antigen-binding protein is an antibody or ScFv that binds to the same epitope on the substituted CH3 polypeptide as an antibody which comprises a heavy chain CDR-1 (HCDR1) having the amino acid sequence of SEQ ID NO:32, an HCDR-2 having the amino acid sequence of SEQ ID NO:33, an HCDR-3 having the amino acid sequence of SEQ ID NO:34, a light chain CDR-1 (LCDR-1) having the amino acid sequence of SEQ ID NO:35, an LCDR-2 having the amino acid sequence of SEQ ID NO:36, and an LCDR-3 having the amino acid sequence of SEQ ID NO:37. 
 
     
     
         74 . The method of  claim 57 , wherein:
 (a) the recombinant antigen-binding protein comprises one or more complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence that is at least 95% identical to SEQ ID NO:38, or of a light chain variable region (LCVR) having an amino acid sequence that is at least 95% identical to SEQ ID NO:39;   (b) the recombinant antigen-binding protein comprises a heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO:32, an HCDR-2 having the amino acid sequence of SEQ ID NO:33, an HCDR-3 having the amino acid sequence of SEQ ID NO:34, a light chain CDR-1 (LCDR-1) having the amino acid sequence of SEQ ID NO:35, an LCDR-2 having the amino acid sequence of SEQ ID NO:36, and an LCDR-3 having the amino acid sequence of SEQ ID NO:37;   (c) the recombinant antigen-binding protein comprises an HCVR having an amino acid sequence that is at least 95% identical to SEQ ID NO:38 and an LCVR having an amino acid sequence that is at least 95% identical to SEQ ID NO:39;   (d) the recombinant antigen-binding protein comprises an HCVR having an amino acid sequence of SEQ ID NO:38 and an LCVR having an amino acid sequence of SEQ ID NO:39;   (e) the recombinant antigen-binding protein is an antibody comprising a heavy chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:40 and a light chain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:41; or   (f) the recombinant antigen-binding protein is an antibody that comprises a heavy chain that has an amino acid sequence identical to SEQ ID NO:40 and a light chain that has an amino acid sequence identical to SEQ ID NO:41.   
     
     
         75 . The method of  claim 57 , wherein the recombinant antigen-binding protein is an ScFv fusion protein comprising:
 (a) (i) a heavy chain variable domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:38; (ii) a light chain variable domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:39, and (iii) a membrane anchor domain;   (b) a heavy chain variable domain that has an amino acid sequence identical to SEQ ID NO:38 and a light chain variable domain that has an amino acid sequence identical to SEQ ID NO:39; or   (c) the amino acid sequence of SEQ ID NO:43.   
     
     
         76 . A method of detecting and isolating cells that produce high levels of a heterodimeric protein, 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 cell expresses the heterodimeric protein, wherein the heterodimeric protein comprises multiple subunits and a first site on the heterodimeric protein resides on a first subunit, and a second site on the heterodimeric protein resides on a second subunit, and wherein (i) the CSCP binds to the first site on the heterodimeric protein to form a CSCP-heterodimeric protein complex inside the host cell, (ii) the CSCP-heterodimeric protein complex is transported through the host cell, and (iii) then displayed on the surface of the host cell;   (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 heterodimeric protein on the surface of the isolated and cultured cell of step (c) with a detection molecule that binds the second site on the heterodimeric protein; and   (e) isolating the cell detected in step (d) that bears the detected heterodimeric protein on its surface.