IP Library › Granted Patent US 12,577,326
Granted Patent B2
US 12,577,326 · App. 17/025,656 · Granted Mar 17, 2026

Recombinant cell surface capture proteins

Inventors: Dipali Deshpande (White Plains, NY); Gang Chen (Yorktown Heights, NY); Darya Burakov (Yonkers, NY); James Fandl (LaGrangeville, NY); Thomas Aldrich (Yorktown Heights, NY); Vishal Kamat (Bergenfield, NJ)
Assignee: Regeneron Pharmaceuticals, Inc.
C07K16/4258C07K16/22C07K16/2863C07K16/42C07K2317/31C07K2317/526C07K2317/64C07K2317/92
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Quick Facts
Patent No.
US 12,577,326
App. No.
17/025,656
Granted
Mar 17, 2026
Kind
B2
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).

Claims (60)

1 . A method of detecting and isolating a cell that stably expresses a bispecific antibody comprising the steps of:

(a) expressing in a host cell a cell surface capture protein (CSCP) comprising a recombinant antigen-binding protein, and a 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 host cell, (ii) the CSCP-bispecific antibody 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 (DM), comprising a labeled recombinant antigen-binding domain, wherein the detection molecule (DM) binds to the second site on the bispecific antibody; and

(c) selecting the host cell which binds the detection molecule;

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 detection molecule (DM) specifically binds to the Fc*.

2 . The method of claim 1 , wherein:

(a) the first subunit of the bispecific antibody comprises a heavy chain domain comprising a wild type CH3 domain; or

(b) the first subunit of the bispecific antibody 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.

3 . The method of claim 2 , wherein the CSCP comprises an ScFv fusion protein comprising (a) a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO:15, (b) a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 16, and (c) a membrane anchor domain.

4 . The method of claim 3 , wherein the membrane anchor domain comprises an amino acid sequence of SEQ ID NO:17 or SEQ ID NO:21.

5 . The method of claim 1 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 bispecific antibody, but does not bind to the CSCP-bispecific antibody complex.

6 . The method of claim 5 , wherein the blocking molecule is a non-human IgG or a human Fc molecule.

7 . The method of claim 1 , wherein the cell is detected and selected by fluorescence activated cell sorting.

8 . The method of claim 1 , wherein the detection molecule is an antibody or an ScFv fragment thereof that specifically binds to the Fc*.

9 . The method of claim 1 , 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.

10 . The method of claim 1 , wherein the recombinant antigen-binding protein binds a polypeptide comprising an amino acid sequence of SEQ ID NO:26.

11 . The method of claim 1 , wherein the recombinant antigen-binding protein comprises Protein A or a functional fragment of Protein A.

12 . The method of claim 1 , wherein the recombinant antigen-binding protein binds the bispecific antibody with a KD of less than about 40 nM as measured in a surface plasmon resonance assay.

13 . The method of claim 1 , wherein the CSCP is an ScFv fusion protein comprising the amino acid sequence of SEQ ID NO: 19.

14 . The method of claim 1 , wherein the detection molecule specifically binds to the Fc*, and wherein the labeled recombinant antigen-binding domain:

(a) comprises an antibody or ScFv that binds a polypeptide comprising an amino acid sequence of SEQ ID NO:42;

(b) 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) 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.

15 . 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*.

16 . 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*.

17 . The method of claim 16 further comprising at least one step selected from the steps:

obtaining the bispecific antibody from the cell culture;

obtaining the bispecific antibody from a culture medium wherein the bispecific antibody is secreted into the culture medium;

isolating the expressed bispecific antibody;

purifying the expressed bispecific antibody; and

further processing or modifying the expressed bispecific antibody.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 20, 2022
From: DESHPANDE, DIPALI; CHEN, GANG; BURAKOV, DARYA; FANDL, JAMES; ALDRICH, THOMAS; KAMAT, VISHAL
To: REGENERON PHARMACEUTICALS, INC.
Reel/Frame 058709/0012 →
Continuity (4)
Continuation 15700404 · Sep 11, 2017
Division 14079686 · Nov 14, 2013
Provisional Application 61726040 · Nov 14, 2012
Related Publication 20210009714A1 · Jan 14, 2021
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