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Recombinant Cell Surface Capture Proteins

a technology capture proteins, which is applied in the field of recombinant cell surface capture proteins, can solve the problems of insufficient available methods, laborious procedures, and inability to detect and/or isolate cells, and achieve the effect of convenient detection and/or isolation of cells

Inactive Publication Date: 2018-05-03
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a method for identifying and isolating cells that produce secreted proteins. The method involves creating cells that express a molecule on the surface of the cell that binds to the secreted protein. These cells are then detected and isolated. The method can be used to identify and isolate cells that produce various types of secreted proteins, such as growth factors, antibodies, and peptide hormones. The method is useful for research and commercial purposes.

Problems solved by technology

These procedures are laborious, inefficient, expensive, and the number of clones that can be analyzed is usually limited to a few hundred.
Moreover, the collection of clone pools or hand-picked colonies risks losing high expression cells, which often grow more slowly, to faster growing low expression cells.
Incorporation of flow cytometry into methods used for the isolation of stable expression cell lines has improved the capability of screening large numbers of individual clones, however, currently available methods remain inadequate for diverse reasons.
Diffusion of the POI between cells of different characteristics was also a problem.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0109]Construction of pTE084.

[0110]pTE084 was constructed by ligating the 1,436 bp Xba I fragment from pCAE100 that encodes the human FcγRI (hFcγRI; GenBank accession number M21091) into the Xba I site of pRG821. The orientation of hFcγRI in desirable plasmids resulting from the ligation was examined by restriction mapping with Not I, Pst I, Eco RI, and Stu I. pTE084 was designed for the high level expression of hFcγRI, the high affinity cell surface receptor for the Fc domain of human IgG. It contains two independent expression cassettes. One cassette is a hFcγRI gene driven by the CMV-MIE promoter, and the second cassette is the neomycin phosphotransferase II (npt) gene, which confers resistance to G418, driven by the SV40 late promoter.

[0111]Construction of a CHO K1 Derivative that Expresses hFcγRI.

[0112]CHO K1 cells (4×106) were transfected with pTE084 using Lipofectamine™ (Life Technologies; Rockville, Md.) following manufacturer's suggestions. The cells were placed in the cult...

example 2

ace Fluorescence Correlates with the Expression Level of 4SC622

[0116]RGC1 cells (4×106) were transfected with pEE14.1-622 and a pool of stable transfectants was obtained after selection for 2 weeks in medium comprised of 10% dialyzed fetal bovine serum, 90% glutamine-free Dulbecco's Modified Eagle's Medium (DMEM), 1× GS supplement, and 25 μM MSX (All reagents were from JRH Biosciences, Lenexa, Kans.). Rat IgG was added to the culture medium to 1 mg / ml 18 hours prior to immunostaining. The cells were trypsinized, washed with PBS, and stained with 1.5 μg / ml of a polyclonal FITC-conjugated anti-human IgG (H+L) F(ab′)2 fragment (Jackson ImmunoResearch Laboratories) for one hour at room temperature following procedures as described for FITC-hFc staining in Example 1. Cell staining was then analyzed by flow cytometry. The distribution of fluorescence suggested that the selected pool contained cells with a wide range of 4SC622 expression levels. Cells in the top 3% (R3 bracket), 7-11% (R5 ...

example 3

of Expression Clones in RGC1: IL-4 Trap

[0117]To directly demonstrate the efficiency in generating clonal cell lines with high level secreted protein production by our methodology, clonal 4SC622 producing cell lines were generated from RGC1. RGC1 cells (4×106) were transfected with pEE14.1-622, and selected for two weeks with 25 μM MSX to obtain a pool of stable transfectants. MSX-resistant cells were pooled and incubated with 1 mg / ml human IgG for 18 hours, prior to staining with PE-AG184. Six cells from the top 5% gate, as determined by flow cytometry analysis of cell surface 4SC622 staining, were isolated and expanded. 4SC622 production from the six clonal lines was determined and compared to 4SC622 production from clones obtained by hand-picking selected colonies followed by dilution cloning and amplification. One RGC1-derived clone, RGC4, produced 4SC622 at 12 pg / cell / day. This level is similar to that of the best 4SC622 producer isolated by hand-picking and analyzing 2,700 clon...

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PUM

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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).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of, and claims priority under 35 U.S.C. § 120, to U.S. patent application Ser. No. 14 / 079,686 filed Nov. 14, 2013, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61 / 726,040 filed on Nov. 14, 2012, which applications are herein specifically incorporated by reference in their entirety.BACKGROUNDSequence Listing[0002]This application incorporates by reference the Sequence Listing submitted in Computer Readable Form as file 8600A_ST25.txt created on Oct. 30, 2013 (86,267 bytes).FIELD OF THE INVENTION[0003]The field of this invention is related to recombinant cell surface capture proteins and a methods for identifying, isolating and enriching cells that produce secreted proteins that contain heterodimers, e.g. bispecific proteins. More specifically, the cell surface capture proteins and methods allow rapid and efficient isolation of high expression recombinant antibody-produc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/42C07K16/22C07K16/28
CPCC07K16/4258C07K16/22C07K16/2863C07K16/42C07K2317/31C07K2317/526C07K2317/64C07K2317/92G01N33/5005G01N33/68C07K2317/622C07K2319/00
Inventor DESHPANDE, DIPALICHEN, GANGBURAKOV, DARYAFANDL, JAMESALDRICH, THOMASKAMAT, VISHAL
Owner REGENERON PHARM INC
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