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Method of selecting for antibodies

A technology of antibodies and antibody mimics, applied in chemical instruments and methods, anti-animal/human immunoglobulins, library screening, etc., can solve problems such as inability to obtain antibodies

Pending Publication Date: 2019-10-25
OXFORD GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, their method cannot obtain antibodies against complex membrane-bound targets (which is most often required), since it requires soluble proteins for biological selection

Method used

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  • Method of selecting for antibodies
  • Method of selecting for antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1: Illustration of Self-Labeling by Cells Expressing Anti-EpCAM Antibodies

[0138] Epithelial cell adhesion molecule (EpCAM) was chosen as a suitable target polypeptide (bait antigen). EpCAM is a glycosylated 30- to 40-kDa type I membrane protein that contains three potential N-linked glycosylation sites.

[0139] HEK293 cells were transfected with the EpCAM expression construct (HEK293 cells are usually EpCAM negative) together with a secreted HA-tagged anti-EpCAM single chain antibody expression construct. After a 24 hour incubation period, cells were stained with a fluorescently labeled anti-HA-tag antibody and analyzed by flow cytometry to determine which cells had self-labeled their membrane EpCAM with the encoded anti-EpCAM antibody.

[0140] result in figure 1 shown in . Cells expressing both the 'bait' antigen (EpCAM) and the scFv were highly fluorescent, whereas all other cells (expressing EpCAM only or scFv only) were not. This shows that scFv-sec...

Embodiment 2

[0141] Example 2: Optimizing stringency to prevent labeling of non-relevant cells

[0142] Two populations of EpCAM-expressing cells were prepared. Some expressed anti-EpCAM antibodies while others expressed green fluorescent protein (GFP). Cells were mixed at different ratios (where antibody-expressing cells were always the fewest) and incubated for different times to visualize immobilized and surface-bound antibody by staining in the red channel. A small number of antibody-expressing cells were always self-labeled first, resulting in cells in the lower right quadrant of the flow cytometry plot (cells were red rather than green, indicating that antibody-producing cells were labeled before non-relevant cells).

[0143] The results are shown in FIG. 2 . Even at a dilution of 1:125, a significant number of cells appeared in the lower right quadrant, representing cells expressing antibodies that bind cell surface antigens.

Embodiment 3

[0144] Example 3: Generation of DRD1 Antibody

[0145] In this example, the target polypeptide (bait) is DRD1; it is expressed in CHO cells (see image 3 ). Target polypeptide (bait) constructs and antibody libraries were cloned into CHO cells using retroviral transfer vectors.

[0146] Target polypeptide (bait) cell lines were generated by integrating the gene for the target polypeptide (bait) along with a selectable marker into the host cell (CHO) genome using a retroviral system (see Figure 4 ). The target polypeptide construct also contains the gene for the Tet repressor protein (TetR). Expression of the target polypeptide (bait) is driven by a doxycycline-inducible promoter.

[0147] CHO cells were infected with a library of retroviral particles encoding a cDNA-based library of human scFv sequences encoding human-like scFv sequences. For scFv libraries, retroviral transfer vectors were modified to contain constitutive promoters (SFFV) and flanking regions of scFv an...

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Abstract

The present invention relates to a method for identifying specific binding partners (e.g. antibodies or antibody mimetics) which bind to a desired target polypeptide. In particular, the method involves expressing a library of antibodies or antibody mimetics in a population of mammalian cells, wherein each cell in the population of cells displays the target polypeptide on the outer surface of the cell, and identifying or isolating cells within the population of cells to which antibodies or antibody mimetics are bound.

Description

technical field [0001] The present invention relates to a method for identifying specific binding partners (eg, antibodies or antibody mimetics) that bind to a desired target polypeptide. Specifically, the methods: involve expressing a library of antibodies or antibody mimics in a population of mammalian cells, wherein each cell in the population of cells displays a target polypeptide on an extracellular surface; and involve identifying or isolating within the population of cells, antibody or cells to which antibody mimics bind. Background technique [0002] Since the invention of hybridoma technology in 1986, monoclonal antibodies have emerged as powerful and multifunctional biotherapeutics, combining target selectivity, efficacy, and favorable biological and delivery half-lives with relative simplicity for large-scale manufacturing. Today, almost fifty monoclonal antibodies are authorized for medical use in the United States and Europe, and many others are in development....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42G01N33/537G01N33/566
CPCG01N33/531G01N33/6854G01N2500/04G01N2500/10C12N15/1037C07K14/705C07K16/30C07K2319/03C07K16/42C40B30/04C40B40/02C40B40/10G01N33/5032G01N33/537G01N33/566G01N33/6845
Inventor 赖安·卡伍德托马斯·佩恩理查德·派克-曼纽
Owner OXFORD GENETICS
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