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US 2019/0153547 A1Published application
Novel cell based assay
AssigneeMedical Diagnostic Laboratories, LLC
InventorYick Loi Raymond Yu
Life of this patent
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Abstract
The present invention relates to cell-based assays involving HER2. The assays use assay cells that are transfected with cassettes containing the HER2 gene of interest and measure the effect of mutations on the activity of HER2, and on their response to inhibitors.
Claims20
A method of determining the activity of a HER2 variant, comprising the steps of:
- a) preparing a cDNA encoding said HER2 variant;
- b) preparing a linear expression cassette containing the HER2 variant cDNA;
- c) transfecting said prepared expression cassette containing said HER2 variant cDNA in an assay cell having a JNK reporter construct comprising a reporter gene cDNA linked to at least one AP-1 binding site, and said cell is capable of expressing HER3;
- d) exposing said transfected cell to a HER3 ligand, wherein said HER3 complexes with said HER2 variant to form a dimer which thereby activates the INK signal construct and generates a signal;
- e) determining the signal activity, wherein a change in signal activity relative to wild type HER2 is indicative of a change in activity of said HER2 variant.
2 more elements
A method of determining the activity of a HER2 variant, comprising the steps of:
- a) preparing a cDNA encoding said HER2 variant;
- b) preparing a linear expression cassette containing the HER2 variant cDNA;
- c) transfecting said prepared expression cassette containing said HER2 variant cDNA in an assay cell having a stably integrated JNK reporter construct comprising a reporter gene cDNA linked to 6 AP-1 binding sites, said assay cell having a stably integrated HER3 expression construct;
- and said assay cell having a HER1 and HER2 double knockout;
- d) exposing said transfected cell to a HER3 ligand, wherein said HER3 complexes with said HER2 variant to form a dimer which thereby activates the JNK signal construct and generates a signal;
- e) determining the signal activity, wherein a change in signal activity relative to wild type HER2 is indicative of a change in activity of said HER2 variant.
3 more elements
An assay cell comprising:
- a) a stably integrated HER3 expression construct;
- b) a double knockout of HER1 and HER2;
- and
- c) a stably integrated JNK signal expression construct comprising a reporter gene cDNA linked to at least one AP-1 sequence.
1 more element
Dependencies are inferred from the claim text.