Cell based assay
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.
1. 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 JNK reporter 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;
f) exposing said transfected cell to a HER3 ligand and a HER2 inhibitor, wherein said HER3 complexes with said HER2 variant to form a dimer which thereby activates the JNK reporter construct and generates a signal; and
g) determining the signal activity, wherein a change in signal activity relative to wild type HER2 is indicative of a change in the sensitivity of said HER2 variant to said HER2 inhibitor.
2. The method of claim 1 , wherein said HER2 variant contains a missense mutation, insertion, or deletion.
3. The method of claim 1 wherein said assay cell has a double knockout of HER 1 and HER2.
4. The method of claim 1 wherein the INK reporter construct comprises a reporter gene cDNA linked to 3 to 12 AP-1 binding sites.
5. The method of claim 1 wherein the JNK reporter construct comprises a reporter gene cDNA linked to 6 AP-1 binding sites.
6. The method of claim 1 wherein said assay cell further comprises a HER3 expression construct.
7. The method of claim 6 wherein said HER3 expression construct is stably transfected.
8. The method of claim 1 wherein said JNK reporter construct is stably transfected.
9. The method of claim 1 , wherein step c) and step d) are performed simultaneously.
10. The method of claim 1 wherein steps a) through f) are performed in 32 hours or less.
11. The method of claim 1 , wherein said HER2 inhibitor is Lapatinib, Trastuzumab, or Pertuzumab.
12. The method of claim 1 , wherein said reporter gene cDNA encodes luciferase, said signal is light emission produced upon addition of a substrate for said luciferase, and a decrease in light emission relative to a control without HER2 inhibitor is indicative of the variant being sensitive to the inhibitor.
13. The method of claim 1 wherein said HER2 variant is obtained from a patient's biological sample selected from the group consisting of blood, serum, and tumor tissue.
14. The method of claim 1 , wherein said HER2 variant is transiently transfected.
15. The method of claim 1 wherein the JNK reporter construct is SEQ ID NO: 31.
16. 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 AK 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 reporter construct and generates a signal; and
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.