Highly sensitive and specific luciferase based reporter assay for antigen detection
AssigneeUniversity of Southern California
InventorPreet M. Chaudhary
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Abstract
Provided herein are methods for detecting an antigen or for detecting expression of a chimeric antigen receptor (CAR). The methods include obtaining a sample from a subject, contacting the sample with a fusion protein comprising a reporter fused to a single chain antibody specific to the antigen or fused to an extracellular domain of an antigen targeted by the CAR or fused to Protein L and assaying the activity of the reporter, wherein presence of reporter activity or increase in reporter activity relative to a reference value is indicative of presence of the antigen or presence of the expression of the chimeric antigen receptor in the sample.
Claims20
A method for detecting expression of a chimeric antigen receptor (CAR), comprising:
- obtaining a sample comprising cells from a subject wherein the cells are expected to have CAR expressed on their surface and in need of determination of expression of the CAR;
- contacting the sample with a fusion protein comprising a luciferase reporter operably linked to an extracellular domain of an antigen targeted by the CAR under conditions such that the fusion protein binds to cells in the sample expressing the CAR;
- wherein the extracellular domain targeted by CAR is linked to the luciferase reporter through a Gly-Gly-Ser (GGS) or Gly-Gly-Ser-Gly (GGSG) peptide linker and wherein the luciferase reporter is selected from the group consisting of NanoLuc (NLuc), MLuc7, PaLuc1 and TurboLuc16 (TLuc);
- washing the sample after the contacting step to remove any unbound fusion protein;
- and
- assaying the activity of the luciferase reporter bound to the cells expressing the CAR;
- wherein presence of luciferase reporter activity or increase in luciferase reporter activity relative to a reference value of cells lacking the CAR is indicative of the expression of the CAR in the sample;
- wherein the method is capable of detecting at least 1000 CAR expressing cells in the background of 1 million non-CAR expressing cells;
- and
- wherein the fusion protein is encoded by a nucleic acid sequence wherein nucleotide sequence encoding the extracellular domain is fused in-frame to nucleotide sequence encoding the GGS or GGSG linker and the luciferase reporter.
Dependencies are inferred from the claim text.