Highly sensitive and specific luciferase based reporter assay for antigen detection
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.
1. 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.
2. The method of claim 1 , wherein the reporter is a non-secretory form of a luciferase.
3. The method of claim 2 , wherein the non-secretory form of luciferase is obtained from copepods, deep sea shrimp or homologs or orthologs thereof or mutants or derivatives thereof.
4. The method of claim 1 , wherein the reporter activity is assayed by exposing the target cells to a luciferase specific substrate.
5. The method of claim 4 , wherein the luciferase-specific substrate is (a) coelentrazine or a derivative thereof, or (b) imidazopyrazinone or a derivative thereof.
6. The method of claim 1 , wherein the CAR is expressed on an immune cell.
7. The method of claim 6 , wherein the immune cell is a T cell, a CD4 T cell, a CD8 T cell, a Treg cells, a naïve T cell, a memory T cells, a central memory T cell, an effector memory T cell or an NK cell.
8. The method of claim 1 , wherein the CAR is expressed on a stem cell.
9. The method of claim 1 , wherein the CAR is expressed on a hematopoietic stem cell.
10. The method of claim 1 , wherein the CAR is expressed on an induced pluripotent stem cell.
11. The method of claim 1 , wherein the target antigen of the CAR is CD19, CD20, CS1/SLAMF7, BCMA, CD123, CD33, MPL, Lym1, Lym2, CD200R, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, FLT3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R α, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.
12. The method of claim 1 , wherein the target antigen of CAR is a complex of a HLA molecule with a peptide antigen.
13. The method of claim 12 , wherein the HLA molecule is HLA-A2.
14. The method of claim 1 , wherein the subject has cancer or an immune disorder.
15. The method of claim 14 , wherein the subject is being assessed for immune therapy.
16. The method of claim 15 , wherein the immune therapy comprises CAR T cells.
17. The method of claim 1 , wherein the fusion protein further comprises a tag.
18. The method of claim 17 , wherein the tag is any one or more of chitin binding protein (CBP), glutathione-S-transferase (GST), polyhistidine (His) tag, FLAG tag, HA tag, Myc tag, V5 tag, AcV5 tag, Streptag or a combination thereof.
19. The method of claim 1 , wherein the reference value is the reporter activity in any one or more of (i) cells that do not express the CAR; (ii) cells that express the CAR but are treated with fusion protein which is not targeted by the CAR; (iii) cells that are not treated with the substrate for the reporter; or (iv) combinations thereof.
20. The method of claim 1 , wherein more than one molecules of the reporter are fused to the extracellular domain of an antigen targeted by the chimeric antigen receptor.