Non-radioactive cytotoxicity assays
Described herein are methods for assessing cytotoxicity of an agent. The methods include providing a target cell that has been engineered to express intracellularly a reporter that is not expressed endogenously by the target cell, exposing the target cell to an agent capable of modulating cytotoxicity and assaying the activity of the reporter, wherein a change in reporter activity relative to a reference value is indicative of the agent being able to modulate cytotoxicity of the target cell.
1. A method for assessing cytotoxicity of an agent comprising:
i) exposing a target cell engineered to express an intracellular non-secretory thermostable reporter to an agent capable of modulating cytotoxicity;
ii) measuring the level of the reporter; and
wherein the reporter is a thermostable beetle luciferase.
2. The method of claim 1 , wherein the reporter is stable under assay conditions of 37° C. for more than 15 min.
3. The method of claim 1 , wherein the beetle luciferase has the amino acid sequence of SEQ ID NO: 50-56 or a sequence with 85-99% identity thereto.
4. The method of claim 1 , where the beetle luciferase has the amino acid sequence of SEQ ID NO: 50-56 or a functionally active homolog, ortholog, mutant, variant or derivative thereof.
5. The method of claim 1 , wherein the beetle luciferase is obtained from Photinus pyralis, Photuris pennsylvanica or Pyrophorus plagiothalamus.
6. The method of claim 1 , wherein the beetle luciferase is any one or more of
Firefly luciferase (FfLuc or Fluc), Photuris pennsylvanica luciferase LucPPe-146-1H2, LucPPe-133-1B2, LucPPe-78-OB10, or LucPPe49-7C6A, Pyrophorus plagiothalamus luciferase LucPpL-81-6G1, or CBGRluc, or functionally active homologs or orthologs or mutants or variants or derivatives thereof.
7. The method of claim 1 , wherein the target cells are present in a cell media wherein the reporter is assayed in a) the cell media containing the target cells; or b) a cell supernatant that is free of the target cells.
8. The method of claim 1 , where the reporter activity is assayed using a substrate; and wherein optionally the substrate is D-luciferin or a synthetic analog thereof with which the luciferase can interact to generate light.
9. The method of claim 1 , wherein the target cell expresses a second reporter.
10. The method of claim 9 , wherein the activity of the first reporter and the second reporter is measured independently, simultaneously or sequentially.
11. The method of claim 9 , where the second reporter is selected from the group consisting of a) a non-secretory luciferase derived from a copepod, a deep-sea shrimp, or a functional variant thereof; and b) alkaline phosphatase or a functional variant thereof.
12. The method of claim 9 , where the second reporter is Guassia princeps luciferase (GLuc), NanoLuc (NLuc), Metrida longa luciferase-7 (MLuc7), Heterorhabdus tanneri luciferase (HTLuc), Pleuromamma abdominalis luciferase-1 (PaLuc1), Pleuromamma abdominalis luciferase-2 (PaLuc2), Metridia pacifica luciferase-1 (MpLuc1), Metridia curticauda luciferase-1 (McLuc1), Metridia asymmetrica luciferase-1 (MaLuc1), Metridia okhotensis luciferase-1 (MoLuc1), Metridia okhotensis luciferase-2 (MoLuc2), Metridia longa luciferase-39 (MLuc39), Pleuromamma scutullata luciferase-1 (PsLuc1), Lucicutia ovaliformis luciferase 1-3 (LoLuc1-3), Heterorhabdus tanneri luciferase-2 (HtLuc2), TurboLuc16 (TLuc), Lucia Luc, Renilla Luc, or a functionally active homologs or orthologs or mutants or variants or derivatives thereof.
13. The method of claim 9 , wherein
i) the substrate for one of the two reporters is D-luciferin or a synthetic analog thereof, with which the luciferase can interact to generate light and the substrate for the other of the two reporters is coelenterazine or an imidazopyrazinone or a synthetic analog thereof, with which the luciferase can interact to generate light; or
ii) the substrate for a one of the two reporters is pNNP and the substrate for the other of the two reporters is coelenterazine or an imidazopyrazinone or a synthetic analog thereof, with which the luciferase can interact to generate light; or
iii) the substrate for one of the two reporters is D-luciferin or a synthetic analog thereof with which the luciferase can interact to generate light and the substrate for the other of the two reporters is pNNP.
14. The method of claim 1 , wherein the target cell expresses two or more reporters and the target cell expressing two or more reporters are mixed together prior to the assay.
15. The method of claim 1 , wherein the agent capable of modulating cytotoxicity is any one or more of an antibody, small molecule, chemical compound, radiation agent, cytotoxic cells, biologics or combinations thereof.
16. The method of claim 15 , where the agent capable of modulating cytotoxicity targets an antigen selected from the group consisting of: Cluster of Differentiation (CD)-19 (CD19); CD-1-91 CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECLI); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); FmsLike Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAlX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gpl00); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ES0-1); Cancer/testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family Member 1A (XAGE-1A); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope Tag, CD34, LAMP1, TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Sialyl Lewis Antigen) Fucosyl-GM1, PTK7, gpNMB, CDH1, DLL3, CD276/B7H3, IL11Ra, IL13Ra2, CD179b, ALK, TCR gamma-delta, NKG2D, CD32 (FCGR2A), Tim1, CSF2RA (GM-CSFR-alpha), TGFbetaR2, VEGFR2/KDR, Lewis Ag, TCR-betal chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, Luteinizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), Chorionic Gonadotropin Hormone receptor (CGHR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, influenza A hemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR or HLA-G.
17. The method of claim 1 , wherein the target cell is exposed to the agent a) in vitro; and/or b) in vivo.
18. The method of claim 1 , wherein the assay is performed a) in vitro and/or b) in vivo.
19. The method of claim 1 , which is practiced using a kit containing one or more components of claim 1 .