Pete Stecha

Luciferase Reporter Assay System for Deciphering GPCR Pathways

The G protein coupled receptors (GPCR) represent the target class for nearly half of the current therapeutic drugs and remain to be the focus of drug discovery efforts. The complexity of receptor signaling continues to evolve. It is now known that many GPCRs are coupled to multiple G-proteins, which lead to regulation of respective signaling pathways downstream. Deciphering this receptor coupling will aid our understanding of the GPCR function and ultimately developing drug candidates. Here, we report the development of four homogenous bioluminescent reporter assays using improved destabilized luciferases and various response elements: CRE, NFAT-RE, SRE, and SRF-RE. These assays allowed measurement of major GPCR pathways including cAMP production, intracellular Ca2+ mobilizations, ERK/MAPK activ-ity, and small G protein RhoA activity, respectively using the same reporter assay format. We showed that we can decipher G protein activation profiles for exogenous m3 muscarinic receptor and endogenous β2-adrenergic receptors in HEK293 cells by using these four reporter assays. Furthermore, we demonstrated that these assays can be readily used for potency rankings of agonists and antagonists, and for high throughput screening.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

Improved Dual-Luciferase Reporter Assays for Nuclear Receptors

Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neoR fusion protein, which provides selection capability (G418 resistance, neoR) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains.

Abstract 5439: Development of a robust reporter-based T-cell activation assay for bispecific therapeutic antibodies in immunotherapy

Bispecific T-cell Engager (BiTE), which simultaneously targets CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, has emerged as a promising immunotherapy approach to treat cancer. Current methods for bispecific antibody potency determination measure T-cell proliferation or cytokine release using primary peripheral blood mononuclear cells. They can be complex and highly variable. Here we report the development of a reporter-based T cell activation assay using two Jurkat cell lines stably expressing luciferase reporter driven by IL-2 promoter or NFAT-response element. Both Jurkat reporter cell lines showed robust reporter signal upon stimulation of crossed-linked CD3 antibody. These cell lines were developed in Thaw-and-Use format and showed similar assay performance as that from the cells fresh-from-culture. When tested with bispecific therapeutic antibody catumaxomab, we showed specific reporter response by co-culturing Jurkat reporter cells with cancer target cells endogenously expressing EpCAM, such as MDB-MA-231 and SK-BR-3 cells. No signal was observed without target cells or with EpCAM negative Raji cells. The assay can measure the relative potency for catumaxomab with good precision. It also can detect changes in biological activity for catumaxomab in stressed stability study, and therefore has appropriate stability-indicating property. In summary, the reporter-based T cell activation assay provides a simple and robust approach to quantitatively measure antibody potency for bispecific antibody. It can potentially serve as a potency bioassay for bispecific therapeutic antibodies during drug development and manufacture. Citation Format: Pete Stecha, Jamison Grailer, Zhi-jie Jey Cheng, Jim Hartnett, Frank Fan, Mei Cong. Development of a robust reporter-based T-cell activation assay for bispecific therapeutic antibodies in immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5439. doi:10.1158/1538-7445.AM2015-5439

Abstract 3635: Improved T Cell activation bioassays to advance the development of bispecific antibodies and engineered T cell immunotherapies

T cells play a central role in cell-mediated immunity and can mediate long-term, antigen-specific, effector and memory responses. In recent years, a variety of immunotherapy strategies aimed at inducing, strengthening or engineering T cell responses have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCR-mediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using anti-CD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and show assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells. Citation Format: Richard L. Somberg, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Jey Cheng. Improved T Cell activation bioassays to advance the development of bispecific antibodies and engineered T cell immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3635. doi:10.1158/1538-7445.AM2017-3635

Abstract 5610: Quantitative cell-based bioassays to advance individual or combination immune checkpoint immunotherapy

Immune checkpoint receptors play a critical role in maintaining immune homeostasis and are genetically and functionally associated with autoimmune disease, cancer and persistent viral infections. Blockade of immune checkpoints (e.g., PD-1 and CTLA-4) has emerged as a promising new approach to enhance anti-tumor immune responses. While immunotherapies directed against PD-1 and CTLA-4 are showing unprecedented efficacy in the treatment of cancer, many patients and tumor types remain refractory to these therapies. This has resulted in a broadening of immunotherapy research and development to include additional immune checkpoint receptors (e.g., LAG-3, TIGIT, CD112R) targeted individually or in combination with other immunotherapy strategies. A major challenge in the development of biologics that target immune checkpoints is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. These assays are cumbersome, highly variable, and fail to yield the quality of data that is required for drug development in a quality-controlled environment. To address this need, we have developed a suite of immune cell line-based bioluminescent reporter bioassays for individual and combination immune checkpoint immunotherapy targets including PD-1 (PD-L1 or PD-L2), CTLA-4, LAG-3, TIGIT, PD-1+TIGIT and more. These assays consist of stable cell lines that express luciferase reporters driven by specific response elements under the precise control of intracellular signals mediated by the T cell receptor and immune checkpoint target(s). These mechanism of action-based bioassays are available in “thaw-and-use” format and demonstrate high specificity, sensitivity and reproducibility. The bioassays are pre-qualified according to ICH guidelines and demonstrate the performance required for use in antibody screening, potency testing and stability studies. Citation Format: Jamison Grailer, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based bioassays to advance individual or combination immune checkpoint immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5610. doi:10.1158/1538-7445.AM2017-5610

Abstract 4878: Quantitative cell-based bioassays for therapeutic development targeting immune checkpoint and co-stimulatory receptors

Immunotherapy harnesses the immune system to fight cancer and has proven to be a very promising therapeutic strategy. Drug targets in cancer immunotherapy include both inhibitory and co-stimulatory immune receptors on T cells or NK cells, in particular. Current approaches to assay immunotherapy biologics rely on primary cells, are highly variable, and are not suitable for a quality control environment during drug development. We have developed a panel of cell-based assays using a bioluminescent reporter platform that can quantitatively determine the potencies of antibodies and ligand proteins targeting immune checkpoint receptors and co-stimulatory receptors including PD-1, CTLA-4, LAG-3, GITR, 4-1BB, OX40 and CD40. For each target, a stable cell line was generated in an immune cell background to stably express an immune checkpoint or co-stimulatory receptor and a luciferase reporter driven by a response element specifically responding to signaling induced by TCR or directly from the immune receptor. These bioassays reflect biological mode-of-action for each class of drug candidate and are able to determine the potencies for on-market biologic drugs including PD-1 antibodies pembrolizumab and nivolumab, and CTLA-4 antibody ipilimumab. The assay signals are robust, specific, and have good repeatability and linearity. Therefore they can serve as valuable tools for drug screening, QC lot release and stability studies in immunotherapy drug development. Citation Format: Jamison Grailer, Pete Stecha, Jun Wang, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based bioassays for therapeutic development targeting immune checkpoint and co-stimulatory receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4878.

Abstract 151: Advancing pathway analysis using bioluminescent reporters

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL To enable investigation of key cellular signaling pathways, Promega has developed a portfolio of bioluminescent reporter gene assays using Firefly and Renilla luciferases. In combination with best-in-class luciferase detection reagents, these genetic reporter systems enable interrogation of important cellular responses involved in cancer, inflammation, and CNS disease. To address specialized customer needs in our industrial and research markets, Promega has a new custom assay service team dedicated to applying these enabling technologies through strategic external research collaborations. The performance of this technology portfolio is presented, including novel applications of luciferase reporters to interrogation of cytokine, stress, and toxicity pathway responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 151. doi:1538-7445.AM2012-151

Abstract 3883: Miniaturizing the GloSensor™ cAMP assay for MC4R screening using the Echo® acoustic liquid handler

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL As biological assays used for HTS and uHTS are miniaturized, it is critical to maintain assay sensitivity while minimizing reagent volumes and maximizing throughput. The GloSensor™ cAMP Assay provides an extremely sensitive and easy to use, real-time luminescent assay format for the interrogation of over expressed or endogenous GPCRs that signal via changes in the intracellular concentration of cAMP. Tipless, touchless transfers with the Echo® liquid handler eliminate the need for costly disposable tips and greatly simplify assay development efforts. Precise and accurate drop placement eliminates cross-contamination. In this work, we demonstrate optimization of the GloSensor cAMP Assay in low-volume format using a stably transfected HEK293 cell line model expressing the melanocortin 4 receptor (MC4R) and a cAMP-sensing variant of firefly luciferase. We show that the Echo liquid handler is able to titrate small molecule and peptide agonists and antagonists, as well as transfer the GloSensor cAMP Reagent, providing robust assay results that are achieved with significantly reduced volumes of cells and compound. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3883. doi:1538-7445.AM2012-3883