Denise Garvin

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 robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of action for many therapeutic antibodies. Classic ADCC assays measure antibody-dependent target cell cytotoxicity induced by primary effector cells that are isolated from human blood. They suffer from high assay variability due to the genetic and immune-status-mediated variation from blood donors. Here we report the development of a robust reporter-based ADCC assay that uses an engineered Jurkat stable cell line as the source of effector cells. These engineered effector cells were further developed as frozen, thaw-and-use format that can be plated for assay immediately after thaw. We demonstrate that frozen, thaw-and-use Jurkat effector cells showed appropriate Fc effector function similar to fresh cells from continuous culture, with added benefits of convenience and consistency. This robust assay is able to measure antibody potency for several therapeutic antibodies targeted to hematopoietic or solid tumors. The assay can distinguish effector functions for different antibody IgG isotypes in two antibody model systems: anti-CD20 and anti-EGFR. It is able to detect changes in ADCC biological activity for heat-stressed rituximab and trastuzumab, demonstrating that it possesses proper stability-indicting property. When compared with a classic PBMC-based ADCC assay, the ADCC reporter assay showed better assay precision and similar correlation of antibody glycosylation with ADCC biological activity for a panel of glyco-modified trastuzumab mixtures. Together these data demonstrate that this robust ADCC reporter assay meets the requirement of a potency bioassay that can quantify antibody Fc effector function in ADCC mechanism of action during drug discovery and development.

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 4693: Fc effector bioassays enable faster and quantitative measurement of ADCC and ADCP mechanisms of action

Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are recognized as important mechanisms of action (MOA) of therapeutic antibodies. Primary peripheral blood mononuclear cells (PBMCs) are routinely used in traditional bioassays to measure potency, stability of antibody drugs in ADCC and ADCP. However these methods are labor intensive and highly variable. Here we report the development of a luciferase-based reporter assay that measures activation of effector cells via cross-linking of Fc receptors with target cell-bound antibodies. We will discuss functional cell-based Fc effector reporter bioassays developed to measure Human FcgRIIIa (V158 and F158 variants), Human FcgRIIa (H131 and R131 variants), Human FcgRI as well as Mouse FcgRIV and FcgRIII. Compared to primary cell-based assays these reporter bioassays are less variable, easier to use and provide more consistent analysis of the important MOAs that are critical to for drug development programs. In qualification studies, performed in accordance with ICH guidelines for antibody screening and characterization, we tested the bioassays for specificity, accuracy, precision and linearity. Citation Format: Zhi-Jie Jey Cheng, Rich Moravec, Aileen Paguio, Denise Garvin, Gopal B. Krishnan, Frank Fan, Mei Cong. Fc effector bioassays enable faster and quantitative measurement of ADCC and ADCP mechanisms of action [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 4693. doi:10.1158/1538-7445.AM2017-4693

Abstract 3740: Paired ADCC reporter bioassays enable quantification and differentiation of antibody Fc-effector activities via V158 and F158 variant FcγRIIIa receptors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to clinical efficacy of a broad range of therapeutic antibodies. FcγRIIIa polymorphisms of individual cancer patients are correlated with clinical efficacy of several antibody drugs. An understanding of in vitro antibody activity via both Fc receptor variants is the key to ultimately understanding drug efficacy in vivo. Yet, with classic ADCC assays that rely on primary effector cells which are highly heterogeneous and variable, it remains challenging to quantitatively measure antibody ADCC activity and evaluate the impact of FcγRIIIa polymorphisms by in vitro ADCC assays. To address this problem, we developed a pair of reporter-based ADCC assays, where two engineered effector cell lines were generated in Jurkat T-cells which stably express a NFAT-RE driven luciferase reporter and either FcγRIIIa/V158 or FcγRIIIa/F158 polymorphism variant. The engineered Jurkat effector cells were further developed in frozen, thaw-and-use format to reduce handling time and minimize assay variability. We compared the V variant ADCC reporter assay with the classic ADCC assay using PBMCs from homozygous 158VV donors, by testing a panel of glyco-modified trastuzumab mixtures. The results demonstrate that the reporter-based ADCC assays using engineered Jurkat effector cells provide ADCC biological activity ranking equivalent to that obtained using classic PBMC-based ADCC assay. When tested side by side, both V variant and F variant ADCC reporter assays appropriately measure the biological activities in ADCC pathway activation of various human and mouse antibody isotypes of rituximab. The two ADCC reporter assays are also able to measure antibody potencies for multiple therapeutic antibodies, in various native target cell systems including suspension and adherent cell lines, and also genetically engineered cell lines such as a membrane-bound TNFα cell line. When tested in the same antibody/target cell system, the V variant ADCC assay showed higher antibody biological activity in ADCC reporter response than the F variant ADCC assay. This result appropriately reflects the reported impact of FcγRIIIa polymorphism on antibody binding and ADCC activity. In summary, the pair of V and F variant ADCC reporter assays provides a valuable approach to quantitatively measure the potency of therapeutic antibodies in ADCC and evaluate the impact of FcγRIIIa polymorphism in drug discovery and development for antibody therapeutics. Citation Format: Zhi-Jie Jey Cheng, Denise Garvin, Aileen Paguio, Rich Moravec, Frank Fan, Teresa Surowy. Paired ADCC reporter bioassays enable quantification and differentiation of antibody Fc-effector activities via V158 and F158 variant FcγRIIIa receptors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3740. doi:10.1158/1538-7445.AM2014-3740

Abstract 2840: Development of a bioluminescent cell-based bioassay to measure Fc receptor functionality in antibody-dependent cellular cytotoxicity

Primary peripheral blood mononuclear cells (PBMCs) are routinely used in traditional bioassays to quantify antibody drug potency in antibody-dependent cellular cytotoxicity (ADCC). These bioassays are labor intensive and have high inherent assay variability. Here, we report the development of a bioluminescent cell-based bioassay which measures activation of effector cells via cross-linking of FcαRIIIA with target cell-bound antibodies. For this, Jurkat T-cell stable cell lines that stably express NFAT-luciferase reporter and human FcαRIIIA were generated to replace primary PBMCs as effector cells in ADCC bioassay. Effector cells were also developed in a frozen, thaw-and-use format, to minimize assay variability due to cell culture and handling. The resultant bioassay using this format demonstrated good assay precision and accuracy in bioassay qualification. Bioassay using the engineered effector cells is robust, specific and is able to quantify the potencies of rituximab and trastuzumab, two monoclonal antibody drugs for cancer. When used to measure effects of Fc glycosylation on effector functions of therapeutic antibodies, a linear correlation was observed between relative antibody activity and the extent of Fc glycosylation. Thus, the bioluminescent cell-based reporter bioassay provides a simple and robust approach to measure potency of therapeutic antibodies in ADCC with high precision. 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 2840. doi:1538-7445.AM2012-2840

Abstract 1768: A bioluminescent cell-based reporter bioassay alternative to ADCC bioassay

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We have developed a bioluminescent cell-based reporter bioassay with reduced variability and complexity compared with traditional antibody-dependent cell-mediated cytotoxicity (ADCC) bioassays, in order that more accurate and precise quantification of therapeutic antibody Fc functionality can be achieved. Traditional bioassays to quantify ADCC are labor intensive, have high inherent assay variability, and routinely use primary NK cells from blood donors. Our bioluminescent cell-based reporter bioassay is based on the same NFAT pathway-based activation of gene transcription which occurs in NK cells after ligation of FcγRIIIA with target cell-bound antibody in a traditional ADCC assay system. In NK cells this leads to activation of cytokine and FasL gene transcription; FasL participates in lysis of target cells. We generated Jurkat T-cell lines which stably express both human FcγRIIIA and an NFAT-luciferase reporter. These cell lines were evaluated as effector cell replacements of primary NK effector cells in bioassays. The resultant reporter bioassay signal was robust and exhibited appropriate specificity, meaning robust signal above background and appropriate dose-response were achieved and were absolutely dependent on specific target and effector cells, and specific antibody. A “best” effector cell clone was selected based on bioassay performance and stability of the clone. Two types of target systems were evaluated: suspension CD20+ B cell targets with rituximab (anti-CD20) and adherent Her2+ cell targets with trastuzumab (anti-Her2). Our reporter bioassay performed well with both target test systems to quantify biological activity of the therapeutic antibodies. With a goal toward obtaining high inter-assay precision of the bioassay, we evaluated growing and freezing cells under specific conditions to identify those which allowed the cells to be ready for bioassay with minimal handling after thaw and we were able to identify such conditions. Additionally, we identified passage range suitable for banking vials of cells to which we can return time after time, obviating the variability that can arise when using continuously cultured cells for bioassay. We further optimized our bioassay using design of experiments (DoE) and established range of effector to target cell ratios over which the assay gave suitable bioassay performance in test systems. A qualification study was performed for characterization of precision, accuracy, linearity across potency range and stability-indicating ability. The bioluminescent cell-based reporter bioassay provides a robust, low variability and easier-to-perform alternative to traditional ADCC bioassays for quantification of Fc effector functionality of therapeutic antibody candidates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1768. doi:10.1158/1538-7445.AM2011-1768