Zhi-jie Jey Cheng

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.

Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.

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 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 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 5440: Novel PD-1 blockade bioassay to assess therapeutic antibodies in PD-1 and PD-L1 immunotherapy programs

Programmed death receptor-1 (PD-1) and its ligand (PD-L1) are among the few important immunotherapy targets for cancer. Current PD1 assays measure cell proliferation or cytokine production in primary T cells which are tedious, have high assay variation and small assay window. To enable quantitative potency measurement for key anti-PD-1 drugs in the market or in clinical trials such as pembrolizumab and nivolumab, as well as anti-PD-L1 drugs in clinical trials such as MPDL3280A and BMS-936559, here we report the development of a robust bioluminescent cell-based PD1 blockade bioassay. For this, we built a PD-1 effector cells in Jurkat cells which stably express human PD-1 and a NFAT-RE-luciferase reporter, and a PD-L1 positive artificial Antigen Presenting Cells (PD-L1+ aAPC) in CHO-K1 cells which stably express PD-L1 and an engineered TCR activator. Once these two cell types were co-cultivated, transcriptional activation of NFAT pathway in PD-1 effector cells, mediated by binding of TCR complex with TCR activator in PD-L1+ aAPC, is significantly suppressed by PD-1/PD-L1 engagement. This inhibition can then be specifically reversed by co-incubation of PD-1 or PD-L1 blocking antibodies in dose-dependent manner, but not by the antibody for other immune checkpoint receptors such as anti-CTLA4 ipilimumab. We further developed both PD-1 effector cells and PD-L1+ aAPC in Thaw-and-Use format so the cells can be plated for assay without the need of cell culture. The resultant PD-1 assay using Thaw-and-Use cells brings the benefit of convenience, low day-to-day variation, and easy lab-to-lab assay transfer. We demonstrate the assay is able to measure relative potency for antibody biologics, and also can detect potency changes for stressed antibody samples. In summary, the reporter-based PD-1 blockade assay provides a valuable tool for both drug screening and characterization in early drug discovery, and lot release and stability study in drug manufacture for therapeutic antibody drug candidates in PD-1 and PD-L1 immunotherapy programs. Citation Format: Zhi-Jie Jey Cheng, Natasha Karassina, Jamison Grailer, Jim Hartnett, Frank Fan, Mei Cong. Novel PD-1 blockade bioassay to assess therapeutic antibodies in PD-1 and PD-L1 immunotherapy programs. [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 5440. doi:10.1158/1538-7445.AM2015-5440

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