Genetic Engineering & Biotechnology News

OCT15 2017

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12 | OCTOBER 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News Andrew Niles and Kevin Kupcho Cell death in vitro can be induced by several distinct mechanisms depending upon the type and potency of the stimulus. Biological variables present a challenge to rapid and ef- ficient characterization of the mode of cell death for a biologic or a new chemical entity during the process of drug discovery. Although assay methods to study cell death pathways have improved in recent years, with respect to sensitivity, miniaturiza- tion, and ease-of-use, kinetics of cell death have been largely ignored in favor of conve- nience as provided by often arbitrary end- point measures. Endpoint measurement ap- proaches miss important information relating to the cytotoxic phenotype. Conversely, real-time (non-endpoint), live- cell assay methods are better suited for charac- terizing the kinetics and magnitude of a cyto- toxic response. Although elegant methods have been devised using electrical impedance and optical imaging, they are limited in throughput and require both specialized instrumentation and training, and costly consumables. We have developed a real-time, plate- based method that is accessible to any labo- ratory with a multimode (i.e., fluorescence and luminescence) reader. The new method is called RealTime-Glo™ Annexin V Apop- tosis and Necrosis Assay. Established Biomarker, New Approach Viable cells ensure their physical and functional integrity through a highly ordered membrane structure consisting of an array of proteins and phospholipids. One such class of proteins is phosphatidylserine (PS), which is actively maintained in an asymmetric ori- entation at the cytosolic membrane surface of healthy cells. During apoptosis induction, PS translocates to the cell surface. In vitro, externalized PS represents a reliable and quantifiable biomarker of apoptosis. Fluorescently labeled annexin V is a tra- ditional imaging agent for detecting PS trans- location because of its highly specific and robust calcium ion-dependent affinity for PS. This technique is limiting, though, as it is restricted to endpoint measurements and re- quires removal of unbound annexin V probe by repeated washes of cell samples prior to analysis by microscopy or flow cytometry. For our improved method, we created an- nexin V-fusion proteins, which act as a com- plementation reporter, and that contains two different subunits of an improved small and highly sensitive NanoBiT ® luciferase enzyme. In the absence of externalized PS, the annexin V-fusion proteins have only modest affinity for each other, and luminescence from ran- dom complementation of the luciferase en- zyme is practically nonexistent. The assay is homogeneous with no re- quirement for washes. When PS exposure occurs during apoptosis, the Annexin V- NanoBiT protein pairs are drawn to close proximity for complementation after bind- ing to PS rafts. Complementation reconsti- tutes a functional luciferase protein reporter molecule. Luminescence, as a result of this pairing, generates a magnitude of response proportional to PS exposure. In cases of secondary necrosis (the even- Continuous Assessment of Cell Death Mechanism of Action Using Annexin V Real-Time, Bioluminescent Apoptosis Assay Drug Discovery Tutorial Insights Discovery & Development The Bill & Melinda Gates Foundation is investing up to $40 million in Immunocore to support development of the company's soluble TCR (T-cell receptor)-based ther- apeutics, ImmTAV® (immune mobilizing monoclonal TCRs against virus) and ImmTAB® (immune mobilizing monoclonal TCRs against bacteria). ImmTAV and Im- mTAB may prove to be effective against tuberculosis, human immunodeficiency virus (HIV), and other infec- tious diseases that pose global health challenges. Namir Hassan, Ph.D., vice president of the infectious disease unit at Immunocore, commented, "We believe the immune system harbors the capacity to resolve prob- lematic infectious diseases, and our TCR-based therapies are well placed to mobilize this process." He adds that the company is focused on providing affordable medicines globally, including in the developing world. The investment in Immunocore is being made as part of the Gates Foundation's program-related invest- ments (PRI) strategy, which aims to stimulate private sector–driven innovation and attract external capital to global health and development initiatives. The col- laboration with the Gates Foundation also represents part of a larger Immunocore initiative to exploit the TCR-based therapeutics platform in non-oncology areas, including infectious diseases and autoimmune disorders. Preclinical data published last year demon- strated the potential for ImmTAV molecules to redirect the immune system to kill HIV-infected cells from pa- tients treated using antiretrovial therapy. U.K.-based Immunocore claims its platform of TCR- based soluble, synthetic bifunctional drugs can address both intracellular and extracellular targets. Lead ImmTAC® programs for cancer therapy are designed to trigger the patient's own immune system to attack and kill cancer cells. The firm's most advanced ImmTAC candidate, MICgp100, targets the melanoma-associated antigen gp100, and is being evaluated in several studies, both as a monotherapy and as a component in combination therapies. Partners in the combination trials include AstraZeneca and Eli Lilly. Another partner, Glaxo- SmithKline, recently announced that it had selected a third target as part of a cancer discovery collaboration, a program that aims to develop ImmTAC molecules against targets that can't be addressed using antibody- based technologies. n Gates Foundation Invests up to $40M in Immunocore to Develop TCR-Based Therapies Figure 2. The progression of an apoptotic response initiated by an antibody-drug conjugate. SKBR3 cells (HER2+) were dosed with trastuzumab emtansine in the presence of a real-time annexin and necrosis detection reagent. The emergence of (A) PS signal at 22 hours and (B) necrosis signal at 30 hours demonstrate the kinetic signature of apoptosis. Figure 1. How the assay works. RealTime-Glo™ Annexin V Apoptosis Assay uses Annexin V fusion proteins containing two complementary subunits (Annexin V-LgBiT and Annexin V-SmBiT) of NanoBiT® luciferase. When in close proximity, the LgBiT and SmBiT subunits come together to form a functional NanoBiT® luciferase. Left: Healthy Cell. Detection reagent contains Annexin V-LgBiT and Annexin V-SmBiT (NanoBiT) fusion proteins and a profluorescent DNA dye. Middle: Early Apoptosis. Luminescence remains low until PS exposure brings the annexin fusion proteins close together, forming a functional NanoBiT® luciferase. Right: Secondary Necrosis. A fluorescent signal is generated upon loss of membrane integrity during late-stage apoptosis, when the DNA dye can enter the cell. Figure 3. A kinetic trace of PS exposure and necrosis. A 1 µg/mL dose of trastuzumab em- tansine evoked time-dependent increases in PS exposure and necrosis biomarkers consistent with an apoptotic mechanism.

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