Although human JURKAT T lymphocytes induced to undergo apoptosis with anti-Fas/APO-1 antibody were observed to rapidly lose reduced glutathione (GSH), increased concentrations of oxidized products were not detectable. Unexpectedly, the reduced tripeptide was instead quantitatively recovered in the incubation medium of the cells. As GSH loss was blocked by bromosulfophthalein and dibromosulfophthalein, known inhibitors of hepatocyte GSH transport, a specific export rather than nonspecific leakiness through plasma membranes is proposed to be responsible. Apoptosis was delayed when GSH-diethylesters were used to elevate intracellular GSH, although the high capacity of the activated efflux system quickly negated the benefit of this treatment. Stimulation of GSH efflux provides a novel mechanism whereby Fas/APO-1 ligation can deplete GSH. We speculate that it enhances the oxidative tonus of a responding cell without requiring an increase in the production of reactive oxygen species.In the current paradigm for apoptotic cell death, stimulation of the proteolytic activity of a family of interleukin 1--converting enzyme (ICE) 1 -like proteases initiates several distinct events within a cell (1-3). These include chromatin fragmentation, plasma membrane blebbing, and an overall cell shrinkage, eventually culminating in either the phagocytosis or lysis of the apoptotic cell. There have also been numerous reports that apoptotic cells accumulate oxidized proteins and lipids and are presumably therefore exposed to some degree of oxidative stress (4). In addition, antioxidants have been widely reported to protect against, or delay, many different forms of apoptosis (5). However, several recent reports of apparently normal apoptosis occurring in very low oxygen environments indicate that reactive oxygen species (ROS) are unlikely to be essential mediators of this type of cell death (6 -8).Human JURKAT T lymphocytes are rapidly induced to undergo apoptosis after exposure to anti-Fas/APO-1 antibody (9 -11). One of the earliest detectable events downstream of ligandreceptor binding is activation after 15 min of the ICE-like protease CPP-32/apopain (12, 13). Plasma membrane blebbing and degradation of the cytoskeleton commence soon after, while chromatin breakdown is first detectable at 45 min with internucleosomal DNA fragments beginning to accumulate after approximately 90 min (14). Gross alterations in plasma membrane permeability develop several hours later. Although it has been reported that these changes occur with similar kinetics independently of ambient oxygen tension (6), the possibility remains that oxygen-independent modification of intracellular redox states are involved in these events. To further understand this controversial area, the glutathione metabolism of JURKAT cells exposed to anti-Fas/APO-1 antibody was investigated. Consistent with results in thymocytes (15, 16), T lymphocytes undergoing apoptosis became dramatically depleted of their reduced glutathione (GSH) coincident with the onset of chromatin fragmen...
Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.
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