Engineering a leucine zipper-TRAIL homotrimer with improved cytotoxicity in tumor cells

Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA, USA.
Molecular Cancer Therapeutics (Impact Factor: 6.11). 07/2009; 8(6):1515-25. DOI: 10.1158/1535-7163.MCT-09-0202
Source: PubMed

ABSTRACT Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. The current suboptimal efficiency and selectivity drugs have therapeutic limitations and induce concomitant side effects. Recently, novel cancer therapies based on the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have emerged. TRAIL, a key component of the natural antitumor immune response, selectively kills many tumor cell types. Earlier studies with recombinant TRAIL, however, revealed its many shortcomings including a short half-life, off-target toxicity, and existence of TRAIL-resistant tumor cells. We improved the efficacy of recombinant TRAIL by redesigning its structure and the expression and purification procedures. The result is a highly stable leucine zipper (LZ)-TRAIL chimera that is simple to produce and purify. This chimera functions as a trimer in a manner that is similar to natural TRAIL. The formulation of the recombinant LZ-TRAIL we have developed has displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. Our results have shown that the half-life of LZ-TRAIL is improved and now exceeds 1 h in mice compared with a half-life of only minutes reported earlier for recombinant TRAIL. We have concluded that our LZ-TRAIL construct will serve as a foundation for a new generation of fully human LZ-TRAIL proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.


Available from: Vladislav Golubkov, May 03, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: It was shown that cancer cells acquired resistance to TRAIL-induced apoptosis in confluent cultures. Recombinant protein izTRAIL induced apoptosis of human carcinoma A431 cells in the first hours after cell plating at a concentration of 3–10 ng/mL, while in confluent cultures these cells acquire resistance to protein izTRAIL even at the concentration of 2 μg/mL. Detachment and suspending of the cells of confluent cultures immediately suppressed the resistance to izTRAIL. The cells of confluent cultures, being resistant to TRAIL-induced apoptosis continue progression through the cell cycle, as evidenced by the DNA cytograms and the Ki67p-GFP reporter system. Thus, the results showed that tumor A431 cells can acquire resistance to TRAIL-induced apoptosis in confluent cultures, while continue progression through the cell cycle, keeping the proliferative potential.
    Biophysics 01/2013; 57(4). DOI:10.1134/S0006350912040045
  • [Show abstract] [Hide abstract]
    ABSTRACT: The effects of TRAIL at various physiological levels were studied on 101/Hf and C3H/Sn mice during acute γ-irradiation with 750 R. An intravenous injection of TRAIL attenuated the effects of γ-irradiation on the body weight, subfractions of blood plasma, horizontal and vertical activity in the open-field test, tissue injury in the small intestine and liver, and content of Hassall's corpuscles in the thymus. The degree of these changes differed in mice of study strains.
    Bulletin of Experimental Biology and Medicine 02/2014; 156(4):452-6. DOI:10.1007/s10517-014-2372-9 · 0.37 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumor activity in a wide range of cancers without deleterious side effects on normal tissues. Several TRAIL derivatives have been developed to improve its pharmacokinetics and therapeutic effects through strategies such as adding a leucine zipper to increase the circulation half-life. To obtain clinical grade LZ-TRAIL for phase I clinical trial, a single batch of 30L bioreactor culture was performed using the Escherichia coli BL21 (DE3) strain expressing the recombinant LZ-TRAIL. A robust LZ-TRAIL production fermentation process was developed, which could be scaled up from 5L-50L, and had a titer of approximately 1.4g/l. A four-step purification strategy was carried out to obtain a final product with over 95% purity and 45% yield. The final material was filter sterilized, aseptically vialed, and stored at 4°C, and comprehensively characterized using multiple assays (vialed product was sterile, purity was 95%, aggregates were <5%, potency revealed a IC50 of 9nM on MDA-MB-231cells, and the endotoxin level was <0.25U/mg). The purity, composition, and functional activities of the molecule were confirmed. In vivo investigations indicated that LZ-TRAIL has better antitumor potency in three Xenograft tumor models compared to TRAIL (95-281). LZ-TRAIL also showed improved pharmacokinetic and safety profiles in cynomolgus monkeys without abnormalities associated with drug exposure. In conclusion, the scalable synthesis of LZ-TRAIL is useful for production of phase I clinical trial material. These preclinical investigations warrant further clinical development of this product for cancer therapy.
    European Journal of Pharmacology 06/2014; DOI:10.1016/j.ejphar.2014.06.002 · 2.68 Impact Factor