The Design Of Of TNF-related Apoptosis-inducing And (TRAIL) Conjugates And Studies On Their Specific Antitumor Effect

1. Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NCITumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is recently considered to be a promising anticancer drug, as active TRAIL trimer can trigger apoptosis through death receptors on the surface of various tumor cells (but not of normal cells). Generally, the existing formulations of recombinant TRAIL are not uniformly effective because of their instability. Moreover, these shortcomings are further aggravated by its short half-life in the bloodstream. On the other hand, various recombinants of human TRAIL have been generated to improve its activity and stability, for example, His-TRAIL, Flag-TRAIL, leucine zipper (LZ)-TRAIL, isoleucine zipper (iLZ)-TRAIL. However, it has been suggested that fusion proteins with exogenous tags may induce side effect on normal human cells, especially on liver cells. Thereby, endogenous protein domains (such as single chain variable fragment, human pulmonary surfactant-associated protein D) were developed to generate TRAIL fusion proteins for improving its activity and stability, and such endogenous domain fused TRAIL revealed a dual function and higher apoptosis activity.A non-collagenous domain (NCI) of human collagen XVIII NCI is composed of a trimerization domain (TD), a hinge region, and the endostatin domain, which is known to efficiently inhibit the tumor growth in animal models and has been launched in the Chinese market in2005. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NCI and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than55%of TRAIL-NC1and90%of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NCI was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460cells.2. Novel conjugation of tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) with Monomethyl Auristatin E for efficient antitumor drug deliveryHomotrimeric TRAIL induces programmed cell death via interacting with its death receptors (DR4and DR5). Decoy receptors (DcR1and DcR2) and soluble receptor (osteoprotegerin) containing no or only truncated death domain thus could not induce apoptosis upon TRAIL binding. However, there are still some TRAIL-resistant tumor cell lines even with expression of DR4and DR5on cell surface, for example:many breast and melanoma cancer cell lines. It has been proposed that the intrinstic reasons for TRAIL resistance included defects in the adaptor protein Fas-associated death domain (FADD) and caspase-8, overexpression of cellular FADD-like interleukin-1β-converting enzyme-inhibitory protein (cFLIP) and so on. Internalization of TRAIL is mediated via both TRAIL death receptors (DR4and DR5) as soon as it has bound with death receptors, and this endocytosis is not required for TRAIL induced apoptosis. To overcome the resistance of cancer cell lines with DR4or DR5to TRAIL, the internalization process is supposed to deliver highly toxic small molecule via conjugation with TRAIL. The toxic small molecule will exert its antitumor activity after release from TRAIL conjugates through enzymatic hydrolysis in lysosome.To demonstrate the strategy, we chose monomethyl auristatin E (MMAE) to conjugate with TRAIL via a linker consisting of two amino acids valine (Val) and citrulline (Cit). MMAE is a synthetic antimitotic agent which inhibits cell division by blocking the polymerization of tubulin. The linker between TRAIL and MMAE is stable in extracellular fluid, but is cleaved by cathepsin once the conjugate has entered tumor cells, thus activating the antimitotic mechanism. Herein, we describe the design, synthesis, and application of a novel TRAIL-linker-MMAE (TRAIL-vcMMAE) conjugate that delivers MMAE to cytoplasm after targeting to death receptors (DR4and DR5) on a tumor cell surface. We demonstrated that TRAIL conjugate is a conceptually viable therapeutic strategy with improved in vitro antitumor activity, cell circle arrest, uptake by tumor cells, receptor-mediated internalization and specific accumulation in tumor to treat TRAIL-resistant tumors. It is believed that the TRAIL conjugates, as well as antibody-drug conjugates (ADCs), will expand the application of toxic chemicals to serve as antitumor drugs. Further studies will focus on in vivo antitumor activity and toxicity of TRAIL-vcMMAE conjugates along with the conjugation of TRAIL with other toxic molecules. 3. Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)In vivo studies in different murine tumor models have suggested that Apo2L/TRAIL, alone or in combination with chemotherapy or radiation therapy, can exert substantial antitumor effects. However, TRAIL (product name:Dulanermin) has (?)een cut off from its pipeline by Amgen and Genentech successively in2011without my pronounced reason. Presumably several shortcomings of TRAIL could be the major easons:poor pharmacokinetics, instability and hepatoxicity.Polyethylene glycol (PEG) modification is regarded as one of the most successful strategies employed to modify native proteins, to obtain increase in body-residence time and stability, and decrease in immunogenicity. There are a variety of enzymes and Droteins that have been approved by FDA (American Food and Drug Administration) after PEGylation, for examples, PEG-asparaginase (Oncaspar) and PEG adenosine deaminase(Adagen). As could be expected, PEG was also applied to the modification of TRAIL by researchers. They have all chosen N-terminal amino conjugation to modify TRAIL among various conjugation strategies, taking advantage of the lower pKa of the N-terminal a-amino groups compared with that of the α-amino group in lysines, to achieve site-specific TRAIL PEGylation. Although their work has exhibited successful site-specifically PEGylated TRAIL with improved in vivo antitumor activity, we seek to find out whether there would be more facile or effective conjugation method for TRAIL PEGylation.Herein, we described the preparation, analysis and evaluation of a PEG-TRAIL, a Cys-SH site-specifically PEGyalted TRAIL mutant. Methoxypoly (ethylene glycol) maleimide (mPEG-MAL) was used to conjugate with TRAIL, as the maleimides react rapidly with reduced reduced thiols (-SH groups) cysteines forming a stable thioether. Mutant N109C was chosen for it has mutated a potential N-linked glycosylation site (Asn-109), and showed good antitumor activity upon conjugation with monomethyl auristatin E (MMAE) in our previous study. In the present study, we have investigated the physicochemical, pharmacokinetic and pharmaceutical characteristics of the cysteine mutated PEG-TRAIL (mPEGMAL-N109C) and evaluated its biological and antitumor activities in vitro and in vivo. We also prepared N-terminal specifically PEGylated TRAIL (mPEGALD-TRAIL114-281) for the sake of comparison, and conducted all the studies above in parallel with the cysteine-mutated PEG-TRAIL. Comparing to mPEGALD-TRAIL114-281, mPEGMAL-N109C exhibited higher in vitro and in vivo antitumor activities, better stability, and longer half-life, indicating site-specific PEGylation of a mutated-cysteine residue would be a better way to modify TRAIL.