ABSTRACT Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive

anticancer agent due to its tumor cell-specific cytotoxicity. However, earlier studies with recombinant TRAIL revealed many shortcomings, including a short half-life, off-target toxicity and

existence of TRAIL-resistant tumor cells. In this study, we developed a novel engineering strategy for recombinant soluble TRAIL by redesigning its structure with the adenovirus knobless

fiber motif to form a stable homotrimer with improved antitumor activity. The result is a highly stable fiber-TRAIL fusion protein that could form homotrimers similar to natural TRAIL. The

VIEWED BY OTHERS USE OF CELL FUSION PROTEINS TO ENHANCE ADENOVIRAL VECTOR EFFICACY AS AN ANTI-CANCER THERAPEUTIC Article 01 July 2020 COMBINED DELIVERY OF IL12 AND AN IL18 MUTANT WITHOUT

IL18BP-BINDING ACTIVITY BY AN ADENOVIRAL VECTOR ENHANCES TUMOR SPECIFIC IMMUNITY Article Open access 28 January 2025 BIOREDUCTIVE PRODRUG PR-104 IMPROVES THE TUMOUR DISTRIBUTION AND TITRE OF

THE NITROREDUCTASE-ARMED ONCOLYTIC ADENOVIRUS ONYX-411NTR LEADING TO THERAPEUTIC BENEFIT Article 26 November 2021 MAIN Cancer therapies aim to induce selective apoptosis in neoplastic cells

with limited concomitant side effects. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, as TRAIL-based therapeutics have been

shown to be more effective inducers of apoptosis in cancer cells than in normal cells.1, 2, 3 Natural TRAIL is a type II membrane protein, and its membrane form is cleaved to produce a

soluble, biologically active cytokine.4 The soluble TRAIL (sTR) protein exists as a homotrimer, and a single zinc atom is chelated by the Cys230 of each monomer. The trimeric structure is a

critical requirement for its biological function.5 TRAIL induces apoptosis by binding to TRAIL-R1/death receptor 4 (DR4) and TRAIL-R2/death receptor 5 (DR5), which are widely expressed in

cancer cells.6, 7 These interactions lead to trimerization of DR4/DR5 and their intracellular death domains and consequent recruitment of an adaptor molecule, Fas-associated death domain,

which then signals tumor cell death via caspase-dependent apoptotic pathways.8, 9 Importantly, TRAIL induces apoptosis in malignant cells via both extrinsic and intrinsic pathways, and its

effect is independent of the functional status of p53,10, 11, 12, 13 thus increasing the probability of the apoptotic outcome.14 However, TRAIL is unstable and has low activity, which are

obvious shortcomings, and these deficiencies are further aggravated by a short half-life in the blood as well as the original and acquired resistance of certain cancers to TRAIL.15 The human

adenovirus type 5 (Ad5) fiber protein is encoded by a single gene that expresses a polypeptide of 581 amino acid (aa) residues and exists as a homotrimer.16 The monomeric fiber protein is

composed of an N-terminal tail of approximately 47 aa residues that interact with the penton base protein of the capsid, a shaft comprising 21 pseudo repeats of 15 aa and an ~180 aa

C-terminal knob for cellular attachment. The formation of the trimeric fiber is less essential for its function and its assembly into virus particles. The fiber shaft at the C terminus was

found to be required for fiber knob trimer formation, since knob proteins containing two or more shaft repeats can form functional trimers.17 Notably, formation of trimerization motifs by

peptide fusion at the fiber C terminus has been reported.18, 19, 20 Another study demonstrated that a series of shortened fibers consisting of 6.5–9 shaft repeats, along with the tail, could

be fused to the N terminus of the trimerization element.21 Here, to obtain a highly stable and active trimeric TRAIL protein while avoiding hepatocyte transduction _in vivo_ by the fiber

shaft,22, 23 several trimerization elements from human Ad5 and fowl Ad1 fiber were first used to engineer the sTR protein. A human Ad5 fiber sTR was reengineered to contain the N-terminal

tail and the shortened shaft repeats (HA5FT) or to contain the C-terminal shaft repeat (HA5ST). A fowl Ad1 sTR (FA1FT) containing the full-length knobless fiber was also constructed. The

results demonstrated that the reengineered HA5ST and FA1FT were stable trimers, displaying improved bioavailability and antitumor activity, whereas HA5FT was a nonfunctional fusion protein.

RESULTS DESIGN, PURIFICATION AND CHARACTERIZATION OF TRIMERIC ADENOVIRUS FIBER-TRAIL FUSION PROTEINS A series of trimeric constructs were generated as follows: the N-terminal tail and the

first two shaft repeats of human Ad5 fiber fused to sTR (HA5FT), the last shaft repeat of human Ad5 fiber fused to sTR (HA5ST) and the fowl Ad1 knobless fiber fused to sTR (FA1FT) (Figure

1a). The amino acid sequences used for the constructs are indicated in Figure 1b. All fusion proteins were purified through Ni-resin by using a His-tag in the pET-28a expression vector.

Owing to the reported toxicity of the polyhistidine tag in normal hepatocytes and keratinocytes24, 25 and lack of the optimum amount of zinc,26 all purified proteins were digested by

thrombin, and zinc was added in the sTR samples. Analysis by SDS-PAGE (Figure 2a) of the purified protein showed single protein bands with a molecular mass of ~22 kDa for sTR, ~44 kDa for

FA1FT, ~30 kDa for HA5FT and ~23 kDa for HA5ST, matching the expected calculated molecular masses of 21.61, 43.48, 29.54 and 23.04 kDa, respectively. Analysis by gradually denatured SDS-PAGE

(GDS-PAGE) indicated that preparations of both FA1FT and HA5FT contained more trimeric proteins than HA5ST and sTR (Figure 2b). High-performance liquid chromatography (HPLC) analysis

indicated that all recombinant proteins were trimeric structures in the native buffer (pH=8.0), and the purity was above 95% (Figure 2c). APOPTOTIC ACTIVITY OF RECOMBINANT TRAIL FUSION

PROTEINS IN DIFFERENT CANCER CELL LINES The cytotoxicity of FA1FT, HA5FT and HA5ST fusion proteins were tested in the SW480 (human colon adenocarcinoma), ZR-75-30 (human breast cancer), Hela

(human cervical cancer) and SMMC-7721 (hepatocellular carcinoma) cell lines (Figure 3a). All cells were treated with sTR, FA1FT, HA5FT or HA5ST at 10-fold dilutions from 100 to 0.01 nM for

16 h. MTT analysis showed that both 10 nM FA1FT and HA5ST induced an ∼50% level of cytotoxicity in all cells. Of note, FA1FT and HA5ST were highly sensitive to the Hela cell line compared

with sTR, which has been reported to not respond to TRAIL treatment.27 As these cancer cells were not sensitive to HA5FT. TRAIL has been reported to induce apoptosis specifically in various

tumor cells without significant toxicity toward normal cells.4 We further examined whether FA1FT and HA5ST would cause cytotoxicity in normal cells; human Chang liver cells and MCF-10A human

mammary cells were used for testing their effects on cell growth. As expected, FA1FT and HA5ST showed little cytotoxicity at a high protein concentration (100 nM) (Figure 3b). To further

corroborate these results, FITC-conjugated Annexin V was used for detection of phosphatidylserine exposed on the membrane of apoptotic cells and for quantification of cells undergoing

apoptosis. The abilities of FA1FT, HA5FT and HA5ST fusion proteins to induce apoptosis were tested in human breast cancer cell ZR-35-30 and MCF7 (TRAIL sensitive) and in human lung cancer

cell A549 (TRAIL resistive).28, 29 Fluorescence activated cell sorting (FACS) analysis of Annexin V-stained cells showed that 61.3 and 66.8% of ZR-75-30 cells became apoptotic following

treatment with FA1FT and HA5ST at 10 nM, respectively, compared with 34.1% with exposure to sTR at the same concentration (Figure 4a). In MCF7 cells, about 30–37% cells became apoptotic

following a same treatment, while about 11% breast cancer cells became apoptotic with the treatment of HA5FT. In human lung cancer cell A549, the cell apoptosis was about 4.3~7% after

treated with these TRAIL agents, respectively. To confirm the apoptosis induced by these recombinant TRAIL proteins in cancer cells via caspase-dependent apoptotic pathways,8, 9 we tested

the caspase-3, -8 and -9 activities in ZR-75-30, MCF7 and A549 cells. (Figure 4b). As expected, a continuous high level of caspase-3 and -8 activities were detected in both ZR-75-30 and MCF7

cells. No obvious signal in A549 cells could be detected. Furthermore, the outstanding apoptosis induced by TRAIL agents in ZR-75-30 cells may attribute to the high level of caspase-9

activities. The extent of chromatin condensation was analyzed by fluorescence microscopy of cells stained with the DNA-binding fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) in ZR-75-30

cultured with either FA1FT or HA5ST at concentrations from 0.1 to 100 nM (Supplementary Figure S1). All these data confirmed the high specific activity of these fusion TRAIL proteins in

TRAIL-sensitive cancer cells. Since HA5FT has lower apoptosis activity, it was not tested in further experiments. ANALYSIS OF RECOMBINANT TRAIL FUSION PROTEIN STABILITY _IN VITRO_ To assess

the physicochemical properties of FA1FT and HA5ST using defined _in vitro_ conditions, we evaluated their stability during storage and repeated freeze/thaw cycles. All recombinant proteins

were analyzed by the same killing activities with different concentrations by using ZR-75-30 cells. sTR (100 nM), FA1FT(50 nM) and HA5ST(50 nM) were incubated at 37 °C in PBS, 4 °C in PBS or

37 °C in plasma for the indicated times. When maintained in PBS, sTR readily lost a significant fraction of its initial killing potential. In contrast, FA1FT and HA5ST were unaffected by

this treatment and retained nearly 100% of activity following a 24 -h incubation period (Figures 5a and b). In order to mimic the intravital condition, we analyzed the stability of FA1FT and

HA5ST in plasma at 37 °C. While sTR lost its killing activity after 1 h, FA1FT and HA5ST retained 60% of the killing activity following a 24- h incubation period (Figure 5c). We also

assessed the effect of repeated freeze/thaw cycles on the stability of FA1FT and HA5ST compared with sTR (Figure 5d). A rapid loss ranging between 90 and 95% of the activity of sTR was noted

following only one freeze/thaw cycle compared with the non-frozen control. In contrast, FA1FT and HA5ST retained biological activity even after undergoing eight free/thaw cycles. These

results indicated that the enhanced killing activity may be attributed to the improved stability of FA1FT and HA5ST, since the binding process was carried out for several hours at the

intravital temperature, conditions under which sTR would have lost its bioactivity. TUMORICIDAL EFFECTS OF RECOMBINANT TRAIL FUSION PROTEINS ON HUMAN BREAST CANCER IN NUDE MICE To

investigate whether the improved tumoricidal activity of FA1FT and HA5ST observed _in vitro_ would be effective _in vivo_, we investigated the antitumor activity of these proteins in nude

mice carrying ZR-75-30 cells. The systemic treatment was initiated after the established solid, vascularized tumors could be clearly discerned. In the first experiment, the regimen was eight

daily intraperitoneal (i.p.) injections of a low protein concentration (0.1 nmol) of sTR, FA1FT or HA5ST. Tumor growth was monitored for 24 days. With this treatment protocol, we observed

significant tumor regression in the HA5ST-treated group compared not only with the sTR group but also with the control group (Figure 6a). Unexpectedly, FA1FT treatment did not induce

regression of tumors. Therefore, we performed a second experiment with a high protein concentration (1 nmol) of sTR and FA1FT (Figure 6b). The treatment strategy was the same as that for the

first experiment. Consequently, under the treatment conditions applied, the FA1FT group presented only a slight, but not statistically significant, tumoricidal activity, compared with the

control group. A local treatment strategy was then applied by injecting 0.5 nmol of FA1FT or sTR in an area close to the established tumors. Both treatments delayed tumor growth, but FA1FT

presented a stronger reduction of tumor size (Figure 6c). ANALYSIS OF PHARMACOKINETICS, DISTRIBUTION AND SAFETY OF RECOMBINANT TRAIL FUSION PROTEINS _IN VIVO_ To evaluate the pharmacokinetic

characteristics of FA1FT and HA5ST in comparison with sTR, serum samples were collected after a single intravenous (i.v.) injection of 1 nmol into nude mice. Plasma was collected and tested

for biological activity at 2 min (baseline), 10, 30, 60, 120, 240 and 360 min post injection. The relative serum concentrations plotted over time fit well to a two-phase exponential decay

regression curve in both cases. Our measurements showed the presence of HA5ST in the bloodstream even 6 h after injection, suggesting that the half-life of HA5ST exceeded 1 h in mice. This

figure is quite favorable when compared with a half-life of only minutes reported earlier for recombinant TRAIL.30 We found that both sTR and FA1FT displayed similar kinetics each with a

serum half-life of ~30 min. At later time points, an obvious and significant difference in bioavailability of HA5ST was detected with a half-life of ~360 min (Figure 7a). Thus, HA5ST was

more efficiently retained in the bloodstream, which may positively affect its therapeutic activity. These results demonstrate that HA5ST has more favorable pharmacokinetic characteristics

compared with those of FA1FT and sTR. The improved stability of HA5ST over sTR and FA1FT, especially _in vivo_, may prove to be a significant advance for researchers as well as clinicians.

In order to investigate the reason for the insufficient tumoricidal activity of FA1FT _in vivo_, biodistributions of FA1FT and HA5ST were analyzed. At the indicated time points post

injection, mice were killed, and tumors and major organs (liver, kidney, spleen and tumor) were excised. Supernatants of the tissue homogenates were analyzed by enzyme-linked immuno sorbent

assay (ELISA) (Figure 7b). The results showed that tumor tissues in HA5ST-treated mice contained more of the administered fusion protein than those of FA1FT-treated mice. Moreover, the FA1FT

proteins appeared to be concentrated in the liver. These results suggested that the remarkably reduced antitumor activity of FA1FT and its weak pharmacokinetic characteristics may be due to

the metabolic detoxification process. The fusion proteins were detected at steady levels in the kidney, which is reasonably expected since it has been reported as a major organ for TRAIL

clearance.30 Histological examinations of liver tissues and kidney tissues 24 h after injecting 1 nmol of sTR, FA1FT or HA5ST showed no detectable changes by hematoxylin and eosin staining

compared with the tissues of PBS-treated mice. The kidney tissues also revealed the absence of acute renal toxicity by sTR, FA1FT or HA5ST injections (Figure 8a). We further evaluated if the

injections of FA1FT and HA5ST led to acute hepatotoxicity in mice by measuring levels of liver alanine aminotransferase and aspartate aminotransferase enzymes in the serum (Figure 8b). Our

results indicated that the levels of both enzymes were close to the normal range. Based on these results, we concluded that our fusion proteins did not cause hepatotoxicity in mice.

DISCUSSION The considerable interest in the use of TRAIL for cancer therapy is due to its unique tumor-directed apoptotic activities.8, 9, 14, 31 Despite its clinical advantages, however,

several pharmaceutical issues, such as the instability of TRAIL under physiological conditions, its inability to form an active trimer and weak pharmacokinetic characteristics, limit its

clinical applications as a cancer treatment.30, 32, 33, 34 To address these obstacles, researchers have focused on the development of reengineered TRAIL with improved tumor specificities and

enhanced trimer-forming abilities,35, 36 such as leucine zipper TRAIL,37, 38 single-chain TRAIL (scFv-TRAIL)39, 40, 41 and covalently linked TRAIL trimer (TR3).37, 42 However, a percentage

of engineered sTR proteins are produced as dimers which may be toxic to normal human hepatocytes, suggesting that more ideal genetic engineering strategies are still needed with safety as a

primary consideration. Endogenous TRAIL exists as a homotrimer, a critical requirement for its biological function. Thus, in order for recombinant TRAIL fusion proteins to function as an

active homotrimer, they must mimic the natural membrane-bound form of the ligand enabling DR4- and particularly DR5-mediated apoptosis induction.5 In this study, recombinant human TRAIL

trimers were generated based on fiber shaft trimerization elements. According to the crystal structures of the adenovirus fiber,43 the knob at the C-terminal of the shaft is similar to sTR

in its trimeric structure. Therefore, the shaft motif would be more veritable in maintaining the stability of natural sTR compared with tagged (e.g., FLAG, His) or other types of fusion

TRAIL proteins. The recombinant TRAIL variant with a fowl Ad1 fiber (FA1FT) and human Ad5 shaft (HA5ST) showed the greatest apoptotic activity in human tumor cell lines and a negligible

toxic effect in normal cells (Figure 3). In addition, FA1FT and HA5ST were found to be more stable than sTR at 37 °C or after freeze/thaw cycles _in vitro_ (Figure 5). Furthermore, HA5ST

displayed a better pharmacokinetic profile than sTR with minimal activity loss (Figure 6). These enhanced physiological stability and better pharmacokinetic profiles than sTR resulted in

improved therapeutic effects in an animal breast tumor model without detectable side effects (Figure 6). We demonstrated potent apoptosis-inducing activities of FA1FT and HA5ST, similar to

that of sTR but with an enhanced stability profile _in vitro_ compared with the latter. In fact, an improved half-life of HA5ST was observed, now exceeding 6 h in mice compared with a

half-life of only half an hour reported earlier for recombinant TRAIL.30 Furthermore, HA5ST did not induce cell death in human primary hepatocytes (Figure 8). Our results clearly indicate

that HA5ST has therapeutic potential for the treatment of cancers. Another important finding of our current work is the fact that the last repeat of the shaft in human Ad5 fiber could be

further genetically modified as a trimerization element. We believe that this genetic approach to trimerization will extend to other TNF family members. However, HA5FT could not induce cell

apoptosis efficiently. This fusion protein was reengineered to contain N-terminal fiber tail and the shortened shaft. We speculate that HA5F domain without C-terminal repeat could not make

TRAIL form an accurate trimmer, which hindered the recognition and combination between TRAIL and TRAIL receptors. Meanwhile, FA1FT was found to have substantial loss of activity _in vivo_

after i.p. injection. In living animals, circulation plays an important role in the metabolism of proteins. Unlike that found with HA5ST, FA1FT did not induce tumor cell apoptosis even at

high protein concentrations (Figure 6b). The local treatment results suggested that FA1FT could sufficiently induce tumor apoptosis _in vivo_ (Figure 6c). The biodistribution analysis

revealed that FA1FT was concentrated in the liver, which we believe was responsible for the remarkably reduced antitumor activity and the weak pharmacokinetic characteristics (Figure 7b).

These unexpected results presumably was due to the shaft sequence, which is a repetitive heparin-binding motif. The human Ad5 shaft domain has been demonstrated to target to hepatic cells

and cytokine release when administered through intravenous injection in mice.44, 45 Although most of the FA1FT proteins were detected in the liver, the fowl Ad1 shaft did not induce cell

death in primary hepatocytes. On the other hand, the fiber-specific neutralizing antibodies (NAbs) are primarily against knob domain in human patients.46, 47, 48 In this study, all of the

TRAIL compounds were engineered with knobless fiber motifs. Therefore, we speculate these recombinant soluble TRAIL proteins could not be neutralized by preexisting NAbs. Moreover, the

truncate shaft motif in HA5ST with a low immunogenicity, although the regimen of eight daily continuous injections of protein may cause antibodies against shaft slightly, it will not

restrain the antitumor activities of HA5ST. The therapeutic strategy of HA5FT was prefer to located injection, which may not cause an immunoreaction efficiently. In summary, we present here

a new method for generating recombinant human TRAIL based on an adenovirus shaft trimerization motif format featuring potent apoptosis-inducing activity and enhanced stability. Additional

modifications demonstrated the potential of HA5ST as an investigative tool and as a platform on which to build cell-targeted anticancer therapeutics. As a consequence, the predicted

advantages of such a concept would be a stronger and more sustained induction of the death receptor pathway in the targeted tumor cells. As TRAIL has been shown to augment the effects of

standard therapies, a tumor-targeted HA5ST protein may further enhance the effectiveness of other chemotherapies while limiting off-target toxicities to patients. Importantly, HA5ST can be

further applied in combination with small compounds in cancer therapy or for inhibiting markers of angiogenesis to gain greater effects than with either agent alone.49, 50, 51 MATERIALS AND

METHODS PREPARATION OF RECOMBINANT TRAIL PROTEINS Fragments corresponding to the N-terminal tail and the shaft region of Ad5 fiber were generated by PCR amplification using the fowl Ad1

fiber or human Ad5 fiber gene as a template. First-strand cDNA of TRAIL (aa 95–281) was synthesized from the total RNA of human peripheral blood leukocytes using avian myeloblastosis virus

reverse transcriptase (Promega, Madison, WI, USA) and random primers. The fusion genes were constructed by overlapping PCR. The primers were (_Nh_ _e_I; restriction site is underlined)

5′-GCTAGCATGACCTCTGAGGAAACCATTTCTAC-3′ (sTR); 5′-GCTAGCATGGCTGACCAGAAAAGGAAGCTG-3′ (FA1FT); 5′-GCTAGCATGAAGCGCGCAAGACCGTC-3′ (HA5FT); 5′-GCTAGCATGGGTGCCATTACAGTAGGAAAC-3′ (HA5ST). The

reverse primer for all constructs (_Hin_dIII; restriction site is underlined) was 5′- AAGCTTTTAGCCAACTAAAAAGGCCC-3′. All sequences were verified. Subsequently, the fragments were cloned into

the pET-28a (+) expression vector (Novagen, Madison, WI, USA) by using _Nh_eI/_Hin_dIII restriction sites. EXPRESSION AND PURIFICATION OF RECOMBINANT TRAIL PROTEINS The other constructed

recombinant plasmids were transformed into competent _Escherichia coli_ BL21 (DE3). The bacteria were cultured in Luria-Bertani (LB) medium (1% bactotryptone, 0.5% yeast extract, 85 mM NaCl)

with vigorous shaking (220 r.p.m.) at 37 °C to a density of OD600=0.8. Thereafter, 1 mM IPTG and 50 mM ZnCl2 were added to induce the expression of the recombinant protein at 20 °C for 16 

h. After induction, the cultures were centrifuged at 6 000 × _g_ for 30 min at 4 °C. One gram (wet weight) of the 1 mM protease inhibitor PMSF was added. Supernatants were collected by

centrifugation at 10 000 × _g_ for 30 min at 4 °C. Harvested cells were lysed, and soluble proteins were purified by Ni-affinity chromatography (by sequential washing with 20 and 50 mmol/l

imidazole and eluting with 250 mmol/l imidazole). A thrombin cleavage site was present in the N-terminal of each target protein. Thus, the His-tag was removed by digesting at 4 °C for 12 h

with thrombin (10 U/mg). Finally, 0.46 mol zinc/mol protein26 and 1 mM DTT were added to the sTR samples. HPLC The purified fusion TRAIL protein samples were applied to the HPLC TSK gel

G5000 (TOSOH, Tokyo, Japan) to determine the purity and molecular mass. The column was equilibrated with two column volumes of 20 mM Na2HPO4 containing 300 mM NaCl (pH 8.0) before loading

100 _μ_l of the final purified sample at 15 ml/h. SDS-PAGE AND GDS-PAGE SDS-PAGE and GDS-PAGE analyses were employed according to procedures previously described52 using 13.5% polyacrylamide

gels. Samples were denatured by boiling at 97 °C in Laemmli buffer (62.5 mM Tris-HCl, 2% SDS, 5% mercaptoethanol, 10% glycerol, 0.002% bromophenol blue) prior to SDS-PAGE analysis. GDS-PAGE

was basically similar to SDS-PAGE, except that the samples were not boiled and the proteins were gradually denatured under the effect of SDS. CELL VIABILITY ASSAY The following cell lines

were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA): SW480, SMMC-7721, ZR-75-30, Hela, MCF7 and A549. All cell lines were cultured in Dulbecco’s modified

Eagle's medium or RPMI1640 (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS; HyClone, UT, USA) plus 100 U/ml penicillin and 100 mg/ml streptomycin at 37

 °C in a humidified 5% CO2. Experimental cells (1 × 104/100 μl) were grown in a 96-well plate, incubated for 16 h and then treated with sTR, FA1FT, HA5FT and HA5ST at the indicated

concentrations. Cell viability was assessed by the MTT assay according to previously described procedures.53 DETECTION OF APOPTOSIS BY FLOW CYTOMETRY USING ANNEXIN V STAINING Early apoptotic

cells were detected using the Annexin V-PI Apoptosis Kit (Beckman Coulter, San Diego, CA, USA). Cells were incubated with sTR, FA1FT, HA5FT or HA5ST for 6 h and washed with PBS. The cells

were then resuspended in binding buffer, stained with Annexin V (0.6 mg/ml) and propidium iodide (PI, 5 mg/ml) for 15 min in the dark at room temperature and analyzed by two-color flow

cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using Cell Quest software. Annexin V-FITC fluorescence was detected in FL-1, and PI was detected in FL-3. ANALYSIS

OF CASPASE-3, -8 AND -9 ACTIVITIES Caspase activities were measured using Caspase Activity Kit (Beyotime, C1115, CII51 and C1157, Jiangsu, China) according to the manufacturer’s

instructions. Briefly, cells were incubated with 10 nM sTR, FA1FT, HA5FT or HA5ST for 6 h and washed with cold PBS. The cells were then resuspended in lysis buffer and left on ice for 15 

min. The lysate was centrifuged at 14 000 r.p.m. at 4 °C for 15 min. Activities of caspase-3, -8 and -9 were measured using substrate peptides Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA,

respectively. The release of _p_-nitroanilide (pNA) was qualified by determining the absorbance at 405 nm. Results were calculated as a percent relative to untreated cells. DAPI ANALYSIS

Cells were incubated with sTR, FA1FT, HA5FT and HA5ST for 6 h. The cells were then fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. For intracellular immunofluorescence,

experiments were performed by permeabilizing the cells in 0.1% Triton X-100 for 8 min. Cells were washed three times in PBS, blocked in 10% FBS in PBS and then incubated with 1 mg/ml DAPI

for 1 h at room temperature. Stained cells were washed three times in PBS before analysis. Fluorescent images were obtained using a Zeiss LSM710 confocal microscope equipped with a × 40

objective. ELISA ANALYSIS For ELISA, flat-bottom Immune FEP-101 96-well plates (JET BIOFIL, Guangzhou, China) were coated overnight with 0.1 _μ_g per well of the anti-TRAIL mAb (Santa Cruz

Biotechnology, Santa Cruz, CA, USA). Plates were washed three times in PBS containing 0.2% Tween20 (PBST), blocked with PBST containing 2% BSA and then incubated in triplicate with diluted

plasma or diluted tissue homogenate supernatant for 2 h at 37 °C. Plates were washed again and incubated with a 1/2000 dilution of a rabbit polyclonal antibody (ab2435; Abcam, Cambridge, MA,

USA) to TRAIL for 2 h at 37 °C. The plates were then incubated with a 1/5000 dilution of a horse radish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech Group, Inc.,

Chicago, IL, USA). After the final wash, plates were developed with 100 _μ_l of 3,3’,5,5’-tetramethylbenzidine (TMB, QIAGEN, Hilden, Germany) for 20 min at room temperature and stopped after

10 min by adding 50 _μ_l of 2M H2SO4. Analysis was performed using double wavelengths 450–630 nm with an EL × 800 Universal Microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

XENOGRAFT MOUSE MODELS Female BALB/c nu/nu mice (7 weeks old) were obtained from Vital River Laboratories (Beijing, China) (VRL). Human breast cancer cells (ZR-75-30; 1 × 106) in log phase

were implanted subcutaneously (s.c.) in the flank of each mouse. Tumor growth was monitored daily, and tumor volume was calculated by the following equation: tumor volume (mm3)=length ×

width2 × 0.5. After 10 days, animals with representative tumors were randomized by tumor size into four groups (_n_=10 per group, tumor volume ~100 mm3). Mice were given eight daily i.p.

bolus doses of sTR, FA1FT, HA5ST (1 nmol/mice/day) or PBS (100 μl/mice/day). For local treatment, mice received eight daily s.c. injections of 0.5 nmol of the various TRAIL fusion proteins

in PBS. Tumor growth was monitored for 20 days in the treatment groups. MOUSE PHARMACOKINETICS Balb/c mice (female, 7 weeks, 20±3 g, 3 mice/group) received an i.v. injection of 1 nmol of the

recombinant proteins in a total volume of 50 _μ_l. At time intervals of 5, 10, 30, 60, 120, 240 and 360 min, blood samples were taken from the tail and incubated on ice. Clotted blood was

centrifuged at 3 000 × _g_ for 20 min, 4 °C, and plasma samples were stored at −80 °C. Plasma concentrations of recombinant proteins were finally determined by ELISA. For data calculation,

relative values of serum concentrations were analyzed with the first value (2 min) being set to 100%. BIODISTRIBUTION ASSAY Balb/c mice (female, 7 weeks, 20±3 g, 3 mice/group) received an

i.p. injection of 1 nmol of the recombinant proteins. At time intervals of 10, 30 min, 1, 2, 4, 6 and 12 h, the kidney, liver, tumor, lungs and spleen tissues were removed from animals and

then frozen in liquid nitrogen immediately. All tissues were dissected and weighed, placed in PBS with 15 mM protease inhibitor PMSF (1 mg in 10 _μ_l) on ice. Samples were homogenized and

centrifuged at 14 000 r.p.m., for 15 min, and the supernatant was aspirated for ELISA analyses. STATISTICAL ANALYSIS Statistical calculations were performed using the GraphPad Prism 5.0

statistical program. ABBREVIATIONS * TNF: tumor necrosis factor * TRAIL: TNF-related apoptosis-inducing ligand * Ad5: human adenovirus type 5 * GDS-PAGE: gradually denatured SDS-PAGE * HPLC:

High performance liquid chromatography * FACS: Fluorescence activated cell sorting * FITC: fluorescein isothiocyanate * MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide *

DAPI: DNA-binding fluorescent dye 4, 6-diamidino-2-phenylindole * i.p.: intraperitoneal * i.v.: intravenous * s.c.: subcutaneously * ELISA: enzyme-linked immuno sorbent assay * AP: alkaline

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ACKNOWLEDGEMENTS This study was supported by funding from the National Nature Science Foundation of China (Nos. 31300756 and 81472816), the Science & Technology Development Plan of Jilin

Province (No. 20140520007JH and 20160519018JH) and the Project funded by China Postdoctoral Science Foundation (No. 2014T70301). We also wish to acknowledge Phuong Thi Sarkis for editorial

support in the preparation of this manuscript. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University,

Changchun, Jilin, China J Yan, L Wang, Z Wang, Z Wang, B Wang, R Zhu, J Bi, J Wu, H Zhang, H Wu, B Yu, W Kong & X Yu * Key Laboratory for Molecular Enzymology and Engineering of the

Ministry of Education, School of Life Sciences, Jilin University, Changchun, Jilin, China W Kong & X Yu Authors * J Yan View author publications You can also search for this author

inPubMed Google Scholar * L Wang View author publications You can also search for this author inPubMed Google Scholar * Z Wang View author publications You can also search for this author

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homotrimer of soluble TRAIL with enhanced stability and antitumor activity. _Cell Death Dis_ 7, e2274 (2016). https://doi.org/10.1038/cddis.2016.177 Download citation * Received: 04 February

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