PSMA and TRAIL-R2 expression was analyzed using a prostate cancer tissue microarray (cat. no. 79562483; Tristar Technology Group LLC). The spots were stained using an anti-PSMA antibody (cat. no. EPR6253; Abcam) and an anti-DR5 antibody (cat. no. ab47179; Abcam). The primary antibodies were incubated for 60 min at 25°C. For secondary detection, Histofine Simple Stain Mouse MAX-PO (R) (cat. no. 414341; Nichirei Biosciences, Inc.) was applied and incubated for 30 min at 25°C. The stained sections were observed using a light microscope at a magnification of ×200. The H-score was calculated as follows: i) The intensity score was determined based on the staining intensity of positive cells among cancer cells (0; no staining, 1; low, 2; middle, 3: high). ii) The positive occupancy rates (%) of cancer cells for each score were calculated in increments of 5%. iii) Each score was multiplied by the positive occupancy rate and the result was used as the H-score=Σ (positive occupancy rates (%) × intensity score).

The variable domains of the heavy chain (VH) and light chain (VL) regions of E11 (26), a non-agonistic antibody TRAIL-R2 clone, were used in the present study. The novel PSMA antibody clones with a VL in common with E11 were generated using phage display with an E11 VL fixed library (27). cDNA extracted from the spleen of PSMA-immunized human antibody transgenic mice (28) was used as the VH gene source in the library by isolating total RNA using ISOGEN (cat. no. 315-02504; Nippon Gene Co., Ltd.), synthesizing cDNA by reverse transcription, and amplifying VH fragments by PCR. M13 phage panning was then performed on PSMAcoated tubes to obtain clones that bound to PSMA. The PSMA-binding clones were in-frame cloned into the tetravalent bispecific antibody expression vector REGULGENT™ that included a signal peptide and human IgG4 (S228P/L235E/R409K) Fc region designed to reduce Fcγ receptor binding, thereby eliminating effector functions (29). The designed gene sequences were synthesized by Azenta Life Sciences and inserted into the expression vector using restriction enzyme digestion and ligation. The tetravalent bispecific antibody PSMA/TRAIL-R2 REGULGENT™ plasmid was generated by fusing anti-TRAIL-R2 Fab to the N-terminus of the anti-PSMA heavy chain. The bivalent bispecific antibody plasmid was constructed based on IgG1 with knobs-into-holes and Fc-silencing (L234A/L235A/G237A) mutations (10,30,31). The two plasmids were transfected into 293F or Expi293F™ (Thermo Fisher Scientific, Inc.) cells using the FreeStyle™ 293 or Expi293™ Expression Systems (cat. no. A14635; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol; briefly, cells were cultured in suspension, transfected with the supplied transfection reagent, and cultured for 5 days before harvesting. Antibodies were purified using Protein A (cat. no. 29048576; Cytiva). After eluting the purified antibodies, the buffer used to dissolve the sample was replaced with phosphate-buffered saline (PBS, cat. no. 14249-24; Nacalai Tesque, Inc.). Subsequently, the monomer fraction was separated from the antibody solution using an NGC™ Chromatography System (Bio-Rad Laboratories, Inc.) with Superdex™ High-Performance Columns (28990944; Cytiva).

The purity of the prepared antibodies was determined using microfluidic chip electrophoresis (LabChip GXII Touch; PerkinElmer, Inc.) and ultra-high-pressure size exclusion chromatography (UHP-SEC). The LabChip analysis was performed using a Protein Express Assay Reagent Kit (cat. no. 760499; PerkinElmer, Inc.); all microfluidic chips were primed and prepared according to the manufacturer's protocol, the antibody samples and ladder were loaded and the run was performed on the LabChip GXII Touch system under standard settings. The non-reducing sample buffer contained N-ethylmaleimide at a final concentration of 6.36 mM and the heat treatment conditions were 70°C for 15 min. The denatured samples were diluted with deionized water and loaded onto a primed chip and the analysis was conducted according to the manufacturer's instructions. Finally, the LabChip^® GX Reviewer program (PerkinElmer, Inc.) was used to automatically calculate the size and concentration of each separated peak. The proportions of monomers, high-molecular-weight species (HMWS) and low-molecular-weight species (LMWS) were determined using UHP-SEC on an ACQUITY UPLC Protein BEH SEC Column [200 À 1.7 µm, 4.6×150 mm (Waters Corporation)]. The mobile phase contained 20 mM sodium phosphate (pH 6.8), 500 mM NaCl and 5% ethanol. The flow rate and detection wavelength were 0.4 ml/min and 215 nm, respectively. Peaks indicative of antibody protein monomers, HMWS and LMWS were analyzed by integrating the area under each eluting peak using LabSolutions Software (Shimadzu Corporation).

Human lung adenocarcinoma (NCI-H2122; ATCC no. CRL-5985), human prostate cancer (PC-3; ATCC no. CRL-1435) and lymph node carcinoma of the prostate (LNCaP) clone fast-growing colony (FGC; ATCC no. CRL-1740) cells were seeded in RPMI 1640 medium (cat. no. 11875093; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (cat. no. 10099141; Thermo Fisher Scientific, Inc.). Human PSMA cDNA was transfected into the cell lines via lipofection using HilyMax (cat. no. H357; Dojindo Laboratories, Inc.) according to the manufacturer's instructions. The cells were incubated with the transfection mixture at 37°C and the medium was replaced with fresh growth medium 2 days after transfection. The transfected cells were then cultured under blasticidin selection to establish stable clones, and subsequent experiments were conducted approximately 1 month after transfection. Nontransfected cells were included as a negative control. The transduced cells were detached by adding 0.25% trypsin/1 mM EDTA to the adhered cells and then counted. Thereafter, the cell suspension was diluted and seeded at a density of one cell per well containing 200 µl of drug selection medium in 96-well plates. Wells in which the cells formed a single colony were expanded. Subsequently, PSMA expression was confirmed using a FACSCanto II Flow Cytometer (BD Biosciences).

PSMA expression was confirmed using the Wes™ capillary-based automated western system (ProteinSimple) system. Cells were lysed in RIPA buffer (MilliporeSigma) at 100 µl per 10⁶ cells, and the lysates were directly analyzed by loading 0.2 µl of each sample into the capillaries. The analysis was performed with the 12-230 kDa Separation Module (cat. no. SM-W001; ProteinSimple) according to the manufacturer's standard protocol. Blocking, separation, immunodetection, and signal development were carried out automatically within the system. For primary detection, an anti PSMA/GCPII (cat. no. 13163-1-AP; Proteintech Group, Inc.) was applied at a concentration of 20 µg/ml and incubated at 25°C under instrument default settings. The anti-Rabbit Detection Module for Wes, Peggy Sue or Sally Sue (cat. no. DM-001; ProteinSimple) were used for secondary detection and for blocking and chemiluminescent visualization. An anti-β-actin antibody provided in the detection module was used as a loading control. Signal acquisition and quantification were performed using Compass software version 6.1.0 (ProteinSimple).

The antigen-binding affinities of the antibodies were measured using a Biacore™ T200 surface plasmon resonance (SPR) system (Cytiva) at 25°C with HBS-EP+ pH 7.4 (Cytiva) as the buffer. Briefly, a Human Antibody Capture Kit (cat. no. BR100839; Cytiva) was used to immobilize the anti-human IgG onto a CM5 sensor chip (Cytiva). Next, the antibodies were injected, followed by TRAIL-R2 (cat. no. 310-19; PeproTech) and PSMA (PSA-H82Qb-25 µg; ACROBiosystems) solution in a single cycle. A dilution series of the antigens was injected for 4 min at a flow rate of 30 µl/min. The dissociation time was 400 sec. After each binding event, the surface was regenerated using 3 M MgCl₂. The sensorgrams were analyzed using Biacore Evaluation software (Cytiva), fitting a 1:1 binding model to determine the following binding kinetics: ka, kd and KD.

Cells were suspended in PBS (cat. no. 14249-24; Nacalai Tesque, Inc.) supplemented with 5% fetal bovine serum (cat. no. 10099141; Thermo Fisher Scientific, Inc.), 1 mM EDTA and 0.1% sodium azide (staining buffer) and added to a 96-well plate. After centrifugation at 340 × g for 3 min at 4°C, the supernatant was removed and each antibody was added to the pellet and incubated for 30 min on ice. After washing, a goat F(ab')2 anti-human IgG (γ chain-specific) R-phycoerythrin conjugate (cat. no. 2043-09; SouthernBiotech) was added and the cells were incubated for 30 min at 4°C. After washing, the cells were suspended in a staining buffer containing 7-aminoactinomycin D staining solution (cat. no. 559925; BD Biosciences) and incubated for 10 min at 25°C. The fluorescence intensity of each cell was measured using a FACSCanto II Flow Cytometer (BD Biosciences). Data were analyzed using FlowJo software version 9.6.4 (BD Biosciences).

Cells were seeded in 96-well plates and cultured overnight at 37°C under 5% CO₂. Anti-2,4-dinitrophenol (DNP), anti-TRAIL-R2 agonistic KMTR2, anti-TRAIL-R2 E11, anti-PSMA (PN7 or PI101) and PSMA/TRAIL-R2 bispecific antibodies diluted with culture medium were added at 50 µl per well and incubated at 37°C for 2 days. Cell Counting Kit-8 solution (cat. no. CK04; Dojindo Laboratories, Inc.) was added (10 µl per well) to the cells and the mixture was incubated for 4 h at 37°C under 5% CO₂. Next, 0.1 M HCl (10 µl per well) was added to stop the reaction. The percent cell viability was calculated as follows: (1-([absorbance at each antibody concentration]-[absorbance of medium only])/([absorbance at an antibody concentration of 0 µg/ml]-[absorbance of medium only])) ×100. Data were expressed as mean ± SE of quadruplicate experiments.

Antibodies were added to PC-3 and PSMA/PC-3 cells, after which activated caspases in the cells were detected using a FAM-FLICA Poly Caspase Assay kit (cat. no. 92; ImmunoChemistry Technologies, LLC). Thereafter, the cells were suspended in a medium containing antibodies and TRAIL/Apo2 ligand (1 µg/ml) and incubated at 37°C for 6 h. After centrifugation at 340 × g for 3 min at 4°C, the supernatant was removed, 50 µl/ml of FLICA supplied with the kit (diluted in medium) were added and the mixture was incubated at 37°C for 30 min at 4°C. After incubation, the cells were washed four times with the apoptosis wash buffer provided with the kit and suspended in 100 µl (per well) of apoptosis wash buffer, after which the fluorescence intensity of each cell was measured using a FACSCanto II Flow Cytometer (BD Biosciences). Data were analyzed using FlowJo software version 9.6.4 (BD Biosciences). The apoptotic rate was calculated as the percentage of FAMFLICA-positive cells (early and late apoptotic cells combined) among the total cell population.

PSMA/PC-3 cells were seeded in 96-well plates and incubated overnight at 37°C to attach to the well. The anti-TRAIL-R2 antibody, apomab (32,33) and PSMA/TRAIL-R2 REGULGENT™ were added at final concentrations of 1 µg/ml each. Antibody crosslinkers [anti-human IgG (γ-chain-specific) antibody; cat. no. I3382; MilliporeSigma] were added to the wells and the cells were cultured at 37°C for 3 days. Thereafter, Cell Counting Kit-8 solution was added and the mixture was incubated for 4 h to determine cell viability. To examine the effect of IVIG on cell death, it was applied before apomab and PSMA/TRAIL-R2 REGULGENT™ were added to the wells.

SCID mice were purchased from CLEA Japan, Inc. A total of 80 mice were used in the present study. The mice were housed under specific pathogenfree conditions at 23±3°C with a 12-h light/dark cycle and 50±20% relative humidity. The body weight of the mice at the start of the experiment ranged from 19-24 g. A xenograft mouse model was developed by injecting NCI-H2122 and PSMA/NCI-H2122 transfectant cells (5×10⁶) subcutaneously into the dorsal flank of 6-week-old male SCID mice. Tumor volume was measured twice a week using the following formula: (length × width²)/2. When the mean tumor volume reached 100 mm³ after 7 days, the mice were randomly assigned to treatment groups using a computer-generated randomization method to ensure unbiased distribution. Each group consisted of five mice. Drug administration was initiated with the following treatments: anti-DNP, anti-TRAIL-R2 agonistic antibody KMTR2 and PSMA/TRAIL-R2 REGULGENT™, diluted in saline containing 0.05 mg/ml Polysorbate 80, administered intravenously through the tail vein at doses of 5 ml/kg once a week. After treatment, tumor volume was measured twice a week to verify efficacy. Data are expressed as mean ± SE. Tumor volumes and animal body weights were measured bi-weekly. The experiments were terminated 2 weeks after the start of treatment. No animal reached the predetermined humane endpoint. Mice were anesthetized by inhalation of 3% isoflurane for ~10 min. The depth of anesthesia was confirmed by assessing reflex responses and respiratory patterns to ensure adequate anesthetic depth. Subsequently, the mice were euthanized by cervical dislocation. The mortality of the experimental animals was verified by the cessation of respiration and heartbeat.

The pharmacokinetic profile of REGULGENT™ was assessed in 6-week-old male SCID mice after a single tail vein injection of 1 or 10 mg/kg of anti-TRAIL-R2 antibody E11, anti-PSMA antibody PI101, or PSMA/TRAIL-R2 REGULGENT™ (PI101/E11). Blood samples were collected at 1, 6, 24, 48, 120 and 168 h post-dose. Serum antibody concentrations were measured by electrochemiluminescence immunoassay using streptavidin-coated plates blocked with PBS containing 1% casein. Biotinylated anti-human antibodies, calibration standards, quality control samples and test samples were incubated in duplicate, followed by HRP-labeled anti-human IgG and ruthenium-labeled anti-HRP detection reagents. Luminescence was read on a SECTOR Imager 2400 (Meso Scale Diagnostics, LLC). Between steps, plates were washed with PBS containing 1% Tween-20.

Human hepatocytes from six donors (cat. no. M00995-P; BioIVT) were seeded in collagen-coated 96-well plates and cultured overnight at 37°C under 5% CO₂. The anti-DNP antibody, anti-TRAIL-R2 agonistic antibody KMTR2 and PSMA/TRAIL-R2 REGULGENT™, diluted in culture medium, were added at 50 µl per well and incubated at 37°C for 4 days. Cell Counting Kit-8 solution was added at 10 µl per well and the plates were incubated for 4 h at 37°C under 5% CO₂. Next, 0.1 M HCl (10 µl per well) was added to stop the reaction. The percent cell death was calculated as follows: (1-([absorbance at each antibody concentration]-[absorbance of medium only])/([absorbance at an antibody concentration of 0 µg/ml]-[absorbance of medium only])) ×100. Data were expressed as mean ± SE of triplicate experiments.

Chimeric PXB mice with a humanized liver were purchased from PhoenixBio Co., Ltd. Chimeric PXB mice with a humanized liver were purchased from PhoenixBio Co., Ltd. A total of 16 mice were used in this study. The mice were housed under specific pathogenfree conditions at 23±5°C with a 12-h light/dark cycle and 55±25% relative humidity. All animals were male, 12-18 weeks old at the start of the study and weighed 18-23 g. The mice were produced by xeno-transplanting human hepatocytes into immunodeficient recipient cDNA-uPA+/-/SCID mice. The PXB mice were assigned randomly to four groups (n=4 per group) and injected intravenously with vehicle, anti-TRAIL-R2 agonistic antibody KMTR2 (1 mg/kg), or PSMA/TRAIL-R2 REGULGENT™ (1 or 10 mg/kg) and monitored for 1 week. Individual body weights were recorded once daily before blood sampling on Day 1, before dosing on Day 1 and before blood sampling on Days 3 and 8. After the completion of serial blood sampling for Day 8, mice were anesthetized by inhalation of 3% isoflurane for ~10 min for induction, followed by maintenance with 2.5% isoflurane. The depth of anesthesia was confirmed by assessing reflex responses and respiratory patterns to ensure adequate anesthetic depth. Subsequently, both the abdominal cavity and the thoracic cavity were opened to access the heart for the terminal blood sampling. The blood was collected from each animal via the heart using disposable needles after which the animals were euthanized by exsanguination. The serum was diluted with saline by a factor of 2.5. The serum human alanine aminotransferase (ALT) concentration was determined using a Human ALT ELISA Kit (cat. no. ab234578; Abcam) (34). Serum ALT/ Aspartate aminotransferase (AST) activities were determined by measuring diaryl imidazole type of leucopigment. A DRI CHEM 7000 (Fujifilm) analyzer was used for these measurements.

For preparation of the antigen–antibody complexes, PSMA/TRAILR2 REGULGENT™, TRAIL/Apo2 ligand, and soluble PSMA were mixed at equal protein amounts to achieve a final concentration of 1 mg/ml and incubated at 4°C for 16 ho to allow complex formation. Antigenantibody complex fractions were then separated using Superdex™ High-Performance Columns (Cytiva). The antigen-antibody complexes were diluted to 0.1 µg/ml in phosphate-buffered saline and crosslinked with 0.1% glutaraldehyde at 4°C for 4 h. Thereafter, sample particles were adsorbed on to a carbon foil and stained with Nano-W^® containing methylamine tungstate (Nanoprobes) and the sample grids were imaged using a TF20 microscope (FEI; Thermo Fisher Scientific, Inc.). The particles were selected semi-automatically using COW (https://www.cow-em.de/) and were used for 2D classification using RELION (https://www2.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page).

Statistical analyses were performed using GraphPad Prism version 8 (Dotmatics). To evaluate the effect of cross-linker on cytotoxic activity, data were analyzed using one-way ANOVA followed by Dunnett's post hoc test. To assess the combined effects of cross-linker and IVIG on cytotoxic activity, two-way ANOVA with Bonferroni's post hoc correction was applied. For all other analyses in which statistical significance is indicated, repeated-measures one-way ANOVA followed by Tukey's multiple comparisons test was used. P<0.05 was considered to indicate a statistically significant difference.

The present study first investigated the expression of PSMA and TRAIL-R2 in human prostate cancer, including hormone-resistant cancer, using a tissue microarray. The H-score was calculated as described in the Materials and methods. The results showed that >90% of prostate cancers that were tested were positive for both PSMA and TRAIL-R2 (Fig. 1A and B). In addition, ~90% of the samples from hormone-resistant cancers were positive for both PSMA and TRAIL-R2. Collectively, double positivity for PSMA and TRAIL-R2 was present in almost all human prostate cancers, including hormone-resistant cancers ((Fig. 1C and D).

Agonistic TRAIL-R2 antibodies are known to induce apoptotic cell death through peripheral blood mononuclear cells expressing FcγR. However, this poses some adverse risks to normal cells, such as hepatocytes, which also express TRAIL-R2 (26,35). Therefore, a non-agonistic TRAIL-R2 antibody, E11, was selected to construct bispecific antibodies. Additionally, mispairing of antibody light chains can occur if bispecific antibodies are generated using two different antibody clones. The present study used a novel tetravalent and symmetric bispecific antibody, namely REGULGENT™, comprised of a fused Fab in the N-terminus of the IgG4 heavy chain and a common light chain (Fig. 2A, left) (36). The present study successfully constructed some PSMA antibody clones that shared a light chain with the TRAIL-R2 antibody E11 and they were generated via phage display using an E11 VL fixed library. For comparison with PSMA/TRAIL-R2 REGULGENT™, a bivalent bispecific antibody was constructed with a common light chain and IgG1 Fc mutations lacking effector function (Fig. 2A, right) (30). Based on previous reports (10), the present study selected effector function-null IgG variants of IgG4 and IgG1 Fc regions to eliminate nonspecific TRAIL-R2 signaling mediated by Fc-dependent mechanisms. This approach allowed the present study to specifically evaluate activity independent of ADCC against PSMA-expressing cells. Both bispecific antibodies were produced using Expi293F™ or HEK293 cells (Fig. 2B) and their purity was analyzed using LabChip and SEC (Fig. 2C and D). The purity was 97.4% for PSMA/TRAIL-R2 REGULGENT™ and 99.3% for the bivalent bispecific antibody. Subsequently, the PSMA/TRAIL-R2 binding of these bispecific antibodies was evaluated using BIAcore SPR (Fig. 2E). The results indicated that both bispecific antibodies bound to both PSMA and TRAIL-R2, with no marked difference in the binding levels.

To investigate the biological activities of the bispecific antibodies, the present study evaluated their abilities to induce cell death in some tumor cell lines. First, PSMA-overexpressing variants of the prostate cancer cell line PC-3 and the human lung adenocarcinoma cell line NCI-H2122 were generated, termed PSMA/PC-3 and PSMA/NCI-H2122, respectively. PSMA expression was confirmed by western blot analysis (Fig. S1). Next, PSMA/TRAIL-R2 expression in PC-3 and PSMA/PC-3 cells was verified using flow cytometry (Fig. 3A). The super-agonistic TRAIL-R2 antibody, KMTR2, induced death in both PC-3 and PSMA/PC-3 cells (Fig. 3B and C), whereas the non-agonistic TRAIL-R2 antibody, E11 and PSMA antibody, PN7, did not elicit any cell death. By contrast, PSMA/TRAIL-R2 REGULGENT™ selectively induced death in PSMA/PC-3 but not in PC-3 cells, with a potency superior to that of the KMTR2 antibody. The efficacy of tumor cell death was the same even if another PSMA antibody, clone PI101, was used in REGULGENT™ instead of PN7 (Fig. S2). These findings implied PSMA-dependent biological activity of PSMA/TRAIL-R2 REGULGENT™. Notably, the bivalent bispecific antibody did not induce death in PSMA/PC-3 cells. Comparable results were obtained for NCI-H2122 cells, PSMA/NCI-H2122 and the LNCaP clone FGC (Fig. 3D-H).

To confirm that the cell death induced by PSMA/TRAIL-R2 REGULGENT™ was apoptotic, activated caspase levels were evaluated using flow cytometry (Fig. 3I). The results showed that PSMA/TRAIL-R2 REGULGENT™ activated caspases only in PSMA/PC-3 cells, suggesting PSMA-dependent apoptotic induction.

The first-generation anti-TRAIL-R2 antibody, apomab, requires a crosslinker to induce apoptosis. Apomab markedly decreased cell viability in a crosslinker dose-dependent manner, whereas PSMA/TRAIL-R2 REGULGENTTM slightly increased cell viability at high crosslinker concentrations (Fig. 4A). To examine the effects of these antibodies on cell death, IVIG was used to mimic the in vivo environment. The efficacy of Apomab was markedly inhibited by IVIG in a dose-dependent manner. On the other hand, PSMA/TRAIL-R2 REGULGENTTM did not markedly affect its activity in the presence of IVIG (Fig. 4B).

To assess whether PSMA/TRAIL-R2 REGULGENT™ exhibited a selective antitumor effect on PSMA-expressing tumors in vivo, NCI-H2122 and PSMA/NCI-H2122 cells were implanted subcutaneously into SCID mice (Fig. 5A). Although images of the excised xenograft tumor were not captured, the tumor was visually confirmed to be normal in volume and size. The maximum diameter of the tumor in all mice was 17.9 mm and the maximum volume was 1,195 mm³. KMTR2 showed a strong antitumor effect in both NCI-H2122- and PSMA/NCI-H2122-bearing mice (Fig. 5B, C and Table SI, Table SII, Table SIII, Table SIV, Table SV, Table SVI). By contrast, PSMA/TRAIL-R2 REGULGENT™ showed a specific and significant antitumor effect in PSMA/NCI H2122- but not in NCI-H2122-bearing mice. The pharmacokinetic profile of PSMA/TRAIL-R2 REGULGENT™ in wild-type mice was favorable compared with that of monoclonal antibodies (Fig. S3). These results indicated that PSMA/TRAIL-R2 REGULGENT™ exerted a potent antitumor effect in a PSMA-dependent manner in vivo.

Following previous reports, the present study evaluated the toxicity in human hepatocytes and chimeric livers derived from PBX mice, which exhibit higher sensitivity to TRAIL-mediated apoptosis compared to hepatocytes from rodents and non-human primates (6). Therefore, human hepatocytes and PXB mice were used to evaluate whether PSMA/TRAIL-R2 REGULGENT™ exerts liver toxicity. Human hepatocytes did not express PSMA but slightly expressed TRAIL-R2 on their cell surface (Fig. 6A). KMTR2 induced cell death in human hepatocytes whereas PSMA/TRAIL-R2 REGULGENT™ did not (Fig. 6B). In vitro toxicity evaluations were performed using hepatocytes from multiple donors and PSMA/TRAIL-R2 REGULGENT™ did not induce cell death in any of the hepatocytes tested (Fig. 6C).

Subsequently, PXB mice were administered a single dose of KMTR2 or PSMA/TRAIL-R2 REGULGENT™ intravenously to assess liver toxicity (Fig. 6D). Compared with vehicle-treated mice, KMTR2-treated mice displayed statistically significant increase in serum human ALT1 level, an indicator of liver toxicity (Fig. 6E). By contrast, no significant changes in the serum human ALT1 levels were observed in mice administered 1 or 10 mg/kg of TRAIL-R2/PSMA REGULGENT™ (Fig. 6E). Previous studies have reported that human ALT is the optimal indicator of hepatotoxicity in PXB mice (34). In addition to human ALT1, the present study also evaluated the activities of AST and ALT. Significant increases in these values were observed in mice administered KMTR2, whereas no elevations were detected in mice treated with PSMA/TRAIL-R2 REGULGENT™ (Fig. S4). Moreover, the body weights of mice did not differ markedly among the treatment groups (data not shown). These results suggested that PSMA/TRAIL-R2 REGULGENT™ does not exhibit hepatotoxic effects in PXB mice when administered intravenously as a single dose of up to 10 mg/kg. By contrast, KMTR2 showed hepatotoxic effects following a single intravenous administration at 1 mg/kg.