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Article Open Access

Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT™, induces selective tumor cell apoptosis without hepatotoxicity

  • Authors:
    • Makoto Nakayama
    • Sayaka Takagi‑Maeda
    • Yusuke Machino
    • Kaito Nihira
    • Miho Inoue
    • Nobuaki Takahashi
    • Katsuaki Usami
  • View Affiliations / Copyright

    Affiliations: Core Research Laboratories, Bio‑Pharmaceutical Center, Research Division, Kyowa Kirin Co., Ltd., Tokyo 194‑8533, Japan, Project and Product Management Group 3, CMC Development, Manufacturing, Kyowa Kirin Co., Ltd., Tokyo 100‑0004, Japan, Global Operational Transformation Kyowa Kirin International Plc., Marlow SL7 1HZ, UK, Innovation Center, Research Division, Kyowa Kirin Co., Ltd., Tokyo 194‑8533, Japan
    Copyright: © Nakayama et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 155
    |
    Published online on: September 15, 2025
       https://doi.org/10.3892/or.2025.8988
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Abstract

Tumor necrosis factor‑related apoptosis‑inducing ligand‑receptor 2 (TRAIL‑R2) can induce apoptosis in various tumors through the oligomerization of TRAIL. Several TRAIL‑R2 agonistic monoclonal antibodies have been tested in clinical trials but have failed owing to a lack of efficacy or severe hepatotoxicity. Although bispecific constructs have been developed to improve TRAIL‑R2 targeting and enhance efficacy against tumors while reducing adverse effects on hepatocytes, the risk of hepatotoxicity still persists. The present study used a TRAIL‑R2 antibody, E11, that does not trigger apoptosis in the absence of crosslinking and constructed a novel tetravalent bispecific IgG4‑based antibody, REGULGENT™, comprised of E11 and a clone that binds to prostate‑specific membrane antigen (PSMA), a specific marker for prostate tumors. PSMA/TRAIL‑R2 REGULGENT™ selectively induced death in PSMA/TRAIL‑R2 double‑positive cells but not in TRAIL‑R2 single‑positive cells in vitro and in vivo. By contrast, a bivalent bispecific antibody did not result in tumor cell death, indicating that tetravalent bispecific antibodies have an important role in inducing tumor cell apoptosis by binding to TRAIL‑R2 in a bivalent manner. Moreover, the present study demonstrated, for the first time to the best of the authors' knowledge, that PSMA/TRAIL‑R2 REGULGENT™ is not hepatotoxic in vitro (primary human hepatocytes) or in vivo (chimeric human hepatocyte‑transplanted PXB mouse model). This finding suggests that tetravalent bispecific therapeutics such as REGULGENT™ can be promising therapeutic agents for TRAIL‑R2‑positive tumors by exerting tumor‑specific activity while avoiding toxicity.
View Figures

Figure 1

PSMA and TRAIL-R2 expression in
patients with prostate cancer. Representative staining of prostate
cancer tissue microarrays. (A) PSMA expression and (B) TRAIL-R
expression. The H scores were calculated based on percent
positivity and intensity score for (C) all spots and (D) spots in
hormone-resistant prostate cancers. The red asterisk represents a
normal gland/duct. PSMA, prostate-specific membrane antigen;
TRAIL-R2, tumor necrosis factor-related apoptosis-inducing
ligand-receptor 2.

Figure 2

Design and production of
prostate-specific membrane antigen and tumor necrosis
factor-related apoptosis-inducing ligand-receptor 2 (PSMA/TRAIL-R2)
bispecific antibodies. (A) Schematic of the Fab-based tetravalent
bispecific antibody (REGULGENT™) and bivalent bispecific antibody
designs. (B) Flowchart of expression and purification of REGULGENT™
and bivalent bispecific antibody. Analytical data of REGULGENT™ and
bivalent bispecific antibody after expression in 293F cells and a
single-step protein A purification using (C) LabChip and (D) size
exclusion chromatography. (E) PSMA or TRAIL-R2 binding of generated
antibodies to recombinant proteins as analyzed by Biacore surface
plasmon resonance. PSMA, prostate-specific membrane antigen;
TRAIL-R2, tumor necrosis factor-related apoptosis-inducing
ligand-receptor 2.

Figure 3

Cancer cell death induced by
prostate-specific membrane antigen and tumor necrosis
factor-related apoptosis-inducing ligand-receptor 2 (PSMA/TRAIL-R2)
bispecific antibodies. (A, D and G) Evaluation of PSMA and TRAIL-R2
expression in various cell lines using flow cytometry. (B, C, E, F
and H) Viability of human tumor cell lines in response to treatment
with PSMA/TRAIL-R2 bispecific antibodies (REGULGENT™ and bivalent
bispecific antibody) in a 96-well cell proliferation assay using
the (B) PC-3 and (E) NCI-H2122 cell lines
(PSMA−TRAIL-R+), (C) PSMA/PC-3 and (F) a
PSMA/NCI-H2122 transfectants and (H) the LNCaP clone FGC cell line
(PSMA+TRAIL-R+). Antibodies were tested in
fivefold dilutions starting at 12.5 nM to 0.004 nM. Data represent
quadruplicate experiments and are shown as means ± SE. The vertical
axes represent cell death (%) and the horizontal axes represent the
antibody concentration (nM). An anti-DNP antibody was used as the
negative control. (I) Caspase activation in PC-3 or PSMA/PC-3 cells
by the TRAIL/Apo2 ligand and PSMA/TRAIL-R2 REGULGENT™ using the
FAM-FLICA Poly Caspase Assay. The solid line shows caspase
activation by the TRAIL/Apo2 ligand, the dotted line shows caspase
activation by TRAIL-R2/PSMA REGULGENT™ and the gray line shows
caspase activation by the anti-DNP antibody used as the negative
control. PSMA, prostate-specific membrane antigen; TRAIL-R2, tumor
necrosis factor-related apoptosis-inducing ligand-receptor 2; DNP,
2,4-dinitrophenol.

Figure 4

Effect of crosslinker and IVIG on
tumor cell death induced by the prostate-specific membrane antigen
and the tumor necrosis factor-related apoptosis-inducing
ligand-receptor 2 (PSMA/TRAIL-R2) bispecific antibody (REGULGENT™).
(A) PSMA/PC-3 cells were seeded onto 96 well plates. Then apomab
and PSMA/TRAIL-R2 REGULGENT™ were added and cultured with
crosslinkers for 3 days. Statistical analysis was performed using
one-way ANOVA with Dunnett's post hoc test to compare each
treatment group at a given concentration with the corresponding
no-antibody (no Ab) control group. **P<0.01, ****P<0.0001,
ns, no significance. (B) IVIG affected the efficacy of crosslinked
apomab and PSMA/TRAIL-R2 REGULGENT™ in a concentration-dependent
manner. Statistical analysis was performed using twowayANOVA with
Bonferroni post hoc test. ****P<0.0001, ns, no significance.
IVIG, intravenous immunoglobulin; PSMA, prostate-specific membrane
antigen; TRAIL-R2, tumor necrosis factor-related apoptosis-inducing
ligand-receptor 2; DNP, 2,4-dinitrophenol.

Figure 5

In vivo PSMA-dependent
antitumor activity of the PSMA/TRAIL-R2 bispecific antibody
(REGULGENT™). (A) Schedule of antibody administration and tumor
measurement in a xenograft model. (B) NCI-H2122 and (C)
PSMA/NCI-H2122 transfectant cells were transplanted subcutaneously
into SCID mice. The mice were grouped 7 days after transplantation
to start receiving the anti-2,4-dinitrophenol (DNP) antibody,
anti-TRAIL-R2 agonistic antibody KMTR2, or PSMA/TRAIL-R2
REGULGENT™. The vertical axes indicate tumor size (mm3)
and the horizontal axes indicate the time (days) after the start of
antibody administration. Plotted values are the means (SEM) of
duplicates of four independent experiments. To determine the
statistical significance, an RM one-way ANOVA with Tukey's multiple
comparisons was performed (**P<0.01, ns, no significance). PSMA,
prostate-specific membrane antigen; TRAIL-R2, tumor necrosis
factor-related apoptosis-inducing ligand-receptor 2; DNP,
2,4-dinitrophenol.

Figure 6

In vitro/in vivo liver
toxicity of prostate-specific membrane antigen and tumor necrosis
factor-related apoptosis-inducing ligand-receptor 2 (PSMA/TRAIL-R2)
bispecific antibodies. (A) PSMA and TRAIL-R2 expression in
hepatocytes was determined using flow cytometry. (B) Hepatocytes
were treated with serially diluted antibodies anti-DNP,
anti-TRAIL-R2 agonistic KMTR2, anti-TRAIL-R2 non-agonistic E11, or
PSMA/TRAIL-R2 REGULGENT™) and incubated for 96 h. Cell viability
was determined using Cell Counting Kit-8. (C) Antibody-induced
toxicity in human primary hepatocytes from six donors (lot numbers:
XVN, TRZ, AKB, EBS, QOQ and OGF). (D) Schedule of in vivo
safety assessment using PXB mice. (E) Serum human ALT1
concentrations in response to anti-TRAIL-R2 agonistic antibody
KMTR2 and PSMA/TRAIL-R2 REGULGENT™ treatment. *P<0.05, ns, no
significance. PSMA, prostate-specific membrane antigen; TRAIL-R2,
tumor necrosis factor-related apoptosis-inducing ligand-receptor 2;
DNP, 2,4-dinitrophenol; ALT, alanine aminotransferase.
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Copy and paste a formatted citation
Spandidos Publications style
Nakayama M, Takagi‑Maeda S, Machino Y, Nihira K, Inoue M, Takahashi N and Usami K: Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity. Oncol Rep 54: 155, 2025.
APA
Nakayama, M., Takagi‑Maeda, S., Machino, Y., Nihira, K., Inoue, M., Takahashi, N., & Usami, K. (2025). Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity. Oncology Reports, 54, 155. https://doi.org/10.3892/or.2025.8988
MLA
Nakayama, M., Takagi‑Maeda, S., Machino, Y., Nihira, K., Inoue, M., Takahashi, N., Usami, K."Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity". Oncology Reports 54.5 (2025): 155.
Chicago
Nakayama, M., Takagi‑Maeda, S., Machino, Y., Nihira, K., Inoue, M., Takahashi, N., Usami, K."Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity". Oncology Reports 54, no. 5 (2025): 155. https://doi.org/10.3892/or.2025.8988
Copy and paste a formatted citation
x
Spandidos Publications style
Nakayama M, Takagi‑Maeda S, Machino Y, Nihira K, Inoue M, Takahashi N and Usami K: Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity. Oncol Rep 54: 155, 2025.
APA
Nakayama, M., Takagi‑Maeda, S., Machino, Y., Nihira, K., Inoue, M., Takahashi, N., & Usami, K. (2025). Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity. Oncology Reports, 54, 155. https://doi.org/10.3892/or.2025.8988
MLA
Nakayama, M., Takagi‑Maeda, S., Machino, Y., Nihira, K., Inoue, M., Takahashi, N., Usami, K."Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity". Oncology Reports 54.5 (2025): 155.
Chicago
Nakayama, M., Takagi‑Maeda, S., Machino, Y., Nihira, K., Inoue, M., Takahashi, N., Usami, K."Novel tetravalent bispecific antibody, PSMA/TRAIL‑R2 REGULGENT&trade;, induces selective tumor cell apoptosis without hepatotoxicity". Oncology Reports 54, no. 5 (2025): 155. https://doi.org/10.3892/or.2025.8988
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