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Antitumor Activity of Dual αB7-H3×αCD3 and αPD-
L1×αCD28 Bispecific T Cell Engager-Armed T Cells Against
Prostate Cancer
Preechaya Naraprasertkul, Yanapith Teerachattrawat*
Faculty of Medicine, Siriraj Hospital, Mahidol University
*Corresponding Author E-mail: yanapith.tee@student.mahidol.edu
Background: Abstract
Prostate cancer (PCa), a highly prevalent tumor in males which frequently develops
treatment resistance and metastasis involving B7-H3 and PD-L1 antigens, leading to
standard treatment inefficacy. Alternatively, immunotherapy has potential against PCa with
enhanced precision and reduced toxicity. Therefore, this study evaluates the antitumor
activity of dual αB7-H3×αCD3 and αPD-L1×αCD28 bispecific T-cell engager (BITE)-armed
T-cells (dual BATs) by assessing cytotoxicity, T-cell activation, and cytokine production in
2D and 3D PCa models.
Methods: Results: The expression levels of B7-H3 and PD-L1 in PCa cell lines were determined using
immunoblot analysis, immunofluorescence assay (IFA), and flow cytometry. Based on prior
studies, bioinformatically designed αB7-H3×αCD3 and αPD-L1×αCD28 BITE cassettes
were constructed to generate stable cells expressing these BITE proteins. The BITE proteins
were validated via immunoblotting and tested for binding to T cells and PCa cell lines. Dual
BATs were generated and evaluated for antitumor activities, including cytotoxic activity,
T cell proliferation, and production of cytolytic molecules. Additionally, their efficacy will be
assessed using a 3D PCa tumoroid model and tissue samples.
B7-H3 and PD-L1 expression levels in four PCa cell lines (DU145, LNCaP P1, VCaP, LN
CaP WT) were quantified, ranging from 89.10%-97.45% and 10.39%-61.77% respectively,
as confirmed by flow cytometry; IFA, and immunoblot analysis. BITE constructs and stable
Lenti-X 293T cells expressing BITEs were successfully generated and validated via
immunoblot analysis. Ongoing work includes the generation of dual BATs and the
evaluation of their antitumor activities using 2D and 3D cancer models.
Conclusion: The highly expressed B7-H3 across PCa cell lines is suitable for therapeutic targets.
Despite moderate PD-L1 expression, its strong association with metastasis and severity
underscores its relevance. Previous studies have demonstrated the effectiveness of
each BITE in activating T cells, suggesting that dual BATs may enhance T cell-mediated
antitumor activity against PCa.
Harmony in health: Innovation for Sustainable Medicine
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