Iranian Journal of Basic Medical Sciences

Iranian Journal of Basic Medical Sciences

Structure-based design of novel and biparatopic anti-PD-L1 nanobodies via CDR grafting and molecular dynamics simulations for enhanced cancer immunotherapy

Articles in Press

Document Type : Original Article

Authors
Mohammad Mehdi Heidari
Elaheh JafariNejad Bidgoli
Zahra Mirshamsi
Mehri Khatami
Nima Amadi Afshar
Mobina Zare
Department of Biology, Yazd University, Yazd, Iran
10.22038/ijbms.2026.96266.20759
Abstract
Objective(s): In recent years, nanobodies (VHH) have become attractive tools for cancer immunotherapy due to their small size, low immunogenicity, and excellent tissue penetration. Historically, researchers have derived nanobodies from camelid immune libraries, a process that requires animal immunization and complex screening. PD-L1 is a critical immune checkpoint molecule that suppresses T-cell activity by interacting with PD-1. Blocking the PD-1/PD-L1 interaction can reactivate the immune system against tumors.
Materials and Methods: In this study, the complementarity-determining region (CDR) grafting approach was applied to engineer a humanized VHH framework by transferring the VH CDRs from the therapeutic antibodies Atezolizumab, Avelumab, and Durvalumab. Based on this strategy, two novel constructs, designated PDL-VHH1 and PDL-VHH2, were generated.
Results: To produce this recombinant protein, the engineered biparatopic nanobody sequence was synthesized and subsequently cloned into the pET-21b(+) vector in an Escherichia coli origami expression system. SDS-PAGE analysis confirmed successful production of the biparatopic nanobody. Indirect ELISA showed that PDL-VHH1 had the highest specific binding signal (OD450=1.82±0.14 at 0.130 µM, compared with 1.21±0.09 for PDL-VHH2 and 0.15±0.03 for the control), and both constructs showed concentration-dependent binding to immobilized PD-L1 across 0.0048–0.1305 µM.
Conclusion: These results underscore PDL-VHH1 as a more promising candidate for future therapeutic and diagnostic applications, while PDL-VHH2 serves as a useful binder with moderate affinity. This study establishes a valuable framework for rapidly, cost-effectively, and efficiently designing in vitro nanobodies that target immune checkpoints.
Keywords
Biparatopic nanobody
CDR grafting
Immune checkpoint blockade
Molecular dynamics simulations
Single-domain antibody Therapeutic antibody design
Subjects
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Iranian Journal of Basic Medical Sciences

Articles in Press, Accepted Manuscript
Available Online from 07 November 2026