Innovative CDR grafting and computational methods for PD-1 specific nanobody design

Abstract

Introduction: The development of nanobodies targeting Programmed Cell Death Protein-1 (PD-1) offers a promising approach in cancer immunotherapy. This study aims to design and characterize a PD-1-specific nanobody using an integrated computational and experimental approach.

Methods: An in silico design strategy was employed, involving Complementarity-Determining Region (CDR) grafting to construct the nanobody sequence. The three-dimensional structure of the nanobody was predicted using AlphaFold2, and molecular docking simulations via ClusPro were conducted to evaluate binding interactions with PD-1. Physicochemical properties, including stability and solubility, were analyzed using web-based tools, while molecular dynamics (MD) simulations assessed stability under physiological conditions. The nanobody was produced and purified using Ni-NTA chromatography, and experimental validation was performed through Western blotting, ELISA, and dot blot analysis.

Results: Computational findings demonstrated favorable binding interactions, stability, and physicochemical properties of the nanobody. Experimental results confirmed the nanobody's specific binding affinity to PD-1, with ELISA and dot blot analyses providing evidence of robust interaction.

Discussion: This study highlights the potential of combining computational and experimental approaches for engineering nanobodies. The engineered PD-1 nanobody exhibits promising characteristics, making it a strong candidate for further testing in cancer immunotherapy applications.

Keywords: ELISA; Western blot; cancer immunotherapy; complementarity-determining region; dot blot; nanobody; programmed cell death protein-1.

FIGURE 1

(A) Multiple sequence alignment of the Caplacizumab nanobody, Cemiplimab, and Pembrolizumab heavy chains. (B) Superimposition of the Caplacizumab nanobody (Brown), Cemiplimab (Oranage), and Pembrolizumab heavy chains (Green). (C) Phylogram of the Caplacizumab nanobody, Cemiplimab, and Pembrolizumab heavy chains. (D) Sequence alignment between Cemiplimab and Caplacizumab, with highlighted CDR grafting sequences in the designed nanobody. (E) Three-dimensional structure of the nanobody predicted by AlphaFold 2, with highlighted CDR sequences, visualized using UCSF Chimera. (F) Superimposition of the constructed nanobody (Cyan), Cemiplimab (Orange), and Pembrolizumab (Green) heavy chains. (G) Ramachandran plot of the nanobody structure, illustrating its conformational quality.

FIGURE 2

(A) PD-1/PD-L1 complex. Visualization of the complex was done by using UCSF Chimera (Pettersen et al., 2004). (B) Amino acid interactions between Chain A (PD-L1) and Chain B (PD-1). (C) PD-1/PD-L2 complex. (D) Amino acid interactions between Chain A (PD-1) and Chain B (PD- L2).

FIGURE 3

(A) NB and PD-1 docking structure. (B) Detailed amino acid interactions between Chain A (Constructed Nanobody as Ligand - NB) and Chain B (PD-1 as receptor). (C) Nanobody blocking PD-1/PD L-1 and PD-1/Pd L-2 interactions Visualization of the complex was done by using UCSF Chimera (Pettersen et al., 2004).

FIGURE 4

Molecular dynamics simulation plots at 300 K (A) RMSD analysis (B) RMSF analysis. (C) H-bonding analysis (D) Rg plot. (E) SASA assessment.

FIGURE 5

Molecular dynamics simulation plots at 310 K (A) RMSD analysis (B) RMSF analysis. (C) H-bonding analysis (D) Rg plot. (E) SASA assessment.

FIGURE 6

SDS-PAGE analysis of nanobody expression and purification. Lane M: Protein ladder; Lane 1: cell lysate after IPTG induction (1:50 dilution); Lane 2: Flow-through from Ni-NTA column (1:50 dilution); Lanes 3–6: Eluted fractions from Ni-NTA purification. The arrow indicates the purified nanobody band.

FIGURE 7

(A) Western blot analysis of nanobody affinity for PD-1. Lane M: Prestained protein ladder; Lane 1: Negative control (BSA); Lane 2: Human PD-1 protein probed with nanobody and detected with anti-His tag HRP-conjugated antibody. (B) Dot blot analysis of nanobody affinity for PD-1.1,2,3,4, C dots are 200ng, 100ng, 50ng, 25ng, Control (BSA 200 ng) respectively.

FIGURE 8

ELISA binding curve of the nanobody against PD-1, showing absorbance at 450 nm across nanobody concentrations from 1.95 nM to 1,000 nM. The curve demonstrates a saturation pattern, indicating high-affinity binding to PD-1.