In this study, Complementarity-Determining Region (CDR) grafting was used to generate Nanobody (Wagner et al., 2018; Mirzaei et al., 2024; Reddy et al., 2024). We are using FDA-approved PD-1 inhibitors (Twomey and Zhang, 2021) for CDR grafting, specifically Pembrolizumab (IMGT/mAb DB ID – 472) (Kwok et al., 2016), Nivolumab (IMGT/mAb DB ID – 424) (Gunturi and McDermott, 2015), and Cemiplimab (IMGT/mAb DB ID – 846) (Markham and Duggan, 2018). Sequences for antibodies and Caplacizumab (IMGT/mAb DB ID – 401) Nanobody were obtained from the IMGT database (Manso et al., 2022). We selected Cemiplimab and Pembrolizumab for sequence alignment with Caplacizumab. The alignment indicated that Cemiplimab shared the highest similarity with Caplacizumab, reducing the likelihood of structural disruption during CDR grafting. Consequently, the CDRs from Cemiplimab were grafted onto the framework regions of Caplacizumab, whereas Nivolumab was excluded due to its association with cytokine release syndrome (Manso et al., 2022; Ciner et al., 2021). The newly designed nanobody was subjected to three-dimensional structural prediction using AlphaFold 2 (Mirdita et al., 2022), and its quality was assessed with a Ramachandran plot using the PROCHECK tool (Laskowski et al., 1993).

The designed nanobody underwent thorough physicochemical analysis using multiple parameters. Initially, its molecular weight, theoretical isoelectric point (pI), stability index, aliphatic index, predicted half-life, and grand average of hydropathicity (GRAVY) were assessed with the ProtParam tool (Bidkar et al., 2014).

The tool Vaxigen v2.0 (Zaharieva et al., 2017) was employed to predict immunogenic epitopes within the Nanobody sequence, providing insights into potential immune responses upon administration. Furthermore, allergy prediction was conducted using the ALLERCATPRO 2 tool (Nguyen et al., 2022), which identifies allergenic regions within protein sequences.

The PD-1/PD-L1 and PD-1/PD L-2 interactions were analyzed by PBDsum online tool (Laskowski et al., 2018) utilizing the crystal structures of their complex obtained from the PDB with the ID 4ZQK, 6UMT (Zak et al., 2015; Wen et al., 2020; Tang and Kim, 2019). By examining this structural model, crucial insights into the binding interface and key amino acids involved in the PD-1/PD-L1 and PD-1/PD-L2 interactions were elucidated.

Molecular docking analysis was carried out using the ClusPro web tool (Desta et al., 2020; Vajda et al., 2017; Kozakov et al., 2013; Kozakov et al., 2017), where the PD-1 and PD-L1 complex (PDB ID: 4ZQK, Chain B) served as the receptor, and the designed nanobody was used as the ligand. A thorough exploration of ligand orientations was ensured by setting the number of ligand rotations to probe at 70,000. Post-docking, the structures interactions were analysed by using PDB sum webtool (Laskowski et al., 2018).

We employed WEBGRO (Simlab, 2022) for macromolecular simulations to assess the stability of the constructed nanobody. The system was solvated with a water model, neutralized, and supplemented with 0.15 M NaCl, applying the GROMOS96 43a1 force field. Energy minimization was achieved in 5,000 steps using the steepest descent method. For equilibration, NVT and NPT simulations were performed. We conducted two 50 ns simulations, each with 1,000 frames, at temperatures of 300 K and 310 K (Poustforoosh et al., 2023). Both simulations were run at 1.0 bar. Stability was assessed through parameters including RMSD, RMSF, Rg, H-bonding, and SASA (Sultana et al., 2024).

The nanobody gene sequence, codon-optimized for E. coli expression, was synthesized and inserted into the pET-28a (+) expression vector (Synbio Technologies, NJ, United States) at the NdeI and XhoI restriction sites (Li et al., 2022). The recombinant vector was transformed into Escherichia coli BL21 (DE3) competent cells (Khirehgesh et al., 2021) using the heat shock method. Briefly, 50 µL of competent cells were mixed with 100 ng of the recombinant plasmid and incubated on ice for 30 min. The mixture was then subjected to heat shock at 42°C for 45 s, followed by rapid cooling on ice for 2 min. SOC medium (950 µL) was added to the cells, and the culture was incubated at 37°C for 1 h with shaking at 220 rpm (Chang et al., 2017).

Transformants were selected by plating on LB agar plates containing 50 μg/mL Kanamycin and incubated overnight at 37°C. Positive colonies were screened by colony PCR using primers specifically designed to amplify the inserted nanobody gene (Gussow and Clackson, 1989; Azevedo et al., 2017). Primers were designed using PrimerBLAST (NCBI) (Kozyreva et al., 2021), with sequences as follows:

Forward Primer: 5′CATTCGATGGTGTCCGGGAT-3'.

Reverse Primer: 5′-TCAGCTTCCTTTCGGGCTTT-3'.

PCR conditions were set as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 53°C for 45 s, and 72°C for 1 min, with a final extension at 72°C for 5 min (Rychlik et al., 1990). PCR products were analyzed on a 1% agarose gel stained with ethidium bromide, and the expected amplicon size (nearly 825 bp) was confirmed by comparison with a DNA ladder and positive control (Amplification product of pure plasmid).

A single positive colony was inoculated into 10 mL of LB broth containing 50 μg/mL Kanamycin and grown overnight at 37°C with shaking at 220 rpm. The overnight culture was then transferred into 1,000 mL of LB broth with Kanamycin and grown until the OD600 reached 0.6–0.8. Expression of the nanobody was induced by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, followed by incubation at 18°C for 16 h with shaking at 180 rpm (Nikkhoi et al., 2017).

Cells were harvested by centrifugation at 6,000 × g for 10 min at 4°C, and the cell pellet was resuspended in lysis buffer (50 mM Tris -HCl, 150 mM NaCl, 1 mM PMSF, 10 mM imidazole, pH 8.0). The cells were lysed by sonication on ice, and the lysate was clarified by centrifugation at 12,000 × g for 30 min at 4°C. The nanobody was then purified using Ni-NTA agarose resin pre equilibrated using 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 30 mM imidazole (Himedia, India). Bound proteins were eluted with elution buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0), and fractions were analyzed by 15% SDS-PAGE. Eluted nanobody fractions were pooled and dialyzed overnight at 4°C in 50 mMTris-HCl, 150 mM NaCl, pH 8.0, with buffer changes to remove imidazole. After dialysis overnight the nanobody was subjected to protein estimation by the Lowry method to quantify protein concentration (Crowe et al., 1994; Zhang et al., 2020; Waterborg, 2009).

To assess the affinity of the nanobody for PD-1, both Western blotting and dot blot assays were conducted, with BSA included as a control in both assays. For Western blotting, human PD-1 Fc recombinant protein (Peprotech, United States, Catalog # 310–40-1 MG) and BSA were separated on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Himedia, India). The membrane was blocked with 5% non-fat dry milk in TBST for 1 h at room temperature. After blocking, it was incubated overnight at 4°C with the purified nanobody as the primary antibody, diluted to 10 μg/mL in TBST (Zhang Y. et al., 2023; Shin et al., 2021).

For the dot blot, a series of PD-1 protein concentrations (200, 100, 50, 25 ng) and BSA as controls were directly spotted onto a nitrocellulose membrane, dried, and blocked similarly. The dot blot membrane was then incubated with the purified nanobody under the same conditions, allowing for a direct binding comparison across different PD-1 concentrations and the BSA control. Following primary incubation, both membranes (from Western and dot blot assays) were washed three times with TBST, then incubated with an anti-His tag HRP-conjugated secondary antibody (1:500 dilution, Invitrogen, United States, Cat no: MA1-21315-HRP) for 1 h at room temperature. After post-incubation washes, the blots were developed using tetramethylbenzidine (TMB) substrate solution (Himedia, India).

The binding activity of the purified nanobody against PD-1 was assessed by ELISA. Ninety-six-well plates were coated with 200 µL of 5 μg/mL recombinant human PD-1 in PBS and incubated overnight at 4°C. The plates were washed three times with PBS containing 0.05% Tween-20 (PBST) and blocked with 5% bovine serum albumin (BSA) in PBST for 1 h at room temperature. After blocking, 200 µL of serially diluted nanobody (starting from 1000 to 1.95 nM) was added to the wells and incubated for 2 h at room temperature. The plates were then washed with PBST and incubated with 200 µL of anti-His tag HRP-conjugated antibody (1:500 dilution, Invitrogen, United States, Cat no: MA1-21315-HRP) for 1 h. After washing, 200 µL of tetramethylbenzidine (TMB) substrate solution was added to each well, and the reaction was stopped with 100 µL of 2 M H2SO4. The absorbance was measured at 450 nm using a microplate reader (Benesphera E21) (Chen et al., 2020).

In the CDR grafting process, the complementarity-determining regions (CDRs) from Cemiplimab were successfully grafted onto the framework regions of Caplacizumab. The resulting 3D model was constructed using AlphaFold2, a state-of-the-art protein structure prediction tool. The quality of the constructed nanobody model was rigorously evaluated using a Ramachandran plot, which is crucial for assessing the conformational angles of the protein backbone, ensuring that the model is structurally sound and free from steric clashes. The Ramachandran plot generated by PROCHECK demonstrated that 94.7% of the residues were located in the most favored regions, 5.3% in the additional allowed regions, with no residues in the generously allowed or disallowed regions. This indicates that the model is of high quality, with no significant structural issues. Specifically, the analysis included 95 non-glycine and non-proline residues, with 17 glycine residues and 3 proline residues, totaling 117 residues.

Figure 1 illustrates the overall process of multiple sequence alignment, CDR grafting and homology modeling, demonstrating the integration of Cemiplimab CDRs into the Caplacizumab framework. The resultant 3D structure generated by AlphaFold2 is visualized using UCSF Chimera (Pettersen et al., 2004), and the quality assessment is confirmed through the Ramachandran plot analysis.

The physicochemical attributes of the designed nanobody, analyzed by the ProtParam tool, revealed the following: a molecular weight (MW) of 12.5 kDa (with his tag ∼13.5 k Da), theoretical pI of 8.05, and instability index of 35.84, representing that the designed nanobody is stable. The aliphatic index was 64.96, and the estimated half-life in E. coli was greater than 10 h and the GRAVY was −0.257, which indicates nanobody potential suitability for practical applications. The number of negatively charged (Asp and Glu) amino acids was 10, while the number of positively charged (Arg and Lys) amino acids was 11.

The immunogenicity and allergy potential of the nanobody were evaluated VaxiJen v2.0 and the ALLERCATPRO 2 tool, respectively. The average immunogenicity score was 0.59 (threshold value is 0.5), indicating that the nanobody is mild immunogenic and it has no allergy potential.

Interaction analysis of the PD-1/PD-L1 and PD-1/PD-L2 complexes using the PDBsum tool revealed detailed insights into the binding interface and key residues involved. The crystal structures (PDB ID: 4ZQK for PD-1/PD-L1 and 6UMT for PD-1/PD-L2) identified several hydrogen bonds, salt bridges, and non-bonded interactions stabilizing these complexes. Notably, residues spanning positions 65–85 and 124–136 in PD-1 were crucial in interacting with PD-L1. These interactions are summarized in Table 1 below and visualized in Figure 2, highlighting the molecular interactions of both complexes.

Molecular docking was performed using the ClusPro web tool, with the PD-1/PD-L1 complex (PDB ID: 4ZQK, Chain B, i. e., PD-1) as the receptor and the designed nanobody as the ligand. The best-docked complex demonstrated strong binding affinity, supported by multiple hydrogen bonds, salt bridges, and hydrophobic interactions at the binding interface. PDBsum analysis highlighted key residues involved in the interaction, summarized in Table 2 below. Figure 3 depicts the detailed molecular interactions between PD-1 and the nanobody, and elucidates the mechanism by which the nanobody inhibits the binding of PD-1 to its ligands, PD-L1 and PD-L2.

The transformation of the pET-28a (+)-nanobody construct into E. coli BL21 (DE3) cells was successful, as indicated by the growth of kanamycin-resistant colonies. Positive colony screening was performed using colony PCR, with primers specific for the nanobody gene. The expected PCR product size was 825 bp, and agarose gel electrophoresis (figure provided in supplementary material) confirmed the presence of this product in selected colonies. This result confirms the successful insertion of the nanobody gene into the expression vector and its stable transformation into the bacterial host.

The expression of the nanobody was induced by IPTG in E. coli BL21 (DE3) cells, and the protein was successfully purified using Ni-NTA affinity chromatography. The purified protein was analyzed by SDS-PAGE, revealing a single band corresponding to the expected molecular weight of the nanobody, approximately 13.5 kDa (Figure 6). This result indicates successful expression, purification, and quantification of the nanobody. Protein quantification was performed after dialysis using the Lowry method, yielding approximately 1 mg of nanobody per liter of culture.

The affinity of the purified nanobody for PD-1 was assessed using Western blot and dot blot assays, as shown in Figure 7. In the Western blot, recombinant human PD-1 (200 ng) was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with the nanobody. A strong signal at ∼63 kDa, detected using an anti-His tag HRP-conjugated antibody, confirmed the nanobody’s specific binding to denatured PD-1.

The dot blot assay evaluated concentration-dependent binding by spotting varying amounts of PD-1 on a nitrocellulose membrane. Strong signals across the protein gradient demonstrated the nanobody’s effective binding to native PD-1. A BSA control confirmed specificity, highlighting the nanobody’s integrity and functionality in both assays.

The binding affinity of the developed nanobody against PD-1, ELISA assay was performed (Figure 8) with a series of nanobody concentrations ranging from 1,000 to 1.95 nM The absorbance at 450 nm increased with nanobody concentration, indicating specific binding to PD-1. A saturation curve was observed, suggesting a high-affinity interaction between the nanobody and PD-1. At lower concentrations (1.95–125 nM), there was a gradual increase in absorbance, while higher concentrations (500 nM and above) approached a plateau, indicating near-maximal binding. These results demonstrate that the nanobody binds to PD-1 with high affinity, validating its potential use for potential therapeutic applications.