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Article

Camelid-Derived Nanobodies Targeting Human Epidermal Growth Factor Receptor: Screening, Expression, and Functional Validation

by
Yunfeng Liu
1,*,
Qiting Huang
1,
Dongna Zhang
1,
Yingjun Wang
1,
Shuaiying Zhao
1,
Jianchuan Wen
1,
Yingying Kong
2,3 and
Jianfeng Xu
1,*
1
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
3
The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
*
Authors to whom correspondence should be addressed.
Antibodies 2026, 15(2), 19; https://doi.org/10.3390/antib15020019
Submission received: 12 January 2026 / Revised: 8 February 2026 / Accepted: 23 February 2026 / Published: 24 February 2026
(This article belongs to the Topic Antibody-Mediated Therapy and Other Emerging Therapies in Cancer Treatment)
Browse Figures
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Abstract

Objectives: The epidermal growth factor receptor (EGFR) is a clinically relevant membrane receptor that is frequently overexpressed or dysregulated in multiple types of cancer, making it an important target for antibody-based strategies. Nanobodies, derived from camelid heavy-chain antibodies, possess favorable properties such as small size, high stability, and strong antigen-binding capacity. This study aimed to generate EGFR-specific nanobodies and to systematically characterize their binding properties and initial functional activity. Methodology: Bactrian camels were immunized with a whole-cell antigen prepared from 293F cells transiently transfected to express full-length human EGFR. A high-diversity phage display nanobody library was constructed from peripheral blood lymphocytes. After two rounds of biopanning against EGFR, positive clones were screened and selected. The identified nanobodies were recombinantly expressed in Escherichia coli and purified. Binding specificity, epitope relationships, and kinetic parameters were evaluated using high-performance liquid chromatography (HPLC), bio-layer interferometry (Octet), and flow cytometry. The effect of selected nanobodies on EGF-induced cell proliferation was evaluated using a CCK-8 assay. Results: Two EGFR-specific nanobodies, Nb2H4 and Nb2B6, were successfully isolated. Both nanobodies exhibited specific binding to EGFR and recognized distinct, non-competing epitopes. Kinetic analyses revealed favorable binding affinities, and flow cytometry confirmed their ability to recognize EGFR in its native cellular context. In addition, Nb2H4 significantly suppressed EGF-induced proliferation in an EGFR-overexpression cell model, indicating preliminary functional activity. Conclusions: This study reports on the successful generation and in vitro characterization of EGFR-targeting nanobodies based on the extracellular domain of EGFR. The identified nanobodies provide useful molecular tools for epitope mapping, structural studies, and the further exploration of EGFR-directed antibody engineering strategies.

1. Introduction

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase of the ErbB family and a key regulator of cell proliferation and survival [1,2]. Ligand binding to the extracellular domain induces receptor dimerization and activation of its intracellular kinase activity [3,4], leading to the initiation of downstream signaling pathways that are critically involved in tumor initiation and progression [5] (Figure 1).
EGFR is frequently overexpressed or aberrantly activated in multiple solid tumors and is associated with aggressive tumor behavior and poor clinical outcomes [6,7]. In particular, EGFR has attracted attention as a potential target in triple-negative breast cancer [8], where classical receptors such as ER, PR, and HER2 are absent [1,9]. In addition, EGFR can form heterodimers with other ErbB family members, further amplifying oncogenic signaling and promoting tumor progression [10,11].
In recent years, anti-EGFR therapeutic strategies have advanced, with monoclonal antibodies (e.g., cetuximab) and small-molecule inhibitors (e.g., gefitinib) widely applied in clinical practice [1,5,12]. However, these conventional agents have limitations, including high molecular weight, poor tissue penetration, and high immunogenicity, which restrict their broad applicability [1,13,14,15].
Nanobodies are single-domain antibody fragments (VHHs) derived from camelid heavy-chain antibodies [16,17,18]. They exhibit compact structure (~15 kDa), high affinity, excellent thermal stability, high solubility, and low immunogenicity [3,7,19,20,21]. Their single-domain structure enables efficient expression in Escherichia coli or yeast systems [22,23,24], facilitating large-scale screening and molecular engineering [25,26,27]. Compared to conventional monoclonal antibodies, nanobodies exhibit better solid tumor penetration and are well-suited for constructing multivalent antibodies [28,29,30,31], bispecific fusion proteins, antibody-drug conjugates (ADCs), and targeted delivery platforms [25,32,33].
Several EGFR-targeting nanobodies have been reported and investigated in different experimental settings [34,35]. For example, the anti-EGFR nanobody 7D12 has been incorporated into a quantum dot-micelle system for targeted delivery to triple-negative breast cancer cells [35], and the nanobody OA-cb6 has been described as a high-affinity binder capable of recognizing EGFR-positive cells [34]. However, despite the availability of these EGFR-targeting nanobodies, most studies have focused on individual candidates and application-driven validation, with limited comparative analysis of binding affinity, epitope relationships, and suitability as modular antibody resources. In particular, paired nanobodies recognizing distinct, non-competing epitopes of EGFR have rarely been systematically generated and experimentally validated.
In this study, we aimed to address this gap by generating and characterizing a defined pair of EGFR-specific nanobodies as well-characterized antibody resources for EGFR-related research and engineering applications. We hypothesized that immunization with cells expressing full-length human EGFR would facilitate the selection of nanobodies recognizing native and conformational epitopes of the receptor. To test this hypothesis, camels were immunized with HEK-293F cell pellets transiently expressing full-length EGFR, followed by the construction and screening of a high-diversity phage display nanobody library, leading to the identification of two EGFR-specific nanobodies, Nb2H4 and Nb2B6. Subsequently, the binding specificity, affinity, and epitope relationships of Nb2H4 and Nb2B6 were systematically characterized using complementary biophysical and cell-based assays. Their ability to recognize EGFR in its native cellular context was further evaluated by flow cytometry, and an initial in vitro functional assessment was performed to examine their effects on ligand-induced EGFR signaling. Together, these analyses establish Nb2H4 and Nb2B6 as a paired nanobody resource and provide the experimental framework for the subsequent sections of this study.
In its resting state, EGFR exists as a monomer. Upon binding of extracellular ligands (e.g., EGF), EGFR undergoes a conformational change that promotes dimerization. Dimerization triggers autophosphorylation of the intracellular tyrosine kinase domains, leading to the activation of downstream signaling pathways, including the RAS-MAPK pathway mediated by Grb2/SOS and the PI3K-Akt pathway. These cascades regulate cell proliferation, survival, and migration [1,6,13].

2. Materials and Methods

2.1. E. coli Strains, Plasmids, and Cells

All Escherichia coli strains, plasmids, and cell lines used in this study are listed in Table 1.

2.2. Plasmid Construction of hEGFR(1-1210)-CMV-FLAG, hEGFR(25–645)-Avi and hEGFR(25–645)-CMV-FLAG

The coding sequence of human EGFR was obtained from the NCBI database. The full-length EGFR (residues 1–1210) and its extracellular domain (residues 25–645) were cloned into a CMV-FLAG expression vector. To facilitate immobilization during phage display screening, an Avi tag (GLNDIFEAQKIEWHE) was fused to the C-terminus of the EGFR extracellular domain, generating the hEGFR(25–645)-Avi-CMV-FLAG construct. All primers used for plasmid construction are listed in Table 2.

2.3. Expression, Purification, and Biotinylation of hEGFR(25–645)-Avi and hEGFR(25–645)-CMV-FLAG

hEGFR(25–645)-Avi and hEGFR(25–645)-CMV-FLAG were expressed in HEK-293F cells using transient transfection and purified following an identical procedure. Cells were transfected using polyethyleneimine, and protein expression was enhanced by sodium n-butyrate supplementation. Culture supernatants were harvested and clarified prior to purification.
Recombinant proteins were purified by anti-FLAG affinity chromatography, followed by size-exclusion chromatography using a HiLoad™ 16/600 Superdex™ 200 pg column (Cytiva, Marlborough, MA, USA). Purified proteins were concentrated and stored at −80 °C. Protein purity was assessed by SDS-PAGE.
For biotinylation, hEGFR(25–645)-Avi was subjected to in vitro biotinylation using BirA according to the reaction conditions listed in Table 3. After buffer exchange into storage buffer, biotinylation efficiency was confirmed by Western blotting using an HRP-conjugated anti-biotin antibody.
Detailed procedures for protein expression, purification, and biotinylation are described in the Supplementary Materials (Supplementary File S1).

2.4. Camel Immunization, Nanobody Library Construction, and Antibody Panning

Healthy adult Bactrian camels were immunized with HEK-293F cell pellets transiently expressing full-length human EGFR. The immunogen was prepared 48 h after transfection, washed with PBS, and formulated with Freund’s adjuvants for subcutaneous administration. After prime immunization and weekly boosts (seven immunizations in total), peripheral blood (~200 mL) was collected three days after the final boost for nanobody library construction.
Peripheral blood mononuclear cells were isolated by Ficoll density-gradient centrifugation. Total RNA was extracted and reverse-transcribed into cDNA. VHH gene fragments were amplified by nested PCR using VHH-specific primers (Table 2) and cloned into the pMECS phage display vector via PstI/NotI sites. The ligation products were electroporated into Escherichia coli TG1 cells to generate a phage display nanobody library with a capacity exceeding 1 × 108 CFU (colony-forming units).
Phage particles were rescued using M13KO7 helper phage and enriched by two rounds of panning against immobilized biotinylated hEGFR(25–645)-Avi on neutravidin-coated plates. Bound phages were eluted by trypsin treatment and used to infect TG1 cells for amplification. After the second round, individual clones were randomly picked and screened for EGFR binding by indirect ELISA using biotinylated hEGFR(25–645)-Avi as the antigen. Positive clones were identified based on OD450 (optical density measured at 450 nm) values relative to control wells, followed by Sanger sequencing. Sequence analysis was performed using IMGT/V-QUEST (IMGT®, Montpellier, France), and clones with diverse CDR3 sequences were selected for further characterization.
Detailed procedures for camel immunization, nanobody library construction and antibody panning are described in the Supplementary Materials (Supplementary File S1).

2.5. Nanobody Purification and Affinity Assay

Following sequence verification, nanobodies were expressed in Escherichia coli WK6 cells and purified by nickel-affinity chromatography followed by size-exclusion chromatography. Purified nanobodies were obtained as monomeric proteins with high purity, as assessed by SDS-PAGE.
Nanobody binding to EGFR and potential epitope overlap were evaluated by size-exclusion chromatography coupled with high-performance liquid chromatography (SEC-HPLC). hEGFR(25–645)-FLAG was incubated with Nb2H4 or Nb2B6 individually or in combination prior to analysis, and complex formation was assessed based on changes in elution profiles.
Binding kinetics and affinities between nanobodies and EGFR were further determined by bio-layer interferometry (BLI) using an Octet RED96 system. Biotinylated hEGFR(25–645)-Avi was immobilized on streptavidin sensors, and nanobody binding was analyzed using a 1:1 interaction model to derive the association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD).
Bio-layer interferometry measurements were performed in a single independent experiment using multiple analyte concentrations, with each concentration measured in triplicate technical repeats.
Detailed experimental procedures for nanobody expression, purification, SEC-HPLC, and BLI analysis are provided in the Supplementary Materials (Supplementary File S1).

2.6. Flow Cytometry Analysis of Nanobody Binding to Tumor Cells

Based on previous studies by Omidfar et al. [14], the epidermal growth factor receptor (EGFR) has been established as a promising therapeutic target in cancer. According to data from The Human Protein Atlas, EGFR mRNA is highly expressed in the human bladder cancer cell line 5637, with a reported expression level of 103.1 normalized transcripts per million (nTPM). To evaluate whether the nanobodies selected in this study can recognize endogenously expressed EGFR, flow cytometry analysis was performed using 5637 cells. Human embryonic kidney 293T (HEK-293T) cells, which exhibit negligible endogenous EGFR expression and were not transfected with any EGFR-expressing plasmids, were used as a negative control to assess binding specificity.
Cells were incubated with candidate nanobodies, followed by detection using an anti-HA (hemagglutinin) primary antibody and a fluorophore-conjugated secondary antibody. Nanobody binding was analyzed by flow cytometry, and specificity was evaluated by comparing the binding profiles between EGFR-positive and EGFR-negative cell lines.
Flow cytometry experiments were performed separately for 5637 and HEK-293T cells. For each cell line, experiments were conducted in two independent runs, with three biological replicates per group (n = 3).
Detailed staining procedures and flow cytometry acquisition parameters are provided in the Supplementary Materials (Supplementary File S1).
For flow cytometry analysis, cells were first gated based on forward and side scatter (FSC/SSC) parameters to exclude debris. Doublets were excluded using FSC-A versus FSC-H gating. Fluorescence thresholds were defined based on secondary antibody-only control samples, and the percentage of positive cells was determined accordingly.

2.7. CCK-8 Assay to Evaluate EGF-Induced Proliferation and the Inhibitory Effect of Nb2H4 in EGFR-Expressing Cells

To evaluate the functional activity of the anti-EGFR nanobody Nb2H4, a CCK-8 assay was performed to assess its effect on EGF-induced cell proliferation. HEK-293T cells transiently expressing full-length human EGFR were used as a model system. Cells were stimulated with recombinant human EGF to induce proliferation, and the inhibitory effect of Nb2H4 was examined by co-incubation with EGF at different concentrations. In parallel, cells treated with Nb2H4 alone in the absence of EGF were included to assess any direct effect of the nanobody on cell viability.
Cell proliferation was quantified using the CCK-8 assay by measuring absorbance at 450 nm. The proliferative response induced by EGF and its modulation by Nb2H4 were evaluated by comparison between treatment groups.
The CCK-8 cell viability assay was performed using six biological replicates per group (n = 6).
Detailed experimental procedures and assay conditions are provided in the Supplementary Materials (Supplementary File S1).

3. Results and Discussion

3.1. Expression, Purification and Biotinylation of hEGFR(25–645)-Avi-CMV-FLAG and hEGFR(25–645)-CMV-FLAG

The extracellular domain of human EGFR (residues 25–645) was successfully expressed in HEK-293F cells as a C-terminally FLAG-tagged protein, with or without an additional Avi tag. Both hEGFR(25–645)-CMV-FLAG and hEGFR(25–645)-Avi-CMV-FLAG were efficiently purified from the culture supernatant by anti-FLAG affinity chromatography, as confirmed by SDS-PAGE analysis (Figure 2A,B).
In vitro biotinylation of the purified hEGFR(25–645)-Avi protein using BirA enzyme was verified by Western blotting using an HRP-conjugated anti-biotin antibody, confirming successful biotin incorporation (Figure 2C).

3.2. Construction of Nanobody Library and Screening of Nanobodies That Bind Specifically to EGFR

Total RNA extracted from peripheral blood mononuclear cells of immunized camels showed good integrity (Figure 3A). VHH gene fragments were successfully amplified by two rounds of nested PCR, yielding products of the expected size (Figure 3B,C), and subsequently cloned into a phage display vector.
The quality of the constructed library was evaluated by colony counting. The anti-EGFR nanobody phage display library exhibited a high capacity and a favorable positive insertion rate, indicating sufficient diversity for downstream screening (Figure 3D).
To enrich EGFR-specific nanobodies, two rounds of biopanning were performed using biotinylated EGFR-Avi as the target antigen. After the second round of selection, a marked enrichment of EGFR-binding phage clones was observed compared with the control group lacking antigen, indicating successful selection of specific binders. Following biopanning, 96 individual clones were randomly selected and screened by ELISA for binding to EGFR. Seventeen clones displayed OD450 values more than fourfold higher than the negative control, indicating specific binding activity (Figure 3E,F). Sequence analysis of these positive clones revealed six nanobody candidates with distinct CDR3 regions (Figure 3G), which were selected for further characterization.

3.3. Expression, Affinity, and Binding Epitope Determination of Nanobodies

The nanobody plasmids Nb2H4 and Nb2B6 were expressed in E. coli WK6 and purified by Ni-NTA affinity chromatography followed by size-exclusion chromatography. SDS-PAGE analysis confirmed efficient expression and high purity of both nanobodies (Figure 4A,B).
SEC-HPLC analysis was performed to assess complex formation between EGFR and the nanobodies Nb2H4 and Nb2B6. In all tested conditions, stable antigen-nanobody complexes were observed, and no peak shifts indicative of competitive binding were detected, supporting the conclusion that Nb2H4 and Nb2B6 recognize distinct, non-competing epitopes on EGFR. Similar chromatographic profiles were obtained in three independent experiments (n = 3) (Figure 4C–E).
Binding kinetics were further characterized by bio-layer interferometry using an Octet system. Biotinylated EGFR-Avi was immobilized on streptavidin biosensors and exposed to nanobodies at multiple concentrations. For Nb2B6, concentration-dependent kinetic fitting yielded an association rate constant (kon) of 1.09 × 106 M−1 S−1 and a dissociation rate constant (koff) of 2.08 × 10−3 S−1, corresponding to an apparent equilibrium dissociation constant (KD) of approximately 1.93 nM. For Nb2H4, concentration-dependent fitting yielded an association rate constant (kon) of 8.1 × 105 M−1 S−1, whereas the dissociation rate was extremely slow and fell below the reliable detection limit of the instrument (koff < 1 × 10−7 S−1). Because bio-layer interferometry approaches its technical limits in accurately resolving very slow off-rates, the binding affinity of Nb2H4 is more appropriately described as an apparent high-affinity interaction in the sub-nanomolar to picomolar range, derived from model fitting (Figure 4F,G).
Together, these results indicate that Nb2H4 and Nb2B6 bind EGFR with high apparent affinity and distinct epitope specificities, supporting their use as well-defined EGFR-targeting nanobody reagents.

3.4. Validation of Nanobody Binding to Tumor Cells (Flow Cytometry)

To evaluate whether the selected nanobodies Nb2H4 and Nb2B6 can recognize endogenously expressed EGFR on the surface of tumor cells, the EGFR-high 5637 bladder cancer cell line was used as the experimental group. HEK-293T cells, which express minimal levels of EGFR, served as the negative control. After incubation with nanobodies, the binding ability and specificity were analyzed by flow cytometry.
The results showed that in 5637 cells, compared to the secondary antibody-only control group (Figure 5A, 0.92% positive cells), both Nb2H4 (Figure 5B) and Nb2B6 (Figure 5C) induced a marked rightward shift in fluorescence, with the proportion of positive cells reaching 17.56% and 24.04%, respectively. These data indicate that both nanobodies effectively recognize EGFR expressed on the surface of 5637 cells. In contrast, in 293T cells, fluorescence signals following treatment with Nb2H4 (Figure 5E) and Nb2B6 (Figure 5F) were nearly identical to the negative control (Figure 5D), with positive rates below 2%, further confirming the high specificity and selectivity of their binding.
Collectively, these results indicate that Nb2H4 and Nb2B6 specifically recognize endogenous EGFR on the surface of EGFR-positive tumor cells, while showing minimal binding to EGFR-low cells.

3.5. Nb2H4 Inhibits EGF-Induced Proliferation of EGFR-Overexpressing 293T Cells

Treatment with Nb2H4 resulted in a concentration-dependent reduction in EGF-induced cell proliferation. Increasing concentrations of Nb2H4 (10–150 nM) progressively decreased cell viability in the presence of EGF, with statistically significant differences observed at concentrations of 60 nM and above compared with the EGF-only group (Figure 6). These results indicate that Nb2H4 attenuates EGF-induced proliferative responses in EGFR-overexpressing cells under the experimental conditions tested.
In the absence of EGF stimulation, treatment with Nb2H4 alone across the same concentration range did not significantly affect cell viability, indicating that Nb2H4 does not induce detectable cytotoxic or proliferative effects under the conditions tested (Figure 6).
Taken together, these data show that Nb2H4 reduces EGF-driven proliferation in an EGFR overexpression model.

4. Conclusions

In this study, a phage display nanobody library targeting human EGFR was successfully constructed, leading to the identification of two EGFR-specific nanobodies, Nb2H4 and Nb2B6. Biophysical and biochemical analyses indicate that both nanobodies bind EGFR with high apparent affinity and recognize distinct, non-competing epitopes. Cell-based assays further showed that Nb2H4 and Nb2B6 are capable of recognizing native EGFR on the surface of EGFR-positive tumor cells. In addition, Nb2H4 was found to reduce EGF-induced cell proliferation in an in vitro EGFR overexpression model.
Taken together, this work provides a set of well-characterized EGFR-targeting nanobodies and an initial assessment of their binding and functional properties at the cellular level. These findings support the utility of Nb2H4 and Nb2B6 as molecular tools for EGFR-related research and warrant further investigation in more physiologically relevant experimental systems.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antib15020019/s1, File S1: Supplementary Methods; Figure S1: Schematic overview of camel immunization, nanobody library construction, and phage display screening.

Author Contributions

Y.L. (first author): Performed the experimental work, Analyzed the data, Wrote the manuscript; Y.W.: Analyzed the data; Q.H.: Analyzed the data; D.Z.: Analyzed the data; S.Z.: Analyzed the data; J.W.: Analyzed the data; Y.K.: Analyzed the data; J.X. (corresponding author): Coordinated analysis of the data. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic illustration of EGFR activation.
Figure 1. Schematic illustration of EGFR activation.
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Figure 2. Expression, purification, and biotinylation analysis of recombinant hEGFR(25–645)-Avi-CMV-FLAG and hEGFR(25–645)-CMV-FLAG proteins. (A) Gel filtration profile of purified hEGFR(25–645)-CMV-FLAG using a HiLoad™ 16/600 Superdex™ 200 pg column (Cytiva, USA). The corresponding SDS-PAGE image is shown in the upper right corner. (B) Gel filtration and SDS-PAGE analysis of hEGFR(25–645)-Avi purified under identical conditions. (C) Western blot analysis of biotinylation. Biotinylation of hEGFR(25–645)-Avi was detected using an HRP-conjugated anti-biotin antibody. The blank group (no protein added) served as a negative control.
Figure 2. Expression, purification, and biotinylation analysis of recombinant hEGFR(25–645)-Avi-CMV-FLAG and hEGFR(25–645)-CMV-FLAG proteins. (A) Gel filtration profile of purified hEGFR(25–645)-CMV-FLAG using a HiLoad™ 16/600 Superdex™ 200 pg column (Cytiva, USA). The corresponding SDS-PAGE image is shown in the upper right corner. (B) Gel filtration and SDS-PAGE analysis of hEGFR(25–645)-Avi purified under identical conditions. (C) Western blot analysis of biotinylation. Biotinylation of hEGFR(25–645)-Avi was detected using an HRP-conjugated anti-biotin antibody. The blank group (no protein added) served as a negative control.
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Figure 3. Nanobody library construction and biopanning of EGFR-specific clones. (A) Agarose gel electrophoresis of total RNA extracted from peripheral blood lymphocytes. Clear 28S and 18S rRNA bands indicate high RNA integrity. (B) First round of nested PCR using cDNA as a template produced a ~750 bp fragment containing the VHH gene region. The red box indicates the expected DNA fragment corresponding to the target gene. (C) Second round of nested PCR using the 750 bp fragment as a template yielded the expected ~400 bp VHH product. (D) Colony PCR analysis of randomly selected clones was performed to estimate the insertion rate of the library. (E,F) ELISA screening of individual clones for binding to EGFR. Black dots represent test clones; red dots indicate negative controls. The red horizontal line denotes the OD450 threshold for identifying positive clones. (G) Alignment and analysis of CDR3 regions revealed nanobody clones with diverse sequences. CDR1, CDR2, and CDR3 regions are highlighted in yellow, blue, and red, respectively.
Figure 3. Nanobody library construction and biopanning of EGFR-specific clones. (A) Agarose gel electrophoresis of total RNA extracted from peripheral blood lymphocytes. Clear 28S and 18S rRNA bands indicate high RNA integrity. (B) First round of nested PCR using cDNA as a template produced a ~750 bp fragment containing the VHH gene region. The red box indicates the expected DNA fragment corresponding to the target gene. (C) Second round of nested PCR using the 750 bp fragment as a template yielded the expected ~400 bp VHH product. (D) Colony PCR analysis of randomly selected clones was performed to estimate the insertion rate of the library. (E,F) ELISA screening of individual clones for binding to EGFR. Black dots represent test clones; red dots indicate negative controls. The red horizontal line denotes the OD450 threshold for identifying positive clones. (G) Alignment and analysis of CDR3 regions revealed nanobody clones with diverse sequences. CDR1, CDR2, and CDR3 regions are highlighted in yellow, blue, and red, respectively.
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Figure 4. Purification, affinity analysis, and epitope characterization of anti-EGFR nanobodies. (A,B) Size-exclusion chromatography (SEC) and SDS-PAGE analysis of purified nanobodies Nb2B6 and Nb2H4, both appearing as monomeric proteins with high purity (~19 kDa). (C,D) High-performance liquid chromatography (HPLC) analysis of nanobody-EGFR binding. Incubation of EGFR with Nb2B6 or Nb2H4 resulted in a notable peak shift, indicating formation of EGFR-nanobody complexes. SEC-HPLC profiles showing EGFR alone, EGFR incubated with Nb2H4 or Nb2B6 individually, and EGFR incubated with both nanobodies. Chromatograms are representative of three independent experiments (n = 3) with similar results. (E) HPLC-based epitope binning to assess potential binding competition. The elution profile of the ternary mixture (EGFR + Nb2B6 + Nb2H4) exhibited a further peak shift compared to binary complexes, suggesting that Nb2B6 and Nb2H4 bind to distinct, non-competing epitopes on EGFR. (F,G) Binding kinetics of Nb2B6and Nb2H4 to EGFR determined using bio-layer interferometry (Octet system). Sensorgrams at serial concentrations (62.5–1000 nM) showed typical association and dissociation curves, consistent with strong binding interactions. Representative BLI sensorgrams obtained using multiple nanobody concentrations, with each concentration measured in triplicate as technical replicates, are shown.
Figure 4. Purification, affinity analysis, and epitope characterization of anti-EGFR nanobodies. (A,B) Size-exclusion chromatography (SEC) and SDS-PAGE analysis of purified nanobodies Nb2B6 and Nb2H4, both appearing as monomeric proteins with high purity (~19 kDa). (C,D) High-performance liquid chromatography (HPLC) analysis of nanobody-EGFR binding. Incubation of EGFR with Nb2B6 or Nb2H4 resulted in a notable peak shift, indicating formation of EGFR-nanobody complexes. SEC-HPLC profiles showing EGFR alone, EGFR incubated with Nb2H4 or Nb2B6 individually, and EGFR incubated with both nanobodies. Chromatograms are representative of three independent experiments (n = 3) with similar results. (E) HPLC-based epitope binning to assess potential binding competition. The elution profile of the ternary mixture (EGFR + Nb2B6 + Nb2H4) exhibited a further peak shift compared to binary complexes, suggesting that Nb2B6 and Nb2H4 bind to distinct, non-competing epitopes on EGFR. (F,G) Binding kinetics of Nb2B6and Nb2H4 to EGFR determined using bio-layer interferometry (Octet system). Sensorgrams at serial concentrations (62.5–1000 nM) showed typical association and dissociation curves, consistent with strong binding interactions. Representative BLI sensorgrams obtained using multiple nanobody concentrations, with each concentration measured in triplicate as technical replicates, are shown.
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Figure 5. Specific binding analysis of nanobodies Nb2H4 and Nb2B6 to EGFR-positive cells by flow cytometry. EGFR-high 5637 bladder cancer cells and EGFR-low HEK-293T cells were incubated with nanobodies Nb2H4 or Nb2B6, and surface EGFR binding was analyzed by flow cytometry. Panels (A–C) show results from 5637 cells, and panels (D–F) show results from HEK-293T cells. (A,D) Secondary antibody-only controls, showing 0.92% and 0.81% positive cells, respectively. (B,C) 5637 cells treated with Nb2H4 and Nb2B6, showing increased proportions of EGFR-positive cells (17.56% and 24.04%, respectively). (E,F) 293T cells treated with Nb2H4 and Nb2B6 exhibited low positive cell percentages (2.06% and 1.94%, respectively), with no evident increase compared with controls. For each cell line, experiments were conducted in two independent runs, with three biological replicates per group (n = 3). The percentage of positive cells was used as the quantitative readout. Cell density is represented using a pseudocolor scale (blue to red indicating increasing event density), and the pink box indicates the gated positive population.
Figure 5. Specific binding analysis of nanobodies Nb2H4 and Nb2B6 to EGFR-positive cells by flow cytometry. EGFR-high 5637 bladder cancer cells and EGFR-low HEK-293T cells were incubated with nanobodies Nb2H4 or Nb2B6, and surface EGFR binding was analyzed by flow cytometry. Panels (A–C) show results from 5637 cells, and panels (D–F) show results from HEK-293T cells. (A,D) Secondary antibody-only controls, showing 0.92% and 0.81% positive cells, respectively. (B,C) 5637 cells treated with Nb2H4 and Nb2B6, showing increased proportions of EGFR-positive cells (17.56% and 24.04%, respectively). (E,F) 293T cells treated with Nb2H4 and Nb2B6 exhibited low positive cell percentages (2.06% and 1.94%, respectively), with no evident increase compared with controls. For each cell line, experiments were conducted in two independent runs, with three biological replicates per group (n = 3). The percentage of positive cells was used as the quantitative readout. Cell density is represented using a pseudocolor scale (blue to red indicating increasing event density), and the pink box indicates the gated positive population.
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Figure 6. Nb2H4 inhibits EGF-induced proliferation of EGFR-overexpressing cells. Cell viability was assessed by CCK-8 assay in HEK-293T cells transiently expressing full-length EGFR. (A) Treatment with EGF (50 ng/mL) significantly increased cell viability compared with the unstimulated control, indicating activation of EGFR-mediated proliferative signaling. Co-treatment with increasing concentrations of Nb2H4 (10–150 nM) resulted in a concentration-dependent reduction in cell viability, with the strongest inhibitory effect observed at 150 nM. In contrast, Nb2H4 alone (same concentration range, without EGF) did not affect cell viability, indicating that Nb2H4 is non-toxic and does not exhibit agonistic activity. Different shades of gray are used for visual clarity only. (B) Dose-response representation of Nb2H4-mediated inhibition of EGF-induced proliferation, plotted as relative cell viability versus Nb2H4 concentration. Data are presented as mean ± SD from six biological replicates (n = 6). Statistical significance at each Nb2H4 concentration was determined by one-way ANOVA followed by Dunnett’s post hoc test, with comparisons made against the EGF-treated group (** p < 0.01; **** p < 0.0001).
Figure 6. Nb2H4 inhibits EGF-induced proliferation of EGFR-overexpressing cells. Cell viability was assessed by CCK-8 assay in HEK-293T cells transiently expressing full-length EGFR. (A) Treatment with EGF (50 ng/mL) significantly increased cell viability compared with the unstimulated control, indicating activation of EGFR-mediated proliferative signaling. Co-treatment with increasing concentrations of Nb2H4 (10–150 nM) resulted in a concentration-dependent reduction in cell viability, with the strongest inhibitory effect observed at 150 nM. In contrast, Nb2H4 alone (same concentration range, without EGF) did not affect cell viability, indicating that Nb2H4 is non-toxic and does not exhibit agonistic activity. Different shades of gray are used for visual clarity only. (B) Dose-response representation of Nb2H4-mediated inhibition of EGF-induced proliferation, plotted as relative cell viability versus Nb2H4 concentration. Data are presented as mean ± SD from six biological replicates (n = 6). Statistical significance at each Nb2H4 concentration was determined by one-way ANOVA followed by Dunnett’s post hoc test, with comparisons made against the EGF-treated group (** p < 0.01; **** p < 0.0001).
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Table 1. The E. coli strains, cells, and plasmids used in this paper.
Table 1. The E. coli strains, cells, and plasmids used in this paper.
E. coli StrainsSource
WK6Biovector NTCC
TG1Lucigen
DH5αBiovector NTCC
PlasmidsSource
hEGFR(1–1210)-CMV-FLAGThis study
hEGFR(25–645)-Avi-CMV-FLAGThis study
hEGFR(25–645)-CMV-FLAGThis study
pMECsBiovector NTCC
CMV-FLAGBiovector NTCC
pMECs-Nb2H4This study
pMECs-Nb2B6This study
Cell linesSource
HEK-293FBiovector NTCC
HEK-293TBiovector NTCC
5637Biovector NTCC
Table 2. Primers used for plasmid construction.
Table 2. Primers used for plasmid construction.
PrimersSequence
hEGFR(1–1210)-CMV-forward primer5′-CTCTAGAATGCGACCCTCCGGGACGGC-3′
hEGFR(1–1210)-CMV-reverse primer5′-GGAAAAAAGCGGCCGCTGCTCCAATAAATTCACTG-3′
hEGFR(25–645)-Avi-forward primer5′-GCTCTAGAGCCACCATGCGACCCTCCGGGACGGCCGGGGCA-3′
hEGFR(25–645)-Avi-reverse primer5′-GGCGGCCGCCTCGTGCCACTCGATCTTCTGGGCCTCGAAGA-3′
hEGFR(25–645)-CMV-forward primer5′-GCTCTAGAGCCACCATGCGACCCTCCGGGACGGCCGGGGCA-3′
hEGFR(25–645)-CMV-reverse primer5′-ATAAGAATGCGGCCGCGGACGGGATCTTAG-3′
VHH-forward primer5′-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3′
VHH-reverse primer5′-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3′
CALL-leader primer5′-GTCCTGGCTGCTCTTCTACAAGG-3′
CALL-CH2 primer5′-GGTACGTGCTGTTGAACTGTTCC-3′
Table 3. Reaction components for in vitro biotinylation of hEGFR(25–645)-Avi.
Table 3. Reaction components for in vitro biotinylation of hEGFR(25–645)-Avi.
ComponentsFinal Concentration/Volume
hEGFR(25–645)-Avi30 nM
BirA enzyme3 nM
10 × Biotin Ligase Buffer A(0.5 M bicine, pH 8.3)250 µL
10 × Biotin Ligase Buffer B(ATP, MgOAc, D-biotin)250 µL
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MDPI and ACS Style

Liu, Y.; Huang, Q.; Zhang, D.; Wang, Y.; Zhao, S.; Wen, J.; Kong, Y.; Xu, J. Camelid-Derived Nanobodies Targeting Human Epidermal Growth Factor Receptor: Screening, Expression, and Functional Validation. Antibodies 2026, 15, 19. https://doi.org/10.3390/antib15020019

AMA Style

Liu Y, Huang Q, Zhang D, Wang Y, Zhao S, Wen J, Kong Y, Xu J. Camelid-Derived Nanobodies Targeting Human Epidermal Growth Factor Receptor: Screening, Expression, and Functional Validation. Antibodies. 2026; 15(2):19. https://doi.org/10.3390/antib15020019

Chicago/Turabian Style

Liu, Yunfeng, Qiting Huang, Dongna Zhang, Yingjun Wang, Shuaiying Zhao, Jianchuan Wen, Yingying Kong, and Jianfeng Xu. 2026. "Camelid-Derived Nanobodies Targeting Human Epidermal Growth Factor Receptor: Screening, Expression, and Functional Validation" Antibodies 15, no. 2: 19. https://doi.org/10.3390/antib15020019

APA Style

Liu, Y., Huang, Q., Zhang, D., Wang, Y., Zhao, S., Wen, J., Kong, Y., & Xu, J. (2026). Camelid-Derived Nanobodies Targeting Human Epidermal Growth Factor Receptor: Screening, Expression, and Functional Validation. Antibodies, 15(2), 19. https://doi.org/10.3390/antib15020019

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