FcIgG-GE11-Melittin as a novel EGFR targeted peptibody with potent cytotoxic activity against cancer cells

Abstract

The overexpression of epidermal growth factor receptor (EGFR) in various cancer types makes it an attractive target for therapeutic intervention. In this study, we designed a novel peptibody, FcIgG-GE11-Melittin, by fusing Melittin, a cytotoxic peptide from bee venom, to an EGFR-targeting peptibody (FcIgG-GE11). The FcIgG-GE11 component ensures specific binding to EGFR-overexpressing cancer cells, while Melittin induces cell death through its lytic activity. We evaluated the efficacy of FcIgG-GE11-Melittin in vitro using EGFR-overexpressing cancer cell lines. Our results demonstrate that FcIgG-GE11-Melittin selectively targets and kills EGFR-positive cancer cells while sparing normal cells. These findings highlight the potential of FcIgG-GE11-Melittin as a targeted therapeutic agent for EGFR-overexpressing cancers.

Introduction

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that, upon activation by its ligands, triggers signaling pathways promoting cell proliferation and survival. Overexpression of EGFR, often due to gene amplification or increased transcription, leads to sustained activation of these pathways, contributing to oncogenesis^1,2.

The EGFR overexpression has been observed in various cancers, including lung, bladder, kidney, ovarian, head and neck, colorectal, breast and pancreatic cancers; making it a promising target for cancer therapy². Recent advances in targeted therapies have focused on developing agents that specifically bind to EGFR and deliver cytotoxic payloads to cancer cells³. One such approach involves the use of peptibodies, which are fusion proteins consisting of a targeting peptide and an antibody Fc region⁴.

Melittin is the principal bioactive component of bee (Apis mellifera) venom. It comprises approximately 50–52% of the venom’s dry weight. Structurally, it is a cationic, amphiphilic peptide with a linear sequence of 26 amino acids and a molecular weight of 2840 Da^5,6. Due to its positive charge, it is able to bind and interact with the negatively charged surfaces of cellular membranes. It disrupts cell membranes, leading to cell lysis and apoptosis^7,8. At low concentrations, Melittin demonstrates potent anti-inflammatory activity; however, at higher doses, it is associated with adverse effects such as pruritus, inflammation, and localized pain⁹.

Melittin demonstrates significant anticancer activity through multiple mechanisms, including the induction of apoptosis, cell cycle arrest, modulation of oncogenic signaling pathways, inhibition of metastasis, and enhancement of chemo- and radiosensitivity¹⁰. Numerous studies have extensively investigated the regulatory role of Melittin in apoptosis, as well as its involvement in apoptotic mechanisms across various malignancies, including breast, ovarian, prostate, and lung cancers⁹.

Recent studies highlight the therapeutic potential of combining membrane-lytic peptides with targeting or chemotherapeutic entities. For example, the hybrid peptide NTP-385, generated by covalently linking LTX-315 to rhodamine B, showed up to 37-fold enhanced activity in adherent cancer cells and induced near-complete melanoma regression in mice by disrupting the nuclear membrane and triggering intrinsic apoptosis¹¹. Similarly, conjugation of the DNA-alkylating drug chlorambucil to an oncolytic peptide produced the compound FXY-3, which demonstrated increased nuclear delivery, stronger DNA damage, and superior antitumor efficacy compared with its individual components¹². These findings support the strategy of pairing oncolytic peptides with targeted delivery or chemotherapeutic mechanisms to improve efficacy.

Although anticancer peptides hold strong therapeutic potential, many linear sequences are rapidly degraded by proteases, resulting in short in vivo half-lives and reduced therapeutic effect. To address this limitation, mirror-image peptides composed of D-amino acids have emerged as attractive alternatives because of their exceptional proteolytic stability and reduced immunogenicity. Recent studies demonstrate that D-configured anticancer peptides retain potent membranolytic activity while displaying greatly prolonged stability in biological environments, leading to enhanced antitumor efficacy in vitro and in vivo^13,14.

Despite Melittin’s considerable promise as an anticancer agent, its clinical application is limited by several major challenges. These include nonspecific hemolytic activity, rapid systemic clearance and degradation, as well as insufficient accumulation within tumor tissues¹⁵. To overcome this limitation, Melittin has been conjugated to targeting moieties, such as nanoparticles or antibodies, to enhance its specificity and reduce off-target effects¹⁶.

To address these challenges, we incorporated Melittin into a targeted peptibody format. The GE11 peptide provides selective binding to EGFR-overexpressing cancer cells, reducing nonspecific cytotoxicity and hemolysis, while fusion to the human IgG Fc region enhances molecular stability and circulating half-life, helping overcome rapid degradation and systemic clearance¹⁷. By combining EGFR-targeting with Melittin’s lytic activity, the FcIgG-GE11-Melittin peptibody is designed to improve tumor accumulation and selectively disrupt cancer-cell membranes.

In this study, we describe the design, production, and in vitro evaluation of FcIgG-GE11-Melittin. We have previously shown that FcIgG-GE11 exhibits strong and specific binding to EGFR and inhibits the proliferation of EGFR-overexpressing cancer cells in a dose- and time-dependent manner, comparable to Cetuximab but with improved stability and reduced off-target effects¹⁸. Its efficacy was comparable to that of Cetuximab, while offering additional advantages such as enhanced stability, cost-effective production, and reduced off-target effects, underscoring its potential as an alternative cancer therapeutic. Based on this, we hypothesized that fusion with Melittin would enable selective lysis of EGFR-positive cancer cells while minimizing toxicity toward normal cells. Here, we report that FcIgG-GE11-Melittin selectively kills EGFR-overexpressing cancer cells in vitro, supporting its potential as a targeted therapeutic for EGFR-positive malignancies.

Results

FcIgG-GE11-Melittin design and production

Figure 1a illustrates the schematic of the FcIgG-GE11-Melittin construct. The expression vector for this fusion protein was introduced into Escherichia coli BL21(DE3) host cells. Protein production was triggered by IPTG, resulting in the formation of FcIgG-GE11-Melittin monomers as inclusion bodies (IBs). After washing and solubilizing these IBs, most of the contaminating DNA was successfully eliminated using anion exchange chromatography in negative mode. The monomeric proteins were then refolded through a dilution-based method, allowing the creation of dimeric FcIgG-GE11-Melittin. Final purification was conducted using affinity chromatography. SDS-PAGE analysis verified the presence of dimer (~ 60 kDa) forms of the peptibody (Fig. 1b). The identity of the purified protein was further confirmed by western immunoblotting with a goat anti-human IgG-Fc fragment HRP-conjugated antibody (Fig. 1c), validating the successful production and purification of the target fusion protein.

Fig. 1.

Design, expression, and characterization of the FcIgG-GE11-Melittin fusion protein. (a) Schematic representation of the FcIgG-GE11-Melittin construct. The gene was designed to include the Fc fragment of human IgG1, two GE11 peptides connected by glycine linkers, and the Melittin peptide at the C-terminus. The right panel illustrates the structural organization of the refolded dimeric fusion protein, highlighting the Fc fragment, GE11 targeting peptides, linkers, and Melittin moieties. (b) SDS-PAGE analysis of protein expression and purification. Lane 1: whole cell lysates from E. coli before induction; Lane 2 and 3: Lysates of E. coli BL21(DE3) cells harboring the construct, 2 and 4 h after IPTG induction, respectively. Lane M: protein molecular weight markers. Lane 4: purified FcIgG-GE11-Melittin showing a distinct band at the expected molecular weight (~ 60 kDa under non-reducing conditions). (c) Western blot analysis confirming the identity of FcIgG-GE11-Melittin using an anti-human IgG Fc-specific antibody. Lane 1: purified fusion protein; Lane M: protein molecular weight markers.

Specific binding of FcIgG-GE11-Melittin to A549 cells

Flow cytometry analysis was conducted to evaluate the binding specificity of the FcIgG-GE11-Melittin construct to A549 cells. As shown in Fig. 2, the green histogram represents cells treated with FcIgG-GE11-Melittin, demonstrating a clear rightward shift in fluorescence intensity compared to the negative control (blue), indicating significant binding of the construct to the cell surface. The red histogram, representing cells treated with Romiplostim (unrelated peptibody), shows minimal fluorescence, similar to the negative control, confirming the lack of non-specific binding. The orange histogram corresponds to cells treated with Cetuximab (positive control for EGFR binding), showing a similar shift to FcIgG-GE11-Melittin group, suggesting that the binding of the construct is mediated through interaction with EGFR. Detection was performed using a FITC-conjugated anti-human IgG Fc-specific antibody, confirming the presence of the Fc-containing fusion proteins on the cell surface. These results indicate that FcIgG-GE11-Melittin specifically binds to EGFR-expressing A549 cells, supporting its potential for targeted delivery.

Fig. 2.

Flow cytometry analysis showing the binding capacity of FcIgG-GE11-Mellitin to EGFR. Flow cytometry analysis was performed to investigate the binding capability of FcIgG-GE11-Mellitin, cetuximab, and romiplostim to the A549 and cell line. The (FITC-conjugate) negative control and romiplostim are shown in blue and red, respectively, the green graph is related to FcIgG-GE11-Mellitin, and the orange one denotes cetuximab. Binding was detected using anti-human IgG-FITC conjugate. The FcIgG-GE11-Mellitin and cetuximab bind specifically to the EGFR-expressing cell line (A549) as shown by the shift in fluorescence value compared to FITC-conjugate negative control and romiplostim.

Selective cytotoxicity of FcIgG-GE11-melittin conjugate in cancer cell lines

To evaluate the cytotoxic effects of Melittin and the FcIgG-GE11-Melittin conjugate on different cell lines, MTT assays were performed on DU145 (prostate cancer cell line), A549 (lung cancer cell line) and SW480 (colorectal cancer cell line) cells. Melittin alone exhibited a strong dose-dependent cytotoxic effect in all tested cancer cell lines, with significant reduction in cell viability observed even at low concentrations. The FcIgG-GE11-Melittin conjugate demonstrated selective cytotoxicity, showing substantial inhibitory effects on A549, DU145 and SW480 cells in a dose-dependent manner. In contrast, treatment with Romiplostim did not result in significant cytotoxicity in any cell line, confirming its lack of anticancer activity. These findings suggest that FcIgG-GE11-Melittin has potential as a targeted anticancer agent, particularly against DU145 and SW480 cells, with minimal off-target toxicity to normal cells (Fig. 3).

Fig. 3.

Cytotoxicity evaluation by MTT assay. Cytotoxicity effects of FcIgG-GE11-Mellitin and Melittin at various concentrations for 24 h on different cell lines was determined by MTT assay. A549 cell line (a); DU145 cell line (b); SW480 cell line (c); MDA_MB-453, as negative control (d). Data represent the means ± standard deviation of independent replicates. ***p < 0.001, ****p < 0.0001 vs. Romiplostim. Three independent experiments were performed.

FcIgG-GE11-melittin exhibits minimal hemolytic activity compared to free Melittin

To evaluate the safety and potential hemolytic activity of the FcIgG-GE11-Melittin construct compared to free Melittin, a hemolysis assay was performed using red blood cells (RBCs). As shown in Fig. 4, Melittin induced a concentration-dependent increase in hemolysis, with significant lysis observed at concentrations above 4 µg/mL, reaching nearly 100% at 5 µg/mL. In contrast, the FcIgG-GE11-Melittin conjugate exhibited minimal hemolytic activity across a broad range of concentrations (up to 200 µg/mL), with hemolysis remaining consistently below 5%. These findings suggest that conjugation of Melittin to the FcIgG-GE11 carrier significantly reduces its nonspecific cytolytic activity toward erythrocytes, indicating a favorable safety profile and reduced systemic toxicity for the targeted construct.

Fig. 4.

Hemolytic activity of Melittin and FcIgG-GE11-Melittin. Red blood cell hemolysis was measured at increasing concentrations of free Melittin (red) or FcIgG-GE11-Melittin fusion protein (blue). Free Melittin induced dose-dependent hemolysis, with complete lysis observed at concentrations above 5 µg/mL. In contrast, FcIgG-GE11-Melittin exhibited negligible hemolytic activity even at concentrations up to 200 µg/mL, indicating reduced cytotoxicity upon conjugation. Results are presented as mean ± standard deviation, and all experiments were performed in duplicate.

Discussion

In this study, a novel fusion protein, FcIgG-GE11-Melittin, was successfully engineered, expressed, and purified. This construct was designed to selectively target and eliminate cancer cells overexpressing the epidermal growth factor receptor (EGFR) while minimizing off-target cytotoxicity. The fusion integrates Melittin—a potent cytolytic peptide—with the EGFR-specific GE11 peptide and the Fc region of human IgG, which facilitates both purification and detection. The GE11 moiety mediates specific binding to EGFR-expressing cells, while Melittin induces cell death through membrane disruption and apoptotic pathways¹⁹. This targeted strategy addresses the systemic toxicity typically associated with free Melittin, thereby enhancing its therapeutic potential²⁰.

Previous studies have investigated Melittin’s anticancer potential, often employing nanoparticle-based delivery systems. For example, Melittin-loaded nanoparticles have been shown to effectively destroy malignant cells while limiting damage to normal tissues²¹. The use of a peptibody for Melittin delivery, as demonstrated here, represents a novel and promising approach²². Furthermore, incorporation of the Fc domain is expected to improve molecular stability and may confer additional immune-mediated benefits, such as antibody-dependent cellular cytotoxicity (ADCC)²³.

High-level expression of the monomeric fusion protein in E. coli was achieved, followed by efficient refolding and purification of the dimeric form. Protein identity and structural integrity were confirmed via SDS-PAGE and Western blot analyses.

Flow cytometry demonstrated that FcIgG-GE11-Melittin binds specifically to A549 lung cancer cells, suggesting its potential as a targeted anticancer therapeutic. A marked rightward shift in fluorescence intensity was observed in treated cells compared to untreated controls, indicating EGFR-mediated binding—likely due to the high affinity of the GE11 peptide. Such specificity is essential for minimizing collateral toxicity and improving therapeutic outcomes. As reported by Hallaji et al.¹⁸, the FcIgG-GE11 construct binds selectively to EGFR-overexpressing A549 cells, evidenced by a pronounced fluorescence shift relative to both the negative control and Romiplostim-treated cells. In contrast, Romiplostim-treated samples showed no shift, indicating an absence of nonspecific binding. High fluorescence intensity observed in Cetuximab-treated cells—a known EGFR-targeting monoclonal antibody—validated the specificity of the detection system and corroborated the binding profile of FcIgG-GE11-Melittin. These findings collectively confirm the construct’s precision in targeting EGFR-positive malignancies.

The results of MTT highlight the contrasting activity profiles of free Melittin and FcIgG-GE11-Melittin. As expected, free Melittin exhibited potent but nonspecific cytotoxicity across all tested cell lines, consistent with its well-known ability to disrupt cellular membranes regardless of receptor expression. While effective at very low concentrations, this broad activity underscores Melittin’s lack of tumor selectivity and its potential systemic toxicity if used directly as a therapeutic.

By contrast, the FcIgG-GE11-Melittin fusion construct demonstrated selective cytotoxic activity, requiring higher concentrations to exert its effects but showing greater preference for EGFR-expressing cells (A549, DU145, and SW480). The reduced potency compared to free Melittin is expected, as the fusion protein restricts Melittin’s free membrane-disruptive action and directs it through receptor-mediated targeting. Importantly, MDA-MB-453 cells, which have low EGFR expression, showed limited sensitivity to the construct, further supporting the targeting role of the GE11 peptide.

The lack of cytotoxicity with Romiplostim confirms that the observed activity of FcIgG-GE11-Melittin is not due to nonspecific effects of the Fc scaffold but is mediated by the targeting and cytolytic moieties. These findings are in line with the hemolysis data, which showed that the fusion construct reduces Melittin’s nonspecific toxicity while preserving anticancer effects.

Taken together, this result demonstrates that FcIgG-GE11-Melittin offers a more tumor-selective therapeutic profile than free Melittin, with activity linked to EGFR expression levels. Although the construct requires higher concentrations than free Melittin to achieve similar cytotoxicity, its markedly reduced nonspecific toxicity suggests a safer and more clinically viable strategy for delivering Melittin in cancer therapy. Notably, the MDA-MB-453 cell line exhibited relative resistance even to FcIgG-GE11-Melittin, showing only partial viability reduction at the highest concentrations. This may reflect low EGFR expression in this triple-negative breast cancer subtype or intrinsic mechanisms conferring resistance to membrane-lytic agents. Further analysis, including EGFR expression profiling and membrane composition studies, will be needed to elucidate the cause of this differential sensitivity. Romiplostim, used here as a negative control, had negligible effects on viability across all concentrations and cell lines, confirming that the observed cytotoxicity was specifically due to Melittin and its fusion construct.

Collectively, these findings underscore the therapeutic potential of targeted peptide-based constructs in oncology. FcIgG-GE11-Melittin effectively combined the potency of Melittin with the selectivity of GE11 targeting, offering a promising strategy for treating EGFR-positive tumors. However, variability in efficacy across cell types emphasizes the need for biomarker-based patient selection and highlights the importance of personalized medicine in future applications. Further studies are warranted to assess the in vivo antitumor efficacy, biodistribution, and safety profile of FcIgG-GE11-Melittin, as well as to explore combinational regimens that might overcome resistance in less responsive tumors such as MDA-MB-453.

The differential antiproliferative effects of Melittin across various cancer cell lines can be attributed to distinct cellular and molecular characteristics²⁴. One determinant is plasma membrane composition, particularly cholesterol, phospholipids, and negatively charged components such as phosphatidylserine²⁵. Melittin, a cationic amphipathic peptide, exhibits strong affinity for negatively charged membranes, which are often more abundant in cancer cells. However, susceptibility varies: DU145 prostate cancer cells, with inhibiting NF-κB activity and upregulating caspase signaling and a lipid profile conducive to Melittin insertion, are more sensitive²⁶, while cells with higher cholesterol content, such as A549, may resist via membrane stabilization²⁴. In addition, variability in PI3K/Akt and MAPK pathway activation, as well as differences in apoptotic integrity, further modulate Melittin’s efficacy. Cells with compromised apoptotic responses or elevated drug resistance proteins may limit Melittin-induced death²⁷. These findings highlight the importance of tumor-specific membrane properties and signaling context in determining therapeutic outcomes, underscoring the need for targeted strategies that consider each cancer type’s molecular landscape.

Supporting evidence from prior research highlights Melittin’s diverse anticancer mechanisms, including ferroptosis and ER stress–mediated apoptosis in A549 lung cancer cells²⁸, NF-κB suppression–induced apoptosis in DU145 prostate cancer cells²⁶, and strong cytotoxic effects in SW480 colorectal carcinoma cells²⁹. Collectively, these findings affirm Melittin’s robust anticancer potential and underscore its promise for continued therapeutic development.

Hemolysis assays revealed that free Melittin induced concentration-dependent red blood cell (RBC) lysis, reaching nearly 100% at 4–5 µg/mL. This aligns with reports of Melittin’s potent, nonspecific membrane-disruptive effects and systemic toxicity. In contrast, FcIgG-GE11-Melittin exhibited minimal hemolytic activity, with hemolysis levels near baseline even at concentrations up to 200 µg/mL. This marked reduction suggests that fusion with the Fc-GE11 scaffold shields erythrocytes from Melittin’s lytic activity. The improved safety profile likely results from receptor-specific internalization into EGFR-expressing cancer cells, thereby limiting nonspecific damage.

Previous studies have similarly reported Melittin’s hemolytic dose 50 (HD50) at ~ 0.44 µg/mL³⁰. However, formulations such as Melittin-loaded nanoparticles significantly reduce hemolysis while preserving anticancer efficacy³¹. These findings support targeted Melittin delivery as a strategy to enhance safety without compromising potency.

In conclusion, FcIgG-GE11-Melittin demonstrates significant promise as a selective anticancer therapeutic. Its ability to bind EGFR-positive cells, induce targeted cytotoxicity, and spare normal tissues—including erythrocytes—underscores the potential of peptide-guided delivery systems for Melittin-based treatment. Nevertheless, further research is needed to evaluate pharmacokinetics, biodistribution, and immunogenicity in vivo³². Future studies should also assess efficacy in EGFR-overexpressing tumor models³³ and investigate possible immune responses to the Fc or Melittin components³⁴.

Materials and methods

Sample collection from human subject

The collection and use of human peripheral blood were approved by the Pasteur institute of IRAN Ethics Committee (Approval No. IR.PII.REC.1401.007). All procedures involving human sample were performed in accordance with the Declaration of Helsinki and Pasteur Institute of IRAN guidelines and regulations. Written informed consent was obtained from the donor prior to blood collection.

Design and production of FcIgG-GE11-melittin

The gene fragment encoding the FcIgG-GE11peptibody¹⁸ was fused at its C-terminus to Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ) via a flexible linker. The DNA sequence was codon-optimized for Escherichia coli expression and synthesized by Biomatik company (Canada). The construct was subsequently subcloned into the pET26b (+) expression vector (Novagen, USA) using the NdeI and HindIII restriction sites.

The construct was expressed in E. coli BL21 (DE3) using 0.2 mM IPTG for 4 h at 37 °C. Finally, E. coli cells were pelleted, and the supernatants were discarded. Protein analysis was carried out using 12% SDS-PAGE, followed by staining with Coomassie Brilliant Blue R-250 (Merck) for visualization³⁵.

Purification and characterization

The bacterial pellet underwent sequential washing with 20 mM Tris-HCl and 20 mM EDTA (pH 8.0) buffers, each step followed by centrifugation at 8000 rpm. Subsequently, the pellet was resuspended in 10 mL of lysis buffer (20 mM Tris-HCl, 5 mM EDTA, 1% v/v Triton X-100, pH 8.0) and incubated at 20 °C for 1 h. Cell disruption was achieved on ice through sonication (MSE Soniprep 150 Plus) for 45 cycles (30 s on, 15 s off; amplitude 10). The lysate was then centrifuged at 8000 rpm for 20 min at 4 °C, and the resulting inclusion bodies (IBs) were stored at − 20 °C. The IBs were solubilized in a buffer containing 8 M urea, 20 mM Tris-HCl, 20 mM NaCl, and 16 mM DTT (pH 8.0) by gentle mixing at 60 rpm for 2 h at room temperature. The solubilized fraction was obtained by centrifugation at 8000 rpm for 30 min and stored at 4 °C for further use. The FcIgG-GE11-Melittin was refolded using the dilution method, with the peptibody solution being added dropwise to the refolding buffer (50 mM Tris-HCl, 200 mM arginine, 2 M urea, 1 mM EDTA, 5 mM cysteine, pH 8.0) and stirred at 60 rpm overnight at 10 °C.

The protein purification was carried out using affinity chromatography with a Protein A resin (Sigma, Aldrich). The column was first equilibrated with a binding buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) to establish optimal conditions for the interaction. The sample was then loaded onto the column, allowing the Fc-mediated binding of the peptibody to the Protein A resin. Elution of the bound protein was performed using an elution buffer (100 mM sodium citrate, pH 3.5). Immediately following elution, the sample was neutralized using a neutralization buffer (1000 mM Tris-HCl, pH 9) to stabilize the protein.

The purified protein was subsequently analyzed by 12% SDS-PAGE to assess purity, followed by immunoblotting for specific detection. Protein concentration was determined using the Bio-Rad Protein Assay and verified by measuring UV absorbance at 280 nm with a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

Cell lines and culture

The human cell lines A549, SW480, DU145 were obtained from the National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI-1640 medium, and 100× penicillin-streptomycin solution was purchased from Bio-IDEA (Tehran, Iran), while fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY, USA).

Specificity and binding assays

Flow cytometry was performed to evaluate the binding of FcIgG-GE11-Melittin peptibodies to EGFR on A549 cells, which are known to overexpress EGFR. Romiplostim, containing a peptide sequence unrelated to GE11, served as a negative control. A549 cells were cultured and aliquoted at 1 × 10⁶ cells per tube. Cells were incubated with FcIgG-GE11-Melittin, Romiplostim or Cetuximab for 1 h at 4 °C, followed by washing and incubation with a FITC-conjugated anti-human IgG antibody for 45 min in the dark at 4 °C. After a second wash, cell pellets were resuspended in PBS and analyzed by flow cytometry within one hour.

In vitro cytotoxicity assay

To assess the cytotoxic effects of the FcIgG-GE11-Melittin peptibody, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was conducted. This experiment included four distinct cell lines: A549, SW480, and DU145 as EGFR-positive cell lines, and MDA-MB-453 as an EGFR-negative cell line. The EGFR expression profile of these cell lines was previously confirmed by our group using real-time PCR analysis¹⁸. In brief, 1 × 10⁴ cells per well were seeded into 96-well plates and exposed to escalating concentrations of the FcIgG-GE11-Melittin, Melittin and Romiplostim, for 24 h at 37 °C. Following the incubation period, 10 µL of MTT solution (5 mg/mL) was added to each well and further incubated for 4 hours at 37 °C. The MTT solution was then removed, and the resulting formazan crystals were dissolved by adding 100 µL of dimethyl sulfoxide (DMSO). Absorbance was subsequently measured at 570 nm, with background correction performed at 630 nm.

Hemolysis assay

To evaluate the hemolytic activity of the FcIgG-GE11-Melittin construct, a hemolysis assay was conducted using human red blood cells (RBCs). Peripheral blood (5 mL) was collected from a healthy donor into a heparinized tube and centrifuged at 500 ×g for 5 min to isolate the RBCs. The collected RBCs were washed three times with phosphate-buffered saline (PBS) to remove plasma components and then resuspended in PBS to achieve a final concentration of 2% (v/v).

Aliquots of the RBC suspension were dispensed into 1.5 mL microcentrifuge tubes, after which various concentrations of Melittin and FcIgG-GE11-Melittin were added. The samples were incubated at 37 °C for 1 h to allow for potential hemolysis. Following incubation, the tubes were centrifuged at 500× g for 5 min, and the supernatants were collected. A volume of 100 µL from each supernatant was transferred to a 96-well microplate, and the absorbance was measured at 540 nm to assess the release of hemoglobin, which is indicative of RBC lysis. PBS was used as the negative control, while 0.2% (v/v) Triton X-100, a non-ionic surfactant known for its membrane-disruptive properties, served as the positive control.

The percentage of hemolysis was calculated using the Eq. (1), and the results were subsequently plotted to generate a hemolysis profile.

1

Author contributions

**M H: ** Investigation, laboratory experiments, data mining, data analysis, writing original draft and editing.**SH F: ** Investigation, laboratory experiments, data mining, data analysis, writing original draft and editing.**M A: ** Investigation, review and editing.**M G: ** Investigation, review and editing.**K P B: ** laboratory experiments (providing melittin toxin produced in his laboratory), review and editing.**P F-E: ** Supervision, research concept development, design, data analysis, writing, review and editing.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Declarations

Competing interests

The authors declare no competing interests.

Ethics statement

All experiments and procedures were approved by the ethics committee of Pasteur Institute of Iran (IR.PII.REC.1401.007) and performed in accordance with the approved guidelines and regulations.