anti-EGFR Nanobody Research Articles

Reprogramming tumor-associated macrophages by a dually targeted milk exosome system as a potent monotherapy for cancer

Tumor-associated macrophages (TAMs) play a key role in inducing an immunosuppressive tumor microenvironment (TME) and cancer immune escape. We previously revealed that PDL1 (a key immune checkpoint) was upregulated in TAMs and induced M2 polarization, highlighting PDL1 in TAMs as a promising cancer therapeutic target. In this study, we developed an engineered milk exosome (mExo) system decorated with M2pep (an M2 macrophage binding peptide) and 7D12 (an anti-EGFR nanobody) (7D12-mExo-M2pep-siPDL1) to specifically deliver siPDL1 into M2 TAMs. A series of in vitro and in vivo assays showed that the dually targeted engineered mExos efficiently delivered siPDL1 into M2 TAMs and repolarized them into M1 macrophages, restoring CD8+ T cell immune activity and remodeling TME. Importantly, systemically administered 7D12-mExo-M2pep-siPDL1 showed efficient single-agent antitumor activity, resulting in nearly 90% tumor growth inhibition in a mouse model of orthotopic epidermal growth factor receptor (EGFR) cancer. Collectively, our study indicates that PDL1 is a promising target for TAM-based cancer immunotherapy, and our engineered mExo-based nanomedicine represents a novel tool for specifically targeting M2 TAMs, distinguishing this novel therapeutic method from other TAM-targeting therapies and highlighting its promising clinical potential.

An off-the-shelf small extracellular vesicle nanomedicine for tumor targeting therapy

Small extracellular vesicles (sEVs) have shown excellent prospects as drug delivery systems for cancer therapy. However, the inherent non-targeting and short blood circulation characteristics severely restrict their practical applications as a delivery system. In addition, post-encapsulating drugs into sEVs also remains challenging. Here, we constructed an engineered cell line that secreted multifunctional sEVs (termed NBsEV204) with 7D12 (an anti-EGFR nanobody) and hCD47 decorations on their surface, as well as high levels of miR-204-5p encapsulation. NBsEV204 exhibited extended blood circulation and improved macrophage-mediated phagocytosis of tumor cells by blocking CD47 signaling. Importantly, NBsEV204 specifically targeted EGFR+ tumor cells and showed robust tumor-suppressive effects both in vitro and in vivo. Overall, this study provides a convenient and feasible method to produce off-the-shelf anticancer sEV nanomedicine, which exhibits tremendous potential for clinical translation.

Engineered synthetic nanobody-based biosensors for electrochemical detection of epidermal growth factor receptor

Two engineered synthetic nanobody-based nanobiocomposite platforms were developed for label-free electrochemical detection of the epithelial growth factor receptor (EGFR) biomarker. Screen-printed carbon electrodes (SPCE) were decorated either with NiO nanoparticles (NPs) or poly(thiophene acetic acid) (PTAA) to link the anti-EGFR nanobody (Nb) and form nanobiocomposites for detecting the EGFR biomarker by electrochemical impedance spectroscopy (EIS). The nanoarchitectures were prepared by in situ electrosynthesis of NiO NPs or PTAA layers at SPCEs. A modified version of the 9G8 Nb (Nb9G8m), specific for the EGFR (anti-EGFR), was designed and produced as the nanobiosensor bioreceptor. This Nb was engineered to provide a hexahistidine tag (6xHis-tag) and a lysine (Lys) dual functionality to form a (6xHis-tag)/Ni2+ or Lys/PTAA interface. The biosensing interfaces were characterized by field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, cyclic voltammetry, and EIS. The nanobody/nanobiocomposite-based biosensors detected EGFR proteins in a linear range from 0.25 to 50 μg mL−1 and 0.5 to 50 μg mL−1, with limits of detection of 0.46 μg mL−1 and 1.14 μg mL−1, for NiO- and PTAA-based platforms, respectively. The biosensing platforms offer high simplicity, specificity, and selectivity to detect EGFR, but Nbs can be readily engineered to detect other (glycol)proteins. Finally, as a proof of concept, the EGFR was detected in several tumor cell lines, differentiating biomarker expression among them.

Programmable half-life and anti-tumour effects of bispecific T-cell engager-albumin fusions with tuned FcRn affinity

Fc-less bispecific T-cell engagers have reached the immuno-oncology market but necessitate continual infusion due to rapid clearance from the circulation. This work introduces a programmable serum half-life extension platform based on fusion of human albumin sequences engineered with either null (NB), wild type (WT) or high binding (HB) FcRn affinity combined with a bispecific T-cell engager. We demonstrate in a humanised FcRn/albumin double transgenic mouse model (AlbuMus) the ability to tune half-life based on the albumin sequence fused with a BiTE-like bispecific (anti-EGFR nanobody x anti-CD3 scFv) light T-cell engager (LiTE) construct [(t½ 0.6 h (Fc-less LiTE), t½ 19 hours (Albu-LiTE-NB), t½ 26 hours (Albu-LiTE-WT), t½ 37 hours (Albu-LiTE-HB)]. We show in vitro cognate target engagement, T-cell activation and discrimination in cellular cytotoxicity dependent on EGFR expression levels. Furthermore, greater growth inhibition of EGFR-positive BRAF mutated tumours was measured following a single dose of Albu-LiTE-HB construct compared to the Fc-less LiTE format and a full-length anti-EGFR monoclonal antibody in a new AlbuMus RAG1 knockout model introduced in this work. Programmable half-life extension facilitated by this albumin platform potentially offers long-lasting effects, better patient compliance and a method to tailor pharmacokinetics to maximise therapeutic efficacy and safety of immuno-oncology targeted biologics.

Identification of a novel anti-EGFR nanobody by phage display and its distinct paratope and epitope via homology modeling and molecular docking

Since EGFR is an important and effective target for tumor therapy in the clinic. Several monoclonal antibodies and nanobodies were proved to target domain III of EGFR. Regarding the increased attention on nanobodies, the present study aimed to generate nanobodies specifically against domain III. After camel immunization, a gene repertoire of sdAb fragments with a diversity of 3×109 clones was produced. Following the construction of two sdAb phage display libraries, the successful epitope binning was carried out to identify the nanobody with the designated epitope. Modelling of the identified nanobody and molecular docking studies illustrated the paratope and epitope. Docking analysis revealed that the paratope focused on CDR2 loop of the identified nanobody. The identified nanobody potently cover part of the epitope of Matuzumab and Nb 9G8, which indicated that it blocked EGFR by preventing dimerization of the receptors.

Anti-EGFR Binding Nanobody Delivery System to Improve the Diagnosis and Treatment of Solid Tumours.

Epidermal Growth Factor Receptor (EGFR) and members of its homologous protein family mediate transmembrane signal transduction by binding to a specific ligand, which leads to regulated cell growth, differentiation, proliferation and metastasis. With the development and application of Genetically Engineered Antibodies (GEAs), Nanobodies (Nbs) constitute a new research hot spot in many diseases. A Nb is characterized by its low molecular weight, deep tissue penetration, good solubility and high antigen-binding affinity, the anti-EGFR Nbs are of significance for the diagnosis and treatment of EGFR-positive tumours. This review aims to provide a comprehensive overview of the information about the molecular structure of EGFR and its transmembrane signal transduction mechanism, and discuss the anti-EGFR-Nbs influence on the diagnosis and treatment of solid tumours. Data were obtained from PubMed, Embase and Web of Science. All patents are searched from the following websites: the World Intellectual Property Organization (WIPO®), the United States Patent Trademark Office (USPTO®) and Google Patents. EGFR is a key target for regulating transmembrane signaling. The anti-EGFR-Nbs for targeted drugs could effectively improve the diagnosis and treatment of solid tumours. EGFR plays a role in transmembrane signal transduction. The Nbs, especially anti- EGFR-Nbs, have shown effectiveness in the diagnosis and treatment of solid tumours. How to increase the affinity of Nb and reduce its immunogenicity remain a great challenge.

Nanobody-Ferritin Conjugate for Targeted Photodynamic Therapy.

Ferritin is an iron-storage protein nanocage that is assembled from 24 subunits. The hollow cavity of ferritin enables its encapsulation of various therapeutic agents; therefore, ferritin has been intensively investigated for drug delivery. The use of antibody-ferritin conjugates provides an effective approach for targeted drug delivery. However, the complicated preparation and limited protein stability hamper wide applications of this system. Herein, we designed a novel nanobody-ferritin platform (Nb-Ftn) for targeted drug delivery. The site-specific conjugation between nanobody and ferritin is achieved by transglutaminase-catalyzed protein ligation. This ligation strategy allows the Nb conjugation after drug loading in ferritin, which avoids deactivation of the nanobody under the harsh pH environment required for drug encapsulation. To verify the tumor targeting of this Nb-Ftn platform, a photodynamic reagent, manganese phthalocyanine (MnPc), was loaded into the ferritin cavity, and an anti-EGFR nanobody was conjugated to the surface of the ferritin. The ferritin nanocage can encapsulate about 82 MnPc molecules. This MnPc@Nb-Ftn conjugate can be efficiently internalized by EGFR positive A431 cancer cells, but not by EGFR negative MCF-7 cells. Upon 730 nm laser irradiation, MnPc@Nb-Ftn selectively killed EGFR positive A431 cells by generating reactive oxygen species (ROS), whereas no obvious damage was observed on MCF-7 cells. Given that ferritin can be used for encapsulation of various therapeutic agents, this work provides a strategy for facile construction of nanobody-ferritin for targeted drug delivery.

Protein labeling approach to improve lysosomal targeting and efficacy of antibody-drug conjugates.

An anti-EGFR nanobody was labeled at the C-terminus with a lysosome-sorting NPGY (Asn-Pro-Gly-Tyr) motif via sortase-mediated ligation to enhance the engagement of the clathrin-mediated endocytosis. The synergistic effects of NPGY motif and nona-arginine peptide were found to induce robust internalization and lysosomal trafficking, which in turn improved anti-tumor activity of an antibody-drug conjugate.

A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics.

Quantum-Dot-Based Theranostic Micelles Conjugated with an Anti-EGFR Nanobody for Triple-Negative Breast Cancer Therapy.

A quantum-dot (QD)-based micelle conjugated with an anti-epidermal growth factor receptor (EGFR) nanobody (Nb) and loaded with an anticancer drug, aminoflavone (AF), has been engineered for EGFR-overexpressing cancer theranostics. The near-infrared (NIR) fluorescence of the indium phosphate core/zinc sulfide shell QDs (InP/ZnS QDs) allowed for in vivo nanoparticle biodistribution studies. The anti-EGFR nanobody 7D12 conjugation improved the cellular uptake and cytotoxicity of the QD-based micelles in EGFR-overexpressing MDA-MB-468 triple-negative breast cancer (TNBC) cells. In comparison with the AF-encapsulated nontargeted (i.e., without Nb conjugation) micelles, the AF-encapsulated Nb-conjugated (i.e., targeted) micelles accumulated in tumors at higher concentrations, leading to more effective tumor regression in an orthotopic triple-negative breast cancer xenograft mouse model. Furthermore, there was no systemic toxicity observed with the treatments. Thus, this QD-based Nb-conjugated micelle may serve as an effective theranostic nanoplatform for EGFR-overexpressing cancers such as TNBCs.

In vivo detection of small tumour lesions by multi-pinhole SPECT applying a (99m)Tc-labelled nanobody targeting the Epidermal Growth Factor Receptor.

The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody 99mTc-D10 for visualizing small tumour lesions with volumes below 100 mm3 by targeting EGFR in orthotopic human mammary MDA-MB-468 and MDA-MB-231 and subcutaneous human epidermoid A431 carcinoma mouse models. Use of nanobody 99mTc-D10 of a size as small as 15.5 kDa enables detection of tumours by single photon emission computed tomography (SPECT) imaging already 45 min post intravenous administration with high tumour uptake (>3% ID/g) in small MDA-MB-468 and A431 tumours, with tumour volumes of 52.5 mm3 ± 21.2 and 26.6 mm3 ± 16.7, respectively. Fast blood clearance with a serum half-life of 4.9 min resulted in high in vivo contrast and ex vivo tumour to blood and tissue ratios. In contrast, no accumulation of 99mTc-D10 in MDA-MB-231 tumours characterized by a very low expression of EGFR was observed. Here we present specific and high contrast in vivo visualization of small human tumours overexpressing EGFR by preclinical multi-pinhole SPECT shortly after administration of anti-EGFR nanobody 99mTc-D10.

Intraoperative fluorescence delineation of head and neck cancer with a fluorescent Anti‐epidermal growth factor receptor nanobody

Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.

Efficient growth inhibition of EGFR over-expressing tumor cells by an anti-EGFR nanobody

Epidermal growth factor receptor (EGFR) is deemed to be one of the main molecular targets for diagnosis and treatment of cancer. It has been identified that EGFR involves in pathogenesis of some forms of human cancers. Monoclonal antibodies targeting EGFR could control the tumor cell growth, proliferation, and apoptosis by suppressing the signal transduction pathways. Nanobodies can be regarded as the smallest intact antigen binding fragments, derived from heavy chain-only antibodies existing in camelids. Here, we describe the identification of an EGFR-specific nanobody, referred to as OA-cb6, obtained from immunized camel with a cell line expressing high levels of EGFR. Utilizing flow cytometry (FACS) and blotting methods, we demonstrated that OA-cb6 nanobody binds specifically to EGFR expressing on the surface of A431 cells. In addition, OA-cb6 nanobody potently causes the inhibition of EGFR over expression, cell growth and proliferation. The antibody fragments can probably be regarded as worthwhile binding block for further rational design of anti-cancer therapy.

Inhibition of Tumor Growth by Targeted Anti-EGFR/IGF-1R Nanobullets Depends on Efficient Blocking of Cell Survival Pathways

The clinical efficacy of epidermal growth factor receptor (EGFR)-targeted inhibitors is limited due to resistance mechanisms of the tumor such as activation of compensatory pathways. Crosstalk between EGFR and insulin-like growth factor 1 (IGF-1R) signaling has been frequently described to be involved in tumor proliferation and resistance. One of the attractive features of nanomedicines is the possibility to codeliver agents that inhibit different molecular targets in one nanocarrier system, thereby strengthening the antitumor effects of the individual agents. Additionally, exposure to healthy tissues and related unwanted side-effects can be reduced. To this end, we have recently developed anti-EGFR nanobody (Nb)-liposomes loaded with the anti-IGF-1R kinase inhibitor AG538, which showed promising antiproliferative effects in vitro. In the present study, we have further evaluated the potential of this dual-active nanomedicine in vitro and for the first time in vivo. As intended, the nanomedicine inhibited EGFR and IGF-1R signaling and subsequent activation of downstream cell proliferation and survival pathways. The degree of inhibition induced by the nanomedicine on a molecular level correlated with cytotoxicity in tumor cell proliferation assays and may even be predictive of the response to nanomedicine treatment in tumor xenograft models. Combination therapy with kinase inhibitor-loaded Nb-liposomes is therefore an appealing strategy for inhibiting the proliferation of tumors that are highly dependent on EGFR and IGF-1R signaling.

Rapid Visualization of Human Tumor Xenografts through Optical Imaging with a Near-Infrared Fluorescent Anti–Epidermal Growth Factor Receptor Nanobody

Given that overexpression of the epidermal growth factor receptor (EGFR) is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.