Nanobody Synthesis to Target Epidermal Growth Factor Receptor (EGFR) in Colorectal Cancer Cells
Abstract
Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with the overexpression of epidermal growth factor receptor (EGFR) playing a critical role in its progression. Current therapies face challenges in targeting EGFR effectively. To synthesize and evaluate nanobodies targeting EGFR in colorectal cancer cells, aiming to improve therapeutic specificity and efficacy. Nanobodies were synthesized using phage display technology and screened for high affinity to EGFR. In vitro studies involved colorectal cancer cell lines (HT-29, SW480, HCT116) to assess binding specificity, internalization, and cytotoxicity. In vivo studies used mouse models implanted with human colorectal tumors to evaluate biodistribution, tumor targeting, and therapeutic outcomes. Synthesized nanobodies demonstrated high binding affinity (KD in nanomolar range) and specificity to EGFR, inhibiting cancer cell proliferation by up to 70% and reducing tumor volume by 65% in mouse models. Stability tests confirmed nanobody resilience under various biological conditions. The study highlights the potential of nanobodies targeting EGFR as an effective therapeutic approach for colorectal cancer, with significant improvements in targeting specificity and tumor reduction. Further clinical trials are necessary to confirm these findings.
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References
Bai, M., Wang, Y., Zhang, C., Wang, Y., Wei, J., Liao, X., Wang, J., Anfossi, L., & Wang, Y. (2023). Nanobody-based immunomagnetic separation platform for rapid isolation and detection of Salmonella enteritidis in food samples. Food Chemistry, 424, 136416. https://doi.org/10.1016/j.foodchem.2023.136416
Bao, C., Gao, Q., Li, L.-L., Han, L., Zhang, B., Ding, Y., Song, Z., Zhang, R., Zhang, J., & Wu, X.-H. (2021). The Application of Nanobody in CAR-T Therapy. Biomolecules, 11(2), 238. https://doi.org/10.3390/biom11020238
Bao, K., Liu, X., Xu, Q., Su, B., Liu, Z., Cao, H., & Chen, Q. (2021). Nanobody multimerization strategy to enhance the sensitivity of competitive ELISA for detection of ochratoxin A in coffee samples. Food Control, 127, 108167. https://doi.org/10.1016/j.foodcont.2021.108167
Beltrán Hernández, I., Angelier, M. L., Del Buono D’Ondes, T., Di Maggio, A., Yu, Y., & Oliveira, S. (2020). The Potential of Nanobody-Targeted Photodynamic Therapy to Trigger Immune Responses. Cancers, 12(4), 978. https://doi.org/10.3390/cancers12040978
Bridoux, J., Broos, K., Lecocq, Q., Debie, P., Martin, C., Ballet, S., Raes, G., Neyt, S., Vanhove, C., Breckpot, K., Devoogdt, N., Caveliers, V., Keyaerts, M., & Xavier, C. (2020). Anti-Human PD-L1 Nanobody for Immuno-PET Imaging: Validation of a Conjugation Strategy for Clinical Translation. Biomolecules, 10(10), 1388. https://doi.org/10.3390/biom10101388
Chen, K., Xue, J., Zhou, Q., Zhang, Y., Zhang, M., Zhang, Y., Zhang, H., & Shen, Y. (2020). Coupling metal-organic framework nanosphere and nanobody for boosted photoelectrochemical immunoassay of Human Epididymis Protein 4. Analytica Chimica Acta, 1107, 145–154. https://doi.org/10.1016/j.aca.2020.02.011
Chen, Y., Duan, W., Xu, L., Li, G., Wan, Y., & Li, H. (2022). Nanobody-based label-free photoelectrochemical immunoassay for highly sensitive detection of SARS-CoV-2 spike protein. Analytica Chimica Acta, 1211, 339904. https://doi.org/10.1016/j.aca.2022.339904
Cohen, T., Halfon, M., & Schneidman-Duhovny, D. (2022). NanoNet: Rapid and accurate end-to-end nanobody modeling by deep learning. Frontiers in Immunology, 13, 958584. https://doi.org/10.3389/fimmu.2022.958584
Cruz-Pacheco, A. F., Monsalve, Y., Serrano-Rivero, Y., Salazar-Uribe, J., Moreno, E., & Orozco, J. (2023). Engineered synthetic nanobody-based biosensors for electrochemical detection of epidermal growth factor receptor. Chemical Engineering Journal, 465, 142941. https://doi.org/10.1016/j.cej.2023.142941
De Beer, M. A., & Giepmans, B. N. G. (2020). Nanobody-Based Probes for Subcellular Protein Identification and Visualization. Frontiers in Cellular Neuroscience, 14, 573278. https://doi.org/10.3389/fncel.2020.573278
De Munter, S., Van Parys, A., Bral, L., Ingels, J., Goetgeluk, G., Bonte, S., Pille, M., Billiet, L., Weening, K., Verhee, A., Van Der Heyden, J., Taghon, T., Leclercq, G., Kerre, T., Tavernier, J., & Vandekerckhove, B. (2020). Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly. International Journal of Molecular Sciences, 21(3), 883. https://doi.org/10.3390/ijms21030883
Del Rosario, J. M. M., Smith, M., Zaki, K., Risley, P., Temperton, N., Engelhardt, O. G., Collins, M., Takeuchi, Y., & Hufton, S. E. (2020). Protection From Influenza by Intramuscular Gene Vector Delivery of a Broadly Neutralizing Nanobody Does Not Depend on Antibody Dependent Cellular Cytotoxicity. Frontiers in Immunology, 11, 627. https://doi.org/10.3389/fimmu.2020.00627
Delfin-Riela, T., Rossotti, M., Alvez-Rosado, R., Leizagoyen, C., & González-Sapienza, G. (2020). Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA. Biomolecules, 10(12), 1652. https://doi.org/10.3390/biom10121652
Fan, R., Li, Y., Park, K.-W., Du, J., Chang, L. H., Strieter, E. R., & Andrew, T. L. (2022). A Strategy for Accessing Nanobody-Based Electrochemical Sensors for Analyte Detection in Complex Media. ECS Sensors Plus, 1(1), 010601. https://doi.org/10.1149/2754-2726/ac5b2e
Hartung, F., Krüwel, T., Shi, X., Pfizenmaier, K., Kontermann, R., Chames, P., Alves, F., & Pardo, L. A. (2020). A Novel Anti-Kv10.1 Nanobody Fused to Single-Chain TRAIL Enhances Apoptosis Induction in Cancer Cells. Frontiers in Pharmacology, 11, 686. https://doi.org/10.3389/fphar.2020.00686
He, Y., Ren, Y., Guo, B., Yang, Y., Ji, Y., Zhang, D., Wang, J., Wang, Y., & Wang, H. (2020). Development of a specific nanobody and its application in rapid and selective determination of Salmonella enteritidis in milk. Food Chemistry, 310, 125942. https://doi.org/10.1016/j.foodchem.2019.125942
Hong, H., Lin, H., Li, D., Gong, L., Zhou, K., Li, Y., Yu, H., Zhao, K., Shi, J., Zhou, Z., Huang, Z., & Wu, Z. (2022). Chemoenzymatic Synthesis of a Rhamnose?Functionalized Bispecific Nanobody as a Bispecific Antibody Mimic for Cancer Immunotherapy. Angewandte Chemie International Edition, 61(38), e202208773. https://doi.org/10.1002/anie.202208773
Huang, C., Ren, J., Ji, F., Muyldermans, S., & Jia, L. (2020). Nanobody-Based high-performance immunosorbent for selective beta 2-microglobulin purification from blood. Acta Biomaterialia, 107, 232–241. https://doi.org/10.1016/j.actbio.2020.02.028
Ishiwatari-Ogata, C., Kyuuma, M., Ogata, H., Yamakawa, M., Iwata, K., Ochi, M., Hori, M., Miyata, N., & Fujii, Y. (2022). Ozoralizumab, a Humanized Anti-TNF? NANOBODY® Compound, Exhibits Efficacy Not Only at the Onset of Arthritis in a Human TNF Transgenic Mouse but Also During Secondary Failure of Administration of an Anti-TNF? IgG. Frontiers in Immunology, 13, 853008. https://doi.org/10.3389/fimmu.2022.853008
Kang, G., Hu, M., Ren, H., Wang, J., Cheng, X., Li, R., Yuan, B., Balan, Y., Bai, Z., & Huang, H. (2021). VHH212 nanobody targeting the hypoxia-inducible factor 1? suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo. Cancer Biology and Medicine, 18(3), 772–787. https://doi.org/10.20892/j.issn.2095-3941.2020.0568
Koklu, A., Wustoni, S., Guo, K., Silva, R., Salvigni, L., Hama, A., Diaz?Galicia, E., Moser, M., Marks, A., McCulloch, I., Grünberg, R., Arold, S. T., & Inal, S. (2022). Convection Driven Ultrarapid Protein Detection via Nanobody?Functionalized Organic Electrochemical Transistors. Advanced Materials, 34(35), 2202972. https://doi.org/10.1002/adma.202202972
Kühne, L., Völker, L. A., Hagmann, H., Hägele, H., Osterholt, T., Eichenauer, D. A., Thomas, A., Breuer, J., Grüttner, B., Gottschalk, I., Kann, M., Benzing, T., Thevis, M., Müller, A. M., & Brinkkoetter, P. T. (2022). First use of the anti?VWF nanobody caplacizumab to treat iTTP in pregnancy. British Journal of Haematology, 196(3). https://doi.org/10.1111/bjh.17833
Liu, B., & Yang, D. (2022). Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools. International Journal of Molecular Sciences, 23(3), 1482. https://doi.org/10.3390/ijms23031482
Ma, L., Gai, J., Qiao, P., Li, Y., Li, X., Zhu, M., Li, G., & Wan, Y. (2020). A novel bispecific nanobody with PD-L1/TIGIT dual immune checkpoint blockade. Biochemical and Biophysical Research Communications, 531(2), 144–151. https://doi.org/10.1016/j.bbrc.2020.07.072
Mei, Y., Chen, Y., Sivaccumar, J. P., An, Z., Xia, N., & Luo, W. (2022). Research progress and applications of nanobody in human infectious diseases. Frontiers in Pharmacology, 13, 963978. https://doi.org/10.3389/fphar.2022.963978
Moeglin, E., Desplancq, D., Stoessel, A., Massute, C., Ranniger, J., McEwen, A. G., Zeder-Lutz, G., Oulad-Abdelghani, M., Chiper, M., Lafaye, P., Di Ventura, B., Didier, P., Poterszman, A., & Weiss, E. (2021). A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress. Cancers, 13(13), 3317. https://doi.org/10.3390/cancers13133317
Moliner-Morro, A., J. Sheward, D., Karl, V., Perez Vidakovics, L., Murrell, B., McInerney, G. M., & Hanke, L. (2020). Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs. Biomolecules, 10(12), 1661. https://doi.org/10.3390/biom10121661
Qin, S., Yu, Y., Guan, H., Yang, Y., Sun, F., Sun, Y., Zhu, J., Xing, L., Yu, J., & Sun, X. (2021). A preclinical study: Correlation between PD-L1 PET imaging and the prediction of therapy efficacy of MC38 tumor with 68Ga-labeled PD-L1 targeted nanobody. Aging, 13(9), 13006–13022. https://doi.org/10.18632/aging.202981
Shi, W., Yang, X., Xie, S., Zhong, D., Lin, X., Ding, Z., Duan, S., Mo, F., Liu, A., Yin, S., Jiang, X., Xu, Z. P. (Gordon), & Lu, X. (2021). A new PD-1-specific nanobody enhances the antitumor activity of T-cells in synergy with dendritic cell vaccine. Cancer Letters, 522, 184–197. https://doi.org/10.1016/j.canlet.2021.09.028
Siebuhr, A. S., Werkmann, D., Bay-Jensen, A.-C., Thudium, C. S., Karsdal, M. A., Serruys, B., Ladel, C., Michaelis, M., & Lindemann, S. (2020). The Anti-ADAMTS-5 Nanobody® M6495 Protects Cartilage Degradation Ex Vivo. International Journal of Molecular Sciences, 21(17), 5992. https://doi.org/10.3390/ijms21175992
Slater, A., Di, Y., Clark, J. C., Jooss, N. J., Martin, E. M., Alenazy, F., Thomas, M. R., Ariëns, R. A. S., Herr, A. B., Poulter, N. S., Emsley, J., & Watson, S. P. (2021). Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer. Blood, 137(24), 3443–3453. https://doi.org/10.1182/blood.2020009440
Su, B., Bei, Z., Pei, H., Xie, X., Sun, Z., Chen, Q., Cao, H., & Liu, X. (2022). Generation of a nanobody-alkaline phosphatase heptamer fusion for ratiometric fluorescence immunodetection of trace alpha fetoprotein in serum. International Journal of Biological Macromolecules, 201, 507–515. https://doi.org/10.1016/j.ijbiomac.2022.01.056
Su, B., Wang, Y., Pei, H., Sun, Z., Cao, H., Zhang, C., Chen, Q., & Liu, X. (2020). Phage-mediated double-nanobody sandwich immunoassay for detecting alpha fetal protein in human serum. Analytical Methods, 12(39), 4742–4748. https://doi.org/10.1039/D0AY01407A
Sun, T., Zhao, Z., Liu, W., Xu, Z., He, H., Ning, B., Jiang, Y., & Gao, Z. (2020). Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B Nanobody–Alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B. Analytica Chimica Acta, 1108, 28–36. https://doi.org/10.1016/j.aca.2020.01.032
Takeuchi, T., Kawanishi, M., Nakanishi, M., Yamasaki, H., & Tanaka, Y. (2022). Phase II/III Results of a Trial of Anti–Tumor Necrosis Factor Multivalent NANOBODY Compound Ozoralizumab in Patients With Rheumatoid Arthritis. Arthritis & Rheumatology, 74(11), 1776–1785. https://doi.org/10.1002/art.42273
Tang, X., Catanante, G., Huang, X., Marty, J.-L., Wang, H., Zhang, Q., & Li, P. (2022). Screen-printed electrochemical immunosensor based on a novel nanobody for analyzing aflatoxin M1 in milk. Food Chemistry, 383, 132598. https://doi.org/10.1016/j.foodchem.2022.132598
Tieu, T., Wojnilowicz, M., Huda, P., Thurecht, K. J., Thissen, H., Voelcker, N. H., & Cifuentes-Rius, A. (2021). Nanobody-displaying porous silicon nanoparticles for the co-delivery of siRNA and doxorubicin. Biomaterials Science, 9(1), 133–147. https://doi.org/10.1039/D0BM01335H
Wang, X., Chen, Q., Sun, Z., Wang, Y., Su, B., Zhang, C., Cao, H., & Liu, X. (2020). Nanobody affinity improvement: Directed evolution of the anti-ochratoxin A single domain antibody. International Journal of Biological Macromolecules, 151, 312–321. https://doi.org/10.1016/j.ijbiomac.2020.02.180
Wu, T., Liu, M., Huang, H., Sheng, Y., Xiao, H., & Liu, Y. (2020). Clustered nanobody–drug conjugates for targeted cancer therapy. Chemical Communications, 56(65), 9344–9347. https://doi.org/10.1039/D0CC03396K
Wu, W., Shi, L., Duan, Y., Xu, S., Shen, L., Zhu, T., Hou, L., Meng, X., & Liu, B. (2021). Nanobody modified high-performance AIE photosensitizer nanoparticles for precise photodynamic oral cancer therapy of patient-derived tumor xenograft. Biomaterials, 274, 120870. https://doi.org/10.1016/j.biomaterials.2021.120870
Xiong, T., Peng, Q., Chen, Y., Li, M., Du, J., Fan, J., Jia, L., & Peng, X. (2021). A Novel Nanobody–Photosensitizer Conjugate for Hypoxia Resistant Photoimmunotherapy. Advanced Functional Materials, 31(37), 2103629. https://doi.org/10.1002/adfm.202103629
Zhang, C., Wu, X., Li, D., Hu, J., Wan, D., Zhang, Z., & Hammock, B. D. (2021). Development of nanobody-based flow-through dot ELISA and lateral-flow immunoassay for rapid detection of 3-phenoxybenzoic acid. Analytical Methods, 13(14), 1757–1765. https://doi.org/10.1039/D1AY00129A
Zhang, Y.-Y., Li, L.-H., Wang, Y., Wang, H., Xu, Z.-L., Tian, Y.-X., Sun, Y.-M., Yang, J.-Y., & Shen, Y.-D. (2022). Ultrasensitive and rapid colorimetric detection of paraquat via a high specific VHH nanobody. Biosensors and Bioelectronics, 205, 114089. https://doi.org/10.1016/j.bios.2022.114089
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