Computational Design and Evaluation of ScFv-IFN-B Fusion Protein against HER1-positive Breast Cancer
Keywords:
Breast cancer, Fusion protein, Human Epidermal Growth Factor Receptor 1 (HER1), Interferon Beta (IFN-β), immunocytokine, Single-chain Variable Fragment (ScFv).Abstract
Objective: To computationally design immunocytokines capable of targeting cancer cell antigens and modulating the immune response, representing a promising therapeutic approach for HER1-positive breast cancer
Methodology: This study was conducted at the National Center of Excellence, Molecular Biology, University of Punjab, Lahore, between 2021 and 2022. This is an in silico study in which a novel immunocytokine was engineered by linking an anti-EGFR/HER1 single-chain variable fragment to interferon-?. In this design, the ScFv moiety targets HER1-positive tumor cells, while the IFN-? domain retains its natural specificity for IFNAR1/IFNAR2 and is intended to provide localized immune-modulatory signaling at the tumor site. Sequence data for the ScFv and for IFN-? were concatenated to yield the ScFv-IFN-? design. Physicochemical metrics, including molecular weight, extinction coefficient, and instability index, were calculated using ProtParam and SOLpro. We predicted the 3D conformation using AlphaFold, followed by refinement with GalaxyRefine. We assessed structural quality using PROCHECK and RAMPAGE. Potential allergenic sites were mapped using AlgPred, and the corresponding mRNA secondary structure was analyzed with RNAfold. Protein-receptor interactions were examined through docking on the HDOCK server.
Results: The fused protein displayed acceptable physicochemical characteristics, retained a plausible and well-folded conformation, maintained a robust mRNA secondary structure, and demonstrated favorable docking poses for both HER1 and interferon receptors. Stability metrics from the dynamic simulations corroborated these findings.
Conclusion: The ScFv–IFN-? fusion protein emerges as a theoretically interesting design for targeted therapy against HER1-positive breast cancer, but the evidence at this stage is purely computational.
References
1. Afzal S, Hassan M, Ullah S, Abbas H, Tawakkal F, Khan MA. Breast cancer; discovery of novel diagnostic biomarkers, drug resistance, and therapeutic implications. Frontiers in molecular biosciences. 2022; 9: 783450.
2. Costa B, Amorim I, Gärtner F, Vale N. Understanding breast cancer: From conventional therapies to repurposed drugs. European Journal of Pharmaceutical Sciences. 2020; 151: 105401.
3. Vafadar A, Taheri-Anganeh M, Movahedpour A, Jamali Z, Irajie C, Ghasemi Y et al. In silico design and evaluation of scFv-CdtB as a novel immunotoxin for breast cancer treatment. International Journal of Cancer Management. 2020; 13(1).
4. Prodi E, Corbellari R, Ghezzi L, Ciambellotti S, Catastini JE, Rappuoli M, et al. Tripokin: A multi?specific immunocytokine for cancer immunotherapy. Int J Cancer. 2025; 156(1): 216-28.
5. Sampson JF, Zhang H, Zhang D, Bi M, Hinthorne A, Syed S et al. CD38-targeted attenuated interferon alpha immunocytokine activates both innate and adaptive immune cells to drive anti-tumor activity. PLoS One. 2025; 20(5): e0321622.
6. Mobark N, Hull CM, Maher J. Optimising CAR T therapy for the treatment of solid tumors. Expert Rev Anticancer Ther. 2025; 25(1): 9-25.
7. Gout DY, Groen LS, van Egmond M. The present and future of immunocytokines for cancer treatment. Cell Mol Life Sci. 2022; 79(10): 509.
8. Park S, Nguyen MQ, Ta HKK, Nguyen MT, Lee G, Kim CJ et al. Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2 (scFv)-PE24B as a Maltose Binding Protein Fusion. Int J Mol Sci. 2021; 22(12): 6483.
9. Rezaie E, Bidmeshki Pour A, Amani J, Mahmoodzadeh Hosseini H. Bioinformatics predictions, expression, purification and structural analysis of the PE38KDEL-scfv immunotoxin against EPHA2 receptor. Int J Peptide Res Therapeut. 2020; 26(2): 979-96.
10. Patel DK, Menon DV, Patel DH, Dave G. Linkers: A synergistic way for the synthesis of chimeric proteins. Protein Expression and Purification. 2022; 191: 106012.
11. Hon J, Marusiak M, Martinek T, Kunka A, Zendulka J, Bednar D et al. SoluProt: prediction of soluble protein expression in Escherichia coli. Bioinformatics. 2021; 37(1): 23-8.
12. Nussinov R, Zhang M, Liu Y, Jang H. AlphaFold, artificial intelligence (AI), and allostery. J Phys Chem B. 2022; 126(34): 6372-83.
13. Laskowski R, MacArthur M, Thornton J. PROCHECK: validation of protein-structure coordinates. 2006.
14. Wang W, Xia M, Chen J, Deng F, Yuan R, Zhang X et al. Data set for phylogenetic tree and RAMPAGE Ramachandran plot analysis of SODs in Gossypium raimondii and G. arboreum. Data in Brief. 2016; 9: 345-8.
15. Arnold M, Morgan E, Rumgay H, Mafra A, Singh D, Laversanne M et al. Current and future burden of breast cancer: Global statistics for 2020 and 2040. Breast. 2022; 66: 15-23.
16. Negrini M, Gramantieri L, Sabbioni S, Croce C. Anticancer Agents Med. Chem. 2011; 11: 500-21.
17. Singh K, Yadav D, Jain M, Singh PK, Jin J-O. Immunotherapy for Breast Cancer Treatment: Current Evidence and Therapeutic Options. Endocrine, Metabolic & Immune Disorders-Drug Targets (Formerly Current Drug Targets-Immune, Endocrine & Metabolic Disorders). 2022; 22(2): 212-24.
18. Bibi F, Rauf A, Khan MA, Ashfaq UA, Qasim M, Masoud MS, et al. Network Pharmacological Study of Papaver Somniferum to Explore the Potential Compounds to Treat Epilepsy. 2025.
19. Agostinetto E, Montemurro F, Puglisi F, Criscitiello C, Bianchini G, Del Mastro L et al. Immunotherapy for HER2-Positive Breast Cancer: Clinical Evidence and Future Perspectives. Cancers. 2022; 14(9): 2136.
20. List T, Neri D. Immunocytokines: a review of molecules in clinical development for cancer therapy. Clin Pharmacol. 2013; 5(sup1): 29-45.
21. Pasche N, Neri D. Immunocytokines: a novel class of potent armed antibodies. Drug Discov Today. 2012; 17(11-12): 583-90.
22. Haji Abdolvahab M, Mofrad MR, Schellekens H. Interferon Beta: From Molecular Level to Therapeutic Effects. Int Rev Cell Mol Biol. 2016; 326: 343-72.
23. Trinh KR, Vasuthasawat A, Steward KK, Yamada RE, Timmerman JM, Morrison SL. Anti-CD20-interferon-? fusion protein therapy of murine B-cell lymphomas. J Immunother. 2013; 36(5): 305-18.
24. Lee CG, Kim T, Hong S, Chu J, Kang JE, Park HG et al. Antibody-based targeting of interferon-beta-1a mutein in HER2-positive cancer enhances antitumor effects through immune responses and direct cell killing. Front Pharmacol. 2021; 11: 608774.
25. Alewine C, Hassan R, Pastan I. Advances in anticancer immunotoxin therapy. Oncologist. 2015; 20(2): 176-85.
26. Keshtvarz M, Salimian J, Yaseri M, Bathaie SZ, Rezaie E, Aliramezani A et al. Bioinformatic prediction and experimental validation of a PE38-based recombinant immunotoxin targeting the Fn14 receptor in cancer cells. Immunotherapy. 2017; 9(5): 387-400.
27. Rybchenko VS, Aliev TK, Panina AA, Kirpichnikov MP, Dolgikh DA. Targeted Cytokine Delivery for Cancer Treatment: Engineering and Biological Effects. Pharmaceutics. 2023; 15(2): 336.
28. Yang X, Zhang X, Fu ML, Weichselbaum RR, Gajewski TF, Guo Y, et al. Targeting the tumor microenvironment with interferon-? bridges innate and adaptive immune responses. Cancer Cell. 2014; 25(1): 37-48.
29. Samavarchi Tehrani S, Gharibi S, Movahedpour A, Goodarzi G, Jamali Z, Khatami SH et al. Design and evaluation of scFv-RTX-A as a novel immunotoxin for breast cancer treatment: an in silico approach. J Immunoass Immunochem. 2021; 42(1): 19-33.
30. Navabi P, Ganjalikhany MR, Jafari S, Dehbashi M, Ganjalikhani-Hakemi M. Designing and generating a single-chain fragment variable (scFv) antibody against IL2R? (CD25): An in silico and in vitro study. Iran J Basic Med Sci. 2021; 24(3): 360-8.
31. Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-69.
32. Santos J, Cardoso M, Moreira IS, Gonçalves J, Correia JD, Verde SC et al. Integrated in sili-co and experimental approach towards the design of a novel recombinant protein containing an an-ti-HER2 scFv. Int J Mol Sci. 2021; 22(7): 3547.
33. Aguilar MF, Garay AS, Attallah C, Rodrigues DE, Oggero M. Changes in antibody binding and functionality after humanizing a murine scFv anti-IFN-?2: From in silico studies to experi-mental
analysis. Mol Immunol. 2022; 151: 193-203.
34. Lima AJF, Hajdu KL, Abdo L, Batista-Silva LR, de Oliveira Andrade C, Correia EM,
Lourenzoni MR. In silico and in vivo analysis reveal impact of c-Myc tag in FMC63 scFv-CD19 protein interface and CAR-T cell efficacy. Computat Struct Biotechnol J. 2024; 23: 2375-2387.
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Journal of Liaquat University of Medical & Health Sciences
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
Submission of a manuscript to the journal implies that all authors have read and agreed to the content of the undertaking form or the Terms and Conditions.
When an article is accepted for publication, the author(s) retain the copyright and are required to grant the publisher the right of first publication and other non-exclusive publishing rights to JLUMHS.
Articles published in the Journal of Liaquat University of Medical & health sciences are open access articles under a Creative Commons Attribution-Noncommercial - Share Alike 4.0 License. This license permits use, distribution and reproduction in any medium; provided the original work is properly cited and initial publication in this journal. This is in accordance with the BOAI definition of open access. In addition to that users are allowed to remix, tweak and build upon the work non-commercially as long as appropriate credit is given and the new creations are licensed under the identical terms. Or, in certain cases it can be stated that all articles and content there in are published under creative commons license unless stated otherwise.
