IJPMBS 2026 Vol.15(4): 107-113
doi: 10.18178/ijpmbs.15.4.107-113
doi: 10.18178/ijpmbs.15.4.107-113
Optimization of a Nanobody Targeting the Tyrosine Kinase Domain of Human EGFR via Hitchhiker-mediated Survival Selection
Chanason Lardarpajid1, Apisitt Thaiprayoon1, Onapa Punpeng2, Aiyaret Kraivisootsup 2,
Duangtip Moonmangmee2, Lueacha Tabtimmai 3, Kiattawee Choowongkomon4, and
Dujduan Waraho-Zhmayev 1,*
1. Biological Engineering program, Faculty of Engineering, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand
2. Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand
3. Department of Biotechnology, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand
4. Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand
Email: champ.chanason1994@gmail.com (C.L.); Nick_mic4@hotmail.com (A.T.); onapa.punp@mail.kmutt.ac.th (O.P.); aiyaret.krai@mail.kmutt.ac.th (A.K.); duangtip.moo@kmutt.ac.th (D.M.); Lueacha.t@sci.kmutnb.ac.th (L.T.) fsciktc@ku.ac.th (K.C.); dujduan.war@mail.kmutt.ac.th (D.W.-Z.)
*Corresponding author
2. Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand
3. Department of Biotechnology, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand
4. Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand
Email: champ.chanason1994@gmail.com (C.L.); Nick_mic4@hotmail.com (A.T.); onapa.punp@mail.kmutt.ac.th (O.P.); aiyaret.krai@mail.kmutt.ac.th (A.K.); duangtip.moo@kmutt.ac.th (D.M.); Lueacha.t@sci.kmutnb.ac.th (L.T.) fsciktc@ku.ac.th (K.C.); dujduan.war@mail.kmutt.ac.th (D.W.-Z.)
*Corresponding author
Manuscript received May 6, 2026; accepted August 18, 2026; published October 20, 2026.
Abstract—The Epidermal Growth Factor Receptor (EGFR) is essential for cell growth, differentiation, and migration, and its overexpression is closely linked to tumor progression and drug resistance. Although conventional EGFR-targeting antibodies and small-molecule inhibitors are effective, they often face limitations such as poor tissue penetration and acquired resistance. Nanobodies offer advantages including small size, high stability, and strong binding specificity. This study aimed to optimize a previously reported anti-EGFR tyrosine kinase domain nanobody, VHH35, by isolating improved variants using the Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins (FLI-TRAP) system. A VHH35 library of approximately 7.68 × 10⁵ clones was generated, leading to the isolation of two mutants, M.5 and M.6, which exhibited enhanced solubility while maintaining specific binding to EGFR-TK. Dot blot analysis confirmed strong EGFR reactivity with minimal nonspecific binding. This work demonstrates a practical strategy for enhancing intracellular nanobody performance using FLI-TRAP, yielding promising candidates for cancer therapeutics.
Keywords—antibody engineering, Epidermal Growth Factor Receptor (EGFR), Escherichia coli (E. coli), Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins (FLI-TRAP), protein isolation strategy, single-domain antibody
Cite: Chanason Lardarpajid, Apisitt Thaiprayoon, Onapa Punpeng, Aiyaret Kraivisootsup, Duangtip Moonmangmee, Lueacha Tabtimmai, Kiattawee Choowongkomon, and Dujduan Waraho-Zhmayev, "Optimization of a Nanobody Targeting the Tyrosine Kinase Domain of Human EGFR via Hitchhiker-mediated Survival Selection," International Journal of Pharma Medicine and Biological Sciences, Vol. 15, No. 4, pp. 107-113, 2026.
Copyright © 2026 by the authors. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
Keywords—antibody engineering, Epidermal Growth Factor Receptor (EGFR), Escherichia coli (E. coli), Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins (FLI-TRAP), protein isolation strategy, single-domain antibody
Cite: Chanason Lardarpajid, Apisitt Thaiprayoon, Onapa Punpeng, Aiyaret Kraivisootsup, Duangtip Moonmangmee, Lueacha Tabtimmai, Kiattawee Choowongkomon, and Dujduan Waraho-Zhmayev, "Optimization of a Nanobody Targeting the Tyrosine Kinase Domain of Human EGFR via Hitchhiker-mediated Survival Selection," International Journal of Pharma Medicine and Biological Sciences, Vol. 15, No. 4, pp. 107-113, 2026.
Copyright © 2026 by the authors. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
