Difference between revisions of "Part:BBa K5271003"

Revision as of 14:51, 28 September 2024

HER2-binding peptide -scrambled

A dual targeting nanobody made up of an EGFR and HER2 nanobody linked by a GSSG linker.

Profile

• Name: Panobody scrambled
• Base Pairs: 753 bp
• Amino acid: 251 a.a
• Origin: Synthetic
• Properties: A scramble peptide sequence that acts as a control to the dual targeting nanobody -Panobody

Usage and Biology

The design of this Biobrick began with an in-house bioinformatics analysis which shows that EGFR and HER2 are both upregulated in pancreatic cancers, and overexpression of these two genes is associated with a worse patient prognosis. The overexpression of EGFR and HER2 is associated with gemcitabine resistance in pancreatic cancer cell lines and patients. These results provided the rationale for targeting both EGFR and HER2 to improve pancreatic cancer treatment and overcome gemcitabine resistance. Based on the dry lab result, we chose EGFR and HER2 as the dual targets for our Biobrick design. To verify our design, we used Alphafold to create a three-dimensional model of Panobody. (Figure 1.) [Jumper et al., 2021] Subsequently, we perform a molecular docking analysis to examine the binding affinity of the dual specific nanobody. (Figure 2.)

Figure 1. Workflow for the genomic bioinformatic dry lab for the design of dual-targeting nanobody – Panobody.

Figure 2. Workflow of molecular docking experiment.

The molecular docking analysis of Panobody revealed distinct binding patterns and interaction profiles with the HER2 and EGFR receptors, highlighting Panobody's superior binding affinity and stability. For the Panobody, the HER2 nanobody demonstrated a robust interaction network through its key residues, notably His5, Asn37, Leu54, Arg57, Glu68, and Glu109 with the HER2 receptor (Fig. 3a). These residues establish strong hydrogen bonds that are crucial for stabilizing the peptide-receptor complex. Additional interactions (salt bridge) with Glu51 further contribute to binding integrity, forming a well-anchored complex. This extensive network of electrostatic interactions underscores the stability and specificity of Panobody in targeting HER2, indicating its potential as a highly effective inhibitor of HER2-mediated pathways. Similarly, the Panobody’s EGFR nanobody displays a strong interaction profile with the EGFR receptor through its critical residues, including Asp190, Thr192, Tyr194, Asp196, Phe202, Thr203, and Trp238, which form essential hydrogen bonds and electrostatic interactions. The presence of π–π stacking interactions, particularly involving the key residue (Tyr194), further enhanced the molecular docking strength, contributing to the robust binding stability of the Panobody-EGFR complex (Fig. 3b). The observed interactions, including additional stabilizing forces from hydrophobic residues, indicate a well-organized and stable binding mode that can effectively modulate the EGFR signaling pathways.

Figure 3. (A) 3D and 2D interaction maps showing the molecular interactions between the HER2 nanobody of the Panobody and HER2 receptor. (B) 3D and 2D interaction maps depicting the molecular interactions between the EGFR nanobody of the Panobody and EGFR receptor.

Design Note

The dual nanobody is made up of two separate nanobodies that specifically target HER and EGFR. These two nanobodies are linked by a bridging linker. Our preliminary result on the linker showed that cysteine residues should be avoid since it potentially reduces the solubility of the dual targeting nanobody in prokaryotic expression system.

Source

The HER2 specific nanobody is an antigen-binding fragments that are derived from Camelus dromedarius heavy-chain antibodies and have advantageous characteristics compared with mAbs and their derived fragments for in vivo targeting [Hamers-Casterman et al., 1993] The EGFR specific nanobody was isolated from a phage library generated from Llama glama lymphocytes that had been immunized with A431 epidermoid carcinoma cells [Roovers et al., 2007]

Reference

• Roovers, R. C., Laeremans, T., Huang, L., De Taeye, S., Verkleij, A. J., Revets, H., ... & van Bergen en Henegouwen, P. M. P. (2007). Efficient inhibition of EGFR signalling and of tumour growth by antagonistic anti-EGFR Nanobodies. Cancer immunology, immunotherapy, 56, 303-317.
• Schmitz, K. R., Bagchi, A., Roovers, R. C., en Henegouwen, P. M. V. B., & Ferguson, K. M. (2013). Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains. Structure, 21(7), 1214-1224.
• D'Huyvetter, M., De Vos, J., Xavier, C., Pruszynski, M., Sterckx, Y. G., Massa, S., ... & Devoogdt, N. (2017). 131I-labeled anti-HER2 camelid sdAb as a theranostic tool in cancer treatment. Clinical cancer research, 23(21), 6616-6628.
• Hamers-Casterman C, Atarhouch T, Muyldermans S. Naturally occurring antibodies devoid of light chains. Nature 1993;363:446–48.
• Vaneycken I, Devoogdt N, Van Gassen N, Vincke C, Xavier C, Wernery U, et al Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J 2011;25:2433–2446.
• Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., ... & Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. nature, 596(7873), 583-589.

Sequence and Features