Part:BBa_K5271010

Panobody

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

Profile

• Name: Panobody
• Base Pairs: 777 bp
• Amino acid: 258 a.a
• Origin: Synthetic
• Properties: A dual targeting nanobody with a molecular weight of 26.9 kDa that specifically bind to EGFR and HER2 in Gemcitabine resistant pancreatic cancer cells.

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.

Expression

The recombinant Panobody is sub-cloned into pET-24d(+) expression vector. The pET-24d(+)-Panobody was transformed into Bl21 (DE3) competent cells (Figure 4.). Only bacteria received the Kanamycin-resistant gene in the recombinant vector survived in the LB agar plate containing kanamycin.

Figure 4. LB agar plate with pET-24d(+)-Panobody transformants [BL21 (DE3)].

The positive colony was picked and grow. Following induction with 0.5 mM IPTG, bacterial cells were cultured at 16°C, 25°C and 30°C, respectively, overnight (Figure 5 - 7). Harvested cells were lysed, and the total cell lysate (referred to as the "Total" fraction) was collected. Post-centrifugation, the supernatant (referred to as the "Soluble" fraction) was isolated. Both total and soluble samples were denatured by heating at 100°C for 5 minutes in the presence of 5X SDS loading buffer. These samples were subjected to SDS-PAGE electrophoresis. The target protein, Panobody, was expected to have a molecular weight (MW) of ~27.9 kDa.

Figure 5. Protein expression of Panobody in 16°C.
Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total Panobody in 16°C with 0.5mM IPTG induction for 6 hours; lane 3, Soluble Panobody in 16°C with 0.5mM IPTG induction for 6 hours; lane 4, Total Panobody in 16℃ with 0.5mM IPTG induction for 24 hours; lane 5, Soluble Panobody in 16°C with 0.5mM IPTG induction for 24 hours; lane 6, Total Panobody in 16°C without IPTG induction for 6 hours; lane 7, Soluble Panobody in 16°C with without IPTG induction for 6 hours; lane 8, Total Panobody in 16°C without IPTG induction for 24 hours; lane 9, Soluble Panobody in 16°C without IPTG induction for 24 hours.

Figure 6. Protein expression of Panobody in 25°C.
Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total Panobody in 25°C with 0.5mM IPTG induction for 6 hours; lane 3, Soluble Panobody in 25°C with 0.5mM IPTG induction for 6 hours; lane 4, Total Panobody in 25℃ with 0.5mM IPTG induction for 24 hours; lane 5, Soluble Panobody in 25℃ with 0.5mM IPTG induction for 24 hours; lane 6, Total Panobody in 25°C without IPTG induction for 6 hours; lane 7, Soluble Panobody in 25℃ with without IPTG induction for 6 hours; lane 8, Total Panobody in 25°C without IPTG induction for 24 hours; lane 9, Soluble Panobody in 25℃ without IPTG induction for 24 hours.

Figure 7. Protein expression of Panobody in 25℃.
Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total Panobody in 25℃ with 0.5mM IPTG induction for 6 hours; lane 3, Soluble Panobody in 25℃ with 0.5mM IPTG induction for 6 hours; lane 4, Total Panobody in 25℃ with 0.5mM IPTG induction for 24 hours; lane 5, Soluble Panobody in 25℃ with 0.5mM IPTG induction for 24 hours; lane 6, Total Panobody in 25℃ without IPTG induction for 6 hours; lane 7, Soluble Panobody in 25℃ with without IPTG induction for 6 hours; lane 8, Total Panobody in 25℃ without IPTG induction for 24 hours; lane 9, Soluble Panobody in 25℃ without IPTG induction for 24 hours.

The SDS-PAGE results showed a prominent band at ~27 kDa in the induced sample of 16°C and 25°C condition, especially in the soluble fraction but 30°C is not favourable for expression of Panobody. This band was absent in the uninduced samples, indicating that the protein expression was successfully induced by IPTG. The expected size of the protein aligns with the observed band, as indicated by the red bracket.

Purification

The Panobody protein, designed to target both EGFR and HER2, was purified using Ni-NTA affinity chromatography. The binding was facilitated by equilibrating the column with the equilibration buffer, while the washing steps removed non-specifically bound proteins. Finally, an imidazole gradient was used to elute the target Panobody protein, as imidazole competes with histidine residues for binding to nickel, thereby releasing the protein from the column. First, the column was equilibrated using equilibration buffer to prepare the resin for protein binding. Then, unbound proteins were removed using washing buffer, ensuring that only His-tagged Panobody remained bound to the column. Next, gradient of imidazole was applied to elute the Panobody. Imidazole displaced the His-tagged Panobody from the nickel, leading to the collection of elution fractions. Finally, eluted fractions were collected and mixed with 5X SDS loading buffer. Samples were then denatured by heating at 100°C for 5 minutes. Samples were loaded onto an SDS-PAGE gel and run at 220 V for 40 minutes to assess protein purity and confirm the presence of the target Panobody.

Figure 8. Purification of the Nanobody using nickel affinity chromatography.
A Chromatographic trace showing Nanobody (blue line) from the 6xHis-tag-chelating affinity column with increasing imidazole concentration (green line).

Figure 9. Panobody expression in selected fractions after Ni-NTA Affinity Chromatography.
Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; Lane 2, Soluble Panobody before going through the column; Lane 3, Flow through of the column (FT); Lane 4, 20mM imidazole washing of the column (W); Lane 5, 60mM imidazole elution of the column (E); Lane 6, 0.2M imidazole elution of the column.

Fast protein liquid chromatography (FPLC) results showed significant protein efflux at both 30% and 100% elution phases, suggesting a successful purification of Panobody. The SDS-PAGE gel analysis confirmed the presence of the target Panobody protein, with distinct bands observed at approximately ~27 kDa, as highlighted by the red bracket. The elution fractions contained a high concentration of Panobody, as confirmed by FPLC and SDS-PAGE analysis. This demonstrates the successful purification of Panobody using Ni-NTA affinity chromatography.

Following purification, the Panobody protein was buffer-exchanged using Amicon Ultra filtration (10 kDa MWCO) into PBS, to remove imidazole and prepare the sample for further characterization. Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) was then performed to confirm the molecular weight of the purified Panobody and validate successful expression and purification.

Figure 10.LC-ESI-MS spectrum of elution fraction after Ni-NTA Affinity Chromatography showing the molecular mass of Panobody (27851 Da).

LC-ESI-MS confirmed the presence of the Panobody in the elution fraction, with a molecular weight of 27,851 Da, validating the effectiveness of the Ni-NTA affinity chromatography.

Proof of Function

After confirmation of successful expression and purification, we proceeded to test the function of Panobody by MTT cytotoxicity assay. Gemcitabine-resistant pancreatic cancer cells-PANC1 (GemR PANC-1) was treated alone with Panobody or in combination with Panobody and gemcitabine.

Figure 11. Combined treatment of Panobody with gemcitabine synergistically inhibited the growth of gemcitabine resistant PANC-1.
Cells were treated with the indicated combination at different doses for 48 hours. Cell viability was measured using MTT assay. Positive value in excess over Bliss indicated a synergistic effect in the combined treatment.

From the MTT cytotoxicity assay, Panobody alone suppressed the cell proliferation of GemR PANC-1 cells. Panobody exerted a maximal synergistic growth suppressive effect in combination with gemcitabine in GemR PANC-1 cells. The results of Bliss analysis demonstrated that our Panobody alone inhibited cell proliferation, while also exhibiting a synergistic growth-inhibiting effect when combined with gemcitabine. In conclusion, These promising initial findings suggest that Panobody represents a potential targeted therapy for pancreatic cancer, complementing gemcitabine treatment.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 154
  Illegal NheI site found at 861
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 521
  Illegal AgeI site found at 653
  
• 1000
  COMPATIBLE WITH RFC[1000]