Part:BBa_K5271011:Design

HER2nb-Fc-EGFRnb

Assembly Compatibility:

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

  Illegal NheI site found at 154
  Illegal NheI site found at 1615
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 643
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 820
  Illegal AgeI site found at 1275
  Illegal AgeI site found at 1407
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Avoid fusing Fc fragment to nanobody when using a prokaryotic system. Insufficient secretion and formation Inclusion bodies were found when expressing the Fc-fused nanobody in E. Coli.

Source

The VHH domain of the 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]

The DNA sequence of the Fc fragment was obtained from part of the Fc region of the heavy chain of immunoglobin gamma (IgG) of human.

The 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]

References

• 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.
• 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.
• Jin, B. K., Odongo, S., Radwanska, M., & Magez, S. (2023). NANOBODIES®: A Review of Generation, Diagnostics and Therapeutics. International journal of molecular sciences, 24(6), 5994.
• Bao, G., Tang, M., Zhao, J., & Zhu, X. (2021). Nanobody: a promising toolkit for molecular imaging and disease therapy. EJNMMI research, 11, 1-13.
• Klint, J. K., Senff, S., Saez, N. J., Seshadri, R., Lau, H. Y., Bende, N. S., ... & King, G. F. (2013). Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli. PloS one, 8(5), e63865.
• De Marco, A. (2020). Recombinant expression of nanobodies and nanobody-derived immunoreagents. Protein expression and purification, 172, 105645.