Part:BBa_K3346002

Usage and Biology

Antibodies have great potential for the design of diagnostics (immunoassays) and therapeutic applications (immunotherapy) [1]. With such high affinity to their target molecules, antibodies can effectively capture target analytes in in vitro samples or in in vivo systems. However, the use of immunoassays and immunotherapy is limited in part because of their high cost [2]. While mass production can help reduce the expenses associated with the use of antibodies in a project design, the cost burden can make it hard for teams with limited resources to design and implement synthetic biology projects that use these molecules. We decided to use synthetic biology to produce the antibodies for immunoassays and immunotherapies to lower the cost and make our diagnostic product more accessible to clinics and laboratories across the globe. This could additionally help other teams planning to use antibodies in diagnostic or therapeutic applications. This BioBrick was designed for efficient use in the modified E. coli strain SHuffle, which has an oxidative cytoplasm and additional chaperone proteins to ensure the proper formation of disulfide bonds [3].

In addition to the alterations to the chassis organism, BioBricks designed for use in E. coli SHuffle must have two specific point mutations to allow for binding to FC𝛾Rs, a family of effector proteins [3]. While FC𝛾R-antibody interaction is not important for the use of antibodies for detection, it is critical for proper immune system recognition in a therapeutic context [4]. This interaction affects the expression level of cytokines and antibody glycosylation. Previous literature had induced E382V and M428I mutations to induce these changes [3]. However, the constant sequence used for this paper was not available for comparison to our desired murine sequence [5]. As such, we hypothesized that introducing the same amino acid substitutions at A382V and R428I may help increase the binding affinity of FC𝛾R to our antibody of choice. However, we do acknowledge the limitations of this assumption as the amino acids found at the 382 and 428 positions in our murine constant sequence were different than those that were listed in literature.

This BioBrick is designed to allow for the insertion of the desired heavy variable chain and entire light chain of the antibody sequence prior to our mutated constant chain (Siltuximab VH Chain BBa_K3346000). This allows for future iGEM teams to insert the VH chain of their desired antibody into this plasmid and produce their antibodies efficiently in E. coli SHuffle. This can be performed using Standard Assembly, as shown in our composite BioBrick producing the full length Siltuximab therapeutic antibody.

This sequence was widely available on the UniProt database [5].

[1] Khan, F. H. (2014). Chapter 25 - Antibodies and Their Applications (A. S. Verma & A. B. T.-A. B. Singh (eds.); pp. 473–490). Academic Press. https://doi.org/https://doi.org/10.1016/B978-0-12-416002-6.00025-0

[2] Hernandez, I., Bott, S. W., Patel, A. S., Wolf, C. G., Hospodar, A. R., Sampathkumar, S., & Shrank, W. H. (2018). Pricing of monoclonal antibody therapies: higher if used for cancer? The American Journal of Managed Care, 24(2), 109–112. http://www.ncbi.nlm.nih.gov/pubmed/29461857

[3] Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T. J., Tuckey, C., Riggs, P. D., Colussi, P. A., Noren, C. J., Taron, C. H., DeLisa, M. P., & Berkmen, M. (2015). Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nature Communications, 6(1), 8072. https://doi.org/10.1038/ncomms9072

[4] Lu, R.-M., Hwang, Y.-C., Liu, I.-J., Lee, C.-C., Tsai, H.-Z., Li, H.-J., & Wu, H.-C. (2020). Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science, 27(1), 1. https://doi.org/10.1186/s12929-019-0592-z

[5] NCBI. Ighg1 immunoglobulin heavy constant gamma 1 (G1m marker) [ Mus musculus (house mouse). https://www.ncbi.nlm.nih.gov/gene/16017