Part:BBa_K3346006

Usage and Biology

Local estrogen production has been linked with the growth of lesions in endometriosis [1]. As such, many researchers have been interested in the connection between estrogen levels and the progression of endometriosis. This area of research has led to the discovery that reducing estrogen levels through low dosage hormone therapy can be beneficial for the reduction of symptoms such as pelvic pain [2]. Therefore, we postulate that a therapeutic antibody targeting estradiol, the prominent form of estrogen, could be a useful treatment for endometriosis patients in the future.

Fab fragments are a popular option for immunotherapy as their small sizes allow for rapid cellular diffusion and their antibody binding domains have high specificities for their target molecules [3]. Past iGEM teams have also worked with Fab fragments to create efficient expression cassettes for the mass production of these therapeutic options in simple organisms. In particular, 2019 Aboa created a periplasmic expression vector for Fab fragments using simple SfiI digestion (BBa_K2941004). Therefore, the production of an anti-estradiol Fab fragment with high efficiency expression in Escherichia coli lends itself to the inexpensive production of a therapeutic option for endometriosis patients. The 57-2 anti-17β-estradiol Fab fragment is a strong choice for immunotherapy as it has been shown to have high production rates in E. coli with high affinity for 17β-estradiol, therefore indicating its ability to reduce estrogen levels in endometriosis [4].

However, there needs to be an efficient way to deliver these immunotherapy options to its target cells. Targeted drug delivery allows for the specific delivery of therapeutic hormones and antibodies to the most relevant physiological sites. In endometriosis, targets for anti-estradiol therapeutic antibodies are the sites of estrogen biosynthesis, which includes ovarian, adipose, and skin fibroblast cells [1]. Since these processes occur intracellularly, cytosolic protein delivery allows therapeutic antibodies to target 17β-estradiol, a prevalent precursor for estrogen in the biosynthesis pathway. Recent research has shown that the addition of an anionic peptide tag, a short amino acid sequence consisting of negatively charged residues, can bind to cationic delivery lipids in the bloodstream to allow for the delivery of therapeutic antibodies into the cytosol [5]. This cytosolic delivery method has been reported at 90% efficiency at low concentrations (500 nM) of antibody, thus demonstrating its potential for use in immunotherapy [5]. The anionic peptide tag contains a linker region for attachment to therapeutic antibodies or fab fragments followed by the 25 aspartate (D25) anionic region, optimized for delivery efficiency and low toxicity levels [5]. A GFP reporter gene is included for detection of cells with successfully delivered antibodies.

This composite part consists of parts BBa_K3346004 (Anti-Estradiol Fab Fragment) and BBa_K3346005 (Anionic Peptide Tag). We recommend the use of the 3A standard assembly using the RFC23 prefixes to allow for proper in-frame assembly.

1. Kitawaki, J., Kado, N., Ishihara, H., Koshiba, H., Kitaoka, Y., & Honjo, H. (2002). Endometriosis: the pathophysiology as an estrogen-dependent disease. The Journal of Steroid Biochemistry and Molecular Biology, 83(1), 149–155. https://doi.org/https://doi.org/10.1016/S0960-0760(02)00260-1

2. Barbieri, R. L. (1998). Endometriosis and the estrogen threshold theory. Relation to surgical and medical treatment. The Journal of Reproductive Medicine, 43(3 Suppl), 287–292. http://www.ncbi.nlm.nih.gov/pubmed/9564663

3. Nelson A. L. (2010). Antibody fragments: hope and hype. mAbs, 2(1), 77–83. https://doi.org/10.4161/mabs.2.1.10786 Pajunen, M., Saviranta, P., Jauria, P., Karp, M., Pettersson, K., Mäntsälä, P., & Lövgren, T. (1997). Cloning, sequencing, expression and characterization of three anti-estradiol-17β Fab fragments. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1351(1), 192–202. https://doi.org/https://doi.org/10.1016/S0167-4781(96)00202-3

4. Wang HH, Tsourkas A. (2019). Cytosolic delivery of inhibitory antibodies with cationic lipids. Proceedings of the National Academy of Sciences, 116(44), 22132-22139.