Acurate targeting is one of the important characteristics for many emerging cancer therapies, which is also our project’s ambition. We designed two fusion proteins. They function like fishing rod, so they are termed as Fishing Rod Proteins (FRPs). The one that targets Fn was named as Bacteria Tageting Rod (BTR), and the other that targets CRC cells was named as Tumor Tageting Rod (TTR). FRPs are displayed on BI-BYE membranes, helping BL targets to the correct places. Of course, their targets are cancer cells and Fn, rather than the fish. Each FRP consists of four parts (Figure1): the signal peptide, helping FRPs land on cell membrane; the membrane protein, the base of FRPs; the linker, helping FRPs become flexible; and the targeting fragments, the most pivotal role in FRPs.

The membrane protein came from GNB/LNB substrate-binding membrane protein (GL-BP), which is derived from Bifidobacterium and can be expressed on BL’s cell surface^[6]. It belongs to ATP-binding cassette transporter (ABC). iGEM17 _TJU_China and iGEM 22_Shanghai_HS also took advantage of it to display proteins

TTR’s targeting fragment is Histone-like protein A (HlpA), which has been proved that can bind to heparan sulfate proteoglycan (HSPG) on CRC cells^[6]. HlpA was also used by LZU_China in 2022. When HlpA is fused with the linker and the GL-BP, TTR can precisely find where the cancer cells are.

The difference between two TTR and BTR lies on the targeting fragments, not only the structures, but the working mechanism. BTR is more special. BTR (BBa_K4990011) is one of our contributions this year. To understand its working mechanism, pilus FadA should be known first. FadA is Fn’s pilus and composed of a number of mFadA monomers. The monomers connect to each other by self-assemblely in a head-to-tail way, and that is the point of our design. Since the self-asselmblely binding force is really strong, we thought it would be a good strategy for Fn targeting. We truncated the key domain, which is related to the self-assembly of mFadA, and termed it B-domain (BBa_K4990002). B-domain is BTR’s targeting fragment. The BI-BYE displayed on B-domain can assemble with the pilus of Fn, which leads to bacteria-bacteria specific adhesion.

All in all, with the guidance of TTR and BTR, BI-BYE is able to find targets easily. Moreover, they can narrow the distance between BI-BYE targets and enlarge safety and therapeutic effects.

LL-37, released by serine proteases from human cationic antimicrobial protein (hCAP18), is the only cathelicidin identified so far in humans. It has a broad spectrum of antimicrobial activity against a wide range of pathogens and has the potential to be an antimicrobial and anti-biofilm agent^[7]. In addition, a growing number of studies demonstrated the impact of LL-37 in the process of tumor growth, and discovered that the loss of LL-37 expression is associated with human CRC, indicating a tumor suppressor role of LL-37 ^[8,9]. Therefore, it was chosen to be the core domain of our cytotoxic peptides.

Based on LL-37, we truncated a 13AA peptide and termed it FK-13(BBa_K4990001), which has better cytotoxicity(Figure). Furthermore, the two targeting fragments with Self-cleaving linkers (BBa_K4990005, BBa_K4990006) fuses with FK-13, so that the cytotoxic peptides can target Fn or CRC more precisely. In total, we designed three cytotoxic peptides(Figure). Bacteria targeting peptide (BTP), the cytotoxic peptide that can only target Fn; Tumor targeting peptide (TTP), the one that can only target CRC cells. And the ultimate form, Dual-Edged Harpoon (DEH, BBa_K4990005), has one type of targeting fragment on the either end of FK-13(figure), so it has the ability to target and kill both Fn and CRC cells.

The expression and secretion of cytotoxic peptides are controlled by the sensing system. Only when BI-BYE is located in TME, will the cytotoxic peptides start to function. The cytotoxic peptides linkers will be cleaved after the cytotoxic peptides enter TME, and release FK-13 to kill targets.

Here is a detailed relationship between our parts, maybe you’ll know more clearly after reading it:

To ensure the delivery safety of BI-BYE, we introduced colon soluble gelatin hollow capsules (mainly composed of gelatin and hydroxypropyl methylcellulose phthalate (HPMCP)). The formulation is sustained-release, colon targeted, so the BL only released when is penetrated in TME.

To reduce the cytotocixity of Cytotoxic Peptides to healthy tissures, we introduce hypoxia-responsive module to control the expression and secretion of Cytotoxic Peptides. The hypoxia-responsive module was inspired by TecMonterey 2013. It contains a FNR expressing box and he native regulatory promoter NirB. Under aerobic conditions, FNR exits as monomers with no transcriptional activity, resulting in non-activation of the promoter NirB and no expression of downstream genes. In anaerobic environments, FNR undergoes dimerization, activating the NirB promoter and initiating downstream gene expression. Since TME is hypoxia, BI-BYE will only start to dual-killing upon reaching the TME, hence, minimising negative effects on healthy cells.

we have two suicide systems to further protect human bodies and environment from being harmed by BI-BYE. The first system is functioned in vivo. Once the engineered bacteria have permeated the tumor microenvironment, the expression of HokB^[10] is automatically activated. The accumulation of HokB is slow due to the effect of the weak RBS(BBa_B0033 ), thereby preventing immediate death of the engineered bacteria. The accumulation of toxin will eventually reach a certain threshold, and killed BL like a time bomb.

As for the suicide system designed for preventing environmental leakage, we implement the MazEF Toxin-Antitoxin System. The expression of antitoxin mazE is regulated by a temperature-sensitive RNA switch (BBa_K3247005: NoChill-19). When the bacteria are expelled from bodies, the temperature reduces (25℃). RNA switch will form a hairpin structure, obstructing the mazE translation, and only the toxin, MazF^[11], will be expressed and kill the BI-BYE .

Other than BI-BYE, we also designed a set of CRC prognostic kit, which patients can test themselves after treatment to assess the changes of Fn and tumor. More details can be seen in Hardware.

[1] Cancer today. (n.d.). Retrieved 20 August 2023, from http://gco.iarc.fr/today/home

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[3] Ou, S., Wang, H., Tao, Y., Luo, K., Ye, J., Ran, S., Guan, Z., Wang, Y., Hu, H., & Huang, R. (2022). Fusobacterium nucleatum and colorectal cancer: From phenomenon to mechanism. Frontiers in Cellular and Infection Microbiology, 12, 1020583. https://doi.org/10.3389/fcimb.2022.1020583

[4] Wang, N., & Fang, J.-Y. (2023). Fusobacterium nucleatum, a key pathogenic factor and microbial biomarker for colorectal cancer. Trends in Microbiology, 31(2), 159–172. https://doi.org/10.1016/j.tim.2022.08.010

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[6] Ho, C. L., Tan, H. Q., Chua, K. J., Kang, A., Lim, K. H., Ling, K. L., Yew, W. S., Lee, Y. S., Thiery, J. P., & Chang, M. W. (2018). Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nature Biomedical Engineering, 2(1), 27–37. https://doi.org/10.1038/s41551-017-0181-y

[7] Ridyard, K. E., & Overhage, J. (2021). The Potential of Human Peptide LL-37 as an Antimicrobial and Anti-Biofilm Agent. Antibiotics (Basel, Switzerland), 10(6), 650. https://doi.org/10.3390/antibiotics10060650

[8] Niemirowicz, K., Prokop, I., Wilczewska, A. Z., Wnorowska, U., Piktel, E., Wątek, M., Savage, P. B., & Bucki, R. (2015). Magnetic nanoparticles enhance the anticancer activity of cathelicidin LL-37 peptide against colon cancer cells. International Journal of Nanomedicine, 10, 3843–3853. https://doi.org/10.2147/IJN.S76104

[9] Porter, R. J., Murray, G. I., Alnabulsi, A., Humphries, M. P., James, J. A., Salto-Tellez, M., Craig, S. G., Wang, J. M., Yoshimura, T., & McLean, M. H. (2021). Colonic epithelial cathelicidin (LL-37) expression intensity is associated with progression of colorectal cancer and presence of CD8+ T cell infiltrate. The Journal of Pathology. Clinical Research, 7(5), 495–506. https://doi.org/10.1002/cjp2.222

[10] Wilmaerts, D., Bayoumi, M., Dewachter, L., Knapen, W., Mika, J. T., Hofkens, J., Dedecker, P., Maglia, G., Verstraeten, N., & Michiels, J. (2018). The Persistence-Inducing Toxin HokB Forms Dynamic Pores That Cause ATP Leakage. mBio, 9(4), e00744-18. https://doi.org/10.1128/mBio.00744-18

[11] Chono, H., Matsumoto, K., Tsuda, H., Saito, N., Lee, K., Kim, S., Shibata, H., Ageyama, N., Terao, K., Yasutomi, Y., Mineno, J., Kim, S., Inouye, M., & Kato, I. (2011). Acquisition of HIV-1 resistance in T lymphocytes using an ACA-specific E. coli mRNA interferase. Human gene therapy, 22(1), 35–43. https://doi.org/10.1089/hum.2010. 001