iGEM AFCM Team's Project Inspiration and Description
Being part of a small sum of participating iGEM Teams from Africa, it has always been a challenging concept to gather engagement and funds for our projects; which is why we choose therapeutic projects that target serious public health concerns.
In the last two years we pushed our effort down the road to find a solution for hepatocellular as well as colorectal carcinoma. This year, alike, we chose another cancer that has been plaguing our country and many others for decades. Bladder cancer (BC) is the most common malignancy among Egyptian males and previously has been attributed to Schistosoma Haematobium infection. It is also the ninth most common cancer throughout the world and is considerably more common in developed than developing countries. In the United States, BC is the fourth most common cancer in males. In Europe, its rates are also relatively high.
Our 2019 project of bladder cancer gained the highest public engagement and collaborations among previous AFCM projects; through several meet ups and social events, we tackled several significant causative and correlative factors for bladder cancer, one of which being smoking.
Scratching through the surface of BC – with its prominence and risk factors providing us with motivational force, we navigated through the core of our project. The idea of utilizing the body’s own defense mechanisms to deter cancer was thrilling to us. We navigated into the realm of chimeric antigen receptor T cells and the various generations of CAR-T cells and their advantages.
Our objectives steered us to consider the immune-modulatory action of bio-engineered immune cells on bladder cancer cell lines. Through siRNA (short interference RNA) introduced to the T–Cells (CD3+ve) on a plasmid vector carrying 3 siRNA cassettes, we silenced 2 genes responsible for CAR-T cells exhaustiveness (TOX and NR4a) and PD-1 to increase the efficacy of the therapy and inhibit tumor blockage for T-cells.
We separated T-cells from urinary bladder cancer patients with lab evidence of Schistosoma haematobium infection; edited and introduced CAR-T cell design for the cells. From end to end, via CRISPR-CAS9 we guided a gRNA targeting the TRAC locus. Then, Homology Directed repair template (HDR) was inserted on a separate donor vector, the HDR carrying the CAR design construct.
The CAR design is based on TRUCK CAR, (Fourth Generation CAR T-cell) which has bispecific 2 antibodies (computationally designed); one targeting bladder cancer specific neo-antigen variant of ESR2 (Estrogen receptor beta 2) which is used by Sch. Haematobium to induce bladder cancer, the other antibody targets the Major egg antigen of the ova) along with the typical CAR constituents (2nd Generation CAR), the signaling domains of the TRUCK CAR have an extra signaling domain carrying Il-18 gene to enhance cytokine anti-tumor activity.
Schistosoma and Bladder cancer
The urinary bladder squamous cell carcinoma is a malignant cancer that arises from urothelium, nearly 90%-95% are urothelial in origin, male have higher incidence than females 2:1. [1,2,3] Schistosomiasis is one of the main causes, so the highest incidence is in the endemic countries like: Egypt, which represents nearly 2/3 of all malignant neoplasms in the bladder. [4]
82% of patients who have bladder carcinoma were found to harbor Schistosoma haematobium eggs in the bladder wall. The Schistosoma eggs start a severe inflammatory response and fibrosis in the bladder wall. S haematobium total antigen has shown that it increases proliferation, migration, and invasion of Schistosoma and decrease recognition and destruction by human immunity.
Our project Storyline:
- T–Cells (CD3+ve) cells will be separated from patient bladder cancer patients with lab evidence of Schistosoma haematobium Infection (ova in bladder tissue).
- T-Cells will be optimized for enhancing activity against solid tumors (bladder cancer) through silencing 2 genes responsible for CAR-T cells exhaustiveness (TOX and NR4a) which encodes 2 transcription factors.
- T-Cells will be also be PD-1 silenced to increase the efficacy of the therapy and inhibit tumor blockage for T-cells.
- The 3 genes will be silenced through siRNA (short interfering RNA) Introduced to the T-Cells on a plasmid vector carrying 3 siRNA cassettes.
5. T cells will be edited to introduce CAR T cell design.
6. CRISPR will be used, Cas9 will cut the T-cell genome guided by a guide RNA targeting TRAC locus.
7. Then, Homology Directed repair template (HDR) will be inserted on a separate donor pcDNA+ vector, the HDR will carry the CAR design construct.
8. The CAR design is based on TRUCK CAR, (Fourth Generation CAR T-cell) which has
(bispecific 2 antibodies (computationally designed); one targeting bladder cancer specific neo-antigen variant of ESR2 (Estrogen receptor beta 2) which is used by Sch. Haematobium to induce bladder cancer, the other antibody targets the Major egg antigen of the ova) along with the typical CAR constituents (2nd Generation CAR), the signaling domains of the TRUCK CAR have an extra signaling domain carrying Il-18 gene to enhance cytokine anti-tumor activity.
9. This design shall be tested on bladder cancer cell-line associated with bilharzia (sch.haematobium) ova.
Our Project Inspiration
Rationale for selecting Dual Targets of Chimeric Antigen Receptor T-cell :
- Literature was mined to define the relation between Schistosoma haematobium infection and bladder ova deposition on development of squamous and transitional bladder carcinoma. We found that deposited ova release estrogen-like metabolites [1,2,3, that act on estrogen receptor through mostly in antagonistic way and keep irritating the bladder cells helping the development of carcinogenesis specially in Egyptian patients [4]. Estrogen receptor as well was found to be an interesting highly expressed intracellular target for bladder cancer [5] Thus, we aimed at targeting this pathway (ESR2-Estrogen receptor beta) in bladder cancer as a potential pathway of schistosomiasis associated bladder cancer.
- We wanted to target the disease at 2 stages; the development stage and disease progression stage, thus we mined the databases of Schistosoma haematobium [6] and found major egg antigen as a crucial target for the ova to provide more guidance to our CAR-T cells and initiate further oxidative stress against irritating ova to bladder epithelium. Thus we aimed at creating an OR gate of bispecific tandem CAR recognition domain for both targets.
Rationale for selecting 4th generation “TRUCKs” (T cells Redirected for Universal Cytokine Killing)
Basically we have been relying upon a second generation CAR, in which intracellular signaling portion consists of the T-cell signaling domain for killing CD3zeta as well as a CD28 costimulatory domain. However, as we aimed at further recognition of schistosoma haematobium ova, we had some doubts during our integrated human practices discussions with Dr. Andrea Savarino about the possibility of T-cell attack for the ova, thus we further mined the literature and found the possibility of designing a fourth generation TRUCK CAR by adding an extra intracellular signaling domain for the CAR construct to make it produce IL-18 which has the ability to modulate reactive oxygen species (ROS) which in turn could have potential ability to attack schistosomiasis ova deposited in patient’s bladders as well as our in-vitro model [7, 8,9].
Improving CAR-T cells activity against solid tumors and Designing short Interference silencing RNAs
We selected TOX and NR4A as they are exhaustiveness-associated transcription factors in which silencing could potentially improve the killing activity of CAR T-cells against solid tumors as bladder cancer in a challenging tumor microenvironments [10]. Another important target for improving CAR activity against solid tumors for silencing is PD-1 to inhibit the inhibitory costimulatory between PD-1 and PDL-1 on tumor cells with considerations to long-term effects on T-cell proliferation[11].
References:
1-Botelho, MC, Soares, R, Vale, N. Schistosoma haematobium: identification of new estrogenic molecules with estradiol antagonistic activity and ability to inactivate estrogen receptor in mammalian cells. Exp Parasitol 2010; 126: 526–535.
2-Adebayo AS, Mundhe SD, Awobode HO, Onile OS, Agunloye AM, Isokpehi RD, et al. (2018) Metabolite profiling for biomarkers in Schistosoma haematobium infection and associated bladder pathologies. PLoS Negl Trop Dis 12(4): e0006452.
3-Botelho, MC, Alves, H, Barros, A. The role of estrogens and estrogen receptor signaling pathways in cancer and infertility: the cases of schistosomes. Trends Parasitol 2015; 31: 246–250.
4-Faysal MH. Squamous cell carcinoma of the bladder. J Urol. 1981 Nov. 126(5):598-9.
5-Ou Z, Wang Y, Chen J, et al. Estrogen receptor β promotes bladder cancer growth and invasion via alteration of miR-92a/DAB2IP signals. Exp Mol Med. 2018;50(11):152. Published 2018 Nov 20.
6-https://schistodb.net/schisto/
7-Chmielewski M, Abken H. CAR T cells transform to trucks: chimeric antigen receptor–redirected T cells engineered to deliver inducible IL-12 modulate the tumour stroma to combat cancer. Cancer Immunol Immunother (2012) 61(8):1269–77.
8- Huang HH, Rigouin C, Williams DL. The redox biology of schistosome parasites and applications for drug development. Curr Pharm Des. 2012;18(24):3595–3611.
9- Kim J, Shao Y, Kim SY, et al. Hypoxia-induced IL-18 increases hypoxia-inducible factor-1alpha expression through a Rac1-dependent NF-kappaB pathway. Mol Biol Cell. 2008;19(2):433–444. doi:10.1091/mbc.e07-02-0182.
10- Seo H, Chen J, Gonzalez-Avalos E, Samaniego-Castruita D, Das A, Wang YH, et al. . TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8(+) T cell exhaustion. Proc Natl Acad Sci USA. (2019) 116:12410–5.
11-Wei J, Luo C, Wang Y, et al. PD-1 silencing impairs the anti-tumor function of chimeric antigen receptor modified T cells by inhibiting proliferation activity. J Immunother Cancer. 2019;7(1):209.