Bispecific fourth generation tanCAR against schistosomiasis associated bladder cancer
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 shistosoma hematobium ova, we had some doubts during our integrated human practices discussions with Dr. Andrea Savarino abou the possibility of T-cell attack for the ova, thus we further mined the literature and found the possibiity 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 schsitomiasis ova deposisited 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].
IDT siRNA designing tool were used to design siRNA against the three targets TOX, NR4A and PD-1. Furthermore, to adjust the energy of the designed siRNA molecules, Vienna RNA [12] was used to adjust the stability of each siRNA molecule for optimum binding in favorable range of structural energies [-18, -23] kcal/mol.
As we plan to inset the 3 siRNAs as composite cassettes on one silencing vector to pre-optimize T-cells prior to CAR insertion using CRISPR-Cas9, we had to overcome DNA synthesis challenges for composite cassettes as they could potentially contain repeats of U6 promoters or hairpin loops. Thus, we adopted the asymmetrical design of siRNAs which have been shown to improve siRNA silencing activity by creating asymmetry between lengths of both arms of siRNA molecules and recomputing their energies using Vienna RNA [12].
Asymmetrical-mutated Design of three siRNAs (left to right) PD-1, TOX and NR4A
Designing of Parts and Fragments
We have planned to create two vectors
- The first vector, Represents a silencing vector to carry triple siRNA cassettes to prepare CAR-T cells for solid tumors.
- The second vector, Represents a donor pcDNA+ vector to carry our pre designed TRUCK CAR construct as a Homology Directed repair Template HDR for CRISPR-Cas9 systems.
Parts and design of siRNA Vector (BBa_K3244025):
This Device was designed by inserting part: BBa_K3244024 which is a cassette for the triple siRNAs, into pSC1C3 as a carrying vector after being modified by adding
Biobrick | Type | Role | Length |
Coding | AmpR | 861 | |
Regulatory | AmpR Promoter | 105 | |
Regulatory | U6 Promoter | 228 | |
Coding | EGFP | 768 | |
Terminator | bGH poly(A) signal | 211 | |
Regulatory | RSV Promoter | 227 |
Parts and design of CAR vector (BBa_K3244029):
We intended to design a fourth generation TRUCK CAR that is based on the anatomy of second generation CARs in addition to intracellular cytokine expression domain. The TRUCK CAR design incorporates composition of 2nd generation CAR in part BBa_K3244027 with cytokine expression domain in part BBa_K3244028 in a form of Homology directed repair template for CRISPR-Cas9 designed insertion systems by flanking the gRNA targeting TRAC region on T-cells with 2 DNA arms L-ARM and R-ARM with desired TRUCK CAR insertion targeting ESR2 and MEA in between.
2nd generation CAR in part BBa_K3244027 Designed as a gBlock to be composited with through standard assembly using EcorI ends.
Il-18 Cytokine expression domain in part BBa_K3244028 Designed as a gBlock to be composited with through standard assembly using EcorI ends.
Final BBa_K3244029 TRUCK CAR HDR ligated to pcDNA+
Bio brick | Type | Role | Length |
Regulatory | CD8 Leader Sequence | 64 | |
Coding | scFv for ESR2 neoantigen | 744 | |
Coding | Gly Ser Linker for TanCAR-T scFvs | 75 | |
Coding | scFv for Schistosoma haematobium Major egg antigen | 723 | |
Coding | CD8 Hinge | 144 | |
Coding | CD28 Trans-Membrane Domain | 81 | |
Coding | CD28 Intracellular signaling domain | 123 | |
Coding | CD3 zeta signalling domain | 339 | |
Regulatory | NFAT-Response Ellement | 117 | |
Regulatory | IL-2 Promoter | 114 | |
Coding | IL-18 | 579 | |
Regulatory | IRES | 625 | |
Coding | GFP | 720 | |
Coding | BZip Leucine zipper | 150 | |
Coding | AZip Leucine zipper | 171 | |
Coding | 2ِA Cleavage | 54 | |
DNA | L-ARM for CAR-T insertion in TRAC using CRISPR | 582 | |
DNA | R-ARM for CAR-T insertion in TRAC using CRISPR | 590 | |
Regulatory | Guide RNA Scaffold for CRISPR insertion of CAR-T cell therapies | 107 |
Universal CAR Safety and Pharmacological Kill switch:
We have also provided computational analysis for interaction of a pharmacological kill switch using tyrosine kinase inhibitor Dasatinib (please refer to our modeling page) which could act as a potential safety key to clinical applications of CAR therapies and avoid potential side effects as cytokine release syndrome (please refer to our modeling page) .
Universal CAR designs have emerged as a potential way to improve target specificity and enhance recognition of CAR T-cells, one example is SUPRA CAR, which could be exemplified by a universal modular car design in which T-cells are edited once and provided by a compelltery segment of a leucine zipper instead of extracellular binding domain, then a freely mobile binding domain is introduced with attachment of the other complementary portion of the leucine zipper, that enables therapist to edit T-cells once and then modulates the recognition portions for different targets as well as limiting edited T-cells action only when recognition occurs [13]. We have provided the design for the cellular portion of the design in part Part:BBa_K3244030
and for the extracellular portion in part Part:BBa_K3244031
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.
12-Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH. (2004) Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101(19):7287-92.
13-Zhao J, Lin Q, Song Y, Liu D. Universal CARs, universal T cells, and universal CAR T cells. J Hematol Oncol. 2018;11(1):132. Published 2018 Nov 27.