To solve the problems existing in the current treatment of RA, we design and build a brand new immunotherapy. FOXP3+ regulatory T cells, which can suppress and regulate immune reaction, are engineered by inserting a chimeric antigen receptor (CAR) using lentiviral vectors to recognize RA related inflammatory cells. At the same time, we insert another receptor to activate the functional stability pathway of regulatory T cells in the inflammatory environment, and thus making it possible for them to be more immunosuppressive and more stable in lesions in hope for a better anti-RA effect. These two redirections of the two different systems on the native regulatory T cell response ensure both the efficacy and efficiency of our novel immunotherapy for RA.
SynNotch is a system that is capable of specifically activating the expression of the USP7 gene in an inflammatory environment, thereby maintaining the activity of Treg cells by stabilizing the FOXP3 proteins.
In our SynNotch system, we retain the functional sequence of the transmembrane domain and the cleavage site of the Notch 1 protein. At the N-terminus, we fuse the extracellular domain of IL17RA with Notch 1 to recognize IL-17A secreted by Th17 cells specifically, so that our regulatory T cells to obtain the ability to response to IL-17A. We also connect the gene of Gal4-vp64 (a fusion protein) in the downstream of Notch 1. In the presence of inflammatory cytokines IL17A, SynNotch protein is cleaved, and thus Gal4-VP64 fusion protein is detached from the cell membrane.
The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part BBa_K ) and then these two proteins combine together, which enable USP7 gene to express with high efficiency. USP7 proteins will deubiquitinate the FOXP3 that has been ubiquitinated, so as to enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. In this way, Treg cells can survive and play a role of immunosuppression.
CAR-CD20 is a module that enables engineered modified T cells to specifically recognize the surface antigen CD20 in B lymphocytes and thus to play anti-inflammatory function. Our CAR-CD20 system is made up of the gene sequences of several proteins.
At the N-terminus, we choose variable region of CD20 monoclonal antibody as the scFv fragment, so that it can accurately identify and bind to B lymphocyte surface antigen CD20. Then, we use two 4-1-BB sequences as a co-stimulatory signal and a CD3Z sequence as a single-stimulatory signal that allow the signal to be delivered to the cell at a high level, thereby activating Treg cells in the presence of CD20 B lymphocytes.
In addition, we attach a red fluorescent protein mCherry to the end of the CAR fusion protein as a report signal for a more convenient detection. By designing this CAR system we equip our Treg with a spear, which can reduce the inflammation level in RA patients.
Based on our design on these two systems, we name our engineered Treg as “Human Engineered Anti-Autoimmune-Diseases Regulatory T cell (HEAD-Treg) System”. We hope it can serve as a mighty warrior, leading the immune system to rebalance itself, to conquer the disease and to reestablish the patient’s health.
Construction of plasmids
Our systems were based on three plasmids that were PLVX-IRES-PURO, PLVX-IRES-NEO and PcDNA3.1 (+). We modified these plasmids and apply them on the connection of the SynNotch fusion protein gene, the CAR-CD20 fusion protein gene, and the UAS-USP7-Promoter-USP7 system. (Look up more details from the Parts section)
1.Roybal KT, Williams JZ, et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 2016. doi: 10.1016/j.cell.2016.09.011.
2.Klebanoff CA, Restifo NP. Customizing Functionality and Payload Delivery for Receptor-Engineered T Cells. Cell 2016. doi: 10.1016/j.cell.2016.09.033.
3.Ellebrecht CT, Bhoj VG, et al. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science 2016. doi: 10.1126/science.aaf6756.
4.Fransson M, Piras E, et al. CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery. J Neuroinflammation. 2012. doi: 10.1186/1742-2094-9-112.
5.MacDonald KG, Hoeppli RE, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. J Clin Invest. 2016. doi: 10.1172/JCI82771. Epub 2016 Mar 21.
6.Roybal KT, Rupp LJ. et al. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. Cell. 2016. doi: 10.1016/j.cell.2016.01.011.
7.Kononenko AV, Lee NC. et al. Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette. Nucleic Acids Res. 2015. doi: 10.1093/nar/gkv124.
8.Müller K, Zurbriggen MD, Weber W. An optogenetic upgrade for the Tet-OFF system. Biotechnol Bioeng. 2015. doi: 10.1002/bit.25562.
9.Khalil DN, Smith EL, et al. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol. 2016 May. doi: 10.1038/nrclinonc.2016.25.
10.Chen Z, Barbi J, et al. The ubiquitin ligase Stub1 negatively modulates regulatory T cell suppressive activity by promoting degradation of the transcription factor Foxp3. Immunity. 2013. doi: 10.1016/j.immuni.2013.08.006.
11.Van Loosdregt J, Fleskens V, et al. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity. 2013. doi: 10.1016/j.immuni.2013.05.018.
12.Wang L, Kumar S, et al. Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3+ T-regulatory Cell Function and Promotes Antitumor Immunity. EBioMedicine. 2016. doi: 10.1016/j.ebiom.2016.10.018.
13.Chen X, Oppenheim JJ. Th17 cells and Tregs: unlikely allies. J Leukoc Biol. 2014 May;95(5):723-731. Epub 2014 Feb 21
14. Ren J, Li B. The Functional Stability of FOXP3 and RORγt in Treg and Th17 and Their Therapeutic Applications. Adv Protein Chem Struct Biol.2017;107:155-189. doi: 10.1016/ bs.apcsb.2016.10.002. Epub 2016 Dec 15.
15. Chen X, Oppenheim JJ. Th17 cells and Tregs: unlikely allies. J Leukoc Biol. 2014 May;95(5):723-731. Epub 2014 Feb 21.
16.Brudno JN, Kochenderfer JN. Chimeric antigen receptor T-cell therapies for lymphoma. Nat Rev Clin Oncol. 2017 Aug 31. doi: 10.1038/nrclinonc.2017.128.
17.Miossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discov. 2012 Oct;11(10):763-76. doi: 10.1038/nrd3794.
18.Mills KH. TLR-dependent T cell activation in autoimmunity. Nat Rev Immunol. 2011 Nov 18;11(12):807-22. doi: 10.1038/nri3095.
19.Burzyn D, Benoist C, Mathis D. Regulatory T cells in nonlymphoid tissues. Nat Immunol. 2013 Oct;14(10):1007-13. doi: 10.1038/ni.2683. Epub 2013 Sep 18.
20.Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008 May 30;133(5):775-87. doi: 10.1016/j.cell.2008.05.009.