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Synthetic Biology Approaches to Engineer T Cells Available online at www.sciencedirect.com ScienceDirect Synthetic biology approaches to engineer T cells 1 1 1 Chia-Yung Wu , Levi J Rupp , Kole T Roybal and 1,2 Wendell A Lim There is rapidly growing interest in learning how to engineer genetically modify and edit cellular systems. Thus this is immune cells, such as T lymphocytes, because of the potential a particularly exciting time: our ability to rationally engi- of these engineered cells to be used for therapeutic neer cells is exponentially growing, as are the potential applications such as the recognition and killing of cancer cells. therapeutic applications of engineered immune cells. At the same time, our knowhow and capability to logically engineer cellular behavior is growing rapidly with the Synthetic biologists seek to understand the design prin- development of synthetic biology. Here we describe how ciples of biological systems by dissecting, rebuilding and synthetic biology approaches are being used to rationally alter repurposing natural and synthetic components [1–6]. The the behavior of T cells to optimize them for therapeutic biomedical relevance of engineered T cells demonstrated functions. We also describe future developments that will be in recent clinical trials is one reason why T cells are important in order to construct safe and precise T cell emerging as an important model system for synthetic therapeutics. biologists. In adoptive immunotherapy, T cells are isolat- Addresses ed from blood, processed ex vivo, and re-infused into 1 Dept. of Cellular & Molecular Pharmacology, University of California patients [7 ,8,9 ] (Figure 1a). Although best known San Francisco, San Francisco, CA 94158, United States 2 for cancer therapy, the application of engineered T cells Howard Hughes Medical Institute, University of California San includes and is not limited to treating intracellular patho- Francisco, San Francisco, CA 94158, United States gens and auto-immunity [10,11] (Figure 1b). Remarkably, Corresponding author: Lim, Wendell A ([email protected]) engineered T cells can safely persist for years in vivo [12,13 ]. Progress towards allogeneic, universal donor T cells is underway, and so are methods of differentiating Current Opinion in Immunology 2015, 35:123–130 induced pluripotent stem cells into T cells [14,15,49]. This review comes from a themed issue on Special section: Both technologies are envisioned to significantly increase Immunological engineering the availability of therapeutic T cells. Edited by Darrell Irvine and Hidde Ploegh For a complete overview see the Issue and the Editorial T lymphocytes and their signaling systems are an ideal test bed for synthetic engineering, thanks to decades of Available online 25th July 2015 rigorous basic research that has generated extensive http://dx.doi.org/10.1016/j.coi.2015.06.015 knowledge on T cell biology. The proliferative capacity 0952-7915/# 2015 Elsevier Ltd. All rights reserved. of T cells also makes it relatively simple to obtain large numbers of cells for experimental and treatment pur- poses. Transient or stable expression of synthetic mole- cules in T cells can be achieved using multiple methods (Box 1) [16–20,50–52]; genome engineering via CRISPR or ZFN approaches carries immense potential for con- Introduction: synthetic biology meets immunology struction of complex circuits involving re-wiring, modify- ing, or disabling endogenous pathways. Finally, T cells Cells are capable of remarkably sophisticated behavior. In provide a rich context for intercellular interactions that is particular, immune cells exhibit a wide range of charac- amenable to engineering and can be used to explore key teristics that are well suited for therapeutic applications. parameters in cell–cell communication and dynamic pop- Research in cell biology and immunology has focused on ulation behaviors [21,22]. dissecting the molecular mechanisms underlying these complex behaviors. However, there is now growing inter- Thus the field of T cell engineering (synthetic immunol- est in understanding how to engineer immune cells to ogy) is rapidly growing. This review will discuss selected carry out controlled and redirected natural behavior and examples T cell engineering and how this field might new, non-natural behaviors. This shift comes from the expand in the future to enhance precision control over convergence of two exciting emerging areas of research. therapeutic T cells. First is the establishment that engineered immune cells can be used as therapeutics to treat cancer or autoimmu- nity. Second is the development of synthetic biology — a Progress in rewiring T cells field in which our understanding of molecular regulatory Detection of disease signals through synthetic T cell receptors. systems has been combined with our increasing ability to T cells normally use their T cell receptor (TCR) to detect www.sciencedirect.com Current Opinion in Immunology 2015, 35:123–130 124 Special section: Immunological engineering Figure 1 (a) (b) transfer 2 expand 3 to patient modified T cells therapeutic aims of modified T cells T cancer (CD4+, CD8+) T detect & kill tumor cells infection (CD4+, CD8+) T detect & eliminate chronic infections T genetically autoimmunity (Tregs) 1 modify prevent autoimmune disease T cells prevent transplant rejection Current Opinion in Immunology Engineering T cells for diverse clinical needs. (a) Overview of adoptive immunotherapy using genetically modified T cells. (b) Current and future applications for engineered T cells. peptides presented by MHC molecules. To harness signaling modules from both the TCR and co-stimulatory T cells in treating disease, it is critical to be able to alter receptors. In this regard antigen-dependent activation of T cells such that they recognize specific, selected disease T cells equipped with such receptors is independent of signals (e.g. a tumor antigen). A streamlined way to MHC and costimulatory interactions. The single antigen modulate a T cell’s specificity for input signals is to input activates two intracellular responses — both signal employ synthetic receptors, which are typically chimeras 1 (antigenic stimulation) and signal 2 (co-stimulation) of of motifs and domains of natural or synthetic origin. T cell activation to produce desired outcomes such as Synthetic TCRs, chimeric antigen receptors (CARs) cytokine production, proliferation, and survival. With the and antibody-coupled T cell receptors re-direct cells to advent of current generation CARs containing costimu- recognize disease associated ligands or antigens on target latory motifs, CAR T cells have shown strong in vivo cells [7 ,9 ,23 ,24 ] (Figure 2a). The first generation of proliferation and durability required for therapeutic these synthetic receptors was developed nearly 20 years efficacy. A tremendously exciting example of current ago and generally only contained signaling modules from CAR therapy is the CD19 CAR T cell trials for treatment the TCR. The current generation of CARs and antibody- of B cell cancers, which highlight the potential of cell- coupled T cell receptors typically combine intracellular based therapeutics to transform cancer therapy [7 ]. Box 1 Clinically Validated Permanent Modification Transient Modification •Retroviral Vectors [17] •RNA transfection [16] •Lentiviral Vectors [17] •DNA-based transposons [18] •Zinc-finger nuclease based gene editing [19] Future/In Development Permanent Modification Transient Modification •CRISPR/TALEN based gene editing [50-52] •Protein transfection (dCas9) [20] Current Opinion in Immunology Methods to engineer T cells. Current Opinion in Immunology 2015, 35:123–130 www.sciencedirect.com Synthetic biology approaches to engineer T cells Wu et al. 125 Figure 2 (a) Redirecting T cells: Detecting disease antigens with synthetic receptors T cell activation SYNTHETIC T CELL RECEPTORS tumor tumor tumor cell INPUT 1 INPUT 2 MHC tumor MHC- co-stim. antigen antigen disease peptide ligand peptide antigen antibody antigen Fc receptor modified scFv T Cell co-stim. ectodomain receptor receptor TCR co-stim.domain co-stim.domain T CD3z domain CD3z domain CD3z domain (TCR ITAMs) (TCR ITAMs) (TCR ITAMs) CD3z domain co-stim. (TCR ITAMs) domain engineered Chimeric Antigen Antibody-Coupled TCR Receptor (CAR) T cell Receptor (b) Overcoming immune supresssion convert suppression override: block suppressive signals to stimulation expression of stimulatory PD-L1 PD-L1 cytokines tumortumor TGFβ TGFβ truncated receptor PD-1 immuno- T suppressive cytokines ITIM co-stim. ITSM domain stimulatory T cytokines immuno- dominant T suppression negative immune stimulation response (c) Engineering migration control chemokine synthetic optogenetic GPCRs GPCRs GPCRs chemokines small (e.g. CXCL2) molecule light T (d) Safety & control of T cells antigen 1 antigen 2 T T T T suicide switches activation switches T inhibitory motif co-inhibitory receptors CAR iCAR Current Opinion in Immunology Diverse synthetic modules have been developed to reprogram therapeutic T cells. (a) Synthetic receptor designs for targeting disease antigens. ITAM: immunoreceptor tyrosine-based activation motif, scFv: single chain variable fragment. (b) Methods for engineering T cells resistant to immunosuppressive microenvironments. ITIM: immunoreceptor tyrosine-based inhibitory motif, ITSM: immunoreceptor tyrosine-based switch motif. (c) Approaches to engineer migration/trafficking control. (d) Engineering safety switches and gated activation in gene modified T cells. www.sciencedirect.com Current Opinion
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