Introduction:: Cell-based therapies can overcome the limitations of current cancer treatments that attack the cell cycle with little specificity, resulting in severe off-target effects. Advancements in chimeric antigen receptor (CAR) T cells and similar systems improve the safety and efficacy of targeted cancer treatments, but still rely on intracellular native signaling mechanisms and offer little downstream control. CAR T cells activate signaling pathways using native parts and leave the activity of cells subject to crosstalk with other signaling pathways and T-cell exhaustion. Optimized integration of native signaling pathways with a synthetic construct has the potential to insulate cellular response from crosstalk with signaling pathways activated by the tumor microenvironment, enabling direct control over the cellular response and inhibition of T cell exhaustion. Modular synthetic constructs have been used to release a synthetic transcription factor in response to interchangeable scFv-ligand binding through protease mediated cleavage, but post-cleavage, this system is irreversible1. Post-translational circuits utilize kinase-phosphatase mediated protein-protein interactions to dictate other protein-protein interactions. Preliminary feasibility and characterization of this platform has been performed by inducing the interaction of split mNG and measuring fluorescence. Here, to provide desired control over cell function, the post translational circuit will be integrated with key points of native pathways important in the immune response to cancer such as NF-κB and Stat6.
Materials and Methods:: Amino acid sequences for IκB, IKK and TAK1 were obtained through a bio data base, Uniprot, and then back translated with optimized human expression codons while avoiding restriction enzyme sites used for cloning. The resulting DNA sequence is functionalized with EcoRI and XbaI restriction enzyme recognition sequences at either the 5’ or 3’ end, respectively. This linear cDNA sequence obtained through Twist Bioscience is digested with these enzymes and a buffer in parallel to circular plasmids encoding for the leucine zipper-substrate-mNG or the SH2-mNG. The results of the digest have compatible sticky-hanging ends, allowing for insertion of the cDNA into the existing plasmids backbone after gel electrophoresis to separate the backbone plasmid from the cut-out mNG DNA. Gel bands are purified via filter columns and ligated using T4 ligase. After incubation, these ligations are transformed into E. Coli, plated on ampicillin treated lysogeny broth (LB) plates. Colonies are screened by PCR using primers specific to the backbone and insert, and ran on a gel to confirm cloning success. Colonies with the correct size amplified region are grown in LB media, and via alkaline lysis, purified DNA is obtained after a 16-hour incubation.
Results, Conclusions, and Discussions:: Feasibility testing is performed with rapamycin, because its interaction is well characterized and relatively inexpensive compared to cytokine inputs. TNF-α, VEGFD, TGF-β, IL-6, IL-8 and IL-10 are all potential targets for a cancer therapeutic. These ligand-scFv interactions provide the capability of specific targeting but must first be optimized in vitro. Each ligand-receptor interaction is unique in regards to steric hindrance and background noise, which can be optimized via varying linker lengths as well as cytosolic phosphatase activity. Receptor dimerization promotes the phosphorylation of the minimal substrate bound to one receptor by a leucine zipper by the kinase active domain linked to the other receptor. Proteins of interest can be linked to an SH2 domain that will selectively bind the phosphorylated minimal substrate, and to the substrate. In preliminary studies, halves of split mNeonGreen were linked to the substrate and SH2 domain. In this case, functionality of the post-translational circuit is characterized by fluorescence measured by flow cytometry. Demonstrating similar results in response to a library of cytokines is the next step in functionalizing this construct as a useful therapeutic. Future work aims to demonstrate functionality and optimization of a library of ligand-scFv combinations.
The NF-κB pathway is well characterized and conserved throughout T Cells and macrophages, so it is currently the primary focus of this study. To address the need for a generalizable approach to control cell function, the post-translational circuit will be functionalized with key components of the NF-κB pathway. An induced protein-protein interaction achieving activation of a native pathway establishes a point of integration between the synthetic construct and the native pathway, isolating cellular response to relevant ligands. NF-κB translocation to the nucleus is contingent upon IκB degradation. Here, I propose activating this transcription factor with one of two points of integration; either by inducing the interaction of IκB and IKK or TAK1 and IKK. The functionality of this platform will be optimized using the characterized rapamycin-binding receptors, in HEK293 cells but the ultimate goal is to activate the NF-κB pathway within immune cells in response to tumor-associated ligands that would naturally inhibit this transcription factors translocation.
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References (Optional): : 1.Daringer et al. ACS Syn Bio. 2014
