Part:BBa_K2946003
Inhibitory CAR
Background
Our lab specialize in immunology and specifically immune-therapy, which means our main occupation is to research and investigate about the uses of immune system components. Therefore, we chose to improve an ITIM (immune tyrosine-based inhibitory motif) of the intracellular domain of KIR3DL1. Killer cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins expressed by natural killer cells and subsets of T cells. KIR proteins are classified by the number of extracellular immunoglobulin domains (2D/3D) and by the length of their cytoplasmic domain (Long/Short). KIR proteins with the long cytoplasmic domain transduce inhibitory signals upon ligand binding via an immune tyrosine-based inhibitory motif (ITIM), while KIR proteins with the short cytoplasmic domain lack the ITIM motif and instead associate with the TYRO protein tyrosine kinase binding protein to transduce activating signals. 2010’s UCSF iGEM team attached this specific part to a scFv and a showed basal expression. As we already mentioned the KIRs are receptors on NK and some of T cells membrane and a ligand binding can delay or activate T/NK cells depending on the KIR type. Higher expression of KIR protein can ensure a better effect, if the KIR has an activating intracellular domain we'll get a T cell engager effect and if the KIR has an inhibitory intracellular domain we'll get a better regulated T cells/NK cells killing effect and can avoid cytokine storm and over reaction of the immune system. In our experiment we substitute the original scFv with a well expressed scFv originated from anti HLA antibody and replaced the original expression system with an RNA-based one which automatically enhanced the expression of our protein of interest.
Experiment
The experiment included a cloning stage of the part and ligation to pGEM4Z 5'UT-eGFP-3'UT-A64 plasmid. This allowed us to use a commercial kit to produce mRNA in-vitro. Following this, mRNA transfections were preformed to K562 cell line by electroporation. The specific fluorescent antibodies were added 24hrs later and then the cells were examined via FACS. In our experiment, we used a well-expressed chimeric antigen receptor (CAR) with a cMyc tag as positive control. We also used a negative control - irrelevant RNA which does not produce a mature protein.
Results
In order to examine the expression level of the ITIM protein on the cell membrane, K562 cells were cultured and then transfected with 10µg mRNA. The expression was confirmed and quantified (% of expression within a population of cells) by FACSCallibur 45 minutes after cells were incubated with an αHuman c-Myc conjugated APC antibody 24hrs post-transfection. The controls for this experiment were irrelevant mRNA and a positive control. The FACS was calibrated according to the irrelevant mRNA transfected cells incubated with the antibody.
Figure 1, the expression of the c-Myc tag was examined and showed a 5.01% background from the irrelevant mRNA, while the positive control showed 93.57% expression. The ITIM protein showed an amazing expression of 85.64% in comparison to the controls.
As shown in the FACS results the expression of our improved part was higher than UCSF 2010 similar part as you can see in the following graph:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 263
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 126
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