What Is It?
Natural killer (NK) cells are an exciting alternative to costly custom T cell immunotherapies. NK cells are innate immune cells with inherent cancer cell-killing capabilities. However, engineering NK cells is difficult due to their heterogeneity and plasticity, requiring further research into realizing their use as cell therapies. Here we present a starter toolkit for engineering NK cells in support of fundamental research towards cell therapies.
The NK cell research toolkit product contains three classes of genes: ligands, surface receptors, and transcription factors. Using these genes, researchers can manipulate NK cells, cancer cells, and “feeder” cells (i.e. cells used to trigger NK cell proliferation). More specifically:
- The ligands contained within this FreeGenes product can be transduced or transfected into:
- non-stimulatory feeder cells, cancer cells or NK cell lines to isolate their effects
- stimulatory feeder cells to enhance their activating effects on NK cells
- The transcription factors can be transduced or transfected into NK cells to alter their behavior or phenotype.
- The activating or inhibitory receptors can similarly be transduced or transfected into NK cells to perturb their behavior.
What Can the NK Cell Toolkit Be Used For?
The genes listed here can be expressed on NK cells, feeder cells, or tumor cells to study individual NK cell signaling pathways, strengths of activation of different receptor-ligand interactions, and whether certain NK cells are more or less resistant to inhibitory cues from cancer cells. The provided genes can be used individually or combined to assess synergistic effects.
The combination of these ligands, receptors, and transcription factors represents a powerful toolbox with which to intelligently explore a myriad of iterations of feeder cell-NK cell-tumor cell pipelines. The NK Cell Research Toolkit enables researchers to examine the effects of distinct molecules on NK cells’ targeting and eradication of discrete tumor cell types, and to design novel chimeric antigen receptors to modify endogenous signaling pathways. Research into NK cells may advance the development of effective off-the-shelf cell therapies, which will drastically lower the cost of immunotherapy and make these life-saving treatments available to more patients.
- PCR amplify the gene of interest out of the supplied backbone
- Clone the gene into a mammalian expression vector of your choice
- Note that these genes do not come with a promoter sequence, so one must be present on your new backbone in order for the gene to be expressed
- Transfect or transduce NK cells, feeder cells, or tumor cells to express the corresponding protein
- Note that if you are transducing, you will need to produce virus - see here: https://www.addgene.org/protocols/lentivirus-production/
- Select the NK cells, feeder cells, or tumor cells that have successfully been transduced or transfected using a fluorescent molecule or selection agent such as blasticidin
- Note that the selection agents or fluorescent proteins are not included in the gene sequences, but the provided sequences all end with the beginning of the P2A self-cleaving peptide sequence so that a blasticidin resistance gene, GFP, or other gene can be incorporated into your vector without altering the function of the protein of interest - see here for more information about P2A: https://en.wikipedia.org/wiki/2A_self-cleaving_peptides and see here for information about blasticidin resistance: https://en.wikipedia.org/wiki/Blasticidin_S
- Test the expansion of NK cells after culture with modified feeder cells and compare to the unmodified feeder cells, or test the cytotoxic activity of engineered versus non-engineered NK cells against engineered or non-engineered tumor cells
Where Can I Find More Information?
Designed By: Nina Horowitz, Stanford University
|Gene||Name||NCBI ID||Freegenes ID|
|IL15P1||Interleukin 15 Precursor 1||NP_000576.1||BBF10K_000502|
|IL15P2||Interleukin 15 Precursor 2||NP_751915.1||BBF10K_000503|
|KIR2DL4IA||killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 isoform A||NP_002246.5||BBF10K_000505|
|KIR2DL4IB||killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 isoform B||NP_001074241.1||BBF10K_000506|
|KIR2DL4IC||killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 isoform C||NP_001074239.1||BBF10K_000507|
|HLAG||Homo sapiens major histocompatibility complex, class I, G (HLA-G), transcript variant 1||NP_001350496.1||BBF10K_000508|
|HLAG1||Homo sapiens major histocompatibility complex, class I, G (HLA-G), transcript variant 1||NP_001371219.1||BBF10K_000509|
|NKG2D||KLRK1 killer cell lectin like receptor K1||NP_031386.2||BBF10K_000510|
|ULBP1I1||UL16 binding protein 1 isoform 1||NP_079494.1||BBF10K_000511|
|ULBP1I2||UL16 binding protein 1 isoform 2||NP_001304018.1||BBF10K_000512|
|ULBP2||UL16 binding protein 2||NP_079493.1||BBF10K_000513|
|CD28H||TMIGD2 transmembrane and immunoglobulin domain containing 2||NP_653216.2||BBF10K_000514|
|B7H7||HHLA2 HERV–H LTR-associating protein 2||NP_009003.1||BBF10K_000515|
|CD40||cluster of differentiation 40||NP_001241.1||BBF10K_000516|
|CD40L||CD154 cluster of differentiation 154||NP_000065.1||BBF10K_000517|
|TGFb||transforming growth factor beta 1||NP_000651.3||BBF10K_000518|
|PDGF-DDtv1||platelet-derived growth factor D transcription variant 1||NP_079484.1||BBF10K_000519|
|PDGF-DDtv2||platelet-derived growth factor D transcription variant 2||NP_149126.1||BBF10K_000520|
|SDC2||HSPG syndecan 2||NP_002989.2||BBF10K_000521|
|2B4||Homo sapiens CD244 molecule||NP_057466.1||BBF10K_000522|
|NKp44||NCR2 natural cytotoxicity triggering receptor 2||NP_004819.2||BBF10K_000523|
|StrepCAR-CD28z||anti-streptavidin chimeric antigen receptor, intracellular CD28z||N/A||BBF10K_000524|
|StrepCAR-41BBz||anti-streptavidin chimeric antigen receptor, intracellular 4-1BBz||N/A||BBF10K_000525|
|StrepCAR-2B4||anti-streptavidin chimeric antigen receptor, intracellular 2B4||N/A||BBF10K_000526|
|CD19CAR-CD28z||anti-CD19 chimeric antigen receptor, intracellular CD28z||N/A||BBF10K_000527|
|CD19CAR-2B4||anti-CD19 chimeric antigen receptor, intracellular 2B4||N/A||BBF10K_000528|
|CD19CAR-41BBz||anti-CD19 chimeric antigen receptor, intracellular 4-1BBz||N/A||BBF10K_000529|
|NR4A1||Nuclear Receptor Subfamily 4 Group A Member 1||NP_001189162.1||BBF10K_000530|
|NR4A2||Nuclear Receptor Subfamily 4 Group A Member 2||NP_006177.1||BBF10K_000531|
|CRTAM||Cytotoxic and regulatory T cell molecule||NP_062550.2||BBF10K_000532|
|CD96||TACTILE T cell activation, increased late expression||NP_937839.1||BBF10K_000533|
|CLEC12A||C-lectin type domain family 12 member A||NP_612210.4||BBF10K_000534|
|CXCR6||c-x-c motif chemokine receptor 6||NP_006555.1||BBF10K_000535|
|RGS1||regulator of G protein signaling 1||NP_002913.3||BBF10K_000536|
|Hobit-i1||Homolog of Blimp-1 in T cells isoform 1||NP_001108231.1||BBF10K_000537|
|Hobit-i2||Homolog of Blimp-1 in T cells isoform 2||NP_001294854.1||BBF10K_000538|
|ID2||inhibitor of DNA binding 2||NP_002157.2||BBF10K_000539|
|GPR183||G-protein coupled receptor 183||NP_004942.1||BBF10K_000540|
|CD49a||ITGA1 Integrin subunit alpha 1||NP_852478.1||BBF10K_000541|
|CD103||ITGAE integrin subunit alpha E||NP_002199.3||BBF10K_000542|
Download all of this information as a CSV from our GitHub.
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Download all of this information as a CSV from our GitHub.