Natural Killer Cell Research Toolkit

Natural Killer Cell Research Toolkit

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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: 

  1. The ligands contained within this FreeGenes product can be transduced or transfected into:
    1. non-stimulatory feeder cells, cancer cells or NK cell lines to isolate their effects
    2. stimulatory feeder cells to enhance their activating effects on NK cells
  2. The transcription factors can be transduced or transfected into NK cells to alter their behavior or phenotype.
  3. 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. 

 

Sample Workflow

  1. PCR amplify the gene of interest out of the supplied backbone
  2. Clone the gene into a mammalian expression vector of your choice
    1. 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
  3. Transfect or transduce NK cells, feeder cells, or tumor cells to express the corresponding protein
    1. Note that if you are transducing, you will need to produce virus - see here: https://www.addgene.org/protocols/lentivirus-production/
  4. 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
    1. 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
  5. 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?

      https://en.wikipedia.org/wiki/Cancer_immunotherapy

      https://en.wikipedia.org/wiki/Chimeric_antigen_receptor_T_cell

      https://en.wikipedia.org/wiki/Natural_killer_cell

      https://en.wikipedia.org/wiki/Antibody


       

      Designed By: Nina Horowitz, Stanford University

      Image Source

      Genes

      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
      IL2 Interleukin 2 NP_000577.2 BBF10K_000504
      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.
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