What Is It?
Immunotherapy is an incredibly promising cancer treatment method that retargets the immune system to kill cancer cells; it can eradicate both a tumor and its distant metastases as well as generate long term immunological memory to prevent relapse. Custom T cell immunotherapeutics are effective but relatively inaccessible as they are produced on a patient by patient basis and cost up to $500,000. An exciting alternative is natural killer (NK) cells, innate immune cells that can be given to patients from other donors and have inherent cancer cell-killing and antibody binding capabilities. Engineering NK cells can be difficult due to their heterogeneity and plasticity, but further research into their use as cell therapies - particularly in combination with clinical antibodies - could make a new class of powerful, inexpensive, off-the shelf immunotherapeutics that is accessible to a wider variety of cancer patients.
This product contains three classes of genes: ligands, surface receptors, and transcription factors. Using these sets of free genes, researchers can manipulate NK cells, cancer cells, and “feeder” cells (used to trigger NK cells to proliferate up to a clinically relevant dose) to express a variety of surface and intracellular proteins.
- The ligands contained within this FreeGenes product can be transduced or transfected into non-stimulatory feeder or cancer cells or NK cell lines to isolate their effects, or into 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.
The combination of all of these ligands and receptors represents a powerful toolbox with which to intelligently and rationally design almost countless iterations of feeder cell - NK cell - tumor cell pipelines; they will enable both basic science and translational researchers to answer fundamental immunology questions about which molecules promote NK cell killing against which types of tumor cells in an unprecedented manner. They can also be used by synthetic biologists or bioengineers to design novel chimeric antigen receptors by combining intracellular and extracellular domains of different receptors to modify endogenous signaling pathways. All of these kinds of research will advance the goal 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.
What Can It 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. These can all be combined in various combinations or used individually to assess synergistic effects as well. The CARs can be used to make NK cells that target surface molecules on B cells or any other cells, as long as an antibody or other targeting molecule can be designed against it.
- 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|
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