KRAS G12D-Specific TCR Lentivirus (Clone 10)

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78937

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Description

KRAS G12D-Specific TCR Lentivirus (Clone 10) are replication incompetent, HIV-based, VSV-G-pseudotyped lentiviral particles ready to transduce nearly all types of mammalian cells, including primary and non-dividing cells. These viruses transduce cells with a TCR (T cell receptor) (clone 10) that specifically recognizes the human antigen KRAS (Kirsten rat sarcoma virus) G12D, and in which the TCR α chain and β chain are linked by P2A. The lentiviruses also transduce a puromycin selection marker (Figure 1). Mouse TCR constant regions were used in this construct to boost expression.

Figure 1. (A) Schematic of the lenti-vector used to generate the KRAS G12D-Specific TCR Lentivirus (Clone 10) and (B) diagram of the construct, showing the components of the KRAS G12D-specific TCR. TRAV and TRAC correspond to the TCR alpha chain variable and constant regions, respectively, whereas TRBV and TRBC correspond to the TCR beta chain variable and constant regions. Mouse TCR constant regions were used in this construct to boost expression.

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Product Data Gallery

Expression of KRAS G12D-specific TCR in Jurkat cells transduced with the KRAS G12D-Specific TCR Lentivirus (Clone 10).

Jurkat T cell activation after transduction with KRAS G12D -Specific TCR (Clone 10), using HLA-C*08:02 K562 Cell line as antigen-presenting cells (APCs).

Product Info

Storage and Usage

Citations

Supplied As

≥2 x 10⁷ TU/ml. The titer will vary with each lot; the exact value is provided with each shipment.   

Materials Required But Not Supplied

Name	Ordering Information
Thaw Medium 2	BPS Bioscience #60184
Growth Medium 2C	BPS Bioscience #79592
Assay Medium 2D	BPS Bioscience #78755
CD8⁺ TCR Knockout NFAT-Luciferase Reporter Jurkat Cell Line	BPS Bioscience #78757
HLA-C*08:02 K562 Cell line	BPS Bioscience #78974
KRAS G12D Peptide (10-18, 9mer)	BPS Bioscience #78967
KRAS Wild Type Peptide (10-18, 9mer)	BPS Bioscience #78968
KRAS G12D Peptide (10-19, 10mer)	BPS Bioscience #78969
KRAS Wild Type Peptide (10-19, 10mer)	BPS Bioscience #78970
PE anti-mouse TCR β chain Antibody	BioLegend #109207
ONE-Step™ Luciferase Assay System	BPS Bioscience #60690
Lenti-Fuse™ Polybrene Viral Transduction Enhancer	BPS Bioscience #78939

Formulation

The lentiviruses were produced in HEK293T cells in medium containing 90% DMEM + 10% FBS. Virus particles can be packaged in custom formulations by special request, for an additional fee.

Background

KRAS (Kirsten rat sarcoma virus) are GTPase proteins. They cycle between a GDP-bound inactive state and a GTP-bound (active) form, in a process regulated by two accessory proteins: GEF (guanine exchange factors) and GAPs (GTPase activating proteins). Once activated KRAS can bind to its effectors and regulate multiple signaling pathways, such as the RAF (rapidly accelerated fibrosarcoma)-MEK (mitogen activated protein kinase)-ERK (extracellular regulated kinase) or the PI3K (phosphoinositide 3-kinase)-AKT (protein kinase B)-mTOR (mammalian target of rapamycin) signaling pathways. KRAS mutations account for about 85% of all RAS mutations and are considered one of the main drivers of human cancer, such as in PDAC (pancreatic ductal adenocarcinoma). One of the amino acids frequently mutated is glycine 12, with the most common form being G12D. Since KRAS are intracellular proteins, they are not amenable to CAR (chimeric antigen receptor)-T cell-based therapies, and the development of inhibitors has also proved challenging. One strategy involves the use of TCR (T cell receptor)-T cells, targeting this antigen. Specific TCR clones have been identified, with a KRAS G12D-specific TCR (clone 9c) preferentially being reactive against KRAS G12D peptide (10-18, 9mer), in comparison with KRAS G12D peptide (10-19, 10mer) and being unable to recognize the wild-type KRAS peptides. On the other hand, a KRAS G12D-specific TCR (clone 10) is preferentially reactive against KRAS G12D peptide (10-19, 10mer), in comparison with KRAS G12D peptide (10-18, 9mer) and it does not recognize wild-type KRAS peptides. Results from a trial using a KRAS G12D HLA-C*08:02 restricted TCR demonstrated the potential of this approach for the treatment of PDAC. The use of neoantigen specific TCR-T therapies, targeting single amino acid mutations, is thus an exciting and promising cancer therapy.

References

Eric T., et al., 2016 N Engl J Med 375:2255-2262.
Leidner R., et al., 2022 N Engl J Med 386:2112-2119.

Storage/Stability

Lentiviruses are shipped with dry ice. For long-term storage, it is recommended to store the virus at -80°C for up to 12 months from date of receipt. Avoid repeated freeze-thaw cycles. Titers can drop significantly with each freeze-thaw cycle.

Growth Media

For the best results, the use of BPS Bioscience validated and optimized media is highly recommended. Other preparations or formulations of media may result in suboptimal performance.

Media Required for Maintaining CD8⁺ TCR Knockout NFAT-Luciferase Reporter Cell Line
Growth Medium 2C (BPS Bioscience #79592): RPMI 1640 medium supplemented with 10% FBS, 1% Penicillin/Streptomycin plus 1 mg/ml Geneticin, 100 µg/ml Hygromycin B.

Media Required for Maintaining HLA-C*08:02 K562 Cell Line
Thaw Medium 2E (BPS Bioscience #79638): RPMI 1640 medium supplemented with 10% FBS, 1% Penicillin/Streptomycin plus 0.5 µg/ml Puromycin.

Media Required for Co-culture Assay
Assay Medium 2D (BPS Bioscience #78755): RPMI 1640 medium supplemented with 1% FBS.

Applications

Use as a positive control for KRAS G12D TCR evaluation and optimize experimental conditions.
Generate KRAS G12D TCR expressing cell pools or stable cell lines, following puromycin selection.

Shipping Temperature

-80°C

Notes

To generate a KRAS G12D-Specific TCR expressing stable cell line, remove the growth medium 48 hours after transduction and replace it with fresh growth medium containing the appropriate amount of puromycin (as pre-determined from a killing curve, https://bpsbioscience.com/cell-line-faq), for antibiotic selection of transduced cells, followed by clonal selection.  

Biosafety
The lentiviruses are produced with a SIN (self-inactivation) lentivector which ensures self-inactivation of the lentiviral construct after transduction and integration into the genomic DNA of the target cells. None of the HIV genes (gag, pol, rev) will be expressed in the transduced cells, as they are expressed from packaging plasmids lacking the packing signal. Although the pseudotyped lentiviruses are replication-incompetent, they require the use of a Biosafety Level 2 facility. BPS Bioscience recommends following all local federal, state, and institutional regulations and using all appropriate safety precautions.

Troubleshooting Guide
Visit bpsbioscience.com/lentivirus-faq for detailed troubleshooting instructions. For further questions, please email [email protected].

Warnings

Avoid freeze/thaw cycles.

Scientific Category

Cancer Therapy Target

Regulatory Status

Research Use Only

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KRAS G12D-Specific TCR Lentivirus (Clone 10)

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