Adoptive transfer of Chimeric Antigen Receptor T cells (CAR-T) is a promising therapy for cancer.  Typically T cells are collected from a patient and transduced with lentivirus to express CARs that specifically recognize antigens on the patient's tumor cells.  CAR-expressing T cells are then infused back into the patient to mount T cell-mediate cytotoxicity against tumor cells. The second-generation (2G) of CAR-T is composed of a single-chain variable fragment (scFv) derived from monoclonal antibody against the antigen and a flexible spacer that allows the scFv to orient in different directions to enable the antigen recognition.  The scFv is fused to a transmembrane (TM) domain and two intracellular signaling domains: one from CD28 that provides co-stimulatory signals for T cell activation and another one from CD3ζ, which is the primary transmitter of signals from endogenous T cell receptors (TCRs). After the antigen recognition, CARs cluster and transmit a proliferative/survival signal to the T cell. EGFR is known as epidermal growth factor receptor, proto-oncogene c-ErbB-1 , receptor tyrosine-protein kinase erbB-1.  It is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTK).  EGFR is overexpressed in a variety of tumors.  Mutations in EGFR gene are associated with lung cancer.  EGFR is the target of several anti-cancer therapeutics. Anti-EGFR CAR-T is engineered to specifically target EGFR and supplied as pre-packaged lentiviral particles that are ready to transduce T cells for stably expressing CARs against EGFR.  

Adoptive transfer of Chimeric Antigen Receptor T cells (CAR-T) is a promising therapy for cancer.  Typically T cells are collected from a patient and transduced with lentivirus to express CARs that specifically recognize antigens on the patient's tumor cells.  CAR-expressing T cells are then infused back into the patient to mount T cell-mediate cytotoxicity against tumor cells. The second-generation (2G) of CAR-T is composed of a single-chain variable fragment (scFv) derived from monoclonal antibody against the antigen and a flexible spacer that allows the scFv to orient in different directions to enable the antigen recognition.  The scFv is fused to a transmembrane (TM) domain and two intracellular signaling domains: one from CD28 that provides co-stimulatory signals for T cell activation and another one from CD3ζ, which is the primary transmitter of signals from endogenous T cell receptors (TCRs). After the antigen recognition, CARs cluster and transmit a proliferative/survival signal to the T cell. EGFR is known as epidermal growth factor receptor, proto-oncogene c-ErbB-1 , receptor tyrosine-protein kinase erbB-1.  It is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTK).  EGFR is overexpressed in a variety of tumors.  Mutations in EGFR gene are associated with lung cancer.  EGFR is the target of several anti-cancer therapeutics. Anti-EGFR CAR-T is engineered to specifically target EGFR and supplied as pre-packaged lentiviral particles that are ready to transduce T cells for stably expressing CARs against EGFR.  

 

Product Details

 

Gene Symbol: EGFR, ERBB, ERBB1, HER1, mENA, PIG61, SA7

 

NCBI Gene ID: 1956 (human)

 

Uniprot Entry: P00533 (human)

 

Construct Details:  The gene encoding anti-EGFR scFv in an orientation of SP (signal peptide) → VL → linker → VH fused with the TM and cytoplasmic domains from CD28 and CD3ζ (see the schematic diagram above) is subcloned into the Lentiviral vector pLTE with an EF1a promoter +/- an antibiotics marker.  The lentivector is co-transfected with the LentiPAK DNA mix (SKU# LP-001) into HEK293 cells to produce high titer lentiviral particles.  Multiple choices of antibiotics selection marker are available.

 

Vector Type: pLTE (lentiviral expression vector containing a heterologous EF1a  promoter +/- an antibiotics selection marker)

 

Formulation: Lentivector encoded and pre-packaged viral particles (typical titer 10⁶ - 10⁷ IFU/ml) in the conditional medium (serum-free) from HEK293 cells that have been transfected with the lentivector gene clone and the LentiPAK DNA mix

 

 

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Important Safety Information: With the safety features in place, our lentiviral vectors and viral particles can be employed in standard Biosafety Level 2 tissue culture facilities and should be treated with the same level of caution as any other potentially infectious agent. Any investigator who purchases our lentiviral/retroviral products & services is responsible for following Biosafety Level 2 requirements on the handling of viral particles. For more information on Biosafety Level 2 agents and practices, please refer to NIH’s “Biosafety Considerations for Research with Lentiviral Vectors”.

Restriction: This product is not transferable or re-sellable.  Customer obtain no right to transfer, assign, or sublicense its use rights, or to transfer, resell, package, or otherwise distribute the product, or to use the product for the benefit of any third party or for any commercial purpose.  Customer may only use the product in compliance with applicable local, state and federal laws, regulations and rules.  Customer may not directly or indirectly use the product or allow the transfer, transmission, export or re-export of all or any part of the product in violation of any export control law or regulation of the united states or any other relevant jurisdiction.  Your use of this product constitutes acceptance of the terms of this limited use agreement.  Please refer to our “terms & conditions” for details.  If you are not willing to accept the limitation of this agreement, G&P Biosciences will accept return of the product for a full/partial refund.

 

Transduction Protocol

 

Pre-packaged lentiviral particles are most advanced gene delivery tools. Each particles contain a fully sequence verified target gene and are psudotyped with the VSV-G glycoprotein, ready for transduction into into a wide range of cell types including hard-to-transfect primary cells and non-dividing cells. They are supplied in 1-mL aliquot(s) of the serum-/antibiotic-free solution suitable for both in vitro and in vivo delivery. They are produced in HEK293 packaging cells with a typical titer of ≥10⁷ IFU/ml using our optimized LentPAK^TM packaging system. The lentiviral particles can be used to transduce subconfluent target cells. Depending on your purpose, you may choose a specific multiplicity of infection (MOI) or test a range of MOIs to determine which gives you the desired results. Transduction can be enhanced by the addition of polybrene, also known as hexadimethrine bromide (typically at 8-10 μg/ml). 

 

Quick Protocol for Transduction

 

Day 1. Seeding Target Cells

For an example, plate target cells in a 10 cm plate at a density of 1 - 5x 10⁵ cells/ml that will produce approximately 60% confluency in 24 hours.

 

Note: other size plates can also be used depending on the nature of your experiment. Adjust the reagent amount according to Table 1

 

Table 1. Seeding Density of Target Cells (1 day prior to transduction)

Vessel Type	Seeding Density	Volume of Media
10-cm dish	1 – 5 x 106	10 mL
6-well plate	0.3 – 1 x 106	2 mL/well
12-well plate	0.15 – 0.5 x 106	1 mL/well
24-well plate	0.6 – 2 x 105	0.5 mL/well
96-well plate	1 – 4 x 104	0.1 mL/well

  

Day 2. Transduction

Remove the growth media from the dish/plate prepared the day before. Replace with 1/2 volume of culture medium containing desired amount of lentiviral particles (at 2 to 20 MOI).  For example, add 4.5 mL of growth medium and 0.5 mL of Lentiviral particles, or simply add 5 mL of Lentiviral particles (for a low titer viral preparation or a high MOI transduction). Add polybrene directly to the media on the target cells at 8 μg/ml. Mix by gentle swirling.

 

Incubate at 37°C with 5% CO2 for 4 - 24 hours, then replace the transduction medium with right amount of growth medium according to table 1 (for example 10 mL for 10-cm dish). Culture the cells for 48 – 72 hours, and transduced cells are ready for downstream analyses.

 

Note: Adjust volumes accordingly for transduction of other plate types.  For example, add 1 ml of growth medium and lentiviral particle mixture for 6-well plate, 0.5 ml for 12-well and 0.25 mL for 24-well except for 96 well, in which 100 μl should be used. The change of transduction medium is often unnecessary with our pre-packaged lentiviral particles.  Simply culture cells for 3-4 days before analysis. 

 

The virus-containing media can be changed in as short as 4 hours after transduction if toxicity of the lentiviral transduction is a concern. Normally reverse transcription and genome integration of the lentivector takes place within 24-36 hours. With our ready-to-use, prepackaged lentiviral particles, the change of media is often unnecessary. The transduction process can be ongoing for 2 - 6 days without significant impact on cell growth/viability. The transduction process can also be repeated if desired. For a lentivector with a fluorescent reporter (such as GFP), FACS can be used to enrich for cells that express GFP. If it contains a drug selectable marker, follow the protocol for the particular drug. For example, puromycin selection is usually carried out at 1-10 μg/mL, depending on the target cells’ sensitivity

 

 

Important Safety Information

With the safety features in place, our lentiviral vectors and viral particles can be employed in standard Biosafety Level 2 tissue culture facilities and should be treated with the same level of caution as any other potentially infectious agent. Any investigator who purchases our lentiviral/retroviral products & services is responsible for following Biosafety Level 2 requirements on the handling of viral particles. For more information on Biosafety Level 2 agents and practices, please refer to NIH’s “Biosafety Considerations for Research with Lentiviral Vectors”.

 

Troubleshooting

 

Poor Transduction Efficiency:

Poor transduction efficiency could be due to a low viral titer. While lentivectors can accommodate relatively large inserts (up to 8 kb) comparing to other systems, there is still a packaging limit. In general, lentivectors could transfer up to 8-kb gene inserts. However, any gene insert larger than 4kb will dramatically decreases packaging efficiency, which would results in a lower viral titer. To overcome low titers, concentrate viral particles or lower target cell density for transduction. If the transduction efficiency is low despite a high titer of virus, the total volume of transduction media on the target cells may be too high. Transduction can be carried out in a volume of media that just covers the cells; this may increase the exposure of the cells to the virus. Low speed spin (e.g., 1500 rpm for 15-30 min) may help increase the likelihood of virus-cell interactions.  Pseudotyping of the transducing virus to target more abundant receptors on a particular cell type is another potential approach.

 

Transduction Kills Target Cells:

It is possible that MOI may be too high. Perform the transduction with a lower MOI or less viral particles. Packaging cell medium may not be compatible for target cell growth. Either dilute the virus in target cell-compatible medium or concentrate and resuspend the virus in medium compatible with the target cell growth. Change the transduction media containing the virus as early as 4 hrs after transduction.

 

 

 FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

 

Storage

 

The product is shipped at 4°C for immediate use.  Upon receipt, centrifuge the vial briefly before opening.  Store at –80°C or lower and the product is stable for 3 months.  Avoid repeated freeze-thaw cycles.

 

 

The product should be employed in a Biosafety Level 2 tissue culture facility.

 

 

 

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

 

References

 

 

 

Additional supporting documents, including PDS, COA and MSDS are available upon request.

 

 

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Restriction: This product is not transferable or re-sellable.  Customer obtain no right to transfer, assign, or sublicense its use rights, or to transfer, resell, package, or otherwise distribute the product, or to use the product for the benefit of any third party or for any commercial purpose.  Customer may only use the product in compliance with applicable local, state and federal laws, regulations and rules.  Customer may not directly or indirectly use the product or allow the transfer, transmission, export or re-export of all or any part of the product in violation of any export control law or regulation of the united states or any other relevant jurisdiction.  Your use of this product constitutes acceptance of the terms of this limited use agreement.  Please refer to our “terms & conditions” for details.  If you are not willing to accept the limitation of this agreement, G&P Biosciences will accept return of the product for a full/partial refund.