Production of human CAR-NK cells with lentiviral vectors and functional assessment in vitro

Summary Although natural killer (NK) cells have become a promising immune effector cell for chimeric antigen receptor (CAR)-based therapy, generating human CAR-NK cells with high transgene efficiency has been challenging. In this protocol, we describe how to generate CAR-NK cells with transduction efficiencies >15% from healthy donor ex vivo expanded NK cells using third generation lentiviral vectors (LVs). We also show how to assess CAR-NK cell anti-tumor function in vitro using a flow cytometry-based killing assay. For complete details on the use and execution of this protocol, please refer to Portillo et al. (2021).


SUMMARY
Although natural killer (NK) cells have become a promising immune effector cell for chimeric antigen receptor (CAR)-based therapy, generating human CAR-NK cells with high transgene efficiency has been challenging. In this protocol, we describe how to generate CAR-NK cells with transduction efficiencies >15% from healthy donor ex vivo expanded NK cells using third generation lentiviral vectors (LVs). We also show how to assess CAR-NK cell anti-tumor function in vitro using a flow cytometry-based killing assay. For complete details on the use and execution of this protocol, please refer to Portillo et al. (2021).

BEFORE YOU BEGIN
Note: Within each section of the protocol we have indicated when a reagent/equipment can be substituted with a similar product, otherwise use the provided reagent/equipment.

Culture K562-mb-IL21 feeder cells
Timing: Minimum 2 weeks before NK cell expansion from PBMCs This section describes how to culture the K652 feeder cells expressing membrane bound IL-21 (K562-mb-IL21) that are used to generate expanded NK cells. The K562-mb-IL21 cells were generated by retroviral transduction with truncated CD19, CD64, CD86 and CD137L and by transduction using the Sleeping Beauty transposon expressing mbIL-21 (Denman et al., 2012;Singh et al., 2011). The sequence of mb-IL21 is owned by Kiadis Pharma (https://www.kiadis.com/) and the sequence is expressed from the MNDU3 promoter (Singh et al., 2011). The K562-mb-IL21 cells are available upon request through a Material Transfer Agreement (MTA).
a. Add 10 mL of complete RPMI media to 15 mL falcon tube. b. Remove frozen K562-mb-IL21 cryovial from liquid nitrogen tank. c. Place K562-mb-IL21 cryovial in a 37 C water bath immediately after retrieving from liquid nitrogen tank. As soon as there is one ice crystal remaining, transfer cells drop wise into 10 mL of complete RPMI media. d. Centrifuge tube at 300 3 g for 5 min. e. Remove supernatant and count cells with Trypan Blue.
b. Determine total number of K562-mb-IL21 cells needed to replenish desired number of PBMCs at a 2:1 ratio.
Example: If expanding a starting number of 2.5 million PBMCs you need to irradiate 5 million K562mb-IL21 cells. Depending on the number of NK cells you would like to expand, irradiate the corresponding amount of K562-mb-IL21 plus 2-5 million extra cells to account for pipetting error.
a. Count PBMCs with Trypan Blue. b. Add 2.5 million PBMCs and 5 million irradiated K562-mb-IL21 cells into a 50 mL falcon tube and top up with complete RPMI media to a final volume of 0.5 3 10 6 cells/mL based on the starting PBMC cell number. c. Supplement with 100 IU/mL of IL-2. 17. Incubate the co-culture at 37 C and 5% CO 2.
Note: Starting with 2.5 million PBMCs usually yields a sufficient amount of expanded NK cells as this method typically induces a $10 3 -10 4 fold expansion after 21 days (Denman et al., 2012).
18. Replace media on co-culture every other day.
a. Transfer co-culture to a 50 mL falcon tube and centrifuge at 300 3 g for 5 min. b. Remove supernatant and add 5 mL of fresh complete RPMI media, keeping the same total volume used in the replenishing day. c. Supplement with 100 IU/mL of IL-2. d. Place co-culture back into tissue culture flask and incubate at 37 C and 5% CO 2. 19. Every 7 days, count NK cells in the culture and add freshly irradiated K562-mb-IL21 cells at a 2:1 ratio. See Troubleshooting 1 if low viability or low expansion is observed.
Note: Prior to proceeding with lentiviral transduction, ensure that the expanded NK cells are >75% CD3-CD56 superbirhgt (Troubleshooting 2). 1. Thaw low passage (P16 or less) cryopreserved HEK293T cells in a 37 C water bath. a. Add 10 mL of complete DMEM media to 15 mL falcon tube. b. Remove frozen HEK293T cryovial from liquid nitrogen tank. c. Place HEK293T cryovial in a 37 C water bath immediately after retrieving from liquid nitrogen tank. As soon as there is one ice crystal remaining, transfer cells drop wise into the 15 mL falcon tube. d. Centrifuge tube at 300 3 g for 5 min. e. Remove supernatant and count cells with Trypan Blue. 2. Resuspend 1.5-2 million HEK293T cells in 20 mL of complete DMEM media and transfer to a T-150 tissue culture flask. Culture flasks at 37 C and 5% CO 2 .

Culture SKBR3 breast cancer tumor cells
Note: Always start lentivirus prep with freshly thawed HEK293T cells to ensure higher viral yield. Around 2-3 80% confluent T-150 flasks are needed for one batch of lentiviral prep.
3. 24 h prior to transfection, harvest HEK293T cells by washing flasks with 10 mL warm PBS and adding 3 mL warm 13 Trypsin-EDTA(0.05%). Place flasks in the incubator for 2-5 min and neutralize trypsin with 33 the volume of warm complete DMEM media.
Note: Cells should be $80% confluent three days post-thaw. HEK293T cells are very loosely adherent thus do not require a long incubation time with Trypsin. Use Trypsin to harvest cells to prevent cell clumping.
4. Transfer cell suspension to a 50 mL falcon tube and centrifuge at 300 3 g for 5 min. Resuspend cell pellet with warm complete DMEM media and count cells using Trypan Blue.
Note: Use antibiotic-free complete DMEM media to plate cells prior to transfection as it may interfere with Lipofectamine 2000 reagent.
5. Plate 9 million HEK293T cells in 15 mL of antibiotic-free complete DMEM media per 15 cm NUNC tissue-culture treated plate. Incubate plates at 37 C and 5% CO 2 . a. Re-seed a T-150 flask with 1.5-2 million cells in 20 mL of complete DMEM media for lentiviral titration.
Note: Typically, three 15 cm plates are required to obtain sufficient viral particles per prep, scale up as needed. Carefully place plates into incubator ensuring that plates are even to make sure media covers each plate evenly.
Note: Measure concentration of all plasmids used prior to transfection using a spectrophotometer that measures the concentration and purity of nucleic acids.

7.
For each plate, prepare two 15 mL falcon tubes with 4 mL of Opti-MEM media labeled Tube A and Tube B. 8.
Prepare transfection mixture by following schematic in Figure 2. 9.
Slowly add the DNA and Lipofectamine mixture to each plate drop wise making sure to cover the entire plate area. Swirl plate carefully and slowly to evenly distribute the mixture. 10. Incubate plates at 37 C and 5% CO 2 .
Alternatives: Lipofecatamine (lipid based transfection agent) can be substituted with chemical methods such as calcium phosphate for the transfection. However, the amount of total ll OPEN ACCESS DNA added should be optimized using this method. Refer to Brown et al. (2020) and Follenzi and Naldini (2002) for protocols on LV production using calcium phosphate.
11. 12-16 h after transfection, carefully aspirate media and replace with 15 mL warm antibioticcontaining complete DMEM media supplemented with 1 mM of sodium butyrate.
Note: Sodium butyrate is added as it has been shown to enhance HIV-1 derived vector production (Olsen and Sechelski, 1995).
CRITICAL: During this step, there may be lift off the HEK293T cells from the plate which can decrease viral yield. To avoid this ensure that DMEM media is warmed up to 37 C and aspirate media as slowly as possible. Only work with up to three plates at a time.
Note: From this point forward, you will be working with virus samples. Ensure you are following proper BSL-2 guidelines when handling lentivirus. 13. 36-48 h after transfection, lentivirus released in the supernatant can be harvested for viral concentration.
CRITICAL: In our experience, the use of ultracentrifugation to concentrate the virus cannot be replaced with other methods of viral concentration such ultrafiltration. We have found that viral preps obtained from ultrafiltration do not efficiently transduce NK cells.
Note: Pre-cool the SW-32 Ti ultracentrifuge rotor and buckets at 4 C before starting the harvest.
14. Aspirate viral containing supernatant from the 15 cm plates and transfer to 50 mL falcon tubes. 15. Centrifuge the 50 mL falcon tubes at 2000 3 g for 10 min at 4 C to sediment any collected cells. 16. Filter supernatant using a 150 mL 0.45 mM PES filter to remove cell debris.
Alternatives: A filter with a pore size of 0.22 mM can also be used to improve purity. However, a size of 0.45 mM is used in this protocol to avoid any loss of viral particles during the filtration.
17. Add filtrate into 38.5 mL ultracentrifuge tubes and use cold PBS to bring tube within 0.5 cm of the edge to prevent centrifuge tube from collapsing. 18. Carefully balance the centrifuge tubes before placing into the ultracentrifuge buckets.
CRITICAL: Ultracentrifuge buckets containing tubes must be balanced within 0.02g.
19. Centrifuge buckets at 130,000 3 g for 1 h 40 min at 4 C with minimal deceleration. 20. Carefully remove ultracentrifuge tube from ultracentrifuge bucket and pour the supernatant into a 20% bleach bucket. 21. Keep the ultracentrifuge tube inverted and blot the excess supernatant onto paper towels for up to 1 min to prevent viral pellet from drying out. 22. Immediately place the ultracentrifuge tube on ice and let it sit for 10 min to begin viral resuspension.
CRITICAL: Avoid the formation of bubbles during resuspension of the viral pellet. Keep virus on ice at all times until placing in the À80 C freezer.
Note: From this point forward only use micropipette filter tips when handling the virus.
23. Add between 50-100 mL of cold PBS and start to slowly scrape the bottom of the ultracentrifuge tube using a micropipette filter tip.
Note: Set the micropipette to a low volume ($40 mL) and do not go to the second stop while resuspending to avoid formation of bubbles.
Note: The volume of PBS added to resuspend the viral pellet can be adjusted depending on your experience with the particular LV. For example, you can add a higher volume of PBS to lentivirus preps that have higher yields. If you consistently obtain a low viral yield see Troubleshooting 3.
24. Using the same micropipette filter tip, slowing resuspend the pellet by gently pipetting up and down and wash the sides of the ultracentrifuge tube. 25. Once viral pellet is fully resuspended aliquot virus into 1.5 mL microcentrifuge tubes and immediately place into À80 C freezer. a. Prepare one aliquot of 13 mL to use for the lentiviral titration. 26. Decontaminate anything that has come into contact with the virus using 70% ethanol.

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Note: Minimize the production of aerosols when working in BSL2 conditions.

Timing: 4 days; variable flow time
The lentivirus stock should be titrated using HEK293T cells prior to NK cell transduction. To determine the titer, HEK293T cells are first transduced with serial dilutions of the lentivirus stock and they are then stained to measure the expression of the transduction marker by flow cytometry. The titer is calculated using the following formula: Titer (TU/mL)= ((# of HEK293T cells transduced 3 % Transgene-positive cells 3 Dilution factor)/100). The expected titer for the lentivirus containing the HER2 CAR construct used in this study is between 3 3 10 7 -2 3 10 8 TU/mL. CRITICAL: The lentiviral constructs used in this study contain the NGFR transduction marker. Lentivirus with a titer >3 3 10 7 TU/mL is necessary for NK cell transduction (Troubleshooting 4).

Harvest HEK293T cells from a T-150 flask and count using Trypan Blue.
Note: Perform two separate counts and take the average to ensure the cell count is accurate.
CRITICAL: The titration is dependent on accurately plating 30,000 cells per well.
28. Plate 30,000 HEK293T cells per well in a 24-well tissue culture plate.
a. Resuspend cells to 6 3 10 4 cells/mL using complete DMEM media. b. Up to 6-wells will be needed for each lentivirus batch, using dilutions 10 À2 to 10 À6 and one control well with no virus. c. Resuspend the cell suspension 3 times and add 500 mL per well. 29. Incubate plate at 37 C and 5% CO 2 for at least 3 h to ensure HEK293T cells have adhered. 30. Prepare lentivirus serial dilutions following Figure 3. a. Prepare dilutions using 1.5 mL microcentrifuge tubes in complete DMEM media. b. Mix each dilution thoroughly by pipetting or vortexing. 31. Thoroughly mix and add 500 mL of each dilution per well. 32. Add 500 mL of complete DMEM media to the no virus control well. 33. Incubate plate at 37 C and 5% CO 2 for 72 h.
Alternatives: We find that assessing transgene expression after 72 h post HEK293T cell transduction is adequate to obtain a determination of LV titer. However, if a highly accurate titer is needed waiting 5 days post transduction could minimize the proportion of HEK293T cells that express the transgene but has not been successfully integrated. Pause point: Samples may be stored at 4 C for up to 5 days after fixation before sample acquisition by flow cytometry. However, we recommend acquiring samples within 24-48 h. 52. Decontaminate anything that has come into contact with the virus using 70% ethanol.
Note: Minimize the production of aerosols when working in BSL2 conditions.

Timing: 3 days
This step describes the process of transducing expanded NK cells with 3 rd generation LVs. Prior to NK cell transduction, determine the number of NK cells you need to transduce to perform downstream functional assays. In addition, you need to determine the amount of lentivirus needed to transduce desired number of wells at the desired MOI. Typically an MOI of 5 results in sufficient transduction efficiency. This is based on the LV titers typically obtained with our construct after titration on HEK293T cells. However, the MOIs used should be optimized depending on the size of your CAR lentiviral construct.
Note: Primary NK cells should be expanded by co-cultured with irradiated K562 mb-IL21 feeder cells at a 2:1 ratio for at least 2 weeks before transduction. 53. A day before transduction, replenish expanded NK cells with irradiated K562 mb-IL21 cells at a 1:1 ratio.

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a. Seven days after the last replenishment, count NK cells with Trypan Blue. b. Resuspend the co-culture to a final concentration of 0.5 3 10 6 cells/mL in complete RPMI media supplemented with 100 IU/mL of IL-2. 54. Plate 200 mL of the co-culture into a 96-well U bottom plate for a total of 1 3 10 5 cells per well. 55. Incubate plate for 12-18 h at 37 C and 5% CO 2 . 56. 18-24 h after plating, centrifuge 96-well U bottom plate at 300 3 g for 10 min.
a. During the centrifugation, prepare the NK cell transduction media. 57. Carefully remove the supernatant from each well, and resuspend cells with 90 mL of NK cell transduction media using a multichannel pipette.
CRITICAL: Prepare fresh NK cell transduction media each time.
58. Place plate in the incubator (37 C and 5% CO 2 ) while you prepare the viral suspension.
CRITICAL: Pre-warm the centrifuge to 32 C prior to thawing your virus so that you can perform the spinfection process as soon as possible.
59. Prepare viral suspension by diluting the stock with the appropriate amount of cold PBS to add 10 mL of viral suspension per well at an MOI of 5.
Note: Thaw your lentivirus containing the CAR construct and the control vector on ice. If the lentivirus titre is <5 3 10 7 TU/mL do not dilute in PBS.
Example: To calculate the amount of lentivirus needed to transduced 5 wells at an MOI of 5 use the following calculation (use virus titer obtained from the HEK293T titration): ((1 3 10 5 3 MOI 3 # of wells)/virus titer(TU/mL)) 3 1000 = Total amount of virus needed (mL) 60. Once the virus suspension is prepared, obtain 96-well plate from the incubator and add 10 mL of virus suspension per well. 61. Add 10 mL of PBS to the non-transduced control wells. 62. Immediately centrifuge the plate at 1000 3 g for 45 min at 32 C as the spinoculation step.
Note: In our experience, we find that while spinoculation is not necessary for lentiviral transduction of human T cells, it does enhance the transduction efficiency of expanded NK cells. However, this step may be skipped depending on the size and titer of your lentiviral construct but should be optimized. 63. Gently resuspend the wells using a micropipette and incubate plate for 12-18 h at 37 C and 5% CO 2. 64. The next day, centrifuge the plate at 300 3 g for 10 min. 65. Carefully remove the supernatant and resuspend cells to a final volume of 100 mL using complete RMPI media supplemented with 100 IU/mL of IL-2. 66. Three to four days after transduction, HER2 CAR-NK cells can be stained to assess HER2 CAR transgene expression.

Assessing HER2 CAR expression by flow cytometry
Timing: 3-4 h set up and staining; variable flow time This procedure describes how to assess expression of the HER2 CAR transgene after NK cell lentiviral transduction. The HER2 CAR transgene used in this study is under the EF1a promoter and the transduction marker is under the CMV promoter, the construct is described in Hammill et al. (2015).
67. Pool wells containing transduced HER2 CAR-and control NK cells (non-transduced and control vector transduced) into a 5 mL polystyrene tube. 68. Wash cells with 2 mL of complete RPMI media and centrifuge at 300 3 g for 5 min. 69. Carefully discard the supernatant and count cells with Trypan Blue. 70. Transfer 100,000 cells of the HER2 CAR-, control vector-and non-transduced NK cells to a 96-well U bottom plate for staining with the surface stain cocktail.
71. Prepare additional control wells using 100,000 cells per well. a. Plate one well with the HER2 CAR-NK cells HER2 Fc chimera protein control. b. Plate four wells with the non-transduced NK cells for the unstained, viability dye compensation and fluorescence minus one (FMO) control wells.
Note: FMOs for the IgG Fc PE and NGFR APC can be included but the transgene positive gates should be set based on the fully stained non transduced NK cell control sample.
72. Resuspend the remainder of the cells to a final concentration of 1 3 10 6 cells/mL in complete RMPI media supplemented with 100 IU/mL IL-2. a. Incubate at 37 C and 5% CO 2 until functional assays.
Note: Functional assays should be performed within 1-2 weeks after initial transduction as we have found transgene expression is downregulated over time and CAR-NK cells cannot be further expanded.
73. Spin the plate at 300 3 g for 5 min to pellet the cells. 74. Remove supernatant and wash cells with 200 mL of PBS. 75. Spin the plate 300 3 g for 5 min and discard PBS. a. Prepare a 1:1000 dilution of viability dye (Fixable Viability Stain 510) in PBS. 76. Add 100 mL of the viability dye and incubate at 4 C for 30 min in the dark.
a. Prepare the HER2 Fc chimera protein mix during the incubation by adding 2.5 mL of recombinant ErbB2/HER2 Fc chimeric protein to 25 mL of FACS buffer per test. 77. Spin the plate at 300 3 g for 5 min and discard supernatant. 78. Wash cells with 200 mL of FACS buffer, spin the plate at 300 3 g for 5 min and discard supernatant. 79. Add 25 mL of the HER2 Fc chimera protein dilution and incubate in the dark for 30 min at 20 C-22 C. a. Do not add HER2 Fc chimera protein to the extra HER2 CAR-NK cell control well. 80. Spin the plate at 300 3 g for 5 min and discard supernatant. 81. Wash cells with 200 mL of FACS buffer, spin the plate at 300 3 g for 5 min and discard supernatant. 82. Add 50 mL of the surface stain master mix and incubate 4 C for 30 min in the dark.
Alternatives: Anti-CD3 APC-H7 can be swapped out of this panel with Anti-CD3 PerCp-Cy5.5 and used at the same dilution, in that case you can use the fixable viability dye eBioscience Fixable Viability Dye eFluor 780 instead of Fixable Viability Stain 510.
83. Spin the plate at 300 3 g for 5 min and discard supernatant. 84. Wash cells with 200 mL of FACS buffer, spin the plate at 300 3 g for 5 min and discard supernatant.
CRITICAL: NK cells transduced with lentivirus must be fixed prior to sample acquisition by flow cytometry.
85. Add 100 mL of 2% PFA mixture and incubate at 4 C for 30 min in the dark. 86. Spin the plate at 300 3 g for 5 min and discard supernatant. 87. Wash cells with 200 mL of FACS buffer, spin the plate at 300 3 g for 5 min and discard supernatant.

Resuspend cells to 200 mL of FACS buffer.
Pause point: Samples may be stored at 4 C for up to 5 days after fixation before sample acquisition by flow cytometry. However, we recommend acquiring samples within 24-48 h.
Note: Prepare single color compensation controls using compensation beads prior to sample acquisition by flow cytometry.

Transfer cells to appropriate tubes for flow cytometry and perform sample acquisition.
Note: Ensure that HER2 CAR-NK cells have a minimum of 15% CAR transgene expression before functional assays (Troubleshooting 5).
Flow cytometry-based HER2 CAR-NK cell cytotoxicity assay This step describes how to prepare target cells for the flow cytometry-based cytotoxicity assay and how to plate the effector and target cells. We recommend using 100,000-200,000 target cells but this number can be reduced to 50,000 cells. We recommend to include at least three Effector:Target (E:T) ratios in the cytotoxicity assay to obtain a killing curve.
90. Label a 96-well plate layout with all of your conditions, follow Figure 4 as an example.
Note: Due to the high susceptibility of SKBR3 breast cancer cells to expanded NK cell killing we chose the 1:1, 0.5:1 and 0.25:1 E:T ratios to assess in vitro anti-tumor function. The following protocol uses 100,000 target cells per well.
91. Prepare effector NK cells for plating. a. Transfer cells to a 15 mL falcon tube and centrifuge at 300 3 g for 5 min. b. Carefully remove supernatant and count cells with Trypan Blue. c. Resuspend NK cells with complete RPMI media to a final concentration needed to add 100 mL of effector cells at the highest E:T ratio.
Example: If the highest E:T ratio is a 1:1, resuspend the effector NK cells to a final concentration of 1 3 10 6 cells/mL in order to add 100,000 NK cells in 100 mL.

Set effector NK cells aside while you prepare the target cells.
Optional: One day before the cytotoxicity assay, split SKBR3 target cells 1:2 to ensure high tumor cell viability on the day of the assay.

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Optional: Carefully collect $150 mL of the cell-free supernatants using a multichannel pipette and transfer to a new 96-well U or V bottom plate. Place plate in À20 C freezer for cytokine analysis.
114. Wash wells using 200 mL of PBS, spin plate at 300 3 g for 5 min and discard supernatant.
a. Prepare a 1:1000 dilution of viability dye using eBioscience Fixable Viability Dye eFluor 780 in PBS.
CRITICAL: CFSE has an excitation of 492 nm and emission of 517 nm, stain the cytotoxicity assay using a fixable viability dye which does not overlap. We use eBioscience Fixable Viability Dye eFluor 780 which has an excitation of 633 nm and emission of 780 nm. In our experience, this panel does not require compensation or FMO controls.
115. Add 100 mL of fixable viability dye and incubate at 4 C for 30 min in the dark. 116. Spin plate at 300 3 g for 5 min and discard supernatant. 117. Wash wells with 200 mL of FACS buffer, spin plate at 300 3 g for 5 min and discard supernatant. 118. Add 100 mL of 1% PFA mixture and incubate at 4 C for 1 h in the dark. 119. Spin plate at 300 3 g for 5 min and discard supernatant. 120. Wash wells with 200 mL of FACS buffer, spin plate at 300 3 g for 5 min and discard supernatant. 121. Resuspend plate with 200 mL of FACS buffer.
Pause point: Samples may be stored at 4 C for up to 5 days after fixation before sample acquisition by flow cytometry. However, we recommend to acquire samples within 24-48h.
122. Transfer samples to appropriate tubes for flow cytometry and perform sample acquisition.

EXPECTED OUTCOMES
The expected purity of the expanded CD3-CD56 superbright NK cells is demonstrated in Figure 5.
The expected titer for the lentivirus containing the HER2 CAR construct in this study is between 3 3 10 7 and 2 3 10 8 TU/mL, use Figure 6 as a guide to set the transgene-positive gates after the lentiviral titration.
The transduction efficiency of the CAR construct can vary between donors, generally we obtain at least 15% transduction efficiency, see Figure 7 for an example.

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To determine the percent-specific lysis of the control and HER2 CAR-expressing effector NK cells first determine the percentage of dead target cells using the gating strategy defined in Figure 8. Calculate percent-specific lysis using the following formula: % specific lysis = ((% tumor cell death -% basal tumor cell death)/(100 -% basal tumor cell death)) 3 100

LIMITATIONS
The following protocol can be used to generate human CAR-NK cells with transduction efficiencies ranging between 15 and 80%. However, a main limitation is that transgene expression is downregulated over time thus functional assays should be performed within one to 2 weeks of CAR-NK cell generation. Protocols that use retrovirus instead of lentivirus or include sequences that prevent promoter silencing in their CAR plasmid may yield CAR-NK cells with stable transgene expression (Colamartino et al., 2019;Liu et al., 2020). Additionally, other promoters such as SFFV may lead to greater transduction efficiencies however we only used the EF1alpha and CMV promoters in our studies.
Another limitation is that the transduction protocol has been optimized for use with human NK cells that have been expanded using genetically engineered K562 feeder cells. This protocol may require To determine the lentivirus titer use the formula: Titer (TU/mL)= ((30,000 3 % Transgene-positive cells X Dilution factor)/100). Set the transgene-positive gate on the sample that was stained but did not obtain any lentivirus and use the dilution factor which results in >10% transgene-positive cells. Samples were acquired using the LSRFortessa Flow Cytometer and analyzed with FlowJo Software.

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STAR Protocols 2, 100956, December 17, 2021 further optimization for the transduction of cytokine activated NK cells which are typically more difficult to transduce. However, we have also used the protocol to successfully generate CAR-NK cells from the NK-92 human NK cell line as described in Portillo et al. (2021).

TROUBLESHOOTING Problem 1
Low viability and slow expansion of NK cells from starting PBMCs (Before you begin steps 14-19).

Potential solution
Using fresh PBMCs as opposed to cryopreserved PBMCs to expand the NK cells usually yields a higher viability and expansion rate. In addition, changing the media and adding IL-2 every 2 days is crucial to maintain a good viability of the rapidly proliferating NK cells in culture. Do not use IL-2 aliquots that have been thawed and stored at 4 C for longer than 2-3 days.

Problem 2
A non-specific CD3+ T cell population has expanded in the expanded NK cell culture (Before you begin step 19).

Potential solution
Perform a CD3+ positive selection using commercial isolation kits to remove the contaminating CD3+ T cells and continue replenishing the culture. Additionally, CD3+ cells can also first be depleted from the starting PBMCs using an immunomagnetic CD3+ isolation kit. If the CD3+ T cell population continues to expand, use PBMCs from a different donor.

Potential solution
The transfer vector containing your desired transgene might be too large to efficiently generate 3 rd generation lentivirus using this protocol. We find that lentiviral titer and LV production efficiency decreases as the size of the transgene packaged in the LV increases. In this case, the 2 nd generation lentivirus packaging system could be used since all the packaging genes are encoded within only one vector as opposed to two like in the 3 rd generation system. Using the 2 nd generation system has shown to increased viral yields 50-fold (Cribbs et al., 2013). However, 3 rd generation LVs are preferred for clinical applications due to the added biosafety aspect.

Potential solution
If the LV titer is lower than 1 3 10 7 TU/mL after ultracentrifugation there was an issue during LV production. Ensure that HEK293T cells have a viability between 90 and 95% at every plating step. We recommend that HEK293T cells are no more than 80% confluent during the transfection step to prevent cells lifting off the plate during the media change the next day as this can highly impact viral titer. It is also critical that your starting plasmids have high purity for successful HEK293T transfection. Using commercially available plasmid isolation kits such as the PureLinkä HiPure Plasmid Maxiprep Kit (Thermo Fisher) is recommended to obtain high purity. You may also need to optimize the amount of plasmids used for the transfection.

Problem 5
A low transduction efficiency (<15%) is obtained after NK cell transduction (Transduction efficiency will be determined after step 89).

Potential solution
In our experience we find that lentivirus preps with a titer >3 3 10 7 TU/mL are necessary to yield CAR-NK cells with high transgene expression. In addition, we have found that using expanded NK cell cultures that have not been expanded beyond 8 weeks typically yield higher transduction efficiencies. The MOIs needed to transduce NK cells may also need to be optimized. The CAR lentiviral construct used in this study is $10 Kb, and we find MOIs 3-8 are sufficient to yield >15% transduction efficiencies. However, higher MOIs may be needed for larger lentiviral constructs. We also find that transgene efficiency can also be donor dependent.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Ali A. Ashkar (ashkara@mcmaster.ca).

Materials availability
The study did not generate new unique reagents.

Data and code availability
No data or code was generated or analyzed in this protocol.