Funding and support for the project were provided by Wilhelm Sander Stiftung, project 2020.017.1 to M.H., and J.L.; Deutsche Forschungsgemeinschaft (Collaborative Research Center/Transregio 124 Pathogenic fungi and their human host: Networks of interaction-FungiNet; project A8 to M.H. and H.E.).

All experiments described here must be conducted under sterile conditions. In this protocol, we show two conditions: (I) Mock transfection without DNA that will serve as control and (II) CAR transfection using the previously described Af-CAR6. The Af-CAR plasmid contains a truncated epidermal growth factor receptor (EGFRt) sequence downstream of the CAR, which will be used as a transfection marker (Supplemental Figure S1). For each condition, 4 &#215; 106 NK-92 cells are required.
1. Culturing the NK-92 cells
<ol>
	<li>One or two days before the transfection, prepare fresh cell culture medium with the following composition: 75% (v/v) alpha-MEM Medium (with nucleosides) + 12.5% (v/v) heat-inactivated fetal bovine serum (FBS) + 12.5% (v/v) heat-inactivated horse serum + 2 mM L-glutamine.</li>
	<li>Re-suspend at least 107 NK-92 cells at a cell density of 5 &#215; 105 cells/mL.</li>
	<li>Add IL-2 to the cell suspension at a concentration of 150 UI/mL.</li>
	<li>Culture the cells in a humidified incubator at 37 &#176;C with 5% CO2. 
	NOTE: IL-2 can be used at a final concentration of 5 ng/mL.</li>
</ol>
2. Non-viral transfection
<ol>
	<li>Check the quality of the cells under the microscope. A suspension of cell clusters and a clear background indicate a healthy cell culture.</li>
	<li>In a 6-well plate, add 2 mL of cell culture medium per well for each condition. Place the plate in a humidified incubator at 37 &#176;C with 5% CO2. 
	NOTE: Antibiotics can reduce cell viability after transfection. Keep the medium antibiotic-free.</li>
	<li>Count the cells and check the cell viability using (e.g., Trypan Blue staining).</li>
	<li>Transfer 8 &#215; 106 NK-92 cells to a 15 mL conical bottom tube. Centrifuge at 200 &#215; g for 5 min with an acceleration and deceleration of 3. Use this program for all the following centrifugation steps.</li>
	<li>During the centrifugation, prepare two 1.5 mL reaction tubes. Pipette the DNA for Af-CAR (8 &#181;g of Plasmid-DNA and 4 &#181;g of SB100X Mini Circle, Table 1) into one of the tubes. Keep the second one DNA-free to be a Mock control.</li>
	<li>When the centrifuge is ready, gently discard the supernatant and fill the tube containing the cells with up to 15 mL of prewarmed Phosphate Buffered Saline (PBS).</li>
	<li>Centrifuge (step 2.4).</li>
	<li>During the centrifugation, prepare one transfection system tube for each condition by adding 3 mL of electrolytic buffer in each tube. Put the first tube in the pipette station.</li>
	<li>When the centrifuge is ready, gently discard the supernatant and resuspend the cell pellet in 200 &#181;L of resuspension buffer (100 &#181;L/4 &#215; 106 cells). 
	NOTE: Avoid leaving the cells in resuspension buffer for more than 15-30 min, as this lowers cell viability and transfection efficiency.</li>
	<li>Add 100 &#181;L of the cell suspension into each of the two reaction tubes. Mix gently. 
	NOTE: Avoid air bubbles during pipetting as air bubbles cause arcing, which leads to cell death.</li>
	<li>Pipette the DNA-cell mixture from the first reaction tube with the transfection system pipette and insert the pipette vertically into the tube placed in the pipette station. 
	NOTE: Ensure that the tip fits properly.</li>
	<li>Set the first pulse at 1,650 V and pulse time for 20 ms and press Start.</li>
	<li>After completion, set the second pulse at 500 V and pulse time for 100 ms. Press Start.</li>
	<li>After completion, slowly remove the pipette from the pipette station and immediately transfer the cells into one well of the prepared culture plate containing 2 mL of pre-warmed cell culture medium (see step 2.2). Gently move the plate circlewise to ensure that the cells are evenly distributed.</li>
	<li>For the Mock sample, take 100 &#181;L of the cell suspension from the DNA-free reaction tube (see step 2.9) and repeat the same procedure from step 2.11 to step 2.14, using a new tube and a new pipette tip.</li>
	<li>Incubate the plate in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>After 30 min of incubation, add IL-2 at a final concentration of 150 UI/mL.</li>
</ol>
3. Checking the transfection efficiency using flow cytometry
<ol>
	<li>After 1 week of cultivating and splitting the cells every second day, take 1-5 &#215; 105 cells of each condition into a polystyrene round-bottom tube.</li>
	<li>Wash the cells by adding 1 mL of buffer (PBS + 0.5% (v/v) FBS + 2 mM EDTA); centrifuge and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 50 &#181;L of buffer. Add 1 &#181;L of 7-AAD to detect the dead cells.</li>
	<li>Add 5 &#181;L of anti-tEGFR-Alexa fluor 647 antibody to stain the transfected cells.</li>
	<li>Incubate for 20 min at 4 &#176;C.</li>
	<li>Wash the cells (see step 3.2).</li>
	<li>Resuspend the cell pellet in 200 &#181;L of buffer.</li>
	<li>Measure the samples on a flow cytometer. 
	NOTE: If the transfection efficiency is not satisfying, proceed with the enrichment of transgene-expressing cells.</li>
</ol>
4. Enrichment of transgene-expressing cells by magnetic-activated cell sorting (MACS)
NOTE: For optimal results, start the protocol with 8-10 &#215; 106 cells. All centrifugation steps are conducted at 4 &#176;C.
<ol>
	<li>Prepare buffer as described in step 3.2. Dilute the cells at a concentration of 1-2 &#215; 106/100 &#181;L with the buffer in a 15 mL conical bottom tube.</li>
	<li>Add 1 &#181;L of anti-EGFRt-biotin per 1-2 &#215; 106 cells, and gently swirl the tube to ensure an evenly distributed solution.</li>
	<li>Incubate for 25 min at 4 &#176;C, then fill the tube with up to 10 mL of buffer.</li>
	<li>Centrifuge. Discard the supernatant.</li>
	<li>Add 8 &#181;L/1 &#215; 106 cells of cold buffer.</li>
	<li>Add 2 &#181;L of anti-biotin magnetic beads per 1 &#215; 106 cells.</li>
	<li>Incubate for 15 min at 4 &#176;C.</li>
	<li>Wash by filling up to 10 mL with buffer.</li>
	<li>Centrifuge. Discard the supernatant and resuspend the cells in 500 &#181;L of buffer.</li>
	<li>Place an LS column in the MACS magnet and wash with 3 mL of buffer.</li>
	<li>As soon as the buffer has completely migrated through the column, add the cell suspension.</li>
	<li>Wash the column 3x, each with 3 mL of buffer.</li>
	<li>Remove the column from the magnet and place it in a clean 15 mL conical bottom tube.</li>
	<li>Add 5 mL of buffer to the column. Using the plunger, flush out the EGFR-positive cells as quickly as possible.</li>
	<li>Culture the cells for a few days; then, check the efficiency by flow cytometry.</li>
</ol>
5. Functional testing
NOTE: For the upcoming experiment, prepare and use the following medium: RPMI + 20% FBS.
<ol>
	<li>Day 1 - Seeding Aspergillus fumigatus conidia
	<ol>
		<li>Prepare an A. fumigatus conidia suspension at a concentration of 2.5 &#215; 105 conidium/mL in the medium.</li>
		<li>In a 96-well culture plate, pipette 100 &#181;L of the conidia suspension per well and incubate at 25 &#176;C for 16 h. 
		NOTE: After the incubation period, the conidia will germinate (Figure 1).</li>
	</ol>
	</li>
	<li>Day 2 - Co-culture with CAR NK-92 cells
	<ol>
		<li>Wash the Mock and Af-CAR NK-92 cells 2x with the medium.</li>
		<li>Co-culture with the fungus at an effector-to-target ratio of 2:1 by adding 5 &#215; 104 cells in 100 &#181;L of medium per well (Figure 2).</li>
		<li>For positive control, add 1 ng of PMA and 100 ng of ionomycin per well in 100 &#181;L of medium. Use cells in medium only as a negative control.</li>
		<li>Incubate the plate at 37 &#176;C in a humidified CO2 incubator for at least 6 h.</li>
		<li>Centrifuge the plate at 300 &#215; g for 5 min.</li>
		<li>Harvest 100 &#181;L of each well; avoid touching the bottom. Transfer the supernatants to reaction tubes. 
		NOTE: Make sure that only the supernatant is pipetted and not the grown fungus. If in doubt, add another centrifugation step.</li>
		<li>Perform IFN-&#611; ELISA following the manufacturer&#39;s instructions or store the samples at -80 &#176;C until use.</li>
	</ol>
	</li>
</ol>

Sleeping Beauty Approach to Obtain CAR-NK Cells for Targeting Fungal Pathogens

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcome+and+prognostic+factors+associated+with+invasive+pulmonary+aspergillosis:+An+11-year+follow-up+report+from+Taiwan.">Clinical outcome and prognostic factors associated with invasive pulmonary aspergillosis: An 11-year follow-up report from Taiwan.</a> PLoS One. 12 (10), e0186422 (2017).</li><li>Meis, J. F., Chowdhary, A., Rhodes, J. L., Fisher, M. C., Verweij, P. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+T+cells+to+target+carbohydrate+to+treat+opportunistic+fungal+infection.">Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.</a> Proc Natl Acad Sci U S A. 111 (29), 10660-10665 (2014).</li><li>Kumaresan, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+lentiviral+vector-based+approach+to+generate+chimeric+antigen+receptor+T+cells+targeting+Aspergillus+fumigatus.">A novel lentiviral vector-based approach to generate chimeric antigen receptor T cells targeting Aspergillus fumigatus.</a> mBio. 15 (4), e0341323 (2024).</li><li>Lu, H., Zhao, X., Li, Z., Hu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+CAR-T+cells+to+CAR-NK+cells:+A+developing+immunotherapy+method+for+hematological+malignancies.">From CAR-T cells to CAR-NK cells: A developing immunotherapy method for hematological malignancies.</a> Front Oncol. 11, 720501 (2021).</li><li>Robertson, M. J., Ritz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+clinical+relevance+of+human+natural+killer+cells.">Biology and clinical relevance of human natural killer cells.</a> Blood. 76 (12), 2421-2438 (1990).</li><li>Ziegler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD56+is+a+pathogen+recognition+receptor+on+human+natural+killer+cells.">CD56 is a pathogen recognition receptor on human natural killer cells.</a> Sci Rep. 7 (1), 6138 (2017).</li><li>Esmaeilzadeh, A., Hadiloo, K., Jabbari, M., Elahi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+of+chimeric+antigen+receptor+(car)+t+versus+car+nk+cell+for+immunotherapy+of+solid+tumors.">Current progress of chimeric antigen receptor (car) t versus car nk cell for immunotherapy of solid tumors.</a> Life Sci. 337, 122381 (2024).</li><li>Zhang, J., Zheng, H., Diao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cells+and+current+applications+of+chimeric+antigen+receptor-modified+NK-92+cells+in+tumor+immunotherapy.">Natural killer cells and current applications of chimeric antigen receptor-modified NK-92 cells in tumor immunotherapy.</a> Int J Mol Sci. 20 (2), 317 (2019).</li><li>Hu, Y., Tian, Z. G., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+(CAR)-transduced+natural+killer+cells+in+tumor+immunotherapy.">Chimeric antigen receptor (CAR)-transduced natural killer cells in tumor immunotherapy.</a> Acta Pharmacol Sin. 39 (2), 167-176 (2018).</li><li>Hudecek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+non-viral:+The+Sleeping+Beauty+transposon+system+breaks+on+through+to+the+clinical+side.">Going non-viral: The Sleeping Beauty transposon system breaks on through to the clinical side.</a> Crit Rev Biochem Mol Biol. 52 (4), 355-380 (2017).</li><li>Amberger, M., Ivics, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latest+advances+for+the+Sleeping+Beauty+transposon+system:+23+years+of+insomnia+but+prettier+than+ever.">Latest advances for the Sleeping Beauty transposon system: 23 years of insomnia but prettier than ever.</a> BioEssays. 42 (11), 2000136 (2020).</li><li>Ingegnere, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+car+nk+cells:+A+new+non-viral+method+allowing+high+efficient+transfection+and+strong+tumor+cell+killing.">Human car nk cells: A new non-viral method allowing high efficient transfection and strong tumor cell killing.</a> Front Immunol. 10, 957 (2019).</li><li>Stacey, K. J., Ross, I. L., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+DNA-dependent+cell+death+in+murine+macrophages.">Electroporation and DNA-dependent cell death in murine macrophages.</a> Immunol Cell Biol. 71 (Pt 2), 75-85 (1993).</li><li>Monjezi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+CAR+T-cell+engineering+using+non-viral+Sleeping+Beauty+transposition+from+minicircle+vectors.">Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors.</a> Leukemia. 31 (1), 186-194 (2017).</li><li>Moscarelli, J., Zahavi, D., Maynard, R., Weiner, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next+generation+of+cellular+immunotherapy:+Chimeric+antigen+receptor-natural+killer+cells.">The next generation of cellular immunotherapy: Chimeric antigen receptor-natural killer cells.</a> Transplant Cell Ther. 28 (10), 650-656 (2022).</li></ol>

M.H.: Inventor on patent applications and granted patents related to CAR technology, licensed in part to industry. Co-founder and equity owner T-CURX GmbH, W&#252;rzburg. Speaker honoraria: BMS, Janssen, Kite/Gilead, Novartis. Research support: BMS. M. B. reports travel support from pharmaceutical company "Eli Lilly and company," which is unrelated to this study. The other authors do not have any conflicts of interest.

The provided protocol offers a simple and reliable method for creating CAR-NK cells that target fungal pathogens using the non-viral Sleeping Beauty transposon system. This method allows for the stable and permanent insertion of large transgenes, which enables the expansion of cells to obtain the necessary quantities for further analysis14,16.
Several key factors can impact the success of the transfection, including cell number and via...

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus, with the average person inhaling between 100 and 1,000 conidia on a daily basis1. In patients with acquired or congenital immunodeficiencies, such as those undergoing chemotherapy or who are suffering from leukemia, A. fumigatus can result in severe invasive apsergillosis. Each year, more than 200,000 people worldwide contract aspergillosis. Despite the use of antimycotics for prevention and therapy with azole derivatives, the mortality rate remains between 30% and 95%2,3. Additionally, the treatment has significant side effects, and 7% of A. fumigatus cultures are already azole-resistant4.
Therapy with chimeric antigen receptor (CAR)-engineered T cells has shown clinical success in treating malignant diseases and achieved promising preclinical results in combating infectious diseases such as invasive aspergillosis5,6,7,8. Our previously described Af-CAR-T cells targeting a protein in the cell wall of A. fumigatus hyphae have shown promising results in preclinical models6.
The reduced risk of graft versus host disease, cytokine release syndrome, and neurotoxicity makes CAR-NK cells a valuable alternative to CAR-T cells as an &#34;off-the-shelf&#34; treatment for acute infections9. Additionally, NK cells have been shown to play a crucial role in the antifungal immune response. When stimulated with A. fumigatus, NK cells secrete cytokines such as IFN-&#611; and chemokines like CCL-3 and CCL-4 and release cytotoxic granules10,11.
The NK-92 cell line is a popular source of NK cells for genetic engineering, offering the advantage of rapid proliferation and expansion in relatively simple culture conditions12. Several preclinical studies have demonstrated the therapeutic potential of CAR-transduced NK-92 cells in treating both hematological and solid tumors. Numerous clinical trials are ongoing to investigate their safety, efficacy, and potential use as &#34;off-the-shelf&#34; cellular therapy13.
Achieving high transfection efficiencies and stable transgene expression in NK cells is challenging. While viral methods promise high efficiencies, they carry the risk of insertion mutations with subsequent oncogenesis9,14. The latest generation of Sleeping Beauty (SB) transposon vectors offers a safe, potent, and economically viable system for stable gene transfer, overcoming limitations of viral and transient non-viral gene delivery methods15.
Therefore, this protocol outlines a novel methodology for the generation of Af-CAR-NK-92 cells, which can potentially serve as an &#34;off-the-shelf&#34; therapeutic option for patients with life-threatening invasive aspergillosis. The efficacy of this approach is exemplified by the increased production of IFN-&#611; upon stimulation with A. fumigatus hyphae.

<table><tbody><tr><td>15 ML conical bottom tubes</td><td>Greiner Bio-One</td><td>188271</td><td>PP, 17/120 MM, CELLSTAR, STERILE</td></tr><tr><td>50 ML conical bottom tubes</td><td>Greiner Bio-One</td><td>227270</td><td>PP, 30/115 MM, CELLSTAR, STERILE</td></tr><tr><td>6-and 96-Well- Cell culture plate</td><td>Corning Inc. LifeSciences</td><td>83-3736/ 83-3474</td><td>TC-treated Multiple Well Plates, Flat bottoms, Treated for optimal cell attachment, Sterilized by gamma irradiation, Nonpyrogenic</td></tr><tr><td>7-AAD (7-Aminoactinomycin D)</td><td>BD Biosciences</td><td>559925</td><td>eady-to-use nucleic acid dye for the exclusion of nonviable cells in flow cytometric assays</td></tr><tr><td>Alexa Fluor 647 anti-human EGFR Antibody</td><td>Biolegend</td><td>352918</td><td>Clone AY13</td></tr><tr><td>Anti-Biotin MicroBeads UltraPure</td><td>Miltenyi Biotec</td><td>130-105-637</td><td>UltraPure MicroBeads conjugated to monoclonal mouse anti-biotin antibodies</td></tr><tr><td>Aspergillus fumigatus</td><td>ATCC</td><td>46645</td><td></td></tr><tr><td>Biosphere Filter Tips (20 and 100 &amp;mu;L)&amp;nbsp;</td><td>Sarstedt</td><td>70.3030.365 / 70.3030.275</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td>&amp;nbsp;Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture, endotoxin, tested</td></tr><tr><td>ELISA MAX Deluxe Set Human IFN-&amp;gamma;</td><td>Biolegend</td><td>430104</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt solution (EDTA)</td><td>Sigma-Aldrich</td><td>E7889</td><td>for molecular biology, 0.5&amp;nbsp;M in H&lt;sub&gt;2&lt;/sub&gt;O, DNase, RNase, NICKase and protease, none detected, 0.2 &amp;mu;m filtered.</td></tr><tr><td>FACS clean</td><td>BD Biosciences</td><td>340345</td><td></td></tr><tr><td>FACS Flow Sheath Fluid</td><td>BD Biosciences</td><td>342003</td><td></td></tr><tr><td>FACS Rinse Solution</td><td>BD Bioscience</td><td>340346</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td>sterile-filtered, suitable for cell culture, heat inactivated before use</td></tr><tr><td>Gibco, MEM &amp;alpha;, nucleosides</td><td>Thermofisher Scientific</td><td>22571020</td><td>Phenol Red, Ribonucleosides, Deoxyribonucleosides, Sodium Bicarbonate</td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050122</td><td>Sterile-filtered, heat inactivated before use</td></tr><tr><td>Human IL-2 IS, research grade</td><td>Miltenyi Biotec</td><td>130-097-742</td><td>Research grade. The ED50 is &amp;le;0.3 ng/mL, corresponding to an activity of &amp;ge;3 &amp;times; 10&lt;sup&gt;6&lt;/sup&gt; IU/mg.</td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I0634-1MG</td><td>from Streptomyces conglobatus, used as positive control for cytokine secretion&amp;nbsp;</td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td>W368401</td><td>For cell culture</td></tr><tr><td>MACS LS Column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>&amp;nbsp;Columns and plungers, sterile packed</td></tr><tr><td>NK-92</td><td>DSMZ</td><td>ACC 488</td><td></td></tr><tr><td>Neon Transfektionssystem 100&amp;nbsp;&amp;mu;L-Kit</td><td>Thermo Fisher Scientific</td><td>MPK10096</td><td>Resuspension Buffer R, Electrolytic Buffer E2, 100 &amp;mu;L Neon Tips, Neon Electroporation Tubes</td></tr><tr><td>Phorbol-12-myristat-13-acetat (PMA)</td><td>Sigma-Aldrich</td><td>P1585-1MG</td><td>used as positive control for cytokine secretion&amp;nbsp;</td></tr><tr><td>Polystyrene round-bottom tube, 5 mL</td><td>BD Bioscience</td><td>S-452400</td><td>used for flow cytometry</td></tr><tr><td>Reaction tube, 1.5 ML</td><td>Greiner Bio-One</td><td>7,26,90,001</td><td>PP, transparent, cap attached, with injected graduation. Sterilized before use.&amp;nbsp;</td></tr><tr><td>RPMI 1640 Medium, GlutaMAX</td><td>Thermo Fisher Scientific</td><td>72400021</td><td>GlutaMax I,&amp;nbsp; Phenol Red, HEPES, Sterile-filtered</td></tr><tr><td>TipOne 1000 &amp;mu;L XL Graduated Filter Tip</td><td>StarLab</td><td>S1122-1730</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD FACSCalibur</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator C60&amp;nbsp;</td><td>Labotect</td><td></td><td></td></tr><tr><td>Heraeus Multifuge 3SR</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>HydroFLEX microplate washer&amp;nbsp;</td><td>Tecan</td><td></td><td></td></tr><tr><td>NanoQuant Infinite M200 Pro</td><td>Tecan</td><td></td><td></td></tr><tr><td>Neon Transfection System</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Pipettes&amp;nbsp;</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Quadro MACS Separator&amp;nbsp;</td><td>Miltenyi Biotec</td><td></td><td></td></tr></tbody></table>

engineering chimeric antigen receptor-natural killer cells targeting fungal infections using the non-viral sleeping beauty transposon system

Chimeric antigen receptor (CAR)-based cell therapies have shown impressive efficacy in the treatment of hematological malignancies. Recently, these therapies are being developed for infectious diseases, yet studies targeting fungal infections remain scarce. To identify optimal targets and optimize cellular products, we developed a method to engineer chimeric antigen receptor-natural killer (CAR-NK) cells and evaluated their response to stimulation by fungi. This paper describes a straightforward and robust method for generating CAR-NK cells tailored to fungal targets using the non-viral Sleeping Beauty transposon system. NK-92 cells are transfected with the hyperactive transposase SB100X vectorized as minicircle DNA (MC) along with a plasmid-encoded CAR transposon. Transfection efficiency is assessed 1 week later using flow cytometric analysis. Prior to functional testing, the cells expressing the transgene are enriched using magnetic-activated cell sorting and cultured for 1 more week. To evaluate antigen-specific activation, the engineered cells are co-cultured with Aspergillus fumigatus germ tubes for at least 6 h. Subsequently, the concentration of the secreted interferon-gamma (IFN-&#947;) is measured using an enzyme-linked immunosorbent assay.

The entire process of generating Af-CAR NK-92 cells, including transfection, recovery, enrichment, and expansion, takes approximately 14 days. After transfection, the cells are cultured and split every second day. Cell viability may decrease during the first splitting two days post transfection. Typically, the cells recover 4 days post transfection and begin proliferating, with a doubling time of 48 h.
Following recovery, transfection efficiencies are measured and are expected to reach 10-20% ...

Engineering Chimeric Antigen Receptor-Natural Killer Cells Targeting Fungal Infections Using the Non-viral Sleeping Beauty Transposon System

We outline a transfection protocol for producing chimeric antigen receptor-natural killer (CAR-NK) cells targeting fungal pathogens using the non-viral Sleeping Beauty transposon system. To assess antigen-specific activation, we co-cultured the engineered cells with Aspergillus fumigatus germ tubes and measured IFN-&#947; secretion.

Chimeric antigen receptor (CAR)-based cell therapies have shown impressive efficacy in the treatment of hematological malignancies. Recently, these ...

engineering-chimeric-antigen-receptor-natural-killer-cells-targeting

Research

JoVE Journal

Immunology and Infection

702 Views.  Universitätsklinikum Würzburg. Ons onderzoek richt zich op het ontwikkelen van een kant-en-klare CAR cellulaire therapie voor invasieve schimmelinfecties. We streven ernaar om de beste doelen, CAR-ontwerpen en combinatie van immuuncellen te identificeren om optimale therapeutische resultaten te bereiken. Recente ontwikkelingen, waaronder ons eigen werk, zijn gericht op de ontwikkeling van CAR T-celtherapieën gericht op Aspergillus fumigatus.Deze gemanipuleerde cellen hebben veelbel...

Video: Engineering van chimere antigeenreceptor-natuurlijke killercellen die zich richten op schimmelinfecties met behulp van het niet-virale Sleeping Beauty Transposon-s

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

Cancer Research

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

Medicine

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Biology

A Technique for Gene Editing in Natural Killer Cells Using CRISPR Cas9

Source: Naeimi Kararoudi, M., et al. <a href="https://app.jove.com/v/58237">Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for Cas9 ribonucleoprotein-mediated genetic modification of primary natural killer (NK) cells. A Cas9 ribonucleoprotein, consisting of a Cas9 endonuclease bound to a guide RNA (gRNA) formed by base pairing a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), is introduced into primary natural killer cells via electroporation. The ribonucleoprotein targets and cleaves the host DNA at the target site, leading to gene knockout via modification of the target gene through cellular repair machinery.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Human NK Cell Purification and ...

Engineering Chimeric Antigen Receptor-Natural Killer Cells Targeting Fungal Infections Using the Non-viral Sleeping Beauty Transposon System

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