Reference Effector cell Effector cell (extended description) Medium for NK cell culture Target Vehicle Method/Condition Cancer type Source of scFv Backbone Promotor Leader sequence scFV Order Linker hinge spacer Trans membrane Activation signal CAR generation Result: CAR positive (%) + detection tool Detection time point Clinial or   pre-clinical Country of First author ClinicalTrials.gov number DOI
Imai (Blood 2005) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL AIM-V or RPMI 1640, 10% FCS + 10  IU/mL IL-2 CD19 RV (RD114) plate-coated 100μg/mL fibronectin or 50 μg/mL Retronectin, 4μg/mL polybrene, MOI 4-6 ALL PCR from human cDNA library  pMSCV-IRES-GFP MSCV LTR CD8a VL-VH — CD8a CD8a DAP10 / CD137-CD3ζ / CD3ζ 1 and 2 69%（43%~93%）goat anti mouse Fab WB 7 to 11 days after transduction Pre-clinical  USA https://doi.org/10.1182/blood-2004-12-4797
Altvater (Clin Cancer Res 2009) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL RPMI 1640 and AIM V + rhIL-2 (40 IU/mL), rhIL-12(10 ng/mL), and rhIL-18 (50 ng/mL) CD19, GD2 RV 4 μg/cm2 Retronectin B-ALL, neuroblastoma  — SFG-IRES-GFP SFG — — — — 2B4/CD3 CD3ζ / 2B4 / 2B4-CD3ζ 1 and 2 16.7 ± 7.6% for CD19-ζ, 16.2 ± 8.8% for CD19-2B4, 12.9 ± 7.1% for CD19-truncated 2B4, 24.0 ± 7.4% for CD19-2B4ζ, and 18.0 ± 8.4% for CD19-t2B4ζ.  GFP or Goat anti mouse IgG Fab — Pre-clinical  Germany https://doi.org/10.1158/1078-0432.ccr-08-2810
Li (Cancer Gene Therapy 2010) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL AIM-V or RPMI 1640, 10% FCS+ 10  IU/mL IL-2 CD19 mRNA  MaxCyte GT system 100 mg/ml mRNA with 1~3X108 cells/mL  B-CLL mouse scFv pVAX1 — CD8a VL-VH — CD8a CD8a CD137-CD3ζ 2 Expanded NK >60%; Unstimulated NK > 52% Based on Fab FACS (no difference on viability and CAR expression); Viability NK >50% 1 day after Electroporation, 20-30% decrease on Day2 Pre-clinical  USA https://doi.org/10.1038/cgt.2009.61
Shimasaki (Cytotherapy 2012) PB-NK primary PB-NK cell K562-mIL-15/4-1BBL  SCGM or RPMI 1640, 10% FCS + 100  IU/mL IL-2 CD19 mRNA  MaxCyte GT System, 150–200 μg/mL  mRNA B-cell malignancies  mouse scFv mRNA — CD8a VL-VH — CD8a CD8a CD137-CD3ζ 2 40.3% in freshly purified ( n=18) and 61.3% in expanded ( n=31) NK cells on anti mouse Fab fragment 24 hours after electroporation  Pre-clinical  Singapore https://doi.org/10.1016/j.jcyt.2016.06.013
Boissel (Leukemia Lymphoma 2012) PB-NK / CB-NK PB-NK freshly isolated / CB-NK cell MACS enriched freshly isolated RPMI 1640  + 1000 U/mL I IL-2 GFP, CD19, CD20  mRNA / LV mRNA: BioRad GenePμLser II, 10ug, 2 X 106 cells, 250μL MEM medium. 300 mV, 150μF, 200W
LV: 8/15 μg/mL polybrene, MOI 150		 B-CLL mouse scFv mRNA: pXT7
LV: pCL20c-IRES-GFP		 MSCV hIg heavy-chain VH-VL GS-linker CD8a  CD3ζ  CD3ζ 1 mRNA: <10%, Low viability
LV: 8-68% CAR+		 24 h after electroporation or 48-72 h after transduction Pre-clinical  USA https://doi.org/10.3109/10428194.2011.634048
De Oliveira (Hum Gene Ther 2013) HSC HSC differentiated NK cells alpha-MEM  CD19 LV (2nd gen) MOI 100 B-cell malignancies  mouse scFv  CCLc-MNDU3-EGFP MND — VL-VH — Human IgG4 CD4/CD28 CD3ζ / CD28-CD3ζ 1 and 2 >20% CAR, goat antihuman IgG1
Fc, qPCR copy number		 — Pre-clinical  USA https://doi.org/10.1089/hum.2012.202
Chu (Cancer Immunol Res 2015) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL RPMI 1640, 10% FCS + 40  IU/mL IL-2 CD20 mRNA  Lonza Amaxa Nucleofector II Device with U-001 program B-cell malignancies  PCR from human cDNA library  pcDNA3 mRNA — VL-VH — CD8a CD8a CD137-CD3ζ 2 >66.7% in K562 expanded NK cells (16 hours) anti Mouse IgG 16 hours after electroporation  Pre-clinical  USA https://doi.org/10.1158/2326-6066.cir-14-0114
Suerth (J Mol Med 2016) PB-NK + NKL NKL NK cell line, Primary PB-NK cell,  1000U/mL IL-2  SCGM  + 5% Human AB serum CD19 RV (alpha / gamma) and LV (VSV-G, 3rd gen.) 4 μg/mL protamine sulfate + Retronectin coated, MOI 1- 30  pre-B ALL codon optimized/ humanized scFv pMPSV-IRES-GFP MPSV GMCSF-R alpha chain — — — CD28 CD28-CD137-CD3ζ 3 >60% EGFP 6 days after transduction Pre-clinical  Germany https://doi.org/10.1007/s00109-015-1327-6
Oelsner (Int J Cancer 2016) CIK primary CIK cells RPMI 1640  + 1000 U/mL IFN-g + 500 U/mL IL-2 + 100ng/mL anti-CD3 antibody CD19 LV (VSV-G, 2nd gen) 8 μg/mL polybrene pre-B ALL  codon-optimized pHR’SIN-cPPT-SIEW IRES-EGFP SFFV hIg heavy-chain — (G4S)x3 modified CD8a CD28 CD28-CD3ζ / CD3ζ 1 and 2 49.8% ± 24.3% EGFP    82.2% ± 3.2% CAR+ Cmyc Tag  7 Days after transduction  Pre-clinical  Germany https://doi.org/10.1002/ijc.30217
Chu (OncoImmunol 2017) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL RPMI 1640, 10% FCS + 40  IU/mL IL-2 CD20 mRNA  Lonza Amaxa Nucleofector II Device with U-001 program Burkitt lymphoma PCR from human cDNA library  pcDNA3 mRNA — VL-VH — CD8a CD8a CD137-CD3ζ 2 >95% in K562 expanded NK cells, anti Mouse IgG 24 hours after electroporation  Pre-clinical  USA https://doi.org/10.1080/2162402X.2017.1341031
Kailayangiri (OncoImmunol 2017) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL RPMI 1640 or AIM V + rhIL-2 (40 IU/mL), rhIL-12 (10 ng/mL), and rhIL-18 (50 ng/mL) GD2 RV 4 μg/cm2 Retronectin Ewing sarcoma  — SFG-IRES-GFP SFG — — — hCH2CH3 CD28 CD137-CD3ζ / t2B4-CD3ζ / CD137-t2B4-CD3ζ / t2B4-CD137-CD3ζ 2 and 3  >20% of anti human IgG Fc NK cells  — Pre-clinical  Germany https://doi.org/10.1080/2162402X.2016.1250050
Kloess (Hum Gene Ther 2017) PB-NK Primary NK cells expanded with autologous feeder cells X-VIVO 10 or CellGro
TexMACS , and NK MACS ) NK MACS +5% human AB serum+2% Supplement		 CD123 RV (alpha) 4 μg/mL protamine sulfate + Retronectin coated, MOI 1  AML scFv condon optimized  pMPSV-IRES-GFP MPSV GMCSF-R alpha chain — — — CD28 CD28-CD137-CD3ζ 3  Day3 CD123CAR His+
levels of 22.9% (range 17.8–25.7%) ; but median
expression decreased to 11.5% (range 5.9–18.8%)
on day 9 post transduction cMyc+ 		 3 days after transduction Pre-clinical  Germany https://doi.org/10.1089/hum.2017.157
Liu (Leukemia 2018) CB-NK CB-NK cells expaned K562-mb15-41BBL SCGM + 200  IU/mL IL-2 CD19 RV  RetroNectin pre-coated B-cell malignancies  humanized scFv SFG SFG — — — — CD28 CD28-CD3ζ 2 66.6% % (47.8%-87.4%), goat anti-human IgG (H+L) antibody 10 days after transduction Pre-clinical USA https://doi.org/10.1038/leu.2017.226
Oei (Cancer Immunol Res 2018) PB-NK PB-NK  SCGM +10% FCS +20 ng/mL IL-15 CD19 mRNA  BTX 830 Square Wave electroporator, 25ug mRNA in 400μL Serum free- SCGM 4 mm cuvette 500V 2ms B-cell malignancies  — pCIpA102 — — VH-VL — IgG1-CH2-CH3/ CD8a CD8a CD28-OX40-CD3ζ / OX40-CD3ζ / CD28-CD3ζ / CD137-CD3ζ 2 and 3 >80% on Day1, undetectable on Day 4 after electroporation, anti-CH2-CH3 FC  Day1, Day2, Day3 and Day4 after mRNA electroporation Pre-clinical  USA https://doi.org/10.1158/2326-6066.CIR-17-0207
Yu (Mol Ther 2018) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL — GPC3 LV (VSV-G, 2nd gen) 8 mg/mL polybrene + 6 mM BX795 Hepatocellular Carcinoma humanized scFv pRRLSIN-EGFP EF1α CD8a — — CD8a CD28 CD28-CD3ζ 2 >47%, anti Human Fab FACS — Pre-clinical  China https://doi.org/10.1016/j.ymthe.2017.12.012
Li (Cell Stem Cell 2018) iPSC / NK-92 NK-92/ iPSC-NK 56.6 % DMEM-high glucose, 28.3 % HAMS/F12 + 15 % heat-inactivated human AB serum Mesothelin piggyBac transposon Lonza 4D-nucleofector device Ovarium cancer Mouse IgG  pKT2-mCAG-IRES-GFP mCAG CD8a — — CD8a NKG2D / CD28 / CD16 / NKp44 / NKp46 2B4-CD3ζ / CD28-CD137-CD3ζ / DAP10-CD3ζ / CD137-CD3ζ / 2B4-DAP12-CD3ζ / 2B4-DAP10-CD3ζ / CD137-2B4-CD3ζ / CD3ζ 1, 2 and 3 100% GFP and mouse IgG Fab, FACS sorting — Pre-clinical  USA https://doi.org/10.1016/j.stem.2018.06.002
Kloess (Hum Gene Ther 2019) PB-NK / NK-92 Primary NK cells and NK-92 cell line RPMI1640 for NK-92; NK MACS + 5% human AB serum + 2% Supplement for primary NK CD123 RV (alpha) 4 μg/mL protamine sulfate + Retronectin coated, MOI 1- 3  AML scFv condon optimized  pMPSV-IRES-GFP MPSV GMCSF-R alpha chain — — — CD28 CD28-CD137-CD3ζ 3 before sorting 25%; After GFP sorting anti-HA GFP+ 61.7 % anti His 7 days after transduction Pre-clinical  Germany https://doi.org/10.1089/hum.2018.247
Oberschmidt (Hum Gene Ther Meth 2019) PB-NK Primary NK cell isolated byCD3 depletion and CD56 selection, expanded with soluble IL-2, IL-15 and IL-21 NK MACS + 5% human AB serum + 2% Supplement CD123 RV (alpha) 4 μg/mL protamine sulfate + Retronectin coated, MOI 1- 3  AML scFv condon optimized  pMPSV-IRES-GFP MPSV GMCSF-R alpha chain — — — CD28 CD28-CD137-CD3ζ 3 His+GFP+ 11,2% (5%~12%) — Pre-clinical  Germany https://doi.org/10.1089/hgtb.2019.039
Colamartino (Front Immunol 2019) PB-NK PB-NK cell expanded  K562-mIL-15/4-1BBL NK-MACS  CD19, CD22 LV (BaEV / VSV-G, 2nd gen) 8 μg/mL protamine sulfate +Retronectin coated, MOI 1-10 Lymphoblastoma — pHRSIN-SFFV SFFV  — — — CD28 CD28 CD28-CD137-CD3ζ 3 CD22 antigen Fc (38.3 ± 23.8%) day 3 or day 5 after transduction Pre-clinical  Canada https://doi.org/10.3389/fimmu.2019.02873
Ingegnere (Front Immunol 2019) PB-NK / NK-92 NK-92 cell line and Primary PB-NK cell activated with IL-2 and with autologous irradiated PBMC feeder cells RPMI1640 + 10%FCS + 600U/mL IL-2 CD19 Plasmid Neon Transfection System, NK-92 1650 V and 20 ms +  500 V and 100 ms. Resting NK 2050 V and 20 ms + 500 V and 100 ms. B-ALL mouse scFv pEGFP- pLV-pCEP4YPet — — — — CD8a CD8a CD3ζ/CD137-CD3ζ 1 and 2 >20% truncated CD34  day 1 or day 15 after transfection Pre-clinical  Canada https://doi.org/10.3389/fimmu.2019.00957
Herrera (Sci Rep 2019) CB-NK / PB-NK CB-NK and PB-NK cultured with IL-2 and IL-15 for 8 days RPMI1640 + 10% AB serum + 500U/mL IL-2 CD19 LV (VSV-G, 3rd gen) — B-ALL — pCCL — — — — — — CD137-CD3ζ 2 PB-NK CAR-NKs 47.46% (62.6–20.2%); CB-NK CAR-NK 46.8% (range 79.7–18.1%), Fab staining 7 days after transduction Pre-clinical  Spain https://doi.org/10.1038/s41598-019-55239-y
Bari (Front Immunol 2019) PB-NK Primary PB-NK cell activated by IL-2 and IL-15 NK-MACS CD19 LV (BaEV / VSV-G, 2nd gen) 10 μg/mL Vectofusin-1 B-ALL — — EF1a — — — CD8 CD8 CD137-CD3ζ 2 CD22 antigen Fc (38.3 ± 23.8%) day 3 after transduction Pre-clinical  USA https://doi.org/10.3389/fimmu.2019.02001
Wang (Blood Advance 2020) PB-NK PB-NK with IL-15 first day, followed by expansion on irradiated K562 + 200U/mL IL-2  SCGM + 10% FCS serum BCMA, CD123 RV Transduction on Day 4 after expansion, spinfection on Retronectin coated plates Multiple Myeoloma — SFG SFG — — — CD8a stalk CD8a  CD3ζ 1 Truncated CD19 (>60%), Truncated CD34 (>60%) 8 days after transduction Pre-clinical  USA https://doi.org/10.1182/bloodadvances.2020001510
Liu (NEJM 2020) CB-NK CB-NK expanded on K562-mbIL-15-41BBL SCGM 200  IU/mL IL-2 CD19 RV  RetroNectin pre-coated B-cell malignancies  humanized scFv SFG SFG — — — — CD28 CD28-CD3ζ 2 49.0% (22.7%-66.5%), goat anti-human IgG (H+L) antibody 10 days after transduction Pre-clinical / Clinical USA NCT03056339 https://doi.org/10.1056/NEJMoa1910607
Muller (Front Immunol 2020) PB-NK PB-NK negative enrichment by CD3/CD19 depletion For transduction X-VIVO 10 + 5% human plasma + IL-15, for long term culture of NK cells: NK MACS medium + 5% plasma CD19 LV (VSV-G / RD114, 3rd gen), RV (alpha) 25 µg/ml Retronectin coated or 10 μg/mL vectofusin-1, MOI=1, 5, 10 and 50 B-ALL mouse scFv LV-RRL.PPT.SF.GFPpre    RV-AlphaSIN.noTATA SFFV hIg heavy-chain — (G4S)x3 CD8a  CD28 CD28-CD3ζ 2 LV: Retronectin were 28.7% (MOI 5) and with Vectofusin-1 34.0%
(MOI 10). Higher MOI did not enhance the transduction. Alpha LV: vectodusin-1 higher transduction efficiency than LV, but retronectin not ehance the alpha-RV. Based on cMyc expression 		 72 hours after transduction Pre-clinical  Germany https://doi.org/10.3389/fimmu.2019.03123
Quintarelli (Leukemia 2020) PB-NK PB-NK activated with
Activation/Expansion Kit (Miltenyi Biotec) IL-2+IL15		 NK MACS + 5% human AB serum + 2% Supplement CD19 RV Retronectin B-ALL mouse scFv — — — — — CD8a  CD8a  CD137-CD3ζ 2 27,78 % (14,6-50%) CD34 expression 3~30 days after transduction  Pre-clinical  Italy https://doi.org/10.1038/s41375-019-0613-7
Ueda (Cancer Sci 2020) iPSC iPSC StemPro-34 GPC3 LV — Ovarium cancer (metastatized) mouse scFv  pLVSIN (G2 CAR)  UBC — VH-VL (G4S)x3 CD8a CD8a CD28-CD137-CD3ζ 3 71.9% on EGFR expression — Pre-clinical  Japan https://doi.org/10.1111/cas.14374
Liu (Cell Proliferation 2020) PB-NK PBMC-NK NK cell-specific  (Dakewe) EGFR LV (2nd gen) — Breast cancer — Lenti-EF1a- EF1a — — — CD8 CD28 CD137-CD3ζ 2 75% on EGFR antigen expression 3 days after transduction Pre-clinical  China https://doi.org/10.1111/cpr.12858
Gang (Blood 2020) PB-NK PB-NK — CD19 LV (VSV-G, 3rd gen) — B-cell malignancies  — CCL-MND MND — — — — CD8a CD137-CD3ζ 2 14,8% with CD19 antigen and GFP  2 days after transduction Pre-clinical USA https://doi.org/10.1182/blood.2020006619
Yang (Mol Ther Methods Clin Dev 2020) PB-NK PB-NK RPMI 1640 + 200U/ml IL-2 CD19 RV Retronectin B-cell malignancies  — SFG retroviral backbone SFG — VH-VL — IgG1-CH2-CH3/ CD8a CD28 CD137-CD3ζ 2 >70%, anti Human IgG (H+L) Fab Day4, 7, 11 and 14 after transduction Pre-clinical USA https://doi.org/10.1016/j.omtm.2020.06.014
Wilk (Blood Advances 2020) PB-NK PB-NK RPMI 1640 + 200U/ml IL-2 CD19 mRNA  polyanionic compound  B-cell malignancies  — mRNA — — — — — — — — >10% CAR+  18 hours after transfection Pre-clinical USA https://doi.org/10.1182/bloodadvances.2020002355
Daher (Blood 2020) CB-NK CB-NK expanded by K562-mbIL-15-41BBL SCGM + 200  IU/mL IL-2 CD19 RV RetroNectin pre-coated B-cell malignancies  humanized scFv SFG SFG — — — — CD28 CD28-CD3ζ 2 49.0% (22.7%-66.5%), goat anti-human IgG (H+L) antibody 10 days after transduction Pre-clinical /Clinical study USA https://doi.org/10.1182/blood.2020007748
Jamali (Front Immunol 2020) PB-NK / NK-92 / NKL NK-92, NKL, PB-NK X-Vivo 10 CD19 LV (3rd gen) 8 μg/ml polybrene B-cell malignancies  humanized scFv SINpWPT-EF1a EF1a — VL-VH — CD8a 4-1BB CD137-CD3ζ 2 >90% after sorting, CD19 antigen staining — Pre-clinical  / Clinical study USA https://doi.org/10.3389/fimmu.2020.02028
Gurney (Haematologica 2020)  PB-NK PB-NK NK MACS + 5% human AB serum + 500U/mL IL-2 CD38 mRNA Maxcyte™ GT Flow Transfection System AML anti-CD38 heavy chain with germline light chain SFG SFG — VL-VH (G4S)x3 — CD8a CD28-CD3ζ 2 PB-NK 42~45% Fab and Protein L 18 hours after transfection Pre-clinical  Ireland https://doi.org/10.3324/haematol.2020.271908
Tseng (Nat Commun 2020) NK-92MI, PB-NK NK-92MI, PB-NK alpha-MEM CD147, GPC3 RV, LV (2nd gen) 0.5 µg/ml RetroNectin Liver mouse antibody SFG-RV/ pHR-LV SFG-RV/PGK-LV — — — hIgG1 CD28 CD28-CD137-CD3ζ 3 x CAR-NK92MI sorting out > 95% Fab positive, PB-NK 80% Fab Pre-clinical  USA https://doi.org/10.1038/s41467-020-18444-2
Abbreviations: — denotes details that were not mentioned in the original publiction. PB-NK: peripheral blood NK cells. CB-NK: cord-blood NK cells. iPSC: induced pluripotent stem cell. scFv: single chain variable fragmenet. LV: lentivirus. RV: retrovirus. G4S: 4-glycine serine (linker).