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KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape

Nature Medicine volume 28, pages 2133–2144 (2022)Cite this article

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Abstract

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a ‘don’t kill me’ signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.

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Fig. 1: CAR19-mediated trogocytosis in NK cells cocultured with CD19+ tumor targets.
Fig. 2: Impact of antigen-induced self-engagement on CAR-NK effector cell phenotype and function.
Fig. 3: Impact of TROG-antigen acquisition on CAR-NK cell phenotype and function in vivo.
Fig. 4: A lower level of CAR-mediated TROG-antigen expression was associated with improved clinical response to CAR-NK cell-based immunotherapy.
Fig. 5: Expression of an iCAR by NK cells reduced fratricide and exhaustion induced by aCAR.
Fig. 6: AI-CAR-expressing NK cells showed superior in vivo antitumor activity.

Data availability

The human gene database GeneCard (genecards.org) was used to design the CAR constructs such as KIR2DL1 (GC19P067381), LIR-1 (GC19P067376), LAIR-1 (GC19M054351), NKG2A (GC12M021112) and CD300A (GC17P074466). Transcriptome data used in the generation of Extended Data Fig. 7g are available through the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession GSE190976. Source data are provided with this paper.

Code availability

No custom code was developed.

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Acknowledgements

This work was supported in part by the generous philanthropic contributions to The University of Texas MD Anderson Cancer Center Moon Shots Program, and The Sally Cooper Murray endowment; and by grants (grant nos. 1 R01CA211044-01, 5 P01CA148600-03, P50CA100632-16 and R01GM143243) from the National Institutes of Health (NIH), CPRIT (grant no. RP180466), Stand Up To Cancer Dream Team Research (grant no. SU2C-AACR-DT-29−19) and the Leukemia Specialized Program of Research Excellence (SPORE) (grant no. P50CA100632); by a grant from the NIH to the MD Anderson Cancer Center Advanced Technology Genomics Core (ATGC) Facility (grant no. CA016672); and by the grant (no. P30 CA016672) from the NIH to the MD Anderson Cancer Center Flow Cytometry and Cellular Imaging Core Facility that assisted with the CyTOF studies. We thank E. Gokdemir, B. T. Whitfield and J. Lu for their technical assistance.

Author information

Authors and Affiliations

  1. Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Ye Li, Rafet Basar, Guohui Wang, Enli Liu, Judy S. Moyes, Li Li, Lucila N. Kerbauy, Nadima Uprety, Pinaki P. Banerjee, Luis Muniz-Feliciano, Tamara J. Laskowski, Emily Ensley, May Daher, Mayra Shanley, Mayela Mendt, Sunil Acharya, Bin Liu, Alexander Biederstädt, Hind Rafei, Xingliang Guo, Luciana Melo Garcia, Paul Lin, Sonny Ang, David Marin, Richard E. Champlin, Elizabeth J. Shpall & Katayoun Rezvani

  2. UTHealth Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Ye Li

  3. Human Genome and Stem Cell Research Center, Department of Genetics and Evolutionary Biology, Biosciences Institute, University of Sao Paulo, Sao Paulo, Brazil

    Lucila N. Kerbauy

  4. Department of Stem Cell Transplantation and Cellular Therapy, Hospital Israelita Albert Einstein, Sao Paulo, Brazil

    Lucila N. Kerbauy

  5. Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, USA

    Mohsen Fathi, Ali Rezvan & Navin Varadarajan

  6. Department of Medicine III: Hematology and Oncology, Technical University of Munich, Munich, Germany

    Alexander Biederstädt

  7. Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Ken Chen

  8. Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Laura Bover

  9. Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Laura Bover

Authors
  1. Ye Li
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  2. Rafet Basar
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Contributions

Y.L. and K.R. conceptualized the study, completed the experimental design, and interpreted and analyzed data. Y.L. directed in vitro experiments with assistance by G.W. Y.L., E.L., R.B., L.L. and L.N.K. executed animal works. Y.L., R.B., L.L. and N.U. performed mass cytometry experiments and analyses. Y.L., M.F., A.R. and N.V. executed single-cell imaging and analysis. P.P.B. collected clinical information. Y.L. drafted the original manuscript; B.L., A.B., H.R., X.G., L.M.G., P.L., S.A., D.M., M.D., M.S., M.M., S.A., L.B., R.E.C., E.E., N.V., K.C. and E.J.S. commented on the manuscript, which was reviewed and edited by J.S.M., T.J.L., L.M.-F., Y.L. and K.R.

Corresponding author

Correspondence to Katayoun Rezvani.

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Competing interests

R.B., E.L., L.N.K., P.P.B., S.A., D.M., M.D., R.E.C., E.J.S., K.R. and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Takeda Pharmaceutical. S.A., D.M., R.B., E.L., L.N.K., E.J.S., K.R. and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Affimed GmbH. K.R. participates on the Scientific Advisory Board for GemoAb, AvengeBio, Virogin Biotech, GSK, Caribou Biosciences, Navan Technologies and Bayer. N.V. is a founder of CellChorus. The remaining authors declare no competing interests.

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Nature Medicine thanks Adelheid Cerwenka, Nathan Singh and Evan Weber for their contribution to the peer review of this work. Primary Handling Editors: Saheli Sadanand and Joao Monteiro, in collaboration with the Nature Medicine team.

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Extended data

Extended Data Fig. 1 CAR-mediated CD19 trogocytosis on NK cells in vitro.

(a) Gating strategy to distinguish NK cells and Raji cells in co-culture experiments. Expression of GFP/CD56/CAR was used to identify CAR-NK cells (GFP−CD56+CAR+), Raji cells genetically modified to express GFP (CD19+GFP+CD56−CAR−), and CAR-NK/Raji doublets (CD19+GFP+CD56+CAR+). Expression of CD19 on CAR-NK cells after co-culture with Raji cells was compared to that of control CAR-NK cells cultured alone. Inset numbers indicate the percentage (%) of cells within the indicated gated regions. (b) Representative Amnis® images showing surface protein expression of CD56/CAR/CD19, and intracellular expression of F-actin/GFP in CAR-NK cells cultured alone, Raji cells cultured alone, or CAR-NK/Raji doublets (CAR-NK cell engaged with Raji cell). Cells were identified by DAPI nuclear stain; scale bar indicates 7 μm. Representative images show trogocytic CD19 (tCD19) expression on CAR-NK cells following engagement with Raji targets, as assessed by Amnis® imaging flow cytometry. Data are shown for singlet CAR-NK cells cultured alone (negative control), CAR-NK cells engaged with Raji cells (d: CAR-NK/Raji doublet), or singlet CAR-NK cells after 5 min co-culture with Raji cells. The geometric mean fluorescent intensity (gMFI) of CD19 was indicated for each condition (lower panels). Inserted image shows representative cells for each culture condition. (c) Amnis® imaging showing the % of tCD19 expression on singlet CD56+ CAR-NK cells cultured alone, CAR-NK cells engaged with Raji cells, or the tCD19+ fractions of singlet CAR-NK cells after 5 min co-culture with Raji cells (left); and % of co-localized tCD19 and CAR molecules on singlet NK cells (right; n = 25 objects). (d) Flow cytometric analyses show expression of CD19/CD20/CD22 at the protein level (left) and the mRNA level (middle) in CAR-NK cells cultured alone, Raji cells cultured alone, and CAR-NK cells co-cultured with Raji cells for 5 mins (representative of 3 donors). Inset numbers indicate percentages of cells within the indicated gated regions. Bar graphs show the summary data for each marker (right). P values were determined by two-tailed Student’s t-test in panel c, or two-tailed Student’s paired t test in panel d. Data were shown by mean + s.e.m. Each circle represents an individual cell.

Source data

Extended Data Fig. 2 CAR19-mediated acquisition of tCD19 on NK cells from targeted Raji cells.

(a) UMAP analyses of CAR-NK cells collected after co-culture with RajiCD19+ cells. Phenotypic signatures of all collected CAR-NK cells were evaluated by mass cytometry, and data from 10,000 cells derived from 3 donors were merged to create a single UMAP map, where the analysis generated eight distinct color-coded clusters that represented the different subsets of NK cells. Marker expression for each NK cell subset was shown in UMAP plots. (b) Contour plots showing the UMAP cluster prevalence of CAR-NK cells alone, 30 min, 1 hr, 3 hrs, or 6 hrs after co-culture with Raji cells. The percentage of TROG+ CAR-NK cells (upper panel), and tCD19 expression are presented for each subset for the different conditions (lower panel). (c) Real-time images representing the co-culture of CAR19-NK cells (green) with RajiCD19-mCherry cells (red). Black arrows indicate events of cell apoptosis; yellow arrows indicate the immunologic synapse-like structure; white arrows indicate CAR19-NK cells with evidence of mCherry transfer; scale bar indicates 10 μm. (d) Flow cytometric analyses represent co-expression of CD19 and mCherry on singlet RajiCD19-mCherry cells cultured either alone (grey, representative for 3 samples) or CAR19-NK cells after co-culture with RajiCD19-mCherry cells for 5 mins only (green, representative for 5 donors); the following graph shows the correlation between mCherry (as determined as gMFI) and CD19 (as determined as the number of molecules per cell) for each singlet cell. (e) CD19 (as determined as the number of molecules per cell), and gMFI of mCherry expression on singlet RajiCD19-mCherry cells at different time points during co-culture with CAR19-NK cells (n = 3 donors). P values were determined by two-tailed Pearson’s correlation coefficient in panel d, two-tailed one-way ANOVA in panel e. Data were assessed by flow cytometry in panels d and e, and shown by mean + s.e.m.

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Extended Data Fig. 3 Self-engagement of TROG+ CAR-NK cells resulted in NK cell fratricide.

(a and b) Schematic illustrating the single-cell time-lapse imaging cytotoxicity assay. Time was recorded over 5 hours (T0 –T300min) from the start of co-culture, (a) where one single cell, or (b) two cells of non-TROG-antigen expressing fresh CAR-NK cells (control; grey) or CAR-NKTROG+ cells (green) were incubated in each nanowell. For the duration of the assay, the amount of time taken to detect Annexin V influx in the sorted CAR-NK cell was determined as the time taken to induce cell apoptosis. The following Kaplan-Meier curves show the percent (%) of apoptosis in CAR-NK cells during incubation. (c) Schematic representation of the experimental plan: TROG+ CAR19-NK cells were purified, and their phenotypic signature were evaluated before and 5 hrs post-culture by mass cytometry. Data from 10,000 cells from 3 donors per condition were merged to create a single UMAP map with eight distinct color-coded clusters that represented the different subsets of CAR-NK cells. Marker expression for each NK cell subset is shown. (d) UMAP plots showing the expression of TROG-antigen (tCD19; left) on CAR-NK cells before and after 5 hrs of culture with their sibling cells; the contour plots show the prevalence of each CAR-NK cell subset before and after 5 hrs of culture, with the percentage of each subset also indicated (right). P value was determined by Log-rank test in panels a,b.

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Extended Data Fig. 4 Repeated challenge of CAR19-NK cells with autoNKCD19+ cells result in CAR-NK cell hyporesponsiveness.

(a) tSNE analysis of live hCD45+GFP−CD56+CD3− CAR19-NK cells 4 days following a second round of antigen challenge with autoNKgCD19+gGFP+ cells; controls include CAR19-NK cells cultured alone or co-cultured with autoNKgGFP+ cells for 4 days (lacking CD19 expression). The phenotypic cell signature was evaluated by CyTOF and merged to create a single t-SNE map (10,000 cells from 3 pooled donors per condition). Expression of CAR19 (green) and tCD19 (orange) is determined against NT-NK cell controls. Inset numbers indicate the percentages (%) of CAR and tCD19 expression on CAR19-NK cells. (b) Kaplan-Meier plots showing the percentage (%) of apoptotic Raji cells following co-culture with CAR-NK cell isolated after the 1st round (2 days, left) or 3rd round (6 days, right) of re-challenge with autoNKgCD19+/GFP+ cells compared to fresh CAR-NK cells. Assays were performed in microwells with E:T ratio of 1:1; data were pooled from two donors. (c) Schematic representation of single-cell time-lapse imaging cytotoxicity assay, where a single CAR-NK cell was cultured with two Raji cells. Annexin V influx in Raji cells defined cell apoptosis. The bar plots show the percentage of CAR-NK cells isolated at different time points after re-challenge with autoNKgCD19+/GFP+ cells that succeeded in lysing one (light green) or two (dark green) Raji cells. Fresh CAR-NK cells (pre) were used as control (n = 4 donors for pre, 3 donors for 2nd, and 2 donors for 1st and 3rd challenge). (d-f) Incucyte analyses showing the percentage (%) of caspase 3/7 events in Raji cells co-cultured with CAR19-NK cells isolated after (d) the 1st round, (e) 2nd round, or (f) 3rd round of re-challenge with autoNKgCD19+/GFP+ cells. CAR-NK cells isolated after each round of re-challenge and cultured for 24 hrs in complete media supplemented with 100 U/mL IL-2 (referred to as ‘rested’ cells) were used as controls (representative of 3 donors). P values were determined by Log-rank test in panel b, two-tailed Student’s paired t test in panel c, or two-tailed two-way ANOVA in panels d,e,f, n.s.: not significant. Data were shown by mean + s.e.m.

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Extended Data Fig. 5 CAR19-NK cell trogocytosis and reciprocal reduction in CD19 antigen expression on tumor cells in vivo.

(a) Schematic illustration of timeline using a mouse model engrafted with Raji cells. Mice received three dose levels of Luc/GFP-expressing CD19+ Raji cells (0.2×105, 1×105, or 5×105), respectively, followed by a single infusion of CAR19-NK cells (1×107) or NT-NK cells alone (1×107) as control, (b) Graphs showing the intensity of bioluminescence imaging (BLI) over time; black: Raji cells only; blue: Raji cells with NT-NK cell infusion; green: Raji cells with CAR19-NK cell infusion. (c) tCD19 expression (indicated as TROG+/TROG− ratio) on singlet hCD45+GFP−CD56+CD3− on CAR-NK cell products gated on CAR19-expressing (green) vs. CAR-negative fractions (light blue) in peripheral blood samples collected at different time points after infusion (n = 15 mice). (d) CD19 expression on Raji cells, shown as the count of molecules per cell (grey) in peripheral blood (left, n = 15 mice), and organs [spleen, liver, bone marrow (BM), and blood] of mice at the end time point (right, n = 24 mice) after CAR19-NK cell infusion. (e) CD19 expression on Raji cells, shown as the count of molecules per cell in organs harvested at the end time point after NT-NK cell infusion (n = 10 mice). P values were determined by two-tailed Student’s t test for analyses, and two-tailed one-way ANOVA in panel d. Data were assessed by flow cytometry in samples with cell objects of interest >20 counts, and shown by mean + s.e.m, or medium (min/max) in boxplot. Each circle represents an individual mouse sample and outliers are indicated as dark dots.

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Extended Data Fig. 6 In vivo trogocytosis was associated with limited persistence of CAR-NK cells.

(a-b) The proportion of CAR19-expressing vs. CAR-negative live NK cells (a) in the peripheral blood of mice at different time points after CAR-NK cell infusion (left, n = 15 mice), and (b) in different organs at the end time point (right, n = 24 mice). (c) tCD19 expression on NK cells in the CAR19-expressing vs. the CAR-negative NK cell fractions and their viability based on TROG-antigenpositivity (Q2: CAR19-NKTROG+, Q3: CAR-negative NKTROG+) in cells harvested from different organs collected at the end time point (representative of n = 24 mice). Inset numbers indicate the percentages of cells within the indicated gated regions. (d) tCD19 expression (indicated as TROG+/TROG- ratio) on singlet hCD45+GFP−CD56+CD3−CAR19-NK vs. CAR-negative NK cells harvested from organs at the end time point after CAR-NK cell infusion (left, n = 24 mice) and in mice treated with NT-NK cells alone (right: dark blue, n = 10 mice). (e) Percentage (%) of viable NKTROG+ (tCD19+, left) and viable NKTROG− cells (middle) for CAR19-NK cells vs. CAR-negative NK cells harvested from organs at the end time point after infusion of CAR-NK cells (n = 24 mice), or in mice treated with NT-NK cells (right: dark blue, n = 10 mice). P values were determined by two-tailed Student’s paired t test in panel a, two-tailed one-way ANOVA in panel b, two-tailed Student’s t test in panels d and e. Data were assessed by flow cytometry in samples with cell objects of interest >20 counts, and shown by mean + s.e.m, or medium (min/max) in boxplot. Each circle represents an individual mouse sample, and outliers were indicated as dark dots.

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Extended Data Fig. 7 In vivo trogocytosis was associated with poor viability of CAR-NK cells.

(a) Schematic illustration of the timeline using a mouse model of lymphoma, engrafted with 0.2×105 luc/GFP−expressing CD19+ Raji cells and treated with a single infusion of CAR19-NK cells or NT-NK cells as control. Blood, BM, spleen and liver were harvested for analysis at two weeks (day 13-15), three to four weeks (day 20-27), or at the end time point (day 29-34) after infusion. (b) Tumor burden was assessed weekly by BLI. The BLI intensity is shown for each mouse after infusion with CAR-NK cells (green) or NT-NK cells (blue). Untreated mice were used as controls (black). (c) Heatmap showing expression levels of phenotypic and functional markers on fractions of TROG+ (tCD19+) and TROG− live hCD45+GFP−CD56+CD3−NK cells at different timepoints post-infusion. The expression level for each marker is represented by the color grey (low) - orange (high) and the size. (d) The phenotypic cell signature for each condition was evaluated by mass cytometry and merged to create a single t-SNE map. Expression of tCD19 (orange) and CAR19 (green) on hCD45+GFP-CD56+CD3- NK cells was determined based on their expression on NT-NK cell controls. (e) Violin plots showing expression of tCD19 on NK cells within each cluster harvested from mice treated with CAR19-NK cells (left); cisplatin levels within the TROG+ vs. TROG− fractions for each cluster (right) are shown. (f) Violin plots showing expression of tCD19 on NT-NK cells within each cluster (left); cisplatin levels within the TROG+ vs. TROG− fractions for each cluster (right) are shown. (g) Gene signature for total hCD45+ cells at different time points during the treatment course. The t-SNE maps, generated with the Seurat package in R, show color-coded expression levels for CD19 and MS4A1 (Raji cells), NKG7 and FCGR3A (NK cells) for each cluster. P values were determined by two-tailed Wilcoxon matched pairs test in panels e and f. Data were assessed by mass cytometry and shown in violin graph with the indicated median.

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Extended Data Fig. 8 iCAR design, expression and impact on primary human NK cell signaling and function.

(a) Schematic diagram of the viral vector design for the different anti-CD19 iCARs; TM: transmembrane; SE: signaling endodomain. (b) Cell surface expression of aCAR19, 19scFv, or iCAR19 constructs by flow cytometry in transduced primary human NK cells. Dot plots are representative of three different donors. Inset numbers indicate the percentage of CAR-expressing cells within the indicated gated regions. (c and d) Flow cytometric expression of (c) phos-SHP1 (pSHP1) and (d) phos-Syk/Zap70 in NK cells expressing CAR19, 19scFv or the different iCAR19 constructs (n = 5 donors) in response to stimulation with CD19+ Raji cells; the following bar graphs show their expression, determined by gMFI normalized to the unstimulated NK cell population. (e-h) CD107a (left), TNF-α (middle), and IFN-γ (right) production by NK cells transduced with CAR19, 19scFv, or the different iCAR19 constructs in response to 6-hr stimulation with (e) CD19+ K562 cells (K562gCD19+), (f) K562, (g) RajiCD19+, or (h) CD19− RajiCD19-KO target cells (n = 5 donors). (i and j) Incucyte analyses of percentage (%) caspase 3/7 events in (i) RajiCD19+ and (j) autoNKgCD19+ target cells after co-culture with NK cells expressing CAR19, 19scFv or the different iCAR19 constructs, (representative for three donors). (k and l) Cumulative population doubling (PD) of NK cells expressing CAR19, 19scFv or the different iCAR19 constructs over 70 days of culture with (k) IL-2 only or with (l) IL-2 plus weekly uAPC stimulation (n = 3 donors). (m) tCD19 expression on NK cells transduced with CAR19, 19scFv or the different iCAR19 constructs, presented as ratio of TROG+/TROG− cells at different time points during co-culture with RajiCD19+ cells (n = 3 donors). P values were determined by two-tailed one-way ANOVA in panels c-h, or two-tailed two-way ANOVA in panels i-m. Data were assessed in flow cytometry in panels b,c,d,e,f,g,h,m, and shown by mean + s.e.m. Each symbol represents an individual donor.

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Extended Data Fig. 9 AI-CAR expressing NK cells exert superior in vivo anti-tumor activity in SKOV3 ovarian cancer models.

(a) Schematic illustration of the timeline using a mouse model of SKOV3gCD19+ ovarian cancer. Seven days later, mice received a single infusion of 1×107 NK cells expressing aCAR19/CS1scFv (green), or aCAR19/iCAR-CS1 (orange), or no NK cells (tumor only) (n = 5 mice/group). (b and c) Tumor burden as determined by weekly BLI, (b) representative images at select time points are shown; (c) normalized intensity of BLI for each treatment group over the treatment course; dashed lines represent data for each mouse. (d) Kaplan-Meier curves showing survival of mice after NK cell infusion. (e and f) (e) tCD19 (indicated as TROG+/TROG− population) and (f) viability of the TROG+ fraction (NKtCD19+) in the peripheral blood of mice at Days 5, 15, and 30 after infusion of aCAR19/CS1scFv (green) or aCAR19/iCAR-CS1 (orange) NK cells (n = 5 mice/group). (g) Percentage (%) of live GFP−CD3−CD56+CAR19+ NK cells in the peripheral blood of mice at Days 5, 15, and 30 after infusion of aCAR19/CS1scFv (green, left) or aCAR19/iCAR-CS1 (orange, right) NK cells (n = 5 mice/group). (h) Live NKCAR19+ NK cell counts in the peripheral blood of mice at Days 5, 15, and 30 after infusion of aCAR19/CS1scFv (green) or aCAR19/iCAR-CS1 (orange) NK cells (n = 5 mice/group). (i) Representative images showing H&E and IHC staining with anti-Luciferase, anti-hCD45, or anti-hROR1 antibodies on sections from the mesentery tissue of SKOV3ROR1+ grafted mice treated with aCAR-ROR1/CS1-scFv NK cells or aCAR-ROR1/iCAR-CS1 NK cells. Inserted numbers indicate hCD45+ cell count per 0.1 mm2. Black arrows indicate ROR1 expression on tumor cells; blue arrows indicate ROR1 expression on NK cells; scale bar indicates 100 μm. P values were determined by two-tailed Student’s t test in panels c, e,f,h,i,g, Log-rank test in panel d; n.s.: not significant. Date was pooled from two independent experiments in panels c and d, where NK cells were derived from different donors, or assessed by flow cytometry in panels e,f,g,h and shown by mean + s.e.m. Each circle represents an individual mouse sample.

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Extended Data Fig. 10 Model of AI-CAR NK cell function.

(a) aCAR-NK cell-mediated trogocytosis results in a decrease in antigen density on tumor cells, and promotes CAR-NK cell fratricide and hyporesponsiveness; (b) Engineering NK cells to express both an aCAR against a tumor antigen and a KIR-based inhibitory CAR (iCAR) against an NK self-antigen prevents TROG-induced self-recognition and TROG-antigen mediated fratricide of CAR-NK cells, while preserving their on-target tumor recognition and cytotoxicity.

Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and Videos 1–3.

Reporting Summary

Supplementary Video 1

Antigen-induced self-engagement results in CAR-NK cell fratricide.

Supplementary Video 2

Repeated antigen-induced self-engagement results in CAR-NK cell hyporesponsiveness against K562 cells.

Supplementary Video 3

Repeated antigen-induced self-engagement results in CAR19-NK cell hyporesponsiveness against Raji cells.

Supplementary Data

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Li, Y., Basar, R., Wang, G. et al. KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape. Nat Med 28, 2133–2144 (2022). https://doi.org/10.1038/s41591-022-02003-x

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