Abstract
Resistance to chimaeric antigen receptor (CAR) T cell therapy develops through multiple mechanisms, most notably antigen loss and tumour-induced immune suppression. It has been suggested that T cells expressing multiple CARs may overcome the resistance of tumours and that T cells expressing receptors that switch inhibitory immune-checkpoint signals into costimulatory signals may enhance the activity of the T cells in the tumour microenvironment. However, engineering multiple features into a single T cell product is difficult because of the transgene-packaging constraints of current gene-delivery vectors. Here we describe a cell-sorting method that leverages leucine zippers for the selective single-step immunomagnetic purification of cells co-transduced with two vectors. Such ‘Zip sorting’ facilitated the generation of T cells simultaneously expressing up to four CARs and coexpressing up to three ‘switch’ receptors. In syngeneic mouse models, T cells with multiple CARs and multiple switch receptors eliminated antigenically heterogeneous populations of leukaemia cells coexpressing multiple inhibitory ligands. By combining diverse therapeutic strategies, Zip-sorted multi-CAR multi-switch-receptor T cells can overcome multiple mechanisms of CAR T cell resistance.
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Data availability
The genomic integration study dataset is available on the Sequence Read Archive database via the accession number PRJNA1052637. The scRNAseq data are available on the GEO database via the accession number GSE253563. The main data supporting the findings of the study are available within the article and its Supplementary Information. The raw data generated during the study are available from the corresponding authors on reasonable request. Source data are provided with this paper.
Code availability
Jupyter notebook and Conda environment files are available at https://github.com/sj1233/James_et_al_Zip-sort.
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Acknowledgements
This research was supported by the National Cancer Institute (NCI) award P30-CA008748 MSK Cancer Center Support Grant/Core Grant and National Heart, Lung, and Blood Institute (NHLBI) award number R01-HL147584. Additional funding was received from Comedy vs Cancer, the Parker Institute for Cancer Immunotherapy as well as the Paula and Rodger Riney Multiple Myeloma Research Initiative. S.E.J. is supported by a K08 career development award from the NCI (award number K08-CA252157). S.E.J. was also supported by a Young Investigator award from the American Society for Clinical Oncology, an Amy Program Award from the Be the Match Foundation and a Bridge Scholar award from the Parker Institute for Cancer Immunotherapy. S.C. was supported by a Research Fellowship from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). A.L.L. was supported by the Sweden America foundation, SSMF and DKMS. S.D. is supported by the MSK Leukaemia SPORE Career Enhancement Program and the Gerstner Physician Scholar program. J.U.P. is supported by an NHLBI NIH award (K08-HL143189) and the MSKCC Cancer Center Core Grant NCI P30-CA008748. S.A.V. is supported by a K08 career development award (NCI K08-CA237731) and the Parker Institute for Cancer Immunotherapy. C.A.K. was supported in part by NIH R37-CA259177, R01-CA286507, P50-CA217694 and P30-CA008748; Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research; The Center for Experimental Therapeutics at MSKCC; The MSK Technology Development Fund; The Parker Institute for Cancer Immunotherapy; The Sarcoma Center at MSKCC; The Damon Runyon Cancer Research Foundation CI-96-18; the Tow Center for Developmental Oncology and Cycle for Survival. We acknowledge the use of the Integrated Genomics Operation Core, funded by the NCI Cancer Center Support Grant (CCSG; P30 CA08748), Cycle for Survival and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology.
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S.E.J., L.J. and M.R.M.v.d.B. conceived the study and designed experiments. S.E.J., L.J., S.A.V., C.A.K. and M.R.M.v.d.B. developed methodologies. S.E.J., S.C., B.D.N., J.S.F., L.J., A.P.B., A.R., H.K.E., A.M., D.M., K.B.N., A.L., N.L., A.M.R. and A.P. performed experiments. S.E.J., S.C., B.D.N., A.G.M., J.K.E., F.D.B., A.I.K., A.L.L., T.F., S.D. and J.U.P. analysed data. S.E.J. and S.C. wrote the manuscript with assistance from all authors. M.R.M.v.d.B. supervised the study.
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S.E.J., L.J., and M.R.M.v.d.B. are co-inventors on patent applications related to this manuscript (‘Leucine zipper-based compositions and methods of use’, nos. US20210171601A1 (USA), EP3836944A4 (Europe), WO2020037178A1 (WIPO), CA3109630A1 (Canada) and CN112930186A (China); ‘Cell sorting systems and methods of use’, nos. US20210179686A1 (USA), EP3837287A4 (Europe), WO2020037181A2 (WIPO), CA3109635A1 (Canada) and CN112996819A (China)). A.P.B. has consulted for Bristol Myers Squibb and Cancer Study Group, LLC, and has received honoraria from OncLive. F.D.B. is a founder of Biocept and has intellectual property licensed to Novartis. J.U.P. reports research funding, intellectual property fees and travel reimbursement from Seres Therapeutics as well as consulting fees from Da Volterra, CSL Behring and MaaT Pharma. He serves on an Advisory board of, and holds equity in, Postbiotics Plus Research. He has filed intellectual property applications related to the microbiome. S.A.V. has received funding from Bristol-Meyers Squibb and has received consulting fees from Koch Disruptive Technologies and Generate Biomedicine. C.A.K. has previously filed intellectual property applications related to the FasDNR featured in this manuscript. C.A.K. is a scientific co-founder and holds equity in Affini-T Therapeutics. C.A.K. has previously consulted for or is on the scientific and/or clinical advisory boards of: Achilles Therapeutics, Affini-T Therapeutics, Aleta BioTherapeutics, Bellicum Pharmaceuticals, Bristol Myers Squibb, Catamaran Bio, Cell Design Labs, Decheng Capital, G1 Therapeutics, Klus Pharma, Obsidian Therapeutics, PACT Pharma, Roche/Genentech, Royalty Pharma and T-knife. M.R.M.v.d.B. has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; has received royalties from Wolters Kluwer; has consulted, received honoraria from or participated in advisory boards for Seres Therapeutics, Vor Biopharma, Rheos Medicines, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Ceramedix, Lygenesis, Pluto Therapeutics, GlaxoSmithKline, Da Volterra, Thymofox, Garuda, Novartis (spouse), Synthekine (spouse), Beigene (spouse), Kite (spouse); has intellectual property licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a non-profit organization). MSKCC has institutional financial interests relative to Seres Therapeutics.
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Extended data
Extended Data Fig. 1 Covalently linked blocking-zipper inhibits extracellular leucine zipper pairing.
a, Zip-sorting system vector maps. b, Vector map and diagram for EE-Thy1.1-P2A–BFP vector. c, Flow cytometry analysis of C1498 cells co-transduced with FLAG–RR-CBR–GFP and EE-Thy1.1-P2A–BFP vectors. Arrows depict combined intracellular + extracellular pairing (orange) and extracellular pairing (blue). d, Comparison of FLAG–zipper staining on dual-transduced (EGFP+ BFP+) and capture-zipper single-transduced (EGFP− BFP+) C1498 cells from four independent biological replicate co-transductions with FLAG–RR-CBR–GFP and EE-Thy1.1-P2A–BFP vectors as in panel c. Data are mean ± SEM of replicate samples. e, Maps of non-blocked EE-Thy1.1-P2A–BFP and blocked RR-EE-Thy1.1-P2A–BFP capture-zipper vectors. f, Diagrams and flow cytometry analysis of co-culture of single-transduced FLAG–zipper-secreting FLAG–RR-CBR–GFP C1498 with C1498 cells single-transduced with either (top) non-blocked EE-Thy1.1-P2A–BFP or (bottom) blocked RR-EE-Thy1.1-P2A–BFP capture-zipper vectors at depicted cell ratios for 48 h. FLAG staining represents extracellular pairing of FLAG–RR zippers on single-transduced capture-zipper+ C1498 cells, which capture FLAG–RR zippers secreted into the media by single-transduced FLAG–RR-secreting C1498 cells. g, FLAG–RR zipper surface expression on C1498 cells expressing blocked or non-blocked capture zippers depicted in panel f. n = 1 transduction for each cell line and n = 3 co-culture experiments. Data are mean ± SEM of triplicate samples. Error bars were too small to depict. h, Flow cytometry contour plots and histograms of unsorted, mixed populations of C1498 cells co-transduced with FLAG–RR-CBR–GFP and RR-EE-Thy1.1-P2A–BFP vector variants with capture zippers containing different repulsive mutations in blocking-zippers (See Supplementary Table 1). “EE” blocking-zipper is engineered to be fully repulsive against EE capture-zipper and maximally attractive towards the FLAG–RR zipper. Remaining mutants contain varying numbers of repulsive mutations “2 or 3” in the N-terminal “N” or middle “M” regions of the blocking-zipper. Representative of n = 3 separate transductions. i, Diagram depicting predicted effect of repulsive amino acid substitution on zipper binding affinity. j, Maps for non-blocked and blocked EGFRt-based capture-zipper vectors. k, Flow cytometry analysis of C1498 cells dual-transduced with FLAG–RR-CBR–GFP and either (top) non-blocked EE-EGFRt-P2A–BFP or (bottom) blocked capture-zipper RR-EE-EGFRt-BFP. l, Zip-sort of FLAG–RR-CBR–GFP/RR-EE-EGFRt-P2A–BFP C1498 cells. Representative of n = 2 transductions and Zip-sorts.
Extended Data Fig. 2 Zip-sorted dual-CAR T cells demonstrate CAR-dose-dependent upregulation of ROS and inhibitory receptor expression.
a, Flow cytometry analysis of BM185-ffluc-Thy1.1-Neo cell lines expressing combinations of CD19 and CD20. b, Schematic depicting BM185 syngeneic mouse model of antigen-loss escape in CAR T cell immunotherapy for acute lymphoblastic leukaemia. c–e. Sublethally irradiated BALB/c mice were injected with 1:1 mixture of BM185-CD19/BM185-CD20 at 1×105 BM185/mouse (50x increased dose vs. Figure 3g) and treated with Zip-sorted BALB/c CAR T cells on day 2. c, Leukaemia BLI (ffluc) from two combined experiments. Log-transformed BLI values were compared using a Vardi test to compare AUC values with FDR correction for multiple comparisons. d, Day 10 BLI images from representative experiment. e, Survival, compared via log-rank test. f, PD-1 expression on resting Zip-sorted BALB/c CAR T cells. g, Linear regression analysis of unstimulated T cell PD-1 expression vs. number of signalling-competent CARs expressed, n = 3 donors. h, Immunophenotype analysis of unstimulated Zip-sorted BALB/c CAR T cells; mean ± SEM from n = 3 donors. i, Total cellular ROS (CM-H2DCFDA) analysis of resting Zip-sorted CAR T cells. j, Z-score normalized mean CM-H2DCFDA MFI ± SEM results of n = 3 biological replicates. k, Mitochondrial superoxide (Mitosox Red) analysis of Zip-sorted CAR T cells. l, Z-score normalized mean Mitosox Red MFI ± SEM results of n = 3 biological replicates. Statistical differences for ROS and mitochondrial superoxide were compared using one-way ANOVA, with Tukey’s test.
Extended Data Fig. 3 CAR T cell culture with NAC or dasatinib and ITAM attenuation enhances the antileukaemia activity of dual-CAR T cells.
a,b. BM185-CD19/BM185-CD20 antigen-loss escape model. BALB/c mice were treated with Zip-sorted dual-CAR BALB/c T cells cultured with 1 μM dasatinib (2 days), 10 mM NAC (3 days), or DMSO (3 days). a, Leukaemia BLI from two combined experiments. Log-transformed BLI AUC values were compared using a Vardi test with FDR correction. b, Survival. c, NFAT, AP-1, or NFκB EGFP reporter analysis of unstimulated BALB/c T cells gated on dual-CAR positive or CAR-negative population following 24 h culture with 1 μM dasatinib, 10 mM NAC, or DMSO. Representative of n = 2 donor experiments, with mean ± SEM of triplicate wells. Statistical comparison via two-way ANOVA, with Tukey’s test. d,e, PD-1 and TOX expression in Zip-sorted dual-CAR or delta/delta BALB/c T cells following 24 h co-culture with BM185-CD19 or no targets. Mean ± SEM of triplicate samples were compared with a two-way ANOVA, with Tukey’s test. f, Intracellular flow cytometry analysis of TCF1 and TOX expression in Zip-sorted dual-CAR or delta/delta BALB/c T cells cultured for 2 days with 1 μM dasatinib or DMSO. Representative experiments from n = 2 donors. g, Diagram depicting ITAM mutations in 1XX CAR. h, Survival of BALB/c mice injected with BM185-CD19/BM185-CD20 (1:1) and treated with Zip-sorted dual-CAR WT CD3ζ or 1XX ITAM mutant dual-CAR T cells, from three combined experiments. i, Survival of BALB/c mice injected with dual-target-antigen expressing BM185-CD19-CD20 leukaemia and treated with WT CD3ζ or 1XX dual-CAR T cells, from three combined experiments. Survival curves were compared via log-rank tests or pairwise log-rank test, with FDR correction.
Extended Data Fig. 4 Coexpression of BCL-2 with dominant-negative receptors or combination with a single switch receptor transiently enhances antileukaemia activity of dual-CAR T cells.
a, (Left) Diagram and maps for vectors encoding combinations of the Zip-sorting system, CD19 and CD20 dual-CAR, BCL-2 D34A caspase-cleavage resistant mutant, iC9, 3N-mutant blocked zipper-tagged PD-1-DNR (DNR; dominant-negative receptor), and FasDNR. 3N mutant blocking-zipper was used to increase affinity-tagged zipper surface expression (See Extended Data Fig. 1h). Tandem hCD20 mimotopes were added as expression tag and as a target for cell Rituximab-mediated depletion. (Right) Flow cytometry analysis of Zip-sorted BALB/c T cells dual-transduced with vectors as depicted in legend. Numbers in flow plots refer to post Zip-sort purity percentage for each marker. b, Maps for vector set encoding Q2-RR-iC9 and 3N zipper-tagged PD-1-CAR. c, Expression of PD-1-CAR and surface bound Q2-RR zipper. d, PD-L1 expression on BM185-CD20-PD-L1 clone. e, Luciferase-based 24 h target lysis assay using Zip-sorted Q2-RR-iC9/3N-EE-PD-1-CAR BALB/c T cells or non-transduced T cells. Data are mean ± SEM of triplicate wells for a representative experiment from n = 2 donor experiments. f–i, Sublethally irradiated BALB/c mice were injected with a 1:1 mixture of BM185-CD19 and BM185-CD20 and treated with Zip-sorted CD45.1+ congenic BALB/c T cells. f, Leukaemia BLI (ffluc) from single experiment. g, Flow cytometry analysis of CAR T cells in peripheral blood on day 9. h, Linear regression analysis of leukaemia BLI signal vs. blood CD45.1+ T cell concentration. i, Survival. j–l, Experimental setup as in panel f, but mice were treated with dual-CAR T cells incorporating FasBB switch receptor instead of FasDNR. j, Leukaemia BLI from three combined experiments. k, Flow cytometry analysis of CAR T cells in peripheral blood on day 10, combined from two experiments. l, Survival from three combined experiments. Log-transformed BLI AUC values were compared using a Vardi test with FDR correction. Blood T cell counts were compared with a two-tailed t-test. Survival was compared with log-rank or pairwise log-rank comparison with FDR correction.
Extended Data Fig. 5 Multi-Switch receptor arrays enhance T cell activity in response to inhibitory ligands.
a,b, Flow cytometry analysis of Zip-sorted dual-CAR BALB/c T cells ± FasBB switch receptor co-cultured for 24 h with BM185-CD19 (E:T = 1:1) or left unstimulated. Data are mean ± SEM (biological replicates) of triplicate wells from n = 3 donor experiments. c, Vector maps for dual-CAR and switch receptor configurations as depicted in the table. d–g, Flow cytometry analysis of Zip-sorted dual-CAR, dual-CAR FasBB, and dual-CAR multi-Switch BALB/c T cells stimulated for 24 h with BM185-CD19 (E:T = 1:2) or left unstimulated. Data are mean ± SEM (biological replicates) of triplicate wells from n = 4 donor experiments. Statistical comparisons were calculated using two-way ANOVA, with Tukey’s test. h, Flow cytometry analysis of BM185-ffluc-Thy1.1-Neo cell lines engineered to express FasL, PD-L1, or CD200. i–k. Sublethally irradiated BALB/c mice were injected with 1:1:1 mixture of BM185-CD19-FasL, BM185-CD20-PD-L1, and BM185-CD20-CD200 and treated with Zip-sorted dual-CAR BALB/c T cells, pre-cultured for 2 days with 1 μM dasatinib. i, Leukaemia BLI, and j, mouse survival, respectively, from one experiment. Survival differences were compared via log-rank test. k, Flow cytometry analysis of inhibitory ligand expression of single-ligand-positive BM185 lines harvested from bone marrow of mice reaching humane endpoints. PD-L1 expression difference was compared via a two-tailed t-test. Data are mean ± SEM (biological replicates). l, Live-cell microscopy (Incucyte) analysis of EGFP-labelled dual-CAR and dual-CAR triple-Switch Zip-sorted BALB/c T cells co-cultured with iRFP713+ BM185 target cell lines as depicted (E:T = 1:1), with repetitive target addition on days 0, 1, and 2. Representative of n = 3 donor experiments. Data are mean ± SEM of triplicate samples. m, Live-cell microscopy (Incucyte) analysis of NFκB-EGFP-reporter labelled dual-CAR and dual-CAR triple-Switch Zip-sorted BALB/c T cells co-cultured with iRFP713+ BM185 target cell lines as depicted at E:T = 1:9. Representative of n = 2 donor experiments. Values represent mean ± SEM of triplicate samples.
Extended Data Fig. 6 Coexpression of switch receptors attenuates inhibitory receptor upregulation, ROS production, and endoplasmic reticulum stress response activation.
a, MAGIC110 imputed gene expression violin plots of inhibitory receptor and transcription-factor genes for dual-CAR ± multi-Switch T cells related to Fig. 5. Differentially expressed genes were compared via Wilcoxon test prior to imputation. b, Flow cytometry analysis of dual-CAR ± multi-Switch T cells 24 h following stimulation with BM185-CD19. Representative of n = 2 donor experiments. c, Dual-CAR ± multi-Switch T cells were stained with CM-H2DCFDA to measure total ROS two days following the last TCR stimulation. Left, representative flow cytometry plots from one of two donors. Right, bar graphs depicting mean CM-H2DCFDA values from triplicate wells from n = 2 donors. Replicate values were compared via one-way ANOVA, with Tukey’s test. Data are mean ± SEM. d, ER stress score gene set (GOBP_RESPONSE_TO_ENDOPLASMIC_RETICULUM_STRESS) comparison for dual-CAR ± multi-Switch T cells related to Fig. 5. Single-cell ER stress score values were compared via one-way ANOVA, with Tukey’s test. e, STRING115 analysis combining functional and physical protein interaction networks corresponding to the union of GO:0034976 ER stress response genes significantly upregulated (adjusted P < 0.05) in CD4+ or CD8+ dual-CAR T cells compared with dual-Switch and triple-Switch T cells. Gene nodes without connecting edges were excluded. Thickness of edges indicates strength of data support. f, Heat map depicting gene scaled expression of selected ER stress response genes in the IRE1α and ATF6 ER stress pathways. g, Full GSEA pathway illustrations for Fig. 5g.
Extended Data Fig. 7 Dual-CAR T cells eliminate cognate targets and promote antigen-negative escape of leukaemia mixture with high antigen heterogeneity.
a, C1498 acute myeloid leukaemia cell line was modified to singly express murine CD19, CD20, CD79bΔ (CD79b extracellular ___domain fused to CD28TM and CD3ζΔ; to promote surface expression without requiring CD79a coexpression), and BAFF-R. C1498 also was modified to express CBR, hCD8, and puroR. b–e, Albino B6 mice were sublethally irradiated and injected with either 1:1 mixture of C1498-CD19 and C1498-CD20 or a 1:1:1:1 mixture of C1498-CD19, C1498-CD20, C1498-CD79b, and C1498-BAFF-R and treated with dasatinib-cultured Zip-sorted dual-CAR 1XX T cells or left untreated. b, Leukaemia BLI from single experiment. Log-transformed BLI AUC values were compared using a Vardi test with FDR correction. c, Survival. Differences in survival were compared with a log-rank test. d, Target antigen expression of representative C1498 leukaemia harvested from bone marrow at time of euthanasia for leukaemia progression. Gated on hCD8+ C1498. e, Target antigen expression on bone marrow leukaemia obtained from mice reaching humane endpoints. Percentage CD20+ C1498 fraction of total bone marrow C1498 was compared with two-tailed t-test. Data are mean ± SEM of biological replicates.
Extended Data Fig. 8 Multi-Switch receptor arrays enhance the activity of triple-CAR T cells.
a, Diagrams and vector maps for triple-CAR ± dual-Switch receptor arrays. b, Flow cytometry analysis of Zip-sorted triple-CAR and triple-CAR dual-Switch albino B6 T cells. Numbers in flow plots represent post-sort purity percentage for each construct. c, 24 h luciferase-based target lysis assay with Zip-sorted BAFF-R/CD79b/CD20 triple-CAR BALB/c T cells with targets as depicted. Data are mean ± SEM of triplicate wells. d–f, Sublethally irradiated albino B6 mice were injected with 1:1:1 ratio of C1498-CD20, C1498-CD79b, C1498-BAFF-R and treated with dasatinib-cultured triple-CAR ± dual-Switch T cells co-transduced with a membrane-bound Gaussia luciferase (gLuc) vector: gLuc-PD-1H-CD24-GPI-P2A-EGFP for T cell BLI. d, C1498 BLI (CBR). e, Survival; from a single experiment. f, T cell BLI (Gaussia). Data are mean ± SEM of biological replicates. g,h, Experimental setup as in panels d–f, but with stress test 0.4 × 106 T cell dose and T cell BLI not performed. g, C1498 BLI from two combined experiments (left) and images from representative experiment (right). h, Survival. Log-transformed BLI AUC values were compared using a Vardi test with FDR correction. Survival differences were compared via pairwise log-rank test, with FDR correction.
Extended Data Fig. 9 Quad-CAR T cells including a single-___domain antibody-based CD19-CAR demonstrate cognate target lysis, but exhibit limited antileukaemia activity in vivo.
a, Diagram and vector maps for quad-CAR receptor array. b, Flow cytometry analysis of CAR expression. c, 24 h luciferase-based target lysis assay with quad-CAR B6 T cells and targets as listed. Data are mean ± SEM of triplicate wells for representative experiments from n = 2 donors. d–g, Sublethally irradiated albino B6 mice were injected with 1:1:1:1 ratio of C1498 singly expressing (hCD19, CD20, CD79bΔ, BAFF-R) and treated with dasatinib-cultured quad-CAR albino B6 T cells co-transduced with Gaussia luciferase vector gLuc-PD-1H-CD24-GPI-P2A-EGFP. d, C1498 BLI from three combined experiments with different BLI imaging timing. BLI AUC values were compared using a Vardi test with FDR correction. e, Survival. Differences in survival were compared with a log-rank test. f, Representative day 20 T cell BLI (Gaussia) and day 21 C1498 BLI (CBR) images. g, Antigen expression analysis from C1498 harvested from bone marrow of mice reaching humane endpoints. Percentage hCD19+ C1498 was compared with two-tailed t-test. Data are mean ± SEM of biological replicates.
Extended Data Fig. 10 Immunophenotype profiling of quad-CAR and quad-CAR triple-Switch T cells.
a–n, Resting and BM185-CD20-stimulated quad-CAR and quad-CAR triple-Switch T cells (as depicted in Fig. 6), were analysed by multiparameter spectral flow cytometry. Cells were stimulated at 1:1 E:T ratio with BM185-CD20 for 24 h or left unstimulated without IL-2. a, t-Distributed Stochastic Neighbour Embedding (t-SNE) projection with flowSOM metaclusters depicting combined stimulated and unstimulated CD4+ quad-CAR and quad-CAR triple-Switch T cells (n = 3 concatenated technical replicates from triplicate wells). b, tSNE projections illustrating flowSOM metaclusters separated by stimulated and unstimulated quad-CAR and quad-CAR triple-Switch T cell populations. c, tSNE projections depicting endogenous PD-1 and PD-1-OX40 switch receptor expression. d, Population distributions of stimulated (top) and unstimulated (bottom) T cell products in metaclusters. e, tSNE projections of selected protein expression for combined stimulated and unstimulated quad-CAR and quad-CAR triple-Switch T cells. f, Heat map depicting Z-score-transformed median fluorescence intensity expression values depicted for proteins in stimulated and unstimulated quad-CAR and quad-CAR triple-Switch T cells. g, Histograms depicting protein expression for selected markers. h–n, Concurrent analysis of CD8+ T cell populations as in panels a–g. Data are representative of n = 2 donor experiments with triplicate wells. d,k, Mean of technical replicates and individual data replicates are depicted.
Supplementary information
Supplementary Information
Supplementary figures.
Supplementary Table 1
Mutant RR12EE345L zipper sequences.
Supplementary Table 2
Zip-sort clonal abundance of most frequent chromosomal integrations.
Supplementary Table 3
Vector integration sites.
Supplementary Table 4
Construct sequences.
Supplementary Table 5
Element sequences.
Supplementary Table 6
Antibodies.
Supplementary Table 7
Vector integration study primer sequences.
Supplementary Data 1
Source data for Supplementary Figures 1–4.
Supplementary Video 1
CAR T cell cluster formation and target elimination.
Source data
Source data for Figs. 1–7 and Extended Fig. 1–10
Source data and statistics.
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James, S.E., Chen, S., Ng, B.D. et al. Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors. Nat. Biomed. Eng 8, 1592–1614 (2024). https://doi.org/10.1038/s41551-024-01287-3
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DOI: https://doi.org/10.1038/s41551-024-01287-3
