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CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape

Abstract

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1,2,3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4,5,6,7,8,9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.

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Data availability

All in vivo data can be found in the Source Data. The RNA-sequencing data are available from Gene Expression Omnibus (GEO) under accession number GSE126753. The proteomics data are available from PRIDE database under accession number PXD012833. Other data generated for this manuscript are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank R. Soni, B. Sénéchal, B. J. Safford, P. Vedantam, F. Tamzalit and G. Gunset for logistical and technical assistance. We also thank the SKI Cell Therapy and Cell Engineering, Molecular Cytology, Flow Cytometry, Integrated Genomics Operation, Microchemistry and Proteomics, Antitumor Assessment and Animal Core Facilities for their expert assistance. This work was supported by the Lake Road Foundation, the Lymphoma and Leukaemia Society, the Pasteur-Weizmann/Servier award and the NCI Cancer Center Support Grant P30 CA008748. SKI cores were in part supported by the Tow Foundation, Cycle for Survival, the Marie-Josée and Henry R. Kravis Center for Molecular Oncology and NCI grant P30 CA08748. A.D. and T.G. were supported by fellowships from The Canadian Institutes of Health Research and the Alexander S. Onassis Public Benefit Foundation, respectively.

Reviewer information

Nature thanks Wolfgang Schamel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

M. Hamieh designed the study, performed experiments, analysed and interpreted data, and wrote the manuscript. A.D., A.C., S.J.C.v.d.S., T.G., J.M.-S., J.E., Z.Z. and B.M.W. performed experiments. M.M.M. and Z.L. performed and analysed proteomics experiments. K.M.C. performed statistical analysis. M. Huse and R.C.H. designed experiments and interpreted data. X.W. and I.R. generated and provided clinical experimental materials. M.S. designed the study, analysed and interpreted data, and wrote the manuscript.

Correspondence to Michel Sadelain.

Ethics declarations

Competing interests

Memorial Sloan Kettering has submitted patent applications based in part on results presented in this manuscript (PCT/US18/68134, which has been licensed to several companies; M. Hamieh, J.M.-S. and M.S. are listed among the inventors; US provisional application no. 62/807,181; M. Hamieh and M.S. are listed among the inventors). R.C.H. reports stock ownership in Merck. I.R. and M.S. report research funding from Juno Therapeutics, Fate Therapeutics, Takeda Pharmaceuticals and Atara Biotherapeutics. I.R. serves on the scientific advisory board of Flow Design and M.S. on those of Berkeley Lights and St Jude Children Research Hospital.

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Extended data figures and tables

Extended Data Fig. 1 In vitro and in vivo characterization of 19-28ζ and 19-BBζ CAR T cells.

a, Schematic representation of the CD19 CARs. EC, extracellular domain; scFv, single chain variable region specific for anti-CD19; TM, transmembrane domain. b, c, Percentage of CAR+ T cells (left) and MFI of T cell surface CAR expression (right). b, CAR expression analysis performed 4 days after T cell transduction (n = 9 donors). c, CAR expression analysis performed 7 days after stimulation on NIH/3T3 cells expressing CD19 (n = 10 donors). d, Luciferase-based 18-h cytotoxic assay using NALM6 target cells at several E:T ratios (n = 3 donors). e, Kaplan–Meier survival analysis of NALM6 cell-bearing mice treated with 1 × 106, 0.4 × 106 or 0.2 × 106 transduced CAR T cells. Data are pooled from two independent experiments for the 0.2 × 106 CAR T cell dose. Corresponding bioluminescence imaging data are presented in Fig. 1a. f, Representative FACS profile of NALM6 and CAR T cells. NALM6 cells are GFP+; CAR T cells are LNGFR+. Absolute counts of NALM6 cells (left) and CAR T cells (right) at day 14 after infusion (n = 4 mice per group). g, Left, EOMES/T-bet ratio of CAR T cells. Right, fraction of CAR T cells expressing PD-1, LAG-3 and TIM-3 at day 14 after T cell infusion (n = 4 mice per group). In f and g, cells were isolated from the bone marrow of mice treated with 0.2 × 106 CAR T cells. h, Representative FACS profile of cells obtained by the time of relapse, 39 days after T cell infusion (19-28ζ, representative of 3 mice; 19-BBζ, representative of 9 mice). NA, not available (no detectable cells). P values were determined by two-sided Mann–Whitney U-test (b, c, f and g), or log-rank Mantel–Cox test (e). Data are mean ± s.e.m. Source data

Extended Data Fig. 2 Leukaemic response and relapse patterns associated with SJ25C1 and FMC63 scFv-based CARs.

a, Schematic representation of the CD19 CARs using the scFv SJ25C1 or FMC63. b, c, Kaplan–Meier survival analysis of mice bearing NALM6 cells treated with 0.2 × 106 transduced CAR T cells. n = 5–7 mice per group pooled from two independent experiments. (One of the groups shown in b is one of two experiments merged in Fig. 1a.) d, Representative FACS profile of cells isolated by the time of relapse (data are representative of n = 3 mice per group). e, f, Representative FACS profile of surface CD19 expression on NALM6 cells isolated by the time of relapse (data are representative of n = 3 mice per group). e, Mice treated with SJ25C1-28ζ or FMC63-28ζ. f, Mice treated with SJ25C1-BBζ or FMC63-BBζ. g, Representative FACS profile of PD-1, LAG-3 and TIM-3 surface expression on BBζ CAR T cells isolated from relapsed mice (data are representative of n = 3 mice per group). P values were determined by log-rank Mantel–Cox test (b, c). Source data

Extended Data Fig. 3 CD19 trogocytosis in vitro and in vivo.

a, Left, representative FACS profile of CD19 staining on CAR T cells isolated by the time of relapse from bone marrow or extramedullary tumour sites of mice treated with 19-BBζ CAR T cells. Right, MFI of CD19 on CAR T cells (19-BBζ, n = 9 mice; control, n = 4 mice). b, Cd19 mRNA expression in NALM6 cells of relapsed mice treated with 19-BBζ CAR T cells (fold change D0 versus NALM6 in vitro = 0.97; adjusted P = 0.919; fold change D6 vs D0 = 0.625, adjusted P = 0.23; n = 7 independent samples). c, MFI of CD19 on the segregated NALM6 cells in a transwell assay. NALM6 cells alone were plated at the top of the plate and NALM6 cells with CAR T cells at the bottom. Data were collected on days 1, 4, 7 and 14 (n = 3 independent samples). d, Left, diagram illustrating the co-culture of NALM6 cells and CAR T cells. Percentage and MFI of CD19 on NALM6 cell surface (middle) and on CAR T cell surface (right). Data were collected at 0, 1, 2 and 4 h after co-culture gated on live singlet cells (n = 4 donors). e, MFI of CD19 on NALM6 cell surface after 2 h of co-culture with 19-28ζ (left) or 19-BBζ (right) CAR T cells treated with the F-actin inhibitor latrunculin A (n = 5 donors). f, Intensity of heavy amino acid CD19-derived peptides detected in the lysates of trog+ and trog FACS-sorted cells after 1 h of co-culture with NALM6 cells expressing CD19–mCherry. CD19–mCherry-expressing NALM6 cells are used as a control (n = 2 donors). g, MFI of CD81 on NALM6 cell surface co-cultured with anti-CD19 CAR T cells (n = 3 donors). h, MFI of CD22 on NALM6 cell surface co-cultured with anti-CD19 CAR T cells (data are representative of three donors, n = 3 independent samples). i, MFI of CD22 on cell surface of residual NALM6 retrieved from mouse bone marrow 14 days after 19-BBζ CAR T cell treatment (0.2 × 106 CAR T cell dose, n = 3 mice). ns, non-significant. P values were determined by two-sided Mann–Whitney U-test (a, e and i), binomial false discovery rate-adjusted (b), or two-way ANOVA (d). Data are mean ± s.e.m. Source data

Extended Data Fig. 4 Trogocytosis occurs with several targets and cell types.

a, Top, MFI of CD19 on the cell surface of NALM6, SUP-B15, Raji and SK-OV-3-CD19+ cells co-cultured with 19-BBζ CAR T cells. Bottom, MFI of CD19 on CAR T cell surface (n = 3 donors). b, MFI of CD19 on NALM6 cells co-cultured with 19-28ζ CAR T cells from patients with ALL and CLL (n = 4 patient samples). c, MFI of CD22 on NALM6 cell surface (top) and CD22 CAR T cells (bottom). BCMA CAR T cells are used as control. d, MFI of BCMA on KMS-12-BM cell surface (top) and BCMA CAR T cells (bottom). CD22 CAR T cells are used as a control. e, MFI of MSLN on A549 MSLN+ cell surface (top) and MSLN CAR T cells (bottom). CD19 CAR T cells are used as a control. In ce, n = 3 independent samples, and data are representative of three donors. Data are mean ± s.e.m.

Extended Data Fig. 5 Therapeutic response and relapse patterns after CD22 CAR T cell treatment.

a, Tumour burden was monitored using bioluminescence imaging (average radiance (photons s−1 cm−2 sr−1)) in mice bearing NALM6–GFP luciferase treated with 0.2 × 106 CAR T cells 4 days later (n = 7 mice per group). b, Absolute count of CAR T cells. c, Quantification of CD22 molecules on NALM6 cell surface. d, Expression of PD-1, LAG-3 and TIM-3 on CAR T cell surface. bd, Cells were obtained from mice bone marrow at day 21 (relapse time, n = 5 mice per group). e, Quantification of CD22 molecules on NALM6 surface 6 days after ex vivo culture (CD22-28ζ and CD22-BBζ, n = 5 independent samples per group, NALM6, n = 3 independent samples) . NA, not available (no detectable cells). P values were determined by two-sided Mann–Whitney U-test. Data are mean ± s.e.m. Source data

Extended Data Fig. 6 Functional effects of CD19 acquisition by CAR T cells.

a, Diagram illustrating transduction of T cells with retroviral vector encoding CD19 under a PGK-100 promoter. Representative flow cytometry profile of CD19 expression on transduced T cells (n = 6 donors). b, Percentage of PD-1, LAG-3 and TIM-3 expression in trog+ and trog fractions (n = 4 donors). c, Diagram illustrating co-expression of CD19 and CAR on T cells. T cells were transduced with SFG vector expressing CD19 followed by CAR transduction 24 h later. After transduction, cells were left in culture for 6 days. d, Absolute count of CAR T cells (n = 6 donors). e, Percentage of T cells expressing of PD-1, LAG-3 and TIM-3 (n = 6 donors). P values were determined by two-sided Mann–Whitney U-test. Data are mean ± s.e.m.

Extended Data Fig. 7 Internalized CD19–CAR complex after trogocytosis.

a, Left, MFI of mCherry gated on CAR T cells after co-culture with NALM6 cells expressing CD19–mCherry. Right, MFI of goat anti-mouse IgG (GAM) gated on CAR T cells (n = 3 independent samples, data are representative of three donors). b, Left, representative FACS profile of CAR T cells stained with GAM and LNGFR in the presence or absence of NALM6 cells (data are representative of n = 4 mice per group). Right, MFI of GAM on CAR T cells isolated from mice bone marrow or extramedullary tumour sites by the time of relapse (n = 4 mice per group). c, MFI of GAM on CAR T cells co-cultured with autologous blasts derived from refractory/relapsed patients with ALL and CLL (n = 4 patient samples). d, Representative confocal microscopy images of NALM6 cell–CAR T cell conjugates. Data are representative of three donors (ten cells per experiments). e, Diagram illustrating CD19 trogocytosis by CAR T cells. P values were determined by two-sided Mann–Whitney U-test. Data are mean ± s.e.m. Source data

Extended Data Fig. 8 Generation of NALM6 cell lines with graded CD19 expression.

a, Genotype of edited CD19 locus in NALM6med (top) and NALM6low (bottom) cells. NALM6med and NALM6low cells were obtained by monoallelic and biallelic disruption of the Cd19 gene, respectively, using the CRISPR–Cas9 system. One of the alleles encodes a stop codon (asterisk); the second allele codes for a variant CD19 containing an amino acid substitution (Mut-1, 2 or 3aa). b, Prediction of the cleavage of CD19 protein sequence of the functional allele (Mut-3aa) in NALM6low cells (top) and the wild-type (WT) protein sequence (bottom) using signal 4.1 server. Cd19 gRNA was designed to target Cd19 exon 1, next to the site of cleavage of signal peptide. Data were obtained by deep sequencing. c, MFI of CD19 on the surface of NALM6 cells (NALM6wt), NALM6med cells, NALM6low cells and CD19-knockout NALM6 cells (NALM6KO) (n = 3 independent experiments). d, MFI of CD22 on the surface of NALM6 cells, NALM6med cells and NALM6low cell (n = 3 independent experiments). Data are mean ± s.e.m.

Extended Data Fig. 9 T cell cooperativity and antigen density determine CAR T cell killing capacity.

Tumour lysis frequency in wells containing a ratio of one CAR T cell:one tumour cell (R1:1 E:T) and one CAR T cell:two tumour cells (R2:1 E:T). a, c, d, NALM6 cells (a), NALM6med cells (c) and NALM6low cells (d). b, Estimated and observed killing percentage in wells containing one CAR T cell and two NALM6 cells (R2:1 E:T). Estimated percentage of killing is calculated as described in the Methods. P values were determined by two-sided two-sample test of proportions (a, c and d) and one-sided two-sample test of proportions (b), n = 5 donors (a), and n = 3 donors (c, d). In b, P < 0.1 is used as significance level (n = 5 donors). Data are mean ± s.e.m. Primary data in this figure are presented in Fig. 3.

Extended Data Fig. 10 Modelling of anticipated outcomes after single or combinatorial CAR T cell treatment schemas.

ah, Antigen-positive relapse (ad) versus tumour control scenarios (eh) after single or combinatorial CAR T cell targeting. a, Antigen high relapse of residual tumour cells in the absence of CAR T cells. b, Antigen-low relapse in the presence of insensitive or exhausted CAR T cells. c, Tumour relapse after sequential combinatorial targeting failing against tumour cells with low antigen densities. Tumour rejection could overcome the relapse scenarios (ac) by achieving a higher effector:target ratio (eg), after higher T cell dose infusion or greater post-infusion expansion, operating through additive or cooperative tumour elimination. In d and h, combinatorial targeting is mediated by dual-targeted CAR T cells, which may still fail in the face of low antigen densities (d) depending on the co-stimulatory combinations (h). In all cases, low antigen density may be either constitutively low or actively lowered after CAR T cell-mediated trogocytosis. Antigen-negative escapes are not represented. Other, yet undiscovered, escape mechanisms may exist.

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Further reading

Fig. 1: Trogocytic antigen extraction promotes tumour escape.
Fig. 2: Antigen density reduction differentially impacts CAR T cell activity.
Fig. 3: CAR T cell cooperativity augments insufficient clonal tumour-lytic potential.
Fig. 4: Rational combinatorial targeting overcomes antigen-low tumour escape.
Extended Data Fig. 1: In vitro and in vivo characterization of 19-28ζ and 19-BBζ CAR T cells.
Extended Data Fig. 2: Leukaemic response and relapse patterns associated with SJ25C1 and FMC63 scFv-based CARs.
Extended Data Fig. 3: CD19 trogocytosis in vitro and in vivo.
Extended Data Fig. 4: Trogocytosis occurs with several targets and cell types.
Extended Data Fig. 5: Therapeutic response and relapse patterns after CD22 CAR T cell treatment.
Extended Data Fig. 6: Functional effects of CD19 acquisition by CAR T cells.
Extended Data Fig. 7: Internalized CD19–CAR complex after trogocytosis.
Extended Data Fig. 8: Generation of NALM6 cell lines with graded CD19 expression.
Extended Data Fig. 9: T cell cooperativity and antigen density determine CAR T cell killing capacity.
Extended Data Fig. 10: Modelling of anticipated outcomes after single or combinatorial CAR T cell treatment schemas.

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