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Generation of T-cell-receptor-negative CD8αβ-positive CAR T cells from T-cell-derived induced pluripotent stem cells

Nature Biomedical Engineering volume 6, pages 1284–1297 (2022)Cite this article

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An Author Correction to this article was published on 12 February 2024

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Abstract

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of ‘off-the-shelf’ CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR– CD8αβ+ CAR T cells that perform similarly to CD8αβ+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αβ+ T cells for a broad range of immunotherapies.

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Fig. 1: DLL4 supports in vitro αβTCR-T-cell development of WT-TiPS but not CAR-TiPS.
Fig. 2: TRAC-controlled 1928z-1XX CAR expression facilitates DP T-cell development.
Fig. 3: CAR regulation influences Notch and TCR target gene induction.
Fig. 4: 4-1BBL co-stimulation enhances CD8αβ TRAC-1XX-iT proliferation and function.
Fig. 5: CD8αβ TRAC-1XX-iT cells resemble peripheral-blood-derived CD8αβ T cells.
Fig. 6: TRAC-1XX-iT have improved persistence and function over CAR-iT cells.
Fig. 7: TRAC-1XX-iT cells cure a systemic NALM6 tumour model without inducing graft-versus-host disease.

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Data availability

The RNA-sequencing data are available from the Gene Expression Omnibus under accession number GSE210364. Source data for tumour growth are provided with this paper. The raw and analysed datasets generated during the study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank G. Gunset for logistical and technical assistance, M. Sættersmoen for advice on NK-cell culture, A. Iyer for support with statistical analyses and E. Ortiz for support in cell culture. We thank the SKI Cell Therapy and Cell Engineering Facility, the Flow Cytometry core facility, Integrated Genomics Operation, Antitumor Assessment and Animal Core Facilities for their expert assistance. We also thank Y.-S. Lai, C.-W. Chang, A. Witty, B.-H. Yang, M. Ribadi, J. Huffman, H. Shaked, R. Bjordahl and B. Whitlock (Fate Therapeutics Inc.) for technical contributions. This work was supported by the Tri-I Stem Cell Initiative, the Tow Foundation, Cycle for Survival, the Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Fate Therapeutics Inc., and NCI grant P30 CA08748. S.J.C.v.d.S. and M.T. were supported by a New York Stem Cell Foundation Druckenmiller Fellowship.

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  1. Justin Eyquem

    Present address: Gladstone-UCSF Institute of Genomic Immunology, Gladstone Institutes, San Francisco, CA, USA

Authors and Affiliations

  1. Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Sjoukje J. C. van der Stegen, Pieter L. Lindenbergh, Roseanna M. Petrovic, Hongyao Xie, Mame P. Diop, Vera Alexeeva, Yuzhe Shi, Jorge Mansilla-Soto, Mohamad Hamieh, Justin Eyquem, Annalisa Cabriolu & Michel Sadelain

  2. Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Sjoukje J. C. van der Stegen, Pieter L. Lindenbergh, Roseanna M. Petrovic, Hongyao Xie, Mame P. Diop, Vera Alexeeva, Yuzhe Shi, Jorge Mansilla-Soto, Mohamad Hamieh, Justin Eyquem, Annalisa Cabriolu & Michel Sadelain

  3. Department of Hematology, Cancer Center Amsterdam, Amsterdam UMC, VU Amsterdam, Amsterdam, the Netherlands

    Pieter L. Lindenbergh & Maria Themeli

  4. Cell Therapy and Cell Engineering Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Xiuyan Wang & Isabelle Riviere

  5. Fate Therapeutics Inc, San Diego, CA, USA

    Ramzey Abujarour, Tom Lee, Raedun Clarke & Bahram Valamehr

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Contributions

S.J.C.v.d.S. designed the study, performed experiments, analysed and interpreted data, and wrote the manuscript. P.L.L. and R.M.P. performed experiments, analysed and interpreted data. H.X. performed RNA-sequencing analysis. M.P.D., V.A., Y.S., M.H., J.M.-S., J.E. and A.C. performed experiments. X.W. and I.R. generated and provided clinical experimental materials. R.A., T.L., R.C. and B.V. generated TiPS lines, developed iCD34 methodology. M.T. and I.R. contributed to experimental design and data analysis. M.S. designed the study, analysed and interpreted data and wrote the manuscript.

Corresponding author

Correspondence to Michel Sadelain.

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Competing interests

R.A., T.L., R.C. and B.V. are employees of Fate Therapeutics Inc. and have equity in the company. M.S. reports research funding from Takeda Pharmaceuticals, Atara Biotherapeutics and Fate Therapeutics. M.S. served on the scientific advisory board of St Jude Children’s Research Hospital. Memorial Sloan Kettering has licensed intellectual property on which M.S. is a named inventor to Fate Therapeutics. M.S. does not receive consultation fees or forms of remuneration. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Laurent Poirot, Axel Schambach and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 T lymphoid commitment of hES, FiPS and TiPS on OP9-mDLL1.

a, Flow cytometric analysis of pluripotency marker expression on H1, FiPS and WT-TiPS b, Flow cytometric analysis of T lymphoid markers of H1, FiPS and WT-TiPS during differentiation on OP9-mDLL1 at indicated timepoints. Plots depicting CD7/CD5 are gated on live CD45+ cells, plots depicting CD3/TCRαβ, CD4/CD8α and CD8α/CD8β are gated on live CD45+CD7+ cells. CD3/TCRαβ and CD4/CD8α at D40 are as presented in Fig. 1b.

Extended Data Fig. 2 Generation, validation, and differentiation of TRAC–/–-TiPS.

a, CRISPR/Cas9-targeted integration of EF1a-GFP-P2A-Puromycing-bGHpA (G2AP) expression unit into the TRAC locus. Top, TRAC locus; middle, plasmid containing the G2AP expression unit flanked by homology arms; bottom, edited TRAC locus. ‘FWD’ and ‘REV’ indicate the location of the forward and reverse primers used in b. b, PCR validation of G2AP integration into the TRAC locus of TiPS clones. c, Flow cytometric analysis of pluripotency marker expression on TRAC–/–-TiPS. Gated on live cells. d, T lymphoid makers of WT-TiPS and TRAC–/–-TiPS during differentiation on OP9-mDLL1 at the indicated timepoints. Gated on live CD45+ cells. D40 is as presented in Fig. 1c.

Extended Data Fig. 3 Early T lymphoid commitment of WT-TiPS and CAR−TiPS on human Notch ligands.

a, SFG γRV plasmid design to transduce human Notch ligands (DLL1, DLL4, JAG1 or JAG2) into parental OP9 cells. b, Notch ligand expression on engineered OP9 lines. Filled grey histogram corresponds to stained parental OP9 cells, and open black histogram to transduced OP9 cells. c, DTX1 induction in WT-TiPS by OP9 expressing indicated Notch ligand. D20 differentiating WT-TiPS cells were co-cultured with indicated OP9. DTX1 induction was measured by ddPCR, relative to endogenous RPL13A. The fold change was calculated relative to 0 h. Data shown is average of n = 2 technical replicates. d, g, Flow cytometric analysis of T lymphoid commitment marker expression (CD7, CD5, TCRαβ and CD56) of WT-TiPS (d) and CAR−TiPS (g) differentiated on OP9 expressing indicated human Notch ligands. Gated on live CD45+ cells. e, Flow cytometric analysis of pluripotency marker expression on CAR-TiPS. Gated on live cells. f, Phosphorylated-ERK1/2 levels in WT-TiPS (blue) and CAR-TiPS (red) on D35 (n = 3 technical replicates, p = 0.0014). h, Phenotype (left panels) and apoptosis levels (right panels) of WT-TiPS (top) and CAR-TiPS (bottom) from D27 – D35 of differentiation on OP9-DLL4. Percentage of apoptotic cells in each T lineage developmental stage was based on percentage live Annexin-V+ cells. * P < 0.05, ** P < 0.01, *** P < 0.001, Welch’s 2-sample two-sided t test, data are means ± s.d (f).

Extended Data Fig. 4 CD8αβ single positive CAR+ iT cell development.

WT-TiPS were differentiated on OP9-DLL4 and transduced to express the 1928z CAR at D35 utilizing γRV SFG-1928z-P2A-LNGFR. Cells were expanded for 7 days in expansion media supplemented with IL-2. a, CD4/CD8αβ expression prior to transduction (D35) and on D42 in LNGFR+ cells, LNGFR- cells and untransduced control cells which remained in differentiation on OP9-DLL4. Gated on live CD45+ cells. b, Cytotoxic activity of CAR+ iT cells in a 18 h bioluminescence assay, using FFLuc-NALM6 as target cells (n = 3 technical replicates, data are mean ± s.d). c, CRISPR/Cas9-targeted integration of CAR transgene into the TRAC locus. Top, TRAC locus; middle, plasmid containing the CAR transgene cassette flanked by homology arms; bottom, edited TRAC locus. d, f, PCR validation of CAR integration into the TRAC locus of TRAC-1928z-TiPS (d) and TRAC-1XX-TiPS (f) clones. e, g, Pluripotency marker expression on TRAC-1928z-TiPS (e) and TRAC-1XX-TiPS (g), gated on live cells.

Extended Data Fig. 5 T lineage commitment of TRAC-CAR-TiPS.

a, T lineage commitment marker expression (CD7/CD5, CD4/CD8α, CD8α/CD8β) of WT-TiPS (left), TRAC-1928z-TiPS (middle) and TRAC-1XX-TiPS (right) on OP9-DLL4 at the indicated timepoints. CD7/CD5 is gated on live CD45+ cells, others are gated on live CD45+CD7+ cells. b, Flow cytometric analysis of T cell phenotype markers of D35 DP TRAC-1XX-iT cells. Gated on live CD45+CD7+CD4+CD8αβ+ cells. c, Intracellular and cell-surface expression of CD3 and TCRαβ on D35 TRAC-1XX-iT cells.

Extended Data Fig. 6 Tonic ITAM phosphorylation in CAR+ T cells.

a, Representative flow cytometry plot of CAR expression and pITAM1 (top panel) or pITAM3 (bottom panel) in PBMC-derived T cells expressing γRV-1928z, TRAC-1928z or TRAC-1XX (gated on live CAR+), or in control TRAC–/– cells (gated on live CAR-). b, c, Percentage of pITAM1+ (b) and pITAM3+ (c) in the populations shown in a (n = 4–5 biological replicates, data are means ± s.d.).

Extended Data Fig. 7 DP TRAC-1XX-iT cell mature to CD8αβ SP iT cells on 3T3-CD19-41BBL.

a, c, Flow cytometric analysis of D42 cells matured on 3T3-CD19 (a) or 3T3-CD19-41BBL (c). Gated on live CD45+CD7+ cells. b, Flow cytometric analysis of D35 and D42 phenotypes of stimulated DP TRAC-1XX-iT cells. D35 TRAC-1XX-iT cells were sorted for a CD4+CD8αβ+ DP phenotype, stimulated on 3T3-CD19-41BBL and expanded for seven days. Gated on live CD45+CD7+ cells. d, Fold Expansion and T cell phenotype marker expression of TRAC-1XX-iT cells matured on 3T3-CD19-41BBL (3T3) or recombinant CD19-Fc. e, 4 h cytotoxicity assay of 3T3-CD19-41BBL stimulated TRAC-1XX-iT cells in response to NALM6-CD19+ (red) an NALM6-CD19–/– (blue) as target cells (n = 3 technical replicates, data are means ± s.d.) f, CD19 expression on primary CLL cells. Filled grey histogram are unstained CLL cells, open red histogram are stained CLL cells.

Extended Data Fig. 8 Comparison of CD8αβ TRAC-1XX-iT cells and peripheral blood lymphocytes.

a, Representative examples of lymphoid phenotype marker expression in CD8αβ TRAC-1XX-iT (CD8αβ iT, red), CD8αβ αβTCR-T (CD8, blue), CD4 αβTCR-T (CD4, orange), γδTCR-T (γδ, green) and NK cells (NK, purple). CD8αβ TRAC-1XX-iT cells are the same as represented in Fig. 5a. b, Variability of lymphoid phenotype marker expression in CD8αβ TRAC-1XX-iT cells (n = 3-4 biological replicates, data are means ± s.d). Biological replicates shown are samples utilized in RNA analysis (Fig. 5b,c). c, Principal Component Analysis comparing TRAC-1XX CD8αβ αβTCR-T cells (CD8, n = 4), TRAC-1XX CD4 αβTCR-T cells (CD4, n = 3), γRV-1XX γδTCR-T cells (γδ, n = 4), γRV-1XX NK cells (NK, n = 4) and CD8αβ+ TRAC-1XX-iT cells (iT CD8αβ, n = 4).

Extended Data Fig. 9 Functional comparison of TRAC-1XX-iT, CAR-iT and CD8 TRAC-1XX.

Functional comparison of healthy-donor peripheral blood TRAC-1XX CD8αβ αβTCR-T (CD8 TRAC-1XX), CAR-iT and TRAC-1XX-iT cells. CD8 TRAC-1XX cell doses represent number of CAR+ cells utilized in the assay. a, CAR and CD3 expression in CD8 TRAC-1XX, CAR-iT and TRAC-1XX-iT cells (black line) compared to unstained control (grey filled histogram). b, 18-h Incucyte cytotoxicity assay with NLR+ CD19–/– NALM6 target cells (n = 3 technical replicates). c, 4 h intracellular cytokine detection in T cells stimulated with NALM6 CD19+ target cells (at a 1:1 E:T), PMA/Ionomycin, NALM6 CD19–/– target cells (at a 1:1 E:T) unstimulated controls (n = 3 technical replicates). d, Twenty-four h cytokine secretion using NALM6-CD19–/– as target cells at a 1:1 E:T (n = 11-18 biological replicates, left panel) or unstimulated control (n = 11-18 biological replicates, right panel). e, Schematic representation of the NALM6 in vivo tumour model. f, Kaplan-Meier analysis of tumourfree survival (2×106 TRAC-1XX-iT vs 2×106 CD8 TRAC-1XX p = 0.0062, 2×106 TRAC-1XX-iT vs 1×105 CD8 TRAC-1XX p = 0.0034). * P < 0.05, ** P < 0.01, *** P < 0.001, Chi-Square test (b) log-rank Mantel-Cox test (f). All data are means ± s.d.

Extended Data Fig. 10 TRAC-1XX-iT function compared to healthy donor peripheral blood-derived CD8 TRAC-1XX T cells.

In vivo functional comparison of healthy-donor peripheral blood TRAC-1XX CD8αβ αβTCR-T (CD8 TRAC-1XX), TRAC-1XX-iT cells. CD8 TRAC-1XX cell doses represent number of CAR+ cells utilized in the assay. a, CAR and CD3 expression in CD8 TRAC-1XX and TRAC-1XX-iT cells (black line) compared to unstained control (grey filled histogram). b, Enumeration of tumour cells in the bone marrow and T cells in bone marrow, spleen and blood 6 days or 12 days after T-cell infusion (n = 2-3 mice, T cell in bone marrow day 12, CD8 TRAC-1XX vs TRAC-1XX-iT p = 0.0161, T cells in spleen day 12 CD8 TRAC-1XX vs TRAC-1XX-iT p = 0.0052). c, Phenotype of CD8 cells prior to infusion (day 0, n = 1) and of cells derived from the bone marrow on day 6 (n = 3 mice) and 12 (n = 3 mice). * P < 0.05, ** P < 0.01, *** P < 0.001 Welch’s 2-sample two-sided t test (b). All data are means ± s.d.

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van der Stegen, S.J.C., Lindenbergh, P.L., Petrovic, R.M. et al. Generation of T-cell-receptor-negative CD8αβ-positive CAR T cells from T-cell-derived induced pluripotent stem cells. Nat. Biomed. Eng 6, 1284–1297 (2022). https://doi.org/10.1038/s41551-022-00915-0

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