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Clonal expansion of T memory stem cells determines early anti-leukemic responses and long-term CAR T cell persistence in patients

Nature Cancer volume 2, pages 629–642 (2021)Cite this article

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Abstract

Low-affinity CD19 chimeric antigen receptor (CAR) T cells display enhanced expansion and persistence, enabling fate tracking through integration site analysis. Here we show that integration sites from early (1 month) and late (>3 yr) timepoints cluster separately, suggesting different clonal contribution to early responses and prolonged anti-leukemic surveillance. CAR T central and effector memory cells in patients with long-term persistence remained highly polyclonal, whereas diversity dropped rapidly in patients with limited CAR T persistence. Analysis of shared integrants between the CAR T cell product and post-infusion demonstrated that, despite their low frequency, T memory stem cell clones in the product contributed substantially to the circulating CAR T cell pools, during both early expansion and long-term persistence. Our data may help identify patients at risk of early loss of CAR T cells and highlight the critical role of T memory stem cells both in mediating early anti-leukemic responses and in long-term surveillance by CAR T cells.

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Fig. 1: Immunophenotypic characterization of patients with long-lasting CAR T cells in the product and after infusion over time.
Fig. 2: Immunophenotypic characterization of patients with short-living CAR T cells in the product and after infusion over time.
Fig. 3: Distribution and abundance of ISs collected in the product and after infusion over time in Pt4 and Pt6.
Fig. 4: Diversity of ISs in different T cell subtypes over time from Pt4.
Fig. 5: Diversity of ISs in different T cell subtypes over time from Pt6.
Fig. 6: Tracking of IS in different T cell subtypes over time in Pt4 and Pt6.
Fig. 7: Diversity of ISs in different T cell subtypes over time from Pt10 and Pt17.
Fig. 8: Comparison of IS distribution pre- and post-infusion.

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Data availability

The fastq files relative to sequencing of IS amplicons generated for this study are available through the NCBI repository (https://submit.ncbi.nlm.nih.gov/), BioProject accession number PRJNA718947. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

No new code was generated or used in this manuscript.

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Acknowledgements

This work was funded the JP Moulton Foundation and supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. P.J.A. is a recipient of an NIHR Research Professorship which also supported S.G. M.P. is supported by the National Institute of Health Research University College London Hospital Biomedical Research Centre. A.J.T. is a recipient of a Wellcome Trust Senior Fellowship. The work of L.B. was supported by the Wellcome Trust (grant no. 104807/Z/14/Z, Principal Research Fellowship awarded to A.J.T.) and the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK. L.B. is also currently Director of R&D for the gene therapy company AVROBIO located in Cambridge, MA, USA (none of what is presented in this work is supported by or relates to AVROBIO). The work of L.B. was also in part performed using the resources of the Gene Therapy Program of the Dana Farber/Boston Children’s Cancer and Blood Disorders Center.

Author information

Authors and Affiliations

  1. Molecular and Cellular Immunology Section, UCL Great Ormond Street Institute of Child Health, London, UK

    Luca Biasco, Natalia Izotova, Christine Rivat, Rachel Richardson, Aleks Guvenel, Adrian J. Thrasher & Persis J. Amrolia

  2. Gene Therapy Program, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, USA

    Luca Biasco

  3. Molecular Haematology Section, UCL Great Ormond Street Institute of Child Health, London, UK

    Sara Ghorashian

  4. Department of Haematology, University College London Hospital, London, UK

    Rachael Hough

  5. Department of Bone Marrow Transplant, Royal Manchester Children’s Hospital, Manchester, UK

    Robert Wynn

  6. CRUK UCL Cancer Trials Centre, University College London, London, UK

    Bilyana Popova & Andre Lopes

  7. University College London Cancer Institute, London, UK

    Martin Pule

  8. Department of Bone Marrow Transplantation, Great Ormond Street Hospital, London, UK

    Persis J. Amrolia

Authors
  1. Luca Biasco
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Contributions

L.B. designed and co-supervised the study, performed computational analyses and wrote the manuscript. N.I. and C.R. performed cell isolation and molecular insertion sites retrieval. S.G. and R.R. collected and provided clinical sample material for analysis. A.G. cryopreserved study samples and performed flow cytometric staining. R.H. and R.W. were Principal Investigators for the clinical study. B.P. wrote study documentation and provided trial management. A.L. provided statistical analysis for the study. M.P. generated the CAR construct and participated in its preclinical characterization. A.J.T. conceived the idea and participated in the experimental design and data analysis. P.J.A. supervised the study as PI and wrote the manuscript.

Corresponding author

Correspondence to Persis J. Amrolia.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks Justin Eyquem, Alena Gros and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.' after the Supplementary information section.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Immunophenotyping of CD3+ cells in Pt4 and Pt6.

Additional FACS plot on CD62L+CD45RA+CD95 expression in CAR+ T cells, subtype frequencies within total CD3+ cells and relative summary of data on the CD3+ T cell composition overtime for Pt4 (top panels) and Pt6 (bottom panels). Percentages of CD62L/CD45RA compartments are showed inside each gate. Longitudinal contribution of each subpopulations to the CD3+ cell compartment is shown below for each patient according to the color code legend on the right.

Source data

Extended Data Fig. 2 Integration sites collected from Pt4 and Pt6.

Summary of number of IS (grey bars) and relative sequencing reads (white bars) in Pt4 (top panel) and Pt6 (bottom panel) (d = days after treatment).

Source data

Extended Data Fig. 3 Distribution of integration sites collected from Pt4 and Pt6.

Distribution of IS with respect to TSS (left plots) or gene content of the loci (right plots) in the cell product (red) or after infusion at early or late timepoints after treatment (blue) for Pt4 (top panels) and Pt6 (bottom panels).

Extended Data Fig. 4 Gene categories relative to integration sites collected from Pt4 and Pt6.

Plots on the left show word clouds of hit genes in the cell product (in red) and after infusion at early or late timepoints after treatment (in blue) for Pt4 (top panels) and Pt6 (bottom panels). Relative gene enrichment analysis for top biological processes of hit genes relative to each word cloud in the product (red bars) or after infusion (blue bars) is shown on the right plots (significance reported as -log10 binomial p-value from one-tailed tests decreasing from top to bottom).

Extended Data Fig. 5 Relative abundance and diversity of integration sites collected from Pt4 and Pt6.

Scattered dot plots showing relative abundance of IS in the product (a) and at early (b) or late (c) timepoints after treatment in Pt4 (plots on the left) and Pt6 (plots on the right) (d = days after treatment, m = months after treatment). Mean percent abundance is shown as a dotted line for each sample. Number of events in each dataset is equal to what reported in Extended Data Fig. 2. d) Longitudinal plots showing Gini/Simpson Diversity Index (left y-axis) of IS overtime in TSCM (orange lines) and in TCM/TEM (dark blue) and in all T cells (green lines). The grey lines show the percentage of CAR cells overtime (right y-axis).

Source data

Extended Data Fig. 6 Correlation of integration sites and number of cells collected from Pt4 and Pt6.

Longitudinal plots showing number of IS collected (a) and number of cells collected (b) in TSCM (orange lines) and in TCM/TEM (dark blue) at early timepoints for Pt4 (left panels) and Pt6 (right panels). The plot in (c) shows the correlation observed between number of cells collected (y-axis) and clonal diversity for all samples and both patients (x-axis, Shannon Diversity Index) shown as blue dots. Interpolation with best fit curve and R squared value are shown in black.

Source data

Extended Data Fig. 7 Integration sites collected from Pt10 and Pt17.

Summary of number of IS (grey bars) and relative sequencing reads (white bars) in Pt10 (top panel) and Pt17 (bottom panel) (d = days after treatment).

Source data

Extended Data Fig. 8 Sharing of integration sites in the product with the CAR T populations in Pt10 and Pt17 after infusion.

Ring plots showing relative contribution from each subtype (coloured section of the ring) to the pool of IS detected in the product and at early timepoints in Pt10 (left) and Pt6 (right). The total number of IS captured both in the product and after infusion is shown inside each plot. The relative percentage of IS belonging to each T cell subtype of the product that were shared with samples after infusion is shown in white inside each section of each ring plot.

Supplementary information

Source data

Source Data Fig. 1

Raw datasets relative to figure panels.

Source Data Fig. 2

Raw datasets relative to figure panels.

Source Data Fig. 4

Raw datasets relative to figure panels.

Source Data Fig. 5

Raw datasets relative to figure panels.

Source Data Fig. 6

Raw datasets relative to figure panels.

Source Data Fig. 7

Raw datasets relative to figure panels.

Source Data Fig. 8

Raw datasets relative to figure panels.

Source Data Extended Data Fig. 1

Raw datasets relative to figure panels.

Source Data Extended Data Fig. 2

Raw datasets relative to figure panels.

Source Data Extended Data Fig. 5

Raw datasets relative to figure panels.

Source Data Extended Data Fig. 6

Raw datasets relative to figure panels.

Source Data Extended Data Fig. 7

Raw datasets relative to figure panels.

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Biasco, L., Izotova, N., Rivat, C. et al. Clonal expansion of T memory stem cells determines early anti-leukemic responses and long-term CAR T cell persistence in patients. Nat Cancer 2, 629–642 (2021). https://doi.org/10.1038/s43018-021-00207-7

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