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Synthetic dual co-stimulation increases the potency of HIT and TCR-targeted cell therapies

Nature Cancer (2024)Cite this article

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Abstract

Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies.

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Fig. 1: Synthetic co-stimulatory molecule (80BB) enhances 19-HIT T cell antitumor function.
Fig. 2: 80BB provides dual 4-1BB and CD28 co-stimulation required for optimal antitumor function.
Fig. 3: CTLA4 binding to 80BB enhances 19-HIT T cell antitumor function.
Fig. 4: 80BB enhances TCR-engineered T cell control of subcutaneous melanomas.
Fig. 5: 80BB-endowed TILs exert improved antitumor function in vitro and in vivo.

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Data availability

The single-cell RNA-sequencing data are available from the Gene Expression Omnibus under accession code GSE223211. 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

Code used to analyze the single-cell RNA-sequencing data in this manuscript is available at https://github.com/Sadelain-Lab/Dobrin-et-al-NC-2024.git.

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Acknowledgements

We thank G. Gunset and M. Lopez for logistical and technical assistance. We thank the Sadelain lab for helpful comments and discussion. We thank J. Khan for helpful discussion on mouse models. We thank the Cell Therapy and Cell Engineering Facility, the Antitumor Assessment Facility, the Animal Core Facility, the Flow Cytometry Core Facility and the Integrated Genomics Operation, which are, in part, supported by NCI Cancer Center Support grant P30 CA08748, Cycle for Survival and the Marie–Josée and Henry R. Kravis Center for Molecular Oncology, for their expert assistance. This work was supported by the Lake Road Foundation, the Lymphoma and Leukemia Society, the Pasteur–Weizmann/Servier award and the Leopold Griffuel award (M.S.) and the Canadian Institutes of Health Research (DFSA fellowship; A.D.). The mouse illustration in Figs. 1e, 4c and 5f and Extended Data Fig. 6d was generated using Servier Medical Art, Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

Author information

Author notes

  1. Mohamad Hamieh

    Present address: Department of Pediatrics and Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA

Authors and Affiliations

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

    Anton Dobrin, Pieter L. Lindenbergh, Yuzhe Shi, Karlo Perica, Hongyao Xie, Nayan Jain, Michel Sadelain & Mohamad Hamieh

  2. Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Anton Dobrin

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

    Anton Dobrin, Karlo Perica, Michel Sadelain & Mohamad Hamieh

  4. Cell Therapy Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Karlo Perica

  5. Thoracic Oncology Service, Division of Solid Tumour Oncology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Andrew Chow

  6. Department of Medicine and Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA

    Jedd D. Wolchok

  7. Department of Pharmacology and Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA

    Taha Merghoub

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Contributions

A.D. designed the study, performed experiments, analyzed and interpreted the data and wrote the paper. P.L.L., Y.S. and N.J. performed experiments and contributed to data analysis. K.P. and H.X. analyzed and interpreted the data. A.C., J.D.W. and T.M. generated and provided materials. M.S. designed the study, analyzed and interpreted the data and wrote the paper. M.H. designed the study, performed experiments, analyzed and interpreted the data and wrote the paper.

Corresponding author

Correspondence to Michel Sadelain.

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Competing interests

Memorial Sloan Kettering has submitted a patent application based, in part, on results presented in this manuscript (A.D., M.H. and M.S. are listed among the inventors). M.S. reports research funding from Fate Therapeutics, Takeda Pharmaceuticals, Atara Biotherapeutics and Mnemo Therapeutics. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Cell engineering diagram and extended characterization of 19-HIT80BB cells.

a, Diagram illustrating genetic T cell engineering strategy of 19-HIT and 19-HIT80BB cells. The alpha and beta chains of HIT directed against CD19 were targeted into the TRAC locus as previously described37. 80BB construct was delivered using a γ-retroviral vector. b, Flow cytometry profile of HIT expression on 19-HIT (blue) and 19-HIT80BB (red) measured using a goat anti-mouse antibody (GaM). c, 48 hr serial in vitro cytotoxicity assay using NALM6 targets. Cells were plated at an effector: target ratio of 1:2. n = 7 independent co-cultures of cells from a healthy donor. d, 18 hr cytotoxicity assay with NALM6 and NALM6CD19 KO. n = 3 independent co-cultures of cells from a healthy donor, representative of 4 donors. e, Human IL-2, TNFα and IFNɣ supernatant concentration 24 hr post co-culture with NALM6 target cells at E:T ratio of 1:2. n = 4 independent co-cultures of cells from a healthy donor, representative of 3 donors. f, Serial in vitro cytotoxicity assay with fresh NALM6 cells added every 2 days, starting with sorted CD4 (left) and CD8 T cells (right) at a ratio of 1 effector: 2 targets. Each line is an independent co-culture of cells from a healthy donor. g, Flow cytometry profiles of CD80, CD86 and PD-L1, 4-1BBL and CTLA4 on NALM6 cells. h, Kaplan–Meier survival analysis of NALM62000 (2000 CD19 mol/cell, left) or NALM6200 (200 CD19 mol/cell, right) -bearing mice treated with 1 × 105 (left) or 4 × 105 (right); respectively, 19-HIT + T cells. n = 5 mice/group. i, j, Absolute count of total (i, left), CD4 (i, middle), CD8 (i, right) T cells and absolute count of NALM6 cells (j) were isolated from bone marrow of NALM6 bearing mice seventeen days post 1 × 105 19-HIT or 19-HIT80BB cell treatment. n = 4 mice/group treated with 1 × 105 19-HIT + T cells/mouse. P values were determined by two-tailed t-test (d, e), Mann-Whittney test (i, j), or log-rank Mantel-Cox test (h). Data are mean ± sem.

Source data

Extended Data Fig. 2 UMAP projections and Suerat clustering of 19-HIT and 19-HIT80BB cells.

a, UMAP projection of 2591 cells isolated from bone marrow of NALM6 bearing mice nine days post 19-HIT or 19-HIT80BB treatment. Cells are coloured based on Suerat assigned cluster identities. Clusters 0, 1, 2, 3 and 5 are composed of 19-HIT80BB cells. Cluster 4 is predominantly composed of 19-HIT cells. b, Feature and violin plots of CD8A (left), CD4 (right) transcripts. c, Dot plot of top 10 differentially enriched genes in each cluster. Colour of dot indicates expression level, and size of dot indicated percentage of cells expressing marker. d, Feature and violin plots of MKI67 (left), IL7R (right). For violin plots, cells are plotted segregated by cluster and by treatment type.

Extended Data Fig. 3 Feature plots of selected inhibitory, exhaustion and cytotoxicity genes.

a, b Feature plots of inhibitory and exhaustion (a) and cytotoxicity (b) genes corresponding to violin plots in Fig. 1f.

Extended Data Fig. 4 Extended characterization of 80BB dependence on 4-1BB and CD28 signaling.

a, Left, representative flow cytometry profiles of 19-HIT, 19-HIT80BB, 19-HIT80ΔBB and 19-HITCD80 stained for CD80. Representative of 4 independent healthy donors. Right, summary of CD80 MFI and percentage from 3 independent donors analyzed at same flow cytometry settings. b, T cells were engineered with LNGFR (control), 80BB or 80ΔBB and unstimulated or stimulated with anti-CD3 beads for 1 hr at a 4:1 bead to T cell ratio. Phospho PLCyY783, total PLCy, phospho-AKTS473, total ATK, phospho-p65S536, total p65, phospho-ERKT202/Y204 and total ERK were measured by western blot. Western blots representative of two technical replicates. c, Flow cytometry plots of CD28-Fc binding assay. T cells were incubated with increasing concentrations of CD28-Fc, and then stained with PE-anti-Fc. d, Left, representative flow cytometry plot of 19-HIT and 19-HIT CD28KO cells 5 days post CRISPR Cas9 editing. Right, summary of decrease of CD28 surface expression indicating CD28 KO in 9 donors. e, Left, flow cytometry plot of unstained cells, 19-HIT TRBC KO, 19-HIT CD28 KO cells, and 19-HIT CD28 KO + ΔCD28 OE cells. Right, summary of CD28 MFI after ΔCD28 overexpression in 19-HIT cells. Each dot is an independent healthy donor. f, Human IL-2 and IL-13 supernatant concentration 24hrs post co-culture of NALM6 target cells with 19-HIT and 19-HIT80BB cells at an effector: target ratio of 1:100. g, Ratio of human IL-2 and IL-13 secreted by 19-HIT and 19-HIT80BB cells after 24 hr co-culture with Nalm6CD28 relative to NALM6wt. f, g, n = 4 independent co-cultures of cells from a healthy donor, representative of 3 donors. P values were determined by two-tailed t-test (f, g). Data are mean ± sem.

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Extended Data Fig. 5 Extended characterization of 80BB and CTLA4 interactions.

a, Flow cytometry plots of CTLA4-Fc binding assay. Cells were incubated with increasing concentrations of CTLA4-Fc, and then stained with PE-anti-Fc. b, Ratio of human IL-2 and IL-13 secreted by 19-HIT and 19-HIT80BB cells after 24 hr co-culture with Nalm6CD28 relative to Nalm6. n = 4 independent co-cultures of cells from a healthy donor, representative of 3 donors. c, Serial in vitro cytotoxicity assay using NALM6 or NALM6ΔCTLA4. Fresh target cells were added every 2 days. Each line is an independent co-culture of cells from a healthy donor, representative of 3 donors. d, Left, trace from inference of CRISPR Edits (ICE) tool (Synthego) highlighting extent of CTLA4 knockout. Right, summary of extent of CTLA4 editing indicating CTLA4 knockout evaluated by ICE from 9 donors. e, Serial in vitro cytotoxicity assay of 19-HIT, 19-HIT80ΔBB, or 19-HIT80BB with TRBC, CD28 or CTLA4 genes CRISPR edited. Fresh NALM6 target cells were added every 2 days. Each line is an independent co-culture of cells from a healthy donor, representative of 3 donors. P values were determined by two-tailed t-test (b). Data are mean ± sem.

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Extended Data Fig. 6 80BB does not lead to by-stander cell activation nor increased CRS compared to a clinical-licensed CAR.

a, Representation of in vitro by-stander activation assay. Labelled 19-HIT T cells were plated onto wells with 19-HIT cells or 19-HIT80BB cells, or wells coated with CD28 superagonist antibody (CD28 SA) in the absence of target cells. b, c, Quantification of cell-surface CD69 on bystander 19-HIT cells (b) and levels of IL-2, IFNɣ and TNFα in the supernatant (c) 24hrs after exposure to wells containing 19-HIT, 19-HIT80BB or coated with CD28SA. n = 4 independent co-cultures of cells from a donor, representative of 2 donors. d, Left, Diagram depicting in vivo cytokine release syndrome (CRS) model. Right, weight change of tumour-bearing mice after 19-HIT (n = 8 mice), 19-HIT80BB (n = 4 mice), 1928z CAR (n = 6 mice) or no (n = 4 mice) T cell transfer. Weight per mouse is normalized to starting weight before T cell infusion. 10% cut-off for CRS is illustrated with a dotted line. e, f, Frequency of diarrhea in days (e) and serum cytokine levels 18hrs (f) after T cell infusion into tumour-bearing Scid Beige mice. P values were determined by two-tailed t-test (b, c, f) and Chi-square test(e). Data are mean ± sem.

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Extended Data Fig. 7 Extended characterization of ESO80BB TCR T cells in a subcutaneous melanoma model.

a, Diagram depicting TCR T cell engineering. The alpha and beta chains of an NY-ESO-1 directed TCR were targeted into the TRAC locus as previously described34. 80BB was delivered using the SFG γ-retroviral vector. b, c, d, e, Analyses of T cells isolated from the tumours or spleens of mice implanted with subcutaneous SK-Mel-37 and treated with ESO-TCR or ESO-TCR80BB. n = 4 mice per group per time point b, CD4+ Tetramer+ T cell counts isolated from tumours or spleens. c, counts of CD8+Tetramer+ T stem cell memory (Tscm, CCR7+, CD45Ra+), CD8+Tetramer+ T central memory cells (Tcm, CCR7+, CD45Ra-), CD8+Tetramer+ T effector memory cells (Tem, CCR7-, CD45Ra-) CD8+Tetramer+ T effector cells (Teff, CCR7-, CD45Ra+) isolated from spleens. d, Percentage of Tetramer+ CD8+ spleen-isolated T cells expressing Granzyme B, IFNɣ, IL-2 TNF-a after PMA/Ionomycin stimulation. e, Frequency of Tetramer+ CD8+ spleen-isolated T cells expressing PD-1, Lag3, Tim3 or PD-1 and Tim3 double positive cells. P values were determined by two-tailed t-test (c, d, e). Data are mean ± sem.

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Extended Data Fig. 8 Flow characterization of post-rapid expansion protocol patient-derived tumour infiltrating lymphocytes.

a, Flow profile of transduced TIL transduced for 80BB (left) and LNGFR (right). b, c, d, Flow profile of post-rapid expansion protocol TILLNGFR (blue) and TIL80BB (red) cells stained for 80BB ligands CD28 and CTLA4 (b), differentiation markers CCR7, CD62L, CD45Ra (c), exhaustion markers PD-1, Lag3, Tim3 (d). e, Fraction of cells killed after 18 hrs of co-culture of transduced TIL and autologous tumour cells. n = 3 independent co-cultures from cells expanded from 1 patient-derived tumour sample. Data are mean ± sem.

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Dobrin, A., Lindenbergh, P.L., Shi, Y. et al. Synthetic dual co-stimulation increases the potency of HIT and TCR-targeted cell therapies. Nat Cancer (2024). https://doi.org/10.1038/s43018-024-00744-x

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