Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. June, C. H. & Sadelain, M. Chimeric antigen receptor therapy. N. Engl. J. Med. 379, 64–73 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Chandran, S. S. & Klebanoff, C. A. T cell receptor-based cancer immunotherapy: emerging efficacy and pathways of resistance. Immunol. Rev. 290, 127–147 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. DeRenzo, C. & Gottschalk, S. Genetically modified T-cell therapy for osteosarcoma: into the roaring 2020s. Adv. Exp. Med. Biol. 1257, 109–131 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Ecsedi, M., McAfee, M. S. & Chapuis, A. G. The anticancer potential of T cell receptor-engineered T cells. Trends Cancer 7, 48–56 (2021).

    CAS  PubMed  Google Scholar 

  5. Foy, S. P. et al. Non-viral precision T cell receptor replacement for personalized cell therapy. Nature 615, 687–696 (2022).

  6. Sadelain, M., Riviere, I. & Riddell, S. Therapeutic T cell engineering. Nature 545, 423–431 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. van der Stegen, S. J., Hamieh, M. & Sadelain, M. The pharmacology of second-generation chimeric antigen receptors. Nat. Rev. Drug Discov. 14, 499–509 (2015).

    PubMed  PubMed Central  Google Scholar 

  8. Hamieh, M., Mansilla-Soto, J., Rivière, I. & Sadelain, M. Programming CAR T cell tumor recognition: tuned antigen sensing and logic gating. Cancer Discov. 13, 829–843 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Helsen, C. W. et al. The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity. Nat. Commun. 9, 3049 (2018).

    PubMed  PubMed Central  Google Scholar 

  10. Xu, Y. et al. A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with γ/δ-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release. Cell Discov. 4, 62 (2018).

    PubMed  PubMed Central  Google Scholar 

  11. Baeuerle, P. A. et al. Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Nat. Commun. 10, 2087 (2019).

    PubMed  PubMed Central  Google Scholar 

  12. Liu, Y. et al. Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors. Sci. Transl. Med. 13, eabb5191 (2021).

    CAS  PubMed  Google Scholar 

  13. Birtel, M. et al. A TCR-like CAR promotes sensitive antigen recognition and controlled T-cell expansion upon mRNA vaccination. Cancer Res. Commun. 2, 827–841 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Mansilla-Soto, J. et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat. Med. 28, 345–352 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Cabo, M. et al. Immunostimulatory monoclonal antibodies for oncological indications. Oncoimmunology 6, e1371896 (2017).

    PubMed  PubMed Central  Google Scholar 

  16. Kraehenbuehl, L., Weng, C.-H., Eghbali, S., Wolchok, J. D. & Merghoub, T. Enhancing immunotherapy in cancer by targeting emerging immunomodulatory pathways. Nat. Rev. Clin. Oncol. 19, 37–50 (2022).

    CAS  PubMed  Google Scholar 

  17. Stone, J. D. et al. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control. Cancer Immunol. Immunother. 63, 1163–1176 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Govers, C. et al. TCRs genetically linked to CD28 and CD3ε do not mispair with endogenous TCR chains and mediate enhanced T cell persistence and anti-melanoma activity. J. Immunol. 193, 5315–5326 (2014).

    CAS  PubMed  Google Scholar 

  19. Krause, A. et al. Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes. J. Exp. Med. 188, 619–626 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Omer, B. et al. A costimulatory CAR improves TCR-based cancer immunotherapy. Cancer Immunol. Res. 10, 512–524 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Oda, S. K. et al. A CD200R–CD28 fusion protein appropriates an inhibitory signal to enhance T-cell function and therapy of murine leukemia. Blood 130, 2410–2419 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Anderson, K. G. et al. Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in ovarian cancer enhances therapeutic efficacy. J. Immunother. Cancer 10, e003959 (2022).

    PubMed  PubMed Central  Google Scholar 

  23. Guo, J., Kent, A. & Davila, E. Chimeric non-antigen receptors in T cell-based cancer therapy. J. Immunother. Cancer 9, e002628 (2021).

    PubMed  PubMed Central  Google Scholar 

  24. Prosser, M. E., Brown, C. E., Shami, A. F., Forman, S. J. & Jensen, M. C. Tumor PD-L1 co-stimulates primary human CD8+ cytotoxic T cells modified to express a PD1:CD28 chimeric receptor. Mol. Immunol. 51, 263–272 (2012).

    CAS  PubMed  Google Scholar 

  25. Maher, J., Brentjens, R. J., Gunset, G., Riviere, I. & Sadelain, M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ/CD28 receptor. Nat. Biotechnol. 20, 70–75 (2002).

    CAS  PubMed  Google Scholar 

  26. Imai, C. et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 18, 676–684 (2004).

    CAS  PubMed  Google Scholar 

  27. Cappell, K. M. & Kochenderfer, J. N. A comparison of chimeric antigen receptors containing CD28 versus 4-1BB costimulatory domains. Nat. Rev. Clin. Oncol. 18, 715–727 (2021).

    CAS  PubMed  Google Scholar 

  28. Croft, M. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity? Nat. Rev. Immunol. 3, 609–620 (2003).

    CAS  PubMed  Google Scholar 

  29. Melero, I. et al. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur. J. Immunol. 28, 1116–1121 (1998).

    CAS  PubMed  Google Scholar 

  30. Wang, J. et al. Optimizing adoptive polyclonal T cell immunotherapy of lymphomas, using a chimeric T cell receptor possessing CD28 and CD137 costimulatory domains. Hum. Gene Ther. 18, 712–725 (2007).

    CAS  PubMed  Google Scholar 

  31. Hirabayashi, K. et al. Dual-targeting CAR-T cells with optimal co-stimulation and metabolic fitness enhance antitumor activity and prevent escape in solid tumors. Nat. Cancer 2, 904–918 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Katsarou, A. et al. Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence. Sci. Transl. Med. 13, eabh1962 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhao, Z. et al. Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells. Cancer Cell 28, 415–428 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Salzer, B. et al. Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function. Nat. Commun. 11, 4166 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Ikemizu, S. et al. Structure and dimerization of a soluble form of B7-1. Immunity 12, 51–60 (2000).

    CAS  PubMed  Google Scholar 

  36. Rudd, C. E., Taylor, A. & Schneider, H. CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol. Rev. 229, 12–26 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Chen, J. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530–534 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Good, C. R. et al. An NK-like CAR T cell transition in CAR T cell dysfunction. Cell 184, 6081–6100 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Nam, K. O., Shin, S. M. & Lee, H. W. Cross-linking of 4-1BB up-regulates IL-13 expression in CD8+ T lymphocytes. Cytokine 33, 87–94 (2006).

    CAS  PubMed  Google Scholar 

  41. Kawalekar, O. U. et al. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity 44, 380–390 (2016).

    CAS  PubMed  Google Scholar 

  42. Esensten, J. H., Helou, Y. A., Chopra, G., Weiss, A. & Bluestone, J. A. CD28 costimulation: from mechanism to therapy. Immunity 44, 973–988 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Ward-Kavanagh, L. K., Lin, W. W., Šedý, J. R. & Ware, C. F. The TNF receptor superfamily in co-stimulating and co-inhibitory responses. Immunity 44, 1005–1019 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhao, Y. et al. cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity. Immunity 56, 1187–1203 (2023).

    CAS  PubMed  Google Scholar 

  45. Stephan, M. T. et al. T cell-encoded CD80 and 4-1BBL induce auto- and transcostimulation, resulting in potent tumor rejection. Nat. Med. 13, 1440–1449 (2007).

    CAS  PubMed  Google Scholar 

  46. Menk, A. V. et al. 4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses. J. Exp. Med. 215, 1091–1100 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Nakaseko, C. et al. Cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement delivers an inhibitory signal through the membrane-proximal region in the absence of the tyrosine motif in the cytoplasmic tail. J. Exp. Med. 190, 765–774 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Schraven, B. & Kalinke, U. CD28 superagonists: what makes the difference in humans? Immunity 28, 591–595 (2008).

    CAS  PubMed  Google Scholar 

  49. Giavridis, T. et al. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat. Med. 24, 731–738 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Davila, M. L. et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci. Transl. Med. 6, 224ra225 (2014).

    Google Scholar 

  51. Locke, F. L. et al. Axicabtagene ciloleucel as second-line therapy for large B-cell lymphoma. N. Engl. J. Med. 386, 640–654 (2021).

    PubMed  Google Scholar 

  52. Park, J. H. et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N. Engl. J. Med. 378, 449–459 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Rosenberg, S. A. IL-2: the first effective immunotherapy for human cancer. J. Immunol. 192, 5451–5458 (2014).

    CAS  PubMed  Google Scholar 

  54. van der Leun, A. M. & Schumacher, T. N. An atlas of intratumoral T cells. Science 374, 1446–1447 (2021).

    PubMed  Google Scholar 

  55. Sharma, A. et al. Anti-CTLA-4 immunotherapy does not deplete FOXP3+ regulatory T cells (Tregs) in human cancers. Clin. Cancer Res. 25, 1233–1238 (2019).

    CAS  PubMed  Google Scholar 

  56. Bitra, A. & Zajonc, D. M. Evolution of differential 4-1BB signaling in human and murine immune system. FASEB J. 33, 461.3 (2019).

    Google Scholar 

  57. Porciello, N. et al. A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nat. Commun. 9, 1080 (2018).

    PubMed  PubMed Central  Google Scholar 

  58. Li, G. et al. 4-1BB enhancement of CAR T function requires NF-κB and TRAFs. JCI Insight 3, e121322 (2018).

    PubMed  PubMed Central  Google Scholar 

  59. Kalaitsidou, M. et al. Signaling via a CD28/CD40 chimeric costimulatory antigen receptor (CoStAR), targeting folate receptor α, enhances T cell activity and augments tumor reactivity of tumor infiltrating lymphocytes. Front. Immunol. 14, 1256491 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Bollard, C. M. et al. Adapting a transforming growth factor β-related tumor protection strategy to enhance antitumor immunity. Blood 99, 3179–3187 (2002).

    CAS  PubMed  Google Scholar 

  61. Yamamoto, T. N. et al. T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy. J. Clin. Invest. 129, 1551–1565 (2019).

    PubMed  PubMed Central  Google Scholar 

  62. Roth, T. L. et al. Pooled knockin targeting for genome engineering of cellular immunotherapies. Cell 181, 728–744 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Diskin, B. et al. PD-L1 engagement on T cells promotes self-tolerance and suppression of neighboring macrophages and effector T cells in cancer. Nat. Immunol. 21, 442–454 (2020).

    CAS  PubMed  Google Scholar 

  64. Green, D. R. & Ware, C. F. Fas-ligand: privilege and peril. Proc. Natl Acad. Sci. USA 94, 5986–5990 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Wang, J. et al. A novel adoptive synthetic TCR and antigen receptor (STAR) T-cell therapy for B-cell acute lymphoblastic leukemia. Am. J. Hematol. 97, 992–1004 (2022).

    CAS  PubMed  Google Scholar 

  66. Heemskerk, B. et al. Adoptive cell therapy for patients with melanoma, using tumor-infiltrating lymphocytes genetically engineered to secrete interleukin-2. Hum. Gene Ther. 19, 496–510 (2008).

    CAS  PubMed  Google Scholar 

  67. Zhang, L. et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. Clin. Cancer Res. 21, 2278–2288 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Stadtmauer, E. A. et al. CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365 (2020).

    CAS  PubMed  Google Scholar 

  69. Chesney, J. et al. 883TiP A phase I/II open-label study (IOV-GM1-201) of TALEN-mediated PD-1 inactivated autologous tumor-infiltrating lymphocytes (TIL; IOV-4001) in patients with advanced melanoma and NSCLC. Ann. Oncol. 33, S952 (2022).

    Google Scholar 

  70. Lesch, S. et al. PD-1–CD28 fusion protein strengthens mesothelin-specific TRuC T cells in preclinical solid tumor models. Cell. Oncol. 46, 227–235 (2023).

  71. Dudley, M. E., Wunderlich, J. R., Shelton, T. E., Even, J. & Rosenberg, S. A. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J. Immunother. 26, 332–342 (2003).

    PubMed  PubMed Central  Google Scholar 

  72. Brentjens, R. J. et al. Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts. Clin. Cancer Res. 13, 5426–5435 (2007).

    CAS  PubMed  Google Scholar 

  73. Ghani, K., Cottin, S., de Campos-Lima, P. O., Caron, M. C. & Caruso, M. Characterization of an alternative packaging system derived from the cat RD114 retrovirus for gene delivery. J. Gene Med. 11, 664–669 (2009).

    CAS  PubMed  Google Scholar 

  74. Gallardo, H. F., Tan, C., Ory, D. & Sadelain, M. Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes. Blood 90, 952–957 (1997).

    CAS  PubMed  Google Scholar 

  75. Derré, L. et al. Distinct sets of αβ TCRs confer similar recognition of tumor antigen NY-ESO-1157-165 by interacting with its central Met/Trp residues. Proc. Natl Acad. Sci. USA 105, 15010–15015 (2008).

    PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Ethics declarations

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.

Peer review

Peer review information

Nature Cancer thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

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

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.

Source data

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.

Source data

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.

Source data

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.

Source data

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.

Source data

Supplementary information

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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 5, 760–773 (2024). https://doi.org/10.1038/s43018-024-00744-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s43018-024-00744-x

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer