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Mesenchymal stromal cells with chimaeric antigen receptors for enhanced immunosuppression

Nature Biomedical Engineering (2024)Cite this article

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Abstract

Allogeneic mesenchymal stromal cells (MSCs) are a safe treatment option for many disorders of the immune system. However, clinical trials using MSCs have shown inconsistent therapeutic efficacy, mostly owing to MSCs providing insufficient immunosuppression in target tissues. Here we show that antigen-specific immunosuppression can be enhanced by genetically modifying MSCs with chimaeric antigen receptors (CARs), as we show for E-cadherin-targeted CAR-MSCs for the treatment of graft-versus-host disease in mice. CAR-MSCs led to superior T-cell suppression and localization to E-cadherin+ colonic cells, ameliorating the animals’ symptoms and survival rates. On antigen-specific stimulation, CAR-MSCs upregulated the expression of immunosuppressive genes and receptors for T-cell inhibition as well as the production of immunosuppressive cytokines while maintaining their stem cell phenotype and safety profile in the animal models. CAR-MSCs may represent a widely applicable therapeutic technology for enhancing immunosuppression.

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Fig. 1: MSCs are transduced to express CAR and maintain stem-like features following transduction and stimulation.
Fig. 2: EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells and signalling in vitro.
Fig. 3: EcCAR-MSCs induce superior immunosuppression in tumour and GvHD xenograft models.
Fig. 4: EcCAR-MSCs display antigen-specific activation and trafficking to Ecad+ colonic target tissue in acute GvHD xenograft models.
Fig. 5: Activation of antigen-specific immunosuppressive signalling pathways identified in EcCAR-MSCs.
Fig. 6: Increased cytokine secretion, surface marker expression and subsequent T-cell modulation identified following EcCAR-MSC stimulation.
Fig. 7: CD28ζ signalling domain within EcCAR-MSCs is required for optimal immunosuppressive efficacy.
Fig. 8: EcCAR-MSC safety and clearance profiles across tissues.

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Data availability

The raw sequencing data are available at Gene Expression Omnibus (GEO) via the accession code GSE256355. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This study was partly funded through NSF-GRFP (2021321972, O.S.), RMM (091620TR012, E.L.S.), Mayo Clinic Center for Individualized Medicine (S.S.K.), Mayo Clinic President’s Strategic Initiative Funds (S.S.K.), Mayo Clinic Center for Regenerative Biotherapeutics (S.S.K.), Mayo Clinic Comprehensive Cancer Center (S.S.K.), National Comprehensive Cancer Network (S.S.K.), National Institutes of Health grants (K12CA090628, S.S.K. and R37CA266344-01, S.S.K.), Department of Defense Grant (CA201127, S.S.K.), Predolin Foundation (R.L.S. and S.S.K.) and the generosity of Donald Porteous (S.S.K.).

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Authors and Affiliations

  1. T Cell Engineering, Mayo Clinic, Rochester, MN, USA

    Olivia Sirpilla, R. Leo Sakemura, Mehrdad Hefazi, Truc N. Huynh, Ismail Can, James H. Girsch, Erin E. Tapper, Michelle J. Cox, Kendall J. Schick, Claudia Manriquez-Roman, Kun Yun, Carli M. Stewart, Ekene J. Ogbodo, Brooke L. Kimball, Long K. Mai, Omar L. Gutierrez-Ruiz, Makena L. Rodriguez, Elizabeth L. Siegler & Saad S. Kenderian

  2. Division of Hematology, Mayo Clinic, Rochester, MN, USA

    Olivia Sirpilla, R. Leo Sakemura, Mehrdad Hefazi, Truc N. Huynh, Ismail Can, James H. Girsch, Erin E. Tapper, Michelle J. Cox, Kendall J. Schick, Claudia Manriquez-Roman, Kun Yun, Carli M. Stewart, Ekene J. Ogbodo, Brooke L. Kimball, Long K. Mai, Omar L. Gutierrez-Ruiz, Makena L. Rodriguez, Elizabeth L. Siegler & Saad S. Kenderian

  3. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA

    Olivia Sirpilla, Kendall J. Schick, Carli M. Stewart & Martina Gluscevic

  4. Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN, USA

    Olivia Sirpilla, Ismail Can, James H. Girsch, Claudia Manriquez-Roman, Kun Yun & Carli M. Stewart

  5. Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA

    James H. Girsch, Claudia Manriquez-Roman, Kun Yun & Saad S. Kenderian

  6. Division of Hematopathology, Mayo Clinic, Rochester, MN, USA

    Daniel P. Larson

  7. LifEngine Animal Health Laboratories Incorporated, Rochester, MN, USA

    Alex M. Abel & Wesley A. Wierson

  8. Department of Business Development, Mayo Clinic, Rochester, MN, USA

    Gloria Olivier

  9. Department of Immunology, Mayo Clinic, Rochester, MN, USA

    Saad S. Kenderian

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Contributions

Conceptualization was carried out by O.S. and S.S.K. Methodology was planned and carried out by O.S., M.H., K.J.S., M.J.C., E.L.S., T.N.H., R.L.S. and S.S.K. Investigation was performed by O.S., R.L.S., M.H., E.L.S. and T.N.H. Visualization was carried out by O.S. Formal analysis was performed by O.S. The original draft was written by O.S. and S.S.K. Review and editing was carried out by O.S., E.L.S. and S.S.K. Funding acquisition was performed by O.S., R.L.S., E.L.S. and S.S.K.; and supervision was carried out by S.S.K. All authors edited and approved the final version of the paper.

Corresponding author

Correspondence to Saad S. Kenderian.

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Competing interests

S.S.K. is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between Mayo Clinic, University of Pennsylvania and Novartis) and MustangBio (through Mayo Clinic). M.J.C., R.L.S. and S.S.K. are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen (through Mayo Clinic). M.H. and S.S.K. are inventors on patents in the field of CAR immunotherapy that are licensed to Mettaforge (through Mayo Clinic). S.S.K. receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, LifEngine Animal Health Laboratories Inc. and Lentigen. S.S.K. has participated in advisory meetings with Kite/Gilead, Humanigen, Juno/BMS, Capstan Bio and Novartis. S.S.K. has served on the data safety and monitoring board with Humanigen and Carisma. S.S.K. has served as consultant for Torque, Calibr, Novartis, Capstan Bio, Carisma and Humanigen. O.S., R.L.S., M.H., E.E.T., K.J.S., M.J.C., E.L.S. and S.S.K. have intellectual property in the CAR-MSC technology.

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Peer review information

Nature Biomedical Engineering thanks Hinrich Abken, Kailin Xu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 EcCAR-MSC effects on CART effector functions in in vivo tumor model.

a, Comparison of Ecad−NALM6 and Ecad+ NALM6 tumor flux measurements across control mice without CART19 cell infusion. Displaying mean ± SEM with statistics by 2-way ANOVA (n = 4–5 mice per group). b, c, Relative levels of luc+ Ecad−NALM6 (left) and luc+ Ecad+ NALM6 (right) as measured by luminescence following 24-hour in vitro coculture with UTD-MSCs or EcCAR-MSCs at varying MSC:NALM6 ratios. Data displaying mean ± s.d. with statistics by 2-way ANOVA (n = 2 replicates per group). d, Schema of NALM6 and JeKo-1 tumor models: NSG mice were engrafted with luciferase+ CD19+ Nalm6 or JeKo-1 cells (1 × 106 i.v.) and treated with CART19 (1 × 106 cells i.v.) and irradiated Ecad+ cell line. Mice were then randomized to receive UTD-MSCs or EcCAR-MSCs (1 × 106 cells i.p.) and monitored biweekly for BLI and survival. Image created with BioRender.com. e, Tumor flux following CART19 infusion in Jeko-1 model, comparing EcCAR-MSCs, UTD-MSCs, or no MSC treatment. Data showing mean ± SEM with statistical analysis by 2-way ANOVA (n = 2–5 mice per group) f, Tumor flux measurements across MSC administration groups following CART19 infusion in NALM6 model. Displaying mean ± SEM with statistics by 2-way ANOVA (n = 3–4 mice per group, 2 independent experiments). g, Survival outcomes of EcCAR-MSCs compared to UTD-MSCs and no MSC control groups. Statistics by Kaplan-Meier survival analysis (n = 3–4 mice per group, 2 independent experiments). For all panels, ns=p ≥ 0.05 and significant p values are displayed.

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Extended Data Fig. 2 RNAseq pathway analysis and cytokine secretion.

a, Differentially expressed genes in unstimulated EcCAR-MSCs vs. UTD-MSCs, Ecad-stimulated vs. unstimulated EcCAR-MSCs, Ecad−stimulated vs. unstimulated UTD-MSCs, and Ecad-stimulated EcCAR-MSCs vs. UTD-MSCs. Data displaying significantly unregulated and downregulated gene counts within comparisons with adj. p value < 0.01 and ± 1-log fold change. Transcriptional alterations induced by Ecad stimulation of CAR-MSCs included 2362 significant genes vs. EcCAR-MSC alone and 3032 significant genes vs. Ecad stimulated UTD-MSCs. Transcriptional alterations induced by CAR transduction included 606 significant genes. Transcriptional alterations induced by Ecad stimulation included 206 significant genes. b, Ingenuity Pathway Analysis (IPA) revealed upregulated canonical pathways in unstimulated EcCAR-MSCs vs. UTD-MSCs. Dashed line across x axis represents statistically significant enrichment for all pathways -log(p ≤ 0.05). (n = 3 MSC donors per group). c, Graphical Summaries generated through IPA machine learning algorithm illustrating most significant entities activated in unstimulated EcCAR-MSCs vs. UTD-MSCs and d, stimulated EcCAR-MSCs vs UTD-MSCs. Canonical pathways and activated molecules were used to predict meaningful functional impacts between datasets. e, Additional serum cytokine elevations found in peripheral blood from EcCAR-MSC-treated tumor xenograft mice as compared to UTD-MSC and control. Cytokines include macrophage-derived chemokine (MDC), growth related alpha protein (GRO), granulocyte macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 3 (MCP-3), and FMS-related tyrosine kinase 3 ligand (Flt-3L) in pg/mL. Data showing mean ± s.d. with statistical analysis determined by multiple t tests (n = 4–6 mice per group). For all panels, significant p values are displayed.

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Extended Data Fig. 3 EcCAR-MSC safety profiles within in vivo canine models.

a, Schema for CAR-MSC manufacturing and safety analysis in healthy canine models: EcCARs with cross reactivity to human, mouse, and canine Ecad were lentivirally transduced into human MSCs and expanded in vitro for subsequent i.p. injection into healthy canine subjects. Subgroups were monitored for hematological and organ toxicity for 28 days. Image created with BioRender.com. b, Complete blood count levels displayed as a determinant of hematopoietic safety following administration of EcCAR-MSCs. This includes white blood cells, monocytes, lymphocytes, neutrophils, and platelets with short term (3 day) and long term (28 day) blood level monitoring following in vivo EcCAR-MSC injection as compared to baseline. Displaying mean ± s.d. of blood composition with statistics by 1-way ANOVA (n = 3 subjects per experimental group). c, Total protein, BUN, creatinine, albumin, and alkaline phosphatase levels depicted for safety confirmation with short term (3 day) and long term (28 day) monitoring following in vivo EcCAR-MSC injection. Displaying mean ± s.d. of blood composition with statistics by 1-way ANOVA (n = 3 subjects per experimental group). d, Bodyweight changes in healthy canines following administration of EcCAR-MSCS as compared to control. No significant differences in body weight changes were found between groups. Data showing mean ± s.d. of % weight change from baseline with statistical analysis performed by ordinary 1-way ANOVA, (n = 3 subjects per experimental group) e, Data displaying transverse colonic tissue sections of canines through H&E staining 28 days following administration at 20x and 40x magnification following treatment with human EcCAR-MSCs (left) or control (right). For all panels, ns=p ≥ 0.05.

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Sirpilla, O., Sakemura, R.L., Hefazi, M. et al. Mesenchymal stromal cells with chimaeric antigen receptors for enhanced immunosuppression. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01195-6

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