CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro.
CAR T-cell therapy
CRISPR/Cas9 knockout
Coinhibitory T-cell signaling
Genome editing
IVT-RNA
TGFβ receptor II
Journal
Journal of translational medicine
ISSN: 1479-5876
Titre abrégé: J Transl Med
Pays: England
ID NLM: 101190741
Informations de publication
Date de publication:
27 11 2021
27 11 2021
Historique:
received:
17
08
2021
accepted:
16
11
2021
entrez:
28
11
2021
pubmed:
29
11
2021
medline:
15
12
2021
Statut:
epublish
Résumé
CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. In this study, we hypothesized that knocking out the TGFβ receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFβRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFβRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. Our experiments demonstrated that TGFβRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFβ in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFβRII KO CAR T-cells has been recorded. TGFβRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFβRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. The TGFβRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.
Sections du résumé
BACKGROUND
CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells.
METHODS
In this study, we hypothesized that knocking out the TGFβ receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFβRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFβRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells.
RESULTS
Our experiments demonstrated that TGFβRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFβ in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFβRII KO CAR T-cells has been recorded. TGFβRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFβRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart.
CONCLUSION
The TGFβRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.
Identifiants
pubmed: 34838059
doi: 10.1186/s12967-021-03146-0
pii: 10.1186/s12967-021-03146-0
pmc: PMC8627098
doi:
Substances chimiques
Receptors, Chimeric Antigen
0
Mesothelin
J27WDC343N
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
482Subventions
Organisme : Biotechnology Development Council of the Islamic Republic of Iran
ID : 960404
Organisme : Iran University of Science and Technology
ID : 96011546
Informations de copyright
© 2021. The Author(s).
Références
Porter DL, et al. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365:725–33.
pubmed: 21830940
pmcid: 3387277
Maude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med. 2018;378(5):439–48.
pubmed: 29385370
pmcid: 5996391
Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377(26):2531–44.
pubmed: 29226797
pmcid: 5882485
Newick K, et al. CAR T cell therapy for solid tumors. Annu Rev Med. 2017;68:139–52.
pubmed: 27860544
Wrzesinski SH, Wan YY, Flavell RA. Transforming growth factor-β and the immune response: implications for anticancer therapy. Clin Cancer Res. 2007;13(18):5262–70.
pubmed: 17875754
Massagué J. TGFβ in cancer. Cell. 2008;134(2):215–30.
pubmed: 18662538
pmcid: 3512574
Batlle E, Massagué J. Transforming growth factor-β signaling in immunity and cancer. Immunity. 2019;50(4):924–40.
pubmed: 30995507
pmcid: 7507121
Derynck R, Budi EH. Specificity, versatility, and control of TGF-β family signaling. Sci Signal. 2019. https://doi.org/10.1126/scisignal.aav5183 .
doi: 10.1126/scisignal.aav5183
pubmed: 30808819
pmcid: 6800142
Kloss CC, et al. Dominant-negative TGF-β receptor enhances PSMA-targeted human CAR T cell proliferation and augments prostate cancer eradication. Mol Ther. 2018;26(7):1855–66.
pubmed: 29807781
pmcid: 6037129
Bollard CM, et al. Adapting a transforming growth factor β-related tumor protection strategy to enhance antitumor immunity. Blood J Am Soc Hematol. 2002;99(9):3179–87.
Narayan V, et al. A phase I clinical trial of PSMA-directed/TGFβ-insensitive CAR-T cells in metastatic castration-resistant prostate cancer. J Clin Oncol. 2019;37(7_suppl): TPS347.
Ushiku T, et al. Distinct expression pattern of claudin-6, a primitive phenotypic tight junction molecule, in germ cell tumours and visceral carcinomas. Histopathology. 2012;61(6):1043–56.
pubmed: 22803571
Reinhard K, et al. An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors. Science. 2020;367(6476):446–53.
pubmed: 31896660
Bera TK, Pastan I. Mesothelin is not required for normal mouse development or reproduction. Mol Cell Biol. 2000;20(8):2902–6.
pubmed: 10733593
pmcid: 85523
Morello A, Sadelain M, Adusumilli PS. Mesothelin-targeted CARs: driving T cells to solid tumors. Cancer Discov. 2016;6(2):133–46.
pubmed: 26503962
Holtkamp S, et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood. 2006;108(13):4009–17.
pubmed: 16940422
Zanoni M, et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep. 2016;6(1):1–11.
Gressner AM, et al. Roles of TGF-beta in hepatic fibrosis. Front Biosci Landmark. 2002;7(4):793–807.
Junker U, et al. Transforming growth factor beta 1 is significantly elevated in plasma of patients suffering from renal cell carcinoma. Cytokine. 1996;8(10):794–8.
pubmed: 8980881
Hou AJ, et al. TGF-β-responsive CAR-T cells promote anti-tumor immune function. Bioeng Transl Med. 2018;3(2):75–86.
pubmed: 30065964
pmcid: 6063867
Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–21.
pubmed: 22745249
pmcid: 6286148
Doench JG, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184–91.
pubmed: 26780180
pmcid: 4744125
Golumba-Nagy V, et al. CD28-ζ CAR T cells resist TGF-β repression through IL-2 signaling, which can be mimicked by an engineered IL-7 autocrine loop. Mol Therapy. 2018;26(9):2218–30.
Thomas DA, Massagué J. TGF-β directly targets cytotoxic T cell functions during tumor evasion of immune surveillance. Cancer Cell. 2005;8(5):369–80.
pubmed: 16286245
Tang N, et al. TGF-β inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight. 2020. https://doi.org/10.1172/jci.insight.133977 .
doi: 10.1172/jci.insight.133977
pubmed: 33328388
pmcid: 7819752
Stüber T, et al. Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer. J Immunother Cancer. 2020. https://doi.org/10.1136/jitc-2020-000676 .
doi: 10.1136/jitc-2020-000676
pubmed: 32303620
pmcid: 7204619
Foster AE, et al. Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-β receptor. J Immunother. 2008;31(5):500.
pubmed: 18463534
pmcid: 2745436
Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+ CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor β. J Exp Med. 2001;194(5):629–44.
pubmed: 11535631
pmcid: 2195935
Nakamura K, et al. TGF-β1 plays an important role in the mechanism of CD4+ CD25+ regulatory T cell activity in both humans and mice. J Immunol. 2004;172(2):834–42.
pubmed: 14707053
Chen M-L, et al. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-β signals in vivo. Proc Natl Acad Sci. 2005;102(2):419–24.
pubmed: 15623559
Fahlén L, et al. T cells that cannot respond to TGF-β escape control by CD4+ CD25+ regulatory T cells. J Exp Med. 2005;201(5):737–46.
pubmed: 15753207
pmcid: 2212836
Schmidt A, et al. Comparative analysis of protocols to induce human CD4+ Foxp3+ regulatory T cells by combinations of IL-2, TGF-beta, retinoic acid, rapamycin and butyrate. PLoS ONE. 2016;11(2): e0148474.
pubmed: 26886923
pmcid: 4757416
Schmidt A, Oberle N, Krammer P. Molecular mechanisms of Treg-mediated T cell suppression. Front Immunol. 2012. https://doi.org/10.3389/fimmu.2012.00051 .
doi: 10.3389/fimmu.2012.00051
pubmed: 22969767
pmcid: 3432880
Wright GP, et al. Adoptive therapy with redirected primary regulatory T cells results in antigen-specific suppression of arthritis. Proc Natl Acad Sci. 2009;106(45):19078.
pubmed: 19884493
pmcid: 2776462
Thibault B, et al. Ovarian cancer microenvironment: implications for cancer dissemination and chemoresistance acquisition. Cancer Metastasis Rev. 2014;33(1):17–39.
pubmed: 24357056
Teicher BA. Malignant cells, directors of the malignant process: role of transforming growth factor-beta. Cancer Metastasis Rev. 2001;20(1):133–43.
pubmed: 11831642
Yu Y, et al. Cancer-associated fibroblasts induce epithelial–mesenchymal transition of breast cancer cells through paracrine TGF-β signalling. Br J Cancer. 2014;110(3):724–32.
pubmed: 24335925
Dalal B, Keown P, Greenberg A. Immunocytochemical localization of secreted transforming growth factor-beta 1 to the advancing edges of primary tumors and to lymph node metastases of human mammary carcinoma. Am J Pathol. 1993;143(2):381.
pubmed: 8393616
pmcid: 1887030
Grady WM, et al. Mutational inactivation of transforming growth factor β receptor type II in microsatellite stable colon cancers. Cancer Res. 1999;59(2):320–4.
pubmed: 9927040
Dunn GP, et al. Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol. 2002;3(11):991–8.
pubmed: 12407406
Galliher AJ, Schiemann WP. β 3 integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells. Breast Cancer Res. 2006;8(4):1–16.
Chang ZL, et al. Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nat Chem Biol. 2018;14(3):317.
pubmed: 29377003
pmcid: 6035732
Liu M, et al. TGF-β suppresses type 2 immunity to cancer. Nature. 2020;587(7832):115–20.
pubmed: 33087928
pmcid: 8347705
Li S, et al. Cancer immunotherapy via targeted TGF-β signalling blockade in TH cells. Nature. 2020;587(7832):121–5.
pubmed: 33087933
pmcid: 8353603
Stadtmauer EA, et al. CRISPR-engineered T cells in patients with refractory cancer. Science. 2020;367(6481):7365.
Morgan RA, et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Therapy. 2010;18(4):843–51.
Richman SA, et al. High-affinity GD2-specific CAR T cells induce fatal encephalitis in a preclinical neuroblastoma model. Cancer Immunol Res. 2018;6(1):36–46.
pubmed: 29180536
Lamers CH, et al. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol Therapy. 2013;21(4):904–12.
Beatty GL, et al. Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce antitumor activity in solid malignancies. Cancer Immunol Res. 2014;2(2):112–20.
pubmed: 24579088
Klebanoff CA, Gattinoni L, Restifo NP. Sorting through subsets: which T-cell populations mediate highly effective adoptive immunotherapy? J Immunother. 2012;35(9):651–60.
pubmed: 23090074
pmcid: 3501135