Abstract
The protocol describes the procedure of antigen-specific T cell generation and TCR identification for the use in adoptive T cell therapy. We describe two paths of generating antigen-specific T cells, first, T cell stimulation with autologous dendritic cells pulsed with antigen peptide, second, in vivo T cell stimulation with peptide or DNA by gene gun application in a suitable mouse model followed by in vitro enrichment of peptide-reactive T cells. Peptide-stimulated T cells are sorted by fluorescence-activated cell sorting for CD107α or IFNγ expression and subsequently isolated RNA is used in a 5′ rapid amplification of cDNA ends (RACE )-PCR specific for TCR for TCR chain identification. After retroviral cloning, it is re-expressed on human T cells to test its applicability in adoptive T cell therapy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rosenberg SA, Restifo NP (2015) Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348(6230):62–68
Zacharakis N, Chinnasamy H, Black M et al (2018) Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med 24(6):724–730
Tran E, Robbins PF, Lu Y-C et al (2016) T-cell transfer therapy targeting mutant KRAS in cancer. N Engl J Med 375(23):2255–2262
Wu D, Liu Y, Li X et al (2020) Identification of clonal neoantigens derived from driver mutations in an EGFR-mutated lung cancer patient benefitting from anti-PD-1. Front Immunol 11:1366
Robbins PF, Lu YC, El-Gamil M et al (2013) Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 19(6):747–752
Cohen CJ, Gartner JJ, Horovitz-Fried M et al (2015) Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes. J Clin Invest 125(10):3981–3991
Dudley ME, Wunderlich JR, Shelton TE et al (2003) Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J Immunother 26(4):332–342
Johnson LA, Heemskerk B, Powell DJ Jr et al (2006) Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J Immunol 177(9):6548–6559
Hughes MS, Yu YY, Dudley ME et al (2005) Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 16(4):457–472
Morgan RA, Dudley ME, Wunderlich JR et al (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314(5796):126–129
Davari K, Holland T, Prassmayer L et al (2021) Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy. J Immunother Cancer 9(3):e002035
Wilde S, Geiger C, Milosevic S et al (2012) Generation of allo-restricted peptide-specific T cells using RNA-pulsed dendritic cells: a three phase experimental procedure. Onco Targets Ther 1(2):129–140
Çınar Ö, Brzezicha B, Grunert C et al (2021) High-affinity T-cell receptor specific for MyD88 L265P mutation for adoptive T-cell therapy of B-cell malignancies. J Immunother Cancer 9(7):e002410. https://doi.org/10.1136/jitc-2021-002410
Grunert C, Willimsky G, Peuker CA et al (2022) Isolation of neoantigen-specific human T cell receptors from different human and murine repertoires. Cancers 14(7):1842. https://doi.org/10.3390/cancers14071842
Lu YC, Zheng Z, Robbins PF et al (2018) An efficient single-cell RNA-Seq approach to identify neoantigen-specific T cell receptors. Mol Ther 26(2):379–389
Karpanen T, Olweus J (2017) The potential of donor T-cell repertoires in Neoantigen-targeted cancer immunotherapy. Front Immunol 8:1718
Yamamoto TN, Kishton RJ, Restifo NP (2019) Developing neoantigen-targeted T cell-based treatments for solid tumors. Nat Med 25(10):1488–1499
Harada N, Fukaya S, Wada H et al (2017) Generation of a novel HLA class I transgenic mouse model carrying a knock-in mutation at the β2-microglobulin locus. J Immunol 198(1):516–527
Vitiello A, Marchesini D, Furze J et al (1991) Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex. J Exp Med 173(4):1007–1015
Alexander J, Oseroff C, Sidney J, Sette A (2003) Derivation of HLA-B*0702 transgenic mice: functional CTL repertoire and recognition of human B*0702-restricted CTL epitopes. Hum Immunol 64(2):211–223
Alexander J, Oseroff C, Sidney J et al (1997) Derivation of HLA-A11/Kb transgenic mice: functional CTL repertoire and recognition of human A11-restricted CTL epitopes. J Immunol 159(10):4753–4761
Johnson LA, Morgan RA, Dudley ME et al (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114(3):535–546
Li LP, Lampert JC, Chen X et al (2010) Transgenic mice with a diverse human T cell antigen receptor repertoire. Nat Med 16(9):1029–1034
Gavvovidis I, Leisegang M, Willimsky G et al (2018) Targeting Merkel cell carcinoma by engineered T cells specific to T-antigens of Merkel cell polyomavirus. Clin Cancer Res 24(15):3644–3655
Çakmak-Görür N, Radke J, Rhein S et al (2019) Intracellular expression of FLT3 in Purkinje cells: implications for adoptive T-cell therapies. Leukemia 33(4):1039–1043
Obenaus M, Leitão C, Leisegang M et al (2015) Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice. Nat Biotechnol 33(4):402–407
Poncette L, Chen X, Lorenz FK, Blankenstein T (2019) Effective NY-ESO-1-specific MHC II-restricted T cell receptors from antigen-negative hosts enhance tumor regression. J Clin Invest 129(1):324–335
Yossef R, Tran E, Deniger DC et al (2018) Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy. JCI Insight 3(19):e122467
Parkhurst M, Gros A, Pasetto A et al (2017) Isolation of T-cell receptors specifically reactive with mutated tumor-associated antigens from tumor-infiltrating lymphocytes based on CD137 expression. Clin Cancer Res 23(10):2491–2505
Jahn L, van der Steen DM, Hagedoorn RS et al (2016) Generation of CD20-specific TCRs for TCR gene therapy of CD20low B-cell malignancies insusceptible to CD20-targeting antibodies. Oncotarget 7(47):77021–77037
Gros A, Parkhurst MR, Tran E et al (2016) Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients. Nat Med 22(4):433–438
Jahn L, Hombrink P, Hagedoorn RS et al (2017) TCR-based therapy for multiple myeloma and other B-cell malignancies targeting intracellular transcription factor BOB1. Blood 129(10):1284–1295
Deniger DC, Pasetto A, Tran E et al (2016) Stable, nonviral expression of mutated tumor neoantigen-specific T-cell receptors using the sleeping beauty transposon/transposase system. Mol Ther 24(6):1078–1089
Singh N, Shi J, June CH, Ruella M (2017) Genome-editing technologies in adoptive T cell immunotherapy for cancer. Curr Hematol Malig Rep 12(6):522–529
Schober K, Müller TR, Busch DH (2020) Orthotopic T-cell receptor replacement-an "enabler" for TCR-based therapies. Cell 9(6):1367
Olweus J (2017) Manufacture of CAR-T cells in the body. Nat Biotechnol 35(6):520–521
Wölfl M, Greenberg PD (2014) Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8+ T cells. Nat Protoc 9(4):950–966
Nguyen-Hoai T, Pezzutto A, Westermann J (2015) Gene gun Her2/neu DNA vaccination: evaluation of vaccine efficacy in a syngeneic Her2/neu mouse tumor model. Methods Mol Biol 1317:17–37
Leisegang M, Engels B, Meyerhuber P et al (2008) Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J Mol Med 86(5):573–583
Sommermeyer D, Uckert W (2010) Minimal amino acid exchange in human TCR constant regions fosters improved function of TCR gene-modified T cells. J Immunol 184(11):6223–6231
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rhein, S., Çakmak-Görür, N. (2022). TCR Gene Therapy for Cancer. In: Walther, W. (eds) Gene Therapy of Cancer. Methods in Molecular Biology, vol 2521. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2441-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2441-8_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2440-1
Online ISBN: 978-1-0716-2441-8
eBook Packages: Springer Protocols