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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2019, Vol. 13 Issue (1) : 69-82    https://doi.org/10.1007/s11684-018-0677-1
RESEARCH ARTICLE |
High-affinity T cell receptors redirect cytokine-activated T cells (CAT) to kill cancer cells
Synat Kang1,2, Yanyan Li1, Yifeng Bao1, Yi Li1,2()
1. State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
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Abstract

Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1157–165 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-g and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.

Keywords cytokine-activated T cells      high-affinity T cell receptor      cancer immunotherapy      TCR-CAT     
Corresponding Authors: Yi Li   
Just Accepted Date: 10 January 2019   Online First Date: 18 February 2019    Issue Date: 12 March 2019
 Cite this article:   
Synat Kang,Yanyan Li,Yifeng Bao, et al. High-affinity T cell receptors redirect cytokine-activated T cells (CAT) to kill cancer cells[J]. Front. Med., 2019, 13(1): 69-82.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-018-0677-1
http://academic.hep.com.cn/fmd/EN/Y2019/V13/I1/69
Fig.1  Expansion of CAT or T cells and phenotypes of high-affinity TCR-transduced CAT (TCR-CAT) cells or T cells (TCR-T). (A) Time line of cell expansion, transduction, and functional test. a) CAT cells. PBMCs from consented healthy donors were initially stimulated with IFN-g (day 0), followed by addition of CD3 mAb (day 1). b) T cells. PBMCs from consented healthy donors were activated with CD3/CD28 microbeads. IL-2 was supplemented every 3 d. Other details are provided in the Materials and methods section. (B) Efficiency of cell expression of 1G4 HAT in CAT cells. The expression levels of TCR in CAT cells were confirmed by flow cytometry by staining the cells with anti-human TCR vb13.1 or NY-ESO-1 tetramer.
Fig.2  Antigen-specific recognition by high-affinity TCRs expressed on CAT cells. (A) Cytokine release assay. A total of 2×103 transduced TCR-CAT (32 µmol/L, 1.07 µmol/L, 84 nmol/L, 5 nmol/L, and 26 pmol/L) or nontransduced CAT (NT-CAT) cells were cocultured with peptide-pulsed T2 cells at an E:T ratio of 1:10 for 20 h. The amount of peptide loaded on T2 cells was in serial concentrations of antigen NY-ESO-1157–165 (SLLMWITQV) peptide or an irrelevant peptide SAGE-1 VFSTVPPAFI. (B) Cytotoxicity assays. The effector cells were cocultured with peptide-pulsed T2 cells at serial concentration of the NY-ESO-1 peptide or the irrelevant peptide SAGE-1 for 20 h. The E:T ratio is 5:1 with the constant number of target cells (2×104). Data shown are mean±standard deviation (SD) of three representative tests.
Fig.3  Functions of high-affinity TCRs (1G4 HATs) redirected CAT cell against testis antigens on cancer cells. The enhanced T cell activation was detected after 1G4 HATs transfected CAT cells with the tumor cells at an E:T ratio of 1:10 by ELISPOT assays. (A and C) IFN-g release; (B and D) Granzyme B release. Nontransduced CAT (NT-CAT) cells and transduced-1G4 TCRs (32 µmol/L, 1.07 µmol/L, 84 nmol/L, 5 nmol/L, and 26 pmol/L) CAT cells were stimulated with target cells A375 (HLA-A2+/NY-ESO-1+), Mel624 (HLA-A2+/NY-ESO-1+), Mel526 (HLA-A2+/NY-ESO-1), NCI-H1650 (HLA-A2+/NY-ESO-1), and K562 (HLA-A2+/NY-ESO-1+). (E) Enhanced cytotoxicity of TCR-CAT for target cells. The cytotoxic activities were detected by LDH assay at various E:T ratios of 5:1, 1:1 or 1:2. The target cells A375, Mel624, NCI-H1650, and Mel526 were pre-incubated with effector cells (NT-CAT or TCR-CAT) for 20 h with the constant number of target cells (2×104). Data shown are mean±SD of three representative tests. Asterisks (*) indicate statistical significance (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001) of TCR-CAT compared with NT-CAT.
Fig.4  CD107a expression of activated TCR-CAT cells. Nontransduced-CAT (NT-CAT) and TCR-CAT (32 µmol/L, 1.07 µmol/L, and 84 nmol/L) cells were stimulated with HLA-A2+/NY-ESO-1+ target cells of A375 or Mel624 and HLA-A2+/NY-ESO-1 cells of Mel526 or NCI-H1650 at E:T ratio of 1:1 for 16 h with the constant number of target cells (2 × 104). Percentages of CD107a positive cell population were gated electronically in the channel CD107a (PE) with CD3 (APC). Data shown are mean±SD of three representative tests. Asterisks (*) indicate statistical significance (* P <0.05; ** P <0.01; *** P <0.001) of TCR-CAT compared with NT-CAT.
Fig.5  Enhanced TCR-CAT killing tumor target cells was attenuated by soluble 1G4 HAT. The HLA-A2+/NY-ESO-1+ target cells of U266-B1 or A375 and HLA-A2+/NY-ESO-1 cells of Mel526 were pre-incubated with a final concentration of 20 µg/mL of soluble 1G4 HAT (KD of 26 pmol/L) for 30 min, followed by coculture with 2×103 of CAT cells or TCR-CAT (1.07 µmol/L) and TCR-CAT (32 µmol/L) cells at E:T= 1:10 for 20 h (ELISPOT assay (A)), or with 1×105 of the cells at E:T= 5:1 for 20 h (LDH assay (B)). Data shown are mean±SD of three representative tests. Asterisks (*) indicate statistical significance (*P<0.05; **P<0.01; ***P<0.001) of soluble 1G4 HAT unblocked (black bar) compared with blocked antigen binding on the cells (gray bar).
Fig.6  TCR-CAT cells targeted cancer cell killing measured in real time. A total of 7×103 of targeted cells A375 (HLA-A2+/NY-ESO-1+) or NCI-H1650 (HLA-A2+/NY-ESO-1) were incubated overnight, followed by cocultured with nontransduced CAT (NT-CAT) or TCR-CAT cells (32 µmol/L, 1.07 µmol/L, and 84 nmol/L) at E:T ratio of 5:1 for 48 h. Images were taken at intervals of 2 h. (A) A375 (antigen positive cells). (B) NCI-H1650 (antigen negative cells). Data shown are mean±SD of three representative tests.
Fig.7  Comparison of the activities of TCR-CAT and TCR-T cells transduced with TCR-1.07 µmol/L. The efficiency of TCR-CAT and TCR-T cell transduction with the TCR-1.07 µmol/L showed 31% and 44%, respectively, with staining by anti-mouse TCRβ-C domain mAb. The activities of TCR-CAT and TCR-T cells were measured for secretions of IFN-g (A) and granzyme B (B) at day 14 of culturing. A total of 2×103 of TCR-CAT, TCR-T, CAT, or T cells were cocultured with HLA-A2+/NY-ESO-1+ cells of A375 or Mel624 and HLA-A2+/NY-ESO-1 cells of NCI-H1650 or Mel526 at E:T= 1:10 for 20 h. (C) Cytotoxic activity of TCR-CAT compared with TCR-T cells. The target cells A375, Mel624, NCI-H1650 or Mel526 were pre-incubated with effector cells at E:T= 5:1 for 20 h with a constant number of target cells (2×104). Data shown are mean±SD of three representative tests. Asterisks (*) indicate statistical significance (*P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001) of TCR-CAT or TCR-T cells compared with control nontransduced CAT or T cells (NT-CAT or NT-T). Hashes (#P <0.05, ##P <0.01, ###P <0.001) indicate statistical significant differences between TCR-CAT and TCR-T cells.
1 RAMorgan, ME Dudley, JRWunderlich, MSHughes, JCYang, RMSherry, RERoyal, SLTopalian, USKammula, NPRestifo, ZZheng, ANahvi, CRde Vries, LJRogers-Freezer, SAMavroukakis, SARosenberg. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006; 314(5796): 126–129
https://doi.org/10.1126/science.1129003 pmid: 16946036
2 RAMorgan, N Chinnasamy, DAbate-Daga, AGros, PF Robbins, ZZheng, MEDudley, SAFeldman, JCYang, RMSherry, GQPhan, MSHughes, USKammula, ADMiller, CJHessman, AAStewart, NPRestifo, MMQuezado, MAlimchandani, AZRosenberg, ANath, T Wang, BBielekova, SCWuest, NAkula, FJMcMahon, SWilde, BMosetter, DJSchendel, CMLaurencot, SARosenberg. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother 2013; 36(2): 133–151
https://doi.org/10.1097/CJI.0b013e3182829903 pmid: 23377668
3 PFRobbins, RA Morgan, SAFeldman, JCYang, RMSherry, MEDudley, JRWunderlich, AVNahvi, LJHelman, CLMackall, USKammula, MSHughes, NPRestifo, MRaffeld, CCLee, CL Levy, YFLi, MEl-Gamil, SLSchwarz, CLaurencot, SARosenberg. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011; 29(7): 917–924
https://doi.org/10.1200/JCO.2010.32.2537 pmid: 21282551
4 MPTan, GM Dolton, ABGerry, JEBrewer, ADBennett, NJPumphrey, BKJakobsen, AKSewell. Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells. Clin Exp Immunol 2017; 187(1): 124–137
https://doi.org/10.1111/cei.12828 pmid: 27324616
5 LLLanier, G Yu, JHPhillips. Analysis of FcγRIII (CD16) membrane expression and association with CD3ζ and Fc epsilon RI-γ by site-directed mutation. J Immunol 1991; 146(5): 1571–1576
pmid: 1825220
6 YXing, KA Hogquist. T-cell tolerance: central and peripheral. Cold Spring Harb Perspect Biol 2012; 4(6): a006957
https://doi.org/10.1101/cshperspect.a006957 pmid: 22661634
7 NLiddy, G Bossi, KJAdams, ALissina, TMMahon, NJHassan, JGavarret, FCBianchi, NJPumphrey, KLadell, EGostick, AKSewell, NMLissin, NEHarwood, PEMolloy, YLi, BJ Cameron, MSami, EEBaston, PTTodorov, SJPaston, REDennis, JVHarper, SMDunn, RAshfield, AJohnson, YMcGrath, GPlesa, CHJune, MKalos, DAPrice, AVuidepot, DDWilliams, DHSutton, BKJakobsen. Monoclonal TCR-redirected tumor cell killing. Nat Med 2012; 18(6): 980–987
https://doi.org/10.1038/nm.2764 pmid: 22561687
8 YZhao, AD Bennett, ZZheng, QJWang, PFRobbins, LYYu, Y Li, PEMolloy, SMDunn, BKJakobsen, SARosenberg, RAMorgan. High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines. J Immunol 2007; 179(9): 5845–5854
https://doi.org/10.4049/jimmunol.179.9.5845 pmid: 17947658
9 APRapoport, EA Stadtmauer, GKBinder-Scholl, OGoloubeva, DTVogl, SFLacey, AZBadros, AGarfall, BWeiss, JFinklestein, IKulikovskaya, SKSinha, SKronsberg, MGupta, SBond, L Melchiori, JEBrewer, ADBennett, ABGerry, NJPumphrey, DWilliams, HKTayton-Martin, LRibeiro, THoldich, SYanovich, NHardy, JYared, NKerr, S Philip, SWestphal, DLSiegel, BLLevine, BKJakobsen, MKalos, CHJune. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med 2015; 21(8): 914–921
https://doi.org/10.1038/nm.3910 pmid: 26193344
10 PFRobbins, SH Kassim, TLTran, JSCrystal, RAMorgan, SAFeldman, JCYang, MEDudley, JRWunderlich, RMSherry, USKammula, MSHughes, NPRestifo, MRaffeld, CCLee, YF Li, MEl-Gamil, SARosenberg. A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: long-term follow-up and correlates with response. Clin Cancer Res 2015; 21(5): 1019–1027
https://doi.org/10.1158/1078-0432.CCR-14-2708 pmid: 25538264
11 IGSchmidt-Wolf, P Lefterova, BAMehta, LPFernandez, DHuhn, KG Blume, ILWeissman, RSNegrin. Phenotypic characterization and identification of effector cells involved in tumor cell recognition of cytokine-induced killer cells. Exp Hematol 1993; 21(13): 1673–1679
pmid: 7694868
12 APievani, C Belussi, CKlein, ARambaldi, JGolay, MIntrona. Enhanced killing of human B-cell lymphoma targets by combined use of cytokine-induced killer cell (CIK) cultures and anti-CD20 antibodies. Blood 2011; 117(2): 510–518
https://doi.org/10.1182/blood-2010-06-290858 pmid: 21048157
13 JJMata-Molanes, M Sureda González, B Valenzuela Jiménez, EM Martínez Navarro, A Brugarolas Masllorens. Cancer immunotherapy with cytokine-induced killer cells. Target Oncol 2017; 12(3): 289–299
https://doi.org/10.1007/s11523-017-0489-2 pmid: 28474278
14 GMesiano, M Todorovic, LGammaitoni, VLeuci, LGiraudo Diego, FCarnevale-Schianca, FFagioli, WPiacibello, MAglietta, DSangiolo. Cytokine-induced killer (CIK) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors. Expert Opin Biol Ther 2012; 12(6): 673–684
https://doi.org/10.1517/14712598.2012.675323 pmid: 22500889
15 IGSchmidt-Wolf, RS Negrin, HPKiem, KGBlume, ILWeissman. Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity. J Exp Med 1991; 174(1): 139–149
https://doi.org/10.1084/jem.174.1.139 pmid: 1711560
16 MTodorovic, G Mesiano, LGammaitoni, VLeuci, LGiraudo Diego, CCammarata, NJordaney, FCarnevale-Schianca, SGallo, FFagioli, WPiacibello, ARElia, YPignochino, CDell’aglio, GGrignani, ACignetti, MAglietta, DSangiolo. Ex vivo allogeneic stimulation significantly improves expansion of cytokine-induced killer cells without increasing their alloreactivity across HLA barriers. J Immunother 2012; 35(7): 579–586
https://doi.org/10.1097/CJI.0b013e31826b1fd9 pmid: 22892454
17 SHDu, Z Li, CChen, WKTan, Z Chi, TWKwang, XHXu, S Wang. Co-expansion of cytokine-induced killer cells and Vg9Vd2 T cells for CAR T-cell therapy. PLoS One 2016; 11(9): e0161820
https://doi.org/10.1371/journal.pone.0161820 pmid: 27598655
18 XGao, Y Mi, NGuo, HXu, L Xu, XGou, WJin. Cytokine-induced killer cells as pharmacological tools for cancer immunotherapy. Front Immunol 2017; 8: 774
https://doi.org/10.3389/fimmu.2017.00774 pmid: 28729866
19 YGuo, W Han. Cytokine-induced killer (CIK) cells: from basic research to clinical translation. Chin J Cancer 2015; 34(3): 99–107
https://doi.org/10.1186/s40880-015-0002-1 pmid: 25962508
20 CSHinrichs, SA Rosenberg. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev 2014; 257(1): 56–71
https://doi.org/10.1111/imr.12132 pmid: 24329789
21 AOGure, R Chua, BWilliamson, MGonen, CAFerrera, SGnjatic, GRitter, AJSimpson, YTChen, LJOld, NK Altorki. Cancer-testis genes are coordinately expressed and are markers of poor outcome in non-small cell lung cancer. Clin Cancer Res 2005; 11(22): 8055–8062
https://doi.org/10.1158/1078-0432.CCR-05-1203 pmid: 16299236
22 YTChen, MJ Scanlan, USahin, OTüreci, AOGure, STsang, BWilliamson, EStockert, MPfreundschuh, LJOld. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc Natl Acad Sci USA 1997; 94(5): 1914–1918
https://doi.org/10.1073/pnas.94.5.1914 pmid: 9050879
23 CBarrow, J Browning, DMacGregor, IDDavis, SSturrock, AAJungbluth, JCebon. Tumor antigen expression in melanoma varies according to antigen and stage. Clin Cancer Res 2006; 12(3): 764–771
https://doi.org/10.1158/1078-0432.CCR-05-1544 pmid: 16467087
24 SGnjatic, H Nishikawa, AAJungbluth, AOGüre, GRitter, EJäger, AKnuth, YTChen, LJOld. NY-ESO-1: review of an immunogenic tumor antigen. Adv Cancer Res 2006; 95: 1–30
https://doi.org/10.1016/S0065-230X(06)95001-5 pmid: 16860654
25 PFRobbins, YF Li, MEl-Gamil, YZhao, JA Wargo, ZZheng, HXu, RA Morgan, SAFeldman, LAJohnson, ADBennett, SMDunn, TMMahon, BKJakobsen, SARosenberg. Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J Immunol 2008; 180(9): 6116–6131
https://doi.org/10.4049/jimmunol.180.9.6116 pmid: 18424733
26 SMWhite, M Renda, NYNam, EKlimatcheva, YZhu, J Fisk, MHalterman, BJRimel, HFederoff, SPandya, JDRosenblatt, VPlanelles. Lentivirus vectors using human and simian immunodeficiency virus elements. J Virol 1999; 73(4): 2832–2840
pmid: 10074131
27 LSastry, T Johnson, MJHobson, BSmucker, KCornetta. Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther 2002; 9(17): 1155–1162
https://doi.org/10.1038/sj.gt.3301731 pmid: 12170379
28 ARElia, P Circosta, DSangiolo, CBonini, LGammaitoni, SMastaglio, PGenovese, MGeuna, FAvolio, GInghirami, CTarella, ACignetti. Cytokine-induced killer cells engineered with exogenous T-cell receptors directed against melanoma antigens: enhanced efficacy of effector cells endowed with a double mechanism of tumor recognition. Hum Gene Ther 2015; 26(4): 220–231
https://doi.org/10.1089/hum.2014.112 pmid: 25758764
29 MPTan, AB Gerry, JEBrewer, LMelchiori, JSBridgeman, ADBennett, NJPumphrey, BKJakobsen, DAPrice, KLadell, AKSewell. T cell receptor binding affinity governs the functional profile of cancer-specific CD8+ T cells. Clin Exp Immunol 2015; 180(2): 255–270
https://doi.org/10.1111/cei.12570 pmid: 25496365
30 MRBetts, JM Brenchley, DAPrice, SCDe Rosa, DCDouek, MRoederer, RAKoup. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 2003; 281(1-2): 65–78
https://doi.org/10.1016/S0022-1759(03)00265-5 pmid: 14580882
31 APievani, G Borleri, DPende, LMoretta, ARambaldi, JGolay, MIntrona. Dual-functional capability of CD3+CD56+ CIK cells, a T-cell subset that acquires NK function and retains TCR-mediated specific cytotoxicity. Blood 2011; 118(12): 3301–3310
https://doi.org/10.1182/blood-2011-02-336321 pmid: 21821703
32 YMa, YC Xu, LTang, ZZhang, JWang, HX Wang. Cytokine-induced killer (CIK) cell therapy for patients with hepatocellular carcinoma: efficacy and safety. Exp Hematol Oncol 2012; 1(1): 11
https://doi.org/10.1186/2162-3619-1-11 pmid: 23210562
33 JDStone, AS Chervin, DMKranz. T-cell receptor binding affinities and kinetics: impact on T-cell activity and specificity. Immunology 2009; 126(2): 165–176
https://doi.org/10.1111/j.1365-2567.2008.03015.x pmid: 19125887
34 WCChan, YC Linn. A comparison between cytokine- and bead-stimulated polyclonal T cells: the superiority of each and their possible complementary role. Cytotechnology 2016; 68(4): 735–748
https://doi.org/10.1007/s10616-014-9825-x pmid: 25481728
35 YCLinn, SK Lau, BHLiu, LHNg, HX Yong, KMHui. Characterization of the recognition and functional heterogeneity exhibited by cytokine-induced killer cell subsets against acute myeloid leukaemia target cell. Immunology 2009; 126(3): 423–435
https://doi.org/10.1111/j.1365-2567.2008.02910.x pmid: 18778291
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