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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
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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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.
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.
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