Anti-melanoma activity of T cells redirected with a TCR-like chimeric antigen receptor

Ge Zhang, Lei Wang, Honglian Cui, Xiaomin Wang, Ganlin Zhang, Juan Ma, Huamin Han, Wen He, Wei Wang, Yunfeng Zhao, Changzhen Liu, Meiyi Sun, Bin Gao, Ge Zhang, Lei Wang, Honglian Cui, Xiaomin Wang, Ganlin Zhang, Juan Ma, Huamin Han, Wen He, Wei Wang, Yunfeng Zhao, Changzhen Liu, Meiyi Sun, Bin Gao

Abstract

Genetically modified T cells to recognize tumor-associated antigens by transgenic TCRs or chimeric antigen receptors (CAR) have been successfully applied in clinical trials. However, the disadvantages of either TCR mismatching or the requirement of a surface tumor antigen limit their wider applications in adoptive T cell therapy. A TCR-like chimeric receptor, specific for the melanoma-related gp100/HLA-A2 complex was created by joining a TCR-like antibody GPA7 with the endodomains of CD28 and CD3-ζ chain. This TCR-like CAR, GPA7-28z, was subsequently introduced into human T cells. Retargeted T cells expressing GPA7-28z could exhibit efficient cytotoxic activities against human melanoma cells in vitro in the context with HLA-A2. Furthermore, infusion of GPA7-28z-transduced T cells suppressed melanoma progression in a xenograft mouse model. Redirecting human T cells with TCR-like CARs would be a promising alternative approach to TCR-mediated therapy for melanoma patients, which is also feasible for targeting a variety of other tumor antigens.

Figures

Figure 1. Lentiviral vector design and expression…
Figure 1. Lentiviral vector design and expression of GPA7-28z transgene in human primary T cells.
(a) Schemetic diagram of lentiviral vector used for GPA7-28z expression. GPA7 represents a single domain antibody; hIgG1-Fc, CH2-CH3 hinge domains of human IgG1; CD28, CD28 transmembrane and intracellular domain; CD3-ζ, CD3 ζ chain intracellular domain. (b) Cell surface expression of chimeric antigen receptor GPA7-28z by human primary T cells 72 hours after transduction. CAR-transduced cells were stained with gp100-HLA-A2 tetramer-PE and a mouse anti-human IgG1-FITC to recognize the CAR (filled black histograms). Mock-transduced cell culture was stained as control (open histograms). Representatives of flow cytometry plots of three independent repeats are shown. Frequencies of positive cells are shown as mean ± s.d. (n = 3) in the upper right corner.
Figure 2. Co-culture of GPA7-CD28/ζ transduced PBMC…
Figure 2. Co-culture of GPA7-CD28/ζ transduced PBMC with gp100-pulsed T2 cells.
(a) Schematic illustration of PBMC stimulation, transduction and expansion protocol. After lentiviral transduction, PBMC were cultured and re-stimulated with irradiated antigen-loaded T2 cells from day 4. (b) Cell surface phenotype of transduced T-cell cultures after a round of re-stimulation with gp100-loaded T2 cells. Anti-CD3-PE, anti-CD4-FITC, anti-CD8-FITC and PE-labeled gp100-HLA-A2 tetramer were used for characterization. Control was isotype-stained cells. Frequencies for each population were indicated above panels. A representative of three independent repeats from flow cytometry plots is shown.
Figure 3. GPA7-28z T cells exhibit a…
Figure 3. GPA7-28z T cells exhibit a high functional activity in a peptide-specific and HLA-I restricted manner.
(a) Cytokine secretions of transduced T cells were analyzed for antigen specificity in IFN-γ ELISpot. T2 cells were pulsed with gp100209–217 or control Flu58–66 peptide before incubation with GPA7-28z T cells. Mock-transduced T cells served as a negative control. IFN-γ release by GPA7-28z vs mock-transduced T cells was investigated. Data are presented as mean ± s.d. Asterisks indicate significant differences to control mock-transduced T cells (n = 4; Student's t-test; **P < 0.01). (b) Granzyme B secretion by GPA7-28z and mock-transduced T cells in response to the stimulation with Malme-3m, MEL-624, Primary 1, Primary 2, MEL-888, SK-MEL-28 and B16F10. Data are presented as mean ± s.d. from four replicates (**P < 0.01 versus T-Mock, Student's t-test). A representative experiment of three was shown. (c) IFN-γ release by GPA7-28z T cells compared with that by mock-transduced T cells (control) in response to the exposure to various melanoma cell lines, primary melanoma cell cultures and gp100-pulsed T2 cells. One representative well for each group (left panel) and calculated spot numbers (right panel) are shown. Asterisks indicate significant differences to control mock-transduced T cells (n = 4, **P < 0.01, Student's t-test). A representative experiment of three was shown.
Figure 4. CAR transduced, GPA7-28z T cells…
Figure 4. CAR transduced, GPA7-28z T cells efficiently lyse T2 cells pulsed with gp100 peptides as well as HLA-A2 positive melanoma cells.
(a) Specificity and functional avidity assessment of GPA7-28z T cells. GPA7-28z T cells were incubated with calcein-AM-labeled T2 cells at E/T ratio of 1, in the presence of different concentrations of gp100 peptide () or flu peptide (). Functional avidity was defined as the peptide concentration generating 50% of the maximum specific response. Data are presented as mean ± s.d. (n = 3; Student's t-test). (b) GPA7-28z-mediated cytotoxicity toward gp100 was blocked by a specific antibody to HLA-A2. T2 target cells incubated with gp100 peptide (black), with gp100 peptide and BB7.2 antibody (gray), or T2 cells without peptide, but plus antibody (white) were plated with GPA7-28z T cells in a 4-hour cytotoxicity assay at E/T ratios from 16 to 1. Four replicates were used for each dilution of effector cells. Data are presented as mean ± s.d. (n = 3; one-way ANOVA followed by Tukey's post-hoc test was used for statistic analysis of the data; **P < 0.01). (c) GPA7-28z T cells exert enhanced cytotoxic lysis against HLA-A2-positive melanoma cell lines and primary melanoma cells. Cytotoxic ability of GPA7-28z () and mock-transduced () T cells towards Malme-3m, MEL-624, Primary 1, Primary 2, MEL-888, SK-MEL-28 or B16F10 was compared at different E/T ratio between 1 to 32. Data are presented as mean ± s.d. calculated from three repeated experiments (*P < 0.05 versus T-Mock, Student's t-test).
Figure 5. Anti-tumor efficacy of GPA7-28z-transduced human…
Figure 5. Anti-tumor efficacy of GPA7-28z-transduced human primary T cells in vivo.
SCID-Beige mice were inoculated subcutaneously with 1 × 106 Malme-3m-luc melanoma cells. On day 8 and 13, tumor-bearing mice were locally treated with 5 × 106 GPA7-28z-transduced (n = 6) or mock-transduced (n = 6) human primary T cells. Tumor growth was monitored using an in vivo imaging system (Xenogen IVIS imaging system). (a) Tumor growth was measured as photon emission in a representative cohort of 3 mice from each group on day 5, 10, 16 and 23. (b) Photon emission from luc+ tumor cells was quantified and intensity of bioluminescent signal was presented as mean ± s.d. from 6 mice each for both two cohorts. (c) Survival curves of mice engrafted with Malme-3m-luc tumor cells receiving GPA7-28z or mock-transduced T cells. For intensity of bioluminescent signal, the significant difference between that of GPA7-28z and that of mock-transduced T cell-treated groups was compared using a two-tailed Student's t-test (n = 6; **P < 0.01). For survival curves, data were plotted using the Kaplan–Meier method and statistically analysed by log-rank analysis (n = 6; **P < 0.01).

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