Abstract 5585: TGF-ß insensitive PSMA-specific CD8+ T cells derived from metastatic castration resistant prostate cancer (MCRPC) patients enhance the tumor killing ability
Authors: Qiang Zhang, Timothy Kuzel, Brian Helfand, Ximing J. Yang, Weijun Qin, Chung Lee, Benedito Carneiro, Francis J. Giles
Journal: Cancer Research (2017)

Abstract Introduction: Manufacturing tumor specific CD8+ T cells has been an obstacle for developing prostate cancer (PCa) immunotherapies for men with MCRPC. We report a highly efficient method to expand human TGF-ß insensitivity PSMA-specific CD8+ T cells from MCRPC patients using a FDA approved Cell Processing Work Station (CPWS, Panasonic). Methods: Peripheral blood CD8+ T cells were collected from men with MCRPC by leukapheresis, and cultured in CPWS in TexMACSTM medium with CD-3 Biotin/CD28/Anti-Biotin Beads, 5% human serum, and IL-2. We developed a TβRIIDN-TK-IRES-PZ1 chimeric T cell receptor retroviral construct using an anti-PSMA IgTCR(ζ) gene (PZ1) and a dominant negative TGF-ß type II receptor (TßRIIDN), that could induce CD8+ T cells to be PSMA reactive and insensitive to TGF-ß. PC-3 cells (PSMA negative) or PSMA positive PC-3-PSMA cells (PSMA positive) were exposed to TGF-ß and used as target cells. Cytotoxicity assays were performed by LDH/NADH analysis (target cells: CD8+ T cells at 1:20, 1:50 and 1:100 respectively). Live interactions between CD8+ T cells and PCa cells was recorded by Nikon BiostationIMQ. Apoptosis of PCa was evaluated by immunofluorescence staining for Annexin V and 7-Amino-actinomycin D (7-AAD) respectively. Results: On Day 0, 2x106 CD8 T cells were placed in culture, infected with TßRIIDN-TK-IRES-PZ-1 on Day 8, and expanded 96 fold (1.92x108) by Day 36. The cellular expansion plateaued by Day 40 (1.95x108), when the ratios of CD8+ (95.2%), CD45+ (96.6%) and CD3+ (97.2%) were maintained. The cells strongly expressed PZ1 and TK gene (71.1%), and p-SMAD2 was inhibited 95%. Under TGF-ß treatment for 7 days, the growth of infected CD8+ T cells was not significantly changed, while the naïve CD8+ T control cells were suppressed by 44.8%. PC3-PSMA cells released 25.64 and 4.19 nmol/50ul/105 NADH when co-cultured with infected CD8+ T cells or with naïve CD8+ T cells respectively (p&amp;lt;0.05). There was no significant difference when PC3 cells were co-cultured with infected CD8+ T cells (7.35) or naïve CD8+ T cells (7.59). Infected CD8+ T cells induced 32.0% and 13.7% expression of Annexin V in PC3-PSMA and PC3 cells respectively, while naïve CD8+ T cells induced only 6.9% in both groups (p&amp;lt;0.05). Furthermore, Infected CD8+ T cells induced significant higher expression of 7-AAD in PC3-PSMA (0.688%) compared to naïve CD8+ T cells treatment (0.03%). There were close interactions between infected CD8+ T cells and PC3-PSMA, which was not observed in other groups. Conclusion: Our study supports expansion and retroviral infection generating TGF-ß insensitive PSMA-specific CD8+ T cells ex-vivo within 4 weeks in CPWS without any exogenous specific stimulation signal. These cells can enhance the PSMA expressed PCa killing ability and escape the inhibition by tumor secreted TGF-ß, which have a potential to overcome many of the current barriers of immunotherapies for men with MCRPC. Citation Format: Qiang Zhang, Timothy Kuzel, Brian Helfand, Ximing J. Yang, Weijun Qin, Chung Lee, Benedito Carneiro, Francis J. Giles. TGF-ß insensitive PSMA-specific CD8+ T cells derived from metastatic castration resistant prostate cancer (MCRPC) patients enhance the tumor killing ability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5585. doi:10.1158/1538-7445.AM2017-5585