Abstract 4093: TGF-β insensitivity promotes the proliferation of human CD8+ T cells in metastatic castration resistant prostate cancer (MCRPC) patients
Authors: Zhang Qiang , Helfand Brian , LeFever Ann V. , Lee Chung , Kuzel Timothy Michael 
Published: May 2015

Introduction: Tumor derived TGF-β is a potent immunosuppressant molecule which promotes prostate cancer (CaP) progression. We previously reported that tumor derived TGF-β can suppress the growth of human CD8+ T cells. Here we report a preclinical study on the antitumor response and growth of human CD8+ T cells which were rendered insensitive to TGF-β.

Methods: Whole blood (50 ml/each pt) was collected from three human pts who previously had MCRPC and docetaxel chemotherapy. CD8+ T cells were isolated using the EasySep CD8+ T cells enrichment kit, and then cultured in the presence of clinical grade IL-2 (100unit/ml, Prometheus Labs) and CD-3 Biotin/CD28/Anti-Biotin beads (1:1, Miltenyi Biotec). We developed an TβRIIDN-TK-IRES-anti-PSMA chimeric T cell receptor construct (TβRIIDN-TK-IRES-PZ-1) using an anti-PSMA IgTCR(ζ) gene (provided by Dr. Sadelain) and a dominant negative TGF-β type II receptor (TβRIIDN) and Thymidine kinase (TK). Infection with a retroviral vector encoding this construct could induce T cells to be PSMA reactive, TGF-β insensitive, and undergo TK inducible cell suicide. The purification of CD8 T cells was confirmed by immunofluorescence assay. The transfection of TβRIIDN-TK-IRES-PZ-1 into the packaging cell line GP2-293 was performed by using lipofectamine 2000, and the retrovirus particles were infected into CD8+ T cells. Three days after infection, the expression of TK, anti-PSMA IgTCR(ζ) in CD8+ T cells were evaluated by PCR and RT-PCR. The growth of the CD8+ T cells were evaluated by cell counter every 3 days, and [3H] Thymidine incorporation assay was used to evaluate cell growth under the treatment of TGF-β (10ng/ml, 48hours).

Results: Highly purified CD8+ T cells were isolated from each pt (CD3+: 94.5%; CD8+: 98.6%). Group 1: CD8+ T cells infected with TβRIIDN-TK-IRES-PZ-1 strongly express anti-PSMA IgTCR(ζ) gene and TK gene compared with the uninfected control group (Group 2). On the 7th day after transfection, the infected cells in Group 1 increased 4 fold (from 0.045x106/ml to 0.18x106/ml); however, Group 2 only increased &lt;2 fold (from 0.064x106/ml to 0.12x106/ml). On the 17th day, the cells in Group 1 were expanded 11.5 fold (from 0.675x106 to 7.8x106) and Group 2 only 3.43 fold (from 0.96x106 to 3.3x106). Thymidine incorporation assay demonstrated that under the treatment of TGF-β, there was no significant change in Group 1, while the growth of cells in Group 2 were suppressed by 46.7%.

Conclusion: We have reported that tumor derived TGF-β suppresses functioning and proliferation of CD8+ T cells, which results in immune escape and progression of CaP. Our results indicate that CD8+ T cells rendered insensitive to TGF-β can enhance the proliferation of CD8+ T cells in human samples obtained from pts. In addition, it is possible that in combination with an anti-PSMA IgTCR(ζ) and TK inducible gene, this construct may offer a novel immunotherapy for CaP and men with advanced disease.

Citation Format: Qiang Zhang, Brian Helfand, Ann V. LeFever, Chung Lee, Timothy Michael Kuzel. TGF-β insensitivity promotes the proliferation of human CD8+ T cells in metastatic castration resistant prostate cancer (MCRPC) patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4093. doi:10.1158/1538-7445.AM2014-4093