Abstract LB-42: A novel method of expansion for low numbers of human CD8+ T cells in metastatic castration-resistant prostate cancer (mCRPC) patients in clinic
Authors: Zhang Qiang , Helfand Brian , LeFever Ann , Lee Chung , Kuzel Timothy Michael 
Published: May 2015

Abstract
Introduction: Enrichment and expansion of tumor specific CD8+ T cells is the most important step for developing immunotherapy for prostate cancer (CaP), especially for castration resistant patients with disease recurrence or metastasis. But the expansion is a major challenge. We have reported before that CD8+ T cells rendered insensitive to TGF-ß can enhance the proliferation of
CD8+ T cells in both human and mouse samples obtained from normals. Here we report a pre-clinical study on a high efficient method to expand human CD8+ T cells from mCRPC patients without additional stimulation by tumor specific antigen.
Methods: Whole blood (50 ml/each patient) was collected from two human patients who had mCRPC and were previously treated with docetaxel chemotherapy. Their ages were 83 and 75. . CD8+ T cells were isolated using the EasySep CD8+ T cells enrichment kit, and then cultured in in the medium cocktail consisting of RPMI-1640 without phenol, L-Glutamine (4mM, Sigma G7513), HEPEs Buffer (25mM, Cellgro 25-060-CI), 2-Mercaptoethanol (25mM, Sigma M-7522), 5% Human AB Serum-Sterile Filtered Heated Inactivated (ValleyBiomedical Inc, #HP1022), clinical grade ExpAct Treg Beads (ExpAct Treg kit, CD-3 Biotin/CD28/Anti-Biotin, CliniMACS system)(Bead: Cell ratio of 4:1), IL-2/Proleukin (aldesleukin) (100units/ml, Prometheus Laboratories, NDC 65483-116-07). The medium was changed with fresh IL-2 every 2 days, and CD3 Biotin/CD28/Anti-Biotin beads (Bead: Cell ratio of 1:1) twice weekly. The number of CD8+ T cells were evaluated by cell counter, and the growth curve was generated.
Results: Highly purified CD8+ T cells were isolated from each patient (CD3+: 94.51% ; CD8+ : 98.59%; CD14+: 5.69%; CD56+: 6.14%; CD45+: 91.44%; CD14+: 5.69%). On day 0, 8.25x104 CD8 T cells were isolated, On Day 7, the cells were expanded 31 fold (2.5625x106), and 316 fold (2.615x107) by Day 15. The expansion of cells continued to 8.75x107 on Day 22, and then reached a first peak on Day 25 (1.125x108, 1363 fold where they plateaued through day 48 (1.175x108). The growth curve was followed to a slightly higher second peak by Day 54 (1.475x108), then slowly declined from Day 69 (1.1x108), through Day 78 (1.05x108), back to the same level as Day 25.
Conclusion: Our results indicate that even low amount of human CD8+ T cells from mCRPC previously treated with chemotherapy could be expanded successfully with a suitable level of IL-2 and CD-3 Biotin/CD28/Anti-Biotin bead, even without any exogenous specific stimulation signal, such as prostate specific membrane antigen (PSMA). The CD8+ T cells could be expanded more than 1000 fold, despite a small number of CD8+ T cells at initiation. The period for optimal expansion appears to be approximately 4 weeks when the amplification peak is reached. Our study supports expansion of CD8+ T cells as a potential novel approach for immunotherapy for mCRPC.
Citation Format: Qiang Zhang, Brian Helfand, Ann LeFever, Chung Lee, Timothy Michael Kuzel. A novel method of expansion for low numbers of human CD8+ T cells in metastatic castration-resistant prostate cancer (mCRPC) patients in clinic. [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 LB-42. doi:10.1158/1538-7445.AM2014-LB-42