Abstract 2314: Different stimulation conditions affect the immune phenotype of GD2-specific chimeric antigen receptor-expressing T cells
Authors: Ochs Laurin , Altvater Bianca , Kailayangiri Sareetha , Spurny Christian , Rossig Claudia , Jamitzky Silke 
Published: July 2016

Abstract
The in vivo persistence of chimeric antigen receptor (CAR) modified T cells is a major prerequisite for their antitumor activity and was found to be associated with a less differentiated immune phenotype. Here, we compared two in vitro T cell stimulation conditions: coated anti-CD3/CD28 antibodies [3/28], and Dynabead stimulation of enriched CD3+ T cells [DB]. Peripheral blood T cells from three healthy donors were stimulated with either of the two methods, retrovirally transduced with the GD2-specific CAR GD2-BBz on day 2 or 3, and expanded in RPMI/AIMV medium with 50 IU/ml recombinant human interleukin-2 for 13 days. T cell expansion rates were comparable between the two stimulation conditions and independent of CAR gene expression. Transduction efficiencies, determined by staining with the GD2-CAR-specific antibody Ganglidiomab, were also comparable. The immune phenotype by expression of CD3, CD4, CD8, CD45RO and CD197 was determined by flow cytometry analysis on day 13 or 14 after initial stimulation. The proportions of central memory (TCM), effector memory (TEM) or naïve T cells (TN) within the two types of cultures were noticeably different (Table 1), with a higher proportion of non-transduced CD8+ T cells with a TCM phenotype after DB compared to CD3/CD28 stimulation (p = 0.005 for DB d2, p = 0.01 for DB d3). Compared to non-transduced T cells, CAR-expressing cells of all types of cultures had higher proportions of TCM cells (p = 0.02 for CD4+ T cells, p&lt;0.01 for CD8+ T cells). In conclusion, we found that the stimulation conditions have a strong impact on the T cell phenotype and that retroviral CAR gene transduction can also affect T cell differentiation. The optimal T cell culture conditions for a product with sustained persistence in vivo will ultimately emerge from clinical trials. Table 1:Proportions of T cell subpopulations on day 13 or 14 (medians and ranges)xyTN: CD45RO-/CD197+TCM: CD45RO+/CD197+TEM: CD45RO+/CD197-3/28NT37.9% CD4+ (31.8-41.3) 53.0% CD8+ (20.9-72.9)20.2% CD4+ (20.2-27.0) 6.1% CD8+ (3.7-6.9)35.2% CD4+ (32.8-45.4) 35.5% CD8+ (17.0-66.3)CAR d224.4% CD4+ (15.1-28.8) 32.9% CD8+ (23.9-48.9)33.6% CD4+ (29.9-49.3) 27.7% CD8+ (17.7-38.8)37.3% CD4+ (22.4-47.2) 27.1% CD8+ (25.4-32.7)DBNT33.1% CD4+ (15.0-45.6) 53.8% CD8+ (46.8-53.8)32.6% CD4+ (29.9-45.2) 19.6% CD8+ (17.8-26.3)23.3% CD4+ (16.6-51.7) 12.1% CD8+ (10.6-15.2)CAR d211.5% CD4+ (10.9-27.7) 37.1% CD8+ (24.7-41.860.7% CD4+ (53.0-69.7) 48.0% CD8+ (34.4-65.2)16.5% CD4+ (16.1-26.6) 8.7% CD8+ (5.7-10.3)DBNT33.2% CD4+ (26.5-55.2) 51.6% CD8+ (34.2-62.5)23.0% CD4+ (21.9-40.2) 14.2% CD8+ (11.2-16.9)19.1% CD4+ (17.6-48.6) 15.2% CD8+ (13.0-19.6)CAR d317.5% CD4+ (13.4-34.5) 36.6% CD8+ (28.5-46.5)45.7% CD4+ (36.6-57.5) 38.0% CD8+ (22.2-55.1)22.0% CD4+ (21.4-32.6) 10.8% CD8+ (8.5-19.6)
Citation Format: Laurin Ochs, Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Claudia Rossig, Silke Jamitzky. Different stimulation conditions affect the immune phenotype of GD2-specific chimeric antigen receptor-expressing T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2314.