Abstract 2212: Induction of α5β1 integrin expression rescues geriatric CAR-T cell function
Authors: Guha Prajna , Cunetta Marissa , Somasundar Ponnandai , Espat N J. , Junghans Richard P. , Katz Steven C. 
Published: July 2016

Chimeric antigen receptors expressing T cells (CAR-Ts) are a promising form of immunotherapy for solid tumors. The majority of cancer patients in the United States are over 65 years old. The impact of age-related physiologic changes on cancer therapy and immunotherapy in particular require further study. We tested our hypothesis that CAR-T from geriatric donors (gCAR-T, n = 8, &gt;65 years) are functionally impaired relative to CAR-T from younger donors (yCAR-T, n = 8, 18-45 years). T cells were genetically modified to express CAR specific for carcino-embryonic antigen (CEA), presently in use in our phase I trials. CAR-T cells were expanded ex vivo with IL2 (3000IU/ml) or IL15 (5ng/ml) for 20 days. Higher transduction efficiencies (36.9%+4.3 yCAR-T vs 21.3%+2.2 gCAR-T, p = 0.002) and improved cell expansion (2.5 fold in IL2, p = 0.01, two-tailed t-test) were observed in yCAR-T cells compared to gCAR-T. To define the underlying signaling mechanisms that led to increased proliferation in yCAR-T cells, we examined pERK, pAKT, pSTAT3 and pSTAT5. Following activation with anti-CD3, yCAR-T demonstrated significantly increased levels of pERK (13.4 fold in IL2, p = 0.03 and 2.3 fold in IL15, p = 0.04), pAKT (4.2 fold in IL2, p = 0.04 and 3.0 fold in IL15, p = 0.04), pSTAT3 (1.5 fold in IL2, p = 0.02 and 2.9 fold in IL15, p = 0.01) and pSTAT5 (5.7 fold in IL2, p = 0.02 and 2.1 fold in IL15, p = 0.03) compared to gCAR-T. Furthermore, yCAR-T contained higher proportions of CD4 effector cells (EC) (p = 0.003), CD4 effector memory (EM) cells (p = 0.002) and CD8 effector memory cells (EM) (p = 0.002). In accordance with higher numbers of CD4 and CD8 EM, yCAR-T demonstrated higher levels of CEA specific cytotoxicity compared to gCAR-T (1.3-fold over gCAR-T, p = 0.03), with maximum cytotoxicity observed in IL15 treated yCAR-T cells. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin (1.3+0.1 ng/ml, p = 0.01) and granzyme B (0.4+0.03 ng/ml, p = 0.02). Unmodified geriatric T cells (gT) had decreased α5β1 integrin expression as compared to unmodified young T cells (yT) (yT cells 44.4 +4.2 vs gT cells 22.3 +2.0, p = 0.0003), a known mediator of retroviral transduction. We found that M-CSF or TGF-β1 rescued CAR expression by gCAR-T (yCAR-T 36.9+4.3, gCAR-T 29.9+1.0, gT+M-CSF 41.4+1.0, gT+TGF-β1 40.1+2.0, p&lt;0.002 vs gCAR-T) through restoring α5β1 integrin expression (gT cells +M-CSF 41.0+1.0, gT cells + TGF- β1 31.8+1.4, yT cells 44.41 +4.2, p&lt;0.0005). We performed cytotoxicity assays and demonstrated that M-CSF and TGF-β1 (up to 1.5 fold over untreated gCAR-T, p&lt;0.05) rescued g-CAR-T tumor killing function. Our study suggests that functional impairment of gCAR-T can be mitigated through M-CSF of TGF-β1 treatment to restore α5β1 integrin expression.

Citation Format: Prajna Guha, Marissa Cunetta, Ponnandai Somasundar, N J. Espat, Richard P. Junghans, Steven C. Katz. Induction of α5β1 integrin expression rescues geriatric CAR-T cell function. [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 2212.