Abstract 2298: Cytokine-induced killer cells redirected with anti-CD44v6 chimeric antigen receptor against soft tissue sarcomas
Authors: Leuci Valeria , Casucci Monica , Grignani Giovanni , Rotolo Ramona , Vigna Elisa , Gammaitoni Loretta , Mesiano Giulia , D’Ambrosio Lorenzo , Aglietta Massimo , Bondanza Attilio , Sangiolo Dario 
Published: July 2016

Abstract
Purpose of our study is to explore the anti-sarcoma activity of cytokine-induced killer (CIK) cells engineered with a chimeric antigen receptor (CAR) against the isoform variant 6 of adhesive receptor CD44 (CD44v6). CD44v6 may be an ideal target for immunotherapy as it is a tumor-promoting antigen, associated with the metastatic process and tumor initiating cells. Advanced and metastatic soft tissue sarcomas (STS) are currently incurable and in great need for new therapeutic strategies.
CIK cells are ex vivo expanded T lymphocytes endowed with MHC-independent antitumor activity.
CIK cells are active against STS (Sangiolo et al. Cancer Research 2014) but their function decreases at low effector/target (E/T) ratios with limitations in clinical perspective. We hypothesized that CIK cells may be effective candidates for CAR-based strategies, considering their intense ex vivo expansibility and innate antitumor activity.
Experimental procedures and results. CIK cells were expanded from 9 STS patients and engineered with a lentiviral vector encoding for anti-CD44v6 CAR containing a CD28 signaling domain (Casucci et al, Blood 2013) and the HSV-TK suicide switch. Tumor killing was assessed in vitro against 10 STS (undifferentiated pleomorphic n = 5; Liposarcoma n = 2; Fibrosarcoma n = 1; GIST n = 2), in 2 cases STS targets were autologous.
All 10 STS expressed CD44v6 (Relative Fluorescence Intensity = 4, SE = 1). Mean transduction efficiency was 60% (SE = 6%). Expansion rates and phenotype of CAR-CIK were comparable with unmodified controls (CD3+CD56+ = 50%; CD8 = 66%; NKG2D = 85%).
Anti-CD44v6 CAR-CIK cells efficiently killed STS in vitro. Mean tumor-specific killing, at low E/T ratios, was significantly higher compared with unmodified CIK cells: 98% vs 84% (E/T = 10:1, p&gt;0.05), 89% vs 41% (E/T = 1:1, p = 0.0001), 65% vs 26% (E/T = 1:4 p = 0.0001).
In vitro treatment with Ganciclovir (10 μM) significantly inhibited tumor killing activity of CAR-CIK from 70% to 10% (E/T 1:1, n = 3, p = 0.008).
Blocking experiments (n = 2) with anti-CD44v6 antibody against the same epitope recognized by the CAR (VFF-18) decreased tumor-specific killing from 96% to 43% (E/T = 1:1) and 27% (E/T = 1:2). Adoptive infusion of anti-CD44v6 CAR-CIK cells significantly delayed tumor growth (p = 0.01) and reduced proliferative index (p = 0.001) of established subcutaneous fibrosarcoma xenografts in NOD/SCID mice (n = 3) compared to untreated controls (n = 3) without any sign of toxicity.
Conclusions. Ours is the first report of CAR-engineered CIK cells against solid tumors. This approach significantly potentiates the innate tumor killing ability of CIK cells with a new redirected antitumor specificity. CIK cells may be appealing alternative candidates to conventional T cells for future CAR-based strategies against solid tumors. Our findings support CD44v6 as a valuable target for adoptive cell therapies against currently incurable sarcomas.
Citation Format: Valeria Leuci, Monica Casucci, Giovanni Grignani, Ramona Rotolo, Elisa Vigna, Loretta Gammaitoni, Giulia Mesiano, Lorenzo D’Ambrosio, Massimo Aglietta, Attilio Bondanza, Dario Sangiolo. Cytokine-induced killer cells redirected with anti-CD44v6 chimeric antigen receptor against soft tissue sarcomas. [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 2298.