Abstract 3591: CSPG4 as innovative target for CAR redirected CIK lymphocytes against soft tissue sarcomas
Authors: Leuci Valeria , Rotolo Ramona , Donini Chiara , Mesiano Giulia , Fiorino Erika , Gammaitoni Loretta , Grignani Giovanni , D'Ambrosio Lorenzo , Ferrone Soldano , Dotti Gianpietro , Aglietta Massimo , Sangiolo Dario 
Published: August 2018

Abstract
Purpose of our study is to explore the anti-sarcoma activity of cytokine-induced killer cells (CIK) engineered with a chimeric antigen receptor (CAR) against the Chondroitin sulfate proteoglycan 4 (CSPG4). CIK are ex vivo expanded T lymphocytes endowed with MHC-independent tumor killing potential. The underlying hypothesis is to combine and potentiate the innate antitumor activity of CIK with a new anti-sarcoma specificity by CAR redirection against CSPG4. Soft tissue sarcomas (STS) include multiple histotypes and, in advanced stages, remain mostly incurable. A promising approach in the field is represented by adoptive immunotherapy with genetically redirected lymphocytes that require the identification of relevant tumor targets. CSPG4 may be an innovative candidate in such perspective, as it is highly expressed by several malignancies with restricted presence in normal tissues.
Experimental procedures and results. We exploited a patient derived-platform with CIK and tumor cultures derived from patients. CSPG4 was expressed by 8/8 patient-derived STS cell lines (undifferentiated pleomorphic sarcoma n=3; Liposarcoma n=1; GIST n=4). CAR-CIK were generated starting from PBMC (n=3) engineered with a 2nd-generation anti-CSPG4 CAR, including the 4-1BB co-stimulatory domain. CAR expression ranged between 70%-95%, the expansion rate (ranging from 50 to 100 fold) and phenotype (mean CD3CD56=45%; CD8=76%; NKG2D=83%) of CAR-CIK were comparable with parallel unmodified controls. CAR-CIK included higher rates of differentiated central memory and effector memory cells (36% vs 13% and 36% vs 29%, respectively). Anti-CSPG4 CAR-CIK efficiently killed STS in vitro, without differences among STS histotypes. Mean tumor-specific killing was significantly higher compared with unmodified CIK and notably relevant at low effetor/target (E/T) ratios: 95% vs 63% (10:1), 69% vs 31% (1:1), 54% vs 11% (1:4), 38% vs 8% (1:16), 31% vs 5% (1:32), 24% vs 4% (1:64). CAR-CIK activation, upon recognition of STS, led to intense IFNγ release (12,4 ng/ml), 1 log higher compared with unmodified CIK . CAR-CIK, but not unmodified controls, efficiently killed CSPG4-positive STS lacking NKG2D ligands that are naturally resistant to unmodified CIK (n=2). CAR-CIK presented a low cytotoxicity against normal keratinocytes (20% killing at E/T 3:1) that disappeared at lower but therapeutically effective concentrations (2% E/T 1:1, and 0% E/T 1:2).
Conclusions. Our findings support CSPG4 as a new target for adoptive immunotherapy against advanced sarcomas. CAR redirection highly improves the innate tumor killing ability of CIK that may be a favorable platform for the translation of CAR-based strategies into the field of solid tumors, contrasting the possible heterogeneous expression of CAR-targets. The intense sarcoma killing at very low E/T ratios is representative of realistic scenarios and promising in clinical perspective.
Citation Format: Valeria Leuci, Ramona Rotolo, Chiara Donini, Giulia Mesiano, Erika Fiorino, Loretta Gammaitoni, Giovanni Grignani, Lorenzo D'Ambrosio, Soldano Ferrone, Gianpietro Dotti, Massimo Aglietta, Dario Sangiolo. CSPG4 as innovative target for CAR redirected CIK lymphocytes against soft tissue sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3591.