linfociti killer reindirizzati da anti-CSPG4 CAR contro melanoma metastatico

Purpose of our study was the preclinical exploration of a new adoptive immunotherapy strategy against metastatic melanoma. Our experimental approach is based on immune effectors genetically redirected with a Chimeric-Antigen Receptor (CAR) against the tumor specific target Chondroitin Sulfate Proteoglycan 4 (CSPG4), an attractive target because of its high/selective expression and role in cancer cells. We focused our attention on two promising immune effector candidates for CAR-based strategies: Cytokine-Induced Killer (CIK) cells and the Natural Killer (NK) cell line NK-92 both endowed with intrinsic HLA-independent antitumor potential against a broad range of solid tumors. The underlying hypothesis is to combine and potentiate the innate antitumor activity of CIK and NK-92 with a new anti-melanoma specificity by CAR redirection against CSPG4. Patient-derived CAR+ CIK (n=3) and CAR+ NK-92 (n=2) were successfully generated by transduction with a retroviral vector encoding for antiCSPG4-CAR containing the CD28 signaling domain. The relative high levels of CAR expression (antiCSPG4 CAR+ CIK = 51.4%, antiCSPG4 CAR+ NK-92= 65%) and the significant stability of transduction confirmed CIK and NK-92 as good platforms of CAR engineering. The expansion rates and phenotype of CAR+ CIK (CD3+CD8+ 83.1%; CD3+CD56+ 21.7%) and CAR+ NK-92 were comparable with unmodified controls. We generated patient-derived melanoma cell lines (n=6) from surgical biopsies as functional targets. Their mean CSPG4 membrane expression was 85%; while one melanoma sample (M071) was defective for the membrane expression of HLA class-I molecules. CAR+ CIK and CAR+ NK-92 efficiently killed in vitro melanoma targets (48 hours co-culture). The antimelanoma activity of antiCSPG4-CAR killer lymphocytes, both CIK and NK92, was intense especially at low effector/target (E/T) ratios. The mean melanoma-specific killing was significantly higher compared with paired controls (unmodified effectors): for CAR+ CIK 84% vs 26.5% (E/T 1:1) 76.2% vs 14.9% (E/T 1:2), 59.3% vs 7.8% (E/T 1:4) and for CAR+ NK-92 81.7% vs 61.7% (E/T 1:1), 72.2% vs 51.2% (E/T 1:2), 63.7% vs 49.2% (E/T 1:4). In selected experiments we focused our attention on potential killing melanoma Cancer Stem Cells (mCSC) by CAR+CIK. mCSC were visualized by tumor-engineering with a lentiviral CSC-detector vector encoding the enhanced Green Fluorescent Protein under the control of the stemness promoter oct4. In selected experiments (n=3) we confirmed that CAR+ CIK were capable of killing mCSC. We report first preclinical evidence of CAR+ CIK and CAR+ NK-92's activity against advanced melanoma and propose CSPG4 as an innovative target in this setting. Overall, these findings support the exploitation of CIK and NK-feasible platforms for the translation of CAR-based strategies and might allow the generation of potent cell- based therapeutics for the treatment of metastatic melanoma. The potential efficacy against putative CSC holds perspective potential in contrasting disease relapses.