Metastatic pediatric neuroectodermal solid tumors especially NB, MB, and ES have a dismal prognosis (Perkins et al, PLoS One, 2014; Smoll, Cancer, 2012). Targeted cellular therapy with T or NK cells modified with CARs is a novel approach to chemo-resistant childhood solid tumors (Grupp SA, Clin Cancer Res, 2012; Mackall C,Front Oncol, 2012). NK cells can be significantly expanded by co-culture with genetically engineered K562 cells overexpressing mb-IL21 (Lee D, PLOS, 2012). ROR1 has been identified as a novel target on B cell tumors in which CARs can be developed and utilized for targeted cellular therapy (Hudecek et al., Blood, 2010).


To evaluate the in-vitro cytotoxic activity and function of PBNK expanded ex-vivo with K562 mb-IL21 and nucleofected with mRNA encoding an anti ROR1-CAR against NB, MB, and ES.

Material and Methods:

PBNK were expanded with irradiated K562 Clone 9.mb-IL21 (generously provided by Lee D, MD, PhD, MD Anderson, TX). Ex-vivo expanded PBNK (ExPBNK) cells were electroporated with anti ROR1-CD28-41BBl-CD3ζ-tEGFR-mRNA. The cell surface expression of the ROR1-CAR on NK cells was detected using anti-mouse IgG, F(ab’)2. Cytotoxicity of ROR1 CAR-NK cells was investigated against NB (SKNBE2, SKNFI & SHSY5Y), MB (DAOY) and ES (TC71, EWS 502 & A673) cell lines by DELFIA cytotoxicity assay at an E:T ratio of 10:1. Intracellular staining of CD107a, interferon gamma, perforin and granzyme B was performed using a 10:1 E:T ratio of ROR1-CAR-NK cells against tumor targets and analyzed on the MACSQuant flow cytometer.


NB, MB, and ES cell lines expressed ROR1 (50.2±15.6%), (55.5±5.1%), and (31.5±12%), respectively. Expansion of NK cells was significantly increased 3988 ± 435 fold (p = 0.00001) at day 14 vs day 0. CAR expression after nucleofection was measured by F(ab’)2 staining and showed a significant increase in anti-ROR1-CAR- (88.3±1.7%) vs mock-electroporated NK cell populations (8.1± 6.9%) p = 0.0001 at 36-48 hours. Anti-ROR1-CAR-NK cells exhibited significantly increased lysis of ROR1 expressing tumor cell lines compared to mock NK cells (93±4.6% vs 63.6±7.4%) p = 0.00001 at an E:T of 10:1. Similarly, expression of CD107a (46.1±9.1 vs 27.6±2.4%) p = 0.001, interferon gamma (34.1±11.6 vs 16.7±6.7%) p = 0.003, granzyme B (68.5±8.9 vs 46±7.2%) p = 0.002, and perforin (51.3±7.7 vs 30.3±11.9%) p = 0.002 were significantly increased in anti-ROR1-CAR-NK cells vs Mock-NK cells at 10:1 E:T ratio against the ROR1 expressing targets.


Anti-ROR1-CAR-ex-PBNK cells had significant enhanced cytotoxicity and significantly increased CD107a, interferon gamma, perforin, and granzyme B activity against ROR1 expressing tumors. Future directions include investigating the ex-PBNK anti ROR1-CAR cells in-vivo against ROR1 expressing pediatric solid tumors.

Citation Format: Mona Elmacken, Aradhana Awasthi, Janet Ayello, Carmella VanDeVen,, Wen Luo, Yanling Liao, Stanley Riddell, Mitchell S. Cairo. Neuroblastoma (NB), Medulloblastoma (MB), and Ewing's sarcoma (ES) express ROR1 and can be effectively targeted with NK cells modified to express an anti ROR1 chimeric antigen receptor (CAR). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3155. doi:10.1158/1538-7445.AM2015-3155