Neuroblastoma and Ewing's Sarcoma Associated with ROR1 Expression Can be Effectively Targeted with NK Cells Modified to Express an Anti ROR1 Chimeric Antigen Receptor

Background:

Metastatic solid tumors, especially neuroblastoma (NB) and Ewing's sarcoma (ES) have dismal prognosis (Perkins etal, PLoS One, 2014). Targeted cellular therapy with T or NK cells modified with chimeric antigen receptors (CAR) 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). Riddell and colleagues have identified ROR1 as a novel target on B cell tumors in which CARs can be developed and utilized for targeted cellular therapy (Riddell S, Blood, 2010)

Objective:

To evaluate the in-vitro NK cytotoxic activity and NK cell function following ex-vivo K562 mb-IL21expansion of PBNK nucleofected with anti ROR1-CAR against NB and ES

Material and Methods:

PBNK were expanded with lethally 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-CD3z-tEGFR–mRNA (Anti ROR1-CAR was generously provided by Riddell S, MD, Fred Hutch, WA). Anti-ROR1-CAR-NK expression was detected using anti-mouse IgG, F(ab')₂.  Anti-ROR1-CAR-NK cytotoxicity was investigated against NB (SKNBE2, SKNFI & SHSY5Y) and ES (TC71, EWS 502 & A673) cell lines by DELFIA cytotoxicity assay at 10:1 E: T ratio. Intracellular staining of CD107a, Interferon Gamma, Perforin and GranzymeB was performed using a 10:1 E:T ratio of anti-ROR1-CAR-NK cells against tumor targets & analyzed on the MACSQuant flow cytometer.

Results:

NB and ES cell lines expressed ROR1 (50.2± 15.6%) and (31.5±12%), respectively. Expansion of NK cells was significantly increased 3988 ± 435 fold (p=0.00001) at day 14 vs day 0. Nucleofection success was measured by F(ab')₂  expression and showed a significant  increase in anti- ROR1-CAR- (88.3±1.69%) vs Mock- electroporated NK cell populations (8.1±6.9%) p= 0.0001 at 36-48 hours (Figure 1). ROR1 negative A673 served as negative control.  Anti-ROR1-CAR-NK significantly increased cell lysis compared to Mock NK (93.1±1.9% vs 62.8±5.2%, p= 0.0008) against ROR1expressing cell lines at 10:1 ET ratio (Figure 2). Similarly, expression of CD107a (46.9±2.2 vs 26.9±3%) p=0.0004, Interferon Gamma (36.9±12.4vs15.3±6.9%) p=0.008, GranzymeB (62.8±4.4 vs 46.6±7.7%) p=0.003, and Perforin (50.5±8 vs 32.7±14.1%) p=0.004 were significantly increased in anti-ROR1-CAR-NK vs Mock-NK cells at 10:1 E:T ratio against the ROR1 expressing targets.

Conclusion:

Anti-ROR1-CAR-ex-PBNK cells had significant enhanced cytotoxicity and significantly increased CD107a, Interferon Gamma, Perforin, & GranzymeB activity against ROR1 expressing pediatric solid tumors. Future directions include investigating the ex-PBNK anti ROR1-CAR cells in-vivo against ROR1 expressing pediatric solid tumors.