Enhanced Targeting B Cell Lymphoma/Leukemia (B-L/L) by Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Combined with a Histone Deacetylase Inhibitor, Romidepsin

Background: The outcome for children and adolescents with B-L/L has improved significantly but for patients who relapse or progress, the prognosis is dismal due to chemo-immuno-radiotherapy resistance (Cairo, J Clin Oncol, 2012). Novel therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Ruggeri, Science 2002) but cell number and tumor specific recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Histone Deacetylase Inhibitors (HDACi) represent a new class of anti-tumor agents. They also up-regulate the expression of NKG2D ligands such as MICA/B (Satwani/Cairo, ASBMT 2009)

Objective: We investigated 1) functional activities of anti-CD20 CAR modified PBNK cells following mRNA nucleofection against CD20⁺ B-L/L and 2) the combined effect of CAR⁺ PBNK cells with Romidepsin against B-L/L cells.

Methods: PBMC were expanded with inactivated K562-mbIL15-41BBL cells for 7 days. Expanded CD56⁺CD3^- PBNK (exPBNK) cells were isolated. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced in vitro and nucleofected into exPBNK. CAR expression was detected using specific antibody by flow cytometry. exPBNK cytotoxicity was assessed by europium release assay. Intracellular CD107a degranulation in exPBNK was measured by flow cytometry. CD20⁺ Ramos and CD20^dim NALM-6 cells were treated with 10ng/ml Romidepsin, provided by Celgene. MICA/B and CD20 expression were analyzed by flow cytometry.

Results: exPBNK cells were selected with more than 96% purity. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs after CAR mRNA nucleofection.

exPBNK in vitro cytotoxicity was significantly enhanced by CAR⁺ exPBNK compared to CAR^- exPBNK against CD20⁺ Ramos (p<0.05), Daudi, Raji, two Rituximab resistant cell lines: Raji-2R and Raji-4RH (p<0.001). However, there was no significant difference against CD20^- control cells: RS4;11 and Jurkat.

Consistently, intracellular CD107a degranulation was enhanced in CAR⁺ exPBNK compared to CAR^- exPBNK in response to CD20⁺ Ramos.

MIC expression was significantly increased in Ramos (P<0.05) and in NALM-6 (p<0.001) after romidepsin treatment.

CD20 expression was significantly increased in NALM-6 (p<0.001) after romidepsin treatment.

CAR⁺ exPBNK in vitro cytotoxicity was significantly enhanced against romidepsin treated Ramos (p<0.02) and NALM-6 (p<0.01) compared to untreated targets.

Conclusion: Anti-CD20 CAR expression in exPBNK cells results in significant and specific in vitro cytotoxicity against CD20⁺ B-L/L. Romidepsin increases MICA/B and CD20 expression in B-L/L. CAR exPBNK significantly enhanced cytotoxicity against romidepsin treated B-L/L. Future directions include examining the combination effect of CAR exPBNK and Romidepsin against B-L/L in xenograft mice.