Effectively Targeting Burkitt Lymphoma By ANTI-CD20 Chimeric Antigen Receptor (CAR) Modified Expaneded Natural Killer CELLS Combined with a Histone Deacetylase Inhibitor, Romidepsin in Vitro and in Humanized NSG MICE

Background:

The outcome for patients with Burkitt lymphoma (BL) has improved significantly but for patients who relapse, the prognosis is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012). Our group has successfully engineered expanded peripheral blood Natural Killer cells (exPBNK) with an anti-CD20 chimeric antigen receptor (CAR exPBNK) to target relapsed/resistant CD20⁺ BL cells in vitro and in NSG mice (Chu & Cairo, ASH, 2013). Romidepsin, a histone deacetylase inhibitor, enhances NKG2D ligands expression (Satwani, Chu/Cairo, Cytotherapy 2014).

Objective:

We investigated the combined effect of anti-CD20 CAR exPBNK cells with romidepsin against CD20⁺ BL in vitro and in humanized NSG mice.

Methods:

PBNK cells were expanded with inactivated K562-mbIL15-41BBL cells and purified. Anti-CD20-4-1BB-CD3ζ mRNA was nucleofected into exPBNK (Chu & Cairo, ASH, 2013). Raji, Raji-2R and Raji-4RH (provided by Matthew Barth, MD) cells were treated with 10ng/ml romidepsin, (Celgene Corp). MICA/B expression was analyzed by flow cytometry. NKG2D receptors were blocked using anti-NKG2D antibodies (R & D systems). NK cytotoxicity was assessed by europium release assays.

Romidepsin (2.2mg/kg) or PBS was i.p. injected into Raji-Luc engrafted mice once a week for 3 weeks. 5x10*6 anti-CD20 CAR exPBNK cells or mock exPBNK cells (without CAR expression) were injected into each mouse 24hrs after each romidepsin injection. Tumor regression and/or progression was monitored by tumor volume measurements and by in vivo bioluminescent imaging.

Results:

MICA/B expression was significantly increased in rituximab sensitive Raji, resistant Raji-2R, and Raji-4RH cells (P<0.001) after romidepsin treatment.

 

In vitro cytotoxicity of exPBNK was significantly enhanced against romidepsin-treated tumor targets compared to the untreated target cells at E:T=3:1 (P<0.001).

Blocking NKG2D (99% to 18%) in exPBNK significantly reduced in vitro cytotoxicity against romidepsin treated tumor targets compared to the unblocked exPBNK (P<0.001).

 

In vitro cytotoxicity of anti-CD20 CAR exPBNK cells was significantly enhanced against romidepsin-treated tumor targets compared to the untreated at E:T=3:1 (P<0.001), or compared to the mock exPBNK (P<0.05) (Fig.1A).

In humanized Raji xenograft NSG mice, the romidepsin+CAR exPBNK treated mice significantly extended survival (surviving more than 100 days) compared to the untreated (median 28 days, P<0.001), the mock exPBNK treated mice (median 29 days, P<0.001), the CAR exPBNK treated mice (median 42.5 days, P<0.05), the romidepsin treated mice (median 30 days, P<0.001), and the romidepsin+mock exPBNK treated mice (median 34.5 days, P<0.05) (Fig.1B).

Conclusion: These results suggest the therapeutic potential of the combination of anti-CD20 CAR exPBNK cells and romidepsin against BL in patients with chemo-radio therapy resistance.