Abstract A19: Significantly targeting rituximab resistant B-cell lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR) modified expanded natural killer (NK) cells.
Authors: Chu Yaya , Ayello Janet , Yahr Ashlin , Katz Jared , Lo Lowrence , Flowers Allyson , Cairo Mitchell S. 
Published: August 2014

Abstract
Background: The outcome for children and adolescents with BL has improved significantly but for patients who relapse or progress, the prognosis is dismal due to chemo-immuno-radiotherapy resistance (Cairo, et al, J Clin Oncol, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Czuczman et al generated Rituximab resistant BL cell lines, which provided a good model to study mAb resistance and design novel therapies (Czuczman et al, Clinical Cancer Research, 2008).
Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Ruggeri L et al., Science 2002) but cell number and tumor specific recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Efficient expansion of primary NK cells with K562-mbIL15-41BBL and retargeting expanded NK cells with Anti-CD20 CAR by retrovirus were reported by our group previously (Chu & Cairo, et al, ASH, 2011).
Objective: To generate large-scale, efficiently modified NK cells with low cost, clinical applicable and a non-viral method and to apply these modified NK cells on antibody resistant BL, we investigated the functional activities of anti-CD20 chimeric antigen receptor (CAR+) modified PBNK cells following mRNA nucleofection against rituximab resistant BL cells.
Methods: PBMC were expanded with inactivated K562-mbIL15-41BBL cells for 7 or 14 days. CD56+CD3- expanded PBNK (exPBNK) cells were isolated using NK cell isolation kit. CD56, CD3 and receptor expression were evaluated by flow cytometry. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit. CAR mRNA was nucleofected into exPBNK using Amaxa nucleofector. CAR expression was detected using FITC-conjugated goat anti-mouse IgG, F(ab')2 fragment-specific antibody. exPBNK cytotoxicity was assessed by europium release assay against CD20+ Ramos, Daudi, Raji, two Rituximab resistant cell lines: Raji-2R and Raji-4RH, and CD20- control cells: RS4;11 and Jurkat. CD107a degranulation in exPBNK was measured by flow cytometry.
Results: CD56+CD3- exPBNK cells were significantly expanded by mitomycin C treated K562-mbIL15-41BBL cells at day7. exPBNK cells were selected with more than 96% purity of CD56+CD3-. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs after CAR mRNA nucleofection.
CAR mRNA nucleofection did not affect the expression of exPBNK activating receptors or inhibitory receptors.
exPBNK in vitro cytotoxicity was significantly enhanced by CAR+ exPBNK compared to CAR- exPBNK against CD20+ BL at E:T=10:1 (n&gt;3, p&lt;0.001): Ramos (97.25+ 2.61% vs 82.5+ 4.058%, p&lt;0.05), Daudi (71.5+ 3.26% vs 36.34+ 6.31%), Raji (34.15+ 1.13% vs 6.85+ 2.04%), Raji-2R (43.59+ 1.2% vs 21.34+ 1.75%), and Raji-4RH (37.66+ 1.77% vs 13.25+ 0.7%). However, there was no significant difference against CD20- RS4;11 or Jurkat cells.
Consistently, CD107a degranulation was enhanced in CAR+ exPBNK compared to CAR- exPBNK in response to CD20+ Ramos, however, there was no significant difference in response to RS4;11 or medium.
Conclusion: Anti-CD20 CAR expression in exPBNK cells results in significant and specific exPBNK in vitro cytotoxicity against CD20+ BL and Rituximab resistant cell lines associated with a significant increase in CD107 degradulation. Future directions include examining CAR+ exPBNK cytotoxic activity against CD20+ resistant tumor cells from patients and testing the anti-tumor activity of CAR+ exPBNK against resistant cells in xenograft mice.
Citation Format: Yaya Chu, Janet Ayello, Ashlin Yahr, Jared Katz, Lowrence Lo, Allyson Flowers, Mitchell S. Cairo. Significantly targeting rituximab resistant B-cell lymphoma (BL) by anti-CD20 chimeric antigen receptor (CAR) modified expanded natural killer (NK) cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A19.