Regulation of Cytokines/Chemokines Release and Anti-Tumor Effect of Expanded Natural Killer (NK) Cells By a Novel Fusion of N-820 (2B8T2M), an IL-15 Superagonist with 4 Single-Chain Anti-CD20 Antibody Domains, Against Rituximab Resistant Burkitt Lymphoma (BL)

Background: The prognosis of children with relapsed/refractory B-cell (CD20⁺) non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, BJH, 2018). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab will relapse which limits patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering of NK cells with chimeric antigen receptors (Chu/Cairo et al, Can Imm Res 2015), as well as second-generation engineered anti-CD20 antibodies (Tiwari/Cairo et al BJH 2015). To enhance NK based therapy, our group has successfully expanded the functionally activated peripheral blood NK cells (exPBNK) with irradiated feeder cells (Chu/Cairo, et al, Can Imm Res 2015). To enhance NK cell-based targeted therapy, we had successfully engineered expanded NK cells with chimeric antigen receptors (Chu/Cairo, et al, Can Imm Res 2015) and combined expanded NK with an anti-CD20 targeted IL-15 fusion protein N-820 (2B8T2M) targeting rituximab-sensitive and -resistant BL (Chu/Cairo, et al, ASH 2017). N-820 was generated by fusing ALT-803 to four single-chain antibody domains of rituximab (Liu/Wong, et al, JBC, 2016). ALT-803 is a superagonist of an IL-15 variant bound to an IL-15RαSu-Fc fusion with enhanced IL-15 biological activity (Zhu et al. 2009 J Immunol), longer half-life and increased potency (Han, et al. Cytokine. 2011). N-820 displayed tri-specific binding activity through its recognition of CD20 on tumor cells, activated NK cells to enhance antibody-dependent cellular cytotoxicity (ADCC), and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016).

Objective: To investigate how N-820 modulates the crosstalk between BL tumor cells and expanded NK cells by monitoring cytokines, chemokines and growth factors released and the anti-tumor effects of N-820 on NK cells in BL xenografts.

Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells and isolated as we have previously described with K562-mbIL15-41BBL (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 and N-820 were generously provided by Altor Bioscience. Equal doses of N-820, Rituximab (R), ALT-803, R+ALT-803, obinutuzumab (obinu, generously provided by Christian Klein, PhDfrom Roche) were used for comparison. IgG was used as control. Rituximab-sensitive Raji and -resistant BL cells Raji-2R and Raji-4RH were used as target cells. NK cells were sorted by Beckman Coulter MoFlo XDP high-speed sorter and NK purity was confirmed by flow cytometry. Cytokines, chemokines and growth factors mRNA levels in NK cells were monitored by real-time PCR. Secreted cytokines, chemokines and growth factors were examined by standard ELISAs. Raji-2R-Luc cells were injected into NSG mice. ExPBNK+N-820 and controls were injected to the xenografted NSG mice. The tumor burden was measured with the IVIS-200 system.

Results: We found that ALT-803 and N-820 at equal doses significantly enhanced the expression of NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK compared to IgG controls. Additionally, N-820 significantly enhanced exPBNK cytotoxicity against Raji, Raji-2R and Raji-4RH cells compared to the controls IgG, R, ALT-803, R+ALT-803, or obinu (p<0.001). We further examined 84 genes of cytokines, chemokines and growth factors by real time PCR. Among these 84 genes, N-820 up-regulated 26 genes and down-regulated 22 genes in exPBNK when co-cultured with Raji-2R compared to exPBNK alone. Using ELISAs, we confirmed that N-820 significantly enhanced IFN-g, granzyme B, and GM-CSF (Fig.1A) release from exPBNK against Raji-2R and Raji-4RH compared to IgG and R+ALT-803 (p<0.001). We also found that N-820 significantly reduced MDC (CCL22) (Fig.1B) release from Raji-2R cells compared to IgG (p<0.01) and R+ALT-803 (p<0.05). In NSG mice bearing Raji-2R tumor xenografts, we found that N-820 added to exPBNK significantly reduced tumor burden (p<0.05) and expanded mice survival (Fig.1C) (p<0.05) compared to control groups.

Conclusions: N-820 significantly enhanced exPBNK cytotoxicity, IFN-g, granzyme B, GM-CSF release from exPBNK in-vitro, inhibited MDC release from BL and increased anti-tumor activity against BL xenografts in NSG mice.

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