Regulation of cytokine release and anti-tumor effect of anti-CD20 CAR modified expanded natural killer cells by ALT-803, an IL-15 superagonist

Chimeric antigen receptors (CARs) T-cells have shown promising results in patients with ALL, CLL and DLBCL. However, some patients have developed a life threatening cytokine release syndrome (CRS) with CAR-T-cell therapy. Our group has successfully modified expanded peripheral blood Natural Killer cells (exPBNK) with an anti-CD20 CAR to target rituximab sensitive/resistant CD20+ BL cells in vitro and in NSG mice (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 (Altor BioScience Corporation) 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, better lymphoid tissue retention, increased potency, less toxicity compared to IL-15 (Han, et al. Cytokine. 2011). ALT-803 is currently in several clinical trials including refractory indolent non-Hodgkin’s lymphoma (NCT02384954). To determine the cytokine released and anti-tumor effect of CAR NK cells stimulated by ALT-803 exPBNK cells were isolated using Miltenyi NK cell isolation kit after expansion with K562-mbIL21-41BBL. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was producedin vitro and nucleofected into exPBNK (Chu/Cairo et al, Can Imm Res 2015). ALT-803 was generously provided by Altor BioScience Corporation. ExPBNK were cultured with 0.35 or 3.5ng/ml ALT-803. NK proliferation, cytotoxicity, and cytokine level were examined. NK resistant BL cells Raji and Daudi were used as tumor targets. ALT-803 induced granzyme B, IFN-g, GM-CSF, MIP-1a, RANTES release from CAR exPBNK cells but did not induce IL-2, IL-6, IL-10, IL-12, IL-13, MCP-1, MIP-1b, MMP-9, and TNF-alpha release when CAR exPBNK cells were co-cultured with Raji or Daudi. Furthermore, we demonstrated that the in vitro cytotoxicity of anti-CD20 CAR exPBNK was significantly enhanced by ALT-803 against Raji and Daudi compared to mock-electroporated exPBNK maintained in ALT-803 medium (p < 0.001) and compared to anti-CD20 CAR modified exPBNK maintained in medium without ALT-803 (p < 0.01). Additionally, we observed that the combination of anti-CD20 CAR NK and ALT-803 reduced tumor burden in Raji xenografted mice. CAR exPBNK cells with ALT-803 do not release inflammatory cytokines associated with CRS. ALT-803 significantly enhanced the in vitro cytotoxicity of CAR exPBNK and reduced tumor burden in BL xenografts.