Abstract
Background:
The prognosis of patients with relapsed or progressed 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, 2016). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab relapse which limit patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune 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). Nature Killer (NK) cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors. A variety of activating and inhibitory receptors on the NK cell surface are engaged to regulate NK cell activities. However, NK therapy is limited by several factors, including small numbers of active NK cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of NK cell immunosurveillance. Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells (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 and increased potency (Han, et al . Cytokine . 2011). Recently, a new therapeutic molecule 2B8T2M was generated by fusing ALT-803 to four single-chains of the tumor-targeting monoclonal antibody, rituximab (Liu/Wong, et al, JBC, 2016). 2B8T2M displayed tri-specific binding activity through its recognition of the CD20 molecule 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 examine if 2B8T2M significantly enhances the cytotoxicity of exPBNK against rituximab-sensitive and -resistant BL cells.
Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Denman/Dean Lee, PLoS One, 2012). CD56+CD3- exPBNK cells were isolated using Miltenyi NK cell isolation kit. ALT-803 and 2B8T2M were generously provided by Altor BioScience Corporation. NK receptors expression and cytotoxicity were examined as we previous described (Chu/Cairo, et al, Can Imm Res 2015). IFN-γ and granzyme B levels were examined by standard enzyme-linked immunosorbent assays. Equal doses of Rituximab, ALT-803, Rituximab+ALT-803, obinutuzumab (obinu) were used for comparison. IgG was used as controls. Rituximab-sensitive and -resistant BL cells Raji, Raji-2R and Raji-4RH, were used as target cells.
Results: Both ALT-803 and 2B8T2M at equal doses significantly enhanced NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK cells compared to IgG controls.
2B8T2M significantly enhanced exPBNK cytotoxicity against rituximab-sensitive Raji cells compared to the controls IgG, Rituximab, ALT-803, Rituximab+ALT-803, obinu (2B8T2M vs IgG vs ALT803 vs Rituximab vs ALT803+Rituximab vs obinu=98.34%+0.24% vs 56.5+1.83% vs 62.8+1.1% vs 69.52+1.79% vs 81.85+1.83% vs 81.23+1.8%, p<0.001, E:T=1:1). 2B8T2M also significantly enhanced exPBNK cytotoxicity against rituximab-resistant Raji-2R cells (2B8T2M vs IgG vs ALT803 vs Rituximab vs ALT803+Rituximab vs obinu=90.7%+1.36% vs 20.47+7.7% vs 48.18+8.07% vs 37.44+3.63% vs 51.02+1.42% vs 65.51+3.03%, p<0.001, E:T=1:1) and resistant Raji-4RH cells (p<0.001, E:T=1:1) (Fig.1). Furthermore, we confirmed the enhanced cytotoxicity by measuring IFN-γ and granzyme B production. 2B8T2M significantly enhanced IFN-γ and granzyme B production from exPBNK against rituximab-sensitive Raji and -resistant Raji-2R and Raji-4RH compared to IgG (p<0.001), Rituximab (p<0.001), ALT-803 (p<0.001), Rituximab+ALT-803 (p<0.001), and obinutuzumab (p<0.001).
Conclusions: 2B8T2M significantly enhanced exPBNK activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant BL cells. The in vivo functions of 2B8T2M with exPBNK against rituximab-sensitive and -resistant BL cells using humanized NSG models are under investigation.
Alter: Altor BioScience: Employment, Equity Ownership. Jeng: Altor Bioscience: Employment, Equity Ownership. Rhode: Altor BioScience: Employment, Equity Ownership. Lee: Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director. Wong: Altor BioScience: Employment, Equity Ownership. Cairo: Jazz Pharmaceuticals: Speakers Bureau.
