Optimizing Ex-Vivo Expanded NK Cell- Mediated Cellular Cytotoxicity By Obinutuzumab Combined with NKTR-255 in Burkitt Lymphoma (BL)
Authors: Chu Yaya , Foley Keira , Tian Meijuan , Klose Kayleigh , Ayala-Cuesta Jessica , Lee Dean Anthony , Marcondes Mario , Klein Christian , Cairo Mitchell S. 
Published: December 2023

Background: We have previously reported the success of treating children with Burkitt lymphoma (BL) resulting in a ≥90% long-term EFS utilizing short intensive rituximab containing chemoimmunotherapy (Cairo et al, Blood, 2007; Goldman /Cairo et al, Leukemia, 2012, Goldman /Cairo et al, Br J Haematol., 2014; Goldman /Cairo et al, Leukemia, 2021). However, the prognosis is dismal in children who are refractory or progress after chemoimmunotherapy (Cairo et al Br J Haematol. 2018). The mechanisms associated with this poor prognosis are mainly due to chemoradioimmunotherapy resistance and an immunosuppressive BL microenvironment (TME) (Cairo et al Br J Haematol. 2016; Cairo et al Br J Haematol. 2019) and represent an urgent unmet need. The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully ex-vivo expanded functional and active peripheral blood NK cells (PBNK) with irradiated feeder cells APC (K562-IL21-41BBL) (Chu/Cairo, et al, JITC 2020). We previously demonstrated that obinutuzumab has significantly enhanced ex-vivo expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and NK cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, which may lead to sustained anti-tumor immune response.
Objective: To investigate the anti-BL effects of NKTR-255 on the ADCC of ex-vivo expanded NK cells with obinutuzumab against rituximab-resistant BL in vitro and in vivo using human BL xenografted NSG mice.
Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Lee, et al, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFN-g, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, Front. Immunol, 2022). Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Luciferase expression Raji-4RH cells were xenografted to NSG mice as we previously described (Chu/Cairo, et al, JITC 2021).
Results:
NKTR-255 significantly enhanced the proliferation of ex-vivo expanded NK cells (p&lt;0.0001) at day 7. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab against rituximab-resistant BL cells like Raji-2R (E:T=3:1, p &lt;0.01), and Raji-4RH (E:T=3:1, p&lt;0.01) as compared to the control groups. NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p&lt;0.001, granzyme: p&lt;0.001 and perforin: p&lt;0.001) and Raji-4RH (E:T=3:1, IFN- g: p&lt;0.001, granzyme: p&lt;0.01 and perforin: p&lt;0.01) as compared to controls. Our in vivo study demonstrated that the combination of NKTR-255, Obinutuzumab and expanded NK cells significantly improved the survival of mice xenografted with Raji-4RH compared to controls (Fig.1).
Conclusion:
We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with Obinutuzumab against rituximab-resistant BL cells in vitro with enhanced IFN- g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and Obinutuzumab against rituximab-resistant BL cells using humanized NSG models are very promising. Mechanisms studies of BL relapsed from the combination therapy are under investigation. (Supported by HHOW and St Baldrick grants).