Rituximab Resistant Burkitt Lymphoma Research Articles

Optimizing Ex-Vivo Expanded NK Cell- Mediated Cellular Cytotoxicity By Obinutuzumab Combined with NKTR-255 in Burkitt Lymphoma (BL)

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<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 <0.01), and Raji-4RH (E:T=3:1, p<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<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN- g: p<0.001, granzyme: p<0.01 and perforin: p<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).

Optimizing Ex-Vivo Expanded NK Cell- Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Combined with NKTR-255 in Chronic Lymphocytic Leukemia (CLL), Follicular Lymphoma (FL), and Burkitt Lymphoma (BL)

Background: 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). Rituximab, a monoclonal chimeric anti-CD20 antibody, has been widely used as a chemoimmunotherapeutic regimen in the frontline therapy for patients with CD20+ BL and diffuse large B-cell lymphoma. The addition of rituximab to the CHOP backbone or to standard FAB/LMB therapy has greatly improved outcomes without significantly increasing toxicity in patients with B-NHL (Goldman/Cairo, Leukemia, 2013, Coiffier et al, NEJM, 2002). However, patients who relapse have a poor clinical response to rituximab retreatment. 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 expanded functional and active peripheral blood NK cells PBNKwith irradiated feeder cells to target B-NHL (Chu/Cairo, et al, Can Imm Res 2015). We previously demonstrated that obinutuzumab has significantly enhanced 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 expand natural killer (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, and enhances long-term immunological memory which may lead to sustained anti-tumor immune response. Objective: To investigate the effects of NKTR-255 on the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Dean Lee, 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. IFNg, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, ASH, 2018). MEC-1 (CLL), PGA-1 (CLL), DOHH2 (FL) and Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Results: NKTR-255 significantly enhanced the in vitro cytotoxicity of expanded NK cells when combined with rituximab against MEC-1 (E:T=3:1, p<0.001), PGA-1 (E:T=3:1, p<0.001), and DOHH2 (E:T=3:1, p<0.001) as compared to the control groups (Fig.1A). NKTR-255 also significantly enhanced granzyme and perforin release from expanded NK cells when combined with rituximab against MEC-1 (granzyme: p<0.05; perforin: p<0.001), PGA-1(granzyme: p<0.05; perforin: p<0.05), DOHH2 (granzyme: p<0.05; perforin: p<0.001) as compared to controls. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab agains rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups (Fig.1B). 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<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and 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 anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells using humanized NSG models are under investigation. Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Madakamutil:Nektar Therapeutics: Current Employment. Marcondes:Nektar Therapeutics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Cairo:Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding.

Novel cytokine–antibody fusion protein, N-820, to enhance the functions of ex vivo expanded natural killer cells against Burkitt lymphoma

BackgroundThe prognosis of patients with relapsed or progressive B cell (CD20+) non-Hodgkin’s lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemoradiotherapy resistance. Novel therapeutic strategies are urgently needed....

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. [Display omitted] DisclosuresJeng:Altor: Employment. Alter:NANTCell / Altor Bioscience: Employment. Rhode:NANTCell / Altor Bioscience: Employment, Patents & Royalties: Altor Bioscience. Lee:NantKwest: Employment. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wong:Altor: Employment, Equity Ownership, Patents & Royalties: Altor Bioscience. Cairo:Janssen: Research Funding.

Exogenous Overexpression Of DLEU1 Significantly Induces Programmed Cell Death and Inhibits Cell Proliferation In Burkitt Lymphoma (BL) and Sensitizes Rituximab (RTX) Resistant BL Cells To RTX Induced Apoptosis

BackgroundPediatric Burkitt Lymphoma (PBL) represents over 40% of Non-Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007; Miles/Cairo, BJH, 2012) and the prognosis of PBL has dramatically improved over the past 30 years through the introduction of short intensive multi-agent chemotherapy. (Cairo et al, JCO, 2012). However, the success of this therapeutic approach has been compromised by significant chemotherapy-induced toxicity, requiring the need to identify a less toxic targeted therapy. We previously identified, in a multivariate analysis, that children with Burkitt Lymphoma (BL) and a 13q deletion had a significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leuk., 2009; Nelson/Cairo/Sanger et al, BJH, 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene and a target of c-Myc on the chromosome 13q14.3 region (Dave et al, NEJM, 2006). DLEU1 encodes for proteins of 72 residues and 19 interacting proteins including c-Myc, TUBB2C, RASSF1A and UBR1. We have previously suggested that DLEU1 may function as a tumor suppressor gene in transient DLEU1 overexpressed BL cells (Lee/Cairo, ASH, 2012). However, functions and mechanism(s) following dysregulation of DLEU1 expression in BL are poorly understood. ObjectivesWe proposed that DLEU1 may act as a tumor suppressor gene in PBL, and therefore investigated whether 1) overexpression of DLEU1 in BL cells results in changes in programmed cell death, cell proliferation and cell migration, and 2) sensitizes Rituximab resistant BL cells to apoptosis in PBL. MethodsA stable Raji cell line with DLEU1 overexpression (DLEU1-OE) was established from previous reported (Lee/Cairo et al, ASH, 2012) under G418 selection and all cells including Rituximab (RTX) resistant Raji BL cells (2R) (Barth et al, BJH, 2012) were cultured in RPMI with 10% FBS. Total RNA was isolated by Trizol (Invitrogen) and cDNA was synthesized by cDNA Synthesis Kit (Quantas). Quantitative RT-PCR was performed by CFX96 Real-time (Bio-rad), and MTS and Caspase 3/7 assays (Promega) were employed for measurement of cell proliferation and apoptosis. Statistical significance was performed by one-tailed Student t-test. For classic scratch-wound healing assay, wounded cells were created by scratching cells at 72 hours post-transient transfection with DLEU1 and images of wound areas were taken at 0 and 48 hours after scratching. For transient experiments using 2R, DLEU1 transient overexpressing Raji and 2R cells were treated with Rituximab (20ug/ml) for 48hours and cell proliferation and apoptosis assay were measured as above. ResultsThe expression of DLEU1 mRNA showed a significant increase in DLEU1 stable overexpressing Raji (DLEU1-OE) cells compared to mock control cells (9.0 fold increase, p<0.02). In comparing mRNA expression levels of DLEU1 network genes, there was significant decrease of RASSF1 and TUBB2C mRNA expression (1.2 fold reduction, p<0.02 and 1.3 fold reduction, p<0.03, respectively) in DLEU1-OE cells to mock control cells. DLEU1-OE cells showed a significant decrease in cell proliferation (15% reduction, p<0.03; 19% reduction, p<0.03; and 15% reduction, p<0.01 at 24, 48 and 72 hours, respectively) (Fig 1A). There was significant increase in Caspase 3/7 activity (10.2% increase, p<0.02; 9.7% increase, p<0.05 at 24 and 48 hours, respectively) (Fig 1B). The wounded cells dramatically healed slower than those of control in DLEU1 transient overexpressed 293 cells compared to mock control 293 cells (Fig 2). There were significant increase in Caspase 3/7 activity and significant decrease in cell proliferation in RTX (20ug/ml) treated transient DLEU1 overexpressed 2R (13% increase, p<0.05 and 5% reduction, p<0.02, respectively) compared to RTX treated transient DLEU1 overexpressed Raji. RTX treated transient DLEU1 overexpressed 2R showed significant decrease of expression anti-apoptotic genes, Bcl-xL and Bcl-2 mRNA (<15% reduction, p<0.04 and <20% reduction, p<0.02, respectively). [Display omitted] [Display omitted] ConclusionThese results suggest that 1) overexpression of DLEU1 significantly inhibited cell proliferation, wound healing and induced apoptosis, and 2) we hypothesize that overexpression of DLEU1 in PBL may result in sensitizing BL to chemotherapy induced apoptosis and may result in a potential future therapeutic strategy. Disclosures:No relevant conflicts of interest to declare.