Aradhana Awasthi

A comparative global phosphoproteomics analysis of obinutuzumab (GA101) versus rituximab (RTX) against RTX sensitive and resistant Burkitt lymphoma (BL) demonstrates differential phosphorylation of signaling pathway proteins after treatment

We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

Abstract 2657: PD-L1 blockade enhances T cell cytotoxicity against primary mediastinal B cell lymphoma

Background: Primary Mediastinal B cell Lymphoma (PMBL) represents 2-4% of Non-Hodgkin Lymphomas (NHL) in adolescents and young adults (AYA) (Gerrard/Cairo et al., Blood, 2013). Disease progression, relapse and long-term toxicity remain a concern for patients treated on current chemo-immunotherapy and mediastinal radiotherapy. Alternative therapeutic regimens are urgently needed, especially in patients without an early response to therapy. Programmed Death 1 (PD-1) is a negative co-stimulatory receptor critical for suppression of T-cell activation, with binding of PD-1 and Programmed Death Ligand 1 (PD-L1) resulting in T cell exhaustion (Postow/Wolchok et al., J Clin Oncol, 2015). Gain in 9p associated with amplification of PD-L1 has been reported in up to 60% of PMBL specimens, leading to overexpression of PD-L1 and potential immune cell evasion of PMBL (Rosenwald/Staudt et al., J Exp Med, 2003; Twa/Steidl et al., Blood, 2014). Blockade of PD-1/PD-L1 interaction, therefore, constitute a potentially promising alternative for treatment of resistant PMBL. Objective: In the current study, we sought to investigate whether PD-L1 blockade enhances T cell responses in PD-L1 expressing PMBL. Methods: PD-L1 expression on Karpas 1106p PMBL treated with or without IFN γ was investigated by western blotting and flow cytometry analyses. T cells were isolated from human PBMC followed by activation and expansion using anti-CD2, anti-CD3 and anti-CD28 Biotinylated MACSiBead particles. T cell activation was confirmed by CD25 and CD69 expression using flow cytometry. Activated T cells were incubated with or without anti-PD-L1 (Clone 6E11, Genentech) at a dose of 10 ug/ml together with Karpas 1106p cells treated with or without IFN γ. Cell proliferation was assessed with MTS assays after incubation for 24 hours. Results: We demonstrated that Karpas 1106p cells express a low level of PD-L1. However, following IFN γ treatment (48 hours) there was a significant increase in PD-L1 expression. Anti-PD-L1 had no significant effect on T cell mediated inhibition of cell proliferation in Karpas 1106p cells with low PD-L1 expression. However, anti-PD-L1 and T cell treatment significantly inhibited cell proliferation in IFN γ treated Karpas 1106p cells when compared to T cell treatment alone (32±18% vs. 3±3% at E:T=5:1, p=0.048). Conclusion: PD-L1 blockade enhances T cell cytotoxicity against PMBL, which is dependent on PD-L1 expression level on PMBL cells. Future in-vivo NSG xenograft studies are ongoing. Citation Format: Tishi Shah, Wen Luo, Aradhana Awasthi, Janet Ayello, Jessica Hochberg, Mitchell Cairo. PD-L1 blockade enhances T cell cytotoxicity against primary mediastinal B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2657. doi:10.1158/1538-7445.AM2017-2657

Abstract 3155: Neuroblastoma (NB), Medulloblastoma (MB), and Ewing's sarcoma (ES) express ROR1 and can be effectively targeted with NK cells modified to express an anti ROR1 chimeric antigen receptor (CAR)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Metastatic pediatric neuroectodermal solid tumors especially NB, MB, and ES have a dismal prognosis (Perkins et al, PLoS One, 2014; Smoll, Cancer, 2012). Targeted cellular therapy with T or NK cells modified with CARs is a novel approach to chemo-resistant childhood solid tumors (Grupp SA, Clin Cancer Res, 2012; Mackall C,Front Oncol, 2012). NK cells can be significantly expanded by co-culture with genetically engineered K562 cells overexpressing mb-IL21 (Lee D, PLOS, 2012). ROR1 has been identified as a novel target on B cell tumors in which CARs can be developed and utilized for targeted cellular therapy (Hudecek et al., Blood, 2010). Objective: To evaluate the in-vitro cytotoxic activity and function of PBNK expanded ex-vivo with K562 mb-IL21 and nucleofected with mRNA encoding an anti ROR1-CAR against NB, MB, and ES. Material and Methods: PBNK were expanded with irradiated K562 Clone 9.mb-IL21 (generously provided by Lee D, MD, PhD, MD Anderson, TX). Ex-vivo expanded PBNK (ExPBNK) cells were electroporated with anti ROR1-CD28-41BBl-CD3ζ-tEGFR-mRNA. The cell surface expression of the ROR1-CAR on NK cells was detected using anti-mouse IgG, F(ab’)2. Cytotoxicity of ROR1 CAR-NK cells was investigated against NB (SKNBE2, SKNFI & SHSY5Y), MB (DAOY) and ES (TC71, EWS 502 & A673) cell lines by DELFIA cytotoxicity assay at an E:T ratio of 10:1. Intracellular staining of CD107a, interferon gamma, perforin and granzyme B was performed using a 10:1 E:T ratio of ROR1-CAR-NK cells against tumor targets and analyzed on the MACSQuant flow cytometer. Results: NB, MB, and ES cell lines expressed ROR1 (50.2±15.6%), (55.5±5.1%), and (31.5±12%), respectively. Expansion of NK cells was significantly increased 3988 ± 435 fold (p = 0.00001) at day 14 vs day 0. CAR expression after nucleofection was measured by F(ab’)2 staining and showed a significant increase in anti-ROR1-CAR- (88.3±1.7%) vs mock-electroporated NK cell populations (8.1± 6.9%) p = 0.0001 at 36-48 hours. Anti-ROR1-CAR-NK cells exhibited significantly increased lysis of ROR1 expressing tumor cell lines compared to mock NK cells (93±4.6% vs 63.6±7.4%) p = 0.00001 at an E:T of 10:1. Similarly, expression of CD107a (46.1±9.1 vs 27.6±2.4%) p = 0.001, interferon gamma (34.1±11.6 vs 16.7±6.7%) p = 0.003, granzyme B (68.5±8.9 vs 46±7.2%) p = 0.002, and perforin (51.3±7.7 vs 30.3±11.9%) p = 0.002 were significantly increased in anti-ROR1-CAR-NK cells vs Mock-NK cells at 10:1 E:T ratio against the ROR1 expressing targets. Conclusion: Anti-ROR1-CAR-ex-PBNK cells had significant enhanced cytotoxicity and significantly increased CD107a, interferon gamma, perforin, and granzyme B activity against ROR1 expressing tumors. Future directions include investigating the ex-PBNK anti ROR1-CAR cells in-vivo against ROR1 expressing pediatric solid tumors. Citation Format: Mona Elmacken, Aradhana Awasthi, Janet Ayello, Carmella VanDeVen,, Wen Luo, Yanling Liao, Stanley Riddell, Mitchell S. Cairo. Neuroblastoma (NB), Medulloblastoma (MB), and Ewings sarcoma (ES) express ROR1 and can be effectively targeted with NK cells modified to express an anti ROR1 chimeric antigen receptor (CAR). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3155. doi:10.1158/1538-7445.AM2015-3155