Comparative Analysis on the Anti-Tumor Activity of Venetoclax and Obinutuzumab (VO) Versus Venetoclax and Rituximab (VR) in Primary CLL Cells, Ex Vivo
Authors: Iqbal Madiha , Sharma Aarushi , Manna Alak , Akhtar Sharoon , Sher Taimur , Chanan-Khan Asher A. , Ailawadhi Sikander , Paulus Aneel 
Published: October 2019

Abstract
INTRODUCTION: Treatment of chronic lymphocytic leukemia (CLL) has expanded significantly with the approval of multiple small molecule inhibitors. This is of great significance for patients with adverse cytogenetic features who tend to respond poorly to standard chemo-immunotherapy (CIT). While single agent ibrutinib and venetoclax (V) have shown high rates of overall response, complete remission and minimal residual disease (MRD) eradication rates remain low. This argues for further testing and development of various combination strategies. The MURANO trial compared venetoclax and rituximab (VR) versus bendamustine and rituximab (BR) in patients with relapsed/refractory CLL reporting clear superiority of VR over BR. MRD rates in the bone marrow were reported to be 27.3% for VR versus 1.5% for BR. Given much higher rates of MRD eradication with combination of small molecule inhibitors and monoclonal antibodies (mAb) compared to standard CIT, we performed a comparative investigation into the direct and immune-mediated cytolytic effects of VR versus V + Obinutuzumab (O, type II anti-CD20 mAb) in primary CLL cells and B-lymphoid cell lines.
METHODS: CD19+ B-cells were isolated from PBMCs of CLL patients (N=3). For all experiments using primary CLL cells, concentration of VR and VO was 3nM (V) and 10ug /ml (R, O), respectively. For cell lines, VR and VO was used at 5uM (V) and 10ug /ml (R, O), respectively. Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by VR and VO was assessed in Calcein AM labeled CLL cells or cell lines co-cultured with healthy donor PBMCs (E:T ratio, 40:1); complement-dependent cytotoxicity (CDC) was measured using 10% serum from a healthy human donor.
RESULTS: We assessed the ability of V+/-O or V+/-R to induce apoptotic cell death in the CD20+ BCWM.1 cell line (Waldenström's macroglobulinemia [WM] phenotype) and the MEC-1 cell line (B-PLL phenotype); with CD20- RPCI-WM1 (WM cells, negative control). Notably, Bcl-2 protein is expressed in all the aforementioned cell lines. We observed that single agent V, O and R induced ~30%, 61% and 13.64% annexin V/PI positivity in BCWM.1 cells, respectively. However, a significant degree of cell death was noted in VO-treated cells (~74%) compared to VR-treated cells (~40%) (p<0.01). Next, we examined for apoptosis in MEC1 cells and noted a similar trend; where the VO combination induced markedly more cell death (~71%) than VR (~57%). Contrastingly, in RPCI-WM1 cells neither single agent O or R could elicit >12% annexin V/PI positivity and where the addition of V increased apoptosis by only 3 - 4%. We also examined the apoptotic potential of VO or VR in tumor cells from low, intermediate and high-risk CLL patients. In low and intermediate-risk CLL cells from low and intermediate-risk patients, V alone induced ~30% cell death, which increased significantly with the addition of O (VO) to between 48 - 52%. Contrastingly, the combination of VR did not induce more than 29 - 32% apoptosis. In CLL cells from high-risk patient, we noted that exposure to single agent V induced ~ 28% cell death and in VO-treated cells, this number increased to 47%. We also examined for ADCC and CDC in the same cell lines and primary CLL cells. Despite considerable variability, single agent O and VO treatment of tumor cells resulted in greater ADCC than VR treatment. By contrast, in single agent R or VR-treated cells, more CDC was observed.
CONCLUSION: Our preliminary investigation in VR- and VO-treated cell lines and primary CLL cells suggests the VO combination may be superior to VR in induction of direct tumor cell death. Mechanistic experiments underway will provide further insight and can aid in design of future VO-based clinical studies in CLL.

Disclosures
Ailawadhi: Pharmacyclics: Research Funding; Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy.