Author Manuscript Published OnlineFirst on April 13, 2021; DOI: 10.1158/1535-7163.MCT-20-0864 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Obinutuzumab in combination with chemotherapy enhances direct cell 2 death in CD20-positive obinutuzumab-resistant non-Hodgkin 3 lymphoma cells 4 Takaaki Fujimura, Yoriko Yamashita-Kashima, Natsumi Kawasaki, 5 Shigeki Yoshiura, Naoki Harada, Yasushi Yoshimura 6 Product Research Department, Chugai Pharmaceutical Co., Ltd., Kanagawa, Japan 7 Running title: Obinutuzumab (OBI) with chemotherapy in OBI-resistant cells 8 Keywords: obinutuzumab; CHOP; bendamustine; retreatment; non-Hodgkin lymphoma 9 Corresponding author: 10 Yoriko Yamashita-Kashima, PhD 11 200 Kajiwara, Kamakura 247-8530, Japan 12 Email: [email protected] 13 Tel: +81(467)45-7692 14 Fax: +81(467)45-7643 15 Text: 3785 words, abstract: 203 words, 4 figures, 36 references 16 A conflict of interest disclosure: All authors are employees of Chugai Pharmaceutical 17 Co., Ltd. This research was funded by Nippon Shinyaku Co., Ltd. 18 Author contributions: 1 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 13, 2021; DOI: 10.1158/1535-7163.MCT-20-0864 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 19 T.F. conceived the idea, designed and performed the experiments, analyzed the data, 20 and drafted the manuscript. Y.Y.K., N.H., and Y.Y. established the study concept, 21 supervised the study, and conducted critical revision to the manuscript. N.K., and S.Y. 22 interpreted the results, and reviewed and revised the manuscript. All authors contributed 23 to the final manuscript and approved it for submission. 24 25 2 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 13, 2021; DOI: 10.1158/1535-7163.MCT-20-0864 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 26 Obinutuzumab in combination with chemotherapy enhances direct cell 27 death in CD20-positive obinutuzumab-resistant non-Hodgkin 28 lymphoma cells 29 Abstract 30 Follicular lymphoma (FL) commonly recurs and is difficult to cure. Obinutuzumab is a 31 humanized glycoengineered type II anti-CD20 antibody with a mode of action that 32 includes induction of antibody-dependent cellular cytotoxicity, antibody-dependent 33 cellular phagocytosis, and direct cell death. There is no evidence on the effectiveness of 34 re-treatment with obinutuzumab in patients with prior obinutuzumab treatment. Using 35 obinutuzumab-induced-direct-cell-death–resistant cells, we investigated the efficacy of 36 obinutuzumab re-treatment in combination with chemotherapeutic agents used in FL 37 treatment. Human non-Hodgkin lymphoma (NHL) SU-DHL-4 cells were sustainably 38 exposed to obinutuzumab in vitro, and seventeen resistant clones expressing CD20 and 39 showing 100-fold higher IC50 of obinutuzumab than parental cells were established. The 40 growth inhibition effect of obinutuzumab in combination with bendamustine, 4- 41 hydroperoxy-cyclophosphamide, doxorubicin, vincristine, or prednisolone was estimated 42 using an interaction index based on the Bliss independence model. For each clone, there 43 were various combinations of obinutuzumab and chemotherapeutic agents that showed 44 supra-additive effects. Obinutuzumab combined with doxorubicin enhanced caspase- 45 dependent apoptosis and growth inhibition effect. Obinutuzumab combined with 46 prednisolone enhanced DNA fragmentation and G0/G1 arrest. These combinations also 47 had an antitumor effect in mouse xenograft models. Our results indicate that re-treatment 48 with obinutuzumab, when it is combined with chemotherapeutic agents, is effective in the 49 CD20-positive obinutuzumab-induced-direct-cell-death–resistant cells. 3 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 13, 2021; DOI: 10.1158/1535-7163.MCT-20-0864 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 50 Introduction 51 Follicular lymphoma (FL) is the second most common type of non-Hodgkin lymphoma 52 (NHL), accounting for approximately 35% of NHLs and 70% of indolent lymphomas 53 [1]. FL is usually slow-growing and responds well to treatment; however, it commonly 54 recurs and is difficult to cure. For patients with stage III/IV FL, regimens containing 55 rituximab, a chimeric mouse–human type I anti-CD20 antibody, have become the 56 standard of care [2, 3]. Although the therapeutic outcomes of FL have greatly improved 57 since rituximab was approved, some patients still do not respond adequately to 58 rituximab and others eventually relapse [4]. 59 Obinutuzumab is a humanized, glycoengineered type II anti-CD20 antibody. 60 Because of its type II properties together with glycoengineering, obinutuzumab achieves 61 enhanced direct cell death, antibody-dependent cellular cytotoxicity (ADCC), and 62 antibody-dependent cellular phagocytosis (ADCP) [5, 6]. While the degree to which the 63 induction of direct cell death contributes to the clinical benefit of obinutuzumab is 64 unknown, Herter et al. demonstrated that maximal antitumor activity of obinutuzumab 65 requires not only ADCC/ADCP but also direct cell death in preclinical models [7]. 66 Therefore, it is highly likely that both ADCC/ADCP and type II antibody-mediated 67 direct cell death contribute to the clinical effectiveness of obinutuzumab. 68 On the basis of the randomized phase III GADOLIN [8] and GALLIUM [9] 69 trials, obinutuzumab showed the effectiveness and has been approved in many 70 countries, not only for the treatment of patients with relapsed/refractory FL, but also for 71 patients with previously untreated FL. As obinutuzumab is now employed as the first 72 line therapy against FL, it has become more important to determine which therapies are 73 the best to follow obinutuzumab-containing regimens. In clinical practice, rituximab is 74 widely used to re-treat FL patients who previously received it, and the efficacy of 4 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 13, 2021; DOI: 10.1158/1535-7163.MCT-20-0864 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 75 rituximab re-treatment has also been reported with relapsed/refractory NHLs [10]. 76 However, there is no evidence on the efficacy of re-treatment with obinutuzumab in 77 patients who have already received obinutuzumab, and new scientific data is needed to 78 address this. The purpose of our study was to explore the possibility of obinutuzumab 79 re-treatment for relapsed/refractory FL. Because direct cell death is a characteristic 80 mode of action for type-II CD20 antibodies like obinutuzumab, in the current study we 81 established human NHL cell clones that were resistant to obinutuzumab-induced direct 82 cell death. Then we investigated the effectiveness of obinutuzumab in combination with 83 other agents against these obinutuzumab-resistant cells. 84 Materials and Methods 85 Compounds and cells 86 Obinutuzumab was provided by F. Hoffmann-La Roche (Basel, Switzerland). 87 Doxorubicin, vincristine, bendamustine (Selleck Chemicals LLC, Houston, TX, USA), 88 4-hydroperoxy-cyclophosphamide (4-OOH-CY), an active metabolite of 89 cyclophosphamide (Cayman Chemical, Ann Arbor, MI, USA), and prednisolone 90 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) were used as the combined 91 chemotherapeutic agents. Each of these agents was dissolved in dimethyl sulfoxide 92 (Sigma-Aldrich). N-ethyl-N-nitrosourea (ENU) and Z-VAD-FMK were purchased from 93 Sigma-Aldrich (St Louis, MO, USA) and Promega Corp. (Madison, WI, USA), 94 respectively. 95 SU-DHL-4 cells, a human germinal center B-cell–like diffuse large B-cell 96 lymphoma (GCB-DLBCL) cell line, were obtained from the American Type Culture 97 Collection (ATCC, Manassas, VA, USA) at 2017, and were maintained in RPMI-1640 5 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 13, 2021; DOI: 10.1158/1535-7163.MCT-20-0864 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 98 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 10 99 mM HEPES (Sigma-Aldrich), 0.45% D-glucose (Sigma-Aldrich), and 1 mM sodium 100 pyruvate (Thermo Fisher Scientific, Waltham, MA, USA). All cells were cultured at 101 37°C under 5% CO2, tested Mycoplasma contamination, and passed less than 20 times. 102 Animals 103 Animal procedures were approved by the Institutional Animal Care and Use Committee 104 and the Biosafety Committee at Chugai Pharmaceutical Co., Ltd. Six-week-old female 105 NOG mice (NOD/Shi-scid,IL-2RKOJic) were purchased from CLEA Japan, Inc. 106 (Tokyo, Japan). All animals were allowed to acclimatize and recover from shipping- 107 related stress for more than 5 days prior to the study. Chlorinated water and irradiated 108 food were provided ad libitum, and the animals were kept under a controlled 12-hour 109 light/12-hour dark cycle. 110 Establishment of obinutuzumab-induced-direct-cell-death–resistant clones 111 SU-DHL-4 cells were pre-treated with 100 µg/mL of ENU for 1 day to establish 112 resistant clones more efficiently by inducing random mutations [11] and were then 113 treated with 200 µg/mL of obinutuzumab for 3 weeks. The mean serum trough 114 concentration of obinutuzumab was used as a reference for the concentration [12]. Re- 115 grown cells were single-cell cloned and cultured in medium without obinutuzumab for 116 12 days followed by the exposure of 10 µg/mL of obinutuzumab to eliminate clones that 117 had only temporarily obtained insensitivity to obinutuzumab.