Obinutuzumab in Combination with Chemotherapy Enhances Direct Cell

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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:

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.

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.

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

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

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-2RKOJic) 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. The CD20 expression

118 level of re-grown clones was assessed by flow cytometry and the sensitivity to

119 obinutuzumab-induced direct cell death was assessed by in vitro cell growth inhibition

120 assay.

121 Flow cytometry

122 Cells were labeled with mouse PE conjugated anti-human CD20 antibody or isotype-

123 matched mouse PE conjugated isotype control antibody (BD Biosciences, San Jose, CA,

124 USA). Fluorescence was measured with an LSRFortessa X-20 cell analyzer (BD

125 Biosciences), and analyzed using FlowJo v10 software (Tree Star Inc., Ashland, OR,

126 USA).

127 In vitro cell growth inhibition assay

128 SU-DHL-4 cells and resistant clones were seeded on 96-well plates at 1-2×104

129 cells/well and 1×104 cells/well, respectively, and were treated with obinutuzumab

130 and/or each of the chemotherapeutic agents at the indicated concentrations for 4 days.

131 Cell viability was determined by using CellTiter-Glo 3D Cell Viability Assay (Promega

132 Corp.). In the obinutuzumab treatment, the percentage of cell proliferation was

133 calculated as follows: Proliferation (%) = [(luminescence of treatment well –

134 luminescence of blank well) / (luminescence of non-treatment well – luminescence of

135 blank well)] × 100. In the combination treatments, the percentage of cell proliferation

136 was normalized by the luminescence of the well treated with the corresponding

137 chemotherapeutic agent at the indicated concentration but without obinutuzumab [13].

138 Calculation of the interaction index based on the Bliss independence model

139 The resistant clones were treated with obinutuzumab and/or bendamustine, 4-OOH-CY,

140 doxorubicin, vincristine, or prednisolone at indicated concentrations for 4 days and cell

141 viability was determined as with above. The interaction index based on the Bliss

142 independence model [14] was calculated as follows: log(ratio of cell proliferation of

143 combination treatment relative to non-treatment) – log(ratio of cell proliferation of

144 obinutuzumab treatment relative to non-treatment) – log(ratio of cell proliferation of

145 each chemotherapeutic agent treatment relative to non-treatment). An index of <0, =0,

146 or >0 means a supra-additive effect, additive effect, or sub-additive effect, respectively.

147 Statistical significance was analyzed with student’s t-test for the actual data

148 (combination group) and the additive assumption calculated from each single agent

149 group [15].

150 Immunoblot analysis

151 Cells were seeded on 6-well plates at 1×106 cells/well or 25 cm2 flask at 3×106

152 cells/flask, and treated with obinutuzumab and/or doxorubicin or prednisolone at

153 indicated concentrations. Cells were lysed with cell lysis buffer (Cell Signaling

154 Technology, Inc., Danvers, MA, USA) containing protease inhibitor cocktail (Sigma-

155 Aldrich) and phosphatase inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). Cell

156 lysates were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride

157 membranes by using an iBlot Gel Transfer System (Invitrogen, Carlsbad, CA, USA).

158 Immunoblotting was performed using the following primary antibodies: anti-IRE1, anti-

159 phospho-JNK (pJNK), anti-phospho-Bcl2 (pBcl2), anti-Bcl2, anti-phospho-Rb at

160 Ser807/Ser811 (pRb (Ser807/811)), anti-phospho-Rb at Ser795 (pRb (Ser795)), anti-

161 phospho-Rb at Ser780 (pRb (Ser780)), anti-p27, anti-Skp2, and anti-β actin (Cell

162 Signaling Technology), and anti-phospho-IRE1 (pIRE1), anti-JNK, and anti-Rb (Abcam,

163 Cambridge, MA, USA). Membranes were incubated with horseradish peroxidase–

164 conjugated secondary antibodies (Cell Signaling Technology), followed by

165 chemiluminescence detection.

166 TdT-mediated dUTP nick end labeling (TUNEL) assay

167 Cells were seeded on 6-well plates at 1×106 cells/well and were treated with

168 obinutuzumab and/or doxorubicin and/or Z-VAD-FMK, or with obinutuzumab and/or

169 prednisolone at the indicated concentrations for 3 days. Cells were labeled with the

170 Apo-BrdU TUNEL Assay Kit with Alexa Fluor 488 Anti-BrdU (Thermo Fisher

171 Scientific) in accordance with the manufacturer’s protocol. The fluorescence was

172 measured with an LSRFortessa X-20 cell analyzer and analyzed using FlowJo v10

173 software.

174 Measurement of caspase-3/7 activity

175 Resistant clones were seeded on 96-well plates at 1×104 or 5×103 cells/well,

176 respectively, and were treated with obinutuzumab and/or doxorubicin at the indicated

177 concentrations for 48 hours. Caspase-3/7 activity was measured using a Caspase-Glo

178 3/7 Assay Kit (Promega Corp.) in accordance with the manufacturer’s protocol. The

179 ratio of caspase-3/7 activity was calculated as follows: (luminescence of treatment well

180 – luminescence of blank well) / (luminescence of non-treatment well – luminescence of

181 blank well).

182 Cell cycle analysis

183 Cells were seeded at 2×105 cells/well and were treated with obinutuzumab and/or

184 prednisolone at the indicated concentrations for 48 hours. Cells were labeled with DAPI,

185 and the fluorescence was measured with an Advanced Image Cytometer NucleoCounter

186 NC-3000 (Chemometec, Allerod, Denmark) in accordance with the manufacturer’s

187 protocol. Data was analyzed using FlowJo v7.6.5 software.

188 In vivo antitumor assay

189 Mice were inoculated subcutaneously in the right flank with 5×106 cells/mouse with

190 Matrigel® Matrix (Corning Inc., Corning, NY, USA). Tumor-bearing mice were

191 randomly allocated to the control group or treatment groups. Obinutuzumab (30 mg/kg),

192 control human IgG (HuIgG, 30 mg/kg), doxorubicin (2.5 mg/kg), or its vehicle was

193 administered intravenously. Prednisolone (2 mg/kg) or its vehicle was administered

194 orally. All drugs were treated on the same schedule as in clinical practice. Tumor

195 volume (TV) and body weight were measured twice a week. TV was estimated as

196 follows: TV = ab2/2, where a and b are tumor length and width, respectively.

197 Statistical analysis

198 The statistical significance in in vitro experiments was analyzed with the Tukey’s HSD

199 test. Two-way ANOVA was used prior to Tukey’s HSD test for comparison of dose

200 response curve assay. P<0.05 indicates a significant difference. In in vivo experiments,

201 statistical significance was analyzed by Wilcoxon test. The significant p-values were

202 adjusted for multiple comparisons by the Holm method. All statistical analyses were

203 performed with JMP software (SAS Institute, Cary, NC, USA).

204 Results

205 Establishment of obinutuzumab-induced-direct-cell-death–resistant clones

206 Cells resistant to obinutuzumab-induced direct cell death were established from SU-

207 DHL-4 cells as described in Materials and Methods. Among the obtained clones, 17

208 clones expressing CD20 had a 100-fold higher IC50 for obinutuzumab—with respect to

209 obinutuzumab-induced direct cell death—than the parental cells. These were established

210 as resistant and named SU-DHL-4-OR clones. (Figure 1A and Supplementary Figure

211 S1).

212 Combined effect of obinutuzumab plus chemotherapeutic agent in

213 obinutuzumab-induced-direct-cell-death–resistant clones

214 Next, to investigate the possibility of re-treatment after acquisition of resistance to

215 obinutuzumab, we assessed whether the sensitivity to obinutuzumab in these resistant

216 clones changed when it was combined with chemotherapeutic agents used in the

217 treatment of FL patients. The combination effect was estimated using an interaction

218 index based on the Bliss independence model [14]. This index identifies combinations

219 of agents with a supra-additive effect, whose efficacies when combined were higher

220 than those estimated from the efficacies of the individual agents. The dose of each

221 chemotherapeutic agent was set at the concentration that induced an approximately 30%

222 growth inhibition effect in SU-DHL-4 cells (Supplementary Figure S2), except for

223 prednisolone which was set at a dose of 1 µM with reference to its clinical mean Cmax

224 (approximately 1.79 µM) because it required a higher concentration to show a 30%

225 growth inhibition effect in SU-DHL-4 cells [16-20]. As a result, for each clone, there

226 were several combinations of obinutuzumab plus chemotherapeutic agent that showed

227 supra-additive effects, and the agent producing the highest combination effect varied

228 among clones (Figure 1B). Remarkably, obinutuzumab plus doxorubicin exerted an

229 additive effect or more in all of the resistant clones.

230 Caspase-dependent mechanism enhanced the growth inhibition effect of

231 obinutuzumab in combination with doxorubicin

232 The growth inhibition effect of obinutuzumab combined with doxorubicin was verified

233 at various concentrations using clones SU-DHL-4-OR-1A2 (1A2) and SU-DHL-4-OR-

234 1C4 (1C4), in which the combination effects were stronger than in other clones. In these

235 clones, co-treatment with doxorubicin enhanced the growth inhibition effect of

236 obinutuzumab (Figure 2A). In contrast, the combination only slightly enhanced the

237 inhibitory effect in SU-DHL-4 cells. (Supplementary Figure S3A).

238 A recent report suggested that a Ca2+ signaling-mediated endoplasmic reticulum

239 (ER) stress pathway is involved in obinutuzumab-induced direct cell death in SU-DHL-

240 4 cells [21]. Therefore, we first examined the effect of obinutuzumab plus doxorubicin

241 on the induction of ER stress in clone 1A2. Treatment with either as a single agent

242 increased the level of phospho-IRE1, an ER stress sensor protein, and the combination

243 further increased this level (Figure 2B). Furthermore, the combination increased levels

244 of phospho-JNK and phospho-Bcl2 (Figure 2C), which are downstream molecules of

245 IRE1 [22].

246 Phosphorylation of Bcl2 by JNK is known to inhibit its anti-apoptotic function

247 and to induce apoptosis [22]. Therefore, we next assessed the effect of this combination

248 on the induction of apoptosis by detecting DNA fragmentation using TUNEL assay and

249 caspase-3/7 activity. This combination increased DNA fragmentation more than each

250 single agent in both clones (Figure 2D). Also, while obinutuzumab and doxorubicin

251 alone each increased caspase-3/7 activity, their combination increased it significantly

252 more (Figure 2E). Furthermore, the combination’s enhanced DNA fragmentation and

253 growth inhibition effect were suppressed by Z-VAD-FMK, a pan-caspase inhibitor

254 (Figure 2D, F). On the other hand, the growth inhibition effect of obinutuzumab alone

255 was not suppressed by Z-VAD-FMK, either in resistant clones (Figure 2F) or in

256 parental cells (Supplementary Figure S3C), although caspase-3/7 activity increased, and

257 was suppressed by Z-VAD-FMK (Supplementary Figure S3D).

258 Enhanced induction of apoptosis and G1 arrest with the combination of

259 obinutuzumab plus prednisolone

260 Next, we investigated the effect of combining obinutuzumab with prednisolone, which

261 was the second most common agent to show a strongest combination effect among

262 clones (Figure 1B). In clones SU-DHL-4-OR-2B3 (2B3) and SU-DHL-4-OR-6B3

263 (6B3), in which obinutuzumab plus doxorubicin showed the weakest combination effect,

264 obinutuzumab plus prednisolone resulted in a stronger combination effect than in all

265 other resistant clones except clone 1A2 (Figure 1B). We confirmed that prednisolone

266 increased obinutuzumab’s growth inhibition effect at various concentrations in both

267 clones (Figure 3A), and also in the parental SU-DHL-4 cells (Supplementary

268 Figure S3B).

269 Next, we examined the underlying processes behind this combination’s

270 enhancement of the growth inhibition effect. First, we evaluated the effect of co-

271 treatment with prednisolone on cell death induced by obinutuzumab in clones 2B3 and

272 6B3. While prednisolone alone did not induce DNA fragmentation, the combination

273 increased it more than obinutuzumab alone (Figure 3B). However, unlike with

274 doxorubicin, co-treatment with Z-VAD-FMK did not suppress the enhanced growth

275 inhibition effect of obinutuzumab plus prednisolone (Supplementary Figure S4).

276 Glucocorticoids such as prednisolone induce growth inhibition through G1 arrest

277 [23]. The percentage of cells in the G0/G1 phase was increased by the prednisolone

278 treatment and was significantly more increased by the addition of obinutuzumab,

279 whereas obinutuzumab alone failed to induce G1 arrest in these resistant clones (Figure

280 3C). We also examined the protein levels of cell cycle regulators. This combination

281 decreased the protein level of Skp2, which is a substrate recruiting component of the

282 ubiquitin–proteasome system that targets cell cycle control elements (Figure 3D).

283 Furthermore, p27, a cyclin-dependent kinase inhibitor and a substrate of Skp2, was

284 increased and the level of phosphorylated Rb was decreased by this combination

285 (Figure 3D).

286 In clone SU-DHL-4-OR-7C2 (7C2), in which the combination of obinutuzumab

287 plus prednisolone showed a supra-additive effect but to a lesser extent than that in

288 clones 2B3 and 6B3 (Figure 1B), this combination increased DNA fragmentation more

289 than each single agent, without enhancing G0/G1 arrest (Supplementary Figure S5).

290 Furthermore, in clone SU-DHL-4-OR-1D6 (1D6), in which this combination showed a

291 sub-additive effect (Figure 1B), although the combination slightly decreased the protein

292 level of Skp2, it did not modulate downstream molecules or enhance G0/G1 arrest more

293 than prednisolone, and did not enhance DNA fragmentation more than obinutuzumab

294 (Supplementary Figure S5).

295 In vivo antitumor effect of the combination of obinutuzumab and

296 chemotherapeutic agents in mouse xenograft model

297 We evaluated the antitumor efficacy of the combination of obinutuzumab plus

298 doxorubicin or prednisolone in mouse xenograft models. To evaluate the effect of

299 obinutuzumab-induced direct cell death, as opposed to ADCC, ADCP, or CDC, we used

300 NOG mice which lack NK cells, have dysfunctional macrophages, and display reduced

301 complement activity. Obinutuzumab alone significantly decreased the tumor volume

302 compared with the control group in SU-DHL-4 xenograft model (Supplementary Figure

303 S6), but not in the models with resistant clones (Figure 4A, B). In the 1A2-xenograft

304 model, doxorubicin significantly decreased the tumor volume compared with control

305 group, and the combination of obinutuzumab plus doxorubicin significantly enhanced

306 the antitumor effect compared with each single agent on day 22 (Figure 4A). In the

307 2B3-xenograft model, although neither obinutuzumab nor prednisolone decreased the

308 tumor volume, the combination did show a significant antitumor effect compared with

309 each single agent on day 22 (Figure 4B). Furthermore, in both models, although tumors

310 in combination groups regrew 11-15 days after initial treatment, retreatment using the

311 combination still showed antitumor effects (Figure 4).

312 Discussion

313 Our data demonstrates the efficacy of obinutuzumab against CD20-positive NHL cells

314 resistant to obinutuzumab-induced direct cell death. Remarkably, our data showed that

315 for all resistant cells there were several combinations of obinutuzumab plus

316 chemotherapeutic agents used in the treatment of FL patients that exerted a supra-

317 additive effect. Our results indicated that doxorubicin enhances the growth inhibition

318 effect of obinutuzumab through a caspase-dependent mechanism in the resistant cells.

319 Our results also suggest that the combination of obinutuzumab plus prednisolone

320 enhances the growth inhibition effect by increasing cell death and further enhances it by

321 inducing G0/G1 arrest in the resistant cells. Furthermore, resistant cells retained their

322 resistance to obinutuzumab-induced direct cell death in vivo, and the combination of

323 obinutuzumab plus doxorubicin or prednisolone also significantly enhanced antitumor

324 effect in the resistant clone xenograft models. Moreover, these combination effects were

325 also confirmed in obinutuzumab-refractory cells (Supplementary Figure S7).

326 Our findings suggest that obinutuzumab induces direct cell death independent of

327 caspase in SU-DHL-4 cells (Supplementary Figure S3), and according to a study by

328 Latour, S. et al.[21], obinutuzumab-induced direct cell death occurs via the Ca2+

329 signaling-mediated ER stress pathway in SU-DHL-4 cells. Furthermore, our data

330 indicates that the combination effect of obinutuzumab plus doxorubicin in resistant

331 clones involves caspase-dependent mechanism, likely through the activation of IRE1–

332 JNK–Bcl2 signaling, which is known to be one of the main contributors to ER stress-

333 induced apoptosis in resistant clones (Figure 2) [22, 24]. ER stress kills cancer cells by

334 inducing either caspase-dependent apoptosis or caspase-independent cell death, such as

335 necroptosis, and either of these cell death mechanisms can switch to the other [25-27].

336 Together, our results suggest that, in resistant clones, the mechanism of obinutuzumab-

337 induced cell death via ER stress changes based on the treatment: obinutuzumab alone

338 causes caspase-independent cell death, whereas the combination with doxorubicin

339 causes caspase-dependent apoptosis.

340 While doxorubicin decreases the activity of sarcoplasmic reticulum Ca2+-

341 ATPase, an ATP pump that restores luminal ER calcium levels, and activates ER stress

342 [28, 29], the combination effect of obinutuzumab plus doxorubicin was slight in SU-

343 DHL-4 cells (Supplementary Figure S3A). This could be because each compound

344 works in different phase of cell cycle. Doxorubicin exerts its cytotoxic effect in the

345 G2/M phase, but obinutuzumab induces G0/G1 arrest in SU-DHL-4 cells

346 (Supplementary Figure S3E). We investigated the combination of obinutuzumab and

347 doxorubicin against obinutuzumab-sensitive cells using only one in vitro model, and

348 further study is needed to fully clarify its effect.

349 Unlike in the doxorubicin combination, with prednisolone the caspase inhibitor

350 failed to suppress the enhanced growth inhibition effect in resistant clones

351 (Supplementary Figure S4) despite the promoted cell death (Figure 3B); this suggests

352 that prednisolone strengthens obinutuzumab-induced caspase-independent cell death.

353 The resistant clones subject to stronger supra-additive effects exhibited both enhanced

354 cell death and G0/G1 arrest, while clones subject to weaker supra-additive effects only

355 exhibited enhanced cell death (Figure 3 and Supplementary Figure S5). This suggests

356 that obinutuzumab plus prednisolone enhances growth inhibition primarily by inducing

357 caspase-independent cell death, and that additional enhancement of G0/G1 arrest further

358 increases it.

359 Because NOG mice lack NK cells, have a less efficient complement system, and

360 have dysfunctional macrophages [30], obinutuzumab’s antitumor effect in these mice

361 transplanted with parental SU-DHL-4 cells seems to indicate the efficacy of direct cell

362 death (Supplementary Figure S6). This effect was diminished in mice transplanted with

363 resistant clones (Figure 4). These results indicate that obinutuzumab-induced direct cell

364 death at least partially involves an in vivo antitumor effect and in vivo resistance, which

365 suggests that direct cell death is integral to obinutuzumab’s in vivo mechanism of action.

366 Furthermore, because the combination of obinutuzumab plus doxorubicin or

367 prednisolone showed a significant antitumor effect not only as an initial treatment but

368 also as post-regression treatment, this result contributes to the possibility of an effective

369 clinical treatment option.

370 Rituximab is another anti-CD20 antibody commonly used in the treatment of FL.

371 We investigated the combination effect of rituximab plus chemotherapeutic agents in

372 obinutuzumab-resistant clones. The combination of rituximab plus doxorubicin or

373 prednisolone also significantly inhibited growth compared with each single agent in

374 clones 1A2 and 2B3, in which the combination of obinutuzumab plus doxorubicin or

375 prednisolone showed a supra-additive effect, respectively (Supplementary Figure S8).

376 These results suggest the possibility that not only the combination of obinutuzumab plus

377 chemotherapeutic agent but also that of rituximab plus chemotherapeutic agent is useful

378 after acquisition of obinutuzumab resistance. However, we think the important

379 takeaway from this non-clinical study is the possibility that obinutuzumab can still be an

380 effective treatment option when combined with a chemotherapeutic agent, even after

381 obinutuzumab treatment.

382 In this study, we focused on the efficacy of obinutuzumab against CD20-

383 positive NHL cells resistant or refractory to obinutuzumab-induced direct cell death.

384 The loss or reduction of CD20 expression is known to be one of the mechanisms of

385 resistance to the anti-CD20 monoclonal antibody rituximab [31, 32]. Obinutuzumab is

386 more effective than rituximab at depleting the CD20-low B cell population, and its

387 inability to interact with FcγRIIb is thought to contribute to the lower CD20

388 internalization [33, 34]; however, the loss or reduction of CD20 expression could still

389 be an obinutuzumab-resistant mechanism. At the same time, ADCC and ADCP are also

390 important mechanisms of action for obinutuzumab. Further study is required to

391 investigate how to overcome resistance to obinutuzumab caused by either the reduction

392 of CD20 expression or by resistance to ADCC/ADCP. Furthermore, we could not

393 identify any genetic resistant mechanism in this study. The MoA of obinutuzumab-

394 induced direct cell death in SU-DHL-4 is still unclear and is just beginning to

395 understand that it is different from the more well-known mechanism[21, 35, 36], which

396 makes the analysis of the resistance mechanisms more difficult. In the future, the

397 elucidation of the MoA of obinutuzumab-induced direct cell death along with

398 technological breakthroughs can eventually unravel this resistant mechanism, and it is

399 believed that this will lead to the identification of biomarkers in patients who would

400 benefit from obinutuzumab retreatment.

401 The results of this preclinical study suggests that, combined with chemotherapy,

402 obinutuzumab re-treatment may be a possible option in the clinical treatment of CD20-

403 positive obinutuzumab-resistant lymphoma. Further investigation is needed to confirm

404 the clinical efficacy of obinutuzumab re-treatment. Furthermore, because the

405 chemotherapeutic agent showing the highest combination effect with obinutuzumab

406 differed for each resistant clone, biomarkers to identify the best combination for each

407 patient need to be elucidated.

408 Acknowledgements

409 The authors thank Dr. Yoshiaki Isshiki, Kazushige Mori, Dr. Kaori Fujimoto-Ouchi,

410 and Dr. Osamu Kondoh (Chugai Pharmaceutical Co., Ltd.) for their helpful advice.

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534 Figure legends

535 Figure 1. In vitro combination effect of obinutuzumab plus each chemotherapeutic

536 agent on resistant clones. (A) Each of the cells was treated with various concentrations

537 of obinutuzumab for 4 days, and the obinutuzumab IC50 values for SU-DHL-4 cells and

538 each resistant clone were calculated. # indicates clones for which the IC50 was >200

539 µg/mL. (B) Each clone was treated with obinutuzumab (1 µg/mL) and/or doxorubicin

540 (10 nM), prednisolone (1 µM), 4-OOH-CY (100 nM), vincristine (1 nM), or

541 bendamustine (20 µM) for 4 days. The combination effect was estimated by a

542 interaction index based on the Bliss independence model. An index value of <0, =0, or

543 >0 indicates a supra-additive, additive, or sub-additive effect, respectively. *: P<0.05

544 versus the additive assumption calculated from each single agent by student’s t-test.

545 Figure 2. Enhanced growth inhibition effect of obinutuzumab in combination with

546 doxorubicin through caspase-dependent apoptosis induction in resistant clones.

547 (A) Cell growth inhibition by obinutuzumab (OBI) alone or the combination of OBI

548 plus doxorubicin (DXR) was examined after 4 days of treatment in clone 1A2 and clone

549 1C4. Data points represent mean + SD (n=3). a: P<0.05 versus DXR 0 nM, b: P<0.05

550 versus DXR 2.5 nM, c: P<0.05 versus DXR 5 nM by Tukey’s HSD test with Two-way

551 ANOVA. (B, C) Immunoblots evaluating the phosphorylation levels of IRE1 (B), and

552 JNK and Bcl2 (C) of clone 1A2. Cells were treated with OBI (1 µg/mL) and/or DXR

553 (10 nM) for the indicated times. β Actin was used as a loading control. (D) DNA

554 fragmentation was evaluated by TUNEL assay. Clone 1A2 and clone 1C4 were treated

555 with OBI (1 µg/mL), DXR (10 nM), and/or Z-VAD-FMK (40 µM) for 3 days. (E)

556 Caspase 3/7 activity was measured after 48 hours treatment with OBI (1 µg/mL) and/or

557 DXR (10 nM). Each bar represents mean + SD (n=3). a: P<0.05 versus non-treatment,

558 b: P<0.05 versus treatment with OBI alone, c: P<0.05 versus treatment with DXR alone

559 by Tukey’s HSD test. (F) Cell growth inhibition by OBI, DXR, and/or 40 µM of Z-

560 VAD-FMK (Z-VAD) was examined 4 days after treatment in clone 1A2 and clone 1C4.

561 Data points represent mean + SD (n=3).

562 Figure 3. Enhanced combination effect of obinutuzumab plus prednisolone on cell

563 growth inhibition, cell death induction, and G0/G1 arrest in resistant clones. (A) Cell

564 growth inhibition by obinutuzumab (OBI) alone or the combination of OBI plus

565 prednisolone (PSL) was examined after 4 days of treatment in each clone. Data points

566 represent mean + SD (n=3). a: P<0.05 versus PSL 0 µM, b: P<0.05 versus PSL 0.04

567 µM, c: P<0.05 versus PSL 0.2 µM by Tukey’s HSD test with Two-way ANOVA. (B)

568 DNA fragmentation was evaluated by TUNEL assay. Clone 2B3 and clone 6B3 were

569 treated with OBI (1 µg/mL) and/or PSL (1 µM) for 3 days. (C) Cell cycle was analyzed

570 after 48 hours of treatment with OBI (1 µg/mL) and/or PSL (1 µM) in each clone. Each

571 bar represents mean + SD (n=3, independent assay). Statistical analysis was performed

572 on the percentage of cells in the G0/G1 phase. a: P<0.05 versus non-treatment control,

573 b: P<0.05 versus OBI alone, c: P<0.05 versus PSL alone by Tukey’s HSD test. (D)

574 Immunoblots evaluating the protein levels of cell cycle regulators. Each clone was

575 treated with OBI (1 µg/mL) and/or PSL (1 µM) for 48 hours. β Actin was used as a

576 loading control.

577 Figure 4. The in vivo antitumor effect of obinutuzumab plus chemotherapeutic agents

578 in obinutuzumab-induced-direct-cell-death–resistant cells transplanted mouse

579 xenograft model. (A) In vivo antitumor effect of combination of obinutuzumab 30

580 mg/kg (OBI) plus doxorubicin 2.5 mg/kg in NOG mice bearing clone 1A2 (n=6/group).

581 Control HuIgG and OBI were intravenously treated on day 1, 8, 15, and 22. Vehicle and

582 DXR were intravenously treated on day 1, and 22. a: significant versus control group, b:

583 significant versus obinutuzumab group, c: significant versus doxorubicin group by

584 Wilcoxon test and the Holm method on day 22. (B) In vivo antitumor effect of

585 combination of OBI 30 mg/kg plus prednisolone (PSL) 2 mg/kg in NOG mice bearing

586 clone 2B3 (n=6/group). Control HuIgG and OBI were intravenously treated on day 1, 8,

587 15, and 22. Vehicle and PSL were orally treated on day 1-5, and day 22-26. a:

588 significant versus control group, b: significant versus obinutuzumab group, c:

589 significant versus prednisolone group by Wilcoxon test and the Holm method on day

590 22.

591

Obinutuzumab in combination with chemotherapy enhances direct cell death in CD20-positive obinutuzumab-resistant non-Hodgkin lymphoma cells

Takaaki Fujimura, Yoriko Yamashita-Kashima, Natsumi Kawasaki, et al.

Mol Cancer Ther Published OnlineFirst April 13, 2021.

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