Phase Ib Study of Lumretuzumab Plus Cetuximab Or Erlotinib in Solid Tumor Patients

Author Manuscript Published OnlineFirst on June 9, 2017; DOI: 10.1158/1078-0432.CCR-17-0812 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Lumretuzumab combination treatment

Title:

Phase Ib study of lumretuzumab plus cetuximab or erlotinib in solid tumor patients

and evaluation of HER3 and heregulin as potential biomarkers of clinical activity

Authors:

Didier Meulendijks1#, Wolfgang Jacob2#, Emile E. Voest1, Morten Mau-Sorensen7,

Maria Martinez-Garcia3, Alvaro Taus3, Tania Fleitas5, Andres Cervantes5, Martijn P.

Lolkema4,6, Marlies H.G. Langenberg4, Maja J. De Jonge6, Stefan Sleijfer6, Ji-Youn

Han11, Antonio Calles13*, Enriqueta Felip10, Sang-We Kim12, Jan H.M. Schellens1,16,

Sabine Wilson2, Marlene Thomas2, Maurizio Ceppi2, Georgina Meneses-Lorente8, Ian

James14, Suzana Vega Harring2, Rajiv Dua15, Maitram Nguyen15, Lori Steiner15,

Celine Adessi9, Francesca Michielin9, Birgit Bossenmaier2, Martin Weisser2, Ulrik N.

Lassen7

# Both authors contributed equally to this work.

Author affiliation:

1Department of Clinical Pharmacology, Division of Medical Oncology, The

Netherlands Cancer Institute, Amsterdam, The Netherlands

2Pharma Research and Early Development, Roche Innovation Center Munich,

Penzberg, Germany

3Department of Medical Oncology, Hospital del Mar, Barcelona, Spain

4Department of Medical Oncology, University Medical Center Utrecht, Utrecht, The

Netherlands

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 9, 2017; DOI: 10.1158/1078-0432.CCR-17-0812 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Lumretuzumab combination treatment

5Department of Medical Oncology, CIBERONC, Institute of Health Research

INCLIVA, University of Valencia, Valencia, Spain

6Department of Medical Oncology, Erasmus Medical Center Cancer Institute and

Cancer Genomics, Rotterdam, The Netherlands

7Department of Oncology, Rigshospitalet, Copenhagen, Denmark

8Pharma Research and Early Development, Roche Innovation Center Welwyn,

Welwyn, UK

9Pharma Research and Early Development, Roche Innovation Center Basel, Basel,

Switzerland

10Medical Oncology Department, Vall d'Hebron University Hospital, Vall d'Hebron

Institute of Oncology, Barcelona, Spain

11Center for Lung Cancer, National Cancer Center, Goyang, South Korea

12Department of Oncology, Asan Medical Center, Seoul, South Korea

13START-Madrid, Centro Integral Oncológico Clara Campal, Madrid, Spain

14A4P Consulting Ltd, Discovery Park, Sandwich, UK

15Roche Molecular Systems Inc, Pleasanton, CA, USA

16Utrecht Institute for Pharmaceutical Sciences (UIPS), University Utrecht, Utrecht,

The Netherlands

* Currently working at: Department of Oncology, Hospital General Universitario

Gregorio Marañón, Madrid, Spain

Lumretuzumab combination treatment

Acknowledgments of research support for the study:

This study was funded by F. Hoffmann–La Roche Ltd.

Corresponding author:

Wolfgang Jacob

Address: Pharma Research and Early Development, Roche Innovation Center

Munich, Nonnenwald 2, 82377 Penzberg, Germany

Phone: 0049 8856 60 2736

Fax: 0049 8856 60 79 2736

Mobile: 0049 174 31 80 431

Email: [email protected]

Running header:

Lumretuzumab combination treatments

Key words:

Human epidermal growth factor receptor 3 (HER3), phase I, epidermal growth factor

receptor (EGFR), heregulin, non-small cell lung cancer (NSCLC)

Previous presentation of data:

Poster abstract in Annals of Oncology 25 (Supplement 4): iv147, 2014.

Word count: translational relevance: 155 words; abstract: 233 words; body text:

4570 words; 6 figures/tables; references = 52 3

Lumretuzumab combination treatment

1 Translational Relevance

2 Preclinical models have demonstrated superior anti-tumor activity when human

3 epidermal growth factor receptor 3 (HER3)- and epidermal growth factor receptor

4 (EGFR)-targeting therapies were combined as compared to single agent activity.

5 Furthermore, preclinical and clinical data suggest that response to HER3-targeting

6 therapy is positively correlated to expression of the HER3 ligand heregulin (HRG).

7 Across tumor types, the clinical activity of the anti-HER3 antibody lumretuzumab

8 when given in combination with the EGFR-blocking agents cetuximab or erlotinib

9 was modest with a manageable safety profile. In order to enrich for tumors with

10 higher HRG mRNA levels, we evaluated the combination of lumretuzumab and

11 erlotinib in a dedicated cohort of squamous non-small cell lung cancer (sqNSCLC)

12 patients. Here, neither HRG nor HER3 mRNA expression levels were associated

13 with an increased response rate questioning 1) the relevance of HER3-blockade in

14 combination with EGFR-targeting therapy per se and 2) the relevance of HRG and

15 HER3 as response prediction biomarkers for this therapy.

Lumretuzumab combination treatment

16 Abstract

17 Purpose

18 This study investigated the safety, clinical activity and target-associated biomarkers

19 of lumretuzumab, a humanized, glycoengineered, anti-HER3 monoclonal antibody

20 (mAb), in combination with the EGFR-blocking agents erlotinib or cetuximab in

21 patients with advanced HER3-positive carcinomas.

22 Patients and Methods

23 The study included two parts: dose escalation and dose extension phases with

24 lumretuzumab in combination with either cetuximab or erlotinib, respectively. In both

25 parts, patients received lumretuzumab doses from 400 to 2000 mg plus cetuximab or

26 erlotinib according to standard posology, respectively. The impact of HRG mRNA

27 and HER3 mRNA and protein expression were investigated in a dedicated extension

28 cohort of sqNSCLC patients treated with lumretuzumab and erlotinib.

29 Results

30 Altogether, 120 patients were treated. One dose-limiting toxicity (DLT) in the

31 cetuximab part and 2 DLTs in the erlotinib part were reported. The most frequent

32 adverse events (AEs) were gastrointestinal and skin toxicities, which were

33 manageable. The objective response rate (ORR) was 6.1% in the cetuximab part

34 and 4.2% in the erlotinib part. In the sqNSCLC extension cohort of the erlotinib part,

35 higher tumor HRG and HER3 mRNA levels were associated with numerically higher

36 disease control rate but not ORR.

37 Conclusion

Lumretuzumab combination treatment

38 The toxicity profile of lumretuzumab in combination with cetuximab and erlotinib was

39 manageable but only modest clinical activity was observed across tumor types. In

40 the sqNSCLC cohort, there was no evidence of meaningful clinical benefit despite

41 enriching for tumors with higher HRG mRNA expression levels.

Lumretuzumab combination treatment

42 Introduction

43 HER3 is a key dimerization partner of HER family members which activates several

44 oncogenic signal transduction pathways, particularly the phosphoinositide-3-kinase

45 (PI3K)/Akt pathway [1]. Recent studies indicate that HER3 pathway activation is

46 important in the development of resistance to EGFR and HER2-targeting treatments

47 [2-6]. The role of HER3 as a prognostic marker remains controversial. Increased

48 HER3 protein expression as measured by immunohistochemistry (IHC) has been

49 described as an adverse prognostic factor in many solid tumor types including

50 breast, gastric, lung, ovarian and colon cancers [7-11] and targeting HER3 can

51 sensitize refractory tumor models to EGFR inhibitors [12]. In contrast, higher

52 expression levels of HER3 mRNA were associated with increased progression-free

53 survival (PFS) in HER2-positive metastatic breast cancer treated with pertuzumab

54 plus trastuzumab plus docetaxel [13] and in patients with platinum-resistant ovarian

55 cancer treated with gemcitabine plus pertuzumab [14]. However, adding pertuzumab

56 to chemotherapy in platinum-resistant ovarian cancer patients selected on low HER3

57 mRNA expression did not increase PFS significantly [15]. Autocrine loops involving

58 the HER3 ligand HRG and leading to HER3 activation have been described in

59 squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer

60 (NSCLC) and ovarian cancer [8, 16-20].

61 Lumretuzumab is a humanized, glycoengineered immunoglobulin G1 antibody which

62 selectively binds with high affinity to the extracellular domain of HER3. Prevention of

63 HRG binding to HER3 by lumretuzumab resulted in almost complete inhibition of

64 HER3 heterodimerization and phosphorylation as well as inhibition of tumor growth

65 in cell-line-based mouse xenograft models [21]. In a phase I study, the safety of

66 lumretuzumab was evaluated in patients with advanced solid tumors. No DLTs were

Lumretuzumab combination treatment

67 observed at doses of 100 to 2000 mg every two weeks (q2w, as a flat dose) and a

68 maximum tolerated dose (MTD) was not reached. Downregulation of HER3

69 membranous protein was observed in on-treatment tumor biopsies from 200 mg and

70 higher doses, and a target-independent pharmacokinetic (PK) profile was observed

71 at ≥ 400 mg doses [22].

72 Multiple preclinical models have shown that combinations of anti-HER3 antibodies

73 with anti-EGFR therapies lead to enhanced anti-tumor activity as compared to the

74 single agents and even complete tumor regression has been described in different

75 studies [12, 21, 23, 24]. Preclinical studies have also shown that HER3 pathway

76 activation and overexpression of HRG predicted response to HER3-targeting therapy

77 [25, 26] and preliminary clinical data from early studies with anti-HER3 antibodies

78 are consistent with these reports [22, 27, 28]. We therefore hypothesized that 1) the

79 combination of lumretuzumab with EGFR inhibitors could improve clinical activity

80 versus EGFR inhibitors alone, and 2) higher tumor expression levels of HRG mRNA

81 could indicate improved clinical outcome and could ultimately serve as a predictive

82 biomarker for HER3-targeting therapy.

83 The current study evaluated the safety and clinical activity, and potential biomarkers

84 of lumretuzumab treatment when combined with the EGFR-targeting agents

85 cetuximab or erlotinib. To evaluate the clinical activity of lumretuzumab plus erlotinib

86 in a biomarker-enriched population we recruited an extension cohort of sqNSCLC

87 patients, as sqNSCLC has been shown to express higher HRG mRNA levels

88 compared to non-sqNSCLC [data on file, [29, 30].

Lumretuzumab combination treatment

89 Methods

90 Study design

91 This was a multicenter, phase Ib, open-label, non-randomized, dose-escalation and

92 extension study (ClinicalTrials.gov Identifier: NCT01482377) investigating the safety

93 (including the MTD and/or optimal biological dose [OBD]), PK, pharmacodynamics

94 (PD) and clinical activity of lumretuzumab in combination with cetuximab or erlotinib

95 in patients with metastatic or advanced HER3-positive carcinomas. The study

96 consisted of a dose escalation phase following a standard “3+3” study design and an

97 extension phase conducted for the cetuximab plus lumretuzumab as well as the

98 erlotinib plus lumretuzumab combination.

99 For lumretuzumab monotherapy, PK was linear at dosages ≥400 mg, indicative of

100 target-mediated drug disposition saturation, and clinical PD activity was

101 demonstrated [22]. Therefore, the dose of 400 mg was used as the starting dose for

102 the combination treatments with cetuximab and erlotinib. Two administration

103 schedules were applied: q2w and every 3 weeks (q3w), the latter only applied for a

104 subset of patients in the lumretuzumab plus erlotinib part.

105 Standard doses of cetuximab (400 mg/m2 for the first infusion followed by 250 mg/m2

106 weekly infusions) and erlotinib (daily dose of 150 mg orally) were used.

107 Patients continued treatment until disease progression, unacceptable toxicity or

108 withdrawal of consent.

109 Ethics

110 Local ethics committee approval was obtained and all patients provided written

111 informed consent. The study was conducted in accordance with Good Clinical

Lumretuzumab combination treatment

112 Practice guidelines and the Declaration of Helsinki in nine centers in Spain, the

113 Netherlands, Denmark and South Korea.

114 Patients

115 Patients had to have a histologically confirmed diagnosis of an advanced or

116 metastatic HER3-expressing carcinoma that was refractory to standard treatment or

117 for which no standard therapy existed. Eligible patients were ≥18 years of age, had

118 an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2 and

119 had adequate hematology, blood chemistry, and renal and liver function. Patients

120 eligible for enrollment underwent a fresh (pretreatment) tumor biopsy that was used

121 to assess the level of HER3 protein expression by IHC using central pathology

122 review. Discernible HER3 membrane staining in any neoplastic cell, provided that a

123 minimum of 100 tumor cells were present in the biopsy specimen, was considered

124 diagnostically positive for HER3 protein expression.

125 For the extension phase of the cetuximab combination part, only patients with

126 advanced HER3-expressing SCCHN, NSCLC and colorectal cancer (CRC, proven to

127 be KRAS wildtype and expressing EGFR as per local assessment by the

128 investigator) were recruited. For the extension phase of the erlotinib combination

129 part, only patients with advanced HER3-expressing sqNSCLC and patients with non-

130 squamous lung carcinomas with a documented NRG1 somatic gene fusion

131 (determined by local PCR and sequencing assays) were recruited in order to enrich

132 for patients more likely to benefit from lumretuzumab. Higher expression of HRG

133 mRNA in sqNSCLC as compared to non-sqNSCLC had been demonstrated through

134 mining of data extracted from TCGA and analysis of HRG by quantitative real-time

135 PCR (qRT-PCR) in formalin-fixed, paraffin-embedded (FFPE) tumor specimens from

Lumretuzumab combination treatment

136 the Roche clinical tumor bank and from preclinical HER3 xenograft response

137 modelling studies. In addition, higher HRG levels positively correlated with response

138 to lumretuzumab treatment in preclinical models (data not shown). Recently, gene

139 fusions were identified involving the gene encoding for HRG (NRG1) [31] activating

140 HER3 in an autocrine and paracrine manner as a novel mechanism for HER

141 pathway dysregulation in lung adenocarcinomas [32]. Tumors harboring these NRG1

142 fusions may be particularly susceptible to HER3-targeting therapy. The protocol was

143 therefore amended to allow inclusion of these rare patients to explore signals of

144 clinical activity. Due to the differences in the underlying biology, it was decided to

145 analyze the patients with NRG1-rearranged tumors separately and not include them

146 into the analysis of the sqNSCLC cohort.

147 Responding patients were tested for EGFR mutations with Qiagen therascreen

148 EGFR Pyro assay (Source BioScience, Nottingham, UK).

149 Study drug administration

150 Patients received premedication 30 min prior to the start of the 1st lumretuzumab

151 infusion consisting of paracetamol (500 to 1000 mg p.o.) and diphenhydramine (25

152 to 50 mg p.o. or IV, or an alternative anti-histamine). Corticosteroids were allowed in

153 case of ≥ grade 2 infusion-related reactions (IRRs). Lumretuzumab was

154 administered as an IV infusion. Cetuximab and erlotinib were administered according

155 to standard posology and dose reductions were allowed according to the label. Study

156 drugs could be delayed to assess or treat AEs for up to 14 days. One reduction to

157 the previously assessed lower dose level was allowed for lumretuzumab.

Lumretuzumab combination treatment

158 Tumor response and safety

159 Tumor response assessment using Response Evaluation Criteria in Solid Tumors

160 (RECIST) version 1.1 [33] was conducted at screening and every 8 or 9 weeks

161 thereafter for the q2w or q3w schedule, respectively.

162 Safety assessments included physical (ECOG performance status, vital signs) and

163 laboratory examinations and electrocardiogram (ECG). AEs were defined according

164 to the Common Terminology Criteria for Adverse Events, version 4.0 (CTC-AE v4.0).

165 Definition of dose-limiting toxicity (DLT)

166 A DLT was defined as an AE occurring during the first two cycles of treatment (i.e.

167 28 days) with lumretuzumab that was considered to be study drug-related and was

168 either: Grade 4 neutropenia (i.e. absolute neutrophil count [ANC] < 0.5 x 109 cells/L

169 for a minimal duration of 7 days); Grade 3/4 febrile neutropenia; Grade 4

170 thrombocytopenia; Grade 3 thrombocytopenia associated with bleeding episodes; or

171 Grade ≥ 3 non-hematological toxicity. IRRs and Grade 3 skin and/or epithelial

172 toxicities associated with EGFR inhibition were not considered DLTs for the

173 cetuximab combination. For the erlotinib combination, IRRs, Grade 3 skin and/or

174 epithelial toxicities associated with EGFR inhibition and Grade 3 diarrhea which

175 reduced to < Grade 2 within 14 days by supportive treatment or dose reduction of

176 erlotinib or interruption of erlotinib were not considered DLTs.

Lumretuzumab combination treatment

177 Biomarker assessments

178 Fresh tumor biopsies were collected during screening and on Day 14 of Cycle 1.

179 HER3 protein expression was assessed using an IHC assay and scored semi-

180 quantitatively and reported as an Immuno-Reactive Score (IRS, Range 0 to 300) as

181 described previously [22].

182 HRG mRNA expression was measured by a qRT-PCR assay in FFPE sections

183 obtained from fresh tumor biopsies collected from all patients at screening prior to

184 initiation of treatment. In addition, HER3 mRNA expression was determined using a

185 separate qRT-PCR assay. Both assays were performed at Roche Molecular

186 Systems (Pleasanton, USA).

187 Total RNA was isolated from FFPE tumor tissue sections using the Cobas® RNA

188 isolation kit. Taqman probes were designed to detect HRG or HER3 mRNA and

189 respective reference genes simultaneously. All reagents were prepared at Roche

190 Molecular Systems and qRT-PCR was performed using the Cobas® 4800 system.

191 Calculation of the cycle-to-threshold (Ct) for each fluorescent channel was done

192 using LC480 software and the relative HRG or HER3 mRNA log expression was

193 reported as ΔCt where ΔCt = Ct(Reference) - Ct(Target). Where feasible, biopsies

194 with less than 50% tumor content underwent macro-dissection guided by pathologist

195 annotation of adjacent H&E stained sections. No reference ranges were defined a

196 priori for HRG or HER3 mRNA expression using the initial research-grade assay.

Lumretuzumab combination treatment

197 During the course of the clinical study the HRG mRNA assay was transitioned to a

198 prototype diagnostic assay which differed in that reagents were prepared in a GMP

199 facility, the assay run using a z480 PCR system and calculation of Ct values was

200 performed using validated diagnostic software (Heregulin Expression Test Analysis

201 Package for use with Cobas 4800 System Release 2.1). Pre-dose HRG mRNA

202 expression data derived using both the research and prototype diagnostic assays

203 were available for 37 out of 120 patients. Overall, the research assay reported higher

204 expression than the diagnostic prototype due to differences in the data analysis

205 parameter settings, albeit there was strong correlation between the two assays

206 (Mean Difference 1.53 ΔCt R2 = 0.96).

207 The prototype diagnostic assay was used to define a reference range for HRG

208 expression using 150 primary FFPE tumor biopsy samples from sqNSCLC patients

209 (obtained from a Roche tumor bank and an external vendor). Median HRG log

210 expression corresponded to a ΔCt of -1.92 with a 75th percentile of -0.92. The

211 median was used as a threshold above which tumor biopsies derived from patients

212 were defined as HRG-high.

213 Statistical considerations

214 All patients who received at least one dose of study medication were included in the

215 safety population. Descriptive statistics were used for demographics and safety.

216 In order to explore biomarker levels and correlations, a PD population was also

217 defined, comprising of all patients receiving at least one dose of study medication

218 and providing a PD sample at baseline. Due to the distinct histology – invasive

219 mucinous NSCLC – and potentially distinct biology, lung cancer patients with NRG1

220 gene alterations were included in the sqNSCLC cohort but analyzed separately. For

Lumretuzumab combination treatment

221 some patients, biomarker data were not available, either due to lack of evaluable

222 tumor tissue or for technical reasons. Therefore, for each analysis the number of

223 evaluable patients (n) is specified.

224 Exploratory statistical analyses were used to evaluate differences in biomarker levels

225 for patients with different tumor histologies (non-squamous vs. squamous

226 carcinoma) or lung cancer diagnosis and changes from baseline levels using the

227 Wilcoxon rank-sum test or Wilcoxon signed-rank test as appropriate.

228 The Spearman's rank correlation coefficient (ρ) was used to determine the

229 association between biomarker levels.

230 The information on patients’ histology (squamous and adenocarcinoma) was

231 available for the vast majority of the patients or could be inferred through the tumor

232 type (e.g. patients with CRC have adenocarcinoma): only 4 patients had to be

233 excluded in the analyses of biomarker differences between adeno and squamous

234 carcinoma patients since the histology was either unknown, both histologies were

235 present or the histology was of transitional cell type, although all available data was

236 utilized to assess biomarker correlations.

237 For efficacy, RECIST response was evaluated and summarized by study part

238 (cetuximab/erlotinib combinations), and the objective response from the sqNSCLC

239 patients of the extension cohort was related to biomarker levels via a multinomial

240 logit model. All available patients were included in the analyses, since all dose levels

241 were considered PD-active.

Lumretuzumab combination treatment

242 Results

243 Patients

244 Patient demographics and baseline characteristics are presented in Table 1. In the

245 dose escalation phase of the cetuximab part, 27 patients were enrolled into five dose

246 cohorts, i.e. 400 (n=5), 800 (n=6), 1200 (n=5), 1600 (n=5) and 2000 mg (n=6) of

247 lumretuzumab q2w. In the extension phase of the cetuximab part, 22 patients

248 received 2000 mg. In the dose escalation phase of the erlotinib part, 37 patients

249 were enrolled into four q2w dose cohorts, i.e. 400 (n=6), 800 (n=6), 1600 (n=7) and

250 2000 mg (n=8) of lumretuzumab, and 10 patients were enrolled into two q3w dose

251 cohorts, i.e. 800 (n=7) and 1600 mg (n=3). In the extension phase of the erlotinib

252 part, 34 patients received 800 mg of lumretuzumab q2w (Figure 1). Two of those

253 patients had NSCLC adenocarcinoma with an NRG1 fusion, and 32 were sqNSCLC

254 patients.

255 Overall, 102 patients (85.0%) discontinued the study due to progressive disease or

256 death, six patients (5.0%) were withdrawn due to an AE, six patients (5.0%) refused

257 further treatment, one patient (0.8%) withdrew consent and five patients (4.2%)

258 discontinued for other reasons.

259 Safety

260 Three patients with DLTs were reported, one in the cetuximab part (dehydration

261 Grade 3 in the 800-mg cohort) and two in the erlotinib part (diarrhea Grade 3 and

262 hypokalemia Grade 3 in one patient of the 1600 mg cohort and blood bilirubin

263 increased Grade 3 in one patient of the 2000 mg cohort). The MTD was not reached

264 up to the highest dose tested (i.e. 2000 mg) in both treatment parts. A total of 1452

265 AEs of any grade were reported in 120 patients (100%) (Table 2). Most AEs (87.3%)

Lumretuzumab combination treatment

266 were Grade 1 or 2 in intensity. A total of 184 Grade ≥3 AEs were reported in 88

267 patients (73.3%). No patients in the cetuximab part and three patients (4.2%) in the

268 erlotinib part died from an AE (pneumonia, cerebrovascular accident and hemoptysis

269 with one patient each, all considered unrelated to lumretuzumab treatment). The

270 most frequent AEs included diarrhea (38 patients [77.6%] in the cetuximab part and

271 54 patients [76.1%] in the erlotinib part) and rash (26 patients [53.1%] in the

272 cetuximab part and 32 patients [45.1%] in the erlotinib part). The most frequent

273 ≥Grade 3 AEs included diarrhea (6 patients [12.2%] in the cetuximab part and 11

274 patients [15.5%] in the erlotinib part), hypomagnesemia (5 patients [10.2%] in the

275 cetuximab part and no patients in the erlotinib part) and dermatitis acneiform (5

276 patients [10.2%] in the cetuximab part and 4 patients [5.6%] in the erlotinib part).

277 Diarrhea was the only AE for which a potential dose dependency was observed

278 during dose escalation. In the cetuximab part, 1/6 patients had Grade 3 diarrhea only

279 at the top dose of 2000 mg lumretuzumab. In the erlotinib part, 1/6 patients at 800

280 mg, 3/7 patients at 1600 mg and 2/8 patients at 2000 mg lumretuzumab had Grade 3

281 diarrhea.

282 The severity of diarrhea was increased at 1600 and 2000 mg particularly in

283 combination with erlotinib. Although these diarrhea events did not qualify for DLTs as

284 per protocol, a lower lumretuzumab dose of 800 mg q2w was selected as the

285 recommended phase II dose for the combination with erlotinib whereas a dose of

286 2000 mg q2w was selected for the combination with cetuximab based on the

287 observed safety profile.

288

289 Pharmacokinetics

Lumretuzumab combination treatment

290 Similarly as described for monotherapy with lumretuzumab [22], a linear PK profile

291 was observed with doses of lumretuzumab of ≥400 mg q2w indicating target-

292 mediated drug disposition saturation (data not shown).

293

294 Biomarker Analysis

295 Tumors of all enrolled patients were HER3-positive based on IHC analysis. In line

296 with previously published data [22], membranous HER3 was downregulated in tumor

297 and skin biopsy samples collected on Day 14 of Cycle 1 compared to baseline

298 (Supplementary Table 1, Supplementary Table 2).

299 In baseline tumor samples, HER3 protein and HER3 mRNA were generally more

300 highly expressed in adenocarcinoma compared to those of squamous cell histology

301 (HER3 IHC: Median IRS 2.00 (n=61) and 0.07 (n=50), respectively, p<0.0001; HER3

302 mRNA: Median ΔCt 1.88 (n=61) and -0.27 (n=49), respectively, p<0.0001,

303 Supplementary Figure 1). Furthermore, HER3 protein expression was significantly

304 correlated with HER3 mRNA expression (ρ = 0.61, p<0.001, n = 112). Similarly, for

305 the subset of patients with NSCLC, HER3 protein was more highly expressed in the

306 non-squamous compared to the squamous subtype (HER3 IHC: Median IRS 2.25

307 (n=14) and 0.06 (n=36), respectively, p<0.0001, Supplementary Figure 2).

308 Conversely, analysis of baseline tumor biopsy samples using the research HRG

309 qRT-PCR assay showed an overall higher expression of HRG mRNA in tumors of

310 squamous cell histology compared to those of adenocarcinoma histology (p<0.0001,

311 Supplementary Figure 3). Similarly, within NSCLC samples median HRG mRNA log

312 expression (ΔCt) was higher in sqNSCLC as compared to non-sqNSCLC (p<0.0001,

313 Supplementary Figure 4).

Lumretuzumab combination treatment

314 In addition, HRG mRNA expression across tumor histologies was inversely

315 correlated to HER3 protein (ρ = -0.24, p=0.024, n = 89) and HER3 mRNA (ρ = -0.24,

316 p=0.022, n=91).

317

318 Antitumor activity

319 In the cetuximab part the ORR was 6.1% and the disease control rate (DCR, i.e. the

320 percentage of patients with a best response of stable disease [SD], partial response

321 [PR], or complete response [CR]) was 40.8% (Table 3).

322 Two patients with CRC showed a PR and one patient with SCCHN had a CR

323 (Supplementary Figure 5). The SCCHN patient had not been treated with an EGFR

324 inhibitor previously. The duration of response in this patient was 16.9 months. The

325 patient’s tumor expressed relatively high levels of HRG mRNA (ΔCt: 0.09, median

326 ΔCt in the cetuximab part: -2.44, n = 44) and EGFR protein (IRS: 3.0) and relatively

327 low levels of HER3 mRNA (ΔCt: -1.06, median ΔCt in the cetuximab part: 1.43, n =

328 46) and HER3 protein (IRS: 0.0004) as determined on fresh baseline FFPE tumor

329 biopsies. Overall, there was no detectable relationship between any of the

330 exploratory biomarkers and clinical response defined by RECIST (data not shown).

331 In the erlotinib part, the ORR was 4.2% and the DCR was 43.7% (Table 3).

332 Three patients (4.2%) had a PR, one with ovarian cancer (Supplementary Figure 6)

333 and two with sqNSCLC (Figure 2). The ovarian cancer patient had a duration of

334 response of 19.8 months. This tumor showed high levels of HRG mRNA (ΔCt: -0.36,

335 median ΔCt in the erlotinib part: -1.4, n = 47) and HER3 mRNA (ΔCt: 1.49, median

336 ΔCt in the erlotinib part: 0.28, n = 68]) and HER3 protein expression (IRS: 2.00) as

337 determined on baseline biopsies. All partial responders in this cohort were EGFR

Lumretuzumab combination treatment

338 inhibitor naïve and neither of the two sqNSCLC patients nor the ovarian cancer

339 patient harbored an EGFR-activating mutation.

340 Overall, there was no detectable relationship between any of the analyzed

341 exploratory biomarkers and clinical response according to RECIST (data not shown).

342 In both the cetuximab and the erlotinib part, phosphorylated HER3 and Akt were

343 expressed at low levels in pre-dose FFPE biopsy samples; expression of both were

344 reduced in on-treatment tumor biopsy samples in the cetuximab and erlotinib arm

345 reflecting the mechanism of action of lumretuzumab (Supplementary Table 2). There

346 was no significant downregulation of total membranous EGFR nor a decrease in

347 proliferative activity based on Ki67 staining which is in line with findings from others

348 [34]. Other exploratory biomarkers were unchanged following treatment with

349 lumretuzumab.

350 The two patients with a SLC3A2-NRG1 fusion gene were heavily pretreated with five

351 therapy lines for metastatic disease including prior erlotinib treatment. Both patients

352 had SD as their best response.

353

354 Heregulin and HER3 mRNA levels and anti-tumor activity in the sqNSCLC

355 extension cohort

356 Altogether, 32 patients with sqNSCLC were recruited into the extension cohort of the

357 erlotinib part. HRG mRNA expression data was available for all patients and RECIST

358 response data was available for 29 patients (90.6%). Overall, the ORR was 6.3%

359 and the DCR was 56.3% (Figure 2).

Lumretuzumab combination treatment

360 Twelve patients were considered HRG-high using the median ΔCt as the cut-off,

361 determined from the analysis of an external cohort of 150 FFPE tumor samples from

362 sqNSCLC patients. Among the 12 HRG-high patients, one had a PR as best

363 response with a duration of 3.8 months. Hence, the ORR in the HRG-high population

364 was 8.3%, while the DCR was 75.0% (9/12 patients) and the median PFS (range)

365 was 3.7 (1.0 to 8.0) months, compared to an ORR of 5.0% (1/20 patients, with a

366 response duration of 4.9 months), a DCR of 55.0% (11/20 patients) and a median

367 PFS (range) of 1.8 (0.2 to 6.5) months for the HRG-low patients (Figure 3). In the

368 same cohort, low HER3 mRNA expression (using the median value of the cohort as

369 a threshold to define high and low expression, corresponding to a ΔCt of -0.3) was

370 associated with an ORR of 12.5% (2/16 patients) and a DCR of 43.8% (7/16

371 patients) and median PFS (range) of 1.8 (0.2 to 6.5) months as compared to an ORR

372 of 0%, a DCR of 68.8% (11/16 patients) and a median PFS (range) of 2.9 (0.3 to 8)

373 months for patients with high HER3 mRNA.

374 HRG expression was also confirmed on FFPE tumor biopsies using in-situ

375 hybridization and IHC assays. Relative HRG expression was consistent across

376 assays and no differences were observed in the clinical response relationship (data

377 not shown). In this cohort, there was no evidence that higher HRG or lower HER3

378 mRNA levels were correlated with improved response to lumretuzumab and erlotinib

379 treatment.

Lumretuzumab combination treatment

380 Discussion

381 This study describes the safety, PK, PD and clinical activity of the combination of

382 lumretuzumab, an anti-HER3 mAb, with cetuximab and erlotinib, respectively. The

383 study aimed to detect signals of clinical activity 1) of anti-EGFR/HER3 combination

384 therapy per se, and 2) activity of this combination in a histological entity enriched for

385 higher HRG expression levels, i.e. sqNSCLC.

386 The most common AEs in this study were gastrointestinal and skin toxicities, similar

387 to the well-known side effects of cetuximab and erlotinib. The addition of

388 lumretuzumab may have caused an increase in the incidence of diarrhea compared

389 with the incidence for cetuximab monotherapy (77.6% [any grade] and 12.2% [grade

390 ≥3] compared to 12.7% [any grade] and 1.2% [grade ≥3] for cetuximab monotherapy

391 [35]) and erlotinib monotherapy (76.1% [any grade] and 15.5% [grade ≥3] compared

392 to 55% [any grade] and 6% [grade ≥3] for erlotinib monotherapy [36]). The severity of

393 diarrhea seemed increased at the highest lumretuzumab doses tested, i.e. at 1600

394 mg and 2000 mg. A similar diarrhea side effect profile as in the present study was

395 seen for combination treatments of other HER3-targeting antibodies with cetuximab

396 [37, 38] and erlotinib [38-40] in early clinical studies. Effects might be caused by a

397 disinhibition of HER-modulated chloride channels in colonic epithelial cells leading to

398 secretory diarrhea [41, 42].

399 While not reaching a defined MTD for both combinations, diarrhea was the only AE

400 for which a potential dose dependency was observed. Based on the PK profile and

401 safety assessments, a dose of 2000 mg in combination with cetuximab and a dose of

402 800 mg in combination with erlotinib were determined as the recommended phase II

403 doses.

Lumretuzumab combination treatment

404 Overall, the efficacy seen in the cetuximab (ORR = 6.1%, DCR = 42.8%) and

405 erlotinib (ORR = 4.2%, DCR = 43.6%) combination parts does not exceed what has

406 previously been reported for cetuximab monotherapy [43, 44] or erlotinib

407 monotherapy [45]. Previous clinical studies on combination treatments of HER3-

408 targeting antibodies in combination with erlotinib in NSCLC have demonstrated

409 comparable response rates [40, 46, 47].

410 Subsequently we attempted to assess the clinical activity of lumretuzumab plus

411 erlotinib in a patient population that was more likely to respond to HER3-targeting

412 therapy. Preclinical studies [25, 26] as well as early clinical studies [22, 27, 28, 48,

413 49] reported an association between higher HRG mRNA expression levels and

414 susceptibility to HER3-targeting therapies. Modest but statistically significant

415 prolonged median PFS were reported for HRG-high expressing patients, i.e. a 0.2-

416 month increase in a study reported by Sequist et al. [40] and a 1.6-month increase in

417 a study reported by von Pawel et al. [46].

418 In the present study, we observed higher HRG mRNA expression levels in

419 squamous carcinomas as compared to adenocarcinomas, which is in line with the

420 described analysis from internal tumor bank samples and TCGA data. We therefore

421 chose to enrich for higher HRG mRNA expression in the lumretuzumab plus erlotinib

422 extension cohort by selectively enrolling sqNSCLC patients. However, lumretuzumab

423 plus erlotinib did not demonstrate higher response rates, longer duration of

424 responses or more pronounced disease stabilization than what would have been

425 expected with erlotinib alone, even when taking HRG and HER3 mRNA expression

426 levels into account. Slightly higher DCR and PFS were observed in the HRG- and

427 HER3-high expressing sqNSCLC patients suggesting some biological evidence for

428 the hypothesis of HRG and HER3 expression levels and HER3-blockade. However,

Lumretuzumab combination treatment

429 the short-lived responses and disease stabilization suggest that these tumors are not

430 dependent on HER3 signaling and that alternative signaling pathways may be

431 involved as escape mechanisms. In accordance to our findings, a phase II/III study in

432 NSCLC patients treated with the HER3-targeting mAb patritumab and erlotinib was

433 stopped recently due to a lack of efficacy (ORR of of 2.2% for patritumab plus

434 erlotinib compared to 6.3% for erlotinib alone and median PFS of 1.9 months for

435 patritumab plus erlotinib compared to 2.7 months for erlotinib alone in patients

436 considered to have high HRG levels [47]). Similarly, no correlation was found

437 between HER3 expression and response or clinical benefit in several phase I and II

438 studies testing HER3-tageting therapies [27, 46, 50, 51]. Recently, a dual-action

439 antibody targeting EGFR and HER3 also failed to improve PFS and overall survival

440 compared to cetuximab therapy in SCCHN patients in a randomized phase II trial,

441 even in the HRG-high expressing subpopulation [52].

442 In conclusion, combination treatment of lumretuzumab with cetuximab or erlotinib

443 was manageable but demonstrated modest clinical activity. Apart from two

444 exceptional responders, a patient with SCCHN achieving a prolonged CR and a

445 patient with ovarian cancer achieving a prolonged PR, both showing higher

446 expression levels of HRG mRNA, the present study was not able to identify a robust

447 biomarker signal that could serve as response prediction biomarker for

448 lumretuzumab in the future. Furthermore, in the presented sqNSCLC cohort, even

449 higher HRG or HER3 mRNA expression levels were not associated with increased

450 ORR or clinically meaningful durations of response, refuting our initial hypothesis.

451 Thus, the response and resistance mechanisms to lumretuzumab-based therapy

452 may be multifaceted or simply undetectable in our cohort of sqNSCLC. Together with

453 the results from randomized trials in SCCHN [52] and NSCLC [47], however, our

Lumretuzumab combination treatment

454 data provide further evidence that adding HER3 to EGFR-targeting therapy is not

455 sufficient to derive clinically meaningful benefit.

Lumretuzumab combination treatment

456 Acknowledgements

457 The authors would like to thank the patients and their families for their participation in

458 this study, and the staff at the study sites.

Lumretuzumab combination treatment

459 References

460 1. Campbell MR, Amin D, Moasser MM: HER3 comes of age: new insights into its 461 functions and role in signaling, tumor biology, and cancer therapy. Clin Cancer 462 Res 2010, 16:1373-1383. 463 2. Sergina NV, Rausch M, Wang DH, Blair J, Hann B, Shokat KM, Moasser MM: 464 Escape from HER-family tyrosine kinase inhibitor therapy by the kinase- 465 inactive HER3. Nature 2007, 445:437-441. 466 3. Jain A, Penuel E, Mink S, Schmidt J, Hodge A, Favero K, Tindell C, Agus DB: HER 467 Kinase Axis Receptor Dimer Partner Switching Occurs in Response to EGFR 468 Tyrosine Kinase Inhibition despite Failure to Block Cellular Proliferation. 469 Cancer Research 2010, 70:1989-1999. 470 4. Wang S, Huang X, Lee CK, Liu B: Elevated expression of erbB3 confers 471 paclitaxel resistance in erbB2-overexpressing breast cancer cells via 472 upregulation of Survivin. Oncogene 2010, 29:4225-4236. 473 5. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, 474 Gale CM, Zhao X, Christensen J, et al: MET amplification leads to gefitinib 475 resistance in lung cancer by activating ERBB3 signaling. Science 2007, 476 316:1039-1043. 477 6. Wheeler DL, Huang S, Kruser TJ, Nechrebecki MM, Armstrong EA, Benavente S, 478 Gondi V, Hsu KT, Harari PM: Mechanisms of acquired resistance to cetuximab: 479 role of HER (ErbB) family members. Oncogene 2008, 27:3944-3956. 480 7. Bieche I, Onody P, Tozlu S, Driouch K, Vidaud M, Lidereau R: Prognostic value of 481 ERBB family mRNA expression in breast carcinomas. Int J Cancer 2003, 482 106:758-765. 483 8. Muller-Tidow C, Diederichs S, Bulk E, Pohle T, Steffen B, Schwable J, Plewka S, 484 Thomas M, Metzger R, Schneider PM, et al: Identification of metastasis- 485 associated receptor tyrosine kinases in non-small cell lung cancer. Cancer 486 research 2005, 65:1778-1782. 487 9. Yi ES, Harclerode D, Gondo M, Stephenson M, Brown RW, Younes M, Cagle PT: 488 High c-erbB-3 protein expression is associated with shorter survival in 489 advanced non-small cell lung carcinomas. Modern Pathology 1997, 10:142-148. 490 10. Tanner B, Hasenclever D, Stern K, Schormann W, Bezler M, Hermes M, Brulport M, 491 Bauer A, Schiffer IB, Gebhard S, et al: ErbB-3 predicts survival in ovarian cancer. 492 Journal of Clinical Oncology 2006, 24:4317-4323. 493 11. Beji A, Horst D, Engel J, Kirchner T, Ullrich A: Toward the prognostic significance 494 and therapeutic potential of HER3 receptor tyrosine kinase in human colon 495 cancer. Clin Cancer Res 2012, 18:956-968. 496 12. Yonesaka K, Hirotani K, Kawakami H, Takeda M, Kaneda H, Sakai K, Okamoto I, 497 Nishio K, Janne PA, Nakagawa K: Anti-HER3 monoclonal antibody patritumab 498 sensitizes refractory non-small cell lung cancer to the epidermal growth factor 499 receptor inhibitor erlotinib. Oncogene 2016, 35:878-886. 500 13. Baselga J, Cortes J, Im SA, Clark E, Ross G, Kiermaier A, Swain SM: Biomarker 501 analyses in CLEOPATRA: a phase III, placebo-controlled study of pertuzumab 502 in human epidermal growth factor receptor 2-positive, first-line metastatic 503 breast cancer. J Clin Oncol 2014, 32:3753-3761. 504 14. Makhija S, Amler LC, Glenn D, Ueland FR, Gold MA, Dizon DS, Paton V, Lin CY, 505 Januario T, Ng K, et al: Clinical Activity of Gemcitabine Plus Pertuzumab in 506 Platinum-Resistant Ovarian Cancer, Fallopian Tube Cancer, or Primary 507 Peritoneal Cancer. Journal of Clinical Oncology 2010, 28:1215-1223.

Lumretuzumab combination treatment

508 15. Kurzeder C, Bover I, Marme F, Rau J, Pautier P, Colombo N, Lorusso D, Ottevanger 509 P, Bjurberg M, Marth C, et al: Double-Blind, Placebo-Controlled, Randomized 510 Phase III Trial Evaluating Pertuzumab Combined With Chemotherapy for Low 511 Tumor Human Epidermal Growth Factor Receptor 3 mRNA-Expressing 512 Platinum-Resistant Ovarian Cancer (PENELOPE). Journal of Clinical Oncology 513 2016, 34:2516-+. 514 16. Sithanandam G, Anderson LM: The ERBB3 receptor in cancer and cancer gene 515 therapy. Cancer Gene Therapy 2008, 15:413-448. 516 17. Wilson TR, Lee DY, Berry L, Shames DS, Settleman J: Neuregulin-1-Mediated 517 Autocrine Signaling Underlies Sensitivity to HER2 Kinase Inhibitors in a Subset 518 of Human Cancers. Cancer Cell 2011, 20:158-172. 519 18. Sheng Q, Liu XG, Fleming E, Yuan K, Piao HY, Chen JY, Moustafa Z, Thomas RK, 520 Greulich H, Schinzel A, et al: An Activated ErbB3/NRG1 Autocrine Loop Supports 521 In Vivo Proliferation in Ovarian Cancer Cells (vol 17, pg 298, 2010). Cancer Cell 522 2010, 17:412-412. 523 19. Zhou BBS, Peyton M, He B, Liu CN, Girard L, Caudler E, Lo Y, Baribaud F, Mikami I, 524 Reguart N, et al: Targeting ADAM-mediated ligand cleavage to inhibit HER3 and 525 EGFR pathways in non-small cell lung cancer. Cancer Cell 2006, 10:39-50. 526 20. Shames DS, Carbon J, Walter K, Jubb AM, Kozlowski C, Januario T, AnDo, Fu L, 527 Xiao YY, Raja R, et al: High Heregulin Expression Is Associated with Activated 528 HER3 and May Define an Actionable Biomarker in Patients with Squamous Cell 529 Carcinomas of the Head and Neck. Plos One 2013, 8. 530 21. Mirschberger C, Schiller CB, Schraml M, Dimoudis N, Friess T, Gerdes CA, Reiff U, 531 Lifke V, Hoelzlwimmer G, Kolm I, et al: RG7116, a therapeutic antibody that binds 532 the inactive HER3 receptor and is optimized for immune effector activation. 533 Cancer research 2013, 73:5183-5194. 534 22. Meulendijks D, Jacob W, Martinez-Garcia M, Taus A, Lolkema MP, Voest EE, 535 Langenberg MHG, Fleitas Kanonnikoff T, Cervantes A, De Jonge MJ, et al: First-in- 536 Human Phase I Study of Lumretuzumab, a Glycoengineered Humanized Anti- 537 HER3 Monoclonal Antibody, in Patients with Metastatic or Advanced HER3- 538 Positive Solid Tumors. Clinical cancer research : an official journal of the American 539 Association for Cancer Research 2016, 22:877-885. 540 23. Huang S, Li CR, Armstrong EA, Peet CR, Saker J, Amler LC, Sliwkowski MX, Harari 541 PM: Dual Targeting of EGFR and HER3 with MEHD7945A Overcomes Acquired 542 Resistance to EGFR Inhibitors and Radiation. Cancer Research 2013, 73:824- 543 833. 544 24. Kawakami H, Okamoto I, Yonesaka K, Okamoto K, Shibata K, Shinkai Y, Sakamoto 545 H, Kitano M, Tamura T, Nishio K, Nakagawa K: The anti-HER3 antibody 546 patritumab abrogates cetuximab resistance mediated by heregulin in colorectal 547 cancer cells. Oncotarget 2014, 5:11847-11856. 548 25. Schoeberl B, Faber AC, Li DN, Liang MC, Crosby K, Onsum M, Burenkova O, Pace 549 E, Walton Z, Nie L, et al: An ErbB3 Antibody, MM-121, Is Active in Cancers with 550 Ligand-Dependent Activation. Cancer Research 2010, 70:2485-2494. 551 26. Meetze K, Vincent S, Tyler S, Mazsa EK, Delpero AR, Bottega S, McIntosh D, 552 Nicoletti R, Winston WM, Weiler S, et al: Neuregulin 1 Expression Is a Predictive 553 Biomarker for Response to AV-203, an ERBB3 Inhibitory Antibody, in Human 554 Tumor Models. Clinical Cancer Research 2015, 21:1106-1114. 555 27. Mendell J, Freeman DJ, Feng W, Hettmann T, Schneider M, Blum S, Ruhe J, Bange 556 J, Nakamaru K, Chen S, et al: Clinical Translation and Validation of a Predictive 557 Biomarker for Patritumab, an Anti-human Epidermal Growth Factor Receptor 3 558 (HER3) Monoclonal Antibody, in Patients With Advanced Non-small Cell Lung 559 Cancer. EBioMedicine 2015, 2:264-271.

Lumretuzumab combination treatment

560 28. Juric D, Dienstmann R, Cervantes A, Hidalgo M, Messersmith W, Blumenschein GR, 561 Tabernero J, Roda D, Calles A, Jimeno A, et al: Safety and 562 Pharmacokinetics/Pharmacodynamics of the First-in-Class Dual Action 563 HER3/EGFR Antibody MEHD7945A in Locally Advanced or Metastatic Epithelial 564 Tumors. Clinical Cancer Research 2015, 21:2462-2470. 565 29. The Cancer Genome Atlas [TCGA]. 566 30. Lee ES, Son DS, Kim SH, Lee J, Jo J, Han J, Kim H, Lee HJ, Choi HY, Jung Y, et al: 567 Prediction of Recurrence-Free Survival in Postoperative Non-Small Cell Lung 568 Cancer Patients by Using an Integrated Model of Clinical Information and Gene 569 Expression. Clinical Cancer Research 2008, 14:7397-7404. 570 31. Shin DH, Lee D, Hong DW, Hong SH, Hwang JA, Lee BI, You HJ, Lee GK, Kim IH, 571 Lee YS, Han JY: Oncogenic function and clinical implications of SLC3A2-NRG1 572 fusion in invasive mucinous adenocarcinoma of the lung. Oncotarget 2016, 573 7:69450-69465. 574 32. Fernandez-Cuesta L, Thomas RK: Molecular Pathways: Targeting NRG1 Fusions 575 in Lung Cancer. Clinical Cancer Research 2015, 21:1989-1994. 576 33. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, 577 Arbuck S, Gwyther S, Mooney M, et al: New response evaluation criteria in solid 578 tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009, 45:228-247. 579 34. Fracasso PM, Burris H, Arquette MA, Govindan R, Gao F, Wright LP, Goodner SA, 580 Greco FA, Jones SF, Willcut N, et al: A phase 1 escalating single-dose and 581 weekly fixed-dose study of cetuximab: Pharmacokinetic and pharmacodynamic 582 rationale for dosing. Clinical Cancer Research 2007, 13:986-993. 583 35. Lenz HJ, Van Cutsem E, Khambata-Ford S, Mayer RJ, Gold P, Stella P, Mirtsching 584 B, Cohn AL, Pippas AW, Azarnia N, et al: Multicenter phase II and translational 585 study of cetuximab in metastatic colorectal carcinoma refractory to irinotecan, 586 oxaliplatin, and fluoropyrimidines. Journal of Clinical Oncology 2006, 24:4914- 587 4921. 588 36. Reck M, Mok T, Wolf J, Heigener D, Wu YL: Reviewing the safety of erlotinib in 589 non-small cell lung cancer. Expert Opin Drug Saf 2011, 10:147-157. 590 37. Cleary JM, McRee AJ, Shapiro GI, Tolaney SM, O'Neil BH, Kearns JD, Mathews S, 591 Nering R, MacBeath G, Czibere A, et al: A phase 1 study combining the HER3 592 antibody seribantumab (MM-121) and cetuximab with and without irinotecan. 593 Invest New Drugs 2017, 35:68-78. 594 38. Papadopoulos KP, Adjei AA, Rasco DW, Liu LM, Kao RJ, Brownstein CM, DiCioccio 595 AT, Lowy I, Trail P, Wang D: Phase 1 study of REGN1400 (anti-ErbB3) combined 596 with erlotinib or cetuximab in patients (pts) with advanced non-small cell lung 597 cancer (NSCLC), colorectal cancer (CRC), or head and neck cancer (SCCHN). 598 Journal of Clinical Oncology 2014, 32. 599 39. Sequist LV, Modiano MR, Rixe O, Jackman DM, Andreas K, Pearlberg J, Moyo VM, 600 Harb WA: Targeting EGFR and ERBB3 in lung cancer patients: Clinical 601 outcomes in a phase I trial of MM-121 in combination with erlotinib. Journal of 602 Clinical Oncology 2012, 30. 603 40. Sequist LV, Lopez-Chavez A, Doebele RC, Gray JE, Harb WA, Modiano MR, 604 Jackman DM, Baggstrom MQ, Atmaca A, Felip E, et al: A randomized phase 2 trial 605 of MM-121, a fully human monoclonal antibody targeting ErbB3, in combination 606 with erlotinib in EGFR wild-type NSCLC patients. Journal of Clinical Oncology 607 2014, 32. 608 41. Keely SJ, Barrett KE: ErbB2 and ErbB3 receptors mediate inhibition of calcium- 609 dependent chloride secretion in colonic epithelial cells. Journal of Biological 610 Chemistry 1999, 274:33449-33454.

Lumretuzumab combination treatment

611 42. Van Sebille YZA, Gibson RJ, Wardill HR, Bowen JM: ErbB small molecule tyrosine 612 kinase inhibitor (TKI) induced diarrhoea: Chloride secretion as a mechanistic 613 hypothesis. Cancer Treatment Reviews 2015, 41:646-652. 614 43. Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, Bets D, 615 Mueser M, Harstrick A, Verslype C, et al: Cetuximab monotherapy and cetuximab 616 plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J 617 Med 2004, 351:337-345. 618 44. Vermorken JB, Trigo J, Hitt R, Koralewski P, Diaz-Rubio E, Rolland F, Knecht R, 619 Amellal N, Schueler A, Baselga J: Open-label, uncontrolled, multicenter phase II 620 study to evaluate the efficacy and toxicity of cetuximab as a single agent in 621 patients with recurrent and/or metastatic squamous cell carcinoma of the head 622 and neck who failed to respond to platinum-based therapy. Journal of Clinical 623 Oncology 2007, 25:2171-2177. 624 45. Shepherd FA, Pereira JR, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, Campos D, 625 Maoleekoonpiroj S, Smylie M, Martins R, et al: Erlotinib in previously treated non- 626 small-cell lung cancer. New England Journal of Medicine 2005, 353:123-132. 627 46. Von Pawel J, Tseng J, Dediu M, Schumann C, Moritz B, Mendell-Harary J, Jin XP, 628 Feng WQ, Copigneaux C, Beckman RA: Phase 2 HERALD study of patritumab (P) 629 with erlotinib (E) in advanced NSCLC subjects (SBJs). Journal of Clinical 630 Oncology 2014, 32. 631 47. Paz-Arez L, Serwatowski P, Szczęsna A, Von Pawel J, Toschi L, Tibor C, Morabito 632 A, Zhang L, Shuster D, Chen S, et al: P3.02b-045 Patritumab plus Erlotinib in 633 EGFR Wild-Type Advanced Non–Small Cell Lung Cancer (NSCLC): Part 634 a Results of HER3-Lung Study. Journal of Thoracic Oncology 2017, 12:S1214- 635 S1215. 636 48. Macbeath G, Adiwijaya B, Liu J, Sequist LV, Pujade-Lauraine E, Higgins M, Tabah- 637 Fisch I, Pearlberg J, Moyo V, Kubasek W, et al: 246PA META-ANALYSIS OF 638 BIOMARKERS IN THREE RANDOMIZED, PHASE 2 STUDIES OF MM-121, A 639 LIGAND-BLOCKING ANTI-ERBB3 ANTIBODY, IN PATIENTS WITH OVARIAN, 640 LUNG, AND BREAST CANCERS. Annals of oncology : official journal of the 641 European Society for Medical Oncology 2014, 25:iv82. 642 49. Sarantopoulos J, Gordon MS, Harvey RD, Sankhala KK, Malik L, Mahalingam D, 643 Owonikoko TK, Lewis CM, Payumo F, Miller J, et al: First-in-human phase 1 dose- 644 escalation study of AV-203, a monoclonal antibody against ERBB3, in patients 645 with metastatic or advanced solid tumors. Journal of Clinical Oncology 2014, 32. 646 50. Hill AG, Findlay M, Burge M, Jackson C, Alfonso PG, Samuel L, Ganju V, Karthaus 647 M, Amatu A, Jeffery M, et al: Randomized phase II study of duligotuzumab plus 648 FOLFIRI versus cetuximab plus FOLFIRI in 2nd-line patients with KRAS wild- 649 type (wt) metastatic colorectal cancer (mCRC). Cancer Research 2015, 75. 650 51. Jimeno A, Machiels JP, Wirth L, Specenier P, Seiwert TY, Mardjuadi F, Wang XD, 651 Kapp AV, Royer-Joo S, Penuel E, et al: Phase Ib Study of Duligotuzumab 652 (MEHD7945A) Plus Cisplatin/5-Fluorouracil or Carboplatin/Paclitaxel for First- 653 Line Treatment of Recurrent/Metastatic Squamous Cell Carcinoma of the Head 654 and Neck. Cancer 2016, 122:3803-3811. 655 52. Fayette J, Wirth L, Oprean C, Udrea A, Jimeno A, Rischin D, Nutting C, Harari PM, 656 Csoszi T, Cernea D, et al: Randomized Phase II Study of Duligotuzumab 657 (MEHD7945A) vs. Cetuximab in Squamous Cell Carcinoma of the Head and 658 Neck (MEHGAN Study). Front Oncol 2016, 6:232. 659 660

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661 Figure 1 Flow diagram of study design, patient enrolment and lumretuzumab dose. 662 CRC = colorectal cancer; N = number of patients; NSCLC = non-small cell lung cancer; q2w 663 = every two weeks; q3w = every three weeks

664 a For all patients a biopsy was taken at screening and on treatment 14 days after Cycle 1. 665 Solely for the extension phase of the erlotinib part, no on-treatment biopsy was taken and for 666 these patients, the prototype diagnostic RT-PCR assay was used.

667 Figure 2 Best percentage change from baseline in sum of longest diameters of target 668 lesions for the sqNSCLC patients in the extension phase of the erlotinib part (n = 28). Red 669 bars indicate PD patients, blue bars SD patients and green bars PR patients. Asterisks 670 indicate patients that were classified as HRG-high. Four patients were without target lesion 671 assessment post baseline (2 deaths, 1 clinical progression, 1 disease progression solely 672 based on non-target lesions).

673 Figure 3 Objective response and HRG mRNA log expression in sqNSCLC patients in 674 the lumretuzumab + erlotinib extension cohort (n = 32)

675

Figure 1 Flow diagram of study design, patient enrolment and lumretuzumab dose. CRC = colorectal cancer; N = number of patients; NSCLC = non-small cell lung cancer; q2w = every two weeks; q3w = every three weeks a For all patients a biopsy was taken at screening and on treatment 14 days after Cycle 1. Solely for the extension phase of the erlotinib part, no on-treatment biopsy was taken and for these patients, the prototype diagnostic RT-PCR assay was used.

Figure 2 Best percentage change from baseline in sum of longest diameters of target lesions for the sqNSCLC patients in the extension phase of the erlotinib part (n = 28). Red bars indicate PD patients, blue bars SD patients and green bars PR patients. Asterisks indicate patients that were classified as HRG-high. Four patients were without target lesion assessment post baseline (2 deaths, 1 clinical progression, 1 disease progression solely based on non-target lesions).

Figure 3 Objective response and HRG mRNA log expression in sqNSCLC patients in the lumretuzumab + erlotinib extension cohort (n = 32)

Median Heregulin mRNA Log Expression (sqNSCLC)

Objective Response (RECIST) Response Objective

Heregulin mRNA Log Expression (ΔCt)

* Note: Patients had an unconfirmed PR. Heregulin mRNA log expression determined using the prototype diagnostic assay. NR = not reported, i.e. patients with non-evaluable post-baseline tumor assessments, however,Downloaded all three patients from clincancerres.aacrjournals.org had clinical progression. on September 29, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 9, 2017; DOI: 10.1158/1078-0432.CCR-17-0812 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Lumretuzumab combination treatment

1 Table 1 Baseline patient demographics and characteristics

Cetuximab part Erlotinib part Characteristic N = 49 N = 71

Sex, n (%)

Male 31 (63.3) 46 (64.8)

Female 18 (36.7) 25 (35.2)

Age (years), median 59 (35, 81) 63 (30, 78) (range)

ECOG score, n (%)

0 12 (24.5) 25 (35.2)

1 36 (73.5) 41 (57.7)

2 1 (2.0) 5 (7.9)

Prior chemotherapy, n (%) 46 (93.9) 69 (97.2)

Median number (range)

Prior EGFR-targeting 27 (55.1) 20 (28.2) therapy, n (%)

Prior surgery, n (%) 30 (61.2) 42 (59.2)

Prior radiotherapy 27 (55.1) 41 (57.7)

Tumor type, n (%)

Colorectal 22 13

Head and neck 11 2

Non-small cell lung 10 43 cancer

Carcinoma of unknown 2 1 primary origin

Pancreatic cancer 0 3

Gastric cancer 0 2

Thymus cancer 0 2

Lumretuzumab combination treatment

Other 4 a 5 b

a Other primary tumors included one patient each with esophageal cancer, breast cancer, urothelial pelvic cancer, and spiradenocarcinoma, respectively. b Other primary tumors included one patient each with cervical cancer, esophageal cancer, ovarian cancer, gastro-esophageal junction cancer and anal cancer, respectively.

Lumretuzumab combination treatment

2 Table 2 Summary of adverse events of any grade and of grade ≥3 regardless

3 of relationship to study drug

Adverse event No. of patients having an adverse event (%) Cetuximab part Erlotinib part N = 49 N = 71 All grades Grade ≥3 All grades Grade ≥3 Diarrhea 38 (77.6) 6 (12.2) 54 (76.1) 11 (15.5) Rash 26 (53.1) 4 (8.2) 32 (45.1) 2 (2.8) Dry skin 23 (46.9) 0 26 (36.6) 0 Decreased appetite 21 (42.9) 2 (4.1) 27 (38.0) 5 (7.0) Hypomagnesaemia 19 (38.8) 5 (10.2) 15 (21.1) 0 Fatigue 12 (24.5) 2 (4.1) 26 (36.6) 2 (2.8) Nausea 16 (32.7) 1 (2.0) 16 (22.5) 0 Paronychia 16 (32.7) 2 (4.1) 3 (4.2) 0 Weight decreased 6 (12.2) 0 21 (29.6) 2 (2.8) Mucosal inflammation 14 (28.6) 0 20 (28.2) 1 (1.4) Vomiting 13 (26.5) 1 (2.0) 12 (16.9) 0 Dermatitis acneiform 12 (24.5) 5 (10.2) 18 (25.4) 4 (5.6) Stomatitis 7 (14.3) 2 (4.1) 16 (22.5) 1 (1.4) Hypokalemia 5 (10.2) 2 (4.1) 16 (22.5) 7 (9.9) Dyspnea 6 (12.2) 2 (4.1) 16 (22.5) 3 (4.2) Pyrexia 9 (18.4) 0 15 (21.1) 0 Asthenia 8 (16.3) 1 (2.0) 15 (21.1) 1 (1.4) Conjunctivitis 9 (18.4) 0 4 (5.6) 0 Infusion-related reaction 9 (18.4) 3 (6.1) a 5 (7.0) 0 Anemia 6 (12.2) 3 (6.1) 11 (15.5) 3 (4.2) Cough 7 (14.3) 0 8 (11.3) 0 Rash pustular 7 (14.3) 1 (2.0) 1 (1.4) 0 Urinary tract infection 3 (6.1) 0 9 (12.7) 0 Infection 6 (12.2) 3 (6.1) 3 (4.2) 1 (1.4) Dry mouth 6 (12.2) 0 3 (4.2) 0 Abdominal pain 5 (10.2) 2 (4.1) 6 (8.5) 0 Skin fissures 5 (10.2) 0 3 (4.2) 0 Epistaxis 5 (10.2) 0 1 (1.4) 0 Myalgia 5 (10.2) 0 0 0 Anxiety 5 (10.2) 0 1 (1.4) 0 Dysphonia 0 0 7 (9.9) 0 Palmar-plantar 1 (2.0) 0 7 (9.9) 0 erythrodysaesthesia syndrome Dizziness 4 (8.2) 0 6 (8.5) 0 Dehydration 4 (8.2) 3 (6.1) 5 (7.0) 2 (2.8) Edema peripheral 4 (8.2) 0 4 (5.6) 1 (1.4) Ascites 4 (8.2) 2 (4.1) 2 (2.8) 1 (1.4) Eye infection 4 (8.2) 0 2 (2.8) 0 Nasopharyngitis 4 (8.2) 0 3 (4.2) 0 Pneumonia 4 (8.2) 1 (2.0) 3 (4.2) 2 (2.8)

Lumretuzumab combination treatment

Dysgeusia 4 (8.2) 0 1 (1.4) 0 Blood bilirubin increased 1 (2.0) 1 (2.0) 5 (7.0) 1 (1.4) Rash maculo-papular 1 (2.0) 0 5 (7.0) 0 Constipation 3 (6.1) 0 3 (4.2) 0 Nail infection 3 (6.1) 0 1 (1.4) 0 Erythema 3 (6.1) 0 1 (1.4) 0 Hypocalcemia 3 (6.1) 2 (4.1) 0 0 Musculoskeletal pain 3 (6.1) 0 3 (4.2) 0 Pulmonary embolism 3 (6.1) 3 (6.1) 1 (1.4) 1 (1.4) Dry eye 3 (6.1) 0 2 (2.8) 0 Headache 3 (6.1) 0 2 (2.8) 0 Pneumothorax 3 (6.1) 0 0 0 Angular cheilitis 3 (6.1) 0 0 0 Influenza-like illness 3 (6.1) 0 3 (4.2) 0 Respiratory tract infection 2 (4.1) 0 4 (5.6) 3 (4.2) Blood creatinine increased 2 (4.1) 0 4 (5.6) 0 Hypercalcemia 0 0 4 (5.6) 2 (2.8) Hyponatremia 0 0 4 (5.6) 4 (5.6) Alanine aminotransferase 0 0 4 (5.6) 1 (1.4) increased Pruritus 1 (2.0) 0 4 (5.6) 0 Alopecia 0 0 4 (5.6) 0 Dysphagia 2 (4.1) 2 (4.1) 4 (5.6) 0 Please note: Only adverse events reported by >5% of the patients in the cetuximab or erlotinib treatment part are shown. a For one patient the IRR was considered related to cetuximab and for 2 patients to lumretuzumab. 4

Lumretuzumab combination treatment

5 Table 3 Best overall response to treatment (RECIST)

Number of patients (%) with Cetuximab part Erlotinib part respective assessment N = 49 N = 71

Complete response 1 (2.0) 0 Partial response 2 (4.1) 3 (4.2) Stable disease 17 (34.7) 28 (39.4) Progressive disease 18 (36.7) 30 (42.3) Not evaluable a 3 (6.1) 0 Missing b 8 (16.3) 10 (14.1) Objective response rate 6.1% 4.2% Disease control rate 40.8% 43.7% a Patients had a post-baseline tumor assessment which was not evaluable. All three patients had clinical disease progression. b Patients had a missing best overall response if no post-baseline tumor assessment was available. Of those, four patients in the cetuximab part and five patients in the erlotinib part had clinical disease progression. 6

Phase Ib study of lumretuzumab plus cetuximab or erlotinib in solid tumor patients and evaluation of HER3 and heregulin as potential biomarkers of clinical activity

Didier Meulendijks, Wolfgang Jacob, Emile E. Voest, et al.

Clin Cancer Res Published OnlineFirst June 9, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-0812

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