Phase 1 study of DS‐1205c combined with gefitinib for EGFR mutation‐positive non‐small cell lung cancer

Abstract Background Tyrosine kinase inhibitors (TKIs) are effective for the treatment of non‐small cell lung cancer (NSCLC) patients with activating mutations of the epidermal growth factor receptor (EGFR), but responses are not durable as tumors develop resistance. DS‐1205c is a novel, specific, orally bioavailable, small‐molecule AXL receptor TKI. In preclinical studies, DS‐1205c restored TKI antitumor activity in a TKI acquired‐resistance EGFR‐mutant NSCLC tumor xenograft model. Methods This first‐in‐human, multicenter, open‐label Phase 1 study (registered at ClinicalTrials.gov: NCT03599518) primarily evaluated the safety and tolerability of combination therapy with DS‐1205c and gefitinib in Japanese patients with metastatic or unresectable EGFR‐mutant NSCLC and tumor progression during treatment with EGFR‐TKIs. Patients (n = 20) received DS‐1205c monotherapy (200–1200 mg twice daily [BID]) in a 7‐day safety monitoring period before combination DS‐1205c/gefitinib (250 mg once daily) in 21‐day cycles. Results The observed common treatment‐emergent adverse events (TEAEs) were increased aspartate aminotransferase (35%), increased alanine aminotransferase (30%), rash maculo‐papular (30%), and diarrhea (25%). No serious TEAEs were reported. Plasma concentrations and pharmacokinetic parameters of DS‐1205a (free form of DS‐1205c) were unaffected by concomitant administration of gefitinib. No patient achieved a complete or partial response and 5 patients (25%) had stable disease. Conclusion DS‐1205c was generally safe and well tolerated at all dose levels, but the safety profile of ≤800 mg BID was more favorable than 1200 mg BID. The recommended dose for dose‐expansion cohorts of DS‐1205c in combination therapy with gefitinib was 800 mg BID.


| INTRODUCTION
Established small-molecule tyrosine kinase inhibitors (TKIs) target epidermal growth factor receptor (EGFR) and are effective in non-small cell lung cancer (NSCLC) patients with activating and sensitizing EGFR mutations. These mutations are localized in exons 18-21 of the EGFR gene and have a prevalence of around 10%-20% in Caucasians with NSCLC adenocarcinomas, and approximately 40%-50% in Asians. [1][2][3][4] However, responses to TKIs are not durable as tumors develop resistance. The T790M mutation in exon 20 is the most frequent cause (in 50%-60% of cases) of resistance to the first-(gefitinib, erlotinib) and second-generation (afatinib) EGFR TKIs. 1,2,5 Thirdgeneration TKIs such as osimertinib are effective against activating EGFR mutations and in NSCLC with the EGFR T790M mutation. However, tumors may acquire resistance to third-generation TKIs.
AXL is a member of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases and, along with its ligand growth arrest-specific gene 6 (GAS6), plays an important role in promoting growth, survival, and proliferation in both normal and malignant cells. 6 AXL overexpression in NSCLC and several other solid and hematologic cancers drives a wide range of pathophysiological processes, including epithelial to mesenchymal transition, tumor angiogenesis, decreased antitumor immune response, stem cell maintenance, and resistance to targeted and chemotherapeutic agents. 6-10 DS-1205c is a novel, specific, orally bioavailable, small-molecule inhibitor of AXL which has been developed by Daiichi Sankyo. In pharmacological studies, DS-1205c was a potent AXL inhibitor with half-maximal inhibitory concentration (IC 50 ) values in the low nanomolar range (1.3-3.7 nM). In in vitro studies, 0.1 μM and 1 μM DS-1205c in combination with osimertinib (2 μM), slowed the growth of NCI-H1975 cells, which is a human NSCLC cell line harboring T790M and L858R EGFR mutations, 11 with relative growth rates of 43.1% and 2.2%, respectively. In an EGFR-mutant NSCLC tumor xenograft mouse model, DS-1205c in combination with gefitinib, erlotinib or osimertinib significantly delayed the onset of tumor resistance to TKIs compared with TKI monotherapy (Daiichi Sankyo, data on file). 12 Also, DS-1205c restored antitumor activity of erlotinib in an erlotinib acquired-resistance EGFRmutant NSCLC tumor xenograft mouse model. 12 As the results from these preclinical models demonstrated antitumor activity of DS-1205c in combination with a TKI in EGFR-mutant NSCLC (T790M-negative) tumors, we conducted a first-in-human Phase 1 study of DS-1205c in combination with gefitinib in subjects with metastatic or unresectable EGFR-mutant NSCLC.

| MATERIALS AND METHODS
This was a multicenter, open-label Phase 1 study of DS-1205c in combination with gefitinib in subjects with metastatic or unresectable EGFR-mutant NSCLC and disease progression during treatment with one or more EGFR-TKIs (gefitinib, erlotinib, afatinib, dacomitinib, or osimertinib). The study was registered at Clini calTr ials.gov (NCT03599518).

| Objectives
The primary objective was assessment of the safety and tolerability and determination of the recommended dose of DS-1205c in combination with gefitinib for the expansion cohort in metastatic or unresectable EGFR-mutant NSCLC after disease progression during treatment with EGFR-TKIs.
Secondary objectives were characterization of the pharmacokinetics (PK) of DS-1205a (free form of DS-1205c) following DS-1205c monotherapy, and DS-1205a plus gefitinib combination treatment; and investigation of the antitumor activity of DS-1205c in combination with gefitinib.
Exploratory objectives were evaluation of the effect on corrected QT interval (QTc) of DS-1205c monotherapy and in combination with gefitinib, and identification of biomarkers that correlate with efficacy or toxicity of the combination of DS-1205c plus gefitinib.

| Study participants
All subjects provided written informed consent. Main inclusion criteria were male or female subjects aged ≥20 years with histologically/cytologically confirmed locally advanced or metastatic adenocarcinoma NSCLC which was not amenable to curative surgery or radiation, with Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1, and meeting a clinical definition of acquired resistance to EGFR TKI. 13 Criteria for inhibitor, DS-1205, gefitinib, non-small cell lung cancer, phase I clinical trial  acquired EGFR TKI resistance included a tumor harboring an EGFR mutation (including G719X, exon 19 deletion, L858R, L861Q) known to be associated with EGFR  TKI sensitivity, or experienced clinical benefit from continuous EGFR TKI treatment followed by systemic disease progression defined 15 ; and received gefitinib, erlotinib, afatinib, dacomitinib, or osimertinib for at least 6 weeks with well-controlled related toxicities (less than Grade 3 in severity) at the time of screening. Although the final version of the protocol of the current study (A Multicenter, Open-Label Phase 1 Study of DS-1205c in Combination with Gefitinib in Subjects with Metastatic or Unresectable EGFR-Mutant NSCLC, published on 29 July 2019) required the absence of an EGFR T790M mutation, subjects enrolled in the study before the protocol revision who received osimertinib were enrolled regardless of their EGFR T790M mutation status.
Key exclusion criteria included evidence of small cell histology, or combined small cell and non-small cell histology in an original or screening tumor biopsy performed since progression; previously documented evidence of ALK fusion, ROS1 fusion, BRAF V600E mutation, rearranged during transfection (RET) fusion, human epidermal growth factor receptor 2 (HER2) mutation, or hepatocyte growth factor receptor (MET) exon 14 skipping mutation; treatment with any cytotoxic chemotherapy, investigational agent or other anticancer drug(s) from a previous cancer treatment regimen or clinical study (other than EGFR TKI) within 14 days of the first dose of the current study treatment, or immune checkpoint inhibitor therapy within 30 days of first dose of the current study treatment; major surgery (excluding a vascular access procedure) within 4 weeks of the first dose of the current study treatment; radiotherapy treatment to more than 30% of the bone marrow or with a wide field of radiation within 4 weeks, or palliative radiation therapy within 2 weeks of the first dose of the current study treatment.

| Procedure
Dose escalation involved a run-in period with DS-1205c monotherapy for 7 days (Cycle 0), followed by combination treatment with DS-1205c plus gefitinib (Cycle 1 and beyond). DS-1205c was administered orally at doses of 200, 400, 800, 1000, and 1200 mg twice daily (BID), and in combination with open-label oral gefitinib 250 mg once daily in 21-day cycles.
Safety review meetings (SRMs), consisting of the investigators and the sponsor's medical monitors, reviewed the safety data, including dose-limiting toxicity (DLT) data, to recommend the dose for the next dose-escalation cohort and the recommended dose for the dose-expansion cohort. A Bayesian logistic regression model (BLRM) for inferring the probabilities of DLT at each dose level 16 was applied to guide the recommended dose for the next dose cohort. 17 The final decision for the dose of the next cohort was made based on the evaluation of all safety and PK data.

| Safety
Safety endpoints included treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), DLT, standard clinical laboratory parameters, vital sign measurements, electrocardiogram parameters, physical examination findings (including ECOG PS), left ventricular ejection fraction (ECHO/MUGA findings), and ophthalmologic and pulmonary findings. Adverse events (AEs) were coded using Medical Dictionary for Regulatory Activities (MedDRA) and MedDRA/J version 21.1.

| Pharmacokinetics
PK parameters were calculated for DS-1205a (free form of DS-1205c) and gefitinib in plasma using noncompartmental analysis. For DS-1205a, maximum plasma concentration (C max ), time to reach maximum plasma concentration (T max ), and area under the plasma concentration-time curve (AUC) during the first 10 h (AUC 10h ) were measured on Days 1 and 7 during Cycle 0, and Day 1 in Cycles 1 and 2 except that AUC 8h was estimated on Day 1 in Cycle 1. In addition, trough plasma concentration (C trough ) was measured on Day 7 in Cycle 0, and Day 1 in Cycles 1 and 2. For gefitinib, C max , T max , and AUC 8h were measured on Day 1 in Cycles 1 and 2, and additionally, AUC 0-24h on Day 1 in Cycle 2.

| Efficacy endpoints
The primary efficacy endpoint was the overall response rate (ORR), that is, the number of subjects with best objective response (complete response [CR] or partial response [PR]), divided by the number of subjects who participated in the analysis, which was determined by investigator assessment and based on RECIST version 1.1 criteria. Additional efficacy endpoints included percentage change in size of target lesion(s), duration of response (the time from documented tumor response [CR or PR] to disease progression), disease control rate (DCR: the sum of CR, PR, and stable disease [SD] rates), progression-free survival (PFS), time to response (TTR), and overall survival (OS).

| Biomarkers
Blood samples were collected to measure biomarkers including osteopontin, interleukin-8 (IL-8) and soluble AXL. Tumor tissue samples were used to assess the presence of EGFR mutations and to analyze other exploratory biomarkers including AXL expression by immunohistochemistry (IHC). IHC staining was performed using a rabbit monoclonal antibody (C89E7) against AXL purchased from Cell Signaling Technology.

| Statistical analysis
TEAEs were tabulated by system organ class (SOC) and preferred term (PT). For the other safety and PK endpoints, continuous data were described by mean and standard deviation (SD), median and range, and categorical data were summarized by frequency. The proportionality of the PK parameters, C max and AUC 10h , was evaluated using a power model (see Table 4). For the efficacy endpoints, best overall response was tabulated as a frequency table, and the time-to-event data such as duration of response, PFS, and OS was evaluated using Kaplan-Meier method. All analyses were conducted using SAS® Version 9.4 (SAS Institute).

| Ethical approval
This study was conducted in compliance with ethical principles with their origin in the Declaration of Helsinki, the International Council for Harmonization (ICH) consolidated Guideline E6 for Good Clinical Practice (GCP) (CPMP/ICH/135/95), and applicable regulatory requirements including the Japanese Ministry of Health, Labor and Welfare Ordinance No. 28 of 27 March, 1997 and/ or The Act on Securing Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, Regenerative and Cellular Therapy Products, Gene Therapy Products, and Cosmetics No. 1 of 25 November, 2014; and other applicable local regulations.

| Safety
Overall, 19 (95%) patients experienced one or more TEAE. No serious TEAEs nor TEAEs associated with death were reported.
One subject in Cohort 5 had a TEAE (Grade 2 rash) associated with drug withdrawal. No subjects had TEAEs associated with dose reduction. Seven patients (35%) had TEAEs associated with dose interruption. Of the 19 evaluable patients, DLT was experienced in two patients: one patient in Cohort 3 (800 mg BID) with Grade 3 neutropenia and one in Cohort 5 (1200 mg BID) with Grade 3 nausea, respectively.
TEAEs were Grade 1 (n = 6, 30%), Grade 2 (n = 7, 35%) or Grade 3 (n = 6, 30%). No Grade 4 or 5 TEAEs were observed. Grade 3 TEAEs were observed in 1 patient in Cohort 1 (200 mg BID), which was nausea; in 2 patients in Cohort 3 (800 mg BID), which were increased ALT in 1 patient and decreased white blood cell count and decreased neutrophil count in 1 patient; and in 3 patients in Cohort 5 (1200 mg BID), which were abnormal hepatic function in 1 patient, increased ALT in 1 patient, and nausea in 1 patient. Grade 3 or more TEAEs are summarized in Table 3.

| Pharmacokinetics
The lower limit of quantification for DS-1205a (free form of DS-1205c) was 5 ng/ml. Time courses of mean plasma DS-1205a concentrations for each DS-1205c dose during DS-1205c monotherapy (Cycle 0, Days 1 and 7) and DS-1205c plus gefitinib combination therapy (Cycles 1 and 2, Day 1) are shown in Figure 1. For the first DS-1205c dose (Cycle 0, Day 1), mean DS-1205a C max and AUC 10h levels generally increased along with the increasing DS-1205c dose, reaching a plateau at high dose levels (800-1200 mg BID). By comparing the mean C trough values in each cohort on Cycle 0 Day 7, Cycle 1 Day 1, and Cycle 2 Day 1, it was indicated that plasma DS-1205a concentrations reached steady state within 7 days of twice-daily administration of DS-1205c. C trough values at the abovementioned time points in Cohort 1 were 335, 315, and 408 ng/ml, respectively; 497, 551, and 503 ng/ml, respectively, in Cohort 2; 691, 768, and 643 ng/ml, respectively, in Cohort 3; 437, 486, and 496 ng/ml, respectively, in Cohort 4; and 1040, 1130, and 1090 ng/ml, respectively, in Cohort 5. The PK parameters of DS-1205a on Day 1 in Tumor stage at study entry, n (%)  Cycle 2 were generally similar to those on Day 7 in Cycle 0, suggesting that the PK of DS-1205a was not affected by gefitinib (Table 4). The upper limits of 90% CIs for the slope of the power model were less than 1 for all PK parameters of C max and AUC 10h ( Table 5), suggesting that increases in exposure to DS-1205a were less dose proportional.
Although the mean C max and AUC of gefitinib varied among the cohorts, no specific pattern was observed on Day 1 in Cycle 1 or Day 1 in Cycle 2 (data not shown).

| Efficacy
All 20 patients discontinued the treatments, primarily due to the pathologic progression of the disease defined by RECIST criteria (n = 17; 85%) or clinical progression (Cohort 5; n = 1, 5%). Two patients, one in Cohort 3 and one in Cohort 5, discontinued the treatments due to the withdrawal of consent for the study and AE (see Safety section), respectively.

| Biomarkers and IHC
Following an initial 1 week of DS-1205c monotherapy, plasma levels of soluble AXL increased from the baseline level by approximately 1.5-fold. Although most patients showed increases in soluble AXL plasma levels, increases were not dose dependent and no additional increases were observed during combination therapy with gefitinib. Due to small patient numbers, a correlation between an increase in soluble AXL plasma levels and efficacy was not clear. was observed in fibroblasts (n = 10) and immune cells (n = 13).

| Recommended dose for expansion
Considering all available safety, PK, and exploratory tumor response data, the recommended expansion dose of DS-1205c in combination with gefitinib was determined to be 800 mg BID.

| DISCUSSION
The primary objective of this first-in-human study was to assess the safety and tolerability of DS-1205c in combination with gefitinib in metastatic or unresectable EGFR-mutant NSCLC subjects and to determine the recommended dose of DS-1205c in combination therapy for expansion study. Of the EGFR TKIs, gefitinib was selected because it is known to be generally well tolerated and safety data in Japanese patients have been collected sufficiently. 18 Of the 20 patients in the Safety Analysis Set, most of them (85%) had at least one TEAE. Increases in transaminases (AST and ALT) were the most commonly reported TEAEs, found in about one-third of the patients. The frequency of these events tended to increase with increasing doses of DS-1205c, but no hepatic events (a TEAE of special interest) were observed. No serious TEAEs or TEAEs associated with death were observed in the study.
DS-1205c in combination with gefitinib was well tolerated, although doses of ≤800 mg BID had a more favorable safety profile compared with the dose of 1200 mg BID.  Two patients demonstrated DLT: Grade 3 neutropenia in one patient in Cohort 3 (800 mg BID) and Grade 3 nausea in another in Cohort 5 (1200 mg BID). In addition, one patient in Cohort 5 experienced a TEAE associated with dose interruption, and a second patient in Cohort 5 experienced a TEAE (Grade 2 rash) associated with study discontinuation. Skin reactions, commonly a pustular rash, are very common adverse reactions to gefitinib, but are mainly mild/moderate in severity. Other common adverse reactions include mild/moderate anorexia, diarrhea, vomiting, elevated ALT, nausea, stomatitis and asthenia, which are generally mild in severity. Interstitial lung disease (ILD) is the most serious, relatively common (prevalence of approximately 1%) adverse reaction for gefitinib. [19][20][21] In this study, no ILD was observed. Similarly, no QTcF prolongation (≥Grade 2), another TEAE of special interest, was observed.
In PK analysis, plasma C max and AUC of DS-1205a (free form of DS-1205c) generally increased with increasing doses and tended to reach a plateau at high dose levels (800-1200 mg BID). However, a dose-exposure relationship analysis using power models for C max and AUC suggested that increases in exposure to DS-1205a were not fully dose proportional. Plasma concentrations and PK parameters of DS-1205a were not affected by the concomitant administration of gefitinib. The overall median treatment duration in the current study was 7 weeks.
No specific pattern was observed in the PK analysis of gefitinib on Day 1 in Cycle 1 or Day 1 in Cycle 2. The PK of gefitinib has been studied in patients with solid tumors 22,23 and in advanced NSCLC. 24 In advanced NSCLC, a high day 3/day 8 ratio of plasma trough levels of gefitinib was independently associated with improved PFS. 24 Five out of 20 patients had stable disease and no patient achieved a complete or partial response. The outcome of stable disease is consistent with preclinical studies showing that DS-1205c restored the antitumor activity of erlotinib in an erlotinib acquired-resistance EGFR-mutant NSCLC tumor xenograft mouse model. 12 AXL expression level in tumors was also explored. AXL positivity was shown in 1 out of 17 evaluable tumors before DS-1205c administration. This result was not consistent with previous reports which independently showed about 20% of AXL positivity, 7,25 probably due to differences in the antibody used for IHC. On the other hand, almost all patients showed an increase of soluble AXL by around 1.5-fold in plasma during 7 days of DS-1205c monotherapy. The effect was not dose-dependent, indicating that soluble AXL achieves a plateau at low DS-1205c dosages, with soluble AXL possibly being released from normal tissues. Due to insufficient patient numbers, this study was unable to determine a clear correlation between these exploratory outcomes and clinical efficacy. In particular, it would be intriguing to further evaluate the treatment outcome in patients with AXL-positive tumors as only 1 patient showed tumor AXL positivity in this study. IL-8 and osteopontin were also evaluated as exploratory pharmacodynamic biomarkers, but neither IL-8 nor osteopontin showed significant changes from baseline levels following DS-1205c treatment.
The study has some limitations which reflect the Phase I study design. The sample size was relatively small (20 patients) and, although the study was conducted at 8 sites in Japan, the findings may not be generalizable to the wider population of patients with NSCLC.
gefitinib was chosen at 800 mg BID. Further studies are needed to confirm the safety profile and evaluate the efficacy of this dose, and potentially to identify predictive biomarkers for responsive patients.