Phase 1 study of pembrolizumab (MK-3475; anti-PD-1 monoclonal antibody) in Japanese patients with advanced solid tumors

Summary Background This phase I study evaluated the safety and tolerability, pharmacokinetics and pharmacodynamics, immunogenicity, and antitumor activity of pembrolizumab in Japanese patients with advanced solid tumors. Methods Following an initial dose and a 28-day rest (cycle 1), pembrolizumab was administered as an intravenous infusion at escalating doses (2 or 10 mg/kg) every 2 weeks (Q2W) until disease progression or unacceptable toxicity. Adverse events (AEs) were assessed using CTCAE v4.0, and tumor response was assessed using both RECIST v1.1 and immune-related response criteria (irRC). Full pharmacokinetic sampling was performed during cycle 1. Results Three patients received pembrolizumab at 2.0 mg/kg and seven at 10 mg/kg. No dose-limiting toxicities were observed during cycle 1. Eighty percent of patients experienced drug-related AEs (mostly grade 1 or 2); the most common drug-related AEs were nausea, malaise, pyrexia, and aspartate aminotransferase/alanine transaminase (AST/ALT) elevations (n = 2 each). No drug-related grade 4 or 5 AEs occurred. Immune-related AEs comprised grade 3 ALT elevation (n = 1), grade 3 AST elevation (n = 1), grade 1 pneumonitis (n = 1), and grade 1 thyroid-stimulating hormone elevation (n = 1). The safety and pharmacokinetic profiles of Japanese patients were similar to those previously reported for Caucasian patients. A partial tumor response was observed in one patient with non-small-cell lung cancer (NSCLC) and in one patient with melanoma. Conclusions Pembrolizumab at both 2 and 10 mg/kg Q2W was well tolerated in Japanese patients with advanced solid tumors and showed encouraging anti-tumor activity against melanoma and NSCLC.


Introduction
The PD-1 pathway represents a major immune control switch that may be engaged by tumor cells to overcome active T-cell immune surveillance [1]. The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in various tumors, including melanoma [2][3][4][5]. The high expression of PD-L1 (and to a lesser extent, PD-L2) on tumor cells is correlated with poor prognosis and poor survival in various cancer types, including renal cell carcinoma, pancreatic carcinoma, hepatocellular carcinoma, ovarian carcinoma, and nonsmall-cell lung cancer (NSCLC) [6][7][8][9][10]. Furthermore, it has been suggested that PD-1 regulates tumor-specific T-cell expansion in patients with melanoma [11]. Preclinical in vitro and in vivo experiments have shown that PD-1 and/or PD-L1 blockade using monoclonal antibodies enhances tumor cellspecific T-cell activation, cytokine production, anti-tumor effector mechanisms, and the clearance of tumor cells by the immune system [12][13][14][15][16]. PD-1 and PD-L1 inhibitors have validated PD-1 as an attractive target for clinical therapeutic intervention. PD-1 inhibition was tested in a clinical study of Phase 1 study of pembrolizumab (MK-3475; anti-PD-1 monoclonal antibody) in Japanese patients with advanced solid tumors patients with a range of solid tumor types, and promising clinical activity was noted in multiple tumor types, including melanoma and NSCLC [17].
Pembrolizumab (formerly known as MK-3475) is a potent, highly selective, IgG4-k humanized monoclonal antibody that prevents PD-1 from binding with PD-L1 and PD-L2. This agent was generated by grafting the variable region sequences of a mouse antihuman PD-1 antibody onto a human IgG4-k isotype framework containing a stabilizing S228P mutation of the Fc region. Pembrolizumab exhibited high affinity for the PD-1 receptor, strong inhibition of PD-L1 and PD-L2, and robust activity in a functional ex vivo T-cell modulation assay using human donor blood cells (data on file; Merck & Co., Inc.).
A first-in-human phase 1 study was conducted to evaluate the safety, pharmacokinetics, and pharmacodynamics of pembrolizumab in non-Japanese patients with advanced solid tumors. No dose-limiting toxicities (DLTs) were observed, and the maximum administered dose (MAD) was 10 mg/kg every 2 weeks (Q2W). Pharmacokinetic and pharmacodynamic analyses showed that the lowest dose with the full potential for antitumor activity was 2 mg/kg every 3 weeks (Q3W) [18].
In the present study, the safety and tolerability, pharmacokinetics (PK), and immunogenicity of pembrolizumab were investigated in Japanese patients with advanced solid tumors. The tumor response to pembrolizumab was also evaluated as an exploratory objective.

Patient eligibility
This study was conducted based on the Declaration of Helsinki and the Guidelines for the Clinical Evaluation Methods of Anti-Cancer Drugs in Japan (Japanese Ministry of Health, Labour, and Welfare notification, November 1, 2005). The study was approved by the institutional review board of each study site.
The main eligibility criteria were as follows: a histologically or cytologically confirmed diagnosis of locally advanced or metastatic solid tumors in a patient who had experienced disease progression while on standard therapy or in a patient intolerant of, or not eligible for standard therapy; a patient age of 20 years or older; an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; and adequate hematologic, hepatic, and renal functions. The exclusion criteria included the administration of chemotherapy, radiotherapy, or biological therapy in the 4 weeks (2 weeks for palliative radiotherapy and kinase inhibitors) prior to enrollment; previous treatment with a PD-1, PD-L1, or cytotoxic Tlymphocyte-associated protein 4 inhibitor; untreated and/or unstable central nervous system metastasis; or the presence of autoimmune disease.
All patients provided informed consent, and the study was conducted in accordance with current Good Clinical Practice standards. This study was registered at ClinicalTrials.gov as NCT01840579.

Study design and evaluation
This study was an open-label, non-randomized, phase 1 study of pembrolizumab in Japanese patients with advanced solid tumors that was conducted at two sites in Japan. This study was designed to investigate the safety and tolerability, PK, immunogenicity, and anti-tumor activity of pembrolizumab monotherapy. Pembrolizumab was administered as a 30-min intravenous (i.v.) infusion at a dose of 2 or 10 mg/kg. Dose escalation was conducted using the conventional B3 + 3 design,^with cohorts of three patients sequentially enrolled at pembrolizumab doses of 2 and 10 mg/kg administered on days 1 and 28 and Q2W thereafter until disease progression or intolerable toxicity occurred. The initial 28 days after the first administration (cycle 1) were regarded as the DLT evaluation period. Three or six patients were enrolled at each dose based on the toxicity probability intervals [19]. In the DLT assessments, if none of the three patients or none/one of the six patients had a DLT at a certain dose, that dose was considered to be tolerable.
Adverse events (AEs) were graded using the National Cancer Institute Common Terminology Criteria for AEs, version 4.0. A DLT was defined as any of the following events occurring during cycle 1 (the initial 28 days): grade 4 neutropenia lasting for ≥7 days; grade 3 or 4 neutropenia with a fever >38.5°C and/or infection requiring antibiotic or anti-fungal treatment; grade 4 thrombocytopenia; grade 4 nonhematologic toxicity; or a grade 3 non-hematologic toxicity (laboratory value) persisting for >1 week or requiring medical intervention.
Anti-tumor activity was evaluated at baseline and every 6 weeks according to the Response Evaluation Criteria In Solid Tumors, version 1.1 (RECIST v1.1) and the immunerelated response criteria (irRC) [20].

Pharmacokinetics and immunogenicity (presence of anti-drug antibody)
Blood samples for the PK analyses were collected predose, postdose (<30 min after infusion), and 6, 24, and 48 h after the start of the first infusion; on days 8, 15, 22, and 29 of cycle 1; predose and postdose in cycle 2 and every other cycle thereafter for the first 12 months; and 30 days after the last pembrolizumab dose. The pembrolizumab serum concentrations were quantified using a validated electrochemiluminescent assay (lower limit of quantification, 10 ng/mL). The PK data are described using a noncompartmental approach. Blood samples for anti-drug antibody (ADA) analyses were collected predose and 24 h after the start of the first infusion, predose in cycle 2 and every other cycle thereafter for the first 12 months, and 30 days after the last pembrolizumab dose.
The presence of ADA was determined using bridging electrochemiluminescence and was evaluated using the standard three-step method consisting of a screening test, a confirmation test, and an antibody titer test. ADA was determined to be positive if the result for at least one predose or postdose sample was positive on the confirmation test.

PD-L1 expression
PD-L1 expression was measured using immunohistochemistry performed on formalin-fixed, paraffin-embedded tissue sections at QualTek Clinical Laboratories. PD-L1 positivity was defined as staining in ≥1 % of the tumor cells (modified proportion score) or stroma using immunohistochemistry with the PD-L1 22C3 antibody.

Safety and tolerability
The DLTs occurring during cycle 1 were evaluated. One patient treated with pembrolizumab 10 mg/kg was excluded from the DLT evaluation because the patient discontinued the study because of disease progression during the DLT evaluation period. Therefore, that patient was replaced with a new patient for the DLT evaluation. No DLTs were observed in the three patients treated with 2 mg/kg or in the six patients treated with 10 mg/kg. The drug-related AEs reported for all treatment cycles at both dose levels are summarized in Table 2.
Eighty percent of patients experienced drug-related AEs (mostly grade 1 or 2). The most common AEs related to pembrolizumab treatment were nausea, malaise, pyrexia, elevated alanine transaminase (ALT) levels, and elevated aspartate aminotransferase (AST) levels (2/10 [20 %] for each AE). No drug-related grade 4 or grade 5 AEs occurred in this study. None of the patients discontinued pembrolizumab treatment because of a drug-related AE. One patient (a 53-year-old man) with advanced NSCLC developed grade 3 ALT elevation, grade 3 AST elevation, and grade 1 pneumonitis (onset, day 42 for each); after the discontinuation of pembrolizumab treatment, he developed grade 3 hyponatremia (onset, day 56). The grade 3 ALT/AST elevations resolved after treatment with glycyrrhetinic acid, and the grade 3 hyponatremia resolved with sodium chloride supplement. Overall, one case each of grade 3 ALT elevation, grade 3 AST elevation, grade 1 pneumonitis, and grade 1 thyroid-stimulating hormone (TSH) elevation were reported as immune-related AEs by the investigators.

Pharmacokinetic and immunogenicity evaluation
The mean serum concentration profiles for pembrolizumab are shown in Fig. 1, and descriptive statistics for the PK parameters are given in Table 3 and Fig. 2. The serum concentration of pembrolizumab following the first i.v. administration    in Japanese patients. The PK profiles of pembrolizumab, which has a low clearance, a limited volume of distribution, and a long t 1/2 , are typical of therapeutic antibodies. The trough concentration increased during repeated dosing at 2 or 10 mg/ kg Q2W. Based on the t 1/2 (18 days) and the dose interval (2 weeks), approximately 3.5 months (cycle 8) were considered necessary to achieve a steady state. However, only two patients treated with 2 mg/kg and one patient treated with 10 mg/kg were still receiving pembrolizumab at cycle 8.
Considering the limited PK data available for doses of 2 and 10 mg/kg beyond cycle 8, the steady state trough concentration could not be determined based on the observed PK data. ADA was not detected at any time in screening assays from predose through to cycle 18 (day 140), except in a predose assay performed during cycle 1 in one patient;  C max maximum concentration, CL clearance, Vz the terminal phase volume however, the results of the confirmatory assay were negative for this patient.

Antitumor activity and PD-L1 expression
As an exploratory objective, the tumor response to pembrolizumab was evaluated according to the RECIST v1.1 and irRC criteria. One patient with extramammary Paget's disease was excluded from the tumor response assessment because the patient did not have any measurable lesions at the study baseline. Among the nine patients who were evaluated, partial responses (as determined by an investigator review according to RECIST v1.1) were observed in two patients (22.2 %) treated with pembrolizumab 10 mg/kg Q2W; one patient (a 91-year-old man) had metastatic melanoma (time to response: 46 days), and the other (a 53-year-old man) had NSCLC (time to response: 41 days). The two patients also had partial responses as determined by an investigator review according to irRC. The patient with advanced NSCLC developed immune-related AEs (grade 3 ALT/AST elevations and grade 1 pneumonitis) at the same time as the observation of the partial response. At the time of data cutoff, the patient with advanced metastatic melanoma who had achieved a partial response was continuing to receive pembrolizumab treatment because, despite disease progression according to RECIST v1.1, disease progression was not evident according to irRC (response duration: more than 225 days) (Fig. 3). Tumor tissue from five patients was available to undergo a PD-L1 expression immunohistochemistry assay. PD-L1 expression was positive in tumor samples from two patients and negative in tumor samples from three patients. No responses were observed in the two patients with PD-L1-positive tumors by an investigator review whereas a partial response was observed in one of the three patients with PD-L1-negative tumors.

Discussion
The primary objective of the present study was to investigate the safety and tolerability of single-agent pembrolizumab administered in Japanese patients with advanced solid tumors. The dosing schedules in the present study were selected based on the results of a phase 1 study of pembrolizumab in non-Japanese patients with advanced solid tumors. The previous study showed that the MAD was 10 mg/kg Q2W, and the lowest dose with the full potential for antitumor activity was 2 mg/kg Q3W [18]. No DLTs were observed at either dose in the present study. The most common drug-related AEs were nausea, malaise, pyrexia, and AST/ALT elevations (n = 2 each). One case each of grade 3 ALT elevation, grade 3 AST elevation, grade 1 pneumonitis, and grade 1 TSH elevation were reported as immune-related AEs. The AEs observed in the present study have also been reported for clinical studies of pembrolizumab in non-Japanese patients. Although the number of Japanese patients was limited, the safety profile of pembrolizumab in Japanese patients with advanced solid tumors in the present study was generally consistent with that observed previously in non-Japanese patients. Drug-induced autoimmune-like toxicities have been observed in patients treated with immune checkpoint inhibitors (such as those used in anti-PD-1 therapy) through the infiltration of immune cells into normal noncancerous tissues. Immune-related AEs can affect multiple organs such as the skin, bowel, kidney, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and endocrine tissues. Steroid therapy is recommended for the management of immune-related AEs [21,22]. In the present study, most AEs were of grade 1 or 2 and were manageable by interrupting pembrolizumab treatment and/or medical intervention without steroid therapy.
Pembrolizumab exposure at a dose of 10 mg/kg was generally similar for Japanese and non-Japanese patients with advanced solid tumors (AUC 0-∞ : 3410 vs. 3270 μg day/mL, C max : 250 vs. 256 μg/mL, respectively). The mean CL, t 1/2 , and Vz values were generally similar between the two populations for doses in the range 1-10 mg/kg. In addition, the individual values for AUC 0-∞ , C max , t 1/2 , Vz, and CL for doses in the range 1-10 mg/kg overlapped (Fig. 2) [18]. These results indicated that the PK of pembrolizumab in Japanese patients with advanced solid tumors was generally similar to that in non-Japanese patients with advanced solid tumors.
As an exploratory analysis, the tumor responses were also evaluated. Partial responses were observed in two of the nine patients (22.2 %) treated with 10 mg/kg Q2W by an  Fig. 3 PET-CT images taken before the start of treatment and after cycle 7 show evidence of antitumor activity. A rapid and durable partial response according to irRC was observed in a 91-year-old man with advanced metastatic acral lentiginous melanoma who had active disease in the liver and multiple lymph nodes when treatment with pembrolizumab at 10 mg/kg Q2W was initiated investigator review according to both RECIST v1.1 and irRC criteria. One of the five patients with advanced NSCLC and one of the three patients with advanced melanoma achieved partial responses. A durable response (response duration of more than 225 days according to irRC) was observed in one patient with metastatic acral lentiginous melanoma, which is the most common subtype of melanoma in Asian countries (superficial spreading melanoma is more common in Caucasians). This result is encouraging for the further development of pembrolizumab treatment for melanoma in Japan. Because of the limited number of patients and tumor samples, a relationship between tumor response and PD-L1 expression was not observed in the present study. Recent data have shown that PD-L1 expression, microsatellite instability high, increased mutation burden, and the immune gene expression signature are correlated with the improved efficacy of pembrolizumab [23][24][25][26], indicating the importance of biomarker development to identify patients suitable for anti-PD-1 and anti-PD-L1 therapy. Based on the safety data from this phase 1 study, Japanese patients are currently being enrolled in late-phase global studies of pembrolizumab for the treatment of various types of tumors, such as NSCLC, gastric cancer, head and neck cancer, bladder cancer, and colorectal cancer. Further biomarker analyses in these clinical studies are on-going.
Recent randomized studies of pembrolizumab demonstrated that no clinically meaningful differences in the safety and efficacy of pembrolizumab were evident between 2 mg/kg Q3W and 10 mg/kg Q3W or between 10 mg/kg Q3W and 10 mg/kg Q2W [27][28][29][30][31]. The adm inistration of pembrolizumab at 2 mg/kg Q3W is currently approved in the United States and other countries for the treatment of patients with unresectable or metastatic melanoma or metastatic NSCLC expressing PD-L1.
In conclusion, pembrolizumab at dosages of 2 and 10 mg/ kg Q2W was well tolerated in Japanese patients with advanced solid tumors and showed encouraging anti-tumor activity against melanoma and NSCLC.