Introduction

Blockade of the PD-1/PD-L1 pathway with monoclonal antibodies (mAbs) has been a successful target of cancer immunotherapy in recent years. The approvals of PD-(L)1 inhibitors for treatment of multiple tumor indications worldwide have resulted in a treatment paradigm shift in immuno-oncology therapeutics, providing durable remissions for patients with cancer.

All other approved anti-PD-1/PD-L1 antibodies are administered intravenously (IV). Although some patients receiving these treatments achieve durable responses and long-term survival, the need for maintenance therapy requires repetitive IV infusions in the clinic/hospital. This results in a drain in personal productivity and time along with accumulating health care costs [1]. Subcutaneous (SC) administration of mAbs has become a valuable alternative to IV administration in oncology. Some SC monoclonal antibody formulations (eg, trastuzumab, rituximab and bortezomib) have shown safety and tolerability profiles and efficacy comparable to that of IV administered mAbs in the treatment of cancer [2,3,4,5,6]. Compared with IV administration, SC administration is associated with lower resource utilization and cost, greater patient treatment compliance, thereby providing potential benefit to patients and payers [7, 8]. However, despite the interest in developing SC mAbs, challenges hampering the successful application of SC administration have occurred in drug formulation, with issues such as high viscosity and aggregation, and the inherent immunogenicity of mAbs [9].

Envafolimab, also known as KN035, is a novel recombinant protein of a humanized single-domain anti-PD-L1 antibody fused with a human IgG1 Fc fragment [10]. It is the first and only globally approved subcutaneously injectable anti-PD-L1 antibody for previously treated microsatellite instability high (MSI-H)/mismatch repair deficiency (dMMR) advanced solid tumors in China [11]. The antibody portion of envafolimab was obtained from a focused phage library from peripheral blood mononuclear cells of a human PD-L1-immunized camel. Point mutations introduced in the Fc fragment decreased effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), thus reducing the IgG1-Fc-mediated toxicities. Envafolimab binds human PD-L1 with high affinity (Kd = 3 nM), blocks PD1-PD-L1 interaction, and stimulates T cell activation at concentrations below 0.5 μg/mL, thereby relieving the immunosuppressive effect and enhancing immune surveillance and cytotoxicity by T cells, leading to an anti-tumor immune response [10]. Envafolimab is more water soluble and more stable than conventional mAbs [10]. Because of its high solubility (200 mg/mL), the approved full therapeutic dose of envafolimab (150 mg, QW) can be administered SC in a single injection of 0.75 mL in less than 30 s and without the need for an adjuvant.

Three Phase 1 clinical trials in China, the US, and Japan were designed to evaluate the safety and tolerability of envafolimab, along with its recommended Phase 2 dose, and provide the foundation for future clinical development in different ethnic populations. Results from the study in US patients showed that SC envafolimab had a favorable safety and pharmacokinetic profile, with promising preliminary anti-tumor activity [12]. Results from the pivotal Phase 2 trial in Chinese patients showed that envafolimab was safe and effective in the treatment of previously treated advanced dMMR/MSI-H solid tumors [13]. Here, we report the results of the Phase 1 study in Japanese patients with locally advanced or metastatic solid tumors.

Methods

Patient population

Patients were eligible for inclusion if they were ≥ 18 years, had a histologically or cytologically confirmed solid tumor that was recurrent, metastatic, or refractory to at least one prior line of therapy and had no further standard treatment options. Eligible patients should have adequate renal, hepatic, bone marrow function and an ECOG performance status of 0 or 1. Patients were excluded from the study if they were on any concurrent anticancer treatment, had received prior anti-PD-L1 therapy, had a history of immune-mediated conditions including pneumonitis or interstitial lung disease, or were on active systemic corticosteroid usage > 10 mg/day. Women who were pregnant or lactating were excluded.

Study design and treatment

This was an open-label, multicenter, Phase 1 dose-escalation and dose-expansion clinical trial. In the dose escalation phase, patients received SC envafolimab QW with a DLT evaluation period of 28 days. For the QW schedule, traditional 3 + 3 design was followed and three dose levels of 1.0 mg/kg (n = 3), 2.5 mg/kg (n = 4), and 5.0 mg/kg (n = 3) were planned. The initial starting dose of 1.0 mg/kg QW was chosen based on the well-tolerated safety profile of envafolimab dosing at 0.01–1.0 mg/kg QW demonstrated in the two other phase 1 studies conducted in the United Stated and China. In the dose expansion part-1, 16 patients were treated at 2.5 or 5.0 mg/kg Q2W. After the completion of dose expansion part-1, a fixed dose regimen with a longer dosing interval (Q4W) was investigated, as no DLT was observed at doses ranging from .01-10.0 mg/kg QW and longer than expected half-life observed in the ongoing phase 1 studies. During part-2 of the dose expansion, nine patients received subcutaneous envafolimab 300 mg once every four weeks (Q4W).

Efficacy and safety outcomes

Safety evaluations to assess the primary endpoint of safety and tolerability were performed based on treatment-emergent adverse events (TEAEs) and dose-limiting toxicities (DLTs). Adverse events (AEs) were coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 22.0 and graded using the National Cancer Institute’s Common Terminology Criteria for Adverse Events CTCAE v4.03.

Tumor response was assessed by investigators using the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 every 12 weeks. Response confirmation was done with imaging performed at least 4 weeks after the first documentation of response (complete response (CR) or partial response (PR)).

Pharmacokinetics and Immunogenicity Assessments

In the dose escalation phase and dose expansion phase of the study, full PK sampling was performed after the first dose of Cycle 1 (28 days) and sparse PK samples were collected at pre-dose and around Cmax during the subsequent cycles. PK sampling timepoints are provided in supplementary file (SI. Table 1). Envafolimab concentrations were determined using a validated enzyme-linked immunosorbent assay-based assay. The resulting final-population PK model was used to perform simulations on virtual patients.

Table 1 Patient Demographic and Clinical Characteristics at Baseline
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The immunogenicity of envafolimab was evaluated based on the development of anti-drug antibodies (ADAs) measured using a validated electrochemiluminescence bridging assay. Blood for anti-KN035 antibodies were to have been collected within 24 h before start of injection in Cycle 1, 2, 3 and 4, within 24 h before start of injection in every other subsequent cycle until Cycle 12, and at the mandatory Safety Follow-up Visit.

Statistical analysis

All patients treated with envafolimab were included in the safety analysis. PK and ADAs were analyzed in all patients who received at least one dose of envafolimab and had at least one concentration of envafolimab or ADAs measured. Safety, PK and immunogenicity data were summarized by descriptive statistics. The 95% confidence intervals (95%CIs) for ORR and DCR were calculated using the Clopper-Pearson method. Progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan–Meier product-limit method. Statistical analyses were conducted using SAS version 9.4 (SAS Institute, Cary, NC, USA).

Results

Patient characteristics

From October 2017 to September 2019, 35 patients were enrolled in the study (Table 1). All subjects had received prior systemic anti-tumor drug therapies. The median age was 65 years, and 42.9% were male. Except for 1 patient whose clinical stage assessment could not be completed, all had Stage IV cancer (97.1%). 18 patients (51.4%) had an ECOG performance score ≥ 1. The most common (≥ 8%) types of cancer were esophageal (14.3%), pancreatic (8.6%) and soft tissue sarcoma (8.6%).

Patient disposition

All patients received at least one dose of envafolimab. The median total number of doses was 5 (range:1–118) (SI. Table 2). At the time of data cutoff (March 31, 2020), 33 of the 35 patients (94.3%) had discontinued treatment. The main reason for treatment discontinuation was disease progression (n = 27). 2 patients remained on treatment, at 1.0 mg/kg QW and 300 mg Q4W respectively. The median treatment duration was 9.7 weeks (range: 2 to 120 weeks). Five patients (14.3%) and 2 patients (5.7%) had treatment duration ≥ 6 months and 12 months, respectively.

Table 2 Summary of TEAEs
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Safety and tolerability

As of the data cutoff date, the majority of TEAEs reported during both phases of the study were mild to moderate in intensity and consistent with that reported in previous trials with envafolimab and other PD-L1/PD-1 mAbs (Table 2). The majority were considered related to drug, but did not lead to drug interruption or discontinuation. Overall there was no clear correlation between dose levels and safety. In the dose-escalation phase, no DLTs were reported and a maximum tolerated dose was not reached.

The most common TEAEs (occurring in ≥ 10% of the 35 treated patients in the safety analysis set) reported during both phases included rash, pruritus, pyrexia, nausea, vomiting, decreased appetite, and headache. One patient experienced a Grade 5 TEAE of thrombosis that was not considered related to drug. As expected, no infusion related reactions were reported.

As noted, the majority of reported TEAEs of all grades was considered drug-related (74.3%). The most frequently reported drug-related TEAEs occurring in ≥ 8% of patients included rash, pyrexia, pruritus, nausea, and blood thyroid stimulating hormone increased. Three patients reported Grade 3–4 drug-related TEAEs, including adrenal insufficiency, cerebral infarction, and immune-mediated enterocolitis. No Grade 5 drug-related TEAEs were reported. Four patients (11.4%) experienced drug-related TEAEs leading to treatment discontinuation, including hypoaesthesia, cerebral infarction, hepatic function abnormal, and immune-mediated enterocolitis.

Of the 6/35 (17.1%) patients with serious TEAEs, 4 patients experienced drug-related serious TEAEs of rash, adrenal insufficiency, immune-mediated enterocolitis, and cerebral infarction.

Overall, treatment-emergent adverse events of special interest (TEAESIs) were reported in 25/35 (71.4%) patients in the following 9 categories: gastrointestinal disorders, hepatic disorders, injection site reaction, hypersensitivity reactions, pneumonitis, endocrine disorders, cutaneous reactions, renal disorders, and infectious disorders (Table 2). TEAESIs reported in this study were consistent with that reported for other PD-L1/PD-1 inhibitors; most were drug-related but the majority were mild to moderate in severity. The incidence rate of injection site reactions was 14.3% (5/35), all of which were Grade 1–2.

In total, immune-related adverse reactions (irAEs) of all grades were reported in 13 (37.1%) and Grade 3–4 irAEs were reported in 2 (5.7%) patients. As of the data cutoff date, only 5 categories of irAEs were observed, including immune-related endocrine disorders (8 patients, 22.9%), immune-related skin adverse reactions (4 patients, 11.4%), immune-related neurological adverse events (1 patient, 2.9%), immune-related diarrhea and colitis (1 patient, 2.9%), and immune-related pneumonitis (1 patient, 2.9%). Most irAEs reported in this study were effectively managed with symptomatic treatment, dose interruption or discontinuation, and corticosteroids.

Pharmacokinetics

As shown in Table 3, following a single SC administration of envafolimab, the maximum serum concentration (Cmax) and area under the curve (AUC) increased proportionally over the dose range (1.0–5.0 mg/kg QW, 2.5–5.0 mg/kg Q2W, and 300 mg Q4W).

Table 3 Envafolimab Pharmacokinetic Parameters after the Single Dose Treatment
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Table 4 Best Overall Response Rate (Confirmation required)
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In the dose-escalation phase, the median time to Cmax ranged from 4 to 7 days.

In dose expansion part-1, the median Tmax were 166 h and 118 h and the mean Cmax were 11 μg/mL and 20 μg/mL in the 2.5 mg/kg Q2W and 5.0 mg/kg Q2W dose groups respectively. The AUC up to the last measured concentration (C336h) were 2347 h*μg/mL and 4986 h*μg/mL. No apparent changes in CL/F or Vd/F were found with the dose escalation from 2.5 mg/kg Q2W to 5.0 mg/kg Q2W.

After multiple doses of envafolimab in dose expansion part-1, the mean peak and trough concentrations on C3D1 were 15,533.34 ng/mL and 11,009.71 ng/mL for 2.5 mg/kg Q2W and 47,319.99 ng/mL and 26,486.98 ng/mL for 5.0 mg/kg Q2W, respectively.

In dose expansion part-2, after a single subcutaneous injection of envafolimab in the fixed dose cohort at 300 mg Q4W, the Tmax was 166 h and the mean Cmax was 13,541.86 ng/mL. The median t1/2 was 321 h.

After multiple doses, the steady state tended to be reached on C4D1 in 1.0 mg/kg QW dose group, on C1D22 in 2.5 mg/kg QW dose group, on C2D1 in 5.0 mg/kg QW dose group, and on C3D1 based on simulated serum concentration–time data in 2.5 mg/kg Q2W, 5.0 mg/kg Q2W, and 300 mg Q4W dose groups. The median t1/2 of envafolimab was 321 h after a single dose (C1D1, n = 9) and 430 h after multiple doses (C5D1, n = 1) in subjects of the 300 mg Q4W dose group. Overall, the steady state tended to be reached after 3–4 cycles of multiple doses (SI. Figure 1).

Fig. 1
figure 1

Best Response and Duration of Response in all Treated Patients Abbreviations: PD, progressive disease; PR, partial response; QW, once weekly; Q2W, once every 2 weeks; Q4W, once every 4 weeks; SD, stable disease

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Immunogenicity

Anti-drug antibodies (ADAs) results were available for all 35 patients, of whom 6 had pre-existing ADAs at baseline. Of the 35 patients, 18 (51.3%) patients had a positive ADA at least once post-baseline. Positive ADA measurements were transient in most patients and did not appear to affect PK exposure to envafolimab. As shown in Fig. 2, patients who developed ADA on treatment did not have reduced exposure as measured by dose normalized median trough concentration.

Fig. 2
figure 2

Box-plot for Correlation of Dose Normalized Trough Concentrations to ADA

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The median time to positive ADA measurement was 4.1 weeks (2.0 to 14.0 weeks) and the median duration of positive ADA was 8.14 weeks (0.14 + to 42.14 weeks) for all 18 patients. The incidence of positive ADA was the highest by Cycle 2 Day 1 (42.9%), which subsequently decreased over the time of treatment. By Cycle 6 Day 15, it had decreased to 2.9% for all patients. Additional ADA test results were available for 14 of the 18 patients: 5 had only negative results and 9 had at least one positive result. Of the 9, 3 patients had negative results and 6 patients still had positive results at the time of the last test.

Efficacy

Envafolimab as a single agent demonstrated preliminary durable anti-tumor activity in patients with advanced solid tumors in Japan. Efficacy was observed across different dose regimens (once a week, once every 2 weeks, or once every 4 weeks), and is consistent with the efficacy of other PD-1/PD-L1 antibodies as showed in Table 4.

As of data cutoff, PRs were reported in 5 (14.3%) patients including 4 confirmed PRs (1 each at 1.0 mg/kg QW, 300 mg Q4W, and 2 at 5.0 mg/kg Q2W) and 1 unconfirmed PR (5.0 mg/kg QW). Partial responses were observed in patients with urothelial carcinoma, extrahepatic bile duct cancer, ovarian cancer, soft tissue sarcoma and esophageal cancer. 1 patient with PR was still in continuous response by the time of database lock (Fig. 1). The median duration of response (DoR) was 41.9 weeks (95% CI: 5.1, 43.1). The DoR rates were 80.0% (95% CI: 20.4%, 96.9%) and 53.3% (95% CI: 6.8%,86.3%) at 6 and 9 months, respectively. 8 patients had a best overall response of stable disease (SD) and median duration of SD was 24.4 weeks (95% CI: 11.4, NE). Consistent with that reported with other anti-PD-1/PD-L1 antibodies in previously treated patients with advanced solid tumors, when only confirmed response was included, ORR was 11.4% (95% CI: 3.2%, 26.7%) and DCR was 34.3% (95% CI: 19.1%, 52.2%). The median time to response (TTR) was 11.4 weeks (95% CI: 11.3, 24.6), and approximately 50% of responders had a response at the time of the first response assessment.

As of the data cutoff date, 30 patients had progressed or died, with a median PFS of 2.4 months (95% CI: 1.7, 2.6). 6 deaths were recorded, and the median OS was not reached. The 6-month OS rate was 68.0% (95% CI: 37.5%, 86.0%), and the OS rate at 12 months was 54.4% (95% CI: 21.3%, 78.7%). Considering the limited sample size and number of events in this study, PFS and OS data need to be interpreted with caution.

Discussion

Envafolimab was granted conditional approval by China’s National Medical Products Administration (NMPA) for the treatment of dMMR/MSI-H solid tumor based on a pivotal phase II trial (CN-006; NCT03667170) in November 2021. This is the first study to evaluate the safety, tolerability, and pharmacokinetics of envafolimab in the Japanese population. Results from this study demonstrate that SC administration of envafolimab was well tolerated and had durable antitumor activity in a wide range of doses across different regimens in Japanese patients with previously treated advanced solid tumors. Similar results have been reported in the previously reported Phase 1 studies in US patients (US-001; NCT02827968) and in the Chinese patients (CN-001; NCT03101488) [13].

A total of 35 patients in this study received at least one dose of envafolimab with a median treatment duration of 9.7 weeks (range: 2–120 weeks). 14.3% and 5.7% of patients were on treatment for ≥ 6 and 12 months, respectively.

Overall, the safety results from this trial were consistent with that previously reported in Phase 1 trials with envafolimab and other PD-1/PD-L1 antibodies in Japanese population [21,22,23,24] and non-Japanese population as well as in patients with solid tumors [13,14,15,16]. No DLTs and no new safety signals were reported. Most drug-related TEAEs were mild to moderate and did not lead to treatment discontinuation. No Grade 5 events were reported. The most frequently reported drug-related TEAEs (≥ 8%) included rash, pyrexia, pruritus, nausea, and blood thyroid stimulating hormone increased. The frequency, severity, and type of irAEs reported in this trial were generally consistent with those observed with other PD-1/PD-L1 antibodies, and no irAE events specific to envafolimab were observed. Of note, no immune-related hepatitis and immune-related nephritis were reported in this study [17,18,19,20]. The incidence rate of injection site reactions was 14.3% (5/35), all of which were Grade 1–2.

This study showed preliminary anti-tumor activity of envafolimab in Japanese patients. The objective response rate observed in the current study was in line with the efficacy of other anti-PD-1/PD-L1 antibodies in Phase 1 studies in patients with previously treated patients with advanced solid tumors [14,15,16,17]. Responses to envafolimab were observed across multiple tumor types, including urothelial carcinoma, extrahepatic bile duct cancer, ovarian cancer, soft tissue sarcoma and esophageal cancer (Fig. 1).

Objective responses were observed in five patients including 1 unconfirmed PR across several dose cohorts. The mDoR were 41.9 weeks (5.1 to 43.1 weeks). One of five PR patients were still responding at the time of data cutoff. Stable disease was observed in 8 patients with various tumor types, including urothelial carcinoma, pancreatic cancer, and ovarian cancer. Objective response was first observed at 1.0 mg/kg QW. The proportions of PR did not increase significantly with the increase of dose, an observation also made with other anti-PD(L)-1 antibodies.

SC injection was an effective route of administration for envafolimab with a favorable PK profile. Following SC administration QW, exposure to envafolimab was dose-proportional. Based on the PK results from the US Phase 1 study ([13]), most patients would attain steady state after five cycles and that > 90% of those receiving envafolimab 300 mg once every 3 weeks (Q3W) and 400 mg Q4W would maintain trough concentration above 5 µg/mL, which is at least tenfold higher than the minimum pharmacologically active concentration (0.5 µg/mL [10]). Response was also observed in regimens with longer dosing intervals with PR occurred at 5 mg/kg Q2W and 300 mg Q4W.

Consistent with that observed in other envafolimab Phase 1 trials and other approved PD-1/PD-L1 inhibitors, the safety profile of SC envafolimab in Japanese patients with advanced solid tumors was favorable and well tolerated with efficacy comparable to IV administered treatments. Pharmacokinetics data and preliminary anti-tumor response support dose regimens with longer dosing intervals (administered every 2 weeks or every 4 weeks). As such, envafolimab offers patients a potentially more convenient treatment option than currently available intravenously administered PD-1/PD-L1 inhibitors.

Many clinical trials investigating envafolimab are currently ongoing, including combination studies. Therefore, it is expected that envafolimab may become more widely used for the treatment of various cancer types.