2.1 Study design and participants

A phase I/Ib, open-label, single-arm study was conducted in China (Figure 1). The phase I dose-escalation study (C001 CANCER) using a standard 3 + 3 design determined the maximal tolerance dose (MTD), safety, and PK of DV in patients with HER2-overexpression ABC. Participants received initial intravenous doses of DV of 0.5, 1.0, 1.5, 2.0, and 2.5 mg/kg Q2W, and dose-limiting toxicities (DLTs) were assessed over a 28-day cycle. Since the responses were observed at 1.5, 2.0, and 2.5 mg/kg dose levels in the C001 CANCER study, meanwhile one cohort of the phase Ib study (C003 CANCER) was designed to establish the RP2D of DV and analyze its PK and efficacy in HER2-overexpression ABC with these three dose levels, 15 patients were planned to be enrolled in each group, and the higher dose group was started successively after the lower dose group was enrolled. After the 2.0 mg/kg dose was determined to be RP2D, another cohort of HER2-low ABC patients was included to explore the efficacy and safety of DV.

Eligible patients were at least 18 years old with a life expectancy of more than 12 weeks, had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of 0-1, had histologically or cytologically confirmed invasive breast cancer which was in the locally advanced or metastatic stage as determined by the 7th edition of American Joint Committee on Cancer (AJCC) Tumor Node Metastasis (TNM) staging system, were refractory to standard of care or unable to receive standard of care, had at least one measurable disease based on Response Evaluation Criteria in Solid Tumors (RECIST) criteria, version 1.1 [18], and had adequate organ functions (see the Supplementary Material for full eligibility criteria). Patients were excluded if they had brain or meningeal metastases. HER2 status was determined by local pathology laboratory results according to the 2014 Chinese breast cancer HER2 detection guideline [19]. HER2-overexpression was defined as IHC 3+, IHC 2+/FISH+. HER2-low was defined as IHC 2+/FISH-, IHC 1+/FISH-, or IHC 1+/FISH untested.

All patients provided written informed consent, and the study protocol was approved by independent ethics committees or institutional review boards at each site (approval No. 15-112/1039 [C001 CANCER] and 16-180/1259 [C003 CANCER]). The pooled analyses are in accordance with the two study protocols, the guidelines for Good Clinical Practice and the Declaration of Helsinki.

2.2 Treatment and assessments

Eligible patients received DV (RemeGen Co., Ltd., Yantai, Shandong, China) via intravenous infusion Q2W until progressive disease (PD), unacceptable toxicity, or withdrawal of consent. The dose-escalation phase involved evaluating doses of 0.5, 1.0, 1.5, 2.0, and 2.5 mg/kg Q2W using a 3 + 3 design. The DLTs were grade 4 neutropenia that was observed but effectively managed, grade 3 neutropenia concurrent with infection, and grade 3 nonhematological toxicity. Three doses (1.5, 2.0, and 2.5 mg/kg Q2W) were chosen for the phase Ib study in patients with HER2-overexpression ABC, and only 2.0 mg/kg Q2W was tested in patients with HER2-low ABC.

Spiral computed tomography or magnetic resonance imaging was performed at baseline, every 6 weeks for 24 weeks, and every 12 weeks thereafter. RECIST version 1.1 criteria were used to evaluate disease response and progression. Laboratory assessments were performed Q2W during treatment and 4 weeks after the last treatment.

The safety of DV was assessed by evaluating adverse events (AEs), changes in vital signs and laboratory tests in patients who received at least one dose of DV. The classification of treatment-emergent adverse events (TEAEs) was based on the Medical Dictionary for Regulatory Activities, version 23.0 (www.meddra.org) [20], and they were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0) [21]. In the C003 CANCER study, dexamethasone was administered before DV infusion after the dose-escalation phase to explore its potential in preventing peripheral neuropathy.

2.3 PK analysis

PK analysis of DV involved collecting blood samples during the first three doses at predetermined times up to 336 h post-dose. Phoenix WinNonLin version 8.1 (Pharsight Corp., Mountain View, CA, USA) was used to perform non-compartmental analysis. This analysis determined the peak plasma concentration (C_max), the time to C_max (t_max), the area under the concentration-time curve (AUC), the elimination half-life (t_1/2) and the accumulation ratio (Ra_(AUC)).

2.4 Statistical analysis

The pooled analyses were conducted on the full analysis set and safety set using data from the C001 CANCER and C003 CANCER studies. The full analysis set included patients who had provided informed consent, were enrolled in the phase I/Ib study, and received at least one dose of DV. The safety set consisted of patients who received at least one dose of DV and had available posttreatment safety evaluation data. Clinical activity endpoints were calculated separately for investigator-assessed data.

Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). The efficacy results were analyzed based on the full analysis set, and the safety results were analyzed based on the safety set. Clinical activity endpoints included objective response rate (ORR), disease control rate (DCR), clinical benefit rate (CBR), overall survival (OS), progression-free survival (PFS), percent change of target lesions, and duration of response (DOR). ORR was defined as complete response (CR) plus partial response (PR). DCR was defined as CR, PR, or stable disease (SD) for a minimum of 6 weeks from the first dosing date. CBR was defined as CR, PR, or SD for a minimum of 6 months from the first dosing date. OS was defined as the time from the date of the first dose to the date of death from any cause. PFS was defined as the time from the date of the first dose to the first objective documentation of radiographic PD or death due to any cause, whichever was earlier. DOR was measured from the time at which CR or PR criteria were first met to the first date of objectively documented PD or death due to any cause. PD was defined as an increase in the sum of diameters of target lesions of at least 20% and an absolute increase of at least 5 mm, or the appearance of ≥1 new lesion. The ORR, DCR and CBR were reported with point estimates and two-sided 95% confidence intervals (CIs) using the Clopper–Pearson method. Time-to-event statistics were calculated using the Kaplan-Meier method. No statistical tests or P values were provided in efficacy analyses. Safety evaluations included all AEs and serious AEs observed before October 30, 2021. TEAEs were summarized by grade and dose level. Fisher's exact test was employed to compare the proportions of patients with peripheral neuropathy between groups (with or without dexamethasone pretreatment), and the log-rank test was used to compare the first occurrence time of peripheral neuropathy between the groups. Descriptive statistics summarized the best percent change in the sum of the diameters of measurable tumors, demographic characteristics, and safety data.

3.1 Patient disposition and baseline characteristics

Between March 2016 and March 2018, 24 patients with HER2-overexpression ABC received DV at 5 dose levels (0.5, 1.0, 1.5, 2.0, and 2.5 mg/kg) in the C001 CANCER study (Figure 1). In the C003 CANCER study, 46 patients in the HER2-overexpression cohort received DV at 3 doses (1.5, 2.0, and 2.5 mg/kg) between December 2016 and October 2018, and 66 patients in the HER2-low cohort received DV at a single dose level of 2.0 mg/kg between October 2018 and June 2021.

Pooled analysis of the two studies included 136 patients with HER2-overexpression (n = 70) and HER2-low ABC (n = 66). Of these, 87 patients received the recommended dose of 2.0 mg/kg. The data cutoff date was October 30, 2021. The median follow-up was 5.1 months (range, 0-24.1 months) for patients with HER2-overexpression ABC and 4.3 months (range, 0-27.4 months) for patients with HER2-low ABC, and only 3/136 patients (2.2%) continued to receive DV treatment at date cutoff, and all three patients with HER2-low. The primary reason for treatment termination was disease progression (HER2-overexpression: 55/70 [78.6%]; HER2-low: 53/66 [80.3%]), followed by AEs (HER2-overexpression: 6/70 [8.6%]; HER2-low: 4/66 [6.1%]) (Figure 1).

Baseline demographic and clinical characteristics are presented in Table 1. All patients were female, with a median age of 54 years (range, 26-69 years), and a majority of patients (120/136; 88.2%) had visceral metastases. In the HER2-overexpression ABC group, 49/70 (70.0%) of patients had previously received trastuzumab. For the HER2-low ABC group, there were 35/66 (53.0%) patients with IHC 2+/FISH- and 31/66 (47.0%) patients with IHC 1+. HR positivity was observed in 60/66 (90.9%) patients with HER2-low ABC, with 49/66(74.2%) of patients receiving endocrine therapy.

3.2 Safety

A total of 136 patients received DV. The median number of doses administered was 9 (range, 1-48), and the median treatment duration was 151 (range, 14-834) days. The number of treatment cycles and duration were similar across the 2.0 and 2.5 mg/kg dose groups.

In total, 133/136 (97.8%) of patients experienced at least one TEAE, and 68/136 (50.0%) had an AE grade of 3 or higher (Supplementary Table S1). The most common grade 3 or higher AEs were neutrophil count decreased (24/136, 17.6%), gamma-glutamyl transferase increased (18/136, 13.2%), asthenia (15/136, 11.0%), white blood cell count decreased (13/136, 9.6%), peripheral neuropathy including hypoesthesia (8/136, 5.9%) and neurotoxicity (1/136, 0.7%), and pain (8/136, 5.9%) (Table 2, Supplementary Table S2). Platelet count decreased occurred in 13/136 (9.6%) of patients, with 2/136 (1.5%) of patients experiencing grade 3 or higher. One patient (0.7%) in the 2.5 mg/kg dose group developed a grade 1 left ventricular ejection fraction decreased (Supplementary Table S2).

TEAEs resulted in dose interruption in 50/136 (36.8%) of patients and dose reduction in 23/136 (16.9%) of patients; 8/136 (5.9%) of patients discontinued treatment due to an AE (Supplementary Table S1). Patients receiving DV at the 2.5 mg/kg dose had higher rates of any grade TEAEs, grade 3 or higher TEAEs, dose interruption, and dose reduction (Supplementary Table S1). Additional details are available in Supplementary Tables S3-S5. Seven patients died (Supplementary Table S1), and the causes of death were disease progression, cholestatic jaundice, increased blood bilirubin, pneumonitis, respiratory failure, cardiac tamponade and interstitial lung disease (ILD), with none of the deaths being drug-related.

Peripheral neuropathy, which includes symptoms such as hypoesthesia, neurotoxicity, peripheral sensory neuropathy and pain in extremities, led to treatment discontinuation in 4 patients due to hypoesthesia (3/136, 2.2%) and peripheral sensory neuropathy (1/136, 0.7%) (Supplementary Table S3).

To decrease its incidence, patients in the C001 CANCER and C003 CANCER studies received pretreatment with dexamethasone 10 mg via intravenous drip prior to each DV infusion starting from October 2018. Of the 87 patients receiving the 2.0 mg/kg dose, 31 patients were pretreated with dexamethasone, resulting in a lower incidence of peripheral neuropathy (41.9% vs. 64.3%, P = 0.070) (Supplementary Table S6). No grade 3 or higher cases of peripheral neuropathy occurred in those who received pretreatment compared to patients who did not (0 vs. 8.9%, P = 0.156) (Supplementary Table S6). Dexamethasone also delayed the onset of the first peripheral neuropathy event (227 days with pretreatment vs. 78 days without pretreatment) (Figure 2, Supplementary Table S7). However, the recovery time could not be statistically analyzed due to the short follow-up period after the last dose (4 weeks).

In the C001 CANCER study, DLTs occurred in 3/24 (12.5%) of patients (Supplementary Table S8). These included a grade 4 neutrophil count decrease in 2 patients and a grade 3 alanine aminotransferase increase and aspartate aminotransferase increase in 1 patient. The MTD was not determined, and the highest dose of DV obtained during dose escalation was limited to 2.5 mg/kg to ensure safety and optimal PK characteristics.

3.3 Clinical activity

Clinical activity endpoints for HER2-overexpression and HER2-low ABC, as reported by investigators, are provided in Tables 3, 4. The duration of treatment is depicted in Figure 3.

In the HER2-overexpression ABC group, the confirmed ORR was 32.9% (23/70; 95% CI, 22.1%-45.1%) (Table 3 and Figure 4), with doses of 1.5, 2.0, and 2.5 mg/kg showing ORRs of 22.2% (4/18; 95% CI, 6.4%-47.6%), 42.9% (9/21; 95% CI, 21.8%-66.0%), and 40.0% (10/25; 95% CI, 21.1%-61.3%), respectively. The median DOR was 5.7 months (95% CI, 4.0-7.4 months), and the median PFS was 5.5 months (95% CI, 4.6-6.5 months) (Figure 5), with 1.5, 2.0, and 2.5 mg/kg doses showing PFS of 4.0 months (95% CI, 2.6-7.6 months), 5.7 months (95% CI, 5.3-8.4 months), and 6.3 months (95% CI, 4.3-8.8 months), respectively.

Subgroup analysis of confirmed ORR in HER2-overexpression ABC was conducted based on factors such as age, ECOG PS, visceral metastases, liver metastases, HR status, dose of DV (1.5-2.5mg/kg), prior chemotherapy lines, and trastuzumab prior treatment. No significant differences in confirmed ORR results were observed across these subgroups (Figure 6).

In the HER2-low ABC group, all patients received DV at a 2.0 mg/kg dose, resulting in a confirmed ORR of 33.3% (22/66; 95% CI, 22.2%-46.0%) (Table 4 and Figure 7), a median DOR of 5.6 months (95% CI, 4.3-8.3 months), and a median PFS of 5.1 months (95% CI, 4.1-6.6 months) (Figure 5). Among HER2-low patients, those with HER2 IHC 2+/FISH- had an ORR of 42.9% (15/35; 95% CI, 26.3%-60.6%) and a median PFS of 6.6 months (95% CI, 4.1-8.3 months), while patients with HER2 IHC 1+ exhibited an ORR of 22.6% (7/31; 95% CI, 9.6%-41.1%) and a median PFS of 4.1 months (95% CI, 2.7-5.5 months).

The confirmed ORR in HER2-low ABC was further analyzed based on various factors, such as age, ECOG PS, visceral metastases, liver metastases, HR status, prior chemotherapy lines, prior endocrine therapy, HER2 status, and prior cyclin-dependent kinase 4/6 (CDK4/6) inhibitor treatment. Subgroup analysis revealed no notable differences in confirmed ORR results across these subgroups (Figure 8).

3.4 PK

Blood samples from 65 patients in the C001 CANCER and C003 CANCER studies were analyzed to measure antibody-drug conjugate (ADC), total antibody (TAb), and free MMAE. Figure 9 illustrates the average serum concentration profiles following the first dose within the dose range of 0.5-2.5 mg/kg. TAb and ADC reached their C_max at the end of the infusion, with a median T_max of 0.92-1.50 h, while free MMAE had a median T_max of 24.02-48.02 h. The mean t_1/2 values ranged from 12.86 to 30.75 h for TAb and from 17.38 to 32.71 h for ADC (Supplementary Table S9). Multiple-dose administration of DV showed Ra_(AUC) ratios of 0.93-1.14, 1.11-1.69, and 0.96-1.57 for TAb, ADC, and free MMAE, respectively (Supplementary Table S10).