Open-label, Multicenter, Phase II Study of RC48-ADC, a HER2-Targeting Antibody–Drug Conjugate, in Patients with Locally Advanced or Metastatic Urothelial Carcinoma

Urothelial carcinoma is a common malignancy worldwide. Urothelial cancer accounts for 90% of all bladder cancers and can also be found in the renal pelvis, ureter, and urethra. According to GLOBOCAN 2018, bladder cancer is the 10th most common form of cancer worldwide, with an estimated 549,000 new cases and 200,000 deaths (1). Locally advanced or metastatic urothelial carcinoma (mUC) has a poor prognosis, with only about 5% of patients at stage IV (metastatic) surviving longer than 5 years (1, 2). Platinum-containing chemotherapy is the standard first-line treatment for advanced urothelial carcinoma. The objective response rate (ORR) with platinum-containing chemotherapy is 44.6%–72% historically (2–4). The median overall survival (OS) is 14.0–15.2 months (2–4). Recently, immune checkpoint inhibitors (ICI), an FGFR inhibitor (erdafitinib), and a nectin4-targeting antibody–drug conjugate (enfortumab vedotin) were approved in platinum refractory settings, and avelumab was approved as a maintenance treatment for patients with locally advanced or metastatic urothelial carcinoma with at least disease control on first-line platinum-containing chemotherapy (2).

In bladder cancer, HER2 overexpression strongly correlates with tumor progression and poor prognosis, although HER2 genomic amplification is not a common mechanism (5–8). mAbs and tyrosine kinase inhibitors (TKI) targeting HER2 have failed to show clinical benefits in mUC (9–11). Currently, no HER2-targeting drugs have been approved for the treatment of mUC with HER2 overexpression.

Antibody–drug conjugates are designed for selective delivery of potent cytotoxic drugs to antigen-expressing tumor cells by linking cytotoxins to mAbs. RC48-ADC (disitamab vedotin) is a novel humanized anti-HER2 antibody conjugated with monomethyl auristatin E (MMAE) via a cleavable linker. In phase I studies, RC48 has shown a tolerable safety profile and promising antitumor activities toward solid tumors (12–14). Four patients with mUC were enrolled in one phase I study (13) with 2 patients having confirmed partial responses (PR) and 2 with stable disease (SD) for an ORR of 50% and a disease control rate (DCR) of 100%.

Hence, we conducted a phase II study to assess the efficacy and safety of RC48-ADC in advanced urothelial carcinoma with HER2 overexpression.

This was a multicenter, single-arm, phase II study of RC48-ADC, a recombinant humanized anti-HER2–MMAE conjugate developed by RemeGen, Ltd. Eligible patients were between 18 and 80 years old with central-laboratory confirmed, histologically HER2⁺ urothelial carcinomas that were unresectable, locally advanced, or metastatic. Subjects must have progressed on at least one line of systemic chemotherapy. The subjects must have at least one measurable lesion according to the RECIST version 1.1, with an Eastern Cooperative Oncology Group performance status of 0 or 1 and an expected survival time exceeding 12 weeks. The inclusion and exclusion criteria are listed in the Supplementary Appendix 1 (protocol of study).

All patients provided written informed consent before joining the study. The study protocol was approved by the relevant institutional review board or ethics committee of each study center. The study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice guidelines.

Eligible patients were treated with RC48-ADC at 2.0 mg/kg as an intravenous infusion over 30–90 minutes (60 minutes was recommended) once every 2 weeks until disease progression, intolerable toxicity, death, or withdrawal of consent. Dose was modified and interrupted (up to 28 days) in case of treatment-related adverse events (TRAE) until these events resolved to grade 0/1 or to baseline. Toxicity was managed with supportive care. Survival, follow-ups were performed for all patients every 12 weeks after the last treatment dose.

The clinical response was evaluated by a blinded independent review committee (BIRC) according to RECIST version 1.1 at baseline and every 6 weeks, irrespective of dose delays or interruptions, until documented disease progression or death.

All AEs were monitored and graded according to the Common Terminology Criteria for Adverse Events version 4.03. AEs are described in preferred terms, as defined in the Medical Dictionary for Regulatory Activities (version 20.1).

On the basis of the criteria from other studies evaluating anti-HER2 therapy (9–11), our study targeted patients with HER2 expression by IHC staining method. HER2 3+ and 2+ were defined as HER2⁺. IHC scores were reviewed by an independent pathologist at the central laboratory. The primary antibody used was the Ventana anti-HER2/Neu (4B5) rabbit mAb, and staining was performed with the ultra-View Universal DAB Detection Kit (Roche). The staining scores were assessed according to the HER2 test guidelines for breast cancer (15). Gene amplification of HER2 was evaluated by FISH, which was compliant with the HER2 test guidelines for breast cancer (15). Representative images of the HER2 IHC grading were included in Fig. 1.

The primary endpoint of this study was confirmed ORR assessed by the BIRC. A confirmed response was defined as complete response (CR) or PR demonstrated via CT or MRI according to RECIST 1.1. The secondary endpoints included progression-free survival (PFS), DCR, duration of response (DOR), OS, and safety.

In this single-arm study, the sample size was estimated on the basis of an assumed ORR of 10% for alternative therapy in patients with mUC after progression on at least one line of systemic treatment and an expected ORR of 30% for the investigational drug. With an estimated drop-out rate of 10%, the study planned to recruit 60 patients. An interim analysis was conducted after the 30th subject completed two efficacy assessments. The study could be terminated earlier if the interim data analysis achieved statistical significance.

The 95% confidence intervals (CI) were calculated using the Clopper–Pearson method. The Kaplan–Meier estimate was employed to calculate and plot survival curves. All analyses were performed using SAS 9.4 software.

Individual participant data are available at: http://data.remegen.cn:8888/FolderPublish.aspx?code=Bc68d5a229b4643ebb65b232c73aa6a32.

From December 28, 2017 to November 28, 2018, 133 patients were screened and 43 were enrolled from nine sites. Among 90 patients with screen failure, the majority (71 patients) had ineligible HER2 expression (70 patients with HER2 0/1+ and 1 patient's HER2 status was uncertain). Screening results can be seen in the flowchart (Supplementary Appendix 2).

Recruitment was terminated early as recommended by the regulatory authority based on the interim analyses. Specifically, in the interim analysis, among the initial 30 evaluable patients, the observed ORR was 66.7%, meeting the prespecified statistical requirements and the early stopping criterion for efficacy. At the time of the recommendation, the study had already enrolled 43 patients, which were included in this report.

At baseline, most patients (83.7%) demonstrated visceral metastasis, including 22 patients (51.2%) with metastasis to the lung and 20 patients (46.5%) with metastasis to the liver. Among 43 patients, 41 patients received prior platinum-containing regimens (35 patients with cisplatin, 11 patients with carboplatin, and 5 patients with both cisplatin and carboplatin), and 8 patients received prior programmed cell death-1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy after failure of platinum chemotherapy (7 patients with cisplatin, 1 patient with carboplatin). None of the patients had received prior HER2-targeted therapy. The baseline characteristics of the study population are listed in Table 1.

By the cut-off date of April 14, 2020, the median follow-up (from the first dose to the data cut-off date) was 20.3 months (range, 19.9–21.6 months). As assessed by the BIRC, 22 patients (51.2%) had confirmed objective responses according to RECIST 1.1, including 0 CR and 22 (51.2%) PR. In addition, 17 patients (39.5%) experienced SD as the best response. The ORR was 51.2% (95% CI, 35.5%–66.7%). Decrease of target lesions from baseline was observed in 38 (88.4%) of these patients (Fig. 2A). The DCR was 90.7% (95% CI, 77.9%–97.4%; Table 2). The median DOR was 6.9 months (95% CI, 4.7–10.8; Table 2; Fig. 2B).

Subgroup analyses (Fig. 2C) indicated that the ORR was 51.6% in the 31 patients with only one prior line of chemotherapy and 50.0% in the 12 patients with two or more lines of treatment. Among 20 patients with liver metastasis, the confirmed ORR was 65.0%. Representative images of responses in liver metastases are shown in Fig. 3. Among the 8 patients who had received prior PD-1/PD-L1 inhibitors, the confirmed ORR was 75.0%. The ORR was numerically higher for patients with higher HER2 expression (defined as either HER2 IHC 2+ and FISH+ or IHC 3+; ORR 60.0%) than patients with lower HER2 expression (defined as HER2 IHC 2+ and FISH⁻; ORR 40.0%). Of the 3 patients with IHC 2+ and unknown FISH results, 2 had PR.

The median PFS was 6.9 months (95% CI, 5.6–8.9), with 6-month PFS rate of 59.1% (95% CI, 42.6%–72.3%; Fig. 2D). The subgroup analyses revealed numerically longer PFS for the patients in the second-line setting (7.8 months; 95% CI, 4.0–9.4) than those in third-line setting (6.9 months; 95% CI, 2.1–8.9).

By the data cut-off date, only 1 patient remained on treatment, and 16 patients were in safety/survival follow-up. The median OS was 13.9 months (95% CI, 9.1–NE), and the estimated 12-month OS rate was 55.8% (95% CI, 39.8%–69.1%; Fig. 2E).

The median treatment duration for RC48-ADC was 22.0 weeks (range: 2.0–64.0). The median dose intensity was 94.5% (range: 60.2%–101.8%). As assessed by the BIRC, all 43 patients experienced at least one TRAE (Table 3). The most reported TRAEs were hypoesthesia (26, 60.5%), alopecia (24, 55.8%), leukopenia (24, 55.8%), neutropenia (18, 41.9%), and fatigue (19, 44.2%; Table 3). No grade 4 or 5 TRAEs were observed. Twenty-five (58.1%) patients experienced grade 3 TRAEs. The most reported grade 3 TRAEs were hypoesthesia (10, 23.3%) and neutropenia (6, 14.0%).Treatment discontinuation due to TRAEs occurred in 11 patients (25.6%). The most reported TRAE resulting in drug discontinuation was hypoesthesia (7, 16.3%).

This is the first reported phase II study of HER2-targeting ADC in advanced urothelial carcinoma. In this study, RC48-ADC elicited promising response and PFS in patients with advanced or mUC after progression on at least one prior line of chemotherapy.

Compared with HER2-targeting mAbs or TKIs (7–9), RC48-ADC showed a superior benefit/risk profile. The critical component of RC48-ADC is the MMAE with cytotoxic microtubule inhibition once the drug is delivered to HER2-expressing tumor cells. This effect is similar to preclinical models of HER2-overexpressiong bladder cancer, where T-DM1 had increased antitumor responses compared with trastuzumab (16). In addition, RC48-ADC contained an antibody with a higher HER2 affinity than that of trastuzumab (17), which may lead to increased HER2 selectivity for its antitumor effect.

Second-line treatments in mUC have demonstrated relatively low ORR [13%–24% for the approved ICIs (18), 40% for erdafitinib FGFR inhibitor in patients with specified FGFR alterations (19), and ≤15% for single chemotherapy agents like vinflunine and paclitaxel (20–22)]. The selected population in this study was patients with HER2⁺ expression, which differed from the studies mentioned above. In this study, RC48-ADC demonstrated an ORR of 51.2%, as assessed by the BIRC. Tumor shrinkage was observed in 38 patients (88.4%), demonstrating better disease control than any other approved second-line agent for advanced urothelial carcinoma. A fast-acting RC48-ADC clinical response was observed in this study, with median time to response of 38 days. Most responders (16/22) had achieved partial response at the first efficacy assessment (6 weeks after the first dose). The PFS of 6.9 months observed in our study is numerically longer than the PFS results obtained from second-line ICI treatments (2.1–4.0 months; ref. 18).

Several ADC drugs are under investigation in clinical trials for mUC, including enfortumab vedotin, tisotumab vedotin, ASG-15ME, sacituzumab govitecan, and RC48-ADC. In December 2019, enfortumab vedotin, a nectin-4 targeting ADC, was approved by the FDA for patients with advanced bladder cancer based on the results of a phase II clinical trial (EV-201; ref. 23). Enfortumab vedotin showed an ORR of 44.0% with a median PFS of 5.8 months in patients previously treated with chemotherapy and immunotherapy. Enfortumab vedotin was approved for mUC without the need to screen for nectin-4. In comparison, RC48-ADC focused on patients with mUC with HER2 overexpression. In our study, the ORR and the median PFS in 8 patients refractory to both chemotherapy and immunotherapy were 75.0% and 8.2 months, respectively. The FDA has approved an investigational new drug application of RC48-ADC to be tested in the third-line setting for advanced urothelial cancer (after prior platinum-containing chemotherapy and a PD-1/PD-L1 inhibitor-containing regimen) and granted fast track designation in July 24, 2020. There may be cross-reactivity between the two agents. If patients with a tumor with HER-2 overexpression progressed on enfortumab vedotin, they might still benefit from RC48-ADC. Of note, none of the patients in this study had prior enfortumab vedotin treatment.

Both ADCs and ICIs have been proven effective for advanced urothelial cancer. Furthermore, ADC–ICI combinations have shown promising results. The combination of enfortumab vedotin plus pembrolizumab (a cohort of the EV-103 trial) in first-line cisplatin-ineligible patients with advanced urothelial carcinoma showed an ORR of 73.3% (CR: 15.6%), a DCR of 93.3%, and a median PFS of 12.3 months (2). A phase Ib/II clinical trial exploring the safety and efficacy of RC48-ADC combined with PD-1 inhibitor is ongoing (NCT 04264936).

In this study, RC48-ADC had consistent clinical activity across all subgroups analyzed, including patients with liver metastasis. Chemotherapy or immunotherapy had limited clinical efficacy for patients with urothelial carcinoma with liver metastasis. In our study, RC48-ADC showed an ORR of 65.0% in patients with liver metastasis. Compared with 38.0% ORR for patients with liver metastasis in the EV-201 trial, RC48-ADC seemed to have better clinical activity and disease control in patients with liver metastasis. This novel finding is of significant value because liver metastases are very common for patients with mUC after progression on standard first-line treatment.

In our study, metastatic upper tract urothelial cancer (mUTUC) accounted for 51.2% of the population. A higher incidence rate of UTUC was reported in Asians than Western countries, generally about 20%–36% in most Asian cohorts with mUC (24).Two phase II trials of two PD-1 antibodies originated in China, toripalimab and tislelizumab, for the treatment of mUC after progression of platinum chemotherapy reported to have 47.2% (25) and 47.0% (26) of mUTUC, respectively, similar to our patient population. HER2 overexpression was reported in 9.4%–41.2% of patients with urothelial bladder cancer (UBC; refs. 7, 27) and 18.1%–35.8% of patients with UTUC (28, 29). We did not observe obvious difference in the HER2 expression rate between patients with UTUC and UBC in our study (44.1% vs. 49.2% in the screened patients). In addition, the ORRs were comparable between patients with a primary tumor in the upper urinary tract (52.4%) versus in the lower urinary tract (50.0%; Fig. 2C).

As indicated by this study, HER2-targeting RC48-ADC achieved favorable outcomes in patients with HER2⁺ advanced urothelial carcinoma. In addition, we also observed similar ORR in IHC 2+ and FISH⁻ patients (45.8%). Therefore, it would be of interest to evaluate RC48-ADC in patients with low or negative HER2 expression, because most patients with urothelial carcinoma are HER2 expression negative. Accordingly, a follow-up study (NCT04073602) of RC48-ADC in patients with low HER2 expression is ongoing.

RC48-ADC was well tolerated in our study. Compared with the phase I trial of RC48-ADC in advanced solid tumors (including 7.0% patients with urothelial cancer; ref. 11), TRAEs occurred in this trial were similar, except a higher incidence rate of hypoesthesia in this trial (all grade: 60.5% vs. 44.4%; grade≥3: 23.3% vs. 11.1%). There were two reported fatal AEs, which were considered related to disease progression. No grade 4 TRAEs were reported. Consistent with the mechanism of RC48-ADC, the TRAEs of RC48-ADC seemed to be mostly related to the toxicity of MMAE with regards to cytopenia and alopecia. In current clinical practice, these ADC-associated “chemo-like” toxicities are commonly observed and well managed. In addition, nervous system toxicities were observed and commonly reported as hypoesthesia, pruritus, and peripheral sensory neuropathy in our study. Nonetheless, nervous system toxicities were manageable, as grade 3 toxicities were rare.

This single-arm, phase II proof-of-concept study in Chinese patients was not designed to compare RC48-ADC with other treatment options or to explore treatment response by race or ethnicity. These limitations will be addressed in further research.

In conclusion, this is the first phase II clinical trial to evaluate HER2-targeting ADC in patients with advanced urothelial carcinoma which demonstrated a promising efficacy with a manageable safety profile. Patients with liver metastasis and previously treated by anti-PD-1/PD-L1 therapies also achieved similar responses. The ORR and PFS of this trial show disease efficacy for HER2-expressing mUC, indicating that RC48-ADC has the potential to become a treatment option for mUC with HER2 overexpression.

J. Fang reports other from RemeGen outside the submitted work; in addition, J. Fang has a patent for US10087260B2 issued. J. Guo reports other from MSD, Roche, Pfizer, Bayer, Novartis, Simcere Pharmaceutical Group, Shanghai Junshi Biosciences, and Oriengene outside the submitted work. No disclosures were reported by the other authors.

X. Sheng: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, investigation, methodology, writing-original draft, project administration, writing-review and editing. X. Yan: Resources, investigation, writing-original draft. L. Wang: Resources, investigation, writing-original draft. Y. Shi: Resources, investigation, writing-original draft. X. Yao: Resources, investigation, writing-original draft. H. Luo: Resources, investigation, writing-original draft. B. Shi: Resources, investigation, writing-original draft. J. Liu: Resources, investigation, writing-original draft. Z. He: Resources, investigation, writing-original draft. G. Yu: Resources, investigation, writing-original draft. J. Ying: Investigation, writing-original draft. W. Han: Resources, investigation, writing-original draft. C. Hu: Resources, investigation, writing-original draft. Y. Ling: Investigation, writing-original draft. Z. Chi: Resources, investigation, writing-original draft. C. Cui: Resources, investigation, writing-original draft. L. Si: Resources, investigation, writing-original draft. J. Fang: Conceptualization, methodology, writing-original draft. A. Zhou: Conceptualization, resources, formal analysis, supervision, investigation, methodology, writing-original draft, project administration, writing-review and editing. J. Guo: Conceptualization, resources, formal analysis, supervision, funding acquisition, investigation, methodology, writing-original draft, project administration, writing-review and editing.

This work was supported by grants from the Natural Science Foundation of China (grant number 81672696), Beijing Municipal Administration of Hospitals' Ascent Plan (grant number DFL20181101), and Beijing Municipal Science & Technology Commission (grant number Z161100000516062). The study was sponsored by RemeGen, Ltd.

We thank the patients and their families and caregivers for participating in this study, as well as the investigators, subinvestigators, research nurses, study coordinators, and operations teams for making this study possible. We acknowledge Tian Zhang from Duke Cancer Center for reviewing/editing assistance with the article.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.