Evaluation of RC48-ADC in Combination with PRaG Regimen: An Open-Label,Prospective,Multicentre Study Assessing Efficacy and Safety for Advanced Refractory HER2-Expressing Solid Tumors (PRaG3.0 Study Protocol)

Abstract

Introduction

This study aims to investigate the effectiveness and safety of combining the antibody-drug conjugate (ADC) disitamab vedotin (RC48) with the established PRaG regimen (PD-1 inhibitor, Radiotherapy, and GM-CSF) for treating HER2-expressing (IHC 3+, 2+, or 1+) advanced solid tumors. This novel PRaG3.0 regimen is hypothesized to leverage the tumor radiosensitizing and immunogenic cell death properties of ADCs to amplify the synergistic antitumor effects of radioimmunotherapy, offering a potential paradigm for pan-cancer therapy.

Methods

This study is a prospective, single-arm, open-label, multi-center clinical trial. Patients enrolled have confirmed HER2-expressing solid tumors (IHC 3+, 2+, or 1+) that have progressed after standard treatment or were intolerant to it. Patients received RC48 (2 mg/kg) via intravenous injection on day 1, followed by subcutaneous GM-CSF at 200 µg from days 3 to 7 and interleukin-2 (IL-2) at 2 million IU from days 8 to 12. Radiotherapy was initiated on day 3, targeting one lesion with hypofractionated radiotherapy (2-3 fractions of 5 or 8 Gy). PD-1/PD-L1 antibodies were administered within one week after completing radiotherapy. Treatment was repeated every three weeks, and if there were no target lesions, radiotherapy could be discontinued, with RC48 given for at least six cycles. After achieving a complete tumor response, maintenance therapy with PD-1/PD-L1 antibodies continued until disease progression or intolerable toxicity occurred. The primary endpoint was the objective response rate (ORR).

Results

The study is currently in the recruitment phase (Registration No: NCT05115500). The primary endpoint is the objective response rate (ORR). Secondary endpoints include progression-free survival (PFS), overall survival (OS), disease control rate (DCR), safety, and QoL. Planned enrollment is 62 patients.

Conclusion

The PRaG3.0 protocol represents an innovative approach combining ADC therapy with radioimmunotherapy to address HER2-expressing cancers, including those with HER2-low expression. If successful, this regimen could establish a highly effective combination strategy.

Introduction

Antibody-drug conjugates (ADCs) represent a breakthrough in oncology, offering a highly specific and effective therapeutic approach by combining the tumor-targeting precision of monoclonal antibodies with the potent cytotoxicity of chemotherapeutic agents.^1,2 ADCs have demonstrated efficacy across a variety of HER2-expressing tumors, extending beyond the traditional tissue-of-origin paradigm. ³ HER2 expression, associated with aggressive tumor behavior and poor prognosis, is not limited to breast and gastric cancers but is also found in colorectal, ovarian, pancreatic, and lung cancers.^4,5 Agents like trastuzumab deruxtecan and disitamab vedotin (RC48) have shown significant anti-tumor activity in these malignancies, including those with low HER2 expression, underscoring their versatility and broad applicability.^6–8

Beyond their direct cytotoxic effects, ADCs can induce immunogenic cell death (ICD), leading to tumor antigen release and enhanced immune system activation. This dual mechanism positions ADCs as ideal candidates for combination therapies, particularly with immunotherapeutic approaches like immune checkpoint inhibitors.^9,10 Their ability to target diverse tumor types and stimulate immune responses highlights ADCs as a cornerstone of pan-cancer therapy, paving the way for more effective and precise treatment strategies.

Radiotherapy, with its well-established safety profile, widespread clinical availability, and immune-activating potential, has gained attention as a valuable partner in combination therapies.^11,12 The innovative PRaG therapeutic strategy leverages a combination of treatments to generate an in situ vaccine effect, which stimulates anti-tumor immunity and promotes remodeling of the tumor microenvironment. The regimen's design is rooted in the cancer-immunity cycle hypothesis, targeting three critical stages: (1) the release of tumor antigens is triggered by HFRT, (2) the activation of antigen-presenting cells is promoted by GM-CSF, and (3) the activity of CD8+ T-cells is restored by PD-1 inhibitors, which counteract inhibitory signals. ¹³

Widely implemented in China, PRaG therapy has shown notable clinical outcomes in treating advanced, refractory cancers, including esophageal, gastric, colorectal, pancreatic, lung, and breast malignancies, with demonstrated efficacy in refractory cases such as gastric cancer and ovarian cancer. Initial clinical validation in a phase II trial revealed promising outcomes, including an overall response rate (ORR) of 16.7% and a disease control rate (DCR) of 46.3%. Notably, complete remission was achieved in select patients. ¹⁴

Disitamab vedotin (RC48), a novel humanized anti-HER2 ADC, incorporates monomethyl auristatin E (MMAE) as its cytotoxic payload and exhibits strong HER2 affinity and robust antibody-dependent cell-mediated cytotoxicity (ADCC).^15–17 Beyond its cytotoxic effects, RC48 can induce ICD, promoting widespread release of tumor antigens and enhancing immunotherapy effectiveness by activating effector T cells. These properties make RC48 an attractive candidate for integration into multi-modal combination therapies.^18,19

While RC48 monotherapy and its combination with PD-1 inhibitors have shown efficacy, resistance remains a challenge in refractory solid tumors. The addition of radiotherapy in the PRaG3.0 regimen is specifically designed to overcome this by inducing the “abscopal effect.” Radiotherapy promotes the release of tumor-associated antigens and neoantigens, potentially sensitizing “cold” tumors to the PD-1/ADC combination. GM-CSF further facilitates antigen presentation, while IL-2 enhances T-cell proliferation, creating a stronger synergistic loop than the doublet therapy alone. Therefore, the PRaG3.0 regimen was designed to create a novel synergy by combining RC48-ADC with HFRT, a PD-1/PD-L1 inhibitor, GM-CSF, and IL-2. This multi-component strategy aims to effectively target HER2-expressing cancers, including tumors with low HER2 expression. This approach leverages the complementary mechanisms of ADCs, radiotherapy, and immunotherapy to achieve enhanced anti-tumor effects. To evaluate the clinical potential of this paradigm, an exploratory phase II, open-label, multi-center, single-arm study was conducted, focusing on the efficacy and safety of PRaG3.0 in patients with advanced solid tumors exhibiting HER2 expression.

Methods

Objectives

The primary objective of this study is to investigate the effectiveness of RC48-ADC combined with radiotherapy, PD-1/PD-L1 inhibitor sequential granulocyte-macrophage colony-stimulating factor, and interleukin-2 for HER2-expressing advanced solid tumors. The secondary objective is to assess the safety and toxicity of this treatment. The study also aims to explore a panel of T lymphocyte subsets, tumor-associated cytotoxic T cells, activated cytotoxic T lymphocytes, activated memory T cells, monocytes, dendritic cells, interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-17A, tumor necrosis factor, interferon-γ.

Study Design and Sample Calculation

The PRaG3.0 trial (NCT05115500) was a single-arm, open-label, multicentre, phase II study initiated by the Second Affiliated Hospital of Soochow University. This study was a Phase II, open-label, single-arm, multicenter clinical trial based on Simon's optimal two-stage design. The primary endpoint was ORR, while secondary endpoints were DCR, PFS, OS, and safety/toxicity. This study is currently in the patient recruitment phase. The patient enrollment period for this study is from 30/11/2021 to 31/12/2025. The time of data collection and completion of follow-up is from 31/12/2025 to 31/01/2026 and manuscript publication will in April 2026. The reporting of this study protocol conforms to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 guidelines. ²⁰

The sample size calculation is based on Simon's optimal two-stage design. We assume a null hypothesis objective response rate (ORR) of 15% (H0 = 0.15) based on historical data for standard salvage therapies in this refractory population. The target ORR for the PRaG3.0 regimen is set at 30% (H1 = 0.30). With a one-sided alpha (type I error) of 0.05% and 80% power (beta = 0.20), the design requires 55 evaluable patients. To account for a potential 10% dropout rate, the total enrollment target is set at 62 patients.

Inclusion Criteria

The study inclusion criteria will be as follows:(1) Age ≥18 years; (2) Participants with advanced, confirmed HER2-expressing (IHC3+, 2 + or 1+) solid tumors that had progressed after standard treatment, or standard treatment intolerance were enrolled. Patients must have recurrent or metastatic late-stage solid malignant tumors with a confirmed pathological diagnosis or medical history. Furthermore, HER2 status must be confirmed using an FDA/NMPA-approved IHC kit following ASCO/CAP guidelines. To ensure consistency, HER2 testing results will be verified by [specify: central pathology review OR double-blind review by two senior pathologists, and there must be no guideline-recommended standard treatment options, or the patient must be intolerant or explicitly refuse standard treatments due to personal preference. Additionally, patients should have identifiable measurable metastatic lesions; (3)No occurrences of congestive heart failure, unstable angina, or unstable arrhythmias in the past 6 months;(4) Patient's performance status must be graded 0-3 according to the Eastern Cooperative Oncology Group (ECOG) scoring system, with a life expectancy assessment of ≥3 months; (5) No severe history of hematologic, cardiac, pulmonary, hepatic, renal abnormalities, or immunodeficiencies; (6) One week before enrollment, absolute T-lymphocyte count must be ≥0.5 times the lower limit of normal; neutrophils must be ≥2.0 × 10^9/L; AST and ALT must be ≤3.0 times the upper limit of normal (for liver cancer/liver metastatic cancer, ≤ 5.0 times the upper limit of normal); creatinine must be ≤3.0 times the upper limit of normal;(7) Patients must possess the capability to understand and voluntarily sign the written informed consent form.

Exclusion Criteria

Patients who meet any of the following criteria will be excluded: (1)Pregnant or breastfeeding women.(2)Patients with a history of other malignancies within the past five years, except cured skin cancer and cervical carcinoma in situ.(3)Patients with uncontrolled epilepsy, central nervous system diseases, or psychiatric disorders, which, in the investigator's judgment, could significantly affect the ability to provide informed consent or interfere with medication adherence.(4)Clinically significant (active) heart diseases, such as symptomatic coronary artery disease, New York Heart Association (NYHA) Class II or higher congestive heart failure, severe arrhythmias requiring medication, or a history of myocardial infarction within the past 12 months.(5)Patients requiring immunosuppressive therapy due to organ transplantation.(6)Patients with known major active infections, or significant hematologic, renal, metabolic, gastrointestinal, endocrine dysfunctions, or other uncontrolled serious comorbidities as judged by the investigator.(7)Patients allergic to any components of the investigational drug.(8)Patients with a history of immunodeficiency, including those testing positive for HIV or suffering from other acquired or congenital immunodeficiency diseases, those with a history of organ transplantation, or those requiring long-term oral steroid therapy due to other immune-related diseases.(9)Patients with active acute or chronic tuberculosis (T-spot positive, chest X-ray showing suspicious tuberculosis lesions).(10)Other conditions that the investigator considers inappropriate for inclusion.

Treatment Scheme and Modalities

Enrolled patients were treated using the PRaG 3.0 protocol, those received RC48-ADC(2.0 mg/kg dL, every 3 weeks), then HFRT (2-3 doses of 5-8Gy) was delivered for one metastatic lesion every other day, followed by GM-CSF(200 μg d3-7), sequential IL-2(2million IU d8-12), and PD-1/PD-L1 inhibitor was dosing within one week after completion of HFRT. After RC48-ADC combined with PD-1/PD-L1 inhibitor sequential GM-CSF and IL-2 for at least six cycles, then maintenance with PD-1/PD-L1 inhibitor was administered until disease progression or unacceptable toxicity. The specific treatment protocol is shown in Figure 1 by version1.4.

Figure 1.

Treatment schedule of the PRaG3.0 therapy. This timeline illustrates the treatment schedule for the PRaG3.0 regimen, which is administered every three weeks (q3w) for six cycles. RC48 (ADC): Administered intravenously (IV) at 2 mg/kg on Day 1 of each cycle. Radiotherapy (HFRT): Hypofractionated radiotherapy (2-3 fractions of 5 or 8 Gy) is initiated on Day 3. Radiotherapy is discontinued if there are no appropriate lesions for irradiation or if the normal tissue tolerance dose is reached. GM-CSF: Granulocyte-macrophage colony-stimulating factor (200 µg) is administered subcutaneously (SC) on Days 3-7. IL-2: Interleukin-2 (2 million IU) is administered subcutaneously (SC) on Days 8-12. PD-1/PD-L1 Inhibitor: Administered intravenously within one week after the completion of radiotherapy. Maintenance: After at least six cycles of combination therapy, maintenance therapy with the PD-1/PD-L1 inhibitor continues until disease progression or unacceptable toxicity. Imaging Evaluation (CT/MR): Performed at baseline and every 6 weeks thereafter.

Radiotherapy (HFRT) Target Selection: Radiotherapy targets metastatic lesions based on the following selection criteria: (1) Lesion Characteristics: Measurable lesions are selected, preferably with a diameter of 1-5 cm. (2) Site Prioritization: Lesions in solid organs (eg, liver, lung) are prioritized for irradiation over those in non-solid structures (eg, hollow organs, bone). Within solid organs, liver lesions are prioritized over lung lesions. (3) Irradiation Strategy: A multi-target irradiation strategy is employed [if applicable, add: allowing for the treatment of multiple lesions either simultaneously or sequentially].

Patients received RC48 (2 mg/kg, IV) on day 1, GM-CSF (200 µg, SC) on days 3-7, and IL-2 (2 million IU, SC) on days 8-12. Radiotherapy (2-3 fractions of 5 or 8 Gy) began on day 3, followed by PD-1/PD-L1 antibodies within one week after radiotherapy.

Objective Endpoints and Efficacy Assessment

The primary endpoint was objective response rate (ORR), which was defined as the proportion of participants with partial (PR) or complete (CR) response in evaluable patients in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 determined by investigators. Radiological assessments were performed on average every 6 weeks. Secondary objectives included safety, disease control rate (DCR), progression-free survival (PFS), quality of Life (QoL), and overall survival (OS). ORR was defined as the proportion of patients with complete response (CR) or partial response (PR). DCR was defined as the percentage of patients with CR, PR, or stable disease (SD) from enrollment. OS was calculated from the enrollment date to the date of death or last known alive. PFS was calculated from the enrollment date to disease progression, death, or censored at the last clinical follow-up. QoL will be assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) at baseline and every 2 cycles thereafter. After the conclusion of treatment, all trial participants will undergo survival follow-up every 3 months until death, loss to follow-up, withdrawal of informed consent, or the sponsor decides to terminate the study. The nature, frequency, and severity of adverse events were assessed based on the Common Terminology Criteria for Adverse Events version 5.0 (CTCAE 5.0). Lymphocyte subset counts and cytokine analysis were examined as exploratory endpoints.

Statistical Analysis

Data analysis was conducted using SPSS 18.0 statistical software. Residuals were examined for normality using the Shapiro-Wilk test, with a significance level α>0.05. For variables that met the assumptions of normal distribution, a randomized block analysis of variance was employed; for those that did not, a non-parametric rank-sum test based on a randomized block design was utilized, with a significance level α < 0.05. Comparisons were made for changes in the number of white blood cells, granulocytes, lymphocytes, and their subtypes, and cytokine levels before and after radiotherapy to determine if there were statistically significant differences. Survival time was considered in conjunction with these changes, using Cox regression analysis to assess the impact of changes in white blood cell counts, granulocyte counts, lymphocyte and its subtype counts, and cytokine levels on patient survival rates. Kaplan-Meier analysis was used to compare the survival rates between patients who experienced side effects and those who did not. The relationship between patient survival rates and associated cytokine levels was also analyzed using the Kaplan-Meier method. Subgroup analyses will be performed to explore efficacy variations based on primary tumor type (eg, gastric vs breast) and HER2 expression levels (IHC 1 + vs 2+/3+). Given the limited sample size, these analyses are considered exploratory and descriptive.

Ethics and Dissemination

This study protocol (Version 1.4) was approved by the Ethics Committee of The Second Affiliated Hospital of Soochow University (Address: No. 1055 Sanxiang Road, Suzhou, China), with approval number JD-LK-2021121-A03, September 11, 2025 and it will be conducted in accordance with the Declaration of Helsinki. The study is registered in ClinicalTrails. gov (NCT05115500). The results of the PRaG3.0 study, regardless of the outcome, are intended to be published in a peer-reviewed international medical journal.^21,22 The reporting of the trial's findings will adhere strictly to the guidelines set forth in the Consolidated Standards of Reporting Trials (CONSORT) statement.

Consent to Participate

Prior to being enrolled in the study, every patient must execute a written informed consent form. The required documentation necessitates the signatures of both the study participant and the research professional delegated by the investigator to oversee the consent process. Consent acquisition at each participating center will be managed by the designated study investigator or coordinator. The signed consent form specifically authorizes the collection and use of the participant's data and biological specimens, and its completion must be formally recorded within the patient's medical record.

Discussion

The results of this trial hold significant promise for advancing the treatment landscape of HER2-expressing advanced solid tumors, addressing key challenges associated with tumor heterogeneity and resistance to standard therapies. The PRaG3.0 protocol is built on a synergistic framework that integrates multiple therapeutic modalities. By combining the ADC RC48-ADC with hypofractionated radiotherapy (HFRT), an immune checkpoint inhibitor, GM-CSF, and IL-2, this regimen seeks to create a powerful, multi-pronged antitumor response. The ability of RC48 to induce immunogenic cell death (ICD) is central to this strategy, as it not only enhances its direct cytotoxic potential but also primes the immune system for further activation, making it an ideal partner in this combination approach.^9,15–19

Early data suggesting a favorable objective response rate (ORR) across diverse tumor types, ¹⁴ underscore the potential versatility of this regimen for targeting HER2 expression regardless of tissue origin. This pan-cancer approach aligns with the broader trend of moving toward biomarker-driven therapies. ²³ Additionally, the dual role of HFRT—providing both local tumor control and immune activation—cements its importance as a central component of this strategy. It is particularly crucial for facilitating antigen release and enhancing the efficacy of subsequent immune therapies. ¹¹

However, this complex multi-modal approach introduces potential challenges that require careful consideration. Close monitoring of the safety profile is essential due to the risk of overlapping toxicities, particularly from ADC-related adverse events and immune-related complications. Additionally, the variability in HER2 expression levels (IHC 1+, 2+, 3+) across tumors may influence treatment efficacy, suggesting the need for further stratified analyses to identify optimal patient subgroups.^5,24

However, this study has limitations. The single-arm design prevents direct comparison with a control group, which may introduce selection bias. The diversity of tumor types and HER2 expression levels (heterogeneity) may also impact the uniformity of the results. To address this, we will document the percentage of HER2 staining and correlate it with response rates.

Safety is a priority given the multi-modal nature of the regimen. We have implemented strict monitoring protocols for overlapping toxicities, such as radiation pneumonitis and immune-related adverse events (irAEs). Specific stopping rules and dose-reduction algorithms for RC48 and radiotherapy are defined in the protocol to manage grade 3-4 adverse events.

The exploratory nature of this trial provides a foundation for future research to refine the regimen, including adjustments in dosing schedules, biomarker-guided patient selection, and combination strategies with other novel agents.

As HER2-targeted therapies evolve, this study underscores the importance of leveraging the unique mechanisms of ADCs like RC48 to enhance the effectiveness of radiotherapy and immunotherapy, potentially setting a new standard for personalized cancer treatment. Future studies should focus on long-term outcomes such as overall survival (OS), progression-free survival (PFS), and quality of life, as well as the mechanisms underlying the observed immune modulation. The inclusion of correlative studies examining cytokine levels, immune cell populations, and tumor microenvironment changes will be critical to fully elucidate the biological basis of the observed clinical responses. With continued validation, the PRaG3.0 regimen could establish a transformative framework for treating HER2-positive and HER2-low tumors across multiple cancer types.^21,22

Conclusion

The PRaG3.0 protocol outlines an innovative and investigative treatment strategy that merges ADC therapy (RC48) with radioimmunotherapy. This approach is designed to effectively target HER2-expressing cancers, encompassing tumors with challenging HER2-low expression. The success of this regimen could pave the way for a highly effective combined treatment, leveraging the synergistic mechanisms of RC48 and the PRaG immune-stimulation platform to substantially improve clinical outcomes for patients afflicted with advanced HER2-expressing solid tumors.