This is a Phase I, single-center, open-label, single-arm clinical trial to evaluate the safety and efficacy of personalized neoantigen polyepitope peptide vaccine combined with pembrolizumab in patients with advanced, recurrent or refractory renal cell carcinoma (RCC).

Background: Renal cell carcinoma is one of the most common malignancies of the urinary system. Although pembrolizumab has become a standard first-line treatment, the objective response rate (ORR) of monotherapy is only 20-40%, and most patients eventually develop primary or acquired resistance. Tumor neoantigens are specific antigens produced by tumor-specific gene mutations, with high immunogenicity and tumor specificity, making them ideal targets for tumor immunotherapy. Preliminary clinical studies have shown that neoantigen vaccines can produce synergistic effects when combined with pembrolizumab.

Study Design: This is an investigator-initiated trial (IIT) conducted at Peking University First Hospital. The study will enroll 5-8 patients in two stages: an initial safety assessment cohort (3 patients) followed by an expansion cohort (5 additional patients) if safety criteria are met. The study drug is a personalized neoantigen polyepitope peptide vaccine (Neo-RCC), produced by Mingzhibenyuan Medical Technology (Beijing) Co., Ltd., based on whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) of each patient's tumor tissue. The vaccine is administered via subcutaneous injection in combination with Polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Poly-ICLC) adjuvant, with a priming phase (5 injections on Days 0, 3, 7, 14, 21) and a boosting phase (3 injections on Weeks 6, 12, and 20), totaling 8 injections. Pembrolizumab (200 mg intravenous [IV] every 3 weeks [Q3W]) is administered concurrently as combination therapy.

Primary Objective: To evaluate the safety of the personalized neoantigen peptide vaccine in advanced RCC patients, as measured by the incidence and severity of treatment-emergent adverse events (TEAE) graded by National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0.

Secondary Objectives: To evaluate pharmacokinetic characteristics; to assess efficacy including objective response rate (ORR), duration of response (DOR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1.

Key Eligibility Criteria: Adults (≥18 years) with Stage III or IV, locally advanced, recurrent or metastatic non-surgical RCC who have achieved disease stability for ≥3 months after prior targeted therapy combined with pembrolizumab; measurable disease per RECIST v1.1; Eastern Cooperative Oncology Group (ECOG) performance status 0-3; adequate organ function; and ≥50 tumor gene mutations detectable from biopsy tissue.

Safety Monitoring: A Data Safety Monitoring Board (DSMB) will oversee patient safety. Dose-limiting toxicities (DLT) are defined according to protocol-specified criteria. If ≥2 DLTs occur in the initial cohort, the adjuvant dose will be reduced by 50% and the study will proceed with a de-escalation cohort.

Study Rationale: This study addresses the unmet need for effective second-line or later treatment options for advanced renal cell carcinoma (RCC) patients who have progressed or become refractory after initial immunotherapy. The combination strategy leverages the complementary mechanisms of personalized neoantigen vaccines (which activate tumor-specific T-cell responses) and pembrolizumab (which blocks programmed cell death protein 1 (PD-1)-mediated immune suppression), potentially overcoming PD-1 inhibitor resistance.

Neoantigen Prediction and Vaccine Manufacturing: Tumor tissue and peripheral blood samples are collected for whole exome sequencing (WES) and RNA sequencing (RNA-seq). A proprietary neoantigen prediction platform (neoTrue AI) analyzes sequencing data to identify tumor-specific mutations and predict neoantigen-major histocompatibility complex (MHC) binding affinity. The top 10-30 ranked neoantigen peptides are selected for Good Manufacturing Practice (GMP)-grade synthesis. The median manufacturing period is approximately 12 weeks. Each peptide is 300 μg, mixed with Polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Poly-ICLC) adjuvant (0.5 mg per pool), and administered as 4 pools (left/right axilla and left/right groin) in 250 μL per injection site.

Pembrolizumab Administration: Pembrolizumab 200 mg is administered intravenously (IV) every 3 weeks (Q3W), diluted in 100 mL 0.9% sodium chloride over 30 minutes. Treatment continues for up to 2 years or until disease progression, unacceptable toxicity, or patient withdrawal. During the vaccine manufacturing period (~12 weeks), pembrolizumab may be continued as bridging therapy to maintain disease stability.

Dose-Limiting Toxicity (DLT) Definition: Hematologic: Grade 4 neutropenia (absolute neutrophil count [ANC] <0.5×10⁹/L) lasting >7 days; Grade 4 thrombocytopenia (<25×10⁹/L) within 7 days. Non-hematologic: Grade 3 non-hematologic toxicity lasting >7 days (with specified exceptions); Grade 4 toxicity. Immune-related: ≥Grade 3 immune pneumonitis, colitis, hepatitis; any-grade myocarditis; ≥Grade 2 immune effector cell-associated neurotoxicity syndrome (ICANS) or immune encephalitis; ≥Grade 3 severe skin reactions (Stevens-Johnson syndrome/toxic epidermal necrolysis [SJS/TEN], drug reaction with eosinophilia and systemic symptoms [DRESS]). Other: ≥Grade 2 cytokine release syndrome (CRS) (per American Society for Transplantation and Cellular Therapy [ASTCT] criteria); ≥Grade 3 injection-site reactions requiring surgical intervention; ≥Grade 3 allergic reactions; ≥Grade 3 infections; ≥Grade 3 thromboembolic events; ≥Grade 3 bleeding.

DLT Management: If ≥2 DLTs occur in the first 3 patients during the first 8 weeks (D0-D56), the adjuvant total dose will be reduced by 50% (to 1.0 mg), and 2 additional patients will be enrolled in the de-escalation cohort. If ≥2 DLTs occur in the de-escalation cohort, the study will be terminated. If ≥3 DLTs occur in the expansion cohort, the regimen will be deemed to have unacceptable toxicity.

Efficacy Assessments: Tumor assessments by computed tomography (CT) or magnetic resonance imaging (MRI) per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 are conducted at screening, every 6-8 weeks during treatment, at treatment completion/early termination, and during follow-up. Blinded Independent Central Review (BICR) may be used as sensitivity analysis.

Pharmacokinetic/Immunogenicity Assessments: Peripheral blood mononuclear cells (PBMCs) are collected at baseline, 24-72 hours post-injection, every 2 treatment cycles, and at treatment completion. Neoantigen-specific T-cell responses are quantified by quantitative polymerase chain reaction (qPCR) for interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), granzyme B, and perforin mRNA expression. T-cell receptor (TCR) sequencing is performed to track clonal expansion. Circulating tumor DNA minimal residual disease (ctDNA-MRD) monitoring panels are designed based on each patient's WES data and detected by targeted next-generation sequencing (NGS).

Follow-up Schedule: Long-term follow-up every 3 months for survival status and subsequent anti-tumor treatments.

Risk Mitigation: Comprehensive risk management plans are established for cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), immune-related adverse events (irAEs), infections, injection-related reactions, adjuvant-related risks, pseudoprogression, hyperprogressive disease, and RCC-specific complications (paraneoplastic syndromes, hemorrhage, thromboembolism). A Data Safety Monitoring Board (DSMB) is constituted to oversee safety.

Funding: This study is supported by a research grant from the GenScript Life Science Research Grant Program (2024), with additional support from Mingzhibenyuan Medical Technology (Beijing) Co., Ltd. for neoantigen prediction and peptide synthesis.

Inclusion Criteria:

1. Histologically or cytologically confirmed Stage III or IV, locally advanced, recurrent or metastatic renal cell carcinoma (RCC) not amenable to curative surgery

2. Disease stability for ≥3 months after prior single pembrolizumab therapy, with documented progression or intolerance to standard treatment

3. At least one measurable lesion per RECIST v1.1 (longest diameter ≥10 mm for non-lymph node lesions; short axis ≥15 mm for lymph nodes; or bone lesions confirmed by CT/MRI)

4. Age ≥18 years

5. Estimated life expectancy ≥3 months

6. ECOG performance status 0-3

7. Availability of tumor tissue (biopsy or archival specimen) for whole exome sequencing (WES) and RNA sequencing (RNA-seq), with ≥50 tumor gene mutations detectable

8. Adequate organ function as defined by laboratory parameters within 14 days prior to enrollment:

   8.1 Hematologic: Absolute Neutrophil Count(ANC) ≥1.5×10⁹/L, platelet count ≥100×10⁹/L, hemoglobin ≥90 g/L; 8.2 Hepatic: total bilirubin(TBIL) ≤1.5×Upper Limit of Normal(ULN), Aspartate Aminotransferase(AST)/Alanine Aminotransferase(ALT) ≤2.5×ULN (≤5×ULN if liver metastases present); 8.3 Renal: serum creatinine ≤1.5×ULN or creatinine clearance ≥50 mL/min (Cockcroft-Gault formula); 8.4 Coagulation: international normalized ratio(INR) ≤1.5, activated partial thromboplastin time(APTT) ≤1.5×ULN; 8.5 Cardiac: Left Ventricular Ejection Fractions(LVEF) ≥50% by echocardiography;

9. Ability to comply with study procedures and follow-up schedule;

10. Signed informed consent form;

11. For women of childbearing potential: negative serum pregnancy test (Human Chorionic Gonadotropin sensitivity ≤25 IU/L) within 7 days prior to enrollment; agreement to use effective contraception during study and for 4 weeks after last dose;

12. For men: agreement to use effective contraception during study and for 4 weeks after last dose.

Exclusion Criteria:

1. Pregnant or lactating women

2. Prior treatment with:

   2.1 Two or more lines of immune checkpoint inhibitor (ICI) systemic therapy; 2.2 CTLA-4 inhibitor (e.g., ipilimumab); 2.3 Tumor vaccine therapy (including neoantigen vaccine, dendritic cell vaccine, etc.); 2.4 Chemotherapy specifically for renal cell carcinoma; 2.5 Allogeneic hematopoietic stem cell or solid organ transplantation;

3. Active autoimmune disease or other immune-mediated disorders requiring systemic immunosuppressive therapy;

4. Participation in another investigational drug study within 4 weeks prior to first dose;

5. Active infection including:

   5.1 Known Human Immunodeficiency Virus(HIV) infection; 5.2 Active hepatitis B (HBsAg positive and Hepatitis B Virus-DNA >500 IU/mL) or hepatitis C (HCV-RNA positive); 5.3 Active tuberculosis (T-SPOT or PPD positive with clinical symptoms, or chest CT suggestive of active TB); 5.4 Severe infection requiring intravenous antibiotics within 2 weeks prior to enrollment, or uncontrolled systemic infection;

6. Symptomatic central nervous system (CNS) metastases; exception: patients with treated CNS metastases stable for ≥4 weeks without neurological symptoms and without corticosteroid requirement;

7. Uncontrolled comorbidities including:

   7.1 Symptomatic congestive heart failure (New York Heart Association Class III-IV); 7.2 Unstable angina or myocardial infarction within 6 months; 7.3 Uncontrolled arrhythmia; 7.4 Uncontrolled hypertension (systolic ≥160 mmHg or diastolic ≥100 mmHg despite standard treatment); 7.5 Active peptic ulcer or gastrointestinal bleeding; 7.6 Active interstitial lung disease or pulmonary fibrosis;

8. Other malignancy within 5 years (except cured cervical carcinoma in situ, basal cell carcinoma, or squamous cell skin carcinoma);

9. Live vaccine administration within 4 weeks prior to enrollment or planned during study (inactivated vaccines allowed);

10. Known hypersensitivity to peptide vaccine components, adjuvants (e.g., Montanide ISA-51, Poly-ICLC), or pembrolizumab;

11. Sarcomatoid or rhabdoid RCC as predominant histology (mixed histology allowed if non-pure sarcomatoid/rhabdoid);

12. Any condition that, in the investigator's opinion, would compromise patient safety or compliance;

13. Any other condition that the investigator considers unsuitable for study participation.

IPD and supporting information will be available to qualified researchers upon reasonable request. Interested researchers may contact the Principal Investigator or the study team at Peking University First Hospital to submit a data access request. The request should include a detailed research proposal outlining the scientific rationale, specific data requirements, and planned analyses. Access will be granted following review and approval by the study team, subject to compliance with applicable data protection regulations, patient confidentiality agreements, and institutional policies. De-identified individual participant data, along with the study protocol and statistical analysis plan, may be shared via a secure data transfer mechanism. Data access is contingent upon the execution of a data use agreement and is intended solely for non-commercial research purposes.