In this single-center,open-label, phase I study, the safety and efficacy of PIN in combination with programmed cell death protein antibody (anti-PD1) therapeutic regimen will be evaluated in patients with refractory primary advanced hepatocellular carcinoma(HCC) . A total of 25 to 30 patients are planned to be enrolled and receive PIN plus anti-PD1 combined treatment. It aims to: 1).assess the safety and antitumor effects of the above combined treatment regimen. 2).detect the dynamic changes and molecular characteristics of PIN-induced CD8+ T cells with special phenotype in peripheral blood (PB) and transformation of tumor microenvironment (TME) after the treatment with PIN. 3).evaluate the immunological or clinical predictive biomarkers for toxicity and efficacy.

Currently, systemic therapy for advanced primary HCC mainly relies on targeted therapy and immunotherapy.However,after disease progression, patients often have limited treatment options and poor prognosis.Therefore, no standard therapies beyond second-line are recommended, highlighting the urgent need for innovative treatments.

Several clinical studies have found that oncolytic viruses (OVs) can provide clinical benefits to patients with various malignant tumors, including advanced HCC.To date, there are hundreds of projects in clinical trial stages, especially in recent years, new generations of OVs developed or in clinical stages have shown better safety and stronger anti-tumor capabilities. Through genetic engineering, OVs can express target genes that have anti-tumor effects, such as granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin-12(IL-12),etc, further enhancing their anti-tumor effects. Despite these advances, how to obtain a more durable antitumor immune response and long-term benefits is still an urgent clinical issue.

Previous studies have confirmed that the Newcastle disease oncolytic virus (NDV) can selectively infect tumor cells while sparing normal cells, demonstrating an acceptable safety profile. In this study, investigators have developed a nove PIN . Preclinical studies have shown that combining PIN with anti-PD1 therapy can reverse the immunosuppressive microenvironment and transform "cold" tumors into "hot" tumors, thereby triggering local and systemic anti-tumor immune responses and significantly improving the efficacy of the immune checkpoint inhibitor(ICI). Based on these preclinical findings, investigators are conducting this clinical trial to evaluate the safety and anti tumor activity of the PIN and anti-PD1 combination therapy in vivo.

In this study, 25 to 30 subjects with refractory advanced HCC will be enrolled. The initial dose for the first cycle will be determined as 4e9 or 8e9 viral particles based on the number of injectable lesions, their longest diameter, and the tumor volume capacity.

Following the first cycle of treatment, the subsequent dose and injection sites of PIN will be adjusted based on the permissible volume of the injected tumor mass, according to the following principles:

PIN injection frequency: day 0 and day 3, per 3 weeks for 8 cycles; unless unavailability of injection lesion, disease progression (PD) or serious intolerable adverse events (AEs).

PIN injection dosage:

1. a.For patients with a single injectable lesion with a maximum diameter of <8 cm, the initial cycle's PIN dose is 4e9 viral particles. Subsequent cycles will maintain this dose of 4e9 or increase it to 8e9 viral particles based on the lesion's capacity to accommodate the injection volume; b. For patients with a single injectable lesion with a maximum diameter of ≥8 cm, the initial cycle's PIN dose is 8e9 viral particles. Subsequent cycles will maintain this dose of 8e9 viral particles based on the lesion's capacity to accommodate the injection volume.
2. a.For patients with two injectable lesions, injections will alternate between the two lesions after two cycles. The initial cycle's PIN dose is 4e9 viral particles, and the second cycle will maintain this dose of 4e9 or increase it to 8e9 viral particles based on the tumor volume's capacity; b.For patients with injectable lesions with a maximum diameter of ≥8 cm, the initial cycle's PIN dose is 8e9 viral particles, and subsequent cycles will maintain this dose of 8e9 or decrease it to 4e9 viral particles based on the lesion's capacity.
3. a.For patients with multiple injectable lesions (≥ 3), after 1-2 cycles of injections in each injectable lesion, injections are alternated between lesions. The initial injection dose for each lesion is determined by the size of the lesion; b.For lesions <3 cm, the initial cycle's dose is 4e9 viral particles, and the second cycle will maintain this dose of 4e9 or increase it to 8e9 viral particles based on the tumor volume's capacity; c.For lesions ≥3 cm, the initial cycle's injection dose is selected as 8e9 viral particles, and subsequent cycles will maintain this dose of 8e9 or decrease it to 4e9 viral particles based on the tumor volume's capacity.
4. After injections, if the tumor shrinks by 0.5-1 cm in diameter, the injection dose should be adjusted to 2e9 viral particles until the tumor disappears.

Anti-PD1 infusion frequency: day -3, per 3 weeks for 8 cycles; until unacceptable toxicity occurred or PD.

Objectives:

The primary objective are to assess the safety and adverse event profile of the combination regimen.

The coprimary objective is immune response, assessed by CD8+T cells with special phenotype by Fluorescence Activating Cell Sorter (FACS). The secondary objectives are to evaluate disease control rate (DCR), objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and quality of life.

Inclusion Criteria:

1. Age 18-75 (inclusive).

2. Eastern Cooperative Oncology Group (ECOG) performance status ≤2 and Estimated life expectancy of more than 3 months.

3. Histopathological /cytological or diagnosed clinically confirmed locally advanced or metastatic HCC having undergone treatments recommended by the "Primary Liver CancerDiagnosis and Treatment Guidelines (2024 Edition)" ,which is refractory/relapsed after and/or intolerant of standard therapies (including targeted therapy and immunotherapy) or for which no subsequent standard therapy exists.

4. At least one measurable lesion at baseline according to investigators Response Evaluation Criteria in Solid Tumours 1.1 (RECIST 1.1).

5. Patients with injectable lesions (those suitable for direct injection or injection with the assistance of medical imaging), defined as follows: at least one injectable lesion in the skin, mucous membrane, subcutaneous tissue, lymph node or visceral organ with a longest diameter ≥10 mm.

6. Subjects are willing to accept tumor rebiopsy in the process of this study.

7. Barcelona Clinic Liver Cancer (BCLC) stage ≤C.

8. Adequate organ function as defined by the following criteria:

   • Absolute neutrophil count (ANC) ≥ 1 x 10^9/L, Platelet count ≥50 x 10^9/ L, hemoglobin (Hgb) ≥ 80g/L ;
   • Serum creatinine≤1.5 upper limit of normal (ULN) or creatinine clearance (as estimated by Cockcroft Gault) ≥60 mL/min;
   • Serum aspartate amino transferase (AST) and alanine aminotransferase (ALT), ≤5 x ULN ; Total serum bilirubin ≤3 x ULN);
   • Cardiac ejection fraction ≥ 50%, no evidence of pericardial effusion as determined by an echocardiogram (ECHO), and no clinically significant electrocardiogram (ECG) findings;
   • International Normalized Ratio (INR) ≤ 1.5 times the upper limit of normal (ULN), and Activated Partial Thromboplastin Time (APTT) ≤ 1.5 times ULN;
   • Baseline oxygen saturation >91% on room air.

9. • Patients with chronic or acute hepatitis B virus (HBV) infection [ as characterized by positive hepatitis B surface antigen (HBsAg) and/or hepatitis B core antibodies (anti-HBcAb) with detectable HBV DNA (≥20 IU/ml) ] must receive effective antiviral treatment before enrollment and during the treatment period, and their HBV DNA levels must be dynamically monitored during each treatment cycle.

   • Patients who test positive for anti-hepatitis B core (HBc) with undetectable HBV DNA (<20 IU/ml) do not require anti-viral therapy prior to enrollment.however, these subjects will be tested at every cycle to monitor HBV DNA levels and initiate antiviral therapy if HBV DNA is detected (≥20 IU/ml).
   • Subjects with chronic infection by hepatitis C virus (HCV), who are untreated, are allowed on study. In addition, subjects with successful HCV treatment are allowed, as long as 4 weeks have passed between completion of HCV therapy and start of study drug.

10. Previous treatments must be completed for more than 4 weeks prior to the enrollment of this study, and subjects have recovered to <= grade 1 Toxicity (except for hematological toxicities and clinically non-significant toxicities such as alopecia).

11. Pregnancy tests for women of childbearing age shall be negative; Both men and women agreed to use effective contraception during treatment and during the subsequent 1 year.

12. Voluntarily participate in this clinical trial and sign an informed consent form.

Exclusion Criteria:

1. Subjects are being treated with either corticosteroids (>10 mg daily prednisone equivalent) or other immunosuppressive medications within 14 days of enrollment.
2. Active central nervous system disease involvement (but allow patients with prior brain metastases treated at least 4 weeks prior to enrollment that are clinically stable and do not require intervention), or prior history of Common Terminology Criteria for Adverse Events (CTCAE) Grade ≥3 drug-related Central Nervous System (CNS) toxicity.
3. Presence or suspicion of fungal, bacterial, viral, or other infection that is uncontrolled or requiring intravenous (IV) antimicrobials for management.
4. Any serious underlying medical (eg, pulmonary, renal, hepatic,gastrointestinal, or neurological) or psychiatric condition or any issue that would limit compliance with study requirements.
5. Major surgery or trauma occurred within 28 days prior to enrollment, or major side effects have not been recovered.
6. Received cytotoxic chemicals, monoclonal antibodies, immunotherapy or other intervene within 4 weeks or 5 half-lives before enrollment.
7. Received radiotherapy within 3 months before enrollment.
8. Patients with primary immunodeficiency or autoimmune diseases requiring immunosuppressive therapy.
9. The presence of uncontrollable serous membrane fluid, such as massive pleural effusion or ascites.
10. Previous or concurrent cancer within 3 years prior to treatment start except for curatively treated cervical cancer in situ, non-melanoma skin cancer, superficial bladder tumors [Ta (non-invasive tumor), Tis (carcinoma in situ) and T1 (tumor invades lamina propria)].
11. Known positive test result for human immunodeficiency virus (HIV) or acquired immune deficiency syndrome (AIDS).
12. Prior organ allograft transplantations or allogeneic hematopoietic stem cell transplantation.
13. History of allergy or intolerance to study drug components.
14. Pregnant or breast-feeding. Women of childbearing potential must have a pregnancy test performed within 7 days before the enrollment, and a negative result must be documented.
15. Being participating any other trials or withdraw within 4 weeks.
16. Researchers believe that other reasons are not suitable for clinical trials.