Application of combination of OH2 oncolytic virus and PD-1 inhibitor in preparation of antitumor drugs

Abstract

The invention discloses an application of an OH2 oncolytic virus combined with a PD-1 inhibitor in preparation of an antitumor drug. The OH2 oncolytic virus and the PD-1 inhibitor are jointly applied to tumor treatment, and through the synergistic effect of the OH2 oncolytic virus and the PD-1 inhibitor, the anti-tumor immune response is enhanced, and the treatment effect is improved. According to the combined treatment scheme, tumor cells can be directly killed, recurrence and metastasis of tumors can be inhibited by activating systemic immune response, and a new treatment choice is provided for tumor patients. In the application, the neutrophile granulocyte number level in the blood of a tumor patient is closely associated with the treatment effect of the anti-tumor medicine, the anti-tumor medicine can be used for evaluating the treatment effect, and the anti-tumor medicine is suitable for common tumor adaptations including colorectal cancer, esophageal cancer, head and neck tumor, gastric cancer, biliary tract system tumor, sarcoma, melanoma and other tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and specifically to the use of OH2 oncolytic virus combined with PD-1 inhibitor in the preparation of anti-tumor drugs. Background Art

[0002] Cancer is a major disease that threatens human health and life, long ranking among the top two causes of mortality. Cancer is a large group of diseases that can arise from virtually any organ or tissue in the body. Abnormal cells grow uncontrollably, dividing and expanding without restraint, exceeding their normal boundaries, invading adjacent parts of the body, and / or spreading to other organs. Tumors and malignant tumors are other common names for cancer. There are many types of tumors, including brain tumors, liver tumors, gastrointestinal tumors, and melanoma, depending on their location. Numerous treatment options are available for tumors, with surgery, radiotherapy, and chemotherapy being the most common. While these approaches can control tumor growth to some extent, they suffer from significant side effects, limited efficacy, and the development of drug resistance. In recent years, immunotherapy, as an emerging treatment option, has demonstrated remarkable clinical efficacy by activating or enhancing the patient's own immune system to attack tumor cells. However, the effectiveness of single-agent immunotherapy remains limited by the immunosuppressive state of the tumor microenvironment and the heterogeneity of tumor cells.

[0003] PD-1 (programmed death receptor-1) is an important immune checkpoint molecule primarily expressed on the surface of activated T cells. Binding of PD-1 to its ligand, PD-L1, inhibits T cell activity, leading to immune escape. PD-1 inhibitors, by blocking the PD-1 / PD-L1 pathway and restoring T cell anti-tumor activity, have demonstrated significant clinical efficacy in a variety of tumor types. However, the response rate to monotherapy with PD-1 inhibitors is limited, and some patients develop primary or secondary drug resistance.

[0004] Oncolytic virus (OV) therapy is a new anti-tumor method that uses viruses to attack cells to destroy tumor tissue. Oncolytic viruses are viruses that can infect and destroy tumor cells. They can selectively target and dissolve tumor cells while sparing normal cells. The anti-tumor effect of OV depends on the induction of local and systemic immune responses. OVs preferentially replicate in malignant cells and ultimately induce tumor cell death. In addition, virus-mediated lysis releases tumor-associated antigens, which very effectively stimulate the immune system and promote the induction of anti-tumor immunity. Although some viruses are naturally oncolytic, most OVs are genetically engineered to increase tumor selectivity and improve efficacy. More than 40 OVs from at least 10 different virus families have entered clinical development.

[0005] In recent years, combination therapy strategies have become an important direction in cancer treatment. By combining drugs or treatments with different mechanisms of action, the limitations of single therapy can be overcome and the therapeutic effect can be improved. The combination therapy of PD-1 inhibitors and oncolytic viruses has potential synergistic effects: oncolytic viruses can release antigens by lysing tumor cells, thereby enhancing the immunogenicity of tumors and changing the tumor microenvironment to make them more sensitive to PD-1 inhibitors; while PD-1 inhibitors can relieve the immunosuppressive state of T cells and further enhance the anti-tumor immune response induced by oncolytic viruses. In a phase Ib trial for the treatment of advanced melanoma, OV combined with immune checkpoint inhibitors (ICIs) significantly increased the number of CD8 + T cell infiltration rate and response rate. However, an international randomized, double-blind, multicenter phase III trial from J Clin Oncol showed that the combination of PD-1 inhibitors and T-VEC (a recombinant herpes simplex virus type 1 approved by the FDA and other regulatory agencies) did not significantly improve patient survival outcomes compared with the PD-1 inhibitor control group.

[0006] OH2 is a recombinant herpes simplex virus type 2 (HSV-2) that selectively replicates in tumor cells. OH2 is based on wild-type HSV-2 with the ICP34.5 and ICP47 genes deleted to reduce virulence and enhance oncolytic activity. In addition to these deletions, a human GM-CSF gene expression cassette has been inserted into the OH2 viral genome, mediating GM-CSF protein expression in the tumor microenvironment and enhancing the host immune response. The OH2 virus differs from T-VEC in that its backbone is derived from HSV-2. Preclinical studies have demonstrated that HSV-2 (similar to OH2) exhibits superior tumor suppression compared to HSV-1 (similar to T-VEC) in several tumor-bearing animal models, including breast cancer, colon cancer, and metastatic ovarian cancer. Consequently, OH2 has made progress in the clinic and is currently undergoing late-stage clinical trials. A Phase Ia / Ib clinical trial evaluating the safety and preliminary efficacy of OH2 has been completed in China for patients with unresectable Stage III-IV melanoma, primarily acral melanoma. OH2 oncolytic virus therapy demonstrated a favorable safety profile with no dose-limiting toxicities. OH2 also demonstrated durable anti-tumor efficacy in melanoma patients, particularly those who progressed after PD-1 inhibitor therapy. With the advancement of clinical research, the application of OH2 oncolytic virus in tumor treatment is becoming increasingly widespread, primarily targeting patients with solid tumors, including colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, biliary tract tumors, sarcomas, and melanoma.

[0007] As mentioned above, existing combination therapy studies have not achieved significant improvements in the survival outcomes of cancer patients. Therefore, it is of great clinical significance and application prospects to study the combined application of OH2 oncolytic virus and PD-1 inhibitors, conduct clinical verification, and then develop a combination treatment regimen based on OH2 oncolytic virus and PD-1 inhibitors. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide the use of OH2 oncolytic virus combined with PD-1 inhibitors in the preparation of anti-tumor drugs. This invention proposes for the first time the combined use of OH2 oncolytic virus and PD-1 inhibitors in tumor treatment, which, through the synergistic effect of the two, enhances the anti-tumor immune response and improves the therapeutic effect. This combined treatment regimen can not only directly kill tumor cells, but also inhibit tumor recurrence and metastasis by activating the systemic immune response, providing a new treatment option for cancer patients.

[0009] The present invention provides at least the following technical solutions:

[0010] In a first aspect, the present invention provides the use of OH2 oncolytic virus combined with PD-1 inhibitor in the preparation of anti-tumor drugs.

[0011] In a second aspect, the present invention provides a method for screening cancer patients, comprising the following steps: collecting peripheral blood from cancer patients, detecting a baseline ANC value in the blood, and screening cancer patients based on the baseline ANC value:

[0012] Baseline ANC higher than 4.0×10 9 / L tumor patients are suitable for treatment with OH2 oncolytic virus combined with PD-1 inhibitors;

[0013] Baseline ANC at 4.0×10 9 Patients with tumors below 400 μg / L are suitable for receiving OH2 oncolytic virus monotherapy.

[0014] In a third aspect, the present invention provides the use of a reagent for detecting the number of neutrophils in the preparation of a product for evaluating the therapeutic efficacy of tumors, wherein the tumor treatment method is OH2 oncolytic virus combined with PD-1 inhibitor treatment.

[0015] The present invention has at least the following beneficial effects:

[0016] 1. The examples of this invention demonstrate that the combined use of the OH2 oncolytic virus and PD-1 inhibitors significantly enhances anti-tumor immune responses, improves therapeutic efficacy, and improves patient survival outcomes. This dual mechanism of action reduces the risk of drug resistance associated with single-agent therapies. The OH2 oncolytic virus is tumor-specific and has minimal effects on normal cells; PD-1 inhibitors are widely used clinically and have a high safety profile.

[0017] 2. As verified by the examples of the present invention, the neutrophil count level of tumor patients before treatment is closely correlated with the efficacy of tumor treatment. Therefore, the neutrophil count level can be used as an efficacy predictor to more accurately screen patient groups suitable for treatment with OH2 oncolytic virus combined with PD-1 inhibitors, providing a scientific basis for optimizing treatment plans and improving treatment effects.

[0018] 3. The technical solution provided by the present invention has a simple detection method, low cost, and good stability, and can become a potential predictive tool for clinicians. The tumor treatment solution provided by the present invention is applicable to a variety of solid tumors and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The first test group and the second test group provided in the embodiment of the present invention have an ANC cutoff value of 4.0×10 9 The overall survival comparison results of the patients with ANC>4.0×10 / L were shown in Table 1, where (a) was the high ANC group (ANC>4.0×10 9 / L) of the subjects, (b) is the overall survival curve of the subjects in the first and second test groups (low ANC group (ANC ≤ 4.0 × 10 9 / L) overall survival curve of subjects. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] The present invention's "T-VEC (Talimogene Laherparepvec)", also known as "Imlygic", is a genetically modified herpes simplex virus type 1 (HSV-1). It is the first oncolytic virus drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma patients with skin, subcutaneous and lymph node lesions after initial surgery. T-VEC is injected intratumorally and can selectively replicate in tumor cells, causing tumor cells to rupture and die, while locally releasing GM-CSF (granulocyte-macrophage colony-stimulating factor) in the tumor tissue, which is a white blood cell growth factor that can activate systemic immune responses. The mechanism of action of T-VEC includes inducing tumor tissue dissolution and stimulating a systemic anti-tumor immune response. As an oncolytic virus drug, T-VEC still needs further improvement in the treatment of patients with advanced melanoma or PD-1 resistant melanoma.

[0022] The specific mechanism of action of the "PD-1 inhibitor" of the present invention includes relieving T cell inhibition, enhancing T cell function, and activating immune memory, which has been disclosed in existing research. It should be understood that in the context of the present invention, "PD-1 inhibitor" is a clinical drug and is not limited to a specific antibody drug. PD-1 antibodies that are already on the market or will be approved for marketing in the future should be considered as long as they can block the binding of PD-1 to PD-L1 / PD-L2 and restore the recognition function of T cells to tumor cells, thereby achieving anti-tumor effects through the autoimmune system. It should not be understood to be limited to the Putelizumab (Lepu Biotech Co., Ltd.) selected in the clinical application examples of the present invention. Specifically, the "PD-1 inhibitors" of the present invention include but are not limited to Nivolumab (trade name: Opdivo), Pembrolizumab (trade name: Keytruda), Sintilimab (trade name: Daboshu), Toripalimab (trade name: Tuoyi), Camrelizumab (trade name: Erica), Tislelizumab (trade name: Tislelizumab), and Trade name: BeiGene), Serplulimab (Trade name: Hanszhuang), Durvalumab (Trade name: Imfinzi), Avelumab (Trade name: Bavencio), Cemiplimab (Trade name: Yuto), Pucotenlimab (Trade name: Puyouheng), Penpulimab (Trade name: Aniko). Due to limited space, they will not be listed here one by one.

[0023] The "OH2" or "OH2 virus" of the present invention, which stands for "recombinant oncolytic herpes simplex virus type II," is produced by deleting the neurotoxic gene ICP34.5 and the immunosuppressive gene ICP47 from the viral genome using molecular cloning, DNA (not evaluated) homologous recombination, and inserting a human hGM-CSF expression cassette into the ICP34.5 region. The nucleotide sequence of the expression cassette is shown in SEQ ID NO: 1. The "OH2 virus" strain is named H2d3d4-hGF, and its proposed classification name is Herpes Simplex Virus Type 2. The depository is the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 3600, and the deposit date is February 3, 2010. In the present invention, "OH2" or "OH2 virus," as an oncolytic virus capable of killing tumor cells, should be interpreted as an anti-tumor drug in the specific context of the present invention.

[0024] The term "injection" in this invention refers to sterile solutions (including emulsions and suspensions) prepared from pharmaceuticals for injection, as well as sterile powders or concentrated solutions for preparation into solutions or suspensions prior to use. Injections are rapid and reliable, unaffected by pH, enzymes, food, and other factors, exhibit no first-pass effect, and can exert systemic or localized effects. They are suitable for patients who are not suitable for oral medication or cannot take oral medication. It should be understood that, in this invention, "OH2 injection" consists of "OH2 virus" particles and a buffer to preserve the "OH2 virus."

[0025] The term "OH2 oncolytic virus monotherapy" in this invention should be understood as treatment with only the "OH2 virus" or "OH2 virus preparation," such as treatment with the "OH2 injection." "OH2 oncolytic virus + PD-1 inhibitor combination therapy" and "OH2 oncolytic virus combined with PD-1 inhibitor therapy" both refer to treatments that involve both the "OH2 virus" and a PD-1 inhibitor.

[0026] The "RECIST" of the present invention is a standard for evaluating the efficacy of immunotherapy for solid tumors, and is a set of standards for evaluating the efficacy of anti-tumor treatment. It was first developed by the National Cancer Institute of the United States and the National Cancer Institute of Canada in 2000 (v1.0), and was revised and re-editioned in 2009 (v1.1). The RECIST standard provides a definition of tumor treatment efficacy, including complete remission (CR), partial remission (PR), stable disease (SD) and progressive disease (PD). The "iRECIST" of the present invention is a guideline for evaluating the efficacy of immunotherapy based on the revised RECIST 1.1 standard, specifically for clinical trials of immunotherapy for solid tumors. "iRECIST" solves the limitations of "RECIST 1.1" in immunotherapy by introducing the concepts of "unconfirmed progression" and "confirmed progression", improves the accuracy of evaluating the efficacy of immunotherapy, prolongs the assessment of progression-free survival, and better reflects the patient's survival benefit.

[0027] "OS" in this context stands for Overall Survival. In medical research, particularly in oncology, OS is an important indicator for evaluating therapeutic efficacy, measuring the time from randomization (or start of treatment) to patient death. OS is the gold standard for assessing the long-term efficacy of anti-cancer therapy because it directly reflects the impact of treatment on patient survival.

[0028] The "PFS" of the present invention is the abbreviation of Progression-Free Survival, that is, "progression-free survival", which refers to the time from the start of randomization (or the start of treatment in a single-arm trial) to tumor progression or death from any cause (whichever occurs first). This indicator is very important in tumor clinical trials because it provides an intermediate endpoint for measuring the effect of treatment and can evaluate the treatment effect earlier than overall survival (OS). "iPFS" is the progression-free survival assessed based on the iRECIST standard, and "miPFS" is the median progression-free survival assessed based on the iRECIST standard.

[0029] The "iORR" of the present invention is an indicator related to the evaluation of the therapeutic efficacy of tumors (solid tumors only) based on the iRECIST standard. The "objective response rate (iORR)" refers to the sum of the proportions of partial response (iPR) and complete response (iCR) of immune efficacy. The higher the iORR, the more patients achieved tumor shrinkage using this immunotherapy method.

[0030] "DCR," as used in this invention, stands for Disease Control Rate (DCR), a metric used to assess the effectiveness of cancer treatment. It describes the percentage of patients with advanced cancer who achieve complete remission (CR), partial remission (PR), or stable disease (SD) following treatment. Specifically, DCR is calculated as follows: DCR = (CR + PR + SD) / evaluable number of cases, representing the percentage of patients achieving remission (including complete remission and partial remission) and stable disease after treatment compared to the total number of evaluable cases.

[0031] The "mOS" of the present invention stands for median overall survival, also known as half survival time. It refers to the survival time corresponding to a cumulative survival rate of 0.5, that is, 50% of patients can survive this time period. The median survival time is an indicator for evaluating the efficacy of cancer patients' survival. Generally speaking, the median survival time refers to the time when 50% of patients can still survive after a certain treatment. The median survival time is often used to judge the prognosis of patients with malignant tumors. The longer the median survival time, the longer the overall survival time of patients with malignant tumors. In addition, the length of the median survival time can also judge the effectiveness of new treatment regimens. If it is found in clinical studies that the median survival time of patients is prolonged after receiving a certain treatment regimen, or significantly exceeds the survival time of the current standard treatment regimen, then the new treatment regimen can usually be recommended or applied to clinical treatment, bringing certain benefits to patients, such as increasing the patient's survival rate and improving the patient's quality of life.

[0032] "ANC" in the present invention is the abbreviation of Absolute Neutrophil Count, which refers to the number of neutrophils in the blood. "Baseline ANC" in the present invention refers to the number of neutrophils in the blood of a patient or subject before receiving a tumor treatment regimen.

[0033] In this context, a "cutoff value" refers to a predetermined boundary value (critical value) or threshold used to distinguish between different results (e.g., normal vs. abnormal, negative vs. positive). This value is derived based on statistical principles and large-sample data analysis to determine whether a medical indicator exceeds the normal range, thereby further determining whether intervention or treatment is necessary.

[0034] The “preferred” aspects of the present invention are merely intended to describe implementation methods or examples with better effects, and should be understood as not limiting the scope of protection of the present invention.

[0035] The OH2 oncolytic virus selectively infects tumor cells, replicates intracellularly, and causes cell lysis, releasing tumor antigens and viral particles. The release of tumor antigens activates antigen-presenting cells such as dendritic cells (DCs), initiating specific T cell immune responses. The release of viral particles further stimulates the innate immune response and enhances the immune activity of the tumor microenvironment. By lysing tumor cells and activating the immune response, the OH2 oncolytic virus provides a more immunogenic environment for PD-1 inhibitors. The combined use of the two can significantly improve the therapeutic efficacy of tumors and inhibit tumor recurrence and metastasis.

[0036] Neutrophils are an important type of white blood cells, and they play a vital role in the human immune system. The normal count level of neutrophils is very important for assessing the health status of an individual. In the early stage, the applicant of the present invention only focused on the prediction of the efficacy of OH2 virus in treating melanoma (Chinese patent application number: 202510633072.8, the patent name is "Application of neutrophil count in predicting the efficacy of oncolytic virus treatment of tumors"), and the research on the treatment of tumors with OH2 oncolytic virus combined with PD-1 inhibitors is still blank. There is no precedent for using ANC as a predictor to predict the efficacy of combined treatment or to guide combined treatment of tumors. In this embodiment of the present invention, regardless of cancer indications, a comprehensive analysis of ANC and efficacy evaluation indicators was conducted to verify the role of ANC as a prognostic factor for multiple tumors and to examine its relationship with the clinical response to tumor treatment. The results of the study revealed the biological effects of OH2 oncolytic virus combined with PD-1 inhibitors in tumor treatment and provided support for personalized cancer treatment and prognosis.

[0037] Based on this, an embodiment of the present invention provides the use of OH2 oncolytic virus combined with PD-1 inhibitor in the preparation of anti-tumor drugs.

[0038] In certain embodiments, the PD-1 inhibitor includes at least one of Nivolumab, Pembrolizumab, Sintilimab, Toripalimab, Camrelizumab, Tislelizumab, Serplulimab, Durvalumab, Avelumab, Cemiplimab, Pucotenlimab, and Penpulimab.

[0039] In certain preferred embodiments, the PD-1 inhibitor is Pucotenlimab, with the trade name Puyouheng (HX008), which has been approved for use by the China National Medical Products Administration (NMPA).

[0040] In certain embodiments, the anti-tumor drug prepared is suitable for treating at least one of colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, biliary system cancer, sarcoma, and melanoma.

[0041] In certain embodiments, the prepared anti-tumor drug is used to treat a patient with a baseline ANC level in the blood higher than 4.0×10 9 / L tumor patients, and when the baseline ANC level in the patient's blood is 4.0×10 9 When the level of OH2 is below 1 / L, cancer patients can be treated with OH2 oncolytic virus monotherapy.

[0042] In certain preferred embodiments, the patient's blood is the patient's peripheral blood.

[0043] Based on this, an embodiment of the present invention provides a method for screening cancer patients, comprising the following steps: collecting peripheral blood from cancer patients, detecting a baseline ANC value in the blood, and screening cancer patients based on the baseline ANC value:

[0044] Baseline ANC higher than 4.0×10 9 / L tumor patients are suitable for treatment with OH2 oncolytic virus combined with PD-1 inhibitors;

[0045] Baseline ANC at 4.0×10 9 Patients with tumors below 400 μg / L are suitable for receiving OH2 oncolytic virus monotherapy.

[0046] The above-mentioned tumor patients include patients suffering from at least one of colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, biliary system tumors, sarcoma, and melanoma.

[0047] The above-mentioned PD-1 inhibitors include at least one of nivolumab, pembrolizumab, sintilimab, toripalimab, camrelizumab, tislelizumab, serplulimab, durvalumab, avelumab, cemiplimab, pucotenlimab, and penpulimab. The preferred PD-1 inhibitor is pucotenlimab, which is marketed as Puyouheng (HX008).

[0048] Based on this, an embodiment of the present invention provides the use of a reagent for detecting the number of neutrophils in the preparation of a product for evaluating the therapeutic efficacy of tumors, wherein the tumor treatment method is OH2 oncolytic virus combined with PD-1 inhibitor treatment.

[0049] The above-mentioned tumors include at least one of colorectal cancer, esophageal cancer, head and neck tumors, gastric cancer, biliary system tumors, sarcoma, and melanoma.

[0050] The above-mentioned PD-1 inhibitors include at least one of nivolumab, pembrolizumab, sintilimab, toripalimab, camrelizumab, tislelizumab, serplulimab, durvalumab, avelumab, cemiplimab, pucotenlimab, and penpulimab. The preferred PD-1 inhibitor is pucotenlimab, which is marketed as Puyouheng (HX008).

[0051] The embodiments of the present invention provide a clinical application trial of the OH2 oncolytic virus combined with a PD-1 inhibitor for the treatment of tumors. This clinical trial prospectively analyzed data from subjects who received OH2 monotherapy or OH2 combined with PD-1 immunotherapy. The clinical trial was approved by an independent committee of the clinical institution, and all subjects participating in the treatment signed an informed consent form before enrollment.

[0052] Two immunotherapy test groups were established during the entire clinical trial process, namely: the first test group: subjects receiving OH2 monotherapy (N1=71 multiple tumors); the second test group: subjects receiving OH2+HX008 combined therapy (N2=81, multiple tumors); the clinical data of the applicant's previous study (Chinese patent application number: 202510633072.8, patent name "Application of neutrophil count in predicting the efficacy of oncolytic virus therapy for tumors") were screened out for markers and detection thresholds for predicting the efficacy of OH2 in treating tumors; the effect of OH2 combined with PD-1 immunotherapy for tumors was verified in the embodiment of the present invention, and the screened markers and detection thresholds were further verified through the clinical data of the two test groups. The following are the specific details of the clinical trial.

[0053] Clinical trial materials and methods

[0054] 1. Subject inclusion criteria

[0055] 1.1 Inclusion Criteria

[0056] (1) Aged 18 to 75 years (including the cutoff value), male or female; (2) Patients with a clear diagnosis of malignant tumors by pathology or cytology, such as digestive system tumors, head and neck tumors, soft tissue sarcomas, melanoma, etc.; (3) Patients with advanced (unresectable or recurrent / metastatic) disease who have failed standard treatment (disease progression or intolerance) or lack effective treatment methods; (4) Patients with ECOG general status score 0-1 points; (5) Expected survival time is more than 3 months; (6) Previous anti-tumor treatment (including endocrine, chemo / radiotherapy, targeted therapy) has been completed for more than 4 weeks (patients using nitrosourea and mitomycin chemotherapy should stop the drug for more than 6 weeks), and the adverse reactions of the previous treatment have recovered to level 1; (7) Patients who have undergone major surgery must wait for 4 weeks after surgery; (8) At least one measurable or evaluable lesion; (9) There is a lesion suitable for intratumoral injection (the long diameter of the lesion is at least greater than or equal to 0.5 cm); (10) Patients with asymptomatic central nervous system metastasis or asymptomatic brain metastasis after treatment must have no disease progression by computed tomography (CT) or magnetic resonance imaging (MRI), be stable for at least 3 months, and do not require steroid treatment for at least 4 weeks; (11) Laboratory examination: WBC ≥ 3.0×10 9 / L, ANC≥1.5×10 9 / L,PLT≥100×10 9 / L, Hb ≥ 90g / L; blood BUN and blood creatinine are within 1.5 times the upper limit of normal; TBIL ≤ 1.5 times the upper limit of normal; ALT and AST ≤ 2.5 times the upper limit of normal; patients with liver metastasis do not exceed 5 times the upper limit of normal; coagulation function is normal (PT, APTT are within 1.5 times the upper limit of normal); (12) female subjects and their spouses receive effective contraception during treatment and within 3 months after treatment; (13) subjects with herpes need to wait for 3 months after the end of herpes; (14) voluntarily sign the informed consent form, and good compliance is expected.

[0057] 1.2 Exclusion criteria:

[0058] (1) Subjects with serious medical diseases, including serious heart disease, cerebrovascular disease, uncontrolled diabetes, uncontrolled hypertension, severe infection, active gastrointestinal ulcer; (2) Subjects with clinical symptoms of brain metastasis; (3) Subjects with active infection or unexplained fever >38.5℃ during the screening period and before the first dose; (4) Subjects with congenital or acquired immune deficiency (such as HIV infection) or active hepatitis (Hepatitis B reference: HbsAg, Anti-HBs, HBeAg, Anti-HBc, Anti-HBe, HBV DNA (not evaluated) ≥10 / mL, hepatocyte transaminase, etc.; Hepatitis C reference: HCV antibody and HCV RNA (not evaluated), etc.); (5) lesions cannot meet the requirement of 1 mL injection volume in the tumor; (6) pregnant or lactating women; (7) other experimental drugs or antiviral treatments were used or are being used within 4 weeks before treatment; (8) other clinical studies were participated in within the past 4 weeks; (9) patients allergic to herpes virus and drug ingredients; (10) subjects are known to have a history of psychotropic drug abuse, alcoholism or drug abuse; (11) patients with other malignant tumors within 5 years before enrollment, excluding effectively resected cervical carcinoma in situ, low-risk gastrointestinal stromal tumors, breast cancer, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, and papillary thyroid carcinoma; (12) patients with active autoimmune diseases or a history of autoimmune diseases that may relapse, but patients with the following diseases are not excluded and can be further screened: a. type I diabetes, b. hypothyroidism (if it can be controlled with hormone replacement therapy alone), c. controlled celiac disease, d. skin diseases that do not require systemic treatment (such as vitiligo, psoriasis, alopecia), e. . Any other disease that will not recur without external triggers; (13) Any condition that requires systemic treatment with corticosteroids (prednisone > 10 mg / day or equivalent dose of similar drugs) or other immunosuppressants within 14 days before the administration of the study drug, but patients who are currently or have previously used any of the following steroid regimens can be included: a. Epinephrine replacement steroids (prednisone ≤ 10 mg / day or equivalent dose of similar drugs), b. Topical, ophthalmic, intra-articular, intranasal or inhaled corticosteroids with minimal systemic absorption, c. Short-term (≤ 7 days) use of corticosteroids for prophylaxis (e.g., allergy to contrast agents) or for the treatment of non-autoimmune diseases (e.g., delayed hypersensitivity reactions caused by contact allergens); (14) For the OH2 injection and HX008 combination group, subjects who have previously permanently discontinued treatment with PD-1 / PD-L1 monoclonal antibodies due to serious adverse reactions; (15) Patients who are deemed by the investigator to be unsuitable for participation in this trial for any reason.

[0059] 2. Treatment and assessment methods

[0060] 2.1 Treatment method: OH2 is delivered by intratumoral injection and directly applied to skin or subcutaneous lesions, or used for deep lymph node or organ metastases under ultrasound guidance. Based on the longest diameter (d) of the tumor, the volume injected into each lesion tissue is as follows: when d ≤ 1.5 cm, up to 1 mL; when 1.5 cm < d ≤ 2.5 cm, up to 2 mL; when 2.5 cm < d ≤ 5.0 cm, up to 4 mL; when d > 5 cm, up to 8 mL. Injection of multiple tumor tissues is allowed. There is no limit to the number of lesions that can be injected for each patient, but the maximum volume that can be injected during each visit is 8 mL. The administration method of HX008 is intravenous injection.

[0061] 2.2 Efficacy evaluation: The clinical efficacy of solid tumors is evaluated according to the iRECIST standard.

[0062] 3. Statistical analysis methods

[0063] Descriptive statistical methods are used to describe the baseline characteristics of the patients. As needed, the correlation between ANC and patient characteristics is analyzed by χ 2- test and Mann-Whitney U test. A comprehensive survival analysis is performed on the overall survival (OS) and progression-free survival (PFS). The survival analysis uses the Kaplan Meier method and log-rank test. A univariate Cox proportional hazards regression model is further applied to determine independent prognostic factors of ANC, adjusting for potential confounding variables such as age, gender, ECOG score, previous treatment lines, clinical stage, and treatment duration, etc. The results are presented in the form of hazard ratio (HR) and 95% confidence interval (95% CI).

[0064] 4. Results and analysis

[0065] [[ID=]17] In the embodiments of the present invention, the relationship between the treatment efficacy of patients in each test group and the absolute neutrophil count (ANC), and this ANC value is the baseline ANC, that is, before receiving the treatment regimen, the neutrophil count in the blood of each subject.

[0066] 71 patients with solid tumors (the first test group) treated with OH2 monotherapy from a multi-cohort and multi-indication phase I / II clinical trial (NCT03866525) were included in the analysis and received OH2 monotherapy. Most patients (n = 30) had colorectal cancer, among which the baseline ANC of 30 patients > 4.0×10 9 / L, and the baseline ANC of the other 41 patients ≤ 4.0×10 9 / L. The detailed patient demographics are shown in Table 1. The data show that baseline factors such as age, gender, disease, and disease stage did not reach statistical significance (all P > 0.05), and there was no obvious difference between patients with high and low baseline ANC levels.

[0067] Table 1

[0068]

[0069]

[0070] The present invention also retrospectively analyzed the baseline ANC and efficacy data of 81 patients with various tumors in the second test group (NCT03866525). These 81 patients received the OH2+HX008 combination therapy. Most of the patients (n=31) had colorectal cancer, and 38 of them had a baseline ANC>4.0×10 9 / L, and the baseline ANC of another 43 patients was ≤4.0×10 9 / L. Detailed patient demographic characteristics are shown in Table 2.

[0071] Table 2

[0072]

[0073] The present invention analyzes the baseline ANC of 71 patients in the first test group and 81 patients in the second test group with multiple tumors. The baseline ANC cutoff value is determined to be 4.0×10 9 / L, and the patients’ mOS, miPFS, iORR, and DCR were calculated, as shown in Table 3.

[0074] Table 3

[0075]

[0076]

[0077] From the results in Table 3, we can see that the low ANC group (ANC≤4.0×10 9 / L) of the cohort, the mOS was 10.1 months, miPFS was 1.4 months, iORR was 5.7%, and DCR was 34.3%; in contrast, the high ANC group (ANC>4.0×10 9 In the cohort of subjects with ≥50% ANC (ANC>4.0×10), the mOS was 4.2 months, the miPFS was 1.4 months, the iORR was 4.0%, and the DCR was 20%. mOS (HR=2.6; 95% CI 1.5-4.4), miPFS (HR=1.4; 95% CI 0.8-2.2), iORR (OR=0.7; 95% CI 0.1-10), DCR (OR=0.5; 95% CI 0.1-1.7), the results showed that the high ANC population (ANC>4.0×10 9 The above results show that the cutoff value of baseline ANC is set at 4.0×109 / L, it can accurately predict the best efficacy of OH2 monotherapy in cancer patients, that is, baseline ANC>4.0×10 9 When the ANC was ≤4.0×10 / L, the response rate of patients receiving OH2 monotherapy was low. 9 When the ANC was ≤4.0×10 / L, the patients had a higher response rate to OH2 monotherapy. 9 Patients with tumors with P<0.05 were more likely to benefit from OH2 monotherapy.

[0078] From the results in Table 3, we can also see that the low ANC group (ANC≤4.0×10 9 / L) and high ANC group (ANC>4.0×10 9 There was no significant difference in the efficacy of OH2+HX008 combination treatment in subjects with ≥10% L / L.

[0079] Table 3 compares the high ANC group (ANC>4.0×10 9 It can be seen from the efficacy data of the subjects in the second test group who received OH2+HX008 combination treatment that the efficacy of the subjects in the second test group was significantly better than that of the subjects in the first test group who received OH2 monotherapy. Figure 1 The results show that the baseline of the first and second test groups was higher or lower than the ANC cut-off value (4.0×10 9 / L) when the overall survival was compared, Figure 1 (a) The high ANC group (ANC>4.0×10 9 / L) of the subjects' overall survival curve, Figure 1 (b) The low ANC group (ANC≤4.0×10 9 / L) of the subjects' overall survival curve. Figure 1 The results show that in the high ANC group (ANC>4.0×10 9 / L), the overall survival of the group treated with OH2+HX008 combination therapy was significantly longer than that of the group treated with OH2 monotherapy. Figure 1 The results showed that compared with monotherapy, the high ANC group (ANC>4.0×10 9 / L) showed a high response rate and good prognosis to the OH2+HX008 combination therapy.

[0080] The above results indicate that there is a close correlation between the level of neutrophil count (baseline ANC) in the blood of cancer patients before treatment and the efficacy of OH2. The reason is that, on the one hand, in the clinically non-benefiting patient group, an increase in the baseline ANC level is accompanied by an increase in the number of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). These polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) have a strong immunosuppressive function and can inhibit T cell function, thereby weakening the anti-tumor efficacy of OH2; on the other hand, high levels of neutrophils can inhibit the replication of OH2 by secreting excessive INF-γ, thereby limiting the effect of OH2 in treating tumors.

[0081] In summary, neutrophils are a key indicator of systemic inflammatory response, and their levels are closely related to tumor development and prognosis. They can be used as a marker to predict the efficacy of combined treatment with OH2 and PD-1 inhibitors in tumor patients. The critical value (threshold) for prediction is 4×10 9 / L, that is, the baseline ANC level is higher than 4×10 9 Patients with high ANC levels are recommended to receive OH2+PD-1 immunotherapy, which has a significant effect. 9 Patients with tumors with low ANC levels below 1 / L are recommended to receive OH2 monotherapy.

[0082] The present invention has been described in detail above. Specific examples have been used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is intended only to facilitate understanding of the present invention and its core concepts. It should be noted that those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of OH2 oncolytic virus combined with PD-1 inhibitor in the preparation of anti-tumor drugs.

2. The use according to claim 1, wherein the PD-1 inhibitor comprises at least one of nivolumab, pembrolizumab, sintilimab, toripalimab, carrelizumab, tislelizumab, serotonin, durvalumab, avelumab, sepalizumab, putalumab, and penpulimab.

3. The use according to claim 1, wherein the PD-1 inhibitor is putalimab.

4. The use according to claim 1, wherein the anti-tumor drug prepared is suitable for at least one of colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, biliary system tumors, sarcoma, and melanoma.

5. The use according to claim 1, wherein the prepared anti-tumor drug is used to treat a patient with a baseline ANC level in the blood higher than 4.0×10 9 / L tumor patients. The use according to claim 5 , wherein the patient's blood is the patient's peripheral blood.

7. A method for screening tumor patients, comprising the following steps: Collect peripheral blood from cancer patients and test the baseline ANC value in the blood. Screen cancer patients based on the baseline ANC value: Baseline ANC higher than 4.0×10 9 / L tumor patients are suitable for treatment with OH2 oncolytic virus combined with PD-1 inhibitors; The tumor patients include patients suffering from at least one of colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, biliary system tumors, sarcoma, and melanoma.

8. The method according to claim 7, wherein the PD-1 inhibitor comprises at least one of nivolumab, pembrolizumab, sintilimab, toripalimab, camrelizumab, tislelizumab, serotonin, durvalumab, avelumab, sepalizumab, putalumab, and penpulimab.

9. Use of a reagent for detecting neutrophil count in the preparation of a product for evaluating the therapeutic efficacy of tumors, wherein the tumor is treated with an OH2 oncolytic virus combined with a PD-1 inhibitor; and the tumor includes at least one of colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, biliary system tumors, sarcoma, and melanoma.

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