Application of PLTP as biomarker and target for predicting the effect of liver cancer immunotherapy

Abstract

The application discloses application of PLTP as a biomarker and target point for predicting the effect of liver cancer immunotherapy. Specifically disclosed is application of a biomarker and / or a substance for detecting the biomarker in preparation of a product for predicting or assisting in predicting the effect of liver cancer immunotherapy. The application also discloses application of PLTP and an inhibitor thereof in liver cancer immunotherapy. Experiments show that the PLTP inhibitor can inhibit the occurrence and development of tumors, and is used for preventing or treating liver cancer, and the PLTP inhibitor and a liver cancer immunotherapy drug (such as an immune checkpoint inhibitor) can synergistically enhance the anti-tumor effect of the immune checkpoint inhibitor. The predictive biomarker developed by the application can maximize the effect of the immune checkpoint inhibitor, screen out a population that can benefit from ICIs treatment, and expand the response population of ICIs treatment through multi-target point combination therapy, thereby bringing survival benefits to more patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of PLTP as a biomarker and target for predicting the efficacy of immunotherapy for liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancers worldwide. More than half of HCC patients are diagnosed at an advanced stage, making them ineligible for liver resection or transplantation. Tumor immunotherapy has revolutionized cancer treatment and is considered a strategy for treating and even potentially curing cancer. Immune checkpoint inhibitors (ICIs) are the most thoroughly researched class of immunotherapy drugs to date, working by activating the immune system to kill tumor cells. The most common ICIs are monoclonal antibodies against PD-1 / PD-L1 and CTLA-4, which can be used for patients with various types of cancer. However, only a small percentage of patients achieve long-term and durable efficacy, many develop primary or acquired resistance, and immunotherapy is expensive and has certain toxic side effects. Therefore, researching and developing biomarkers that better predict the efficacy of immunotherapy and accurately identifying patients who can benefit from PD-1 / PD-L1 inhibitors is crucial.

[0003] Compared to targeted therapy, tumor immunotherapy requires a wide range of biomarkers and detection methods to guide clinical practice. This is because different immunotherapies have different mechanisms of action, such as targeting and activating or inhibiting T-cell receptors (CTLA-4 and PD-1). Secondly, multiple immunosuppressive mechanisms exist within the tumor microenvironment. Therefore, biomarkers for immunotherapy are more complex than those for targeted drugs. Currently, many popular biomarkers for predicting immunotherapy efficacy have emerged, such as PD-L1, a protein associated with the tumor inflammatory microenvironment, and tumor mutation burden (TMB), representing neoantigen levels. However, these still have limitations. PD-L1 expression level is the only predictive biomarker approved by the US FDA. However, PD-L1 has limited sensitivity and specificity. The detection of PD-L1 expression levels is influenced by various factors, the variety of antibody types, and inconsistent cut-off values, leading to some inaccuracies in PD-L1 quantification. Therefore, using this single indicator alone cannot fully reflect the immune microenvironment. At the genetic level, TMB can reflect neoantigens and tumor immunogenicity produced by somatic cells to some extent, thus predicting a patient's responsiveness to immunotherapy. However, TMB alone cannot fully characterize the process of antigen production, presentation, and immune response, and it cannot distinguish between treatment-sensitive and insensitive individuals when used alone. Establishing predictive biomarkers can maximize the efficacy of immune checkpoint inhibitors.

[0004] PLTP is a lipid transporter with a boomerang shape. Its N- and C-terminal barrel structures connect to form hydrophobic channels for lipid molecule transport. It transports amphiphilic lipids between circulating lipoproteins and between lipoproteins, cells, and tissues, and is a major factor influencing VLDL, LDL, and HDL plasma levels, turnover, and function. Besides its lipid metabolism function, PLTP is also an important inflammatory factor, participating in various physiological and pathological processes and closely related to dyslipidemia, metabolic syndrome, cardiovascular disease, hypercoagulable states, and infection-induced inflammation. Studies have shown that in chronic obstructive pulmonary disease (COPD), PLTP can inhibit macrophage activation and reduce lung damage caused by inflammatory factors. TCGA-LIHC data analysis shows that patients with high PLTP expression have poor prognosis.

[0005] Therefore, finding more precise biomarkers with clinical translational potential can guide liver cancer patients to receive ICIs treatment, screen out the population that can benefit from ICIs treatment as well as the drug-resistant population, and expand the responding population of ICIs treatment through multi-target combination therapy, bringing survival benefits to more patients. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker that can be used to predict or assist in predicting the efficacy of immunotherapy monotherapy for liver cancer, and / or the application of this biomarker as a target in improving the efficacy of immunotherapy monotherapy for liver cancer. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To achieve the above objectives, the present invention first provides any of the following applications of biomarkers and / or substances for detecting said biomarkers: A1) Application in the preparation of products for predicting or assisting in the prediction of the efficacy of immunotherapy monotherapy for liver cancer; A2) Use in the preparation of products for evaluating or assisting in the evaluation of the efficacy of immunotherapy monotherapy for liver cancer; A3) Application in the preparation of products for predicting or assisting in the prediction of progression-free survival in hepatocellular carcinoma treated with immunotherapy alone; The biomarker may be PLTP.

[0008] Furthermore, the PLTP includes the PLTP protein or the PLTP gene.

[0009] Furthermore, the products include, but are not limited to, reagents, kits, chips, or test strips.

[0010] The preparation includes development and / or screening.

[0011] The nucleotide sequence of the PLTP gene mRNA may be SEQ ID No. 1, and the amino acid sequence of the PLTP protein may be SEQ ID No. 2.

[0012] Furthermore, the substance used to detect the biomarker may include a substance that detects the biomarker using immunohistochemistry (IHC) technology.

[0013] Furthermore, the substance used to detect the biomarker may be a substance used to detect the expression level of PLTP in the tumor tissue (such as baseline tumor tissue) of the liver cancer patient to be tested.

[0014] In the above applications, the substance used to detect the biomarker may include reagents for detecting the expression level or content of PLTP protein.

[0015] In the above applications, the reagent may include antibodies, peptides, proteins, or nucleic acid molecules that bind to PLTP proteins.

[0016] The antibodies that bind to PLTP protein described in this article include antibodies against PLTP protein or their functional fragments (such as antibody variable region Fv, single-chain antibody ScFv, antigen-binding fragments Fab or Fab', F(ab')2, Fab'-SH, etc.).

[0017] The present invention also provides a kit that may include any of the substances described herein for detecting the biomarkers, and the kit may have at least one of the following uses: B1) Predict or assist in predicting the efficacy of immunotherapy monotherapy for liver cancer; B2) To assess or assist in the evaluation of the efficacy of immunotherapy monotherapy for liver cancer; B3) Predict or assist in predicting the duration of progression-free survival in patients receiving immunotherapy monotherapy for liver cancer.

[0018] The kit may be a efficacy prediction kit, efficacy assessment kit, or companion diagnostic kit.

[0019] The test sample for the kit can be a tumor tissue sample.

[0020] Furthermore, the kit may include PLTP monoclonal antibody or PLTP polyclonal antibody, and the kit may also include immunohistochemical reagents.

[0021] The present invention also provides any of the following applications of the biomarker PLTP as a target: C1) Application in the preparation of products for improving the efficacy of immunotherapy monotherapy for liver cancer; C2) Application in the preparation of products for the treatment or adjuvant treatment of liver cancer; C3) Application in the preparation of products that inhibit the occurrence and / or development of liver cancer tumors; C4) in the preparation of products for use in combination with immunotherapy for liver cancer.

[0022] This invention also provides any of the following applications of PLTP inhibitors: D1) Application in the preparation of products for improving the efficacy of immunotherapy monotherapy for liver cancer; D2) Application in the preparation of products for the treatment or adjuvant treatment of liver cancer; D3) Application in the preparation of products that inhibit the occurrence and / or development of liver cancer tumors; D4) in the preparation of products for use in combination with immunotherapy for liver cancer.

[0023] Furthermore, the product may be a reagent or a drug.

[0024] The PLTP inhibitor may have at least one of the following effects: E1) Inhibit or reduce the expression or activity of the PLTP gene; E2) inhibits or reduces the transcription of the PLTP gene into mRNA; E3) inhibits or reduces the translation of the PLTP gene into protein; E4) Inhibits or reduces the activity or function of PLTP protein.

[0025] In the above applications, the PLTP inhibitor may include substances that reduce the expression level or content of PLTP protein, or substances that inhibit the expression of the PLTP gene. The substances may include one or more of the following: nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0026] Furthermore, the inhibition of PLTP gene expression can be achieved through gene mutation, gene silencing, gene knockout, gene editing, or gene knockdown techniques well known to those skilled in the art. For example, RNA interference (RNAi) technology can specifically knock out or shut down the expression of a specific gene; gene editing tools can include CRISPR / Cas9 technology, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs), but are not limited to these. Gene knockdown techniques, which inactivate or silence PLTP gene expression at the post-transcriptional or translational level, are well known to those skilled in the art. These gene knockdown techniques include, but are not limited to, RNA interference, Morpholino interference, antisense nucleic acids, ribozymes, or dominant-negative repressive mutations.

[0027] Gene silencing is well known to those skilled in the art, which utilizes shRNA or siRNA expressed by viruses (such as lentiviruses and adeno-associated viruses) to suppress gene expression.

[0028] The nucleic acid molecules may include shRNA, microRNA, siRNA, and / or antisense oligonucleotides.

[0029] Furthermore, the shRNA (short hairpin RNA), microRNA, siRNA (small interfering RNA), and / or antisense oligonucleotides (such as antisense RNA) are used to suppress the expression of the PLTP gene.

[0030] In the above applications, the nucleic acid molecule may include shRNA, and the shRNA may include any of the following: F1) The shRNA targets and interferes with the expression of the PLTP gene; The encoding DNA sequence of the shRNA described in F2) is SEQ ID No. 3.

[0031] The PLTP inhibitor may also be an antibody, which may be an antibody against PLTP protein or a functional fragment thereof.

[0032] The PLTP inhibitor may also be a lentivirus or adeno-associated virus, which may be a recombinant lentivirus or a recombinant adeno-associated virus expressing shRNA (such as the shRNA shown in SEQ ID No. 3) for knocking down the PLTP gene.

[0033] In this article, the immune monotherapy mentioned can be immune checkpoint inhibitor therapy.

[0034] In this article, the immune monotherapy may include anti-PD-1 immunotherapy or anti-PD-L1 immunotherapy.

[0035] In this article, the drugs used in the immune monotherapy may be PD-1 inhibitors and / or PD-L1 inhibitors.

[0036] The present invention also provides a combination of drugs for the prevention or treatment of liver cancer, the combination of drugs including any of the PLTP inhibitors and liver cancer immunotherapy drugs described herein.

[0037] Furthermore, the immunotherapy drug for liver cancer may be an immune checkpoint inhibitor, which includes PD-1 inhibitors (such as anti-PD-1 antibodies) and / or PD-L1 inhibitors (such as anti-PD-L1 antibodies).

[0038] Furthermore, the immunotherapy drugs for liver cancer include, but are not limited to, pembrolizumab, nivolumab, sintilimab, toripalimab, or durvalumab.

[0039] The purpose of the above applications may be for disease diagnosis, disease prognosis and / or disease treatment, or their purpose may be non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment.

[0040] This invention utilizes immunohistochemistry to detect PLTP expression levels in tumor tissues from liver cancer patients and found that PLTP expression levels are significantly correlated with progression-free survival (PFS) in liver cancer patients receiving ICIs. Based on this, a biomarker was developed to predict or assist in predicting the efficacy of immunotherapy monotherapy for liver cancer. Furthermore, the application of PLTP and its inhibitors in improving the efficacy of immunotherapy monotherapy for liver cancer was investigated using PLTP as a target. The experiments of this invention demonstrate that: 1. PLTP can be used as a biomarker to predict or assist in predicting the efficacy of immunotherapy monotherapy. The efficacy of immunotherapy monotherapy in liver cancer patients can be predicted or assisted in predicting by detecting the expression level of PLTP in the tumor tissue of the patient. The criteria for judgment are: the efficacy of immunotherapy monotherapy in the low PLTP expression group is better or potentially better than that in the high PLTP expression group; the efficacy of immunotherapy monotherapy is reflected in progression-free survival rate or progression-free survival time; at the same follow-up time, the progression-free survival rate of the high PLTP expression group is less or potentially less than that of the low PLTP expression group, or the progression-free survival time is less or potentially less than that of the low PLTP expression group. 2. Reducing PLTP expression in conjunction with immunotherapy monotherapy (such as anti-PD-1 immunotherapy) can significantly inhibit tumor growth, improve the efficacy of immunotherapy monotherapy, and enhance the anti-tumor effect of immunotherapy. That is, the combined use of PLTP inhibitors and liver cancer immunotherapy drugs (such as immune checkpoint inhibitors) can significantly improve the efficacy of immunotherapy monotherapy and enhance the anti-tumor effect of immunotherapy.

[0041] In summary, PLTP can serve as a biomarker for predicting or assisting in predicting the efficacy of immunotherapy monotherapy for liver cancer. The synergistic effect of PLTP inhibitors and immune checkpoint inhibitors can enhance the antitumor activity of immune checkpoint inhibitors. Attached Figure Description

[0042] Figure 1 The relationship between PLTP and patient prognosis was analyzed using a liver cancer database. Figure 1 Figure A shows the relationship between PLTP expression levels and survival rates in liver cancer patients in the TCGA-LIIHC database. Figure 1Figure B shows the TIDE scores for the PLTP high and low expression groups. (*** indicates that the difference reached a significant level (p < 0.001)).

[0043] Figure 2 High expression of PLTP was associated with poorer efficacy of immunotherapy for liver cancer. Among these, Figure 2 Figure A shows the PLTP expression levels in PR, SD, and PD patients; Figure 2 Figure B shows the correlation between PLTP expression levels and progression-free survival in liver cancer patients receiving immunotherapy. Figure 2 The middle C figure shows the PLTP high and low expression groups and CD8. + Percentage of T cell infiltration; Figure 2 The middle D figure shows the PD-L1 levels in the high and low PLTP expression groups. (*** indicates that the difference reached a significant level (p<0.001)).

[0044] Figure 3 To enhance the immunotherapy effect by combining inhibition of PLTP expression in animal models of liver cancer. Figure 3 Figure A shows the tumor volume; Figure 3 Figure B shows the tumor growth in mice of the isotype control group and the anti-PD1 monoclonal antibody group. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] The nucleotide sequence of the PLTP gene mRNA in the following examples is SEQ ID No. 1, and the amino acid sequence of the PLTP protein is SEQ ID No. 2.

[0048] Statistical analysis was performed using SPSS Statistics (version 25.0), GraphPad Prism 8 (GraphPad Software, Inc.), and R (version 3.6.2). The t-test was used to compare data between two groups, and the chi-square test was used to analyze the tumor microenvironment. Survival curves were plotted using the Kaplan-Meier method.

[0049] Example 1: A study on the prognosis and immunotherapy response of PLTP in patients with liver cancer; the correlation of PLTP with the clinical prognosis of liver cancer patients was analyzed using the Tumor Immune Estimation Resource (TIMER) online database, and the results are as follows: Figure 1 As shown in Figure A, the results indicate that the higher the expression of PLTP in the tissues of liver cancer patients, the worse the prognosis of the patients.

[0050] RNAseq data (level 3) and corresponding clinical information for liver cancer were obtained from the Cancer Genome Atlas (TCGA) dataset. The TIDE algorithm was used to predict potential immunotherapy responses. TIDE uses a set of gene expression markers to assess two different tumor immune escape mechanisms: dysfunction of tumor-infiltrating cytotoxic T lymphocytes (CTLs) and rejection of CTLs by immunosuppressive factors. High TIDE scores are associated with poor response to immune checkpoint blockade (ICB) therapy and shorter survival after ICB treatment. Results are as follows: Figure 1 As shown in Figure B, the PLTP high expression group had a higher TIDE score, which meant a worse response to immunotherapy.

[0051] Therefore, PLTP can be used as a biomarker as a prognostic indicator for liver cancer. Patients with high PLTP expression have a poor prognosis and respond poorly to immunotherapy.

[0052] Example 2: Application of PLTP in predicting the efficacy of immunotherapy monotherapy for liver cancer Case selection in this embodiment: This study is a retrospective, non-interventional clinical study. This study has been approved by the Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University, conducted in accordance with the principles of the Declaration of Helsinki, and informed consent has been obtained from all participants.

[0053] This study primarily included patients with hepatocellular carcinoma (HCC) who received anti-PD-1 / PD-L1 antibody therapy at the Fifth Affiliated Hospital of Sun Yat-sen University from September 2019 to August 2022. Inclusion criteria were: 1) clinically diagnosed patients with localized advanced or late-stage HCC; 2) patients receiving immunotherapy for the first time, regardless of the number of lines of treatment. Exclusion criteria were: 1) liver metastases from other tumors; 2) patients without baseline treatment specimens. Enrolled patients received one of the following anti-PD-1 antibodies every two to three weeks: nivolumab (3 mg / kg / dose or 240 mg / dose every two weeks); pembrolizumab, sintilimab, and toripalimab (200 mg / dose every three weeks). Typically, patients underwent enhanced CT scans of the neck, chest, and abdomen every six weeks to assess treatment efficacy, and enhanced MRI of the head was added as needed to further evaluate treatment effectiveness.

[0054] This embodiment includes general patient information such as age at initial diagnosis, sex, ECOG PS score, alcohol consumption status, pathological type, and gene mutation type. Clinical data includes the number of lines of immunotherapy and antibiotic treatment history. Antibiotic treatment history is limited to use from one week prior to immunotherapy to the progression of disease during immunotherapy.

[0055] The efficacy of treatment during the patient's treatment was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). The efficacy evaluation indicators included complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD).

[0056] Progression-free survival (PFS) is defined as the time from the start of immunotherapy monotherapy to disease progression or death.

[0057] All data were obtained through reviewing inpatient medical records and telephone follow-ups. The last follow-up date for this study was March 15, 2023. Surgical tissue specimens were collected from patients prior to immunotherapy. Specimens were immediately harvested according to standard operating procedures and embedded in paraffin blocks after extubation. All tissue specimens were pathologically confirmed as hepatocellular carcinoma. The specimen acquisition and handling procedures were approved by the Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from all specimen providers.

[0058] This study included 26 patients with liver cancer who underwent immunotherapy. Baseline data are detailed in Table 1.

[0059] Table 1. Baseline data of patients receiving immunotherapy for liver cancer

[0060] 1. Detection of PLTP protein expression levels in tissues of 26 liver cancer patients The expression of PLTP protein in the biopsy tissues (baseline tumor tissue) of 26 liver cancer patients was detected by immunohistochemistry (IHC) using Human anti-PLTP monoclonal antibody (Abcam product, Cat#ab282456). The concentration of PLTP antibody used was 1:500. The expression level was expressed by PLTP staining score, with higher scores indicating higher expression levels.

[0061] PLTP protein expression was scored according to the following principle: Staining score (PLTP staining score) = staining intensity × percentage of positive tumor cells × 100. Staining intensity was scored as follows: no staining = 0 (negative), light yellow = 1 (weakly positive), yellow = 2 (moderately positive), and brownish-yellow = 3 (strongly positive). The percentage of positive tumor cells was calculated by examining ten randomly selected fields of view under a high-power microscope (×400). The percentage of positive tumor cells in each field was calculated as the percentage of all tumor cells in that field, and the average of the percentages from the ten fields was used as the positive tumor cell percentage.

[0062] The results are as follows Figure 2 As shown in Figure A and Table 2, PLTP expression was significantly higher in patients with disease progression (PD).

[0063] Table 2. Staining scores of PLTP in tumor tissues and progression-free survival of 20 liver cancer patients.

[0064] In the table above, a 1 in the progress status column 3 indicates a relapse within the follow-up period in column 4, while a 0 indicates no relapse or loss to follow-up within the follow-up period in column 4.

[0065] 2. Survival Curve Based on the optimal cutoff value (PLTP staining score of 6), enrolled patients were divided into a high baseline PLTP expression level group (above 6) and a low baseline PLTP expression level group (below or equal to 6).

[0066] Survival curves were plotted based on progression status and progression-free survival time, and the results are as follows: Figure 2 As shown in Figure B, the analysis results indicate that, under the same follow-up time, the progression-free survival rate was significantly increased in the group with low PLTP expression levels (p < 0.001).

[0067] 3. The relationship between PLTP expression and the tumor immune microenvironment PD-L1 ratio and CD8 + T-cell proportion was assessed based on immunohistochemical staining results. Samples with a tumor proportion score (TPS) >1% were defined as PD-L1+ samples; CD8 + Samples with T cell infiltration >10% are defined as CD8TIL. + Samples. Results showed that the CD8+ expression level was low in the baseline PLTP expression group. + T cell infiltration rate Figure 2 (Figure C) and PD-L1 expression level ( Figure 2 (The middle D diagram) is higher.

[0068] The expression level of PLTP in the aforementioned tumor tissues was reflected by staining scores.

[0069] In summary, the results indicate that the expression level of PLTP in the tumor tissue of liver cancer patients can be used to predict or assist in predicting the efficacy of immunotherapy monotherapy in these patients. The judgment criteria are as follows: Immunotherapy monotherapy was more effective or potentially more effective in patients in the PLTP-low expression group than in patients in the PLTP-high expression group. The efficacy of immunotherapy monotherapy is reflected in progression-free survival rate or progression-free survival time; at the same follow-up time, the progression-free survival rate of the test patients in the PLTP high expression group was less than or less than that of the test patients in the low expression group.

[0070] Therefore, PLTP can be used as a biomarker to assess or assist in assessing the efficacy of immunotherapy monotherapy.

[0071] Example 3: Combining PLTP inhibitors with immunotherapy monotherapy can synergistically enhance the antitumor effect of immunotherapy. PLTP inhibitors can be substances that inhibit PLTP gene expression, silence or knock out the PLTP gene, or substances that inhibit or reduce the content and / or activity of PLTP protein.

[0072] 1. Selection of cell lines and animals The mouse Hepa-1-6 cell line used in this study was obtained from the ATCC Cell Resource Center in the United States.

[0073] The 5- to 6-week-old male C57 mice used in this study were purchased from Zhuhai Beston Biotechnology Co., Ltd. All mice were housed in an SPF-grade environment. All procedures performed in this study strictly adhered to the regulations established by the animal ethics committee.

[0074] 2. Constructing Hepa-1-6 cells with PLTP gene knockdown Based on the mouse PLTP gene sequence and the principles of RNAi, the expression of the PLTP gene was inhibited by knocking down the PLTP gene in Hepa-1-6 cells using a short hairpin RNA (shRNA) sequence. The coding DNA sequence of the designed shRNA is: 5'-GCTGGAGACTATCACCATTCC-3' (SEQ ID No. 3).

[0075] The encoding DNA (SEQ ID No. 3) of the designed shRNA was constructed into a lentiviral cloning vector to obtain a recombinant lentiviral vector named shPLTP, which was provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0076] The recombinant lentiviral vector shPLTP was co-transfected with a packaging plasmid into HEK293T cells, and then packaged in HEK293T cells. After packaging, a recombinant lentivirus expressing shRNA that interferes with the human PLTP gene was obtained, i.e., RNAi virus. After transfecting Hepa-1-6 cells with RNAi virus, PLTP gene knockdown Hepa-1-6 cells were obtained.

[0077] 3. Mouse subcutaneous hepatocellular carcinoma xenograft model Tumor-bearing mouse models were established by subcutaneously transplanting wild-type Hepa-1-6 cells (a mouse liver cancer cell line) and tumor blocks derived from PLTP-knockout Hepa-1-6 cells into 5- to 6-week-old male C57 mice. Three mice were placed in each group, for a total of 12 mice. The six mice with tumor blocks derived from wild-type Hepa-1-6 cells were referred to as wild-type tumor-bearing mice, and the six mice with tumor blocks derived from PLTP-knockout Hepa-1-6 cells were referred to as knockdown tumor-bearing mice.

[0078] The size of the tumor is measured every three days using vernier calipers. The formula for calculating the tumor volume is: Volume = Major Axis × Minor Axis 2 / 2.

[0079] 4. Antitumor treatment of tumor-bearing mice When the maximum diameter of the mouse tumor reached approximately 0.5 cm, the wild-type tumor-bearing mice and the knockdown group tumor-bearing mice were randomly divided into two groups, for a total of four groups, with three mice in each group, and received different treatments: (1) Control group (3 wild-type tumor-bearing mice): On days 1, 4, 7 and 10 after the start of drug administration, each mouse was injected intraperitoneally with 100 μL of phosphate buffered saline solution (product of Wuhan Saiwei Biotechnology Co., Ltd., 1×PBS (0.1M, pH 7.4)).

[0080] (2) PLTP knockdown group (3 tumor-bearing mice in the knockdown group): On days 1, 4, 7 and 10 after the start of drug administration, each mouse was injected intraperitoneally with 100 μL of phosphate buffered saline solution (product of Wuhan Saiwei Biotechnology Co., Ltd., 1×PBS (0.1M, pH 7.4)).

[0081] (3) Anti-PD1 monoclonal antibody group (3 wild-type tumor-bearing mice): On days 1, 4, 7 and 10 after the start of administration, each mouse was injected intraperitoneally with 100 μL of anti-PD1 monoclonal antibody (Innovent Biologics (Suzhou) Co., Ltd.).

[0082] (4) PLTP knockdown combined with anti-PD1 monoclonal antibody group (3 knockdown tumor-bearing mice): On days 1, 4, 7 and 10 after the start of administration, each mouse was injected intraperitoneally with 100 μL of anti-PD1 monoclonal antibody (product of Innovent Biologics (Suzhou) Co., Ltd.).

[0083] Each group consisted of 3 animals, and the drug was administered via intraperitoneal injection. The specific dosage was 10 mg / kg / animal of anti-PD1 monoclonal antibody (product of Innovent Biologics (Suzhou) Co., Ltd.).

[0084] The long and short diameters of the subcutaneous tumors in mice were measured every two days using vernier calipers, and the volume was calculated as long diameter × short diameter. 2 / 2, and growth curves were plotted using tumor volume to observe the survival of mice in each group. The endpoint events were defined as tumor growth reaching a maximum diameter of 2 cm, a weight loss of more than 2 g, or death. At the end of the experiment, mice were euthanized under carbon dioxide anesthesia and dissected to reduce animal suffering.

[0085] The results are as follows: In a mouse subcutaneous hepatocellular carcinoma xenograft model, compared with the control group, the PLTP knockdown group reduced tumor growth rate. Furthermore, the combination of PLTP knockdown and anti-PD1 monoclonal antibody (hereinafter referred to as the dual-antibody treatment group) significantly reduced tumor growth rate and size compared with both the single-agent treatment group (anti-PD1 monoclonal antibody group) and the knockdown group alone (PLTP knockdown group). Figure 3 (Figures A and B in the middle).

[0086] The above results indicate that PLTP knockdown can inhibit tumor growth and achieve the treatment of liver cancer. Furthermore, PLTP knockdown or inhibition of PLTP expression, in combination with anti-PD1 monoclonal antibodies, can significantly inhibit tumor growth, improve the efficacy of immunotherapy monotherapy, and enhance the anti-tumor effect of immunotherapy.

[0087] In summary, PLTP inhibitors can inhibit tumor growth, thereby suppressing tumor occurrence and / or development. They can be used to prevent or treat liver cancer. When used in combination with liver cancer immunotherapy drugs, they can significantly improve the efficacy of immunotherapy monotherapy and enhance the anti-tumor effect of immunotherapy.

[0088] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Any of the following applications of biomarkers and / or substances that detect said biomarkers: A1) Application in the preparation of products for predicting or assisting in the prediction of the efficacy of immunotherapy monotherapy for liver cancer; A2) Use in the preparation of products for evaluating or assisting in the evaluation of the efficacy of immunotherapy monotherapy for liver cancer; A3) Application in the preparation of products for predicting or assisting in the prediction of progression-free survival in hepatocellular carcinoma treated with immunotherapy alone; The biomarker is PLTP; The immunotherapy monotherapy is either anti-PD-1 immunotherapy or anti-PD-L1 immunotherapy.

2. The application according to claim 1, characterized in that, The substances used to detect the biomarkers include reagents for detecting PLTP protein expression levels or PLTP protein content.

3. The application according to claim 2, characterized in that, The reagents include antibodies, peptides, or nucleic acid molecules that bind to PLTP proteins.

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