Molecular marker for evaluating curative effect and prognosis of nasopharynx cancer immunotherapy and therapeutic drug

Abstract

The invention provides a nasopharynx cancer immunotherapy curative effect and prognosis evaluation molecular marker and a nasopharynx cancer immunotherapy prognosis evaluation treatment drug. The molecular marker is MIF. According to the present invention, the research results show that the tumor cells in the group with the ineffective immunotherapy effect secrete more MIF, and the low expression of the MIF is significantly related to the long progression-free survival (PFS); therefore, by detecting the expression level of the MIF, the treatment effect of the nasopharyngeal carcinoma immune checkpoint inhibitor can be evaluated, and the prognosis condition of nasopharyngeal carcinoma patients can be evaluated. Furthermore, the MIF is a target for treatment and synergism of the nasopharyngeal carcinoma immune checkpoint inhibitor, and the MIF inhibitor can be used as a curative effect sensitizer of the nasopharyngeal carcinoma immune checkpoint inhibitor, so that the treatment effect of the nasopharyngeal carcinoma immune checkpoint inhibitor on nasopharyngeal carcinoma is improved. Therefore, the combination with the MIF inhibitor is an effective strategy for treatment synergism of the immune checkpoint inhibitor.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and specifically relates to a molecular marker and therapeutic drug for evaluating the efficacy and prognosis of immunotherapy for nasopharyngeal carcinoma. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in my country. Due to its high incidence in Guangdong Province, it is also known as "Guangdong cancer." With advances in combined radiotherapy and chemotherapy, the prognosis of early-stage NPC has greatly improved, with a five-year survival rate exceeding 95%. However, due to factors such as the hidden site of onset, subtle early clinical symptoms, and a lack of regular NPC screening, the early diagnosis rate for NPC is less than 30%. Over 70% of newly diagnosed NPC cases each year are in the advanced stage, with a five-year survival rate of only approximately 60%. Furthermore, treatment is becoming increasingly difficult, with significant side effects and overall poor efficacy.

[0003] In recent years, cancer immunotherapy has achieved remarkable breakthroughs. Immunotherapy, represented by immune checkpoint inhibitors (such as programmed cell death protein 1 (PD-1) monoclonal antibodies), boasts broad anti-tumor spectrum, long-lasting efficacy in some patients, minimal toxicity, and compatibility with other treatments, significantly improving survival rates for patients with various cancers. In 2018, Honjo Tasuku shared the Nobel Prize in Physiology or Medicine with James P. Allison, the scientist who discovered another immune checkpoint, CTLA-4, for his discovery of the crucial role of PD-1. Cancer treatment has entered a new era of immunotherapy, hailed as the fourth major therapeutic approach for cancer. We previously conducted clinical trials investigating the efficacy and safety of PD-1 antibodies in the treatment of recurrent and metastatic nasopharyngeal carcinoma and lung cancer (Lancet Oncology, 2018; Journal of Clinical Oncology, 2019). The results showed mild toxicity and high tolerability in patients; however, the overall objective response rate was only 20-30%. Furthermore, numerous studies have shown that the efficacy of PD-1 immune checkpoint inhibitors in other cancer types is relatively low, ranging from approximately 10-30%.

[0004] Currently, the main treatment options at home and abroad are combining PD-1 antibodies with conventional treatments such as chemotherapy and radiotherapy, which have shown some improvement in efficacy. Some multi-target immune checkpoint inhibitor combination therapies have been used clinically, but efficacy has not been significantly improved. The key to immunotherapy and a research hotspot are the discovery of markers and new targets that enhance the efficacy of PD-1 antibody therapy, the development of new immunotherapy products, and the development of new immunotherapy regimens. In fact, there are currently no new immunotherapy combination therapies that produce truly synergistic effects for tumors such as nasopharyngeal carcinoma. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a molecular marker and therapeutic drug for evaluating the efficacy and prognosis of immunotherapy for nasopharyngeal carcinoma.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] The first aspect of the present invention provides the use of MIF as a molecular marker in evaluating the therapeutic effect and / or prognosis of nasopharyngeal carcinoma with immune checkpoint inhibitors.

[0008] The second aspect of the present invention provides the use of a reagent for detecting the MIF content in a biological sample in the preparation of a kit for evaluating the therapeutic effect and / or prognosis of nasopharyngeal carcinoma with immune checkpoint inhibitors.

[0009] In some embodiments, the agent is a specific antibody against MIF, preferably a monoclonal antibody.

[0010] The third aspect of the present invention provides the use of an agent that inhibits MIF expression in the preparation of a drug for increasing the therapeutic effect of immune checkpoint inhibitors on nasopharyngeal carcinoma.

[0011] A fourth aspect of the present invention provides the use of an agent that inhibits MIF expression in combination with an immune checkpoint inhibitor in the preparation of a drug for treating nasopharyngeal carcinoma.

[0012] In some embodiments, the agent that inhibits MIF expression is ISO-1.

[0013] In some embodiments, the immune checkpoint inhibitor comprises a PD-1 antibody and / or a PD-L1 antibody.

[0014] A fifth aspect of the present invention provides a pharmaceutical composition for treating nasopharyngeal carcinoma, wherein the pharmaceutical composition comprises an immune checkpoint inhibitor and an agent that inhibits MIF expression.

[0015] In some embodiments, the immune checkpoint inhibitor comprises a PD-1 antibody and / or a PD-L1 antibody; and / or,

[0016] The reagent for inhibiting MIF expression is ISO-1.

[0017] In some embodiments, the immune checkpoint inhibitor and the agent that inhibits MIF expression are each an independent administration unit, or the immune checkpoint inhibitor and the agent that inhibits MIF expression together form a combined administration unit.

[0018] Research has identified a molecular marker, MIF, that is significantly correlated with the efficacy of immune checkpoint inhibitors in nasopharyngeal carcinoma. Tumor cells in the ineffective immunotherapy group secrete more MIF, enhancing interactions with the receptors CD74 / CXCR4 or CD74 / CD44 on microenvironmental immune cells. Low MIF expression is significantly associated with longer progression-free survival (PFS). Therefore, measuring MIF expression levels can be used to assess the efficacy of immune checkpoint inhibitors in nasopharyngeal carcinoma and evaluate the prognosis of nasopharyngeal carcinoma patients.

[0019] Furthermore, compared with MIF inhibitors alone or immune checkpoint inhibitors alone, combined treatment with MIF inhibitors significantly inhibited the growth, size, and weight of NPC xenografts, indicating that MIF is a target for enhancing the efficacy of immune checkpoint inhibitors in NPC. MIF inhibitors can serve as sensitizers for the efficacy of immune checkpoint inhibitors in NPC, increasing their therapeutic efficacy. Therefore, combining treatment with MIF inhibitors is an effective strategy for enhancing the efficacy of immune checkpoint inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The experimental results show that MIF is an effective molecular marker for the efficacy of PD-L1 antibody immunotherapy and patient prognosis in nasopharyngeal carcinoma.

[0021] Figure 2 These are experimental results showing that combining with MIF targeted inhibitors can enhance the effect of PD-L1 antibody immunotherapy for nasopharyngeal carcinoma. DETAILED DESCRIPTION

[0022] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0023] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0024] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0025] The "and / or" mentioned in the present invention describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0026] The following describes the method in conjunction with specific embodiments.

[0027] Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that can promote the secretion or expression of multiple other proinflammatory factors.

[0028] Example 1

[0029] MIF can be used as a specific molecular marker for evaluating the efficacy and prognosis of NPC patients receiving PD-L1 antibody immunotherapy.

[0030] 1. Experimental Methods

[0031] Spatial transcriptomic analysis

[0032] We conducted a prospective NPC clinical trial involving immunotherapy with a PD-L1 antibody and performed spatial transcriptome sequencing and bioinformatics analysis. Nasopharyngeal carcinoma (NPC) biopsy samples were collected from 13 patients (10 in the effective group and 3 in the ineffective group). These samples were formalin-fixed and embedded in paraffin. Tissue sections were 5 μm thick and heated at 42°C for 3 hours. Spatial transcriptomics experiments were performed using the 10X Genomics Visium CytAssist Spatial Gene Expression Kit (Catalog No. PN-1000520) according to the manufacturer's protocol. Sequencing reads were aligned to the human reference genome (UCSC hg38) using the 10X Genomics Space Ranger 1.3.1 pipeline. The feature-barcode matrix for each sample was imported into the R package "Seurat" (version 4.3.0) for normalization, quality control, batch effect correction, dimensionality reduction, and Louvain clustering analysis. Sites expressing fewer than 200 features were excluded from subsequent analysis. Sample-level normalization was performed using the SCTransform function in Seurat (version 4.3.0). Batch effect correction, data integration, and dimensionality reduction were performed according to the instructions in the Seurat tutorial on single-cell RNA sequencing (scRNA-seq) integration (version 4.3.0, https: / / satijalab.org / seurat / articles / integration_introduction.html) without deviation from standard methods.

[0033] Cell type deconvolution and malignancy annotation

[0034] Our previously published NPC single-cell RNA sequencing (scRNA-seq) data (accession number HRA000087, accessible at http: / / bigd.big.ac.cn / gsa-human) were imported into R and processed using the 'CreateSeuratObject' function in Seurat (version 4.3.0). This scRNA-seq dataset served as the reference data, while the spatial transcriptomics data served as the query data. Cell type deconvolution results were added to the Seurat object's metadata for subsequent analysis.

[0035] Ligand-receptor analysis

[0036] To explore cell-type-level cellular interactions from spatial transcriptomics data, we used the CellChat tool to identify significant ligand-receptor (LR) pairs. This analysis inferred cellular interactions at the signaling pathway and receptor-ligand dynamics levels and demonstrated changes in signal intensity between different cell types by visualizing outgoing and incoming signaling patterns.

[0037] Immunohistochemistry (IHC) and evaluation

[0038] Paraffin-embedded tissue sections were collected from 21 nasopharyngeal carcinoma patients before treatment to detect MIF expression. They then underwent PD-L1 antibody immunotherapy and their progression-free survival (PFS) was recorded during follow-up.

[0039] IHC was performed on paraffin-embedded tissue sections from 21 untreated nasopharyngeal carcinoma patients to detect MIF expression. Specimens were cut into 4-μm-thick sections and heated at 65°C for 2 hours. Sections were deparaffinized in xylene, rehydrated through a graded alcohol series, and finally treated in distilled water. To block endogenous peroxidase activity, sections were incubated with 3% hydrogen peroxide solution at room temperature for 15 minutes. Antigen retrieval was performed by heating sections in citrate buffer (pH 6.0; ZSGB-BIO, Beijing, China) in a pressure cooker for 2 minutes. After cooling to room temperature, sections were incubated overnight at 4°C with a diluted primary antibody: anti-MIF antibody (rabbit monoclonal antibody, 1:100; #87501, CST).

[0040] Sections were washed three times with phosphate-buffered saline containing Tween-20 (PBST; ZSGB-BIO) and then incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (DAKO, Santa Clara) for 30 minutes at 37°C. After washing three more times with PBST, sections were stained with 3,3'-diaminobenzidine (DAB) for 2 minutes to visualize target proteins. Sections were then stained with hematoxylin to highlight cell nuclei, rinsed under running water for 2 hours, and dehydrated at 37°C. Finally, sections were mounted with neutral gum (ZSGB-BIO) for preservation.

[0041] Immunohistochemical staining intensity was scored as follows: no staining (0), weak staining (1), moderate staining (2), and strong staining (3). The proportion of tumor cell staining was scored as follows: no staining (0), <25% of cells stained (1), 25-50% (2), 51-75% (3), and 76-100% (4). The immunohistochemical score was calculated by multiplying the staining intensity score by the staining distribution score (total score ranges from 0 to 12). Finally, based on the MIF immunohistochemical score, the cases were divided into two groups: a low MIF expression group (0-8 points) and a high MIF expression group (9-12 points).

[0042] After PD-L1 antibody immunotherapy, the follow-up records were obtained and the PFS of the MIF high expression group and MIF low expression group were analyzed using SPSS software.

[0043] 2. Research Results

[0044] (1) Spatial transcriptomics and ligand-receptor analysis

[0045] Through a prospective clinical trial study of nasopharyngeal carcinoma immunotherapy with PD-L1 antibody (TQB2450), spatial transcriptome sequencing and bioinformatics analysis were performed, and the combined efficacy (10 cases in the effective group vs. 3 cases in the ineffective group) was found to have enhanced interactions between tumor cells and microenvironmental immune cells in the ineffective group ( Figure 1 A~ Figure 1 B) Receptor ligand pair analysis revealed that tumor cells in the ineffective immunotherapy group secreted more MIF, enhancing the interaction with the receptors CD74 / CXCR4 or CD74 / CD44 of microenvironmental immune cells ( Figure 1 C).

[0046] Sequencing expression data further showed that MIF expression in tumor cells of the immunotherapy-ineffective group was significantly increased ( Figure 1 D) The prognosis of patients in the immunotherapy-ineffective group was worse, that is, patients with nasopharyngeal carcinoma with high MIF expression had a worse prognosis after immunotherapy.

[0047] The above results suggest that MIF can be used as a potential molecular marker to evaluate the efficacy of immunotherapy and prognosis of nasopharyngeal carcinoma.

[0048] (2) Immunohistochemistry and prognostic assessment

[0049] To validate the role of MIF in assessing the efficacy and prognosis of NPC immunotherapy, we conducted a study in 21 NPC patients. Paraffin-embedded tissue sections were collected from these 21 patients before treatment to detect MIF expression. These patients then received PD-L1 antibody immunotherapy and their progression-free survival (PFS) was recorded.

[0050] According to the above immunohistochemical staining detection method and grouping criteria, 12 patients were in the MIF high expression group and 9 patients were in the MIF low expression group among 21 nasopharyngeal carcinoma patients. After PD-L1 antibody immunotherapy, the prognosis of patients in the MIF high expression group and the MIF low expression group was analyzed to study the relationship between MIF expression level and patient prognosis.

[0051] Figure 1 E is a representative immunohistochemical staining result of the MIF high expression group and the MIF low expression group. Further analysis of the relationship between MIF expression level and patient prognosis showed that compared with patients in the MIF high expression group, patients in the MIF low expression group had a better progression-free survival, and low MIF expression was significantly associated with a longer progression-free survival (PFS). Figure 1 F).

[0052] The above results indicate that MIF is a molecular marker that is significantly correlated with the efficacy of PD-L1 antibody immunotherapy and the prognosis of patients with nasopharyngeal carcinoma, and can be used to evaluate the efficacy and prognosis of PD-L1 antibody immunotherapy in patients with nasopharyngeal carcinoma.

[0053] Example 2

[0054] MIF-targeted inhibitors effectively improve the therapeutic effect of PD-1 / PD-L1 antibodies in patients with nasopharyngeal carcinoma.

[0055] To explore whether targeted inhibition of MIF can help enhance the efficacy of PD-1 / PD-L1 antibody immunotherapy, we conducted animal experiments by combining the MIF inhibitor ISO-1 with PD-L1 antibody.

[0056] 1. Animal Experimental Methods

[0057] Female PBMC-NSG humanized mice (10 weeks old) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and housed in microisolator cages. Nasopharyngeal carcinoma cell C666 (2×106) cells were suspended in 150 μl of DMEM complete medium containing 20% Matrigel (BD Biosciences) and then subcutaneously injected into 5- to 6-week-old humanized NSG mice. Mice were monitored daily for the development of palpable tumors. When tumors grew to 50-100 mm 3 At 4 hr, treatment (MIF inhibitor ISO-1 [HY-16692, medchemexpress], PD-L1 antibody [TQB2450, Zhengda Tianqing]) was started. The mice were divided into the following groups, with 5 NSG mice in each group: (1) control group: normal saline; (2) PD-L1 antibody group: TQB2450 (15 mg / kg, IP, Q2D×4); (3) MIF inhibitor ISO-1 group: HY-16692 (40 mg / kg, IP, Q2D×8); (4) PD-L1 antibody combined with MIF inhibitor ISO-1 group: HY-16692 (40 mg / kg, IP, Q2D×8) + TQB2450 (15 mg / kg, IP, Q2D×4).

[0058] During the experiment, the size of the tumor was measured using a vernier caliper and calculated according to the formula V = 0.52 × (a 2 × b) to calculate the tumor volume, where a and b are the minimum and maximum diameters of the tumor (in millimeters). 3 The mice were euthanized, and the removed tumors were weighed and photographed.

[0059] 2. Animal Experiment Results

[0060] The results showed that compared with the MIF inhibitor ISO-1 and PD-L1 antibody treatment groups alone, the MIF inhibitor ISO-1 combined with PD-L1 antibody could significantly inhibit the volume growth, tumor size and weight of nasopharyngeal carcinoma transplanted tumors ( Figure 2 ).

[0061] Furthermore, we calculated the tumor inhibition rate of each group using the tumor inhibition rate formula based on the change in tumor volume. The tumor inhibition rate formula is as follows:

[0062]

[0063] The tumor inhibition rates were calculated as follows: the tumor inhibition rate of MIF inhibitor ISO-1 was 0.49, the tumor inhibition rate of PD-L1 antibody was 0.59, and the tumor inhibition rate of the combination drug was 0.90.

[0064] The theoretical combined inhibition rate is predicted using the Bliss Independence formula:

[0065] E Bliss =E A +E B -E A ×E B

[0066] Where: E A = inhibition rate of ISO-1 alone; E B =Inhibition rate of single-agent PD-L1 antibody.

[0067] Get E Bliss =0.79<The actual tumor inhibition rate of combined drug use was 0.90; therefore, MIF inhibitors can effectively improve the therapeutic effect of PD-L1 antibodies in patients with nasopharyngeal carcinoma.

[0068] The above results indicate that MIF is a new target for enhancing the efficacy of PD-1 / PD-L1 antibody immunotherapy in patients with nasopharyngeal carcinoma, and the combination of PD-1 / PD-L1 antibody and MIF targeted inhibitor is an effective strategy for enhancing the efficacy of immunotherapy for nasopharyngeal carcinoma.

[0069] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0070] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of MIF as a molecular marker in evaluating the therapeutic efficacy and / or prognosis of nasopharyngeal carcinoma with immune checkpoint inhibitors.

2. Use of reagents for detecting MIF content in biological samples in the preparation of kits for evaluating the therapeutic efficacy and / or prognosis of nasopharyngeal carcinoma using immune checkpoint inhibitors.

3. The use according to claim 2, characterized in that The reagent is a specific antibody against MIF, preferably a monoclonal antibody.

4. Use of reagents that inhibit MIF expression in the preparation of drugs that enhance the therapeutic efficacy of immune checkpoint inhibitors in nasopharyngeal carcinoma.

5. Use of reagents that inhibit MIF expression in combination with immune checkpoint inhibitors in the preparation of drugs for the treatment of nasopharyngeal carcinoma.

6. The use according to claim 4 or 5, characterized in that The reagent for inhibiting MIF expression is ISO-1.

7. The use according to any one of claims 1 to 6, characterized in that The immune checkpoint inhibitors include PD-1 antibodies and / or PD-L1 antibodies.

8. A pharmaceutical composition for treating nasopharyngeal carcinoma, characterized in that: The pharmaceutical composition includes an immune checkpoint inhibitor and an agent that inhibits MIF expression.

9. The pharmaceutical composition according to claim 8, wherein The immune checkpoint inhibitors include PD-1 antibodies and / or PD-L1 antibodies; and / or, The reagent for inhibiting MIF expression is ISO-1.

10. The pharmaceutical composition according to claim 8 or 9, wherein The immune checkpoint inhibitor and the agent that inhibits MIF expression are each an independent administration unit, or the immune checkpoint inhibitor and the agent that inhibits MIF expression together form a combined administration unit.

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