Extracellular vesicle PD-L2 as a biomarker for evaluating the efficacy of PD-1 therapy in melanoma

Abstract

The present invention relates to the technical field of diagnostic markers, and in particular to a marker for evaluating the efficacy of PD-1 in treating melanoma and its application. Research conducted in the present invention has shown that extracellular vesicle PD-L2 is associated with the clinical response to anti-PD-1 therapy, and its changes are a more reliable biomarker for predicting the prognosis of melanoma patients receiving anti-PD-1 therapy. The baseline level of PD-L2 on the EV membrane and the dynamic changes of PD-L2 on the EV membrane can serve as predictors of the outcome of melanoma immunotherapy and play an important role in evaluating the prognostic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of diagnostic markers, and in particular to a marker for evaluating the effect of extracellular vesicle PD-1 in treating melanoma and its application. Background Art

[0002] Melanoma is a malignant tumor derived from melanocytes. It mostly occurs in the skin, but can also be found in mucous membranes and internal organs, accounting for about 3% of all tumors. In recent years, the incidence and mortality of malignant melanoma have increased year by year, and its age of death is younger than other solid tumors. In addition to early surgical resection, malignant melanoma lacks specific treatment and has a poor prognosis. Therefore, early diagnosis and treatment of malignant melanoma are extremely important. Immunotherapy is one of the fastest-growing tumor treatments in recent years. Among them, certain specific tumors benefit from immune checkpoint inhibitor therapy with PD-1, PD-L1 and CTLA-4 as therapeutic targets, which has reduced mortality and brought new hope to cancer patients. Currently, there are few studies on biomarkers for predicting or evaluating the effect of PD-1 in treating melanoma. In view of this, the present invention is specially proposed. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a marker for evaluating the effect of PD-1 in treating melanoma and its application.

[0004] Specifically, the technical solution of the present invention is as follows:

[0005] In a first aspect, the present invention provides a use of extracellular vesicle PD-L2 in the preparation of a product for predicting or evaluating the effect of immunotherapy for melanoma.

[0006] Preferably, the immunotherapy is PD-1 combined with anti-angiogenic therapy.

[0007] Another preferred embodiment is that the immunotherapy is PD-1 monoclonal antibody therapy.

[0008] In a second aspect, the present invention provides a product for predicting or evaluating the effect of immunotherapy for melanoma, wherein the product comprises a reagent for detecting the expression level of PD-L2 in extracellular vesicles.

[0009] Preferably, the product comprises an ELISA kit for detecting extracellular vesicle PD-L2.

[0010] In a third aspect, the present invention provides a device for predicting or evaluating the effectiveness of immunotherapy for melanoma, the device comprising: a detection module for detecting the content of a marker in a sample to be tested; the marker comprises extracellular vesicle PD-L2; an input module for obtaining the detection results of the detection module; a judgment module for comparing the detection results obtained by the input module with a judgment standard to determine the effectiveness of immunotherapy for melanoma; and an output module for outputting a diagnosis result.

[0011] Preferably, the judgment criteria of the judgment module include: if the level of extracellular vesicle PD-L2 increases after treatment, it is judged that the effect of immunotherapy for melanoma or the prognosis is good.

[0012] Preferably, the sample to be tested is blood, plasma or serum.

[0013] Beneficial effects:

[0014] The present invention provides a marker for evaluating the efficacy of PD-1 in treating melanoma and its application. The marker is extracellular vesicle PD-L2. Research in the present invention has shown that extracellular vesicle PD-L2 is associated with the clinical response to anti-PD-1 monotherapy or combined anti-angiogenic therapy, and its changes are more reliable biomarkers for predicting the prognosis of melanoma patients receiving anti-PD-1 monotherapy or combined anti-angiogenic therapy. Baseline EV membrane PD-L2 levels and dynamic changes in EV membrane PD-L2 can serve as predictors of melanoma immunotherapy outcomes and play an important role in evaluating prognostic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0016] Figure 1 The workflow and characteristic diagram of extracellular vesicles (EVs) described in Example 1 of the present invention are shown below. Figure 1 Figure a is a schematic diagram of the entire research process. Figure 1 Figure b is a schematic diagram of the EV membrane protein array (upper side, used to study the 45 key proteins in the discovery cohort) and the ELISA diagram (lower side, used to measure the concentration of target proteins on the surface of EVs isolated from the validation cohort). Figure 1 Figure c is a representative TEM image showing EVs extracted from melanoma patient plasma, scale bar is 200 nm. Figure 1 Figure d shows the concentration and size distribution of plasma EVs purified using NanoFCM. Figure 1Panel e shows a representative Western blot image of EVs labeling, with CD9, CD81, and Alix as specific EV markers and Calnexin as a negative marker. All lanes were loaded with the same amount of total protein.

[0017] Figure 2 This is the expression profile of EVs membrane proteins in the cohort discovered in Example 1 of the present invention. Figure 2 The heat maps shown in Figures a, b, and c represent the changes in EVs protein expression for each patient in the discovery cohort at baseline (Figure a), 4 weeks after treatment (Figure b), and from baseline to treatment (Figure c), respectively. Figure 2 Figure d in the middle compares the expression levels of EVs membrane proteins in patients with clinical benefit (CB) and non-clinical benefit (NCB), and shows a bar chart of the top 10 proteins.

[0018] Figure 3 The changes in EV membrane protein levels associated with prognosis in the cohort found in Example 1 of the present invention. Among them, Kaplan-Meier survival analysis was used to evaluate progression-free survival (PFS) and overall survival (OS). The baseline EV PD-L2 ( Figure 3 Figures a and b in the middle), EV PD-L2 at 4 weeks after treatment ( Figure 3 After treatment, EV PD-L2 ( Figure 3 The prognostic relevance of the changes in (e and f) was assessed by Kaplan-Meier survival analysis.

[0019] Figure 4 To verify the relationship between EV membrane PD-L2 and prognosis in melanoma patients receiving anti-PD-1 monotherapy in the cohort of Example 1 of the present invention. Figure 4 Figure (a) shows the comparison of EV PD-L2 expression in patients with clinical benefit (CB) and non-clinical benefit (NCB) at baseline. Figure 4 Figures b and c show the PFS (Figure b) and OS (Figure c) results of 68 patients in the validation cohort, respectively. Figure 4 Figure d in the middle shows the comparison of EV PD-L2 expression in patients with clinical benefit (CB) and non-clinical benefit (NCB) after PD-1 treatment. Figure 4 Figures e and f in the middle are the PFS (Figure e) and OS (Figure f) results of the validation cohort, respectively. Figure 4 Figure g shows the comparative results of EV PD-L2 expression in patients with clinical benefit (CB) and non-clinical benefit (NCB) after treatment. Figure 4Figures h and i in the middle are the PFS (Figure h) and OS (Figure i) results of the validation cohort, respectively. DETAILED DESCRIPTION

[0020] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0021] The endpoints and any values of the ranges disclosed in this specification are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0022] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", or "some specific implementation methods" and the like mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0023] In the examples provided herein, if specific techniques or conditions are not specified, the experiments were performed according to those described in literature in the field or according to the product instructions. Reagents or instruments used without manufacturer's indication are conventional products available through regular channels.

[0024] In the examples provided in this specification, unless otherwise specified, the method for EV isolation is:

[0025] EVs were purified by size exclusion chromatography (SEC). 100 μL of 0.7 μm-filtered plasma was separated using an Exosupur column (Echo Biotech, China). The sample was then eluted with 0.01 M PBS, and 2 mL fractions (fractions 3, 4, 5, and 6) were collected according to the manufacturer's instructions.

[0026] In the examples provided in this specification, unless otherwise specified, the characterization method of EVs is:

[0027] The morphology of EVs was examined using transmission electron microscopy (TEM). The concentration and size distribution of purified EVs were measured using nanoFCM (Echo Biotech, China). Surface markers (Alix, CD9, and CD81) and negative markers (Calnexin) of EVs were detected by Western blotting. Antibodies included anti-human Alix (sc-53540, Santa Cruz, USA), anti-human CD9 (ab236630, Abcam, USA), anti-human CD81 (ab79559, Abcam, USA), and anti-human / mouse Calnexin (sc-46669, Santa Cruz, USA). EVs were stained with CellTracker CM-DiI dye (C7000; Thermo Scientific, USA), the plasma membrane was fluorescently labeled, and observed under an Olympus FluoView1000 laser scanning confocal microscope (Olympus Corporation, Japan).

[0028] In the examples provided in this specification, unless otherwise specified, the enzyme-linked immunosorbent assay method is:

[0029] In the validation cohort, all EV samples were randomly pooled to prepare an ELISA standard curve. Based on the standard curve, the appropriate amount of EV sample was diluted to 100 μL with coating buffer (0.1 M, pH 9.6, carbonate solution) and incubated overnight at 4°C. Then, 100 μL of 5% BSA blocking buffer was added, and the cells were washed with PBST for 60 minutes at room temperature. The samples were incubated with anti-PD-L1 (Proteintect, China) and anti-PD-L2 (Invitrogen, USA) antibodies for 2 hours at room temperature. After washing with PBST, goat anti-mouse IgG (ThermoFisher, USA) was added and reacted at room temperature for 1 hour. After incubation with 100 μL of TMB colorimetric solution for 30 minutes, 100 μL of reaction stop solution was added to each well, and the OD value was recorded at a wavelength of 450 nm. For detection of IFN-γ and human IL-2, supernatants from peripheral blood mononuclear cells (PBMCs) and Jurkat cells were harvested and analyzed according to the manufacturer's instructions (BioLegend, USA).

[0030] Example 1

[0031] (1) Study the workflow.

[0032] like Figure 1 Figure A and Figure 1As shown in Figure 2b, this study involved two cohorts (a discovery cohort and a validation cohort). In the discovery cohort, plasma samples from 33 patients with advanced mucosal melanoma who were treated with the anti-PD-1 inhibitor toripalimab and the anti-angiogenesis inhibitor vorolanib were analyzed using an EV membrane protein array to identify EV membrane proteins associated with clinical outcomes. This cohort included 30 baseline plasma samples and 29 samples collected four weeks after the start of treatment. All plasma samples were analyzed using an antibody sandwich expression array to derive the absolute expression profile of EV membrane proteins. The selected EV expression panel included proteins involved in T cell activation, immune-related cytokines, angiogenesis-related ligands and receptors, as well as melanoma-specific and EV-specific markers, providing a detailed view of the tumor microenvironment. In the validation cohort, 68 baseline plasma samples and 67 post-treatment plasma samples (including cutaneous, acral, and mucosal types) from 68 patients with advanced melanoma who were treated with anti-PD-1 monotherapy were analyzed by ELISA to validate the prognostic effect of the target EV membrane protein.

[0033] EVs from plasma samples were purified using SEC. The isolated EVs were characterized by evaluating their size distribution, morphology, and expression of specific markers. Transmission electron microscopy revealed vesicles with a size range of 30–150 nm ( Figure 1 NanoFCM showed a size distribution with an average diameter of 84.94 ± 15.39 nm ( Figure 1 Western blotting confirmed the presence of EV markers Alix, CD81, and CD9, while the negative marker Calnexin was not detected ( Figure 1 Figure e), indicating a high separation efficiency.

[0034] (2) Longitudinal plasma EV membrane protein profiles in patients with mucosal melanoma receiving anti-PD-1 plus antiangiogenic therapy.

[0035] As at baseline, EV membrane protein expression levels varied widely across patients, with some patients showing very high ( Figure 2 However, judging from the time points after treatment and the change levels between baseline and post-treatment, the majority of patients who achieved partial response (PR) and some patients with stable disease (SD) showed an increasing trend in EV membrane protein expression ( Figure 2 These findings suggest that changes in EV membrane protein expression levels reflect the efficacy of anti-PD-1 plus antiangiogenic therapy in mucosal melanoma.

[0036] The study divided patients into a clinical benefit group (CB) and a clinical non-benefit group (NCB) to investigate the relationship between EV membrane proteins and treatment response. The CB group included patients with PR or SD, while the NCB group included patients with progressive disease (PD). To assess the correlation between plasma EV proteins and clinical response, protein expression levels at two time points and changes from baseline to post-treatment were compared between the CB and NCB groups. Figure 2 Panel (d) lists the top 10 proteins with statistically significant differences at each time point. At baseline, EV PD-L2 levels were significantly lower in the CB group than in the NCB group. Comparing changes in EV membrane protein levels between the groups, PD-L2 levels increased in the CB group and decreased in the NCB group. In summary, this study found that EV PD-L2 is associated with clinical response to combined anti-PD-1 and antiangiogenic therapy.

[0037] (3) Changes in EV membrane protein levels are associated with the prognosis of patients with mucosal melanoma.

[0038] Next, Kaplan-Meier survival analysis was used to evaluate the prognostic value of EV membrane proteins. Previous studies have shown that EV PD-L1 can be used as a prognostic biomarker for melanoma. Since PD-L2 is similar to PD-L1 and also binds to PD-1 to mediate immune escape, the present invention preliminarily studied the relationship between PD-L2 expression on plasma EVs and PFS and OS in the discovery cohort. At baseline, patients with high baseline levels of PD-L2 on plasma EVs tended to have a shorter PFS compared with patients with low levels (1.9 months vs. 7.7 months, HR = 1.59, p = 0.3069; P = 0.3069). Figure 3 There was no significant difference in OS (14.9 months vs. 19.2 months, HR = 0.90, p = 0.8431; Figure 3 Four weeks after treatment, patients with higher EV PD-L2 levels had a significantly longer PFS compared with patients with lower EV PD-L2 levels (15.9 months vs. 5.6 months, HR = 0.30, p = 0.0989; Table 5). Figure 3 middle g) and OS (22.9 months vs. 14.9 months, HR = 0.49, p = 0.1964; Figure 3 (middle panel h) showed an improving trend.

[0039] To evaluate the relationship between changes in EV PD-L2 levels and patient survival, the post-treatment level was divided by the baseline level to calculate the fold change (FC), and patients were divided into two groups: FC>0.5 and FC≤0.5. Compared with patients with FC≤0.5, patients with EV PD-L2 FC>0.5 had a significantly longer PFS (11.2 months vs. 1.9 months, HR=0.30, p=0.0073; P<0.001). Figure 3 The OS of the PD-L2 FC>0.5 group was also longer than that of the FC≤0.5 group (19.5 months vs. 14.9 months, HR=0.51, p=0.1663; Figure 3 These findings suggest that changes in EV membrane PD-L2 protein levels are a reliable biomarker for predicting prognosis in melanoma patients receiving combined anti-PD-1 and antiangiogenic therapy.

[0040] (4) The relationship between EV membrane PD-L2 and the prognosis of melanoma patients receiving anti-PD-1 monotherapy.

[0041] The present invention further investigated the relationship between EV membrane PD-L2 in peripheral blood and melanoma prognosis in another cohort of patients receiving anti-PD-1 monotherapy. The validation cohort included 135 plasma samples from 68 patients with different melanoma subtypes, including 26%, 34%, and 18% of cutaneous, acral, and mucosal melanoma, respectively; 22% of patients had melanoma of unknown origin.

[0042] The PD-L2 level on EV membrane was measured by ELISA. At baseline, the EV PD-L2 level of NCB patients was significantly higher than that of CB patients ( Figure 4 Although not statistically significant, patients with higher baseline EV PD-L2 levels tended to have shorter PFS and OS than those with lower levels (mPFS, 3.6 months vs. 5.5 months, HR = 0.79, p = 0.3761; mOS, 16.4 months vs. 26.1 months, HR = 0.60, p = 0.1203; Figure 4 After anti-PD-1 treatment, PD-L2 levels on EV membranes did not differ significantly between CB and NCB patients ( Figure 4 (middle d panel), PFS and OS were comparable between patients with higher and lower PD-L2 levels (mPFS, 5.4 months vs. 4.5 months, HR = 0.84, p = 0.4469; mOS, 24.3 months vs. 23.9 months, HR = 0.94, p = 0.8428; Figure 4 However, when analyzing the changes in EV PD-L2 levels after treatment, it was found that EV PD-L2 levels increased in CB patients, while EV PD-L2 levels decreased in NCB patients ( Figure 4 Compared with patients with decreased PD-L2 levels, patients with increased EV PD-L2 levels had a trend toward prolonged PFS (5.4 months vs. 3.5 months, HR = 0.66, p = 0.2895, Figure 4Notably, patients with elevated EV PD-L2 levels had significantly longer OS than those with decreased levels (30.1 months vs. 19.6 months, HR = 0.49, p = 0.0363, Figure 4 These findings, combining data from two independent cohorts, suggest that baseline EV membrane PD-L2 levels and dynamic changes in EV membrane PD-L2 may serve as predictors of melanoma immunotherapy outcomes.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a reagent for detecting the level and dynamic changes of extracellular vesicle PD-L2 in the preparation of a product for predicting or evaluating the effect of immunotherapy for melanoma, characterized in that: The immunotherapy is PD-1 combined with anti-angiogenesis therapy or PD-1 monoclonal antibody therapy.

2. A device for predicting or evaluating the effect of immunotherapy for melanoma, characterized in that: The device includes: a detection module for detecting the content level and dynamic changes of a marker in a sample to be tested; the marker includes extracellular vesicle PD-L2; the sample to be tested is blood, plasma or serum; an input module for obtaining the detection results of the detection module; a judgment module for comparing the detection results obtained by the input module with the judgment criteria to determine the effectiveness of immunotherapy for melanoma; and an output module for outputting a diagnosis result; the judgment criteria of the judgment module include: if the extracellular vesicle PD-L2 level increases after treatment, it is judged that the immunotherapy for melanoma is effective or the prognosis is good; the immunotherapy is PD-1 combined with anti-angiogenic therapy or PD-1 monoclonal antibody treatment.

Images