Application of extracellular vesicle PD-L2 as marker for evaluating melanoma treatment effect of PD-1

Abstract

The invention relates to the technical field of diagnostic markers, in particular to a marker for evaluating the melanoma treatment effect of PD-1 and application of the marker. Researches show that the extracellular vesicle PD-L2 is related to the clinical response of anti-PD-1 treatment, the change of the extracellular vesicle PD-L2 is a more reliable biomarker for predicting the prognosis of a melanoma patient receiving anti-PD-1 treatment, and the level of PD-L2 on a baseline EV membrane and the dynamic change of PD-L2 on the EV membrane can be used as predictive factors for the result of melanoma immunotherapy. The method plays an important role in prognosis effect evaluation.

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, and can also be seen in mucosa 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 lower than that of 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, and the mortality rate has been reduced, bringing new hope to tumor patients. At present, 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] In order 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 the 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 effect of immunotherapy for melanoma, the device comprising: a detection module, used to detect the content of a marker in a sample to be tested; the marker comprises extracellular vesicle PD-L2; an input module, used to obtain the detection result of the detection module; a judgment module, used to compare the detection result obtained by the input module with the judgment standard to judge the effect of immunotherapy for melanoma; and an output module, used to output the 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 effect of PD-1 in treating melanoma and its application, wherein the marker is extracellular vesicle PD-L2. The present invention's research shows that extracellular vesicle PD-L2 is related to the clinical response of anti-PD-1 monotherapy or combined anti-angiogenic therapy, and its change is a more reliable biomarker for predicting the prognosis of melanoma patients receiving anti-PD-1 monotherapy or combined anti-angiogenic therapy; the baseline EV membrane PD-L2 level and the dynamic changes of PD-L2 on the EV membrane can be used as predictors of the results of melanoma immunotherapy, and play an important role in the evaluation of 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. 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 schematic (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 Panel d shows the concentration and size distribution of plasma EVs purified using NanoFCM. Figure 1Panel e shows a representative Western blot image of EVs markers, 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 after 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 EVs membrane protein levels related to 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 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] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are 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 precise 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, which should be considered as specifically disclosed in this article.

[0022] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means 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 embodiment 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0023] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0024] In the embodiments provided in this specification, unless otherwise specified, the method for EV separation 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 of eluted 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 labeled with fluorescence, 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 ELISA method is:

[0029] In the validation cohort, all EV samples were randomly mixed to prepare the ELISA standard curve. According to the standard curve, the appropriate amount of EV samples was diluted to 100 μL with coating solution (0.1 m pH 9.6, carbonate solution) and incubated at 4 °C overnight. Then 100 μL of 5% BSA blocking solution was added and washed with PBST at room temperature for 60 min. The samples were incubated with anti-PD-L1 (Proteintect, China) and anti-PD-L2 (Invitrogen, USA) antibodies for 2 h at room temperature. After washing with PBST, goat anti-mouse IgG (ThermoFisher, USA) was added and reacted at room temperature for 1 h. After incubation with 100 μL TMB colorimetric solution for 30 min, 100 μL of reaction stop solution was added to each well, and the OD value was recorded at a wavelength of 450 nm. When detecting IFN-γ and human IL-2, the supernatants of 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 Middle a picture and Figure 1As shown in Figure b, this study involved two cohorts (discovery cohort and validation cohort). In the discovery cohort, plasma samples from 33 patients with advanced mucosal melanoma treated with the anti-PD-1 inhibitor toripalimab and the anti-angiogenesis inhibitor vorolanib were analyzed using EV membrane protein arrays to identify EV membrane proteins associated with clinical outcomes. The 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 includes proteins related to 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 skin type, acral type, and mucosal type) from 68 patients with advanced melanoma treated with anti-PD-1 monotherapy were analyzed by ELISA to verify 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 showed that the vesicles were 30-150 nm in size ( 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 The separation efficiency is very high.

[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 levels ( Figure 2 However, judging from the time points after treatment and the change levels between baseline and after treatment, the expression of EV membrane proteins showed an increasing trend in most patients with partial response (PR) and some patients with stable disease (SD) ( 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 clinical benefit group (CB) and 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 evaluate the correlation between plasma EV proteins and clinical response, the protein expression levels at two time points and the changes from baseline to post-treatment were compared between the CB and NCB groups. Figure 2 Figure d in the middle lists the top 10 proteins with statistical differences at each time point according to the significance of the difference. At baseline, the EV PD-L2 level in the CB group was significantly lower than that in the NCB group. When comparing the changes in EV membrane protein levels between different groups, the PD-L2 level in the CB group increased, while that in the NCB group decreased. In summary, the study found that PD-L2 on EVs is associated with the clinical response to anti-PD-1 combined with anti-angiogenic 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, similar to PD-L1, 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; 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 longer PFS compared with patients with lower EV PD-L2 levels (15.9 months vs. 5.6 months, HR=0.30, p=0.0989; Figure 3 middle g) and OS (22.9 months vs. 14.9 months, HR=0.49, p=0.1964; Figure 3 (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 the 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 prolonged PFS (11.2 months vs. 1.9 months, HR=0.30, p=0.0073; Figure 3 Middle k). 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 studied the relationship between EV membrane PD-L2 in peripheral blood and melanoma prognosis in another patient cohort receiving anti-PD-1 monotherapy. The validation cohort included 135 plasma samples from 68 patients with different subtypes of melanoma, of which cutaneous, acral, and mucosal melanoma accounted for 26%, 34%, and 18%, respectively; 22% of patients had melanoma of unknown origin.

[0042] The PD-L2 level on the 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, there was no significant difference in EV membrane PD-L2 levels between CB and NCB patients ( Figure 4 (middle d), 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 tended to have prolonged PFS (5.4 months vs. 3.5 months, HR = 0.66, p = 0.2895, Figure 4It is worth noting that the OS of patients with elevated EV PD-L2 levels was significantly longer than that of patients with reduced 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 embodiments of the present invention.

Claims

1. Application of extracellular vesicle PD-L2 in the preparation of products for predicting or evaluating the effects of immunotherapy for melanoma.

2. The use according to claim 1, characterized in that: The immunotherapy is PD-1 combined with anti-angiogenesis therapy.

3. The use according to claim 1, characterized in that: The immunotherapy is PD-1 monoclonal antibody therapy.

4. A product for predicting or evaluating the effect of immunotherapy for melanoma, characterized in that: The product includes a reagent for detecting the expression level of PD-L2 in extracellular vesicles.

5. The product according to claim 4, characterized in that The product includes an ELISA kit for detecting extracellular vesicle PD-L2.

6. A device for predicting or evaluating the effect of immunotherapy for melanoma, characterized in that: The device includes: a detection module, used to detect the content of a marker in a sample to be tested; the marker includes extracellular vesicle PD-L2; an input module, used to obtain the detection result of the detection module; a judgment module, used to compare the detection result obtained by the input module with the judgment standard to judge the effect of immunotherapy for melanoma; and an output module, used to output the diagnosis result.

7. The device for predicting or evaluating the effect of immunotherapy for melanoma according to claim 6, characterized in that: 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.

8. The device for predicting or evaluating the effect of immunotherapy for melanoma according to claim 7, characterized in that: The sample to be tested is blood, plasma or serum.

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