Application of UBQLN4 detection and inhibitors in immunotherapy and efficacy evaluation

Abstract

The present invention relates to the field of tumor research technology, specifically to the use of UBQLN4 detection reagents in the preparation of detection reagents for measuring PD-L1 expression levels. Furthermore, the present invention relates to UBQLN4 inhibitors and their use in melanoma treatment. The present invention unexpectedly discovered that UBQLN4 and PD-L1 expression are linearly correlated, allowing PD-L1 expression levels to be assessed by detecting UBQLN4. Furthermore, its inhibitors can be used to improve immunotherapy sensitivity.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medicine, and in particular to evaluation indicators for response to PD-1 antibody therapy. Background Art

[0002] Malignant melanoma (MM) is a highly malignant tumor. Treatment options include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Emerging immunotherapies primarily target the interaction between PD-L1 (programmed death ligand 1) and PD-1 (a receptor molecule), hindering tumor cell immune escape. This strategy has achieved promising clinical results in some cancers, particularly melanoma. However, low clinical response rates and high recurrence rates remain limiting factors in the clinical application of immunotherapy in malignant melanoma. Advanced melanoma has become a promising tumor model for testing immune checkpoint modulators. As a key target in immune checkpoint blockade, PD-L1 can inhibit T cell activity through various complex signaling pathways, thereby promoting tumor progression. Tumor PD-L1 expression directly influences the efficacy of immune checkpoint blockade. Despite the rapid development of immune checkpoint blockade, a significant proportion of patients still fail to benefit from PD-L1 / PD-1 antibody immunotherapy. Therefore, finding a new immune checkpoint treatment indicator and adopting a strategy of combining multiple immune checkpoint blockers is an important breakthrough in current basic research and clinical trials. Summary of the Invention

[0003] The first object of the present invention is to provide a use of a UBQLN4 detection reagent in preparing a detection reagent for determining the expression level of PD-L1.

[0004] The second object of the present invention is to provide a use of a UBQLN4 inhibitor for preparing a medicament for reducing UBQLN4 protein content.

[0005] The third object of the present invention is to provide use of UBQLN4 inhibitors in the preparation of drugs for treating melanoma.

[0006] A UBQLN4 detection reagent is used to prepare a detection reagent for measuring PD-L1 expression levels.

[0007] The present study unexpectedly found that UBQLN4 was linearly correlated with PD-L1 expression, and the PD-L1 expression level could be evaluated by detecting UBQLN4.

[0008] In a preferred application of the present invention, the UBQLN4 detection reagent is used to prepare a test reagent for measuring the effect of PD-1 / PD-L1 antibody clinical treatment (ie, PD-1 antibody immunotherapy).

[0009] In the present invention, the linear correlation between UBQLN4 and PD-L1 expression can be used as an indicator of the clinical therapeutic effect of PD-1 / PD-L1 antibodies. The therapeutic effect of PD-1 / PD-L1 antibodies can be evaluated by detecting UBQLN4, providing an effective means of predicting resistance to PD-1 / PD-L1 monoclonal antibody treatment and overcoming drug resistance.

[0010] The present invention uses the UBQLN4 detection reagent to prepare a test reagent for determining the clinical therapeutic effect of PD-1 / PD-L1 antibodies on tumors. The tumor is melanoma.

[0011] The present study found that UBQLN4 expression in melanoma nests is linearly correlated with PD-L1 expression. UBQLN4 expression correlates with response to PD-1 / PD-L1 antibody therapy. Measuring UBQLN4 levels can be used to assess the efficacy of PD-1 / PD-L1 antibody-based clinical treatments for melanoma.

[0012] The UBQLN4 detection reagent is a detection reagent for UBQLN4 protein, for example, it can be a known immunoassay kit.

[0013] Based on the core concept of the linear correlation between UBQLN4 and PD-L1 expression, the present invention also provides UBQLN4 inhibition means and an invention for downregulating PD-L1 expression based on the UBQLN4 inhibition means, thereby improving the clinical treatment sensitivity and efficacy of PD-1 / PD-L1 antibodies.

[0014] For example, the present invention also provides a use of a UBQLN4 inhibitor for preparing a drug for reducing UBQLN4 protein content, wherein the UBQLN4 inhibitor is a small molecule that inhibits UBQLN4 protein expression or a UBQLN4 gene inhibitor.

[0015] Preferably, the small molecule that inhibits UBQLN4 protein expression is albendazole. The present invention innovatively discovered that albendazole can effectively inhibit UBQLN4 protein expression, thereby achieving multiple pharmacodynamic activities of downregulating UBQLN4 protein expression, for example, improving immunotherapy effects.

[0016] In the present invention, the UBQLN4 gene inhibitor is a small interfering RNA;

[0017] Preferably, the sequence of the small interfering RNA is:

[0018] Ubqln4-siRNA1, 5'-GGTCAGGGATGTTCAATAG-3'

[0019] Ubqln4-siRNA2, 5'-CAATAACCCTGAACTCATG-3'.

[0020] The present invention has found that the two sequences can unexpectedly improve the inhibitory effect of UBQLN4 and improve the immunotherapy effect.

[0021] The UBQLN4 inhibitors of the present invention are used to prepare a drug for reducing UBQLN4 protein content. Furthermore, the UBQLN4 inhibitors are used to prepare a drug for reducing UBQLN4 protein content in melanoma cells. Furthermore, the UBQLN4 inhibitors are used to prepare a drug for reducing UBQLN4 protein content in melanoma cells, thereby reducing PD-L1 expression.

[0022] The present invention also provides use of the UBQLN4 inhibitor in preparing a drug for treating melanoma.

[0023] The present invention found that by downregulating UBQLN4 expression in melanoma, the sensitivity of melanoma immunotherapy can be improved, thereby improving the treatment effect of melanoma.

[0024] The beneficial effects of the present invention are:

[0025] 1. The present study found that there is a linear correlation between UBQLN4 and PD-L1 expression levels, and PD-L1 and immunotherapy effects can be evaluated by detecting UBQLN4.

[0026] 2. The present invention also found that albendazole and the small interfering RNA can inhibit the overexpression of UBQLN4 and improve the sensitivity of melanoma to immunotherapy by inhibiting the expression of UBQLN4. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 UBQLN4 and PD-L1 expression in melanoma tumor nests were linearly correlated.

[0028] Immunofluorescence staining of cancer tissue samples with UBQLN4 and PD-L1 antibodies. Statistical analysis shows that (A) a significant positive correlation between UBQLN4 and PD-L1 expression was observed in patient samples. (B) Samples from patients who responded to PD-1 antibody treatment had relatively higher UBQLN4 / PD-L1 expression than samples from non-responders.

[0029] Figure 2 , UBQLN4 expression is associated with clinical response rate of melanoma PD-1 antibody treatment

[0030] The Kaplan-Meier method was used to analyze progression-free survival and median survival in biopsy samples from melanoma patients treated with PD-1 monoclonal antibodies. The total number of patients was 19, and they were divided into two groups based on PD-L1 and UBQLN4 expression. Significance was determined by logistic t-test.

[0031] Figure 3 , Albendazole effectively inhibited UBQLN4 protein expression

[0032] Two melanoma cell lines (A375 and SK-mel-28) were treated with a certain concentration of albendazole, and Western blotting showed that albendazole could reduce the expression of UBQLN4 in cells.

[0033] Figure 4 Specific sequences knock down UBQLN4 expression and reduce PD-L1 protein expression

[0034] The UBQLN4 gene-specific interference sequence was synthesized and the UBQLN4 cell expression level was knocked down. Western blotting found that PD-L1 was significantly downregulated in melanoma cells. DETAILED DESCRIPTION

[0035] The following is a further detailed explanation of this description in conjunction with experiments and experimental data.

[0036] Example 1: UBQLN4 and PD-L1 expression in melanoma nests are linearly correlated

[0037] The experimental steps are as follows:

[0038] Biopsy samples were collected from 19 melanoma patients receiving PD-1 monoclonal antibody therapy (toripalimab injection and Keytruda). Paraffin sections containing the tumor area were selected to ensure the absence of large areas of necrosis. Double fluorescent staining was performed on the paraffin sections to measure UBQLN4 / PD-L1 expression levels.

[0039] 1. Obtain 4 μm paraffin sections, bake at 60°C for 2 h, remove and cool slightly.

[0040] 2. Displace xylene: Filter through 100% alcohol I, 100% alcohol II, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, and 50% alcohol (5 minutes each, dilute the alcohol with double-distilled water to the desired concentration). Wash twice with double-distilled water, 5 minutes each time.

[0041] 3. Antigen retrieval (citrate antigen retrieval solution): Heat in a microwave oven to 100°C, place the paraffin sections in the retrieval solution and heat for 9 minutes.

[0042] Storage solution: 0.1M citric acid solution (A): 21.01g citric acid + 1L distilled water

[0043] 0.1M trisodium citrate solution (B): 29.41g trisodium citrate + 1L distilled water

[0044] Working solution: 9 mL of A solution + 41 mL of B solution + 450 mL of distilled water to obtain 0.01 M citrate buffer

[0045] 4. Cool to below 40°C and wash twice with double-distilled water (on a shaker), 5 minutes each time. Wash with 3% H2O2 (30% H2O2 stock solution diluted to 3% with PBS) for 15 minutes. Finally, wash three times with wash buffer (PBS, 0.1% triton X-100), 5 minutes each time.

[0046] 6. Blocking solution: Add 50 μL of PBS containing 0.3% triton X-100 and 5% BSA to each slide and block at room temperature for 30 minutes. Blot dry with filter paper. Circle the tissue sample using an immunohistochemistry pen. Add the primary antibody against the protein to be detected (PD-L1, Proteintech, Cat# 66248-1-Ig; UBQLN4, Santa Cruz Biotechnology, Cat# 13684) in blocking solution (antibody dilution 1:100) and incubate overnight at 4°C in a humidified chamber. The next day, incubate at room temperature for another 30 minutes. Remove excess antibody with wash buffer three times, 5 minutes each time.

[0047] 7. Incubate with a fluorescent secondary antibody or a biotin-conjugated secondary antibody (same as the Western blot secondary antibody, 4°C, dilute 1:250 in blocking buffer) in a humidified chamber for 2 hours at room temperature. Wash three times with wash buffer for 5 minutes each. Then, incubate with DAPI solution for 10 minutes at room temperature and store in the dark. Detect and capture images using a fluorescence microscope (Nikon, ECLIPSE Ts2R).

[0048] The experimental results are as follows Figure 1 Figure A shows that immunofluorescence staining of UBQLN4 and PD-L1 expression in tumor samples from 19 melanoma patients revealed a positive correlation between UBQLN4 and PD-L1 expression. High UBQLN4 expression in tumor tissue was associated with high PD-L1 expression. Figure B shows that samples from patients who responded to PD-1 antibody therapy had relatively higher UBQLN4 / PD-L1 expression than samples from non-responders.

[0049] Example 2: UBQLN4 expression is associated with clinical response rate to PD-1 antibody therapy in melanoma

[0050] The progression-free survival of clinical melanoma patients (n=19) was evaluated according to the median expression levels of PD-L1 and UBQLN4 using the Kaplan-Meier method, and the significance was determined by the log-T test.

[0051] The experimental results are as follows Figure 2 As shown: The results in the figure show the expression of PD-L1 and UBQLN4 in tissue samples of responders (Responder) and non-responders (Non-responder) to PD-1 antibody treatment; through the intensity and distribution of fluorescence, it was found that the PD-L1 and UBQLN4 protein levels in the Responder sample group were higher than those in the Non-responder sample group.

[0052] Example 3: Albendazole (ABZ, Selleck, Cat# S1640) effectively inhibits UBQLN4 protein expression

[0053] Collect A375 and SK-mel-28 cells in the logarithmic growth phase and adjust the cell suspension concentration to 5×10 5 Cells were plated at 400 μg / mL in a 6-well plate. After attachment, PD-L1 expression was induced with IFN-γ (R&D Systems, Cat#285-IF). Cells were then treated with albendazole at varying concentrations (0.625 and 1.25 μM) for 24 hours. Cells from each treatment group were harvested and total protein extracted. The procedure was as follows: 1. After 24 hours, cells were trypsinized and harvested. RIPA cell lysis buffer (protease inhibitor cocktail and phosphatase inhibitor) was added and mixed. Lyse on ice for 30 minutes, followed by sonication on ice for 30 seconds. The cells were centrifuged at 13,800 rpm at 4°C for 30 minutes, and the supernatant was aspirated to obtain total cellular protein.

[0054] 2. Protein determination by BCA method This experiment uses the Pierce BCA protein concentration assay kit (Thermo Fisher, Cat# 23227) to determine the protein concentration of the sample. The determination method is as follows:

[0055] (1) Prepare the working solution: Prepare an appropriate amount of BCA working solution with a ratio of Reagent A to Reagent B of 50:1, with 200 μL of working solution added to each sample well. Mix thoroughly and set aside.

[0056] (2) Prepare standard protein concentrations: dilute a protein of known concentration at 2 mg / mL with PBS to obtain protein solutions with concentrations of 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL.

[0057] (3) Dilute the protein to be tested 10-fold: dilute the protein to be tested with PBS (for example, if diluted 10-fold, add 5 μL of protein to 45 μL of PBS to obtain a 50 μL system).

[0058] (4) Sample loading: Add the prepared protein samples into 96-well plates, 20 μL per well, and set up 2 replicates for each sample.

[0059] (5) Add working solution: Add 200 μL of the freshly prepared BCA working solution to each sample well, gently shake the 96-well plate to mix, and incubate at 37°C in the dark for 30 min.

[0060] (6) After the incubation, the OD value of the solution was measured using a microplate reader at an excitation wavelength of 570 nm. A protein concentration standard curve was drawn based on the OD value of the standard protein, and the concentration of the protein to be tested was calculated.

[0061] 3. Protein separation by SDS-PAGE electrophoresis: Calculate the volume required to load 20-30 μg of protein. Then, add 2× SDS loading buffer and mix thoroughly with the protein. Denature the protein in boiling water for 5 minutes. After denaturation, place the sample on ice and centrifuge at high speed. Then, load the sample into the wells in order. Mark the lane next to the sample with a protein marker. Electrophoresis is performed at 80V for 30 minutes, followed by 100V for approximately 2 hours. Prepare a NC membrane of appropriate size and transfer it to the membrane at 100V for 90 minutes. Block the membrane with 5% skim milk in PBST solution on a shaker at room temperature for 2 hours. After blocking, incubate the NC membrane with the primary antibody (UBQLN4, Santa Cruz Biotechnology, Cat#13684; PD-L1, Cell Signaling Technology, Cat#13684) diluted to the desired concentration and incubate overnight at 4°C on a shaker. After primary antibody incubation, the membrane was washed three times with PBST at room temperature on a high-speed shaker for 10 minutes each to remove nonspecifically bound primary antibody from the membrane surface. The corresponding secondary antibody, diluted to an appropriate concentration in blocking buffer, was then added and incubated at room temperature for 1.5 hours. After incubation, the membrane was washed three times with PBST on a shaker for 10 minutes each. An appropriate amount of ECL reagent (Beyotime, Cat# P0018AM) was mixed with equal volumes of reagents A and B. The expression of UBQLN4 and PD-L1 was detected using a gel imaging system.

[0062] The experimental results are as follows Figure 3Western blot analysis shows changes in UBQLN4 and PD-L1 protein levels in A375 and SK-mel-28 cells after treatment with different concentrations of albendazole (0.625 and 1.25 μM) for 24 hours in the presence of IFN-γ. The results indicate that UBQLN4 protein levels are significantly downregulated with increasing drug concentration, while PD-L1 also shows a downregulation trend.

[0063] Example 4: Specific sequence knockdown of UBQLN4 expression reduces PD-L1 protein expression

[0064] To further clarify the relationship between UBQLN4 and PD-L1 expression, UBQLN4 gene interference fragment (siRNA) was synthesized, and PD-L1 protein expression was detected when UBQLN4 expression was knocked down. A375 and SK-mel-28 cells in the logarithmic growth phase were collected and the cell suspension concentration was adjusted to 5×10 5 Cells were plated at 400 μg / mL in a 6-well plate. After cell attachment, PD-L1 expression was induced with IFN-γ. RiboFECT CP transfection reagent (RiboBio, Cat# C10511-05) was then used for transfection according to the instructions. 24 hours after cell transfection, total cell protein was extracted using the same procedure as in Example 3. The small interfering RNA sequence used was:

[0065] Control siRNA, 5'-GAAATGTACTGCGCGTGGAGAC-3'

[0066] Ubqln4-siRNA1, 5'-GGTCAGGGATGTTCAATAG-3'

[0067] Ubqln4-siRNA2, 5'-CAATAACCCTGAACTCATG-3'.

[0068] The experimental results are as follows Figure 4 Figure 2: Changes in UBQLN4 and PD-L1 protein levels in A375 and SK-mel-28 cells 24 hours after transfection. Transfection of cells with different siRNA sequences reduced UBQLN4 protein expression to varying degrees. Compared with the NC control group, UbqLN4-siRNA1 and UbqLN4-siRNA2 demonstrated significant knockdown, while PD-L1 also showed a downregulation trend. This further demonstrates that inhibition of UBQLN4 can suppress PD-L1 expression.

[0069] In summary, UBQLN4 expression in melanoma nests is linearly correlated with PD-L1 expression and is associated with response to PD-1 antibody therapy. Therefore, testing patient tumor samples for UBQLN4-PD-L1 expression can assess patient suitability for PD-1 / PD-L1 antibody therapy, thereby improving the response rate to melanoma immune checkpoint therapy and enabling precision medicine to achieve optimal therapeutic outcomes. The use of UBQLN4 protein detection reagents in melanoma drug kits can predict drug efficacy in advance. The use of small interfering RNA (siRNA) to effectively inhibit UBQLN4-PD-L1 expression in tumors could facilitate the development of targeted UBQLN4-PD-L1 inhibitors, providing additional therapeutic strategies for melanoma.

Claims

1. The use of UBQLN4 detection reagent in the preparation of a test reagent for determining the clinical therapeutic effect of PD-1 / PD-L1 antibodies on tumors, characterized in that: The tumor is melanoma.

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