WO2023155896A1

WO2023155896A1 - Use of methylation modification of lysine 162 in human pd-l1 protein in predicting sensitivity of malignant tumor immunotherapy

Info

Publication number: WO2023155896A1
Application number: PCT/CN2023/076866
Authority: WO
Inventors: 王桂华; 胡俊波; 黄昌胜; 李润清
Original assignee: 武汉百英诺生物科技有限公司
Priority date: 2022-02-21
Filing date: 2023-02-17
Publication date: 2023-08-24
Also published as: CN115290895B; CN115290895A

Abstract

Disclosed in the present invention is a use of methylation modification of lysine 162 in a human PD-L1 protein in predicting the sensitivity of malignant tumor immunotherapy, which can be used to prepare products for predicting the sensitivity of malignant tumor immunotherapy; for example, a polyclonal antibody or monoclonal antibody that can specifically recognize methylation modification of lysine 162 in a human PD-L1 protein, a kit containing the polyclonal antibody or monoclonal antibody, or a kit used for amplifying and preparing the polyclonal antibody and monoclonal antibody. The expression level of the human PD-L1 protein in which lysine 162 has undergone methylation modification is detected by means of the polyclonal antibody or monoclonal antibody, then the sensitivity of a sample under test when using anti-PD-1 or anti-PD-L1 immunotherapy is determined, providing a new possible basis for guiding the clinical use of anti-PD-L1 and PD-1 monoclonal antibodies.

Description

The application of methylation modification of lysine 162 in human PD-L1 protein in predicting the sensitivity of malignant tumor immunotherapy

Cross References to Related Applications

This application requires that the application date is February 21, 2022, the application number is 202210158718.8, and the title is "Application of Methylation Modification of Lysine 162 of Human PD-L1 Protein in Predicting the Sensitivity of Malignant Tumor Immunotherapy" Right, the entire content of the above Chinese patent application is hereby incorporated into this application by way of introduction.

technical field

The invention belongs to the technical field of biomedicine and tumor treatment, in particular to the application of the methylation modification of lysine 162 of human PD-L1 protein in predicting the sensitivity of malignant tumor immunotherapy.

Background technique

With the continuous discovery of programmed death receptor ligand (PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) immune checkpoints, immunotherapy techniques for cancer patients have attracted more and more attention. PD-L1 is an immunoglobulin-like immunosuppressive factor present in various cells, including tumor cells, macrophages, dendritic cells (DCs), and cancer-associated fibroblasts (CAFs). The ligand of PD-L1, programmed death receptor (PD-1), is mainly expressed on T cells. PD-L1 on the surface of tumor cells can interact with PD-1 on tumor-infiltrating T lymphocytes (CTLs), thereby inhibiting the proliferation of T cells, weakening the cytokine secretion of T cells, and negatively regulating T cell-mediated cytotoxicity. Ultimately, tumor cells escape the killing effect of the body's immune system on tumor cells.

A number of clinical experiments have shown that the use of anti-PD-L1 monoclonal antibody (mAb) or anti-PD-1 monoclonal antibody can block the interaction of PD-1/PD-L1, so that CTLs can be reactivated, thereby making The body's immune system can kill abnormal tumor cells, which significantly improves the prognosis of cancer patients. According to the National Comprehensive Cancer Network (NCCN) guidelines, patients with PD-L1 expression ≥ 50% in tumors and no tumor driver gene mutations can receive anti-PD-L1 monoclonal antibody monotherapy. However, several clinical studies have shown that the response rate of anti-PD-L1 therapy in various tumors is only 15-45%; in addition, several clinical trials have shown that patients with low expression of PD-L1 can also benefit from anti-PD-L1 -L1 treatment. These clinical evidences show that simply using PD-L1 expression as the basis for judging whether to use PD-L1 monoclonal antibody is still controversial.

Previous studies have shown that the phosphorylation, palmitoylation and glycosylation of PD-L1 can enhance the stability of PD-L1, increase the expression of PD-L1 on the surface of tumor cells, and promote the immune escape of tumor cells. However, the methylation modification and biological function of PD-L1 have not been reported yet.

Contents of the invention

In view of the above deficiencies in the prior art, the present invention provides a methylation modification phenomenon involving the 162th lysine of human PD-L1 protein, which is useful in predicting/evaluating malignant tumor anti-PD-1 or anti-PD- The application in the sensitivity of L1 immunotherapy also provides an antibody that can truly reflect the methylation level of lysine 162 of PD-L1 protein. The antibody provided by the present invention can specifically recognize the 162-position lysine methylation of PD-L1 protein, and can be used to guide the screening of patients with malignant tumors sensitive to anti-PD-1 or anti-PD-L1 immunotherapy, specifically through the following Technical realization.

Application of the methylation modification of lysine 162 of human PD-L1 protein in the preparation of products for predicting the sensitivity of malignant tumor immunotherapy.

Preferably, in the above application method, the product for predicting the immunotherapy sensitivity of malignant tumors is a polyclonal antibody or a monoclonal antibody that specifically recognizes the methylation modification of lysine 162 of human PD-L1 protein. When using the above-mentioned functional role of the methylation modification of lysine 162 of the human PD-L1 protein, you can directly use the polyclonal antibody or monoclonal antibody that can specifically recognize the methylation modification to determine the methylation modification The proportion of expressed human PD-L1 protein, thus judging the use of anti-PD-1 in malignant tumors Or sensitivity to anti-PD-L1 immunotherapy.

Preferably, in the above application method, the product for predicting the immunotherapy sensitivity of malignant tumors is a detection kit containing a polyclonal antibody or a monoclonal antibody that specifically recognizes the methylation modification of lysine 162 of human PD-L1 protein. The kit can be used to directly predict the sensitivity of malignant tumor immunotherapy. The polyclonal antibody or monoclonal antibody contained therein can be used directly. At the same time, the kit may also include conventional buffers, washing solutions, coating solutions and other reagents necessary for detection.

Preferably, in the above application method, the product for predicting the sensitivity of malignant tumor immunotherapy is a kit for PCR amplification of a monoclonal antibody that specifically recognizes the methylation modification of lysine 162 of human PD-L1 protein, Or a kit for obtaining a polyclonal antibody that specifically recognizes the lysine methylation modification at position 162 of human PD-L1 protein by immunizing animals. This kit is different from the kit mentioned in the previous paragraph. This kit is mainly used in the laboratory to prepare and synthesize the corresponding polyclonal antibody or monoclonal antibody through PCR amplification or animal immunization, and then indirectly support the prediction of malignant tumor immunity. Treatment sensitivity.

More preferably, in the above application method, a polyclonal antibody or a monoclonal antibody that specifically recognizes the methylation modification of lysine 162 of the human PD-L1 protein is used to detect the human PD-L1 protein in the sample to be tested. The proportion of human PD-L1 protein expression modified by the methylation of lysine 162; by setting the threshold of the proportion, or the tumor TPS or CPS scoring method, predicting the anti-PD-1 or anti-PD-L1 immunity of malignant tumors Treatment sensitivity.

Further preferably, when using the threshold prediction of the set ratio, if it exceeds the threshold, it is predicted to be insensitive to anti-PD-1 or anti-PD-L1 immunotherapy, and if it is lower than the threshold, it is predicted to be sensitive to anti-PD-1 or anti-PD-L1 immunotherapy . Previous studies have shown that the phosphorylation, palmitoylation and glycosylation of PD-L1 can affect the immune escape of tumor cells. In our study, we found that the methylation of lysine 162 in PD-L1 protein High expression will instead inhibit the combination of PD-L1 and its ligand PD-1, thereby reactivating and promoting T cells to perform their original functions, killing tumor cells, and finally effectively inhibiting the proliferation of tumor cells. Therefore, if the 162nd lysine A of PD-L1 protein The high expression of kylation modification indicates that the proliferation of tumor cells has adapted to the inhibitory effect on PD-L1 and its ligand PD-1, and if anti-PD-1 or anti-PD-L1 monoclonal antibody immunotherapy is used again, it will not be able to produce practical results. significance.

Still further preferably, in the above application method, the threshold value of the expression ratio of human PD-L1 protein modified by methylation at position 162 of lysine is 65%.

More preferably, in the above-mentioned application method, the preparation method of the polyclonal antibody is: firstly synthesizing a polypeptide for simulating the PD-L1 protein methylated at position 162 of lysine, and then injecting the polypeptide into immunized animals for immunization. For sensitization, the serum of immunized animals is extracted, and the polyclonal antibodies in the serum are collected and purified; the monoclonal antibodies are prepared using gene editing technology. On the premise that the amino acid sequence of the PD-L1 protein and the nucleotide sequence encoding and expressing the PD-L1 protein are known, for the methylation modification of lysine 162 of the PD-L1 protein, conventional techniques in the art can be used, Synthesize a polypeptide that can mimic the PD-L1 protein methylated at position 162 of lysine, and then use this polypeptide as an antigen to immunize through animal immunization, extract animal serum and purify to obtain polyclonal antibodies; conventional methods in the field can also be used According to known technology, a gene editing method is used to directly prepare a monoclonal antibody that can specifically recognize lysine methylation.

Through in-depth research on the relationship between the methylation modification of lysine 162 of human PD-L1 protein and the sensitivity of using anti-PD-L1 and PD-1 monoclonal antibody for malignant tumor immunotherapy, the applicant found that by using the specific A polyclonal antibody that specifically recognizes the methylation modification of lysine 162 of human PD-L1 protein can more accurately predict the sensitivity of malignant tumors to anti-PD-1 or anti-PD-L1 immunotherapy. At this stage, TPS or CPS scores in tumor tissue can be used to guide clinical PD-L1 or PD-1 monoclonal antibody treatment; it is possible to provide new evidence for guiding the clinical use of anti-PD-L1 and PD-1 monoclonal antibodies.

Compared with the prior art, the present invention is beneficial in that it has found a method that can more accurately and truly reflect and predict the sensitivity of malignant tumors to anti-PD-1 or anti-PD-L1 immunotherapy, using PD The special function of lysine methylation at position 162 of -L1 protein may provide new evidence for guiding the clinical use of anti-PD-L1 and PD-1 monoclonal antibodies.

Description of drawings

Figure 1 is a schematic diagram of the potential methylation sites found on the PD-L1 protein by protein mass spectrometry analysis;

Figure 2 is a schematic diagram of flow cytometry results of RKO cell lines expressing human wild-type PD-L1 and PD-L1 lysine 162 mutation;

Figure 3 is a schematic diagram of flow cytometry results of Lewis cell lines expressing human wild-type PD-L1 and PD-L1 lysine 162 mutation;

Figure 4 is a schematic diagram of the quantitative detection of the PD-1 protein content on the cells of the RKO WT and RKO K162R mutants obtained by the combination;

Figure 5 is a schematic diagram of the proportion of apoptotic cells detected in RKO WT and RKO K162R cells using the Caspase 3/7 kit;

Figure 6 is the detection results of tumor volume change and tumor tissue weight in the back of mice;

Figure 7 shows the specificity and sensitivity test results of a polyclonal antibody (rabbit serum antibody) that specifically recognizes methylation of lysine 162 of PD-L1;

Figure 8 is the ROC curve of the expression level of PD-L1 protein and PD-L1 lysine 162 monomethylation in each lung cancer sample.

Detailed ways

The technical solution of the present invention will be clearly and completely described below, obviously, the described embodiments are only some embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1: Determination of potential methylation sites of PD-L1 protein

1. Preparation of pDNA3.1-HA-PD-L1 plasmid

The synthetic HA-PD-L1 target gene was cloned into PCDNA3.1 by Aoke Biotechnology Company In the expression vector, obtain the pDNA3.1-HA-PD-L1 plasmid;

2. Identification of PD-L1 protein methylation sites

(1) According to the instructions of turbofect transfection reagent (provided by Thermo Company), transfect pDNA3.1-HA-PD-L1 plasmid in HEK293T cells and express HA-PD-L1 protein, that is, PD with HA tagged protein - L1 protein;

(2) After 3 days of transfection, use Anti-HA magnetic beads (provided by MCE) to isolate the PD-L1 protein in HEK293 cells according to the instructions;

(3) Then, the PD-L1 lysine methylation site was obtained by protein mass spectrometry (machine model: Exactive Plus LC-MS/MS mass spectrometer (Thermo), 2kV, RF=40, 320°C), as shown in the figure As shown in 1, the 75th, 89th, 105th and 162th lysines of PD-L1 protein were methylated.

Example 2: Functional research on the methylation site of PD-L1 protein

Taking the methylation of lysine 162 of PD-L1 protein as an example, the specific function research is as follows:

1. Construct PLKO-AS3W-PD-L1 carrier and PLKO-AS3W-PD-L1 K162R carrier respectively

The synthetic HA-PD-L1 and PD-L1 K162R target genes were cloned into the PLKO-AS3W vector by Aoke Biotechnology Company to obtain the PLKO-AS3W-PD-L1 vector and PLKO-AS3W-PD-L1 K162R vector.

The PLKO-AS3W-PD-L1 K162R carrier, that is, the 162nd lysine of the PD-L1 protein is mutated to arginine, thereby simulating the lack of methylation of the 162nd lysine of PD-L1;

2. Construct colon cancer RKO cell lines and mouse Lewis lung cancer cell lines expressing human wild-type PD-L1 vector, and colon cancer RKO cell lines and mouse Lewis lung cancer cell lines expressing human mutant PD-L1 K162 vector, respectively

(1) Using Crisp/Cas9 technology, use pSpCas9(BB)-2A-Puro(PX459)V2.0 The plasmid (Addgene Company) specifically knocks out the original PD-L1 gene in colon cancer RKO cells, named RKOPD-L1 KO; the specific sgRNA is:

5'-GTCCAGATGACTTCGGCCTT-3';

5'-GGATGACCAATTCAGCTGTA-3';

5'-GACACATTCAGAATATTACC-3';

(2) Using the same Crisp/Cas9 technology, mouse PD-L1 was knocked out in mouse Lewis lung cancer cells. The specific sgRNA is:

5'-GCCAGTGGCAGGTGAGTCTC-3';

5'-GCCTGCTGTCACTTGCTACG-3';

5'-GAGGTTGGACAAGGCTTCCG-3';

(3) Co-transfect HEK293T cells with PLKO-AS3W-PD-L1 vector/PLKO-AS3W-PD-L1 K162R vector, lentiviral packaging plasmid PPAX, and PMG2D. After 96 hours, take the cell culture supernatant to obtain A lentivirus expressing human PD-L1 wild-type (PD-L1WT) and human PD-L1K162R mutant (PD-L1 K162R) gene sequences;

(4) Use lentiviruses containing PD-L1 wild-type and PD-L1 K162R mutant gene sequences to infect the above-mentioned colon cancer cells RKOPD-L1 KO and mouse Lewis lung cancer cells, respectively, to obtain hPD-L1WT and hPD-L1 K162R expressing cells RKO cell lines (named RKO ^hPDL-1WT , RKO ^hPDL-1 ^K162R ) and Lewis cell lines (named Lewis ^hPDL-1WT , Lewis ^hPDL-1 ^K162R ) were used to detect human PD-L1 and The expression of mouse PD-L1, as shown in Figures 2 and 3, shows that the cell line has been successfully constructed.

3. Functional research on the methylation modification of lysine 162 in PD-L1

(1) Loss of lysine methylation at position 162 of PD-L1 protein, which affects the binding ability of PD-L1 and PD-1

The research methods are as follows:

① Firstly, human PD-1 pure protein (provided by SinoBiological Co., Ltd. provided by the company) were added to the RKO cell lines expressing PD-L1WT and PD-L1 K162R, and incubated for 1 hour;

② Use PE-PD-1 antibody (provided by Biolegend) to label the PD-1 protein bound to RKO WT and RKO K162R mutant cells;

③ Using flow cytometry (specific equipment model: Becton-Dickinson FACScan System; Franklin Lakes, NJ, USA), quantitatively detect the PD-1 protein content bound to RKO WT and RKO K162R mutant cells;

The specific detection results are shown in Figure 4, and the results show that the loss of methylation of lysine 162 of PD-L1 enhances the binding ability of PD-L1 and PD-1.

(2) T cell-mediated tumor killing experiment

The research methods are as follows:

① Separate human peripheral blood mononuclear cells (PBMC) using human lymphocyte separation medium (provided by MERCK);

② Use anti-CD3 (0.1ug/ml), anti-CD28 antibody (0.1ug/ml) and interleukin-2 cytokine (100IU/ml) to stimulate PBMC to differentiate into CD8 positive T cells,

③ RKO WT cells and RKO K162R mutant cells were co-cultured with the pre-treated PBMC cells (CD8-positive T cells) respectively. After 96 hours, RKO WT cells were detected using Caspase 3/7 kit (provided by Sangon Biotech). and the proportion of apoptotic cells in RKO K162R cells.

The above detailed experimental steps can refer to the foreign language literature "Eradication of triple negative breast cancer cells by targeting glycosylated PD-L1" (Chia-Wei Li, et al. Cancer cell, 33(2):187-201) published in 2018. Section “T cell-mediated tumor cell killing assay” on page 13.

The test results are shown in Figure 5, from which it can be found that the methylation modification of lysine 162 of the PD-L1 protein can promote the killing of tumor cells by T cells. The left picture of Figure 5 is the three replicates of T-fine The representative figure of cell-mediated tumor killing experiment, the right figure is the statistical result of the proportion of apoptotic cells in three repeated experiments.

(3) Mouse subcutaneous tumor experiment

The experimental animals were derived from C57BL/6 mice (B6/JGpt-Pdcd1em1Cin(hPDCD1)/Gpt) of humanized PD-1 of Jiangsu Jicui Yaokang Biological Co., Ltd. The specific experimental steps are as follows:

① Subcutaneously inject 100ul PBS containing 1× ¹⁰⁶ Lewis ^hPDL-1WT cells or Lewis ^hPDL-1 ^K162R cells into the back of 8-week-old humanized PD-1 C57BL/6 mice, and take them every four days The volume of the tumor on the back of the mouse, the specific calculation method is: volume = 0.5 × longest diameter × shortest diameter × shortest diameter;

②After 20 days, the mice were killed by neck dislocation, and the tumor tissues were separated and weighed.

The specific results of the tumor volume change and tumor tissue weight on the back of the mouse are shown in Figure 6, which verifies that the methylation of lysine 162 of the PD-L1 protein can affect the proliferation ability of its subcutaneous tumor cells .

The applicant also conducted in-depth functional studies on the methylation of lysines 75, 89, 105 and 162 of the PD-L1 protein according to the above research methods. Finally, it was found that only the modification of methylation at position 162 of the PD-L1 protein could affect the binding of PD-L1 to its ligand PD-1. The lysine methylation at positions 75, 89, and 105 of the PD-L1 protein has no effect on the binding of PD-L1/PD-1, so the corresponding specific detection results are related to the technical process and technical effect to be highlighted in the present invention Not big, so omitted.

Example 3: Preparation of a polyclonal antibody that specifically recognizes human PD-L1 protein that is methylated at position 162 of lysine

1. Analysis and design of human PD-L1 protein with lysine 162 methylation

Synthetic polypeptide EGYP (K-Me1) AEV (polypeptide chain structure is EGYPKAEV, wherein lysine K is modified by monomethylation) as immunogen, named WG-00793P, coupled with KLH for immunization, synthesized by abclonal company; at the same time Synthesized non-methylated control peptide (polypeptide chain structure is EGYPKAEV), named WG-00793C, synthesized by abclonal company; used to simulate K162 methylated PD-L1 protein and unmethylated PD-L1 protein.

2. Preparation of artificial immunogen to immunize animals

Concrete preparation process is as follows:

(1) purchase three experimental grade Japanese big-eared white rabbits of SPF grade with a weight of 2.0kg;

(2) Adjuvant-encapsulated antigen: before each immunization, inhale 0.35 mg of KLH-polypeptide PBS complex (polypeptide EGYP (K-Me1) AEV or control peptide EGYPKAEV, powder) into a 1 ml sterile syringe to Inhale the same amount of complete Freund's adjuvant into the other 1ml syringe, connect the two syringes with a sterile plastic hose and pull each other repeatedly until the liquid in the two syringes is completely emulsified, then they can be used for immunization;

(3) Animal sensitization for the first time: In order to increase the sensitization effect, the liquid obtained in step (2) was injected subcutaneously into Japanese big-eared white rabbits (recorded as the 0th day of immunization) by multi-point method (150 μl per point);

(4) The second animal sensitization: After the 7th, 19th, 40th, and 68th days of immunization, prepare 0.35 mg of KLH-polypeptide PBS complex in the same way as in step (2), using the multi-point method (150ul per point) ) was injected subcutaneously in Japanese big-eared white rabbits, and the second, third, fourth, and fifth experimental animals were sensitized. On the 80th day after immunization, the antibody titer and specificity were tested experimentally;

(5) Collect and store antibody serum:

①Fix the limbs of the immunized rabbit with ropes to form a supine position, cross the upper limbs behind the head and fix them, tie the rabbit’s upper incisors with a thin rope and pull it back, and fix the rabbit’s head on the upper limbs;

②Expose the neck, disinfect and cut off the neck hair, cut the skin of the neck about 15cm along the middle of the neck from the suprasternal fossa to the mandible, find the trachea and carefully separate the subcutaneous tissue along the trachea, from the distal end to the larynx, near the heart End to sternocleidomastoid muscle;

③ The pulsating carotid arteries can be seen below the trachea, and the carotid arteries on both sides are carefully separated and fully dissociated;

④Wrap two black silk threads into one side of the artery and separate them (one at the distal end and one at the proximal end), Ligate the distal end of the artery with a silk thread, then clamp the proximal end of the artery with an arterial clip, cut a small opening on the artery wall between the silk thread and the arterial clip with ophthalmological scissors, and quickly insert the pre-made thin plastic tube. And quickly fix the hose and the artery with the proximal silk thread to prevent the hose from coming out and leaking blood;

⑤ Gently open the arterial clip, and place a 50ml centrifuge tube at an angle to catch the arterial blood ejected from the venous vessel until no blood drips out. The other side of the carotid artery can be treated in the same way to increase the amount of bloodletting, and the bloodletting flow rate can be increased. Squeeze the heart when slow to increase blood output;

⑥Place the rabbit serum in a refrigerator at 4°C overnight; absorb the serum for the first time, centrifuge at 12,000 rpm at 4°C for 15 minutes, take the supernatant, which is the antibody serum, and store it in a refrigerator at 20°C.

(6) Rabbit serum antibody purification:

① Preparation of affinity chromatography column

Purification principle: The activated agarose gel binds the antigen to the solid phase carrier by coupling the sulfhydryl group of the antigen protein.

Reagent: Coupling Buffer (50mM tris, 5Mm EDTA-NA), pH=8.5; Wash buffer (1×PBS 320mM sodium chloride);

A. Weigh 10mg of EGYPKAEV polypeptide (control peptide), dissolve it with 2ml of coupling buffer, and take an appropriate volume of protein according to the protein concentration, the total content is about 10mg, which is the material of chromatography column I.

Weigh 10 mg of EGYP (K-Me1) AEV polypeptide and dissolve it with 2 ml of coupling buffer, and take an appropriate volume of protein according to the protein concentration, and the total content is about 10 mg, which is the material of chromatography column II.

Take 2ml of Sulfolink couping gel in the material of the above-mentioned chromatography column I and II, add the antigen to the chromatography column and rotate it on the rotary incubator for 2-3 hours to make the chromatography column 1 and the chromatography column 2 respectively;

② Antiserum purification

A. Put the chromatography column on the chromatography rack for 30 minutes, connect the constant flow pump; balance the flow rate of 40ml 1*PBS at 2.0, 30ml 1×Gly, and 30ml 1×PBS to balance the internal environment of the chromatography column Ⅰ and Ⅱ to medium sex;

B. Sample loading: add 1/20 4M sodium chloride to the serum sample, flow rate 1.3-1.6, and pass through the chromatography column 1 twice;

C. Washing: add 40ml Wash buffer;

D. Sample collection: Add 1×Gly, flow rate 0.5-0.7, collect 8 tubes, 2ml in each tube, 16ml in total;

E. After the chromatographic column I is collected, follow the same steps of B-D to pass through the chromatographic column II;

F. After the chromatography column II is collected, add excess acid to elute the antibody, and then equilibrate to neutrality with 1×PBS;

G. Put the collected antibody into a dialysis bag, mix well and take 30μl for antibody concentration determination, and put the rest into 1×PBS 50% glycerol for dialysis and concentration overnight; then put it into a sterilized 5ml cryopreservation tube,- Store at 20°C.

Example 4: Sensitivity and specificity detection of rabbit serum antibodies

1. Coating

Dilute 100 μl of the PBS complex of unmethylated EGYPKAEV (control peptide) and 100 μl of the PBS complex of methylated EGYP (K-Me1) AEV polypeptide chain to a concentration of 1 μg/ml, and add them dropwise to 1 cm×1 cm of PVDF On the membrane (polyvinylidene fluoride), overnight at 4°C;

2. Closed

Discard the remaining liquid, add 200 μl/well of 5% skimmed milk powder (blocking solution), and incubate at 37°C for 60 minutes;

3. Washing

Add washing solution (TBST) 250ul/well, wash three times, and place for 5min each time;

4. Add the primary antibody (that is, the serum antibody to be tested, the rabbit serum antibody)

Discard the remaining liquid, and add 150 μl/well of the serum antibody to be tested at different dilutions in sequence, and place at 37°C for 120 minutes;

(5) washing

Add 250 μl/well of washing solution (TBST), wash three times, and place for 5 minutes each time;

(6) Add enzyme-labeled antibody (secondary antibody)

Dilute HRP-labeled goat anti-rabbit secondary antibody 1:10000, add 150 μl/well, and place at 37°C 120min;

(7) washing

(8) Color rendering

Add 50 μL/well of ECL chromogenic solution (provided by Thermo Company), and develop according to the instructions; the specific results are shown in FIG. 7 . The results showed that E17404 rabbit serum antibody had better specificity and sensitivity.

Example 5: Using anti-PD-L1 K162me to detect the level of methylation at position 162 of PD-L1 in clinical lung cancer specimens

1. Collected 69 lung cancer samples from patients treated with PD-L1 monoclonal antibody (including 46 patients who were sensitive to PD-L1 monoclonal antibody treatment, and 23 patients who were not sensitive to PD-L1 monoclonal antibody treatment). Clinical tissue samples from lung cancer patients treated with PD-L1 monoclonal antibody were collected under the clinical protocol approved by the hospital's medical ethics committee and with written informed consent.

2. Use the antibody that specifically recognizes the 162-position lysine methylation of PD-L1 prepared in Example 3, and the PD-L1 antibody (product number: 13684; Cell Signaling Technology), and entrust Bioos Biotechnology Co., Ltd. Perform immunofluorescent staining.

3. After immunofluorescence staining, the expression levels of PD-L1 protein and PD-L1 lysine 162 monomethylation in each lung cancer specimen were analyzed using Image J software, and ROC curves were drawn.

As shown in Figure 8, the results of the ROC curve show that: compared to the expression level of PD-L1 protein alone, or compared to the absolute expression level of PD-L1 protein methylation alone, the 162th lysine methyl The ratio of normalized PD-L1 protein can better predict the sensitivity of PD-L1 monoclonal antibody treatment.

Claims

Application of the methylation modification of lysine 162 of human PD-L1 protein in the preparation of products for predicting the sensitivity of malignant tumor immunotherapy.
The application according to claim 1, the malignant tumors are colon cancer and lung cancer.
According to the application according to claim 1, the malignant tumor is lung cancer.
According to the application of claim 1, the product for predicting the immunotherapy sensitivity of malignant tumors is a polyclonal antibody or a monoclonal antibody that specifically recognizes the methylation modification of lysine 162 of human PD-L1 protein.
According to the application of claim 1, the product for predicting the sensitivity of malignant tumor immunotherapy is a detection kit containing a polyclonal antibody or a monoclonal antibody that specifically recognizes the methylation modification of lysine 162 of human PD-L1 protein .
According to the application of claim 1, the product for predicting the sensitivity of malignant tumor immunotherapy is a kit for PCR amplification of a monoclonal antibody that specifically recognizes the methylation modification of lysine 162 of human PD-L1 protein, Or a kit for obtaining a polyclonal antibody that specifically recognizes the lysine methylation modification at position 162 of human PD-L1 protein by immunizing animals.
According to the application according to any one of claims 4-6, the polyclonal antibody or monoclonal antibody that specifically recognizes the 162th lysine methylation modification of the human PD-L1 protein is used to detect the human PD- In the L1 protein, the proportion of human PD-L1 protein expression modified by the methylation of lysine 162; by setting the threshold of the proportion, or the tumor TPS or CPS scoring method, predict the malignant tumor anti-PD-1 or Anti-PD-L1 immunotherapy sensitivity.
According to the application described in claim 7, when using the threshold prediction of the set ratio, if the If it exceeds the threshold, it is predicted to be insensitive to anti-PD-1 or anti-PD-L1 immunotherapy, and if it is lower than the threshold, it is predicted to be sensitive to anti-PD-1 or anti-PD-L1 immunotherapy.
According to the application of claim 8, the threshold value of the expression ratio of human PD-L1 protein modified by the methylation of lysine 162 is 65%.
According to the application according to any one of claims 4-6, the preparation method of the polyclonal antibody is: first synthesize the polypeptide used to simulate the PD-L1 protein methylated at the 162nd lysine, and then inject the polypeptide The immunized animals are sensitized, the serum of the immunized animals is extracted, and the polyclonal antibodies in the serum are collected and purified; the monoclonal antibodies are prepared by gene editing technology.