Antibodies against extracellular vesicle membrane protein PD-L1, their preparation methods and applications

Abstract

The present invention discloses an antibody against extracellular vesicle membrane protein PD-L1, a preparation method and an application thereof. The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO.1, and the amino acid sequence of the light chain variable region is SEQ ID NO.2. The antibody is obtained by immunizing an animal with the extracellular vesicle membrane protein PD-L1, and has high affinity and specificity for the extracellular vesicle membrane protein PD-L1.

Description

Technical Field

[0001] The present invention belongs to the field of immunological testing, and specifically relates to an antibody against extracellular vesicle membrane protein PD-L1 and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles (EVs) are membrane vesicles secreted by cells, carrying characteristic molecules of the mother cells, and can transmit these bioactive molecules to the receptor cells to exert biological functions, and are widely involved in various pathological and physiological processes of the body. Since the biological information molecules carried by extracellular vesicles come from the mother cells, the detection of biological molecules in extracellular vesicles can reflect the characteristics and status of the mother cells, which is the theoretical basis of liquid biopsy based on extracellular vesicles.

[0003] The rise of immunotherapy in recent years has brought hope to patients with advanced malignant tumors. Among them, inhibitors targeting the immune checkpoint programmed apoptosis ligand-1 / programmed death receptor-1 (PD-L1 / PD-1) pathway are currently representative drugs for tumor immunotherapy. Although PD-L1 / PD-1 immunotherapy has significant efficacy and can even completely eliminate tumors to achieve a cure, PD-L1 / PD-1 immunotherapy is not effective for every patient. Clinical trial results show that less than 30% of patients can benefit from PD-L1 / PD-1 immunotherapy. Therefore, it is imperative to find biomarkers that can predict the efficacy of immunotherapy in the early stages of treatment. The latest research has found that at different stages of tumor progression and immunotherapy, the level of PD-L1 carried by extracellular vesicles derived from tumor cells in the peripheral blood of tumor patients shows regular dynamic changes. Therefore, quantitative detection of the level of PD-L1 carried by extracellular vesicles of tumor cells in the body fluids (especially peripheral blood) of tumor patients is expected to become a new marker for liquid biopsy to predict the progression, prognosis and efficacy of immunotherapy of malignant tumors. Summary of the invention

[0004] In order to detect the extracellular vesicle membrane protein PD-L1 secreted by tumor cells, the present invention introduces a PD-L1 overexpression plasmid into tumor cells, causes the tumor cells to overexpress PD-L1, and secrete extracellular vesicles carrying PD-L1 protein on the membrane; immunize animals with the extracellular vesicle membrane protein PD-L1 (hereinafter referred to as Exo-PD-L1) to obtain lymph node cells that secrete anti-extracellular vesicle membrane protein PD-L1 antibodies, fuse the lymph node cells with tumor cells, screen out fused cells that stably express anti-extracellular vesicle membrane protein PD-L1 antibodies (hereinafter referred to as Exo-PD-L1 antibodies) and have good affinity, inject the fused cells into the peritoneal cavity of the animal, extract ascites for purification, and obtain specific and affinity anti-extracellular vesicle membrane protein PD-L1 antibodies.

[0005] The technical solution provided by the present invention is specifically as follows:

[0006] In a first aspect, the present invention provides an antibody against the extracellular vesicle membrane protein PD-L1, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO.1, and the amino acid sequence of the light chain variable region is SEQ ID NO.2.

[0007] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence capable of encoding the heavy chain variable region or the light chain variable region of the above-mentioned anti-extracellular vesicle membrane protein PD-L1 antibody.

[0008] In a third aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

[0009] In a fourth aspect, the present invention provides a host cell, which contains the above-mentioned anti-extracellular vesicle membrane protein PD-L1 antibody, the above-mentioned nucleic acid molecule, or the above-mentioned vector.

[0010] In a fifth aspect, the present invention provides a kit for detecting the extracellular vesicle membrane protein PD-L1, which contains the above-mentioned anti-extracellular vesicle membrane protein PD-L1 antibody.

[0011] In a sixth aspect, the present invention provides the use of the above-mentioned anti-extracellular vesicle membrane protein PD-L1 antibody, the above-mentioned nucleic acid molecule, or the above-mentioned vector, the above-mentioned host cell, and the above-mentioned conjugate in the preparation of a drug or a detection reagent for treating a disease.

[0012] In a seventh aspect, the present invention provides a method for preparing the above-mentioned anti-extracellular vesicle membrane protein PD-L1 antibody, comprising the following steps:

[0013] Immunizing animals with a carrier carrying PD-L1 protein on its membrane to obtain cells that can secrete antibodies against extracellular vesicle membrane protein PD-L1; the carrier is a liposome or an extracellular vesicle;

[0014] The obtained cells are used to produce antibodies against the extracellular vesicle membrane protein PD-L1.

[0015] Based on the above technical solution, the production of antibodies against extracellular vesicle membrane protein PD-L1 using the obtained cells includes:

[0016] fusing the obtained cells with myeloma cells to form a hybridoma cell line;

[0017] Screening out monoclonal cell lines with good specificity and affinity from hybridoma cell lines;

[0018] The screened monoclonal cell line is injected into the abdominal cavity of the animal. When the abdomen of the animal is obviously swollen, the ascites is extracted and purified to obtain the above-mentioned anti-extracellular vesicle membrane protein PD-L1 antibody.

[0019] Based on the above technical solution, the production of antibodies against extracellular vesicle membrane protein PD-L1 using the obtained cells includes:

[0020] The obtained cells were sequenced, RNA was extracted from single B cells, and PCR amplification was performed;

[0021] The amplified products were subjected to high-throughput sequencing analysis, and the screened RNA was used to produce antibodies against the extracellular vesicle membrane protein PD-L1.

[0022] On the basis of the above technical solution, the carrier loaded with PD-L1 on the membrane is obtained by the following method: PD-L1 is embedded in the liposome membrane, or adsorbed on the liposome membrane with the help of electric charge to form a liposome-PD-L1 protein complex, which is the carrier loaded with PD-L1 on the membrane.

[0023] On the basis of the above technical solution, the vector loaded with PD-L1 on the membrane is obtained by the following method: after the cells expressing PD-L1 are crushed, the cell membrane components are collected, and the cell membrane is prepared into phospholipid bilayer membrane structures carrying PD-L1 of varying diameters by extrusion, which is the vector loaded with PD-L1 on the membrane.

[0024] On the basis of the above technical solution, the membrane-loaded PD-L1 vector is obtained by the following method: the PD-L1 overexpression plasmid is transfected into tumor cells, and the extracellular vesicle membrane loaded with PD-L1 in the supernatant of the tumor cells is extracted, which is the membrane-loaded PD-L1 vector.

[0025] As a preferred embodiment of the above technical solution, the tumor cells are Cal-27 cells.

[0026] As a preferred embodiment of the above technical solution, the PD-L1 overexpression plasmid is pLenti-CD274-EGFP.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] 1. The anti-extracellular vesicle membrane protein PD-L1 antibody provided by the present invention has good affinity and specificity in detecting the level of extracellular vesicle PD-L1.

[0029] 2. The method for preparing an antibody against the extracellular vesicle membrane protein PD-L1 provided by the present invention is to prepare fusion cells, inject the fusion cells into the peritoneal cavity of an animal, and regularly extract ascites for purification to obtain specific and affinity anti-extracellular vesicle membrane protein PD-L1 antibodies. The method is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 The secretion of Exo-PD-L1 by Cal-27 OE cells was detected using an inverted fluorescence microscope.

[0032] Figure 2 The particle size distribution of the resuspension was measured by nanotracker (NTA).

[0033] Figure 3 The results of transmission electron microscopy (TEM) observation of the resuspension were shown in Figure 2.

[0034] Figure 4 The results are shown in Figure 2. Western blotting (WB) of the resuspension.

[0035] Figure 5 This is a positive cell line for anti-extracellular vesicle membrane protein PD-L1 screened by ELISA detection of hybridomas.

[0036] Figure 6 The immunofluorescence results of A549 and Exo-PD-L1 antibodies are shown.

[0037] Figure 7 The results are a comparison of the affinity of Exo-PD-L1 and commercially available PD-L1 antibodies.

[0038] Figure 8 This is a map of the PD-L1 overexpression plasmid pLenti-CD274-EGFP. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with examples, which are only intended to better illustrate the technical solution of the present invention rather than to limit the claims. The present invention is not limited to the specific embodiments and implementation schemes described herein. Any technician in the art can easily make further improvements and perfections without departing from the spirit and scope of the present invention, all of which fall within the protection scope of the present invention.

[0040] The present invention utilizes a genetic engineering method to overexpress PD-L1 in tumor cells, thereby increasing the level of Exo-PD-L1 secreted by tumor cells. Compared with the recombinant protein PD-L1, Exo-PD-L1 is highly similar to the extracellular vesicle membrane protein PD-L1 actually detected in terms of protein folding, post-translational modification, etc. (such as glycosylation modification and acetylation modification commonly found in PD-L1 protein), and can better reach the lymph nodes to achieve an efficient immune response. Therefore, the present invention provides a PD-L1 antibody obtained by immunizing an animal with Exo-PD-L1, and uses it to detect the level of Exo-PD-L1 to obtain a detection product with better specificity and stability.

[0041] Example 1

[0042] The method for preparing an antibody against the extracellular vesicle membrane protein PD-L1 provided in this embodiment is as follows:

[0043] 1. Construction and verification of Cal-27 OE cells:

[0044] 1.1 Construction of Cal-27 OE cells for:

[0045] The PD-L1 overexpression plasmid (pLenti-CD274-EGFP) with a GFP tag was transfected into Cal-27 cells. After 48h-72h, the cells were examined with an inverted fluorescence microscope to see if there was green light with the GFP tag. White light cell pictures and cell pictures with GFP green fluorescence were taken, and then they were combined into one picture. The four pictures were compared. If the cells and their surroundings had green light, it proved that the pLenti-CD274-EGFP plasmid was successfully transfected (e.g. Figure 1 ), and the successfully transfected cells were recorded as Cal-27 OE cells.

[0046] 1.2 Verification of whether Cal-27 OE cells secrete Exo-PD-L1 Collect the supernatant of Cal-27 OE cells in the logarithmic growth phase with a cell growth density of about 80%, centrifuge at 10000g for 30 min to remove cells and cell debris, retain the cell supernatant, and concentrate it by centrifugation at 3500g for 30 min using a 10KD ultrafiltration tube; ultracentrifuge at 100000g for 90 min, and resuspend the obtained precipitate in sterile PBS buffer at pH 7.4 to obtain a resuspension, which is stored in aliquots at -20°C for later use.

[0047] The particle size distribution of the resuspension was tested by NTA. The results were as follows: Figure 2 As shown in the figure, the particle size in the resuspension is 150nm, which is the particle size range of extracellular vesicles, indicating that the substances in the resuspension exist in the form of extracellular vesicles. The resuspension was observed by transmission electron microscopy, and the results are as follows Figure 3As shown in Figure 2, a typical 100nm saucer-shaped double-layer membrane structure of extracellular vesicles can be observed. The resuspension was subjected to WB detection, and the results were as follows: Figure 4 The size of the protein is 55kd. The above characterization and protein verification results all show that Cal-27 OE cells secrete extracellular vesicles loaded with PD-L1 protein on the membrane.

[0048] 2. Emulsify and immunize Exo-PD-L1:

[0049] The specific process of Exo-PD-L1 emulsification and immunization is as follows: PBS containing 50-100 μg / mouse Exo-PD-L1 and Freund's complete adjuvant (incomplete) are mechanically emulsified in equal volumes for 15-30 minutes to obtain an emulsifier that does not dissolve when dropped into water; multiple injections (5-6 points) are made subcutaneously on the back of the mouse's neck at 500-700 μL / mouse; immunization is performed 4 times, and the mouse immune serum is tested by ELISA array, and mice with high immune titers are selected. They are immunized by tail vein injection 3 days before fusion to obtain immune lymph node cells.

[0050] 3. Cell fusion technology:

[0051] Use 1mL PEG1450 to fuse the prepared immune lymph node cells with SP2 / 0 myeloma cells at a cell number ratio of 1:5, gently resuspend with HAT medium containing trophoblasts, spread into 96-well plates at 200μL / well, and culture in a 5% CO2 incubator. On the 5th day, if small colonies are observed under a microscope, the hybridoma cell culture medium can be half-changed, and on the 7th day, the medium is fully changed. On the 8th day, 200μL of the hybridoma cell supernatant was taken for ELISA detection (100μL of hybridoma cell supernatant was used for Exo-PD-L1 protein-coated ELISA detection, and 100μL was used for ProG protein-coated ELISA detection), and the affinity of the screened positive cell lines was tested, and the positive cell line with the highest affinity was selected (see Figure 5 ), expand the culture and collect the cell supernatant, perform protein immunoblotting (WB) detection with Cal-27 OE and A549 as samples, subclone the cell lines with the correct size specific bands by limiting dilution method, obtain the cell lines that can stably secrete specific antibodies, and store them in liquid nitrogen tank for a long time. Antibody 4. Preparation of Exo-PD-L1 antibody:

[0052] 5×10 5The cell lines that can stably secrete specific antibodies were inoculated at a density of 100 / mL, and the cell lines in the logarithmic growth phase were collected, resuspended with 0.5-1mL PBS, and injected into the abdominal cavity of mice that had been sensitized with paraffin for 7 days. The abdomen of the mice was obviously swollen and their spirits became worse. The ascites was collected with a 5mL syringe or a 16-gauge needle, and the ascites was collected and centrifuged at 10000rmp for 2min at 4°C to remove the surface oil layer, and the supernatant was aspirated and stored at -20°C to obtain the Exo-PD-L1 antibody.

[0053] 5. Purification of Exo-PD-L1 Antibody:

[0054] 1) Take an affinity chromatography column, wash the column bed with ultrapure water, and then wash the filler with sterile PBS buffer at pH 7.4;

[0055] 2) Filter the ascites supernatant with a 0.45 μm filter, apply the sample at a rate of 0.5 mL / min, and collect the flow-through;

[0056] 3) After loading, elute the column bed with PBS buffer (5-10 column volumes) until the Coomassie Brilliant Blue G250 method detects colorless;

[0057] 4) Wash the column bed with glycine eluent, collect the eluent, quickly add Tris-HCl buffer, shake it continuously to neutralize it to a neutral pH, and obtain an eluent neutralization solution;

[0058] 5) The column bed was washed with sterile PBS buffer at pH 7.4, and the chromatography column was sealed with 20% ethanol and stored at 4°C for future use.

[0059] 6) Place the eluate and neutralization solution into a dialysis bag and dialyze against pH 7.4 PBS buffer placed in a 4°C refrigerator, stirring overnight;

[0060] 7) Take out the dialysate, centrifuge it at 10,000 rpm for 5 min at 4°C, keep the supernatant, add sterile glycerol in equal proportions and store at -20°C.

[0061] 6. Detection of Exo-PD-L1 antibody by western blot:

[0062] 1) Prepare samples. When the density of Cal-27 OE cells and A549 cells reaches 80%, discard the culture medium and add 5 mL of sterile PBS at pH 7.4 to rinse the cells (to wash away the serum and cell debris in the culture medium); add trypsin for digestion. When cells are observed to fall off, quickly add sterile PBS at pH 7.4 and pipette, centrifuge at 1500 rpm for 5 min, and collect the supernatant; add cell lysis buffer, centrifuge, and take the supernatant; dilute the cell lysis buffer with 5× Loading buffer, mix well, boil for 10 min, and then aliquot and store at -20°C;

[0063] 2) 10% electrophoresis gel, constant voltage of 128V, 90min;

[0064] 3) Constant current 300mA, 50-60min, PVDF membrane transfer;

[0065] 4) Block the converted PVDF with 5% skim milk for 2 h, and wash the membrane with TBS 3 times, 5 min each time;

[0066] 5) 5% skim milk with Exo-PD-L1 antibody (1:4000) was placed in a 4°C refrigerator overnight, and the membrane was washed 4 times with TBS, 5 min / time;

[0067] 6) Expose with low-sensitivity ECL chemiluminescent substrate for 1-3 minutes, develop color and take pictures. Figure 4 As shown, Exo-PD-L1 antibody can specifically recognize Exo-PD-L1 in Cal-27 cell samples. 7. Detection of Exo-PD-L1 antibody by immunofluorescence

[0068] Incubate A549 cells secreting Exo-PD-L1 with Exo-PD-L1 antibody to verify whether there is recognition at the histochemical level:

[0069] 1) Cell fixation: A549 cells were placed on about 50% of the confocal culture dish, and 1 mL of 4% paraformaldehyde solution was gently added along the wall. The cells were allowed to stand at room temperature for 30 min, and then washed with 1 mL of sterile PBS, 5 min each time, for a total of 3 washes.

[0070] 2) Cell permeabilization: Use 0.3% Tritone-100 solution at 200 μL / dish for 15-20 min, wash with PBS for 5 min / time, for a total of 3 times.

[0071] 3) Blocking: Goat serum was diluted with sterile PBS, pH 7.4, to obtain a goat serum concentration of 5%, 500 μL / dish.

[0072] 4) Add primary antibody: dilute Exo-PD-L1 antibody (1:100) with 5% goat serum, 100 μL / dish, incubate at 4°C overnight (seal to prevent liquid evaporation), then wash with 1 mL PBS, 5 min / time, for a total of 3 times.

[0073] 5) Add secondary antibody: dilute 594 Goat Anti-Mouse IgG (H+L) 1:100 with 5% goat serum, 100 μL / dish, incubate at room temperature in dark for 1 h, wash with 1 mL PBS in dark for 5 min / time, for a total of 3 washes.

[0074] 6) Nuclear staining: sterile deionized water, DAPI 200 μL / dish, dark-proof for 30 min, 1 mL PBS for washing, 5 min / time, wash 3 times in total. Detect directly or add 1 mL sterile PBS, dark-proof at 4°C, test results are as follows: Figure 6 As shown, the Exo-PD-L1 antibody can recognize Exo-PD-L1 secreted by A549 cells.

[0075] 8. Comparison of affinity between Exo-PD-L1 and PD-L1 antibodies

[0076] The binding ability of Exo-PD-L1 antibody and commercially available PD-L1 antibody to antigen was tested by ELISA. The results are as follows Figure 8 As shown, it indicates that the affinity of Exo-PD-L1 antibody is higher than that of commercially available PD-L1 antibody.

[0077] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods will be described herein.

[0078] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. An antibody against extracellular vesicle membrane protein PD-L1, characterized in that: The amino acid sequence of its heavy chain is SEQ ID NO.1, and the amino acid sequence of its light chain is SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that: It comprises a nucleic acid sequence capable of encoding the heavy chain and light chain of the antibody against the extracellular vesicle membrane protein PD-L1 according to claim 1.

3. A carrier, characterized in that: It contains the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that: The host cell contains the anti-extracellular vesicle membrane protein PD-L1 antibody according to claim 1, the nucleic acid molecule according to claim 2, or the vector according to claim 3.

5. A kit for detecting extracellular vesicle membrane protein PD-L1, characterized in that: An antibody against the extracellular vesicle membrane protein PD-L1 according to claim 1.

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