Cell membrane material with high affinity binding to tumor cell PD-L1 and preparation method and application thereof

Abstract

The present invention discloses a cell membrane material with high affinity for binding to tumor cell PD-L1, and a preparation method and application thereof, belonging to the field of biotechnology. The present invention expresses mutant PD-1 on the surface of host cell membrane by means of genetic engineering, and can obtain a cell membrane material with active targeted binding ability to PD-L1 ligands on the surface of tumor cells. The cell membrane material is used to modify the surface of anti-tumor nanomedicines, so that efficient drug delivery to the tumor site can be achieved; at the same time, by competitively binding to the PD-L1 ligands on the surface of tumor cells, the PD-1 receptors on the surface of T cells are blocked from binding to the PD-L1 ligands, thereby restoring the anti-tumor activity of T cells. Therefore, the construction of this new cell membrane material provides an innovative solution for the development of cell membrane materials with targeted binding to tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a cell membrane material with high affinity for binding to tumor cell PD-L1, and a preparation method and application thereof. Background Art

[0002] With the development of nanotechnology, nanoparticles have been widely studied as drug delivery carriers in the diagnosis and treatment of cancer. However, as foreign substances, most nanoparticles will be recognized and cleared by the body's immune system, and their poor biocompatibility limits the practicality of these nanomaterials. Therefore, there is an urgent need to seek a safe, effective and convenient nano-delivery system that can escape immune system clearance and enhance tumor targeting.

[0003] The biomimetic membrane-mediated nano drug delivery system based on bionic technology uses the cell membrane as a carrier. Without considering the characteristics of the core nanomaterial, it uses the functions of the proteins and polysaccharides on the cell membrane surface to promote the long circulation and targeted delivery of the nanoparticles in the core in the body, which is expected to achieve a new breakthrough in nanotechnology in tumor targeted therapy. Since then, cell membrane biomimetic modified nanoparticles have been widely explored and studied, and how to develop innovative cell membrane materials is a bottleneck problem currently facing research.

[0004] In view of this, this application is hereby filed. Summary of the invention

[0005] The purpose of the present invention is to provide a cell membrane material with high affinity for binding to tumor cell PD-L1 and a preparation method and application thereof, so as to overcome the shortage of existing cell membrane materials and provide a carrier for the delivery of nano-drugs.

[0006] The technical concept of the present invention:

[0007] Cell membrane bionic nanoparticles provide a solution for efficient drug delivery to tumor sites, but how to develop cell membrane materials with targeted binding to tumor cells is a bottleneck problem currently faced. The method of expressing specific proteins that can target and bind to tumor cells on the cell membrane surface through genetic engineering provides a solution for obtaining cell membrane materials with targeted binding to tumor cell surface ligands.

[0008] In order to develop a cell membrane material with specific recognition ability for tumor cells, the inventors adopted the technical concept of expressing specific proteins that can target and bind to tumor cell surface ligands on the cell membrane surface through genetic engineering. Therefore, how to select effective specific proteins that can target and bind to tumor cell surface ligands is very critical. Research literature found that a variety of tumor cells have high expression of programmed death receptor ligand-1 (PD-L1), such as melanoma, non-small cell lung cancer, ovarian cancer, triple-negative breast cancer, renal cell carcinoma, etc. PD-L1 specifically binds to programmed death receptor-1 (PD-1) on the surface of activated T cells, activates the PD-1 / PD-L1 downstream pathway, transmits negative regulatory signals, and then leads to apoptosis of activated T cells and loss of immune activity, causing immune escape. Therefore, the PD-1 / PD-L1 binding site can be used as a target for targeting and binding to tumor cells, and also provides a molecular target for tumor immunotherapy. PD-1 is a type I transmembrane protein composed of 268 amino acids, which is mainly expressed on the membrane surface of mature hematopoietic cells such as CD4+T cells, CD8+T cells, monocytes, macrophages, B cells, dendritic cells, and some non-hematopoietic cells such as endothelial cells, pancreatic islet cells, mast cells, etc. However, the affinity of natural PD-1 to PD-L1 is weak. By designing different PD-1 mutants, a high-affinity PD-1 mutant (M-PD-1) with an affinity nearly 40,000 times higher than that of natural PD-1 / PD-L1 is obtained.

[0009] In summary, by expressing high-affinity PD-1 on the surface of the host cell membrane through genetic engineering, a cell membrane material with enhanced active targeting and binding to the PD-L1 ligand on the surface of tumor cells can be obtained. The surface of nanoparticles can be modified using this cell membrane material to achieve efficient drug delivery to the tumor site; at the same time, by competitively binding to the PD-L1 ligand on the surface of tumor cells, the PD-1 receptor on the surface of T cells is blocked from binding to the PD-L1 ligand, thereby restoring the anti-tumor activity of T cells. Therefore, the construction of this cell membrane material provides an innovative solution for the development of cell membrane materials with targeted binding to tumor cells.

[0010] The present invention is achieved through the following technical solutions:

[0011] In a first aspect, the present invention provides a method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1, comprising:

[0012] Performing site-directed mutagenesis on the full-length base sequence of PD-1 to obtain the base sequence of the mutant M-PD-1;

[0013] The base sequence of the fluorescent marker protein is connected to the base sequence of the mutant M-PD-1 to obtain a fusion base sequence M-PD-1-F;

[0014] Using the expression plasmid to construct a recombinant plasmid containing the fusion base sequence M-PD-1-F;

[0015] Transfecting the recombinant plasmid into host cells and performing resistance screening to obtain recombinant cells that can stably express the mutant M-PD-1 on the cell membrane surface;

[0016] The recombinant cells are expanded and cultured, and after the cells are collected, the cell membranes are extracted.

[0017] Furthermore, in a preferred embodiment of the present invention, at least 10 amino acid positions in the mutant M-PD-1 undergo base mutations;

[0018] Preferably, the base sequence of the mutant M-PD-1 is as shown in SEQ ID NO.1.

[0019] Furthermore, in a preferred embodiment of the present invention, in the process of constructing the fusion base sequence M-PD-1-F, the base sequence of the fluorescent marker protein is connected to the base sequence of the mutant M-PD-1 through a linker;

[0020] Preferably, the base sequence of the linker is as shown in SEQ ID NO.2.

[0021] Furthermore, in a preferred embodiment of the present invention, the fluorescent marker protein includes any one of red fluorescent protein, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein and yellow fluorescent protein.

[0022] Preferably, the fluorescent marker protein is red fluorescent protein, and the base sequence of the fusion base sequence M-PD-1-F is as shown in SEQ ID NO.3.

[0023] Further, in a preferred embodiment of the present invention, in the process of constructing the recombinant plasmid, the expression plasmid includes any one of pCMV3, pCMV6, pcDNA3.1 and pGL4.50;

[0024] Preferably, the fusion base sequence M-PD-1-F is connected to the pCMV3, and the connection sites are Hind III and Xba I.

[0025] Further, in a preferred embodiment of the present invention, in the process of constructing the recombinant cell, the host cell comprises any one of HEK 293T, CHO, NS0, BHK and PER-C6 cells;

[0026] Preferably, the host cells are screened for resistance using hygromycin B;

[0027] Preferably, the concentration of hygromycin B is 380-420 μg / mL.

[0028] In a second aspect, the present invention provides a cell membrane material having high affinity for binding to tumor cell PD-L1, which is prepared according to the above preparation method.

[0029] In a third aspect, the present invention provides a use of the above-mentioned cell membrane material with high affinity binding to tumor cell PD-L1 in the preparation of targeted tumor drugs or diagnostic reagents.

[0030] In a fourth aspect, the present invention provides a targeted drug delivery carrier for treating or diagnosing tumors, which comprises nanoparticles prepared from the above-mentioned cell membrane material, and the tumor is a tumor expressing the PD-L1.

[0031] In a fifth aspect, the present invention provides a tumor-targeting drug, comprising an active drug molecule and the above-mentioned cell membrane material, wherein the cell membrane material is wrapped around the active drug molecule.

[0032] Compared with the prior art, the present invention has at least the following technical effects:

[0033] The present invention uses genetic engineering to express mutant PD-1 on the surface of the host cell membrane, and can obtain a cell membrane material that has the ability to actively target and bind to the PD-L1 ligand on the surface of tumor cells. The cell membrane material is used to modify the surface of anti-tumor nanomedicines, so that the drug can be efficiently delivered to the tumor site; at the same time, the PD-1 receptor on the surface of T cells is blocked from binding to the PD-L1 ligand by competitively binding to the PD-L1 ligand on the surface of tumor cells, thereby restoring the anti-tumor activity of T cells. Therefore, the construction of this new cell membrane material provides an innovative solution for the development of cell membrane materials with targeted binding to tumor cells.

[0034] Compared with natural cell membrane materials, the cell membrane material provided by the present invention that expresses a high-affinity PD-1 mutant on its surface has the following advantages:

[0035] 1) The present invention transforms the cell membrane through genetic engineering technology to obtain a cell membrane material expressing a PD-1 mutant, which has a PD-1 / PD-L1 affinity that is nearly 40,000 times higher than that of a natural cell membrane and is a more powerful cell membrane material;

[0036] 2) The post-processing of the present invention is simple and convenient, and high-quality products can be obtained. Moreover, the method can be scaled up and industrialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The MTT assay of the present invention detected the effects of different concentrations of hygromycin B on the viability of HEK 293T cells;

[0038] Figure 2 a) Western blot detection of M-PD-1 expression in hygromycin-resistant B cell lines; b) CLSM observation of M-PD-1 expression and localization in HEK293T-M-PD-1 cells (red fluorescence signal: Protein; green fluorescence signal: WGAAlexa-Fluor 488, used to label cell membrane dye; blue fluorescence signal: DAPI, used to label cell nucleus dye, scale bar: 20μm)

[0039] Figure 3 1.5% agarose gel electrophoresis was used to detect the PCR products of different groups;

[0040] Figure 4 a) CLSM observation of the expression and localization of M-PD-1 in HEK 293T-M-PD-1 cells of different generations (red fluorescence signal: Protein; Green fluorescence signal: WGAAlexa-Fluor 488, used to label cell membrane dye; Blue fluorescence signal: DAPI, used to label cell nucleus dye, scale bar: 10 μm); b) Western blot detection of M-PD-1 expression in HEK 293T-M-PD-1 cells of different generations;

[0041] Figure 5 The expression of M-PD-1 protein on the surface of CM-M-PD-1 was detected by Western blot;

[0042] Figure 6 a) Western blot detection of PD-L1 expression in 4T1 cells; b) Particle size of CMPNPs; c) TEM image of CMPNPs (scale bar: 200 nm); d) CLSM observation of the binding of CMP NPs to 4T1 cells and e) Quantitative analysis of fluorescence intensity. (Red fluorescence signal: DsRED protein; Green fluorescence signal: WGA Alexa-Fluor 488, used to label cell membrane dye; Blue fluorescence signal: DAPI, used to label cell nucleus dye; n = 3; Scale bar: 10 μm; " ** ” represents P value < 0.01). DETAILED DESCRIPTION

[0043] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0044] The technical solution of the present invention is:

[0045] This embodiment provides a cell membrane material with high affinity for binding to tumor cell PD-L1, and the preparation method thereof includes:

[0046] Step S1: performing site-directed mutagenesis on the full-length base sequence of PD-1 to obtain the base sequence of the mutant M-PD-1;

[0047] The surface of activated T cells contains PD-1. Studies have found that the affinity of natural PD-1 to PD-L1 is weak. In order to further improve the affinity of PD-1 to PD-L1, in this step, the inventors performed site-directed mutagenesis on the full-length base sequence of PD-1. By designing different PD-1 mutants, a high-affinity PD-1 mutant (M-PD-1) with an affinity nearly 40,000 times higher than that of natural PD-1 / PD-L1 was obtained. At least 10 amino acid positions in the mutant M-PD-1 had base mutations;

[0048] Preferably, the base sequence of the mutant M-PD-1 is as shown in SEQ ID NO.1:

[0049] ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGC

[0050] GGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCA

[0051] GCCCTGCTTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACA

[0052] CATCGGAGAGCTTCCATGTAATCTGGCATCGCGAGAGCCCCAGCGGACAGACGGACAC

[0053] ACTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTC

[0054] ACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATG

[0055] ACAGCGGCACCTACGTCTGTGGGGTCATCTCCCTGGCCCCCAAGATCCAGATCAAAGA

[0056] GAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCA

[0057] CCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTG

[0058] GGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCG

[0059] GGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCC

[0060] CTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGA

[0061] AGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGT

[0062] CTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTC

[0063] GGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTC

[0064] Step S2: The base sequence of the fluorescent marker protein is used to connect the base sequence of the mutant M-PD-1 to obtain a fusion base sequence M-PD-1-F.

[0065] In order to facilitate the subsequent detection and positioning of the mutant M-PD-1 expressed on the surface of the host cells, the mutant M-PD-1 was labeled with a fluorescent marker protein. During the specific operation, the C-terminus of the M-PD-1 base sequence was fused with the base sequence of the fluorescent marker protein through a linker.

[0066] Preferably, the base sequence of the linker is: GGGGGTGGAGGCTCT (SEQ ID NO. 2). Using such a flexible base sequence linker for connection is conducive to the separation of the two domains of the fusion protein, so that they can fully fold into their respective natural conformations without interfering with each other.

[0067] The fluorescent marker protein includes any one of red fluorescent protein, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein and yellow fluorescent protein.

[0068] Preferably, red fluorescent protein As a fluorescent marker protein. The advantages of using this red fluorescent protein are that it can be expressed in a variety of organisms, has no cytotoxicity, and no protein aggregation; its excitation and emission wavelengths are longer, with a higher signal-to-noise ratio; it has high light stability and pH stability, high fluorescence conversion efficiency in cells, and is easier to detect; it can be used to detect deeper tissues, and has good application prospects for in vivo biological imaging.

[0069]

[0070] Step S3: constructing a recombinant plasmid containing the fusion base sequence M-PD-1-F using the expression plasmid;

[0071] Furthermore, expression plasmids include pCMV3, pCMV6, pcDNA3.1, pGL4.50 and the like.

[0072] Preferably, the expression plasmid is pCMV3; as a high-copy plasmid, the pCMV3 plasmid can be transfected into host cells by transient transfection, and a cell strain stably expressing the target gene can be obtained by hygromycin B screening.

[0073] Preferably, the fusion base sequence M-PD-1-F is connected to pCMV3, and the connection sites are Hind III and Xba I.

[0074] Step S4: transfecting the recombinant plasmid into host cells and performing resistance screening to obtain recombinant cells that can stably express the mutant M-PD-1 on the cell membrane surface;

[0075] Further, host cells include HEK 293T, CHO, NS0, BHK and PER-C6.

[0076] Preferably, HEK 293T cells are used as host cells. HEK 293T cells are human embryonic kidney cells that continuously express SV40 antigen. These cells are often used in transfection experiments and have a high transfection efficiency.

[0077] Furthermore, the host cells are screened for resistance using hygromycin B to obtain host cells that can stably express the mutant M-PD-1.

[0078] Preferably, HEK 293T cells are selected as host cells, and the minimum lethal concentration of hygromycin B to kill HEK 293T cells is between 380 and 420 μg / mL, and this concentration is used as the concentration of hygromycin B during resistance screening. Preferably, the concentration of hygromycin B is 400 μg / mL.

[0079] The linearized recombinant plasmid pCMV3-M-PD-1-F was transfected into HEK 293T cells by transient transfection, and then resistance screening was performed with hygromycin B to obtain hygromycin B-resistant recombinant cells HEK 293T-M-PD-1. The hygromycin B-resistant cells were inoculated in 96-well plates by limiting dilution to obtain monoclonal cells.

[0080] Subsequently, the expression localization of M-PD-1 protein in monoclonal cells was observed by confocal laser scanning microscopy (CLSM), and the cell line HEK 293T-M-PD-1 expressed on the cell membrane surface was obtained; by extracting the genome of HEK 293T-M-PD-1 cells, it was verified that the fusion base sequence M-PD-1-F had been integrated into the cell genome.

[0081] Step S5: Expanding and culturing the recombinant cells, collecting the cells, and extracting the cell membrane containing the mutant M-PD-1.

[0082] Through the cell subculture method, it was verified that the recombinant cell HEK 293T-M-PD-1 has the ability to stably express M-PD-1 protein.

[0083] The cell membrane is then extracted using the following method to obtain the cell membrane material:

[0084] 1) Using a Dounce homogenizer to disrupt cells and obtain a homogenate;

[0085] 2) The homogenate was subjected to gradient centrifugation to obtain cell membrane material (CM-M-PD-1)

[0086] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0087] Example

[0088] This embodiment provides a cell membrane material having high affinity for binding to tumor cell PD-L1, and a preparation method thereof, comprising:

[0089] (1) Construction of recombinant plasmid:

[0090] The human PD-1 gene sequence number (NM_001040022.1) was retrieved from the NCBI database, and the PD-1 base sequence was subjected to site-directed mutagenesis to obtain base sequences with mutations at 10 amino acid positions. The mutated PD-1 base sequence is shown in SEQ ID NO.1, and the mutated PD-1 amino acid sequence is shown in SEQ ID NO.4. The C-terminus of the mutated PD-1 base sequence was linked to the red fluorescent protein by connecting the sequence GGGGGTGGAGGCTCT (SEQ ID NO.2). The base sequence was fused, and the fused base sequence (M-PD-1-F) was shown in SEQ ID NO.3. The fused base sequence was fully synthesized. Finally, the fused base sequence was connected to the expression plasmid pCMV3, and the connection sites were Hind III and Xba I. The constructed expression plasmid was named pCMV3-M-PD-1-F.

[0091] The mutated PD-1 amino acid sequence is shown in SEQ ID NO.4:

[0092] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS

[0093] ESFHVIWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT

[0094] YVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

[0095] LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL.

[0096] (2) Screening of the minimum lethal concentration of hygromycin B in HEK 293T cells

[0097] HEK 293T cells in the logarithmic growth phase were collected and cultured at 5 × 10 5 Each well was inoculated with 100 μg / well of hygromycin B and cultured in a 5% CO2, 37°C incubator. When the cell growth density reached about 90%, the culture medium was discarded, and fresh culture medium containing 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, and 600 μg / mL hygromycin B was added to each well and cultured for 24 hours. Each group was repeated 3 times. The activity of HEK 293T cells was detected by MTT, and each group was repeated 3 times. The results are shown in Figure 1 shown.

[0098] The activity of HEK 293T cells was detected by MTT. When the concentration of hygromycin B was 400 μg / mL, HEK 293T cells could be completely killed, that is, this concentration was the minimum lethal concentration of hygromycin B to kill HEK 293T cells.

[0099] (3) Screening of Hygromycin-resistant B cell lines

[0100] The pCMV3 vector is used as a constitutive and stable expression plasmid, and hygromycin B can be used to screen and obtain a host cell strain that stably expresses the target protein. First, the expression plasmid pCMV3-M-PD-1-F is digested with endonuclease Bgl II to obtain a linearized expression plasmid; secondly, the linearized expression plasmid is transiently transfected into HEK 293T cells using Lipofectamin 3000 produced by Invitrogen, and the transfected host cells will continue to be cultured for 48 hours, and the culture medium will be removed; then, fresh culture medium with a hygromycin B concentration of 400 μg / mL is added to continue the culture, and the resistance screening is maintained for 2 weeks. Fresh culture medium is replaced every 2 days to obtain a cell strain with resistance to hygromycin B; finally, the hygromycin B-resistant cell strain is inoculated in a 96-well plate by the limiting dilution method, and the red fluorescence signal of the cell is observed by fluorescence microscopy to obtain a monoclonal cell with a red fluorescence signal, and the monoclonal cell is expanded and cultured;

[0101] (4) Western blot detection of M-PD-1 expression in hygromycin-resistant B cell lines

[0102] The cells in the previous step were collected, the cell surface was washed 3 times with sterile PBS buffer, and the total protein was extracted by adding RTPA lysis buffer containing protease inhibitor cocktail. The total protein concentration was determined by BCA protein concentration determination kit, separated by SDS-PAGE electrophoresis, transferred to the membrane, blocked with 5% skim milk powder in a greenhouse shaker for 1 hour, and rabbit anti-human PD-1 monoclonal antibody (1:1000 dilution) and human β-actin monoclonal antibody (1:1000 dilution) were added respectively, and incubated at 4°C overnight; TBST was washed 3 times, 5-10 minutes / time, and then the secondary antibody (1:10 dilution) was added respectively, and incubated at room temperature for 1 hour; TBST was washed 3 times, 5-10 minutes / time; chemiluminescence detection kit was used for development to obtain cell lines with high expression of M-PD-1.

[0103] By Figure 2 a It can be seen that the M-PD-1 protein is expressed in the hygromycin-resistant B cell line. This indicates that a cell line expressing M-PD-1 (HEK 293T-M-PD-1) has been obtained.

[0104] (5) Analysis of M-PD-1 expression sites in HEK 293T-M-PD-1 cells

[0105] The above cells in the logarithmic growth phase were collected and 1×10 4100 cells / well were inoculated in a 6-well plate with a cell slide and cultured in a 5% CO2, 37°C incubator for 48 hours. The culture medium was discarded, and 1 mL of sterile PBS was taken to wash the cell surface 3 times; the cells were fixed with 4% paraformaldehyde, and the cell surface was washed 3 times with sterile PBS; the cell membrane was stained with WGAAlexa-Fluor 488 dye for 10 minutes, and the cell surface was washed 3 times with sterile PBS; finally, the nuclear membrane was stained with DAPI for 10 minutes, and the cell surface was washed 3 times with sterile PBS; the cell slide was taken out, the cell surface was covered on a slide with anti-fluorescence quenching, and the image was taken under a laser confocal microscope (CLSM).

[0106] By Figure 2 b It can be seen that the M-PD-1 protein in HEK 293T-M-PD-1 cells is expressed on the cell membrane surface. This shows that the cell line HEK293T-M-PD-1 that highly expresses the M-PD-1 protein and is expressed on the cell membrane surface was screened and obtained by the above method.

[0107] (6) Genome extraction

[0108] The above cells in the logarithmic growth phase were collected and 1×10 7 Each well was inoculated in a 6-well plate with a cell slide and cultured in a 5% CO2, 37°C incubator for 48 hours. The genome of HEK 293T-M-PD-1 was extracted using the Tiangen genome extraction kit, and full-length primers were designed:

[0109] Forward primer: atgcagatcccacaggcg (SEQ ID NO.5)

[0110] Reverse primer: ttactggaacaggtggtggc (SEQ ID NO.6)

[0111] The full-length sequence of M-PD-1-F was cloned (conditions: denaturation temperature: 95°C, denaturation time: 10s; annealing temperature: 65°C, annealing time: 20s; extension temperature: 72°C, extension time: 1.5min; number of cycles: 35 times). The results are as follows Figure 3 shown.

[0112] By extracting the genome of HEK 293T-M-PD-1 cells, it was verified that the fusion base sequence M-PD-1-F had been integrated into the cell genome.

[0113] (7) Investigation of cell stability expression

[0114] To investigate the ability of HEK 293T-M-PD-1 cells to stably express M-PD-1 protein, the expression of M-PD-1 in cells was detected by fluorescence confocal microscopy and Western blot every 5 generations through cell subculture, and a total of 20 generations were detected.

[0115] The results are as follows Figure 4 As shown in the figure, it can be seen that HEK 293T-M-PD-1 cells can stably express M-PD-1 protein in the 20th generation cells after subculture. This shows that HEK 293T-M-PD-1 cells have the ability to stably express M-PD-1 protein.

[0116] (8) HEK 293T-M-PD-1 cell membrane extraction

[0117] Culture and collect 1×10 10 HEK 293T-M-PD-1 cells were washed with 10 mL of sterile PBS, and the cells were collected by centrifugation at 500 × g. The washing was repeated 3 times. 5 mL of homogenate (HM buffer: 0.25 M sucrose, 1 mM The cells were resuspended in EDTA, 20mM Hepes, pH 7.4, and protease inhibitors were added. The cells were broken on ice using a Dounce Homogenizer, and the process was repeated 100 times to obtain a homogenate. The homogenate was centrifuged for 5 minutes at 4°C and 1,000×g to collect the supernatant. The supernatant was ultracentrifuged for 30 minutes at 4°C and 10,000×g to collect the supernatant. The supernatant was ultracentrifuged for 1 hour at 4°C and 100,000×g to collect the precipitate. The precipitate was resuspended in sterile precooled PBS, and ultracentrifuged for 1 hour at 4°C and 100,000×g. This step was repeated twice, and the final precipitate was collected as the cell membrane material (CM-M-PD-1). The content of M-PD-1 in the cell membrane material was 3550ng per 1.0mg of total protein as detected by ELISA.

[0118] (9) Investigation of M-PD-1 protein in cell membrane materials

[0119] The expression of M-PD-1 protein on the surface of CM-PD-1 was detected by Western blot using the same method as (4).

[0120] The results are as follows Figure 5 As shown in the figure, M-PD-1 protein was detected in the CM-M-PD-1 group, which indicates that CM-M-PD-1 expresses M-PD-1 protein.

[0121] (10) Binding of cell membrane materials to mouse breast cancer 4T1 cells

[0122] In order to investigate the binding of cell membrane materials to tumor cells, mouse triple-negative breast cancer 4T1 cells were selected as the research model.

[0123] First, the expression of PD-L1 in 4T1 cells was detected. Western blot showed that 4T1 cells expressed PD-L1 receptors. The results were as follows Figure 6 As shown in a.

[0124] Then, the cell membrane material CM-PD-1 was prepared into nanoparticles. The specific operation was as follows: the obtained CM-PD-1 was dissolved in a sterile pre-cooled PBS solution, and ultrasonicated for 3 minutes in an ice bath at a power of 80W. The ultrasonicated sample was physically squeezed through a 0.80μm and a 0.22μm filter membrane to obtain a CM-PD-1 nanoparticle solution, and the particle size and morphology of the cell membrane nanoparticles were observed by a particle size analyzer and TEM; the results are shown in FIG. Figure 6 As shown in Fig. 6b and 6c, CMPNPs with an average particle size of 160.1 nm were obtained.

[0125] The cell binding assay was used to detect the affinity of CMPNPs nanoparticles to 4T1 cells. The following experimental groups were set up:

[0126] a) PBS group,

[0127] b) CMPNPs group,

[0128] c) PD-L1 primary antibody + CMPNPs group (PD-L1 primary antibody was added 4 h before adding nanoparticles).

[0129] 4T1 cells in the logarithmic growth phase were taken and 5×10 4 Each well was inoculated in a confocal dish and cultured in a 5% CO2, 37°C incubator for 24 hours. The cells were washed 3 times with sterile PBS, and the corresponding nanoparticle solution was added according to the treatment method of different groups (the protein content in the nanoparticle solution was quantitatively analyzed by the BCA method, and the added protein concentration was 500μg / mL), and gently shaken at room temperature for 4 hours; the supernatant was removed, washed 3 times with sterile PBS, and the cell membrane was stained with WGAAlexa-Fluor 488 dye for 10 minutes; washed 3 times with sterile PBS, and the nuclear membrane was stained with DAPI for 10 minutes; washed 3 times with sterile PBS, photographed under a laser confocal microscope, and the pictures were taken.

[0130] The results are as follows Figure 6As shown in Figures 6d and 6e, in the CMPNPs group, the binding of nanoparticles to 4T1 cells was very obvious. However, in the PD-L1 antibody + CMP NP group, the binding of nanoparticles to 4T1 cells was significantly reduced. The fluorescence intensity of the PD-L1 antibody + CMPNP group was only 25.6% of the CMPNPs group by fluorescence intensity quantitative analysis. Because the PD-L1 antibody binds to the PD-L1 on the surface of 4T1 cells, it hinders the binding of nanoparticles to 4T1 cells. In summary, CMPNPs can effectively bind to 4T1 cells through the surface M-PD-1.

[0131] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1, characterized in that: It includes: The full-length base sequence of the nucleic acid encoding PD-1 is subjected to site-directed mutagenesis to obtain a base sequence encoding a mutant M-PD-1, wherein the base sequence encoding the mutant M-PD-1 is shown in SEQ ID NO.1, and the amino acid sequence of the mutant M-PD-1 is shown in SEQ ID NO.4; The base sequence of the fluorescent marker protein is connected to the base sequence of the mutant M-PD-1 to obtain a fusion base sequence M-PD-1-F; Using the expression plasmid to construct a recombinant plasmid containing the fusion base sequence M-PD-1-F; Transfecting the recombinant plasmid into host cells and performing resistance screening to obtain recombinant cells that can stably express the mutant M-PD-1 on the cell membrane surface; The recombinant cells are expanded and cultured, and after the cells are collected, the cell membranes are extracted.

2. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 1, characterized in that: In the process of constructing the fusion base sequence M-PD-1-F, the base sequence of the fluorescent marker protein is connected to the base sequence of the mutant M-PD-1 through a linker.

3. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 2, characterized in that: The base sequence of the linker is shown in SEQ ID NO.

2.

4. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 1, characterized in that: The fluorescent marker protein includes any one of red fluorescent protein, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein and yellow fluorescent protein.

5. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 4, characterized in that: The fluorescent marker protein is red fluorescent protein, and the base sequence of the fusion base sequence M-PD-1-F is shown in SEQ ID NO.

3.

6. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 1, characterized in that: In the process of constructing the recombinant plasmid, the expression plasmid includes any one of pCMV3, pCMV6, pcDNA3.1 and pGL4.

50.

7. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 6, characterized in that: The fusion base sequence M-PD-1-F is connected to the pCMV3, and the connection sites are Hind III and Xba I.

8. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 1, characterized in that: In the process of constructing the recombinant cell, the host cell includes any one of HEK 293T, CHO, NS0, BHK and PER-C6 cells.

9. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 8, characterized in that: The host cells were screened for resistance using hygromycin B.

10. The method for preparing a cell membrane material having high affinity for binding to tumor cell PD-L1 according to claim 9, characterized in that: The concentration of hygromycin B is 380-420 μg / mL.

11. A cell membrane material having high affinity for binding to tumor cell PD-L1 obtained according to the preparation method according to any one of claims 1 to 10.

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