CN112079928B

CN112079928B - anti-PD-L1 monoclonal antibody

Info

Publication number: CN112079928B
Application number: CN202010990579.6A
Authority: CN
Inventors: 薛童丹; 武鹏程; 孔双泉; 曾跃; 张立帆; 樊敏杰; 张梦思; 马晓飞; 尹长城
Original assignee: Beijing Protein Innovation Co ltd
Current assignee: Beijing Protein Innovation Co ltd
Priority date: 2020-09-19
Filing date: 2020-09-19
Publication date: 2023-08-01
Anticipated expiration: 2040-09-19
Also published as: CN112079928A

Abstract

The invention relates to a monoclonal antibody specifically combined with human PD-L1, and also provides a nucleic acid sequence for encoding the antibody. The invention also relates to a preparation method of the PD-L1 monoclonal antibody and application of the antibody in immunohistochemical detection of concomitant diagnosis.

Description

anti-PD-L1 monoclonal antibody

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a monoclonal antibody specifically combined with human PD-L1, and preparation and application thereof, wherein the application comprises the antibody serving as an in-vitro diagnostic reagent for diagnosing, treating, prognosis evaluating and monitoring an anticancer drug Keystuda of single-drug first-line treatment metastatic non-small cell lung cancer (NSCLC).

Background

PD-L1, also known as apoptosis ligand 1, is one of the ligands for the T cell surface apoptosis receptor 1 (PD-1). PD-L1 is mainly expressed in immune cells and non-hematopoietic cells, especially T cells, B cells, macrophages, etc., and PD-L1 protein is also expressed in many tumor tissues. The combination of PD-L1 and PD-1 can transmit a negative regulation signal to T cells, induce the T cells to enter a resting state, reduce proliferation of CD8+ T cells, limit autoimmunity, negatively regulate inflammatory reaction and protect self tissues, but the immune system can not normally recognize and kill tumor cells, so that immune escape is caused.

PD-L1/PD-1 is used as a popular immune checkpoint molecular target, and a plurality of antibodies against PD-1/PD-L1 have been developed for targeted therapy of tumors, including Kertruda (Pembrolizumab) developed by moxadong, opdivo (Nivolumab, nawuzumab) developed by MeschGuibao BMS, tecentriq (Atezolizumab) developed by Rogowski-Gene Takex, and the like. Many clinical data show that the efficacy of PD-1/PD-L1 antibodies is closely related to the level of PD-L1 expression in patients, from person to person. Therefore, detection of PD-L1 expression levels and tissue specificity is an effective adjunct to PD-1/PD-L1 targeted therapies. The U.S. FDA has already approved a number of companion diagnostic assays for drug therapy, of which 2015 approved PD-L1 IHC 22C3 pharmDx was the first companion diagnostic method developed by Dako corporation to detect PD-L1 expression in non-small cell cancers.

The variety of PD-1 monoclonal antibody drug development is many, the number of varieties on the market in China is 5, and each antibody recognizes the same target protein but different epitopes, so that the concomitant diagnostic antibodies selected for the effectiveness prediction of each antibody can be mutually referred to, but have individuation differences. The invention aims to screen out PD-L1 monoclonal antibodies with high expression, high affinity and good specificity for the concomitant diagnosis of PD-1 monoclonal antibody application by phage display panning (Phage Display Panning) technology, thereby improving the medicine economy and safety.

Disclosure of Invention

The invention provides a monoclonal antibody specifically binding to human PD-L1, which has higher expression level and/or PD-L1 binding specificity compared with the prior art antibody. Specifically, the present invention includes the following aspects:

the first aspect of the invention relates to an anti-PD-L1 monoclonal antibody, or an antigen-binding portion thereof, the heavy chain variable region VH of which consists of the amino acids shown in SEQ ID NO. 1,SEQ ID NO:2,SEQ ID NO:3,SEQ ID NO:7,SEQ ID NO:8,SEQ ID NO:9 and SEQ ID NO. 10; the light chain variable region VL consists of amino acids shown in SEQ ID NO. 4,SEQ ID NO:5,SEQ ID NO:6,SEQ ID NO:11,SEQ ID NO:12,SEQ ID NO:13 and SEQ ID NO. 14.

The second aspect of the present invention relates to a nucleic acid molecule encoding an antibody or antigen binding portion thereof according to claims 1-9.

A third aspect of the invention relates to a recombinant vector comprising a nucleic acid molecule according to claim 9.

In the present invention, the vector may be a cloning vector or an expression vector. The vector may be obtained, for example, by inserting the above-mentioned nucleic acid molecule into a cloning vector or an expression vector, or may be obtained directly by artificial synthesis. In the present invention, the expression vector is, for example, a prokaryotic expression vector, a eukaryotic expression vector or a phage vector.

In an embodiment of the invention, the phage vector is pBPI-RD52, which is based on pCANTAB5E (available from GE company) with the restriction enzyme sites adjusted to replace the NotI site with the SfiI site.

In an embodiment of the invention, the eukaryotic expression vector is pBPIRD1. The vector was engineered from eukaryotic expression vector pcDNA3.4 (available from Thermo Fisher) by inserting an Fc fragment fused downstream of the human IgG1 antibody in an embodiment of the invention.

In the invention, the recombinant vector is obtained after connecting the VH or VL sequence in the phage vector and eukaryotic expression vector.

The fourth aspect of the invention relates to a recombinant cell comprising the recombinant vector of any of the third aspects of the invention.

In the present invention, the cells may be prokaryotic cells or eukaryotic cells. The recombinant cells may be obtained by integrating the above recombinant vectors into cells by, for example, transformation, transfection, or the like. In the present invention, the prokaryotic cells are e.g. E.coli competent cells of various genotypes. In the present invention, the eukaryotic cell is a mammalian cell of human, murine or the like origin.

In an embodiment of the invention, the prokaryotic cell is TG1 ([ F' traD36 proAB lacIqZ.DELTA.M15 ] supE thi-1.DELTA. (lac-proAB) DELTA (mcrB-hsdSM) 5 (rK-mK-).

In an embodiment of the invention, the eukaryotic cell is a HEK293 derived cell line. In an embodiment of the invention, the 293 cells are Expi293 suspension cells that can be grown in a specific medium after acclimation. In an embodiment of the invention, the specific medium is a medium which is free of human or animal origin components, free of proteins, free of serum, and of defined chemical composition.

The fifth aspect of the present invention relates to a monoclonal antibody or antigen-binding portion thereof that specifically binds to human PD-L1, which is prepared from the nucleic acid molecule of any one of the second aspect of the present invention, the recombinant vector of any one of the third aspect, or the recombinant cell of any one of the fourth aspect.

In an embodiment of the invention, the PD-L1 monoclonal antibody is highly expressed in mammalian cells and can specifically bind to human PD-L1.

In another embodiment of the invention, the PD-L1 monoclonal antibody is specifically stained in immunohistochemical detection of tumor tissue sections.

Advantageous effects of the invention

The invention obtains the anti-PD-L1 monoclonal antibody with high expression, high affinity and high titer, and can be used for immunohistochemical detection of PD-L1.

Description of the drawings:

fig. 1: a, eukaryotic expression vector pBPI-RD1 plasmid map; b, eukaryotic expression vector pBPI-RD52 plasmid map

Fig. 2: SDS-PAGE map of PD-L1-Fc fusion protein purification, protein loading of 10. Mu.g, marker loading of 5. Mu.L, lane 1 protein Marker, lane 2 PD-L1 purified protein.

Fig. 3: anti-PD-L1 antibody cell supernatant SDS-PAGE map, supernatant loading 20 μL, marker loading 5 μL, wherein lanes 1-4 are the electrophoresis bands of the reduced samples, concretely, lane 1 is the cell supernatant negative control, lanes 2, 3 are the screened recombinant expressed antibodies A8 and A28, lane 4 is the PD-L1 (22C 3) antibody, and lane 5 is the protein pre-dye molecular weight Marker.

Fig. 4: SDS-PAGE of anti-PD-L1 purified antibody shows that the protein loading amount is 2 mug and the Marker loading amount is 5 mug, wherein lanes 1-3 are electrophoresis bands of reduced samples, specifically, lanes 1, 2 and 3 are high-expression recombinant PD-L1 antibodies screened out, and lane 4 is a protein pre-dyeing molecular weight Marker.

Fig. 5: ELISA results for anti-PD-L1 antibodies ELISA abscissa antibody concentration in μg/mL.

Fig. 6: IHC results for anti-PD-L1 antibody, A is the IHC staining results for control antibody 22C3, B is the IHC staining results for A8

Detailed description of the preferred embodiments

Embodiments of the present invention will be described in detail below with reference to the following examples, which are only for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products, and manufacturers are not noted.

Example 1: human PD-L1 protein expression

Referring to the amino acid sequence and structural feature annotation of accession number Q9NZQ7 in Uniprot database, the cDNA sequence (SEQ ID NO: 15) encoding the extracellular region (SEQ ID NO: 16) of human PD-L1 was synthesized based on the sequence No. NM-014143.4 in GenBank, cloned into cloning plasmid vector pUC57 (Nanjin St Biotechnology Co., ltd.), primers were designed to amplify the target gene from the plasmid vector, digested with EcoRI/XhoI and recovered, cloned into the linearized fragment of eukaryotic expression vector pBPI-RD52 (purchased from Beijing Hua major protein research center Co., ltd.) digested with the same double enzymes, sequenced and identified, plasmid preparation was performed, and reference was made to Expi293 ^TM Expression System Kit (Semer Feishier technology (China) Co., ltd., cat# A14635) were used for the transfection and culture of the Expi293 cells, maintaining the cell viability at 70% or more, and after culturing for 120 hours, purification was performed. Since recombinant PD-L1 Protein carries a human antibody Fc fragment, it can be purified using Protein A according to the procedure provided by the manufacturer using HiTrap rProtein A FF (GE company, cat. No. 17507901).

Example 2: preparation and screening of anti-PD-L1 monoclonal antibodies

BALB/c mice 6-7 weeks old were immunized with the PD-L1 fusion protein expressed in example 1, and the protein was subcutaneously injected, followed by booster immunization every 2 weeks for a total of three times after the primary immunization. Antibody titers in blood samples were determined by ELISA. After the animals reached the appropriate antibody titers, PD-L1 was subjected to the last immunopotentiation. Three days later, mice were sacrificed and bled, and peripheral blood and spleen were collected for mRNA extraction and phage display library construction.

To construct scFv phage display libraries, mouse peripheral blood B cells were isolated, spleen was taken, total RNA was extracted with Trizol kit (Invitrogen Inc), and cDNA was synthesized using reverse transcription kit (Invitrogen Inc). Using the cDNA as a template, light chain variable region and heavy chain variable region fragments were amplified using degenerate primers. Because of the high degree of DNA sequence polymorphism in the leader sequences and variable sequences of the heavy and light chain genes, the design and selection of degenerate primers is also relatively complex, such as whether the amplified region includes a leader signal peptide, whether it includes a constant region, etc., for different purposes in the construction of (scFv/Fab) libraries and cloning of antibody genes (Jones et al, bio/Technology 9:88-89, 1991; kettleborough et al, eur. J. Immunol.23:206-211, 1993; le Boeuf et al, gene 82:371-377, 1989; orlandi et al, proc. Natl. Acad. Sci. USA 86:3833-.3837, 1989). For 5' primers, these primers are typically designed to correspond to the first framework of the variable region (FR 1), and in a few cases also to the leader peptide sequence (L). The 3' primer is typically designed to correspond to the framework 4 (FR 4) region (low polymorphism) or constant region in which conserved homotypic specific sequences are readily identified. Although complex 5 'and 3' primer sets have been designed, they do not always match perfectly the DNA template (Gavilondo-Cowley et al, hybrid mia 9:407-417, 1990; leung et al, bioTechniques 15:286-292, 1993). Thus, the natural sequence of immunoglobulin heavy and light chain genes may be altered by primer artifacts in the FR1 and/or FR4 regions during PCR amplification. The base primer sequences for library construction selected in the present invention are as follows. The primer set for amplifying the heavy chain variable region is shown below, wherein MHV1-MHV10 is an upstream primer, MHVR is a downstream primer, and the downstream primer and each upstream primer pair amplify the heavy chain variable region gene. In PCR, 2. Mu.l of each of the upstream and downstream primers (1. Mu.M final concentration) was added to 20. Mu.l of each of the reaction systems, 2. Mu.l of a 2mM dNTPs mixture, 2. Mu.l of 10 XPCR buffer, andcDNA template 1 μl and 1U hot start Taq DNA polymerase, H added ₂ O makes up 20. Mu.l. The PCR amplification procedure was set to 94℃for 40 seconds, 52℃for 40 seconds, 72℃for 40 seconds, 25 cycles of amplification were performed, and finally 72℃was extended for 3 minutes, and the product was allowed to stand by at 4℃or directly electrophoresed. And (3) taking 20 mu L of PCR products for electrophoresis analysis, separating and cutting gel on 1.5% agarose gel for recovery, mixing the recovered products, carrying out second round amplification by taking MHV-FR1 and MHV-FR4 as primers, wherein the amount of template DNA is 1 mu L (about 200 pg), and the other conditions are the same, wherein the 5 'end of the recovered amplified products is provided with a SfiI cutting point, and the 3' end of the recovered amplified products is provided with a coding sequence of part (GGGGS) 3.

MHV1：5’-ATGCGGCCCAGCCGGCCGATGTGAAGCTTCAGGAGTC-3’

MHV2：5’-ATGCGGCCCAGCCGGCCCAGGTGCAGCTGAAGGAGTC-3’

MHV3：5’-ATGCGGCCCAGCCGGCCCAGGTGCAGCTGAAGCAGTC-3’

MHV4：5’-ATGCGGCCCAGCCGGCCAGGTTACTCTGAAAGAGTC-3’

MHV5：5’-ATGCGGCCCAGCCGGCCGAGGTCCAGCTGCAACAATCT-3’

MHV6：5’-ATGCGGCCCAGCCGGCCGAGGTCCAGCTGCAGCAGTC-3’

MHV7：5’-ATGCGGCCCAGCCGGCCCAGGTCCAACTGCAGCAGCCT-3’

MHV8：5’-ATGCGGCCCAGCCGGCCGAGGTGAAGCTGGTGGAGTC-3’

MHV9：5’-ATGCGGCCCAGCCGGCCGAGGTGAAGCTGGTGGAATC-3’

MHV10：5’-ATGCGGCCCAGCCGGCCGATGTGAACTTGGAAGTGTC-3’

MHVR：5’-GCTACCACCGCCACCTGAGGAGACGGTGACC-3’

Heavy chain heavy-chain amplification primer:

MHV-FR4：5‘-GAGCCGCCGCCGCCGCTACCACCGCCACC-3’

MHV-FR1：5’-ATGCGGCCCAGCCGGCC-3’

the primers used for amplifying the light chain variable region are as follows, wherein MKV1-MKV10 is an upstream primer and MKVR is a downstream primer, and the downstream primer and each upstream primer pair amplify the light chain variable region gene. In PCR, 2. Mu.l of each of the upstream and downstream primers (1. Mu.M final concentration) was added to 20. Mu.l of each reaction system, 2mu.l of a mixture of mM dNTPs, 2. Mu.l of 10 XPCR buffer, 1. Mu.l of cDNA template and 1U of hot start Taq DNA polymerase were added, H was added ₂ O makes up 20. Mu.l. The PCR amplification procedure was set to 94℃for 40 seconds, 52℃for 40 seconds, 72℃for 40 seconds, 25 cycles of amplification were performed, and finally 72℃was extended for 3 minutes, and the product was allowed to stand by at 4℃or directly electrophoresed. And (3) taking 20 mu L of PCR products for electrophoresis analysis, separating and cutting gel on 1.5% agarose gel for recovery, mixing the recovered products, performing second round amplification by using MKV-FR1 and MKV-FR4 as primers, wherein the amount of template DNA is 1 mu L (about 200 pg), the other conditions are the same, the 5 'end of the recovered amplified products is provided with a coding sequence of part (GGGGS) 3, and the 3' end is provided with a second SfiI cutting point.

MKV1：5’-GCGGCGGCGGCGGCTCTGGTGGCGGTGGTAGC

MKV1：GGTGGCGGTGGTAGCGATGTTTTGATGACCCAAACT-3

MKV2：5’-GGTGGCGGTGGTAGCGATATTGTGATGACGCAGGCT-3’

MKV3：5’-GGTGGCGGTGGTAGCGATATTGTGATAACCCAG-3’

MKV4：5’-GGTGGCGGTGGTAGCGACATTGTGCTGACCCAATCT-3’

MKV5：5’-GGTGGCGGTGGTAGCGACATTGTGATGACCCAGTCT-3’

MKV6：5’-GGTGGCGGTGGTAGCGATATTGTGCTAACTCAGTCT-3’

MKV7：5’-GGTGGCGGTGGTAGCGATATCCAGATGACACAGACT-3’

MKV8：5’-GGTGGCGGTGGTAGCGACATCCAGCTGACTCAGTCT-3’

MKV9：5’-GGTGGCGGTGGTAGCCAAATTGTTCTCACCCAGTCT-3’

MKV10：5’-GGTGGCGGTGGTAGCGACATTCTGATGACCCAGTCT-3’

MKVR：5’-GATCTCCAGCTTGGTCCC-3’

MKVFR1:CGGCGGCGGCGGCTCTGGTGGCGGTGGTAGC

MKVFR4:CCTTGGCCTATGCGGCCGTTAGATCTCCAGCTT

The heavy chain and light chain variable regions recovered by amplification are extended into single-chain antibody sequences through overlapping PCR, double ends are provided with SfiI tangential points, recovered are digested by SfiI, and are connected into a SfiI linearized vector pBPI-RD52.. Transforming the constructed plasmid library into largeIn enterobacteria TG 1. The stock was scraped from the large plate and inoculated into 2YTAG broth. Will be about 10 ¹² Helper phages for pfu plaque-forming proteins were added to TG1 samples containing scFv gene libraries and incubated for 1 hour at 37 ℃ in shaking. Ampicillin was added at 100. Mu.g/ml, and the culture was shaken overnight at 30 ℃. Cells were centrifuged at 4000rpm for 15 min at 4℃and the resulting supernatant was mixed with 5ml 20% PEG 8000/2.5M NaCl and incubated on ice for 30 min. Phage were then pelleted by centrifugation at 8000rpm for 20 minutes at 4"C. Phage were suspended in 1.5ml PBS containing l% BSA, shaken on a vortex mixer, and centrifuged at 13000rpm for 5 minutes to obtain a pellet. The supernatant was stored at 4℃or used directly for the following biological screening.

The immune tube was coated with PD-L1 antigen protein, diluted to a concentration of 2. Mu.g/mL in PBS pH7.4, 2 mL/tube, and coated overnight at 4 ℃. The next day, the cells were blocked with 2% of filtered sterilized skim milk powder, 4 mL/tube, and blocked at 37℃for 2h. PBS-T was rinsed 3 times. Phage diluted with blocking solution at 1000 times the stock volume was added, 1 mL/tube, and incubated at 37℃for 2h. PBS-T was rinsed 10 times and unbound phage were washed away. 1mL of 0.2M glycine at pH2.2 was added for elution for about 8min, and 1M Tris at pH9.0 was added for neutralization to give the first round of elutriation product. The titer of the panning product of the first round was determined, the positive rate was determined by colony PCR, and amplification was performed in preparation for the second round of panning. Repeating the operation, and three rounds of panning are carried out together, so that phage with certain affinity can be obtained. The results of each round of panning are shown in the following table:

the third round of panning products were infested into TG1 and ampicillin resistant plates were coated to obtain monoclonal antibodies. And (3) selecting a certain number of monoclonal bacteria to shake bacteria, infecting auxiliary phage, and obtaining phage supernatant.

ELISA plates were coated one day in advance, and PD-L1 antigen was diluted with PBS to a concentration of 2. Mu.g/mL, 100. Mu.L per well, and coated overnight at 4 ℃. The next day, the skim milk powder sterilized by 2% filtration was taken inThe wells were blocked at 300. Mu.L per well and at 37℃for 2h. PBS-T was rinsed 3 times. Phage supernatant was added at 100. Mu.L each and incubated at 37℃for 2h for binding. PBS-T was rinsed 3 times. 100. Mu.L of anti-E-tag antiserum was added at 1:1000 dilution per well and incubated for 2h at 37 ℃. PBS-T was rinsed 3 times. 100 μl of goat anti-rabbit antibody diluted 1:10000 was added to each well and incubated at 37deg.C for 1h. PBS-T was rinsed 3 times. Developing, mixing the developing solution with solution A and solution B at a ratio of 1:1, mixing 100 μl of each well, developing for a certain time, and adding 50 μl of 2N H ₂ SO ₄ The color development was terminated and the absorbance was measured at a wavelength of 450 nm. And screening out strains with high affinity according to the absorbance value, and carrying out sequencing identification. 8 scFv sequences were identified from the high binding clones and 4 scFv antibodies were selected for further characterization.

Example 3: expression and purification of anti-PD-L1 monoclonal antibodies

The 4-strain antibody light chain variable region gene fusion mouse IgG1 constant region sequence is cloned to pcDNA3.4 vector through EcoRI/XhoI cleavage site, and the heavy chain variable region gene fusion mouse kappa constant region is cloned to pcDNA3.4 vector through EcoRI/Eco47III cleavage site.

PD-L1 chimeric antibodies were transiently expressed in the Expi293F cells using Gibco transfection reagents according to the manufacturer's instructions. Briefly, using the ExpiFectamine 293Transfection Kit (Gibco), expi293F cells were transfected with the resulting vector at 25. Mu.g/25 mL total DNA transfected with Expi293F cells at 37℃with 5% CO ₂ After 4-5 days of incubation at 125RPM, the cell culture supernatant was collected, centrifuged at 3500RPM for 5 minutes, and filtered through a 0.22 μm filter to remove cell debris. Cell expression supernatants were tested qualitatively and semi-quantitatively by SDS-PAGE, antibodies with higher expression levels than PD-L1 22C3 were selected (as shown in FIG. 3, 2 antibodies A8 and A28 were expressed significantly higher than PD-L1 22C 3), purified using a pre-equilibrated Protein A affinity column (GE), and further characterized by 20mM citric acid, pH3.4 buffer (eluted, ultrafiltration concentrated, and then antibody stored in PBS buffer (pH 7.0) at concentrations determined by NanoDrop) and one of the highest expression antibodies A8 was selected.

Example 3 affinity detection of anti-PD-L1 antibodies against PD-L1 antigen

Detecting the affinity of the antibody by ELISA method, simply speaking, coating 200 ng/hole of eukaryotic expression PD-L1 antigen by PBS, and coating at 4 ℃ overnight; the wells were discarded, 0.1% PBST, 300. Mu.L/well washed 3 times; 1% BSA, blocking for 2h at 37 ℃; discarding the liquid in the hole, washing the hole for 3 times with 0.1% PBST, diluting the PD-L1 antibody to 2 mug/mL with a blocking solution, sequentially diluting 5 times, adding 6 concentrations of diluted samples into the corresponding hole, and incubating at 37 ℃ for 1h; removing liquid in the wells, washing 3 times with 0.1% PBST at 300 μl/well, diluting goat anti-mouse-HRP at 1:20000, incubating at 37deg.C for 1h at 100 μl/well; 0.1% PBST, 300. Mu.L/well washed 3 times, TMB developed, incubated at room temperature for 8min, added 2N H ₂ SO ₄ The end was reached with an microplate reader OD450 nm. As a result, the EC50 of A8 was equivalent to that of the PD-L1 22C3 antibody, which was about 0.027. Mu.g/mL, as shown in FIG. 5.

EXAMPLE 4 immunohistochemical detection of anti-PD-L1 antibodies

Taking a small piece of normal human placenta tissue, fixing by 10% formalin (3.7% -4% formaldehyde), washing, dehydrating, transparent, dipping wax, embedding paraffin, freezing, slicing and dewaxing conventionally. Anti-dilution, horseradish peroxidase (HRP) -labeled universal secondary antibody, diaminobiphenyl (DAB) substrate, substrate dilution, etc. were purchased from metasequoia gold bridge.

The method for detecting PD-L1 expression and localization in sample tissues by immunohistochemistry comprises dewaxing tissue sections to water, antigen restoration, endogenous peroxidase blocking, dripping the antibody prepared in the example 3 into the sample at a ratio of 1:200 as a primary antibody and PD-L1 (22C 3) purchased from Dako as a control antibody, and incubating overnight at 4 ℃. The buffer is washed 3 times for 5min each time. The universal HRP enzyme-labeled secondary antibody was added dropwise and incubated for 30 minutes at room temperature. The buffer is washed 3 times for 5min each time. DAB color development, counterstaining, dehydration and sealing, and observing the dyeing condition under a microscope.

As shown in FIG. 6, the PD-L1 protein is highly expressed on the cell membrane of the placenta tissue of a normal human, and both the A8 antibody and the PD-L1 (22C 3) antibody can specifically dye with strong yang on the cell membrane without staining cytoplasm.

While specific embodiments of the invention have been described in detail, various modifications and alternatives to those details could be made in light of all the teachings disclosed, and such modifications are intended to be within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

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gaattcgcca ccatgaggat atttgctgtc tttatattca tgacctactg gcatttgctg 60

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acaattgaat gcaaattccc agtagaaaaa caattagacc tggctgcact aattgtctat 180

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cagcatagta gctacagaca gagggcccgg ctgttgaagg accagctctc cctgggaaat 300

gctgcacttc agatcacaga tgtgaaattg caggatgcag gggtgtaccg ctgcatgatc 360

agctatggtg gtgccgacta caagcgaatt actgtgaaag tcaatgcccc atacaacaaa 420

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ctgagaatca acacaacaac taatgagatt ttctactgca cttttaggag attagatcct 660

gaggaaaacc atacagctga attggtcatc ccagaactac ctctggcaca tcctccaaat 720

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130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg

225 230 235

Claims

1. An isolated PD-L1 monoclonal antibody, or an antigen-binding portion thereof, characterized by comprising a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3 sequences, wherein:

the VH-CDR1 sequence is: GYTFTSYW (SEQ ID NO: 1);

the VH-CDR2 sequence is: INPSSGYT (SEQ ID NO: 2);

the VH-CDR3 sequence is: ARSGWLIHGDYYFDF (SEQ ID NO: 3);

the VL-CDR1 sequence is: QSLLNSRTRKNY (SEQ ID NO: 4);

the VL-CDR2 sequence is: WAS (SEQ ID NO: 5);

the VL-CDR3 sequence is: QQSYDIVT (SEQ ID NO: 6).

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region further comprises framework sequences VH-FR1, VH-FR2, VH-FR3, VH-FR4, thereby forming a VH sequence according to the following structure:

VH-FR1-VH-CDR1-VH-FR2-VH-CDR2-VH-FR3-VH-CDR3-VH-FR4。

3. the monoclonal antibody of claim 2, wherein:

the VH-FR1 sequence is: QVHLQQSGAELAKPGASVKMSCKAS (SEQ ID NO: 7);

the VH-FR2 sequence is: IHWVKQRPGQGLEWIGY (SEQ ID NO: 8);

the VH-FR3 sequence is: EYNQKFMDKATLTADKSSTTAYMQLSSLTSEDSAVYYC (SEQ ID NO: 9);

the VH-FR4 sequence is: WGQGTTLTVSS (SEQ ID NO: 10).

4. The monoclonal antibody of claim 1, wherein the light chain variable region further comprises framework sequences VL-FR1, VL-FR2, VL-FR3, VL-FR4, thereby forming a VL sequence according to the following structure:

VL-FR1-VL-CDR1-VL-FR2-VL-CDR2-VL-FR3-VL-CDR3-VL-FR4。

5. the monoclonal antibody of claim 4, wherein:

the VL-FR1 sequence is: DIVMSQSPSSLAVSAGEKVTMTCKSS (SEQ ID NO: 11);

the VL-FR2 sequence is: LAWYQQKPGQSPKLLIY (SEQ ID NO: 12);

the VL-FR3 sequence is as follows: TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ ID NO: 13); the VL-FR4 sequence is: FGAGTKLELK (SEQ ID NO: 14).

6. The monoclonal antibody of claim 1, further comprising a murine IgG1 heavy chain constant region and a murine kappa light chain constant region.

7. A composition comprising the monoclonal antibody, or antigen-binding portion thereof, of any one of claims 1-6.

8. A nucleic acid encoding the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-6.