Anti-PD-L1 antibody and application thereof

Abstract

The invention discloses an anti-PD-L1 antibody and application thereof, and belongs to the technical field of monoclonal antibodies. The number of the anti-PD-L1 antibodies is five, the five anti-PD-L1 antibodies are respectively an anti-PD-L1 antibody 203-64, an anti-PD-L1 antibody 203-69, an anti-PD-L1 antibody 203-10, an anti-PD-L1 antibody 203-59 and an anti-PD-L1 antibody 203-9, and amino acid sequences of VH and VL of the anti-PD-L1 antibodies and nucleotide sequences of coding genes of the anti-PD-L1 antibodies are shown as SEQ ID NO.1-20. The invention also discloses an application of the anti-PD-L1 antibody in preparation of anti-tumor drugs. The anti-PD-L1 antibody disclosed by the invention has good affinity with a human PDL1 protein and a monkey PDL1 protein, and can be used for treating tumors. According to the present invention, the completely humanized single-chain antibody specifically bound with PD-L1 is enriched, the potential new antibody drug is provided for the clinical treatment of tumors, and the high potential application value and the high potential significance are provided.

Description

Technical Field

[0001] The present invention relates to anti-PD-L1 antibodies and applications thereof, and belongs to the technical field of monoclonal antibodies. Background Art

[0002] PD1, or programmed death-1, is a transmembrane protein on T cells, mainly expressed on the surface of activated T cells, B cells and macrophages, and is a negative regulator of T cell proliferation. It can inhibit the function of T cells and induce apoptosis after binding to the receptors PD-L1, or programmed cell death 1ligand 1, and PD-L2, or programmed cell death 1ligand 2.

[0003] PD-L1, also known as surface antigen cluster of differentiation 274 (cluster of differentiation 274, CD274) or B7 homolog 1 (B7 homolog1, B7-H1), is a protein encoded by the CD274 gene. PD-L1 is highly expressed in a variety of tumor cells such as lung cancer, melanoma, colon cancer, breast cancer, gastric cancer, etc., and can interact with PD-1 on the surface of tumor-infiltrating lymphocytes, thereby inhibiting lymphocytes from killing tumors and causing tumor escape. Anti-PD-L1 antibodies can specifically bind to PD-L1 and block the PD-L1-mediated signaling pathway, thereby relieving the inhibitory function of the immune system and achieving the purpose of treating cancer or other related diseases. Therefore, the development of anti-PD-L1 antibodies has good clinical application value. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides several anti-PD-L1 antibodies and applications thereof, belonging to the technical field of monoclonal antibodies.

[0005] The present invention is achieved through the following technical solutions:

[0006] There are 5 types of anti-PD-L1 antibodies, namely anti-PD-L1 antibody 203-64, anti-PD-L1 antibody 203-69, anti-PD-L1 antibody 203-10, anti-PD-L1 antibody 203-59, and anti-PD-L1 antibody 203-9.

[0007] The amino acid sequence of the heavy chain variable region (VH) of the anti-PD-L1 antibody 203-64 is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO.4;

[0008] The amino acid sequence of VH of the anti-PD-L1 antibody 203-69 is shown in SEQ ID NO.6, and the amino acid sequence of VL is shown in SEQ ID NO.8;

[0009] The amino acid sequence of VH of the anti-PD-L1 antibody 203-10 is shown in SEQ ID NO.10, and the amino acid sequence of VL is shown in SEQ ID NO.12;

[0010] The amino acid sequence of VH of the anti-PD-L1 antibody 203-59 is shown in SEQ ID NO.14, and the amino acid sequence of VL is shown in SEQ ID NO.16;

[0011] The amino acid sequence of VH of the anti-PD-L1 antibody 203-9 is shown in SEQ ID NO.18, and the amino acid sequence of VL is shown in SEQ ID NO.20.

[0012] An isolated nucleic acid encoding the above-mentioned anti-PD-L1 antibody.

[0013] Furthermore, the nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-64 is shown in SEQ ID NO.1, and the nucleotide sequence encoding VL is shown in SEQ ID NO.3;

[0014] The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-69 is shown in SEQ ID NO.5, and the nucleotide sequence encoding VL is shown in SEQ ID NO.7;

[0015] The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-10 is shown in SEQ ID NO.9, and the nucleotide sequence encoding VL is shown in SEQ ID NO.11;

[0016] The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-59 is shown in SEQ ID NO.13, and the nucleotide sequence encoding VL is shown in SEQ ID NO.15;

[0017] The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-9 is shown in SEQ ID NO.17, and the nucleotide sequence encoding VL is shown in SEQ ID NO.19.

[0018] A recombinant expression vector comprises the isolated nucleic acid.

[0019] A transformant comprises the above-mentioned recombinant expression vector.

[0020] Use of the above-mentioned anti-PD-L1 antibodies, isolated nucleic acids, recombinant expression vectors, and transformants in the preparation of anti-tumor drugs.

[0021] Furthermore, the tumor is selected from any one or more of lung cancer, melanoma, colon cancer, breast cancer, and gastric cancer.

[0022] The present invention screens out a fully human single-chain antibody that specifically binds to PD-L1 from a fully human phage library, has good affinity with human PDL1 protein and monkey PDL1 protein, and can be used to treat tumors. The present invention enriches fully human single-chain antibodies that specifically bind to PD-L1, provides a potential new antibody drug for the clinical treatment of tumors, and has high potential application value and significance.

[0023] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Schematic diagram of phage ELISA results.

[0025] Figure 2 : Schematic diagram of periplasmic ELISA identification results.

[0026] Figure 3 : Schematic diagram of the binding of each candidate molecule to human PDL1 protein.

[0027] Figure 4 : Schematic diagram of the binding of each candidate molecule to monkey PDL1 protein. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0029] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0030] Experiment 1 Screening of anti-PD-L1 antibodies

[0031] 1. Experimental Methods

[0032] 1.1 Construction of a fully human phage antibody library

[0033] 1.1.1 Collect B cells from healthy individuals and extract total RNA. Amplify the VH and VL genes of the antibody by RT-PCR, perform electrophoresis, and purify and recover the VH and VL fragments.

[0034] 1.1.2 The recovered VH and VL fragments are connected to the phage vector through genetic engineering technology, and the constructed vector is transferred to Escherichia coli by electroporation. The phagemid vector can replicate itself in the successfully transduced host bacteria. When the host bacteria grow to the logarithmic phase, helper phage is added at an MOI of 10 for infection, and the phage is amplified by culturing overnight, and the phage library is obtained by centrifugal precipitation.

[0035] 1.2 Phage antibody library screening

[0036] Here are the steps:

[0037] (1) Coat 10 μg / ml PDL1 protein on an ELISA plate and incubate at 4°C overnight; inoculate the stored Escherichia coli TG1 and culture at 37°C and 220 rpm overnight.

[0038] (2) Transfer overnight grown E. coli TG1 at a ratio of 1:100 until the OD600 reaches 0.4.

[0039] (3) Remove the antigen solution from the ELISA plate, wash it with PBS three times, add 2% skim milk powder and block it at room temperature for 1 hour. At the same time, block the phage antibody library with 2% skim milk powder solution at room temperature for 1 hour.

[0040] (4) Add the blocked phage library to the wells containing the antigen and incubate at room temperature for 1 hour; the volume of the phage library added should be 10 11 ~10 12 .

[0041] (5) Remove the phage solution and wash the ELISA plate 15 times with PBST.

[0042] (6) Add 200 μl of pH 2.0 elution buffer, leave at room temperature for 10 minutes, and immediately add 60 μl of pH 8.0 Tris-HCl buffer for neutralization.

[0043] (7) Collect the eluted phages and take 250 μl to infect 10 ml of TG1 bacterial solution grown to the logarithmic phase. Let it stand at 37°C for half an hour, and then put it in a shaker at 37°C, 220 rpm, for 30 minutes.

[0044] (8) Take 10 μl of bacterial solution and dilute it 100 times, 1000 times, and 10,000 times respectively, and then inoculate it on a 2YT plate with resistance to calculate the phage titer.

[0045] (9) The remaining bacterial suspension was inoculated onto a 2YT plate (containing 100 μg / ml ampicillin and 1% glucose) and cultured in a 37°C incubator for 16 hours.

[0046] (10) Scrape all the colonies from the overnight plate using 2YT medium (100 μg / ml ampicillin and 1% glucose) and place in a 37°C incubator until the OD600 reaches 0.4.

[0047] (11) Add helper phage and place in an incubator at 37°C for 30 min and 220 rpm for 60 min.

[0048] (12) The bacterial precipitate was collected by centrifugation and cultured overnight in 2YT culture medium (100 μg / ml ampicillin, 50 μg / ml kanamycin and 0.1% glucose) at 30°C and 220 rpm.

[0049] (13) Centrifuge the overnight culture and collect the supernatant, which will serve as the phage for the second round of screening.

[0050] (14) Repeat the above screening process for at least 3 rounds until the phage titer is significantly improved.

[0051] 1.3 Enzyme-linked immunosorbent assay (ELISA)

[0052] Here are the steps:

[0053] (1) Dilute the antigen with PBS and coat the plate with 100 μl per well at 4°C overnight.

[0054] (2) After washing the ELISA plate once with PBST, 200 μl of 2% skim milk powder was added and blocked at room temperature for 1 hour.

[0055] (3) Wash the ELISA plate three times with PBST, add the diluted primary antibody, and incubate at room temperature for 1 hour.

[0056] (4) Wash the ELISA plate 4 times with PBST.

[0057] (5) Add diluted HRP-labeled secondary antibody and incubate at room temperature for 1 hour.

[0058] (6) Wash the ELISA plate 4 times with PBST.

[0059] (7) Add 100 μl / well TMB substrate solution, allow color to develop for 10 to 15 minutes, and then terminate the color development reaction with 2 M sulfuric acid.

[0060] (8) Read the plate at 450 nm on an ELISA reader and analyze the results.

[0061] 2. Experimental results

[0062] 2.1 Screening of antibodies binding to PDL1 protein

[0063] In this study, a fully human antibody (scFv) library constructed by our laboratory was used to screen the target of human PDL1. In this experiment, a PDL1 protein with a concentration of 10 μg / ml was used as an antigen and coated on an ELISA plate. After three rounds of screening, the titer of phages was significantly enriched.

[0064] The monoclonal clones were randomly selected for phage ELISA. The specific steps included: the coating concentration of human PDL1 protein was 1 μg / ml, 100 μl per well, coating at 4°C overnight, washing once with PBST, adding skim milk powder to block, then adding Phage supernatant, incubating at room temperature for 1 hour, washing 4 times with PBST, adding secondary antibody for incubation, washing and color development, and detecting OD450 with an enzyme marker. The results are as follows Figure 1 shown.

[0065] Positive clones were selected and sent for sequencing, which showed that 5 independent clones (203-9, 203-10, 203-59, 203-64, 203-69) were highly enriched. The gene sequences and amino acid sequences of VH and VL of these 5 antibodies are shown below.

[0066] The gene sequence of VH of 203-64 is as follows (direction 5'-3'), as shown in SEQ ID NO.1:

[0067] GAGCTGCAGGAGTCGGGCCCAGGACTGGTGAGGCCTTCGGAGACCCTGTCACTCACCTGCAGTGTCTCTCGTGGGTCCGTCAGTCAAAACTATTGGAGCTGGTTCCGGCAGTCCCCAGGGAAGGCATTGGAGTGGATTGGCTTTGTCCACTATAGTGGGGTCGCCAAATACGCC CCCCCCCTCACGGGTCGAGTCACCATCTCCCACGACCCGTCCAAGGCGGAAGTCTCCCTGAGGTTGACTTCTGTGACCGCCGCAGACACGGCCCGCTACTTCTGTGCGCGACATCTTCAGTGGGACAACCCAATCGATGTCTGGGGCCAGGGGACCACGGTCACCGTCTCGAGT.

[0068] The amino acid sequence of VH of 203-64 is shown below, as shown in SEQ ID NO.2:

[0069] ELQESGPGLVRPSETLSLTCSVSRGSVSQNYWSWFRQSPGKALEWIGFVHYSGVAKYAPPLTGRVTISHDPSKAEV SLRLTSVTAADTARYFCARHLQWDNPIDVWGQGTTVTVSS.

[0070] The gene sequence of VL of 203-64 is as follows (direction 5'-3'), as shown in SEQ ID NO.3:

[0071] CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGCTGTCAGTAATCG GCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCAGCTACAGGCGGCACCACCCCCTATGTCTTCGGAACTGGGACCAAAGTCACCGTCCTA.

[0072] The amino acid sequence of VL of 203-64 is shown below, as shown in SEQ ID NO.4:

[0073] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYAVSNRPSGVSNRFSGSKSGNTASLT ISGLQAEDEADYYCSSATGGTTPYVFGTGTKVTVL.

[0074] The gene sequence of VH of 203-69 is as follows (direction 5'-3'), as shown in SEQ ID NO.5:

[0075] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACGTGCACTGTCTCTGGTGGCTCCATCAGAAGTGGTGGTTCCTACTGGACTTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATACATCGATTACAGAGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCCTCGGACACGTCTAGGAACCAGCTCTCCCTGAAGCTGACCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGGGAAGAACGACGTTCGGGGAAATACAGTATGGACGTCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGT。

[0076] The amino acid sequence of VH of 203-69 is shown below, as shown in SEQ ID NO.6:

[0077] QVQLQESGPGLVKPSQTLSLTCTVSGGSIRSGGSYWTWIRQHPGKGLEWIGYIDYRGSTYYNPSLKSRVTISSDTS RNQLSLKLTSVTAADTAVYYCAREERRSGKYSMDVWGQGTLVTVSS。

[0078] The gene sequence of VL of 203-69 is shown below (direction 5'-3'), as shown in SEQ ID NO.7:

[0079] GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGTAGGTCTAGTCAAAGCCTCGTAGCCAGTGATGGAAACACGTACTTAAATTGGCTTCACCAGAGGCCAGGCCAGCCTCCAAGACTCCTAATTTACAAGATTTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGGTGGAAGCTGAGGATGTCGGGGTTTTTTACTGCATGCAAGCTACACAAATTCCTCAGACGTTCGGCCAAGGGACACGACTGGAGATTAAA。

[0080] The amino acid sequence of the VL of 203-69 is shown below, as shown in SEQ ID NO.8:

[0081] DIVMTQTPLSSPVTLGQPASISCRSSQSLVASDGNTYLNWLHQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDF TLKISRVEAEDVGVFYCMQATQIPQTFGQGTRLEIK。

[0082] The gene sequence of the VH of 203-10 is shown below (direction 5'-3'), as shown in SEQ ID NO.9:

[0083] CAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAGCATGAGGCATGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATTTGCAAATGGACAGCCTGAGAGCCGAAGACACGGCTGCGTATTACTGTGCGAGAGCGACGTTTGGCAGTGGCTCAGGTGAATATTTCCAGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGT。

[0084] The amino acid sequence of VH of 203-10 is shown below, as shown in SEQ ID NO.10:

[0085] QVQLVETGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVASMRHDGSEKYYVDSVKGRFTISRDNAK NSLYLQMDSLRAEDTAAYYCARATFGSGSGEYFQDWGQGTLVTVSS.

[0086] The gene sequence of VL of 203-10 is as follows (direction 5'-3'), as shown in SEQ ID NO.11:

[0087] GAAATTGTGCTGACTCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCGACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGC ATCTACCCGGGCATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATTGTGGCAGTTTATCACTGTCAGCAATATTATAGTAGTCCCCGACTTTTGGCCAGGGGACCAAAGTGGATATCAAA.

[0088] The amino acid sequence of VL of 203-10 is shown below, as shown in SEQ ID NO.12:

[0089] EIVLTQSPDSLAVSLGERATINCKSSQSVLYSSDNKNYLAWYQQKPGQPPKLLIYWASTRASGVPDRFSGSGSGT FTLTISSLQAEDVAVYHCQQYYSSPPTFGQGTKVDIK.

[0090] The gene sequence of VH of 203-59 is as follows (direction 5'-3'), as shown in SEQ ID NO.13:

[0091] GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGGAGCCTGGGGCCTCAGTGAAAGTTTCGTGCAAGGCATCTGGATACACCTTCATCAGATACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTAGTGGTAGCACAAGCTACGCAGA GACGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTATATGGACCTGAGCAGCCTGAGATCTGAGGACACGGCCGTCTATTACTGTGCGAGAGATGGGGGATACGGCTATGGGGGAAGTGGGGTCATGGACGTCTGGGGCAAAGGGACCCTGGTCACCGTCTCGAGT.

[0092] The amino acid sequence of VH of 203-59 is shown below, as shown in SEQ ID NO.14:

[0093] EVQLVQSGAEVKEPGASVKVSCKASGYTFIRYYIHWVRQAPGQGLEWMGIINPSSGSTSYAETFQGRVTMTRDTST STVYMDLSSLRSEDTAVYYCARDGGYGYGGSGVMDVWGKGTLVTVSS.

[0094] The gene sequence of VL of 203-59 is as follows (direction 5'-3'), as shown in SEQ ID NO.15:

[0095] GACATCGTGATGACCCAGTCTCCAGGCTCCCTGGTTGTGTCTCTGGGCGACAGGGCCACCATCAACTGCAAGTCCAGCCAGACTATTTTCTCCAGCACCCACAATAAGGACTCCTTAGGTTGGTATCAGGTGAAACCAGGACAGTCTCCTAAACTACTCATTTTCGACAC ATCCATCCGGGGATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCACCAATATGAACATCTTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA.

[0096] The amino acid sequence of VL of 203-59 is shown below, as shown in SEQ ID NO.16:

[0097] DIVMTQSPGSLVVSLGDRATINCKSSQTIFSSTHNKDSLGWYQVKPGQSPKLLIFDTSIRGSGVPDRFSGSGSGT FTLTISSLQAEDVAVYYCHQYEHLPLTFGGGTKVEIK.

[0098] The gene sequence of VH of 203-9 is as follows (direction 5'-3'), as shown in SEQ ID NO.17:

[0099] CAGCTGCAGCTGCAGGAGTCGGGGGGAGCCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCTCCTTCGGGATGTATGAAATGAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGCGTTTCCTACATTAGTGCAACCGGGAATACCATACACTACGC AAACTCTGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCGAGAGGTCGCTGTATCTGCAAATGAACAGTCTGGGAGCCGAGGACACGGGTGTTTTATTTCTGTGCGAGAGGCCCCGGGAGGTATTACTACCACGGGATGGACGTCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGT.

[0100] The amino acid sequence of VH of 203-9 is shown below, as shown in SEQ ID NO.18:

[0101] QLQLQESGGALVQPGGSLRLSCAGSGFSFGMYEMNWVRQAPGKGLECVSYISATGNTIHYANSVKGRFTVSRDNAE RSLYLQMNSLGAEDTGVYFCARGPGRYYYHGMDVWGQGTLVTVSS.

[0102] The gene sequence of VL of 203-9 is as follows (direction 5'-3'), as shown in SEQ ID NO.19:

[0103] GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTACTCAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCAAAGCTGCTCATTTACTGGGC ATCTACCCGGGAATCTGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCACTTTATTACTGTCACCAATATTATACTCTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA.

[0104] The amino acid sequence of VL of 203-9 is shown below, as shown in SEQ ID NO.20:

[0105] DIVMTQSPDSLAVSLGERATINCKSSQSVLLSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGT FTLTISSLQAEDVALYYCHQYYTLPWTFGQGTKVEIK.

[0106] The five clones were induced to express, and the periplasmic protein (scFv) was extracted. Periplasmic ELISA was performed for identification. The specific steps were as follows: coating concentrations of 1 μg / ml human PDL1 protein (h-PDL1), monkey PDL1 (cyno-PDL1) and 1 μg / ml irrelevant protein (NC), 100 μl per well, 4°C overnight, washed once with PBST, added skim milk powder for blocking, and then added periplasmic supernatant for incubation at room temperature for 1 hour, washed 4 times with PBST, added secondary antibody for incubation, washed and colored, and detected OD450 with an enzyme marker. The results are as follows Figure 2 As shown, 203-9, 203-10, 203-59, 203-64, and 203-69 can all specifically bind to human PDL1 protein, and 203-10, 203-59, 203-64, and 203-69 can bind to monkey PDL1 protein.

[0107] Experiment 2: Activity detection of candidate antibody molecules binding to proteins

[0108] The absorbance value of the antibody binding to PDL1 protein at different concentrations was detected by enzyme-linked immunosorbent assay (ELISA) to reflect the binding activity of the candidate molecule antibody to PDL1 protein. The specific steps were as follows: human PDL1 protein (h-PDL1) and monkey PDL1 (cyno-PDL1) with a coating concentration of 1 μg / ml were added, and different concentrations of periplasmic supernatant were added for incubation at room temperature for 1 hour. After washing with PBST, the secondary antibody was added for incubation, and the color was washed and developed. The OD450 was detected by an enzyme marker. The results are as follows: Figure 3 , Figure 4 shown.

[0109] like Figure 3 As shown, 203-9, 203-10, 203-59, 203-64, and 203-69 showed different degrees of binding to human PDL1 protein, and the relevant binding data are shown in Table 1. Among them, 203-64 had the strongest binding to human PDL1 protein, and 203-69, 203-10, 203-59, and 203-9 weakened in turn.

[0110] Table 1

[0111] 203-9 203-10 203-59 203-64 203-69 Bmax 3.248 2.747 2.519 2.52 2.582 Kd 5.453 0.5973 2.951 0.2832 0.4701

[0112] like Figure 4 As shown, the four candidate molecules 203-64, 203-69, 203-10, and 203-59 showed different degrees of binding with monkey PDL1 protein. The relevant data are shown in Table 2. Among the four molecules, 203-64 had the strongest binding with monkey PDL1 protein, and 203-10, 203-69, and 203-59 weakened in turn.

[0113] Table 2

[0114] 203-10 203-59 203-64 203-69 Bmax 2.569 2.525 2.333 2.36 Kd 0.5809 7.253 0.4351 0.6922

[0115] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. An anti-PD-L1 antibody, characterized in that: It is the anti-PD-L1 antibody 203-64, the amino acid sequence of its VH is shown in SEQ ID NO.2, and the amino acid sequence of its VL is shown in SEQ ID NO.

4.

2. An anti-PD-L1 antibody, characterized in that: It is the anti-PD-L1 antibody 203-69, the amino acid sequence of its VH is shown in SEQ ID NO.6, and the amino acid sequence of its VL is shown in SEQ ID NO.

8.

3. An anti-PD-L1 antibody, characterized in that: It is the anti-PD-L1 antibody 203-10, the amino acid sequence of its VH is shown in SEQ ID NO.10, and the amino acid sequence of its VL is shown in SEQ ID NO.

12.

4. An anti-PD-L1 antibody, characterized in that: It is the anti-PD-L1 antibody 203-59, the amino acid sequence of its VH is shown in SEQ ID NO.14, and the amino acid sequence of its VL is shown in SEQ ID NO.

16.

5. An anti-PD-L1 antibody, characterized in that: It is the anti-PD-L1 antibody 203-9, the amino acid sequence of its VH is shown in SEQ ID NO.18, and the amino acid sequence of its VL is shown in SEQ ID NO.

20.

6. An isolated nucleic acid, characterized in that: It encodes the anti-PD-L1 antibody according to any one of claims 1 to 5.

7. The isolated nucleic acid according to claim 6, characterized in that: The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-64 is shown in SEQ ID NO.1, and the nucleotide sequence encoding VL is shown in SEQ ID NO.3; The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-69 is shown in SEQ ID NO.5, and the nucleotide sequence encoding VL is shown in SEQ ID NO.7; The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-10 is shown in SEQ ID NO.9, and the nucleotide sequence encoding VL is shown in SEQ ID NO.11; The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-59 is shown in SEQ ID NO.13, and the nucleotide sequence encoding VL is shown in SEQ ID NO.15; The nucleotide sequence encoding VH of the anti-PD-L1 antibody 203-9 is shown in SEQ ID NO.17, and the nucleotide sequence encoding VL is shown in SEQ ID NO.

19.

8. A recombinant expression vector, characterized in that: Comprising the isolated nucleic acid of claim 6 or 7.

9. A transformant, characterized in that: Comprising the recombinant expression vector according to claim 8.

10. Use of the anti-PD-L1 antibody according to any one of claims 1 to 5, the isolated nucleic acid according to claim 6 or 7, the recombinant expression vector according to claim 8, or the transformant according to claim 9 in the preparation of an anti-tumor drug.

Images