Anti-pd-l1 antibodies and uses thereof

Abstract

The application discloses anti-PD-L1 antibodies and application thereof, and belongs to the technical field of monoclonal antibodies. The anti-PD-L1 antibodies are five kinds, 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, the amino acid sequences of VH and VL and the nucleotide sequences of the coding genes are shown in SEQ ID NO. 1-20. The application of the anti-PD-L1 antibodies in the preparation of anti-tumor drugs. The anti-PD-L1 antibodies have good affinity with human PDL1 protein and monkey PDL1 protein, and can be used for tumor treatment. The application enriches the full human single-chain antibodies specifically combined with PD-L1, provides potential new antibody drugs for clinical treatment of tumors, and has high potential application value and significance.

Description

TECHNICAL FIELD

[0001] The present application relates to anti-PD-L1 antibodies and uses thereof, and belongs to the technical field of monoclonal antibodies. BACKGROUND

[0002] PD1, namely 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. After binding with the receptor PD-L1, namely Programmed cell death 1 ligand 1, and PD-L2, namely Programmed cell death 1 ligand 2, the function of T cells can be inhibited, and apoptosis can be induced.

[0003] PD-L1 is also known as surface antigen cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), which is a protein encoded by CD274 gene. Various tumor cells such as lung cancer, melanoma, colon cancer, breast cancer, gastric cancer, etc. have high expression of PD-L1, which can interact with PD-1 on the surface of tumor-infiltrating lymphocytes, thereby inhibiting the killing of lymphocytes to tumor, leading to tumor escape. Anti-PD-L1 antibody can specifically bind to PD-L1, block the signal pathway mediated by PD-L1, thereby relieving the inhibition function of the immune system, achieving the purpose of treating cancer or other related diseases. Therefore, the development of anti-PD-L1 antibody has good clinical application value. SUMMARY

[0004] In view of the above prior art, the present application provides several anti-PD-L1 antibodies and uses thereof, which belong to the technical field of monoclonal antibodies.

[0005] The present application is realized by the following technical solutions:

[0006] The anti-PD-L1 antibody has five kinds, which are respectively named as 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, wherein,

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

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

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

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

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

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

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

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

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

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

[0018] A recombinant expression vector comprising the above-mentioned isolated nucleic acid.

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

[0020] The application of the anti-PD-L1 antibody, the isolated nucleic acid, the recombinant expression vector and the transformant in preparing an anti-tumor drug.

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

[0022] The application screens a full human source single-chain antibody specifically combined with PD-L1 from a full human source phage library, has good affinity to human source PDL1 protein and monkey source PDL1 protein, and can be used for tumor treatment.

[0023] Various terms and phrases used in the application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF 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 source PDL1 protein. DETAILED DESCRIPTION

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

[0029] The instruments, reagents, materials and the like involved in the following examples are all conventional instruments, reagents, materials and the like existing in the prior art if not specifically stated, and can be obtained through regular commercial channels. The experimental methods, detection methods and the like involved in the following examples are all conventional experimental methods, detection methods and the like existing in the prior art if not specifically stated.

[0030] Experiment 1: Screening of anti-PD-L1 antibody

[0031] 1. Experimental method

[0032] 1.1 Construction of full human source phage antibody library

[0033] 1.1.1 Collect B cells of healthy people, extract total RNA. Amplify VH and VL genes of antibody respectively by RT-PCR, electrophorese, and purify and recover VH and VL fragments.

[0034] 1.1.2 Link the recovered VH and VL fragments to phage vector by genetic engineering technology, transfer the constructed vector to E. coli by electric transfer, and the phagemid vector can self-replicate in the successfully transduced host bacteria. When the host bacteria grow to logarithmic phase, add helper phage to infect at MOI equal to 10, culture overnight to amplify the phage, and obtain the phage library by centrifugal precipitation.

[0035] 1.2 Screening of phage antibody library

[0036] The steps are as follows:

[0037] (1) Coat 10 μg / ml of PDL1 protein on an enzyme-labeled plate at 4°C overnight; inoculate the preserved E. coli TG1, and culture at 37°C, 220 rpm overnight.

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

[0039] (3) Remove the antigen solution on the enzyme-labeled plate, wash with PBS for 3 times, and then add 2% skimmed milk powder to block at room temperature for 1 hour, while blocking the phage antibody library with 2% skimmed milk powder solution at room temperature for 1 hour.

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

[0041] (5) Remove the phage solution, and wash the enzyme-labeled plate with PBST for 15 times.

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

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

[0044] (8) Take 10 μl of bacterial liquid, dilute by 100 times, 1000 times and 10000 times respectively, and inoculate on resistant 2YT plates, for calculating the phage titer.

[0045] (9) The rest of the bacterial solution was inoculated on 2YT plate (containing 100 μg / ml ampicillin and 1% glucose) and incubated at 37°C for 16 hours.

[0046] (10) The clones on the overnight plate were scraped off and incubated at 37°C until the OD600 reached 0.4.

[0047] (11) The helper phage was added and incubated at 37°C for 30 minutes, and then incubated at 220 rpm for 60 minutes.

[0048] (12) The bacteria were collected by centrifugation and incubated in 2YT medium (containing 100 μg / ml ampicillin, 50 μg / ml kanamycin and 0.1% glucose) at 30°C and 220 rpm overnight.

[0049] (13) The supernatant was collected by centrifugation of the overnight culture, which was used as the phage for the second round of screening.

[0050] (14) The above screening process was repeated for at least 3 rounds until the titer of the phage was significantly improved.

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

[0052] The steps are as follows:

[0053] (1) The antigen was diluted with PBS and coated on the ELISA plate at 100 μl per well, and incubated at 4°C overnight.

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

[0055] (3) After washing the ELISA plate 3 times with PBST, the diluted primary antibody was added and incubated at room temperature for 1 hour.

[0056] (4) The ELISA plate was washed 4 times with PBST.

[0057] (5) The diluted HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour.

[0058] (6) The ELISA plate was washed 4 times with PBST.

[0059] (7) 100 μl / well of TMB substrate solution was added, and the color development was stopped after 10-15 minutes by adding 2M sulfuric acid.

[0060] (8) The plate was read on a microplate reader at 450 nm wavelength, and the results were analyzed.

[0061] 2. Experimental results

[0062] 2.1 Screening of antibodies binding to PDL1 protein

[0063] In this study, the full human antibody (scFv) library constructed in our laboratory was used to screen against the target of human PDL1. In this experiment, PDL1 protein with a concentration of 10 μg / ml was used as an antigen and coated on an enzyme-labeled plate. After three rounds of screening, the titer of phage was obviously enriched.

[0064] Randomly selected monoclonal phage ELISA was performed, and the specific steps included coating human PDL1 protein at a concentration of 1 μg / ml per well of 100 μl at 4°C overnight, washing once with PBST, adding skimmed milk powder for blocking, then adding phage supernatant, incubating at room temperature for 1 h, washing 4 times with PBST, adding secondary antibody, washing and developing, and detecting OD450 with an enzyme-labeled instrument. The results are shown in Figure 1 .

[0065] The positive clones were selected for sequencing, and it was shown that five independent clones (203-9, 203-10, 203-59, 203-64, 203-69) were enriched at a higher level. The VHand VLgene sequences and amino acid sequences of the five antibodies are shown below.

[0066] The VHand VLgene sequences of 203-64 are shown below (direction 5'-3'), as shown in SEQ ID NO. 1:

[0067] GAGCTGCAGGAGTCGGGCCCAGGACTGGTGAGGCCTTCGGAGACCCTGTCACTCACCTGCAGTGTCTCTCGTGGGTCCGTCAGTCAAAACTATTGGAGCTGGTTCCGGCAGTCCCCAGGGAAGGCATTGGAGTGGATTGGCTTTGTCCACTATAGTGGGGTCGCCAAATACGCCCCCCCCCTCACGGGTCGAGTCACCATCTCCCACGACCCGTCCAAGGCGGAAGTCTCCCTGAGGTTGACTTCTGTGACCGCCGCAGACACGGCCCGCTACTTCTGTGCGCGACATCTTCAGTGGGACAACCCAATCGATGTCTGGGGCCAGGGGACCACGGTCACCGTCTCGAGT.

[0068] The VHand VLgene sequences of 203-64 are shown below (direction 5'-3'), as shown in SEQ ID NO. 1:

[0069] ELQESGPGLVRPSETLSLTCSVSRGSVSQNYWSWFRQSPGKALEWIGFVHYSGVAKYAPPLTGRVTISHDPSKAEV SLRLTSVTAADTARYFCARHLQWDNPIDVWGQGTTVTVSS.

[0070] The genetic sequence of the VL of 203-64 is shown below (direction 5’-3’), as set forth in SEQ ID NO. 3:

[0071] CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGCTGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCAGCTACAGGCGGCACCACCCCCTATGTCTTCGGAACTGGGACCAAAGTCACCGTCCTA.

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

[0073] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYAVSNRPSGVSNRFSGSKSGNTASLT ISGLQAEDEADYYCSSATGGTTPYVFGTGTKVTVL.

[0074] The genetic sequence of the VH of 203-69 is shown below (direction 5’-3’), as set forth in SEQ ID NO. 5:

[0075] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACGTGCACTGTCTCTGGTGGCTCCATCAGAAGTGGTGGTTCCTACTGGACTTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATACATCGATTACAGAGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCCTCGGACACGTCTAGGAACCAGCTCTCCCTGAAGCTGACCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGGGAAGAACGACGTTCGGGGAAATACAGTATGGACGTCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGT.

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

[0077] QVQLQESGPGLVKPSQTLSLTCTVSGGSIRSGGSYWTWIRQHPGKGLEWIGYIDYRGSTYYNPSLKSRVTISSDTS RNQLSLKLTSVTAADTAVYYCAREERRSGKYSMDVWGQGTLVTVSS.

[0078] The genetic sequence of the VL of 203-69 is shown below (direction 5’-3’), as set forth in SEQ ID NO. 7:

[0079] GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGTAGGTCTAGTCAAAGCCTCGTAGCCAGTGATGGAAACACGTACTTAAATTGGCTTCACCAGAGGCCAGGCCAGCCTCCAAGACTCCTAATTTACAAGATTTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGGTGGAAGCTGAGGATGTCGGGGTTTTTTACTGCATGCAAGCTACACAAATTCCTCAGACGTTCGGCCAAGGGACACGACTGGAGATTAAA.

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

[0081] DIVMTQTPLSSPVTLGQPASISCRSSQSLVASDGNTYLNWLHQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDF TLKISRVEAEDVGVFYCMQATQIPQTFGQGTRLEIK.

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

[0083] CAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAACTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAGCATGAGGCATGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATTTGCAAATGGACAGCCTGAGAGCCGAAGACACGGCTGCGTATTACTGTGCGAGAGCGACGTTGGCAGTGGCTCAGGTGAATATTTCCAGGACTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGT.

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

[0085] QVQLVETGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVASMRHDGSEKYYVDSVKGRFTISRDNAK NSLYLQMDSLRAEDTAAYYCARATFGSGSGEYFQDWGQGTLVTVSS.

[0086] The nucleic acid sequence of the VL of 203-10 is shown below (direction 5’-3’), as set forth in SEQ ID NO. 11:

[0087] GAAATTGTGCTGACTCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCGACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGCATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATCACTGTCAGCAATATTATAGTAGTCCCCCGACTTTTGGCCAGGGGACCAAAGTGGATATCAAA.

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

[0089] EIVLTQSPDSLAVSLGERATINCKSSQSVLYSSDNKNYLAWYQQKPGQPPKLLIYWASTRASGVPDRFSGSGSGTD FTLTISSLQAEDVAVYHCQQYYSSPPTFGQGTKVDIK.

[0090] The nucleic acid sequence of the VH of 203-59 is shown below (direction 5’-3’), as set forth in SEQ ID NO. 13:

[0091] GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGGAGCCTGGGGCCTCAGTGAAAGTTTCGTGCAAGGCATCTGGATACACCTTCATCAGATACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTAGTGGTAGCACAAGCTACGCAGAGACGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTATATGGACCTGAGCAGCCTGAGATCTGAGGACACGGCCGTCTATTACTGTGCGAGAGATGGGGGATACGGCTATGGGGGAAGTGGGGTCATGGACGTCTGGGGCAAAGGGACCCTGGTCACCGTCTCGAGT.

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

[0093] EVQLVQSGAEVKEPGASVKVSCKASGYTFIRYYIHWVRQAPGQGLEWMGIINPSSGSTSYAETFQGRVTMTRDTSTSTVYMDLSSLRSEDTAVYYCARDGGYGYGGSGVMDVWGKGTLVTVSS.

[0094] The genetic sequence of the VL of 203-59 is shown immediately below (direction 5’-3’), as set forth in SEQ ID NO. 15:

[0095] GACATCGTGATGACCCAGTCTCCAGGCTCCCTGGTTGTGTCTCTGGGCGACAGGGCCACCATCAACTGCAAGTCCAGCCAGACTATTTTCTCCAGCACCCACAATAAGGACTCCTTAGGTTGGTATCAGGTGAAACCAGGACAGTCTCCTAAACTACTCATTTTCGACACATCCATCCGGGGATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCACCAATATGAACATCTTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA.

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

[0097] DIVMTQSPGSLVVSLGDRATINCKSSQTIFSSTHNKDSLGWYQVKPGQSPKLLIFDTSIRGSGVPDRFSGSGSGTD FTLLTIS SLQAEDVAVYYCHQYEHLPLTFGGGTKVEIK.

[0098] The gene sequence of the VH of 203-9 is shown below (direction 5’-3’), as set forth in SEQ ID NO. 17:

[0099] CAGCTGCAGCTGCAGGAGTCGGGGGGAGCCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCTCCTTCGGGATGTATGAAATGAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGCGTTTCCTACATTAGTGCAACCGGGAATACCATACACTACGCAAACTCTGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCGAGAGGTCGCTGTATCTGCAAATGAACAGTCTGGGAGCCGAGGACACGGGTGTTTATTTCTGTGCGAGAGGCCCCGGGAGGTATTACTACCACGGGATGGACGTCTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGT.

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

[0101] QLQLQESGGALVQPGGSLRLSCAGSGFSFGMYEMNWVRQAPGKGLECVSYISATGNTIHYANSVKGRFTVSRDNAE RSLYLQMNSLGAEDTGVYFCARGPGRYYYHGMDVWGQGTLVTVSS.

[0102] The genetic sequence of the VL of 203-9 is shown below (direction 5’-3’), as set forth in SEQ ID NO. 19:

[0103] GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTACTCAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCAAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCTGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCACTTTATTACTGTCACCAATATTATACTCTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA.

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

[0105] DIVMTQSPDSLAVSLGERATINCKSSQSVLLSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTD FTLTISSLQAEDVALYYCHQYYTLPWTFGQGTKVEIK.

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

[0107] Experiment two: detection of the activity of the candidate antibody molecules binding to the protein

[0108] The absorbance value of the antibody combined with PDL1 protein of different concentrations was detected by enzyme-linked immunosorbent assay (ELISA) to reflect the binding activity of the candidate antibody to PDL1 protein. The specific steps are as follows: coating human PDL1 protein (h-PDL1) and monkey PDL1 (cyno-PDL1) with a concentration of 1 μg / ml, blocking, then adding different concentrations of periplasmic supernatant for incubation, room temperature for 1 h, washing with PBST, then adding secondary antibody for incubation, washing, developing, detecting OD450 with an enzyme label instrument, and the results are shown in Figure 3 、 Figure 4

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

[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] As shown in Figure 4 , 203-64, 203-69, 203-10, and 203-59 showed different degrees of binding to monkey PDL1 protein. The relevant data are shown in Table 2. Among them, 203-64 showed the strongest binding to monkey PDL1 protein, followed by 203-10, 203-69, and 203-59.

[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 implement and use the claimed embodiments, rather than to limit the scope of the disclosure disclosed herein. Modifications obvious to those skilled in the art will be within the scope of the appended claims.​

Claims

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

4.

2. An isolated nucleic acid, characterized in that: The anti-PD-L1 antibody 203-64 is encoded by the nucleotide sequence shown as SEQ ID NO.

1.

3. The isolated nucleic acid of claim 2, wherein: The anti-PD-L1 antibody 203-64 is encoded by the nucleotide sequence shown as SEQ ID NO.

1.

4. A recombinant expression vector, characterized by: The anti-PD-L1 antibody 203-64 is encoded by the nucleotide sequence shown as SEQ ID NO.

1.

5. A transformant characterized in that: The anti-PD-L1 antibody 203-64 is encoded by the nucleotide sequence shown as SEQ ID NO.

1.

6. The use of the anti-PD-Ll antibody of claim 1 in the preparation of an antitumor drug, characterized in that: The tumor is selected from any one or two or more of lung cancer, melanoma, colon cancer, breast cancer, and gastric cancer.

7. Use of the isolated nucleic acid of claim 2 or 3 for the manufacture of a medicament for the treatment of a neoplasm, characterized in that: The tumor is selected from any one or two or more of lung cancer, melanoma, colon cancer, breast cancer, and gastric cancer.

8. Use of the recombinant expression vector according to claim 4 for the preparation of a medicament for the treatment of tumors, characterized in that: The tumor is selected from any one or two or more of lung cancer, melanoma, colon cancer, breast cancer, and gastric cancer.

9. Use of the transformant according to claim 5 for the manufacture of an antitumor medicament, characterized in that: The tumor is selected from any one or two or more of lung cancer, melanoma, colon cancer, breast cancer, and gastric cancer.

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