A high-affinity human anti-PD-L1 antibody and its application

Abstract

The present invention discloses a high-affinity anti-PD-L1 human antibody and its application. The antibody comprises a light chain variable region and a heavy chain variable region; wherein the light chain variable region comprises an amino acid sequence as shown in FIG.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a high-affinity anti-PD-L1 human antibody and its application. Background Art

[0002] Programmed cell death ligand-1 (PD-L1) is a 40kDa type I transmembrane protein in the B7 family and the primary ligand that binds to PD-1. The amino acid structure of PD-L1 is similar to that of PD-1, containing both IgC and IgV domains. PD-L1 is expressed in hematopoietic cells (T cells, B cells, macrophages, dendritic cells, and mast cells) and non-hematopoietic healthy tissue cells (vascular endothelial cells, pancreatic islet cells, and endothelial cells, among others). Recent studies have revealed that PD-L1 is highly expressed on tumor cells and on host immune and stromal cells within the tumor microenvironment, leading to immune escape. Tumor immune escape is crucial for tumorigenesis and a key breakthrough in tumor immunotherapy. Therefore, developing blocking antibodies targeting immune checkpoints to block the binding between PD-1 and PD-L1 is considered an effective approach for cancer treatment.

[0003] Antibody preparation has gone through three stages: polyclonal technology, monoclonal technology, and antibody library technology. Compared with monoclonal and polyclonal technologies, antibody library technology is much simpler to operate at the genetic level. Gene sequences can be analyzed, modified, and processed to improve antibody properties or impart new properties to antibodies, breaking through the previous limitations of anti-antigen molecule design. However, compared with natural antibodies, antibodies derived from antibody libraries have lower affinity, making it difficult to meet various practical needs. In particular, antibodies from natural antibody libraries without immunization have an affinity only comparable to the level of antibody affinity acquired by animals after a primary immune response. Therefore, in order to improve antibody affinity in vitro, various strategies are adopted to simulate the in vivo affinity maturation process and to mutate antibody genes accordingly based on the principle of in vivo antibody affinity maturation.

[0004] With the continuous development of genetic engineering technology, genetically engineered antibodies have been widely used in drug development in the fields of treatment, diagnosis, and detection. Among genetically engineered antibodies, small molecule antibodies, such as single-chain antibody fragments (scFv), double-chain antibodies (diabodies), and Fab antibodies, have smaller molecular weights, which enable them to have enhanced tissue penetration, lower immunogenicity, ease of construction and expression, and the ability to be used in combination with other functional molecules. These advantages give them broad application prospects in basic medical research and tumor diagnosis and treatment. Compared with monoclonal antibodies, scFvs have lower affinity and long-term stability, and are more likely to aggregate due to their small size. Their rapid clearance from the blood may be a disadvantage for therapeutic applications. Another small molecule antibody, Fab, is one of the most successful antibody fragments in clinical practice. However, compared with intact antibodies, the stability of Fab fragments is weaker, which limits the application of Fab antibody drugs.

[0005] Diabodies are small molecule antibodies constructed on the basis of single-chain antibodies. They possess moderate molecular weight and bivalency, making them one of the best tumor-targeting antibody molecules. They also possess strong tumor tissue penetration and moderate blood clearance. Summary of the Invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a high-affinity humanized anti-PD-L1 antibody. Through in vitro affinity maturation of phage-displayed antibodies, a gene sequence for a humanized anti-PD-L1 antibody with enhanced affinity is obtained. A disulfide-bond-stabilized humanized anti-PD-L1 double-chain antibody containing two antigen-binding sites is then constructed. This is based on a Fab antibody, constructed through the construction of a diabody and the introduction of two cysteines through site-directed mutagenesis, thereby introducing a disulfide bond to create a ds-Diabody. This covalent bond binds the two peptide chains, greatly improving stability.

[0007] Another object of the present invention is to provide an application of the high-affinity anti-PD-L1 humanized double-chain antibody.

[0008] The object of the present invention is achieved by the following technical solutions: a high-affinity anti-PD-L1 human antibody, comprising a light chain variable region and a heavy chain variable region;

[0009] The light chain variable region contains three CDR regions, which are: amino acid sequences such as SLRSYY The LCDR1 shown in the amino acid sequence is as follows GKN The LCDR2 and amino acid sequences shown are as follows NSRDVRLKWV LCDR3 shown;

[0010] The heavy chain variable region contains three CDR regions, which are: amino acid sequences such as GGSISSYY The HCDR1 and amino acid sequences shown are IKQDESTK The HCDR2 and amino acid sequences shown are as follows ARVWWHAEIASNDY HCDR3 shown.

[0011] The amino acid sequence of the light chain variable region is preferably as follows (SEQ ID NO.11):

[0012] QDPVVSVALGQTVRITCQGD SLRSYY ASWYQQKPGQAPVLVIY GKN NRPSGIPDRFSGSSSGNTASLTITG AQAEDEADYYC NSRDVRLKWV FGGGTKLTVLG;

[0013] The amino acid sequence of the heavy chain variable region is preferably as follows (SEQ ID NO.12):

[0014] EVQLLESGPGLVKPSETLSLTCTVS GGSISSYY WSWIRQPPGKGLEWVAN IKQDESTK NYVDSVKGRFTIS RDNAKNSLYLQMNSLRAEDTAVYYC ARVWWHAEIASNDY WGQGTLVTVSS.

[0015] The nucleic acid encoding the high-affinity anti-PD-L1 human antibody can be determined according to the codon principle.

[0016] The sequence of the nucleic acid encoding the light chain variable region is preferably as follows (SEQ ID NO.9):

[0017] CAGGACCCTGTTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCA

[0018] GAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAA

[0019] AACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGA

[0020] CCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACGTGCGCTTGAAG TGGGTGTTCGGC GGAGGGACCAAGCTGACCGTCCTAGGT;

[0021] The sequence of the nucleic acid encoding the heavy chain variable region is preferably as follows (SEQ ID NO.10):

[0022] GAGGTGCAGCTGCTCGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCA

[0023] CTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCGGGGAAGGGACT

[0024] GGATGGGTGGCCAACATAAAGCAAGATGAAAGTACGAAAAACTATGTGGACTCTGTGAAGGGCCG

[0025] ATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAG

[0026] GACACGGGCTGTGTATTACTGTGCGAGAGTTTGGTGGCACGCCGAGATCGCCTCCAACGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

[0027] The gene encoding the light chain variable region consists of 309 bases and encodes 103 amino acids. The variable region contains three CDR (complementarity determining cluster) regions: CDR1 encodes 6 amino acids, CDR2 encodes 3 amino acids, and CDR3 encodes 10 amino acids. The variable region sequence is highly homologous to the human immunoglobulin light chain IGLV3-19*01, reaching 99.62%.

[0028] The gene encoding the heavy chain variable region consists of 363 bases, encoding 121 amino acids. The variable region contains three CDR (complementarity determining cluster) regions: CDR1 encodes 8 amino acids, CDR2 encodes 8 amino acids, and CDR3 encodes 14 amino acids. The variable region sequence is 85.4% homologous to the human immunoglobulin heavy chain IGHV3-7*01.

[0029] A method for preparing a gene encoding a high-affinity anti-PD-L1 human antibody comprises the following steps:

[0030] (1) According to the hotspot mutation principle, mutation hotspots are screened in the CDR3 regions of the original light chain and the original heavy chain to obtain light chain gene sequences containing mutation hotspots and heavy chain gene sequences containing mutation hotspots; based on the light chain gene sequences containing mutation hotspots and the heavy chain gene sequences containing mutation hotspots, primers containing NSS mutation bases are designed to amplify the original light chain gene and the original heavy chain gene, respectively, to obtain light chain genes containing mutation hotspots and heavy chain genes containing mutation hotspots; the genes obtained above are double-digested and ligated with the restriction site of phagemid pComb3XSS to obtain a recombinant phagemid vector;

[0031] (2) Transform the recombinant phagemid into XL1-Blue by electroporation, spread on plates, observe colony growth, calculate the bacterial library content, and obtain a phage antibody mutation library;

[0032] (3) Using PD-L1 as an antigen, solid-phase screening is performed using the phage antibody mutation library constructed in step (2) to obtain humanized anti-PD-L1 antibody genes with improved relative affinity.

[0033] The nucleotide sequence of the original light chain gene described in step (1) is shown in SEQ ID NO.1.

[0034] The nucleotide sequence of the original heavy chain gene described in step (1) is shown in SEQ ID NO.2.

[0035] The nucleotide sequence of the light chain gene containing the mutation hotspot described in step (1) is shown in SEQ ID NO.5.

[0036] The nucleotide sequence of the heavy chain gene containing the mutation hotspot described in step (1) is shown in SEQ ID NO.6.

[0037] Step (1) is preferably as follows:

[0038] ① Design primers:

[0039] Primer 1: 5′-C GAGCTC CAGGACCCTGTTGTG-3′;

[0040] Primer 2: 5′-AACTCCCGGGAC NNSNNSNNSNNS TGGGTGTTCGGC-3′;

[0041] Primer 3: 5′-GCCGAACACCCA SNNSNNSNNSNN GTCCCGGGAGTT-3′;

[0042] Primer 4: 5′-GC TCTAGA ACCTAGGACGGTCAG-3′;

[0043] Primer 5: 5′-GAGGTGCAGCTG CTCGAG TCTGGCCCAGGACTG-3′;

[0044] Primer 6: 5′-TGTGCGAGAGTTTGG NNSNNSNNSNNSNNS GCC NNSNNS GACTACTGGGGCCAG-3′;

[0045] Primer 7: 5′-CTGGCCCCAGTAGTC SNNSNN GGC SNNSNNSNNSNNSNN CCAAACTCTCGCACA-3′;

[0046] Primer 8: 5′-GG ACTAGT TGAGGAGACGGTGACCAGGGT-3′;

[0047] ② The light chain CDR3 region was subjected to site-directed random mutagenesis by PCR, and Sac I and Xba I restriction sites were introduced.

[0048] A. Using the original light chain gene fragment as a template, primers 1 and 3 were used for PCR amplification to introduce a Sac I restriction site and four hotspot mutations;

[0049] B. Using the original light chain gene fragment as a template, primers 2 and 4 were used for PCR amplification to introduce an Xba I restriction site and four hotspot mutations;

[0050] C. Splicing the products of steps A and B by overlap extension PCR to obtain a light chain recombinant gene containing two restriction sites and four hotspot mutations;

[0051] ③ The heavy chain CDR3 region was subjected to site-directed random mutagenesis by PCR, and Xho I and Spe I restriction sites were introduced.

[0052] D. Using the original heavy chain gene fragment as a template, primers 5 and 7 were used for PCR amplification to introduce an Xho I restriction site and seven hotspot mutations;

[0053] E. Using the original heavy chain gene fragment as a template, primers 6 and 8 were used for PCR amplification to introduce a Spe I restriction site and seven hotspot mutations;

[0054] F. Splicing the products of steps D and E by overlap extension PCR to obtain a light chain recombinant gene containing two restriction sites and seven hotspot mutations;

[0055] ④ Double-digest the product of step ② and the phagemid vector pComb3XSS using restriction endonucleases Xba I and Sac I, and ligate the two together using T4 DNA ligase to obtain the recombinant pComb3XSS-VL plasmid.

[0056] ⑤ Double-digest the product of step ③ and the recombinant plasmid of step ④ with restriction endonucleases Xho I and Spe I, and ligate the two together with T4 DNA ligase to obtain the recombinant pComb3XSS-Fab plasmid.

[0057] The transformed E. coli host in step (2) is XL1-Blue.

[0058] The number of solid phase screening in step (3) is preferably 4 rounds.

[0059] The solid phase screening process described in step (3) is a phage display process, and the affinity of the phage antibody mutants with significantly improved binding is screened by the ammonium thiocyanate elution method.

[0060] A disulfide bond-stabilized anti-PD-L1 human double-chain antibody, whose amino acid sequence (SEQ ID NO.7) is as follows: QDPVVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDVRLKWVFGCGTKLTVLGGGGSEVQLLESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKCLEWVANIKQDESTKNYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVWWHAEIASNDYWGQGTLVTVSS.

[0061] The gene encoding the disulfide-stabilized anti-PD-L1 humanized double-chain antibody can be obtained according to the codon principle, and its nucleotide sequence (SEQ ID NO. 8) is preferably as follows:

[0062] CAGGACCCTGTTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCA

[0063] GAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAA

[0064] AACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGA

[0065] CCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACGTGCGCTTGAA

[0066] GTGGGTGTTCGGCTGTGGGACCAAGCTGACCGTCCTAGGTGGCGGTGGCTCGGAGGTGCAGCTGCTC

[0067] GAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCT

[0068] CCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCGGGGAAGTGTCTGGAGTGGGTGGCCAA

[0069] CATAAAGCAAGATGAAAGTACGAAAAACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGA

[0070] GACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATT

[0071] ACTGTGCGAGAGTTTGGTGGCACGCCGAGATCGCCTCCAACGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

[0072] The sequence encoding the disulfide bond-stabilized anti-PD-L1 humanized double-chain antibody is based on the genes encoding the light chain variable region (VL, SEQ ID NO.9) and heavy chain variable region (VH, SEQ ID NO.10) of the Fab antibody with high affinity for PD-L1. The VL region of the antibody is substituted by the VH region of the antibody by overlapping PCR. L The 100th amino acid Gly and V HA site-directed mutation of amino acid Gly to Cys at position 44 introduced a covalent disulfide bond. A gene encoding a linker peptide (amino acid sequence GGGGS) was used to connect the mutated light and heavy chain variable regions to construct a VL-GGGGS-VH gene segment. This segment was recombined with a yeast expression vector (pPICZαA) and then expressed in Pichia pastoris GS115 as a disulfide-stabilized Diabody.

[0073] The method for preparing the disulfide bond-stabilized anti-PD-L1 humanized double-chain antibody comprises the following steps:

[0074] (1) Using the nucleic acid encoding the light chain variable region shown in SEQ ID NO.9 and the nucleic acid encoding the heavy chain variable region shown in SEQ ID NO.10 as templates, the 100th amino acid Gly in the light chain variable region and the 44th amino acid Gly in the heavy chain variable region were site-directedly mutated to Cys by overlapping PCR and PCR, and the light chain variable region and heavy chain variable region genes were connected by a connecting peptide gene fragment to construct a V L -GGGGS-V H gene fragments;

[0075] (2) V obtained in step (1) L -GGGGS-V H The gene fragment was cloned into an expression vector, transferred into host cells for expression, and purified to obtain a disulfide bond-stabilized humanized anti-PD-L1 double-chain antibody.

[0076] Step (1) is preferably as follows:

[0077] ① Design primers:

[0078] Primer 1': 5'-CCG GAATTC CAGGACCCTGTTGTGTCTGTG-3′;

[0079] Primer 2': 5' -CGAGCCACCGCCACC TAGGACGGTCAGCTTGGTCCC ACA GCCGAACACCCA-3′;

[0080] Primer 3': 5'- GGTGGCGGTGGCTCG GAGGTGCAGCTGCTCGAGTCT-3′;

[0081] Primer 4′: 5′-CCCCCGGGGAAG TGT CTGGAGTGGGTGGCC-3′;

[0082] Primer 5′: 5′-GGCCACCCACTCCAG ACACTTCCCCGGGGG-3′;

[0083] Primer 6': 5'-A GCGGCCGC CTATTA ATGATGATGATGATGATG TGAGGAGACGGTGAC-3′;

[0084] ②PCR site-directed mutation of amino acid Gly at position 100 of the light chain variable region to Cys, and amplification of the gene fragment VL-GGGGS was obtained:

[0085] A. Using the nucleic acid encoding the light chain variable region shown in SEQ ID NO. 9 as a template, PCR amplification was performed with primers 1' and 2' to obtain a V sequence in which the amino acid at position 100 was mutated from Gly to Cys. L , and introduced EcoR I restriction site;

[0086] ③PCR site-directed mutation of amino acid Gly at position 44 of the heavy chain variable region to Cys, and amplification of gene fragment V H -GGGGS:

[0087] B. Using the nucleic acid encoding the heavy chain variable region shown in SEQ ID NO. 10 as a template, PCR amplification was performed with primers 3' and 5' to obtain a V variant in which the 44th amino acid Gly was mutated to Cys. H -GGGGS;

[0088] C. Using the nucleic acid encoding the heavy chain variable region as shown in SEQ ID NO. 10 as a template, PCR amplification was performed with primers 4' and 6' to obtain VH-GGGGS with a Not I site and a histidine tag;

[0089] D. Splice the products obtained in step B and step C to obtain a spliced ​​product; then use the spliced ​​product as a template to perform PCR amplification with primers 3' and 6' to obtain a complete V H -GGGGS;

[0090] ④PCR splicing and amplification of gene fragment V L -GGGGS-V H : The products finally obtained from step ② and step ③ are spliced ​​to obtain a spliced ​​product; then, the spliced ​​product is used as a template to perform PCR amplification with primers 1' and 6' to obtain the complete VL-GGGGS-VH.

[0091] The expression vector described in step (2) includes a prokaryotic expression vector and a eukaryotic expression vector; preferably, it is a eukaryotic expression vector, more preferably, it is a yeast expression vector, and most preferably, it is pPICZαA.

[0092] The host cell in step (2) includes prokaryotic host cells and eukaryotic host cells; preferably, a eukaryotic host cell, more preferably a yeast host cell, and most preferably, Pichia pastoris GS115.

[0093] The use of the above-mentioned disulfide bond-stabilized anti-PD-L1 humanized double-chain antibody in the preparation of anti-tumor drugs.

[0094] The present invention has the following advantages and effects compared to the prior art:

[0095] (1) PD-L1 is a human protein. As a tumor immune target, it is closely related to tumors, so it cannot be used directly to develop vaccines. However, the disulfide bond-stabilized anti-PD-L1 double-chain antibody prepared by the present invention is a fully human antibody that solves the human anti-mouse antibody (HAMA) reaction that is unavoidable with mouse monoclonal antibodies, and can be used to directly develop antibody drugs for human treatment.

[0096] (2) The disulfide-bond-stabilized humanized anti-PD-L1 double-chain antibody prepared by the present invention is a dimer formed by covalently linking two single-chain antibodies through disulfide bonds. It contains two antigen-binding sites, is bivalent, and has good affinity. Secondly, a disulfide bond is introduced into the ds-Diabody, so that the two peptide chains are covalently bonded, which greatly enhances the stability of the antibody and has broad application prospects in clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 This is the gel electrophoresis result of the light chain gene fragment of the phage antibody mutation library targeting PD-L1 after site-directed random mutagenesis PCR amplification; among them, lane M is the nucleic acid molecular weight standard; lane 1 is the upstream fragment of the light chain obtained by PCR amplification after site-directed random mutagenesis; lane 2 is the downstream fragment of the light chain obtained by PCR amplification after site-directed random mutagenesis; lane 3 is the fragment after Overlap PCR splicing of the upstream and downstream fragments of the light chain; lane 4 is the full light chain fragment obtained by PCR amplification.

[0098] Figure 2 This is the gel electrophoresis result of the heavy chain gene fragment of the phage antibody mutation library targeting PD-L1 after site-directed random mutagenesis PCR amplification; among them, lane M is the nucleic acid molecular weight standard; lane 1 is the upstream fragment of the heavy chain obtained by PCR amplification after site-directed random mutagenesis; lane 2 is the downstream fragment of the heavy chain obtained by PCR amplification after site-directed random mutagenesis; lane 3 is the fragment after Overlap PCR splicing of the upstream and downstream fragments of the heavy chain; lane 4 is the full heavy chain fragment obtained by PCR amplification.

[0099] Figure 3The figure shows the gel electrophoresis results after double enzyme digestion of phagemid pComb3XSS and light chain gene; lane M is a nucleic acid molecular weight standard; lane pComb is a fragment of phagemid after double enzyme digestion with SacI and XbaI; lane VL is a fragment of light chain gene fragment after double enzyme digestion with SacI and XbaI.

[0100] Figure 4 The figure shows the gel electrophoresis results after double enzyme digestion of the heavy chain gene of the recombinant pComb3XSS-VL plasmid; among them, lane M is a nucleic acid molecular weight standard; lane VH is the fragment of the heavy chain gene fragment after double enzyme digestion with XhoI and SpeI; lane pComb-VL is the fragment of the heavy chain gene fragment after double enzyme digestion with XhoI and SpeI.

[0101] Figure 5 This is the Phage-ELISA test result of the fourth round of screening of the phage antibody mutation library using PD-L1 as the antigen.

[0102] Figure 6 This is a graph showing the results of relative affinity determination of phage antibody mutants using the NH4SCN elution method.

[0103] Figure 7 This is a gel electrophoresis result of the disulfide bond-stabilized anti-PD-L1 humanized double-chain antibody gene fragment constructed by site-directed mutagenesis PCR amplification; among them, lane M is a nucleic acid molecular weight standard; lane 1 is the light chain gene fragment obtained by PCR amplification after site-directed mutagenesis; lanes 2 and 3 are the full light chain gene fragment obtained by PCR amplification after site-directed mutagenesis and the introduction of a five-amino acid short peptide; lanes 4 and 5 are the upper half of the heavy chain gene fragment obtained by PCR amplification after site-directed mutagenesis; lanes 6 and 7 are the lower half of the heavy chain gene fragment obtained by PCR amplification after site-directed mutagenesis; lanes 8 and 9 are the full heavy chain gene fragment obtained by Overlap PCR splicing the upper and lower heavy chain gene fragments; lanes 10 and 11 are the full ds-Diabody gene fragment obtained by Overlap PCR splicing the light chain and heavy chain gene fragments.

[0104] Figure 8 This is a graph showing the results of PCR identification of the recombinant yeast expression vector pPICZαA-ds-Diabody culture solution; wherein, lane M is a nucleic acid molecular weight standard; lanes 1-15 are ds-Diabody target fragments after PCR of the recombinant yeast expression vector pPICZαA-ds-Diabody culture solution.

[0105] Figure 9The figure shows the agarose gel electrophoresis results of using colony PCR to identify the pPICZαA-ds-Diabody recombinant vector transformed with Pichia pastoris GS115; wherein, lane M is a nucleic acid molecular weight standard; lane P is a PCR amplification of the gene fragment of the GS115-pPICZαA empty vector; lane P is a PCR amplification of the gene fragment of the GS115-pPICZαA empty vector; lane - is a PCR amplification of the gene fragment of the pPICZαA empty vector; lane + is a PCR amplification of the gene fragment of the pPICZαA-ds-Diabody recombinant plasmid vector; and lanes 1-17 are PCR amplifications of the gene fragments of the pPICZαA-ds-Diabody transformant.

[0106] Figure 10 This is a Western blot result of the yeast expression supernatant of the disulfide-stabilized anti-PD-L1 humanized diabody after reducing gel electrophoresis SDS-PAGE; among them, lane NC is the band of the yeast expression supernatant of the pPICZαA empty vector; lanes 1-3 and 5-17 are protein bands of the yeast expression supernatant of pPICZαA-ds-Diabody from different transformants.

[0107] Figure 11 This is a graph showing the activity of the anti-PD-L1 human double-chain antibody ds-Diabody binding to the antigen PD-L1 detected by indirect ELISA.

[0108] Figure 12 The figure shows the SDS-PAGE gel electrophoresis results of the expressed disulfide-stabilized anti-PD-L1 human double-chain antibody after purification by Ni SepharoseTM 6ⅹFastFlow; lane M is the protein molecular weight standard; lane 1 is the purified anti-PD-L1 human double-chain antibody ds-Diabody.

[0109] Figure 13 This is a graph showing the activity of the anti-PD-L1 human double-chain antibody ds-Diabody binding to the antigen PD-L1 detected by indirect ELISA.

[0110] Figure 14 This is an analysis chart of the stability of anti-PD-L1 humanized double-chain antibody ds-Diabody binding to the antigen PD-L1 detected by indirect ELISA. DETAILED DESCRIPTION

[0111] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0112] Example 1

[0113] Construction of a phage antibody mutation library targeting PD-L1 and screening of anti-PD-L1 Fab antibodies:

[0114] (1) Construction of an antibody mutation library targeting PD-L1

[0115] The Fab antibody gene of anti-PD-L1 (SEQ ID NO.1 and SEQ ID NO.2) screened from the existing natural phage antibody library in the laboratory was used as a template to construct a phage antibody mutation library. In in vitro affinity maturation, fixed hotspots in the CDR region are most commonly used for site-directed mutagenesis, and most hotspots are the two fixed nucleotide sequences AGY or RGYW (R = A or G, Y = C or T, W = A or T). SnapGene software was used to analyze the gene sequence. According to the hotspot mutation principle, it was found that there were 3 hotspots in the light chain CDR3 region gene (SEQ ID NO.3), involving 4 amino acids, and 4 hotspots in the heavy chain CDR3 region gene, involving 7 amino acids (SEQ ID NO.4).

[0116] The anti-PD-L1 Fab antibody gene sequence is shown below:

[0117] Light chain nucleotide sequence (SEQ ID NO.1):

[0118] CAGGACCCTGTTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCA

[0119] GAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAA

[0120] AACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGA

[0121] CCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAG;

[0122] Heavy chain nucleotide sequence (SEQ ID NO.2):

[0123] GAGGTGCAGCTGCTCGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCA

[0124] CTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCGGGGAAGGGACT

[0125] GGATGGGTGGCCAACATAAAGCAAGATGAAAGTACGAAAAACTATGTGGACTCTGTGAAGGGCCG

[0126] ATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAG

[0127] GACACGGCTGGTATTACTGTGCGAGAGTTTGGGGTACCAGTGGCTGGGCCGGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

[0128] The antibody sequences obtained after analysis are shown below:

[0129] Amino acid sequence of the light chain variable region (SEQ ID NO.3):

[0130] QDPVVSVALGQTVRITCQGD SLRSYY SWYQQKPGQAPVLVIY GKN NRPSGIPDRFSGSSSGNTASLTITGA

[0131] L-CDR-1L-CDR-2

[0132] QAEDEADYYC NSRDXXXXWV FGGGTKLTVLG;

[0133] L-CDR-3

[0134] Amino acid sequence of the heavy chain variable region (SEQ ID NO.4):

[0135] EVQLLESGPGLVKPSETLSLTCTVS GGSISSYY WSWIRQPPGKGLEWVAN IKQDESTK NYVDSVKGRFTIS

[0136] H-CDR-1H-CDR-2RDNAKNSLYLQMNSLRAEDTAVYYC ARVWXXXXXAXXDY WGQGTLVTVSS;

[0137] H-CDR-3

[0138] Among them, X in the CDR3 region is a mutation hotspot site, and X can be any amino acid, preferably valine, arginine, leucine, lysine, tryptophan, histidine, alanine, glutamic acid, isoleucine, serine or asparagine.

[0139] First, the light chain gene of the Fab antibody gene (nucleotide sequence shown in SEQ ID NO.1) was used as a template to amplify the light chain gene by PCR, and a SacⅠ restriction enzyme site, an XbaⅠ restriction enzyme site and random mutations at four amino acid sites were introduced.

[0140] Reaction 1): Design primers

[0141] Primer 1: 5′-C GAGCTC CAGGACCCTGTTGTG-3′(Sac I);

[0142] Primer 3: 5′-GCCGAACACCCA SNNSNNSNNSNN GTCCCGGGAGTT-3′ (mutation site, N=A\T\C\G; S=G\C);

[0143] Reaction system ① is:

[0144] 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of Primer 1 and Primer 3 (10 μmol / L), and 1 μL of Fab antibody gene template were added; sterile water was added to make the total volume 50 μL.

[0145] The PCR amplification procedure is:

[0146] 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 10 s, 30 cycles; 72℃ extension for 1 min. The reaction products were electrophoresed on 1.5% agarose gel (see the results). Figure 1 ), gel recovery and purification to obtain the upstream fragment of VL.

[0147] Reaction 2): Design primers

[0148] Primer 2: 5′-AACTCCCGGGAC NNSNNSNNSNNS TGGGTGTTCGGC-3′ (mutation site, N=A\T\C\G; S=G\C); Primer 4: 5′-GC TCTAGA ACCTAGGACGGTCAG-3′(XbaⅠ);

[0149] Reaction system ② is:

[0150] 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of Primer 2 and Primer 4 (10 μmol / L), and 1 μL of pComb3XSS-Fab recombinant vector were added; the total volume was made up to 50 μL with sterile water.

[0151] The PCR amplification procedure is:

[0152] 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 10 s, 30 cycles; 72℃ extension for 1 min. The reaction products were electrophoresed on 1.5% agarose gel (see the results). Figure 1 ), gel recovery and purification to obtain the downstream fragment of VL.

[0153] Reaction 3): Design primers

[0154] Primer 1: 5′-C GAGCTC CAGGACCCTGTTGTG-3′(SacⅠ);

[0155] Primer 4: 5′-GC TCTAGA ACCTAGGACGGTCAG-3′(XbaⅠ);

[0156] Reaction system ③ is:

[0157] Step 1: 2.5 μL each of the upstream and downstream fragments of VL and 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.); the total volume is 50 μL.

[0158] Step 2: 1 μL of the spliced ​​product; 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of primer 1 and primer 4 (10 μmol / L); and sterile water to make up the total volume to 50 μL.

[0159] The PCR amplification procedure is:

[0160] Step 1: Pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s; and extension at 72°C for 1 min. The reaction products were electrophoresed on a 1.5% agarose gel (see the results). Figure 1 ), gel recovery and purification, to obtain the splicing product of the upper and lower parts of the light chain.

[0161] Step 2: Pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s; and extension at 72°C for 1 min. The reaction products were electrophoresed on a 1.5% agarose gel (see the results). Figure 1 ), gel recovery and purification, to obtain the recombinant light chain gene after site-directed random mutagenesis.

[0162] Then, the heavy chain gene of the Fab antibody gene (nucleotide sequence shown in SEQ IN NO. 2) was used as a template to amplify the heavy chain gene by PCR, and a Spe I restriction enzyme site, an Xho I restriction enzyme site and random mutations at 7 amino acid sites were introduced.

[0163] Reaction 4): Design primers

[0164] Primer 5: 5′-GAGGTGCAGCTG CTCGAG TCTGGCCCAGGACTG-3′(XhoⅠ);

[0165] Primer 7: 5′-CTGGCCCCAGTAGTC SNNSNN GGC SNNSNNSNNSNNSNN CCAAACTCTCGCACA-3′ (mutation site, N=A\T\C\G; S=G\C);

[0166] Reaction system ④ is:

[0167] 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of Primer 5 and Primer 7 (10 μmol / L), and 1 μL of pComb3XSS-Fab recombinant vector were added; the total volume was made up to 50 μL with sterile water.

[0168] The PCR amplification procedure is:

[0169] 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 10 s, 30 cycles; 72℃ extension for 1 min. The reaction products were electrophoresed on 1.5% agarose gel (see the results). Figure 2 ), gel recovery and purification, to obtain the upstream fragment of VH.

[0170] Reaction 5): Design primers

[0171] Primer 6: 5′-TGTGCGAGAGTTTGG NNSNNSNNSNNSNNS GCC NNSNNS GACTACTGGGGCCAG-3′ (mutation site, N=A\T\C\G; S=G\C);

[0172] Primer 8: 5′-GG ACTAGT TGAGGAGACGGTGACCAGGGT-3′(SpeⅠ);

[0173] Reaction system ⑤ is:

[0174] 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of Primer 6 and Primer 8 (10 μmol / L), and 1 μL of pComb3XSS-Fab recombinant vector were added; the total volume was made up to 50 μL with sterile water.

[0175] The PCR amplification procedure is:

[0176] 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 10 s, 30 cycles; 72℃ extension for 1 min. The reaction products were electrophoresed on 1.5% agarose gel (see the results). Figure 2 ), gel recovery and purification to obtain the downstream fragment of VH.

[0177] Reaction 6): Design primers

[0178] Primer 5: 5′-GAGGTGCAGCTG CTCGAG TCTGGCCCAGGACTG-3′(XhoⅠ);

[0179] Primer 8: 5′-GGACTAGTTGAGGAGACGGTGACCAGGGT-3′ (Spe I);

[0180] Reaction system ⑥ is:

[0181] Step 1: 2.5 μL each of the upstream and downstream fragments of VH and 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.); the total volume is 50 μL.

[0182] Step 2: 1 μL of the spliced ​​product; 45 μL of Gold Mix (Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of primer 5 and primer 8 (10 μmol / L); and sterile water to make up the total volume to 50 μL.

[0183] The PCR amplification procedure is:

[0184] Step 1: Pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s; and extension at 72°C for 1 min. The reaction products were electrophoresed on a 1.5% agarose gel (see the results). Figure 2 ), gel recovery and purification, to obtain the splicing product of the upper and lower parts of the heavy chain.

[0185] Step 2: Pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s; and extension at 72°C for 1 min. The reaction products were electrophoresed on a 1.5% agarose gel (see the results). Figure 2 ), gel recovery and purification, to obtain the recombinant heavy chain gene after site-directed random mutagenesis.

[0186] The recombinant light chain gene and phagemid vector pComb3XSS were then double-digested with Sac I and Xba I and ligated to obtain the recombinant pComb3XSS-VL plasmid vector, which was then electroporated into Escherichia coli XL1-Blue to construct a light chain mutant library.

[0187] Enzyme digestion reaction: pComb3XSS vector was double-digested with Sac I (Takara) and Xba I (Takara), and the vector fragment was recovered using an enzyme digestion product recovery kit; PCR-amplified VL fragment was double-digested with Sac I (Takara) and Xba I (Takara), and the VL fragment was recovered using an enzyme digestion product recovery kit. The results of agarose gel electrophoresis of the enzyme digestion products are shown in Figure 3 .

[0188] Ligation reaction system (10 μL): 1 μL of pComb3XSS digestion product (50 ng / μL), 5 μL of VL digestion product (50 ng), 2 μL of ligation buffer, and 1 μL of T4 DNA ligase (Takara). Add deionized water to a final volume of 10 μL. Ligation is carried out overnight at 16°C. The ligation product is transformed into Escherichia coli XL1-Blue (prepared in the laboratory) by electroporation, and single colonies are selected. Mutation rates are assessed by PCR in the culture medium. Gently scrape the growing bacterial lawn and collect it. Plasmid extraction is performed to obtain the recombinant light chain mutant library.

[0189] Finally, the recombinant heavy chain gene and the recombinant phagemid vector pComb3XSS-VL were double-digested with Xho I and Spe I and ligated to obtain the recombinant pComb3XSS-Fab plasmid vector, which was then electroporated into Escherichia coli XL1-Blue to construct a Fab antibody mutation library.

[0190] Enzyme digestion reaction: The recombinant pComb3XSS-VL vector was double-digested with Xho I (Takara) and Spe I (Takara), and the vector fragment was recovered using an enzyme digestion product recovery kit; the PCR-amplified VH fragment was double-digested with Xho I (Takara) and Spe I (Takara), and the VH fragment was recovered using an enzyme digestion product recovery kit. The results of agarose gel electrophoresis of the enzyme digestion products are shown in Figure 4 .

[0191] Ligation reaction system (10 μL): 1 μL of recombinant pComb3XSS-VL digestion product (50 ng / μL), 5 μL of VH digestion product (50 ng), 2 μL of ligation buffer, and 1 μL of T4 DNA ligase (Takara). Add deionized water to a final volume of 10 μL. Incubate the ligation reaction overnight at 16°C. Transform the ligation product into Escherichia coli XL1-Blue (prepared in the laboratory) by electroporation, select a single colony, and analyze the mutation rate using PCR. Gently scrape the bacterial lawn to collect the Fab antibody mutant library.

[0192] The nucleotide sequence of the gene encoding the light chain variable region containing the mutation site (SEQ ID NO.5) is shown below:

[0193] CAGGACCCTGTTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTAT GGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGAC NNSNNSNNSNNS TGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT;

[0194] The nucleotide sequence of the gene encoding the heavy chain variable region containing the mutation site (SEQ ID NO.6) is shown below:

[0195] GAGGTGCAGCTGCTCGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCGGGGAAGGGACTGGAGTGGGTGGCCAAC ATAAAGCAAGATGAAAGTACGAAAAACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTTTGG NNSNNSNNSNNSNNS GCC NNSNNS GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

[0196] (2) Rescue and titration of phage antibody mutant library targeting PD-L1:

[0197] An appropriate amount of Fab antibody mutant library bacteria was inoculated into 2×YT liquid medium (containing 10 μg / mL ampicillin, 10 μg / mL tetracycline, and 2% glucose). After culture to the logarithmic growth phase, helper phage VCSM13 was added at a multiplicity of infection (MOI) of 20. The cells were first incubated at 37°C in a water bath for 30 minutes, followed by shaking at 37°C with 100 rpm for 30 minutes. The cells were then pelleted by centrifugation at 4000 g for 20 minutes and resuspended in an equal volume of 2×YT liquid medium (containing 10 μg / mL ampicillin, 10 μg / mL tetracycline, and 50 μg / mL kanamycin). The cells were shaken and cultured overnight at 30°C. The next day, the overnight culture was collected and centrifuged at 10,000 g for 20 minutes at 4°C. The supernatant was transferred to a clean, sterile centrifuge tube, and 1 / 5 volume of 20% PEG8000 / 2.5M NaCl solution was added. The cells were concentrated on ice for 1 hour. The concentrated bacterial solution was centrifuged at 10,000 g for 20 min at 4°C, the supernatant was discarded, and the phage precipitate was resuspended with 1 mL of PBS solution (pH 7.4, 0.01 mol / L), filtered through a 0.22 μm filter, and dispensed into sterile centrifuge tubes on ice and stored at 4°C.

[0198] The concentrated phage antibody library was diluted using the doubling dilution method and infected with E. coli XL1-Blue in the logarithmic growth phase. The clones were then plated on 2×YT plates containing 10 μg / mL ampicillin. The rescued phage antibody library was titrated to determine its capacity.

[0199] (3) Four rounds of solid-phase screening of a phage antibody mutation library targeting PD-L1:

[0200] A. PD-L1 (Suzhou Jinan Protein Technology Co., Ltd.) was diluted to 50 μg / mL with 0.05 mol / L carbonate buffer at pH 9.6, and coated on an ELISA plate. The plate was incubated at 4°C overnight. The next day, 200 μL of 5% (w / v, g: mL) skim milk powder prepared with PBS (0.01 M, pH 7.4) containing 0.05% (v / v) Tween 20 was added. The plate was blocked at 37°C for 2 h. The blocking solution was discarded, and 100 μL of phage antibody library (approximately 10 13CFU) and incubate at 37°C for 2 hours. Discard the residual phage antibody solution and wash five times with PBST (10 times in the second round and 20 or more times thereafter). Add 100 μL of Gly-HCl elution buffer (0.1 M, pH 2.2) to each well and incubate at room temperature for 10 minutes, pipetting thoroughly. Immediately add 6 μL of 2 M Tris solution and mix gently to neutralize the eluted phage antibodies.

[0201] B. Add the collected phage antibody eluate to 2 mL of E. coli XL1-Blue in the logarithmic growth phase and incubate at 37°C for 30 minutes to allow the phage antibodies to fully infect the E. coli. Take 100 μL of the diluted solution and spread it on a 2×YT plate (containing 10 μg / mL ampicillin) to determine the phage antibody library capacity. The remaining solution is spread on a 2×YT plate (containing 10 μg / mL ampicillin, 10 μg / mL tetracycline, and 2% glucose). The next day, collect the solution using 2×YT liquid medium (2% glucose). After determining the bacterial concentration, add 50% glycerol at a volume ratio of 1:1 to obtain the screened phage antibody bacterial library.

[0202] C. Take an appropriate amount of the bacterial suspension recovered from step B and inoculate it into 2×YT liquid medium (containing 10 μg / mL ampicillin, 10 μg / mL tetracycline, and 2% glucose). Cultivate until the logarithmic growth phase. Add helper phage VCSM13 at a multiplicity of infection (MOI) of 20 and incubate with shaking at 30°C overnight. The next day, collect the supernatant and perform conventional PEG precipitation. The resulting secondary phage antibody library is ready for the next round of screening.

[0203] D. Perform three rounds of panning using the same method (repeat steps A to C) to obtain positive phage Fab antibody clones.

[0204] (4) Preparation of phage antibodies and detection of anti-PD-L1 specificity:

[0205] Colonies were randomly selected from the plates in the fourth round of screening and cultured overnight at 37°C in 2×YT medium (containing 10 μg / mL ampicillin, 10 μg / mL tetracycline, and 2% glucose) to obtain bacterial suspension. The next day, 20 μL of the aforementioned bacterial suspension was transferred to 400 μL of 2×YT medium (containing 10 μg / mL ampicillin, 10 μg / mL tetracycline, and 2% glucose) and cultured at 37°C until the logarithmic growth phase. Helper virus VCSM13 was added at a multiplicity of infection (MOI) of 20. The culture was allowed to stand at 37°C for 30 minutes, then shaken at 100 rpm at 37°C for 30 minutes. After centrifugation at 4000 g for 10 minutes, the bacterial pellet was resuspended in an equal volume of 2×YT-ATK liquid medium and cultured overnight at 30°C. The supernatant was collected to obtain phage antibodies.

[0206] The ELISA test was performed according to the following procedure: the antigen (i.e., PD-L1) was diluted to 10 μg / mL, and the ELISA plate was coated with 100 μL of the diluted antigen. After coating overnight at 4°C, the liquid in the well was discarded. 200 μL of 5% skim milk powder prepared with PBS (0.01M, pH 7.4) containing 0.05% Tween 20 was added for blocking, and then the prepared phage antibody was added. The plate was incubated at 37°C for 2 hours, washed 5 times with PBST, and a 1:10000 diluted HRP-anti-M13 mouse monoclonal antibody (Sino Biological) was added for reaction. After washing, TMB substrate was added for color development, and the A450 value was read. The results are as follows. Figure 5 As shown, among the 185 mutant antibodies, compared with the parent Fab antibody (NC), some mutant antibodies have enhanced affinity for PD-L1, while some mutant antibodies have reduced affinity for PD-L1.

[0207] (5) Determination of relative affinity of positive phage antibodies:

[0208] After sequencing analysis, the positive phage antibodies are mass-produced using the same method as step (4).

[0209] The antigen (i.e., PD-L1) was diluted to 10 μg / mL, and 100 μL of the diluted antigen was used to coat the ELISA plate. After coating overnight at 4°C, the liquid in the well was discarded. 200 μL of 5% skim milk powder prepared with PBS (0.01M, pH 7.4) containing 0.05% Tween 20 was added for blocking, and then the prepared positive phage antibody was added. Each clone was repeated in 8 wells, incubated at 37°C for 2h, and washed 5 times with PBST. Then 100 μL of different concentrations of NH4SCN (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 mol / L) was added to each well and incubated at room temperature for 15 minutes. After washing 5 times with PBST, a 1:10000 diluted HRP-anti-M13 mouse monoclonal antibody (Yi Qiao Shenzhou) was added for reaction. After washing, TMB substrate was added for color development, and the A450 value was read. The results are as follows. Figure 6 As shown, after in vitro affinity maturation, the relative affinity of the phage antibodies was improved to varying degrees compared with the parent Fab antibody (NC). Subsequent experiments selected the mut42 mutant antibody strain and performed sequencing analysis on it.

[0210] The nucleotide sequence of the gene encoding the light chain variable region of the mutated antibody strain mut42 (SEQ ID NO.9) is as follows: CAGGACCCTGTTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGAC GTGCGCTTGAAG TGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT;

[0211] The nucleotide sequence of the gene encoding the heavy chain variable region of the mutated antibody strain mut42 (SEQ ID NO.10) is as follows: GAGGTGCAGCTGCTCGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCGGGGAAGGGACTGGAGTGGGTGGCCAACATAAAGCAAGATGAAAGTACGAAAAACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTTTGG T GGCACGCCGAGATC GCC TCCAAC GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

[0212] The amino acid sequence of the Mut42 light chain is as follows:

[0213] QDPVVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSSGNTASLTITGAQAEDEADYYCNSRDVRLKWVFGGGTKLTVLG;

[0214] The amino acid sequence of the Mut42 heavy chain is as follows:

[0215] EVQLLESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWVANIKQDESTKNYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVWWHAEIASNDYWGQGTLVTVSS.

[0216] (6) Expression and purification of anti-PD-L1 Fab antibodies

[0217] The mutated antibody strain mut42 was inoculated into 10 mL of 2×YT medium (containing 10 μg / mL ampicillin, 10 μg / mL tetracycline, and 2% glucose) and cultured overnight at 37°C to obtain a bacterial suspension. The next day, the suspension was transferred to a 400 mL 2×YT plate (containing 10 μg / mL ampicillin) at a 1:100 ratio and cultured at 37°C until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mM and induced at 28°C, 180 rpm, for 9 hours. The suspension was harvested and centrifuged at 12,000 rpm for 10 minutes, and the supernatant was carefully discarded. The pellet was resuspended in PBS (1 / 10 of the total volume) and placed in an ice-water bath for sonication. The parameters were set at 25W, 5 seconds on, and 5 seconds off, and sonication was continued until the liquid became clear and transparent. After ultrasonic disruption, the solution was centrifuged at 12000 rpm for 10 minutes, the supernatant was retained and filtered through a 0.45 μm filter to remove large molecules. The sample was then purified using NiSepharose™ 6× Fast Flow: first rinsed with water, then rinsed and equilibrated with equilibration buffer (PBS containing 20 mM imidazole, pH 7.4), then loaded at a flow rate of 250 μL / min. After loading, the sample was rinsed with equilibration buffer (PBS containing 20 mM imidazole, pH 7.4), and then the impurities were eluted with PBS containing 200 mmol / L imidazole. The target protein was eluted with PBS containing 300 mmol / L imidazole. Finally, the collected target protein was ultrafiltered through a 10KD ultrafiltration tube and replaced with PBS (0.015 mol / L, pH 7.4) to obtain the purified anti-PD-L1 Fab antibody.

[0218] Example 2

[0219] The disulfide-bond-stabilized anti-PD-L1 humanized diabody of the present invention is based on the VL gene and VH gene fragments encoding the anti-PD-L1 Fab antibody (mut42) prepared in Example 1. By overlap PCR and PCR, the 100th amino acid Gly of the antibody VL and the 44th amino acid Gly of the VH were site-directedly mutated to Cys to introduce a covalent disulfide bond. At the same time, a gene fragment encoding a connecting peptide (amino acid sequence of GGGGS) was used to construct a VL-GGGGS-VH gene fragment (length 708 bp). The two expressed VL-GGGGS-VH (molecular weight 52.14 KD) molecules were covalently linked by disulfide bonds to form a disulfide-bond-stabilized diabody.

[0220] The preparation method of the disulfide bond-stabilized anti-PD-L1 humanized double-chain antibody of the present invention comprises the following steps:

[0221] (I) Preparation of VL-GGGGS-VH.

[0222] (1) PCR site-directed mutation of amino acid Gly at position 100 of VL to Cys was performed, and the gene fragment VL-GGGGS was obtained by amplification:

[0223] Reaction 1): Design primers:

[0224] Primer 1': 5'-CCG GAATTC CAGGACCCTGTTGTGTCTGTG-3'(EcoR I)

[0225] Primer 2': 5'- CGAGCCACCGCCACC TAGGACGGTCAGCTTGGTCCC ACA GCCGAACACCCA-3' (GGGGS short peptide and Cys mutation site)

[0226] Reaction system ① is:

[0227] 2×T8 High-Fidelity Master Mix 25 μL (Beijing Qingke Biotechnology Co., Ltd.), 2 μL each of primer 1' and primer 2' (10 μmol / L), 2 μL of mut42 mutant antibody strain gene template, and 19 μL of sterile deionized water; the total volume was 50 μL.

[0228] The PCR amplification procedure is:

[0229] The reaction was denatured at 98°C for 2 minutes, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 10 seconds, and extension at 72°C for 10 seconds, followed by extension at 72°C for 1 minute. The reaction product was electrophoresed on a 1.5% agarose gel, recovered, and purified to obtain the recombinant VL-GGGGS fragment.

[0230] (2) PCR site-directed mutation of amino acid Gly at position 44 of VH to Cys was performed, and amplification of the partial gene fragment VH-GGGGS was obtained:

[0231] Reaction 2): Design primers:

[0232] Primer 3': 5'- GGTGGCGGTGGCTC GGAGTGCAGCTGCTCGAGTCT-3' (GGGGS short peptide)

[0233] Primer 5': 5'-GGCCACCCACTCCAG ACA CTTCCCCGGGGG-3' (Cys mutation site)

[0234] Reaction system ② is:

[0235] 2×T8 High-Fidelity Master Mix 25 μL (Beijing Qingke Biotechnology Co., Ltd.), 2 μL each of primer 3' and primer 5' (10 μmol / L), 2 μL of mut42 mutant antibody strain gene template, and 19 μL of sterile deionized water; the total volume was 50 μL.

[0236] The PCR amplification procedure is:

[0237] The reaction was denatured at 98°C for 2 minutes, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 10 seconds, and extension at 72°C for 10 seconds, followed by extension at 72°C for 1 minute. The reaction product was electrophoresed on a 1.5% agarose gel, recovered, and purified to obtain the recombinant VH upper half fragment.

[0238] Reaction 3): Design primers:

[0239] Primer 4': 5'-CCCCCGGGGAAG TGT CTGGAGTGGGTGGCC-3′ (Cys mutation site);

[0240] Primer 6': 5'-A GCGGCCGC CTATTA ATGATGATGATGATGATG TGAGGAGACGGTGAC-3' (Not I and 6×His-tag).

[0241] Reaction system ③ is:

[0242] 2×T8 High-Fidelity Master Mix 25 μL (Beijing Qingke Biotechnology Co., Ltd.), 2 μL each of primer 4' and primer 6' (10 μmol / L), 2 μL of mut42 mutant antibody strain gene template, and 19 μL of sterile deionized water; the total volume was 50 μL.

[0243] The PCR amplification procedure is:

[0244] The reaction product was subjected to 1.5% agarose gel electrophoresis, gel recovery, and purification to obtain the recombinant VH lower half fragment.

[0245] Reaction 4): PCR splicing and amplification of the gene fragment VH-GGGGS.

[0246] Primer 3': 5'- GGTGGCGGTGGCTC GGAGTGCAGCTGCTCGAGTCT-3' (GGGGS short peptide);

[0247] Primer 6': 5'-A GCGGCCGC CTATTA ATGATGATGATGATGATG TGAGGAGACGGTGAC-3' (Not I and 6×His-tag).

[0248] Reaction system ④ is:

[0249] Step 1: 2×T8 High-Fidelity Master Mix 12.5 μL (Beijing Qingke Biotechnology Co., Ltd.), 2 μL each of the upper and lower VH fragments, and 8.5 μL of sterile deionized water; the total volume is 25 μL.

[0250] The PCR amplification procedure is:

[0251] Pre-denaturation at 98°C for 2 min; 15 cycles of denaturation at 98°C for 10 s, annealing at 63°C for 10 s, and extension at 72°C for 10 s; and extension at 72°C for 1 min.

[0252] Step 2: 25 μL of 2×T8 High-Fidelity Master Mix (Beijing Qingke Biotechnology Co., Ltd.), 2 μL each of primer 3' and primer 6' (10 μmol / L), 4 μL of VH splicing reaction solution, and 17 μL of sterile deionized water; the total volume was 50 μL.

[0253] The PCR amplification procedure is:

[0254] Denaturation was performed at 98°C for 2 minutes, followed by 15 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 10 seconds, and extension at 72°C for 10 seconds, followed by extension at 72°C for 1 minute. The reaction product was electrophoresed on a 1.5% agarose gel, recovered, and purified to obtain the complete VH-GGGGS recombinant fragment.

[0255] (3) PCR splicing and amplification of gene fragment VL-GGGGS-VH

[0256] Reaction 5): Design primers:

[0257] Primer 1': 5'-CCG GAATTC CAGGACCCTGTTGTGTCTGTG-3'(EcoR I);

[0258] Primer 6': 5'-A GCGGCCGC CTATTA ATGATGATGATGATGATG TGAGGAGACGGTGAC-3' (Not I and 6×His-tag).

[0259] Reaction system ⑤ is:

[0260] Step 1: 2×T8 High-Fidelity Master Mix 12.5 μL (Beijing Qingke Biotechnology Co., Ltd.), 2 μL each of the VL-GGGGS and VH-GGGGS fragments, and 8.5 μL of sterile deionized water; the total volume is 25 μL.

[0261] The PCR amplification procedure is:

[0262] Pre-denaturation at 98°C for 2 min; 15 cycles of denaturation at 98°C for 10 s, annealing at 63°C for 10 s, and extension at 72°C for 10 s; and extension at 72°C for 1 min.

[0263] Step 2: 25 μL of 2×T8 High-Fidelity Master Mix (Beijing Qingke Biotechnology Co., Ltd.), 2 μL each of primer 1' and primer 6' (10 μmol / L), 4 μL of splicing reaction solution, and 17 μL of sterile deionized water; the total volume was 50 μL.

[0264] The PCR amplification procedure is:

[0265] The reaction product was subjected to 1.5% agarose gel electrophoresis, gel recovery, and purification to obtain the recombinant VL-GGGGS-VH fragment.

[0266] The electrophoresis results of the above fragments are as follows Figure 7 shown.

[0267] (2) Construction of the recombinant expression vector pPICZαA-ds-Diabody.

[0268] ①Use EcoRⅠ and NotⅠ restriction endonucleases to double-digest the expression vector pPICZαA and gene VL-GGGGS-VH fragment respectively.

[0269] Enzyme digestion system (50 μL):

[0270] 1 μg of pPICZαA or VL-GGGGS-VH fragment, 5 μL of 10× Buffer H, 5 μL of BSA, 2 μL of NotⅠ (4-12 U / μL), 2 μL of EcoRI (8-20 U / μL), and deionized water were added to a final volume of 50 μL;

[0271] The enzyme digestion was carried out at 37°C for 8 h, and the digestion reaction products were subjected to 1.5% agarose gel electrophoresis, gel recovery and purification to obtain the expression vector pPICZαA and VL-GGGGS-VH with EcoRI and NotⅠ restriction enzyme cleavage sites, respectively.

[0272] ② Connect the gene fragment VL-GGGGS-VH obtained from enzyme digestion in ① to the expression vector pPICZαA after enzyme digestion.

[0273] Ligation reaction system (20 μL):

[0274] VL-GGGGS-VH 200 ng, pPICZαA 450 ng, 10× Buffer 2 μL, T4 DNA ligase (350 U / μL) 1 μL, deionized water to a final volume of 20 μL;

[0275] The ligation reaction was carried out at 16°C for 12 h, and the ligation reaction product was transformed into Escherichia coli DH5α competent cells and inoculated on a low-salt LB agarose culture plate (10 g tryptone, 5 g NaCl, 5 g yeast extract, adjusted to pH 7.4, and dilute to 1 L with deionized water) containing 200 μg / mL Zeocin overnight. Single colonies were randomly picked for PCR amplification of the bacterial solution to preliminarily identify the construction of the recombinant expression vector. The results showed that the recombinant expression vector pPICZαA-ds-Diabody was successfully constructed (see Figure 2). Figure 8 shown).

[0276] (3) Expression and western blot identification of disulfide-stabilized anti-PD-L1 humanized double-chain antibody.

[0277] (1) Transformation of Pichia pastoris GS115 with the recombinant expression vector pPICZαA-ds-Diabody

[0278] First, streak the Pichia pastoris GS115 stored at -80°C on a YPD plate, invert and culture at 28°C for 2-3 days until a white round single colony grows. Then select a well-separated single colony of Pichia pastoris and inoculate it into 5mL of YPD liquid medium at 29°C and 230rpm for activation. The next day, take 1mL of the overnight culture solution and inoculate it into 100mL of YPD liquid medium and culture it at 29°C and 230rpm until the logarithmic growth phase of Pichia pastoris, that is, OD600 The value is 1.3-1.6. Place the yeast liquid in a 50mL centrifuge tube, centrifuge at 4℃ and 4000rpm for 5min to collect the bacteria, discard the supernatant, and aspirate the residual liquid. Add 40mL of pre-cooled sterile water to resuspend, wash the bacteria, centrifuge at 4℃ and 4000rpm for 5min, discard the supernatant; and repeat this step once. Add 10mL of pre-cooled sterile water to wash the bacteria, centrifuge at 4℃ and 4000rpm for 5min, and discard the supernatant. Add 10mL of pre-cooled 1M sorbitol solution to resuspend the bacteria, centrifuge at 4℃ and 4000rpm for 5min, and discard the supernatant. Finally, add 200μL of ice-cold 1M sorbitol solution to resuspend the bacteria, divide on ice, 80μL per tube, which is GS115 competent cells, ready for use.

[0279] The recombinant expression vector pPICZαA-ds-Diabody with the correct sequence was inoculated into low-salt LB medium supplemented with 25 μg / mL Zeocin and cultured at 37°C with shaking at 230 rpm for 16 hours. The recombinant plasmid pPICZαA-ds-Diabody was then extracted using a plasmid extraction kit. The recombinant plasmid was linearized with the restriction endonuclease Sac I using the following digestion system: 40 μL of the recombinant expression vector pPICZαA-ds-Diabody, 2 μL of Sac I restriction enzyme, 5 μL of 10× H Buffer, and deionized water to a total volume of 50 μL. Digestion was performed overnight at 37°C. The linearized pPICZαA-ds-Diabody plasmid was subjected to 1.5% agarose gel electrophoresis, and the completely linearized plasmid band was recovered.

[0280] Transform the linearized recombinant plasmid into competent Pichia pastoris cells by electroporation (electrotransformer parameters: voltage 1.5 kV, capacitance 25 μF, resistance 400 Ω). Immediately after electroporation, resuspend the cells in 900 μL of pre-chilled 1 M sorbitol solution and transfer the transformed cells to a sterile, clean 1.5 mL centrifuge tube. Incubate at 28°C for 2 h. Finally, spread 300 μL of the culture onto a YPD plate containing 200 μg / mL Zeocin and incubate at 28°C for 3-4 days until transformants appear.

[0281] White transformants with good growth were selected and positive transformants were identified by colony PCR using pPICZαA universal primers 5'AOX1 and 3'AOX1. Figure 9 ) showed that all 17 transformants were able to amplify the target band, indicating that they were all positive transformants.

[0282] 5′AOX1 primer: 5′-GACTGGTTCCAATTGACAAGC-3′;

[0283] 3'AOX1 primer: 5'-GCAAATGGCATTCTGACATCC-3'.

[0284] (2) Expression, Western blot, and ELISA identification of disulfide-stabilized anti-PD-L1 humanized double-chain antibody

[0285] The 17 positive transformants were inoculated into test tubes containing 5 mL of YPD liquid medium and cultured at 29°C and 230 rpm overnight. The next day, the overnight bacterial solution was inoculated into a test tube containing 3 mL of BMGY medium at a volume ratio of 1:100 and cultured at 29°C and 230 rpm until the OD 600 If the value is 4-6, add 5 mL of BMMY medium to the test tube and incubate at 29°C, 230 rpm, and shaking. Add methanol to the culture medium every 24 hours to a final concentration of 1% (v / v) for 96 hours. Finally, collect the bacterial suspension into a 15 mL centrifuge tube and centrifuge at 4°C, 8000 rpm, and collect the supernatant of induced expression.

[0286] The induced expression supernatant was concentrated with TCA. 1 mL of the supernatant was transferred to a 1.5 mL centrifuge tube and 100 μL of 100% TCA was added. Mix thoroughly and allow to precipitate at -20°C overnight. Centrifuge at 12,000 g for 10 minutes and discard the supernatant. Then, add 1 mL of anhydrous ethanol, resuspend and mix thoroughly, and centrifuge at 12,000 g for 10 minutes at 4°C. Discard the supernatant. Repeat this process once to ensure complete removal of TCA. Finally, dry the protein pellet at room temperature for 30 minutes to allow the ethanol to evaporate. Redissolve it in 50 μL of ddH2O, add 5x the concentration of loading buffer, mix thoroughly, boil in boiling water for 10 minutes, and store at -20°C until needed.

[0287] Take 25 μL of the concentrated sample and perform molecular cloning. After reducing SDS-PAGE, perform Western blot identification. The results are ( Figure 10 ) showed that after reducing SDS-PAGE, there was a protein band at a molecular weight of 30 kDa, indicating that the ds-Diabody was successfully constructed.

[0288] The yeast induced expression supernatant was used for indirect ELISA identification with PD-L1 as the antigen. The results were as follows: Figure 11 As shown, different clones showed different antigen-binding activities, which may be related to the expression level of ds-Diabody of the clone.

[0289] (IV) Purification and SDS-PAGE identification of disulfide-stabilized humanized anti-PD-L1 double-chain antibody.

[0290] The yeast expression supernatant of clone 8 was collected and precipitated with 50% saturated ammonium sulfate. After being placed at 4°C overnight, the supernatant was centrifuged at 8500 rpm for 20 min, and the supernatant was discarded. 8 mL of PBS (pH 7.4, 0.01 M) was added to the precipitate to dissolve it. After dialysis with PBS, the precipitate was filtered with a 0.45 μm filter membrane and then filtered with Ni Sepharose. TM Purification was performed using 6×Fast Flow: first rinse with water, then rinse and equilibrate with equilibration buffer (PBS containing 20mM imidazole, pH 7.4), then load the sample at a flow rate of 250μL / min, rinse with equilibration buffer (PBS containing 20mM imidazole, pH 7.4), then elute the impurities with PBS containing 200mmol / L imidazole, and elute the target protein with PBS containing 300mmol / L imidazole. Finally, the collected target protein was ultrafiltered using a 30KD ultrafiltration tube and replaced with PBS (0.015mol / L, pH 7.4). The purified product was identified by 12% SDS-PAGE electrophoresis (e.g. Figure 12 The results showed that there was a protein band at a molecular weight of 30 kDa.

[0291] Example 3

[0292] The following is a test of the biological activity of the disulfide-stabilized anti-PD-L1 humanized diabody prepared according to the above method. The test method and results are as follows:

[0293] (I) Indirect ELISA was used to detect the binding activity of disulfide-stabilized anti-PD-L1 humanized double-chain antibody to the antigen PD-L1.

[0294] Recombinant human PD-L1 (Jinan Protein Technology Co., Ltd.) was diluted to 1 μg / mL with 0.05 mol / L carbonate buffer, pH 9.6. 100 μL per well was coated on an ELISA plate and incubated at 37°C for 3 h. The wells were then discarded and washed three times with PBST (PBS containing 0.015 mol / L, pH 7.4, 0.05% Tween-20 by volume). After blocking with 5% skim milk-PBST by mass, a certain concentration of ds-Diabody prepared in Example 2 and anti-PD-L1 Fab antibody were added at 100 μL / well, incubated at 37 ° C for 1 h, washed 3 times with PBST, and then 100 μL of anti-His-tag mouse monoclonal antibody (Mingyan Bio) diluted at 1:5000 was added to each well, incubated at 37 ° C for 1 h, washed 3 times with PBS-T, and 100 μL of HRP-labeled goat anti-mouse polyclonal antibody (Beijing Solaibao Technology Co., Ltd.) diluted at 1:5000 was added to each well, incubated at 37 ° C for 0.5 h, washed 5 times with PBST, and TMB was added for color development. The A450 value was measured. PBS was used as a negative control in the experiment.

[0295] The results are as follows Figure 13 As shown, compared with Fab antibody (mut42 mutant antibody), ds-Diabody can better bind to PD-L1 antigen specifically and has good affinity.

[0296] (II) Stability testing of disulfide-stabilized anti-PD-L1 humanized diabody

[0297] A certain concentration of purified ds-Diabody and Fab (mut42 mutant antibody) was placed in PBS containing 0.2% bovine serum albumin (BSA) and incubated at 37°C for 0h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h; at each time point, 100 μL of the incubated antibody was taken per well and added to an ELISA plate coated with PD-L1 at 100 ng / well, incubated at 37°C for 1h, washed three times with PBST, and then 100 μL of a 1:5000 diluted anti-His-tag mouse monoclonal antibody was added to each well of the ds-Diabody incubated plate, incubated at 37°C for 1h, washed three times with PBST, and 100 μL of a 1:8000 diluted HRP-labeled goat anti-mouse polyclonal antibody was added to each well and incubated at 37°C for 45min. Incubation: Add rabbit anti-human IgG-Fab-HRP labeled antibody at a dilution of 1:5000 to each well of the Fab antibody-incubated plate and incubate at 37°C for 45 min. Finally, wash five times with PBST and develop color with TMB, and measure the A450 value.

[0298] The results are as follows Figure 14 As shown in the figure, the activity of Fab decreased to 50% after 6 hours of incubation and was only about 6% after 96 hours, indicating loss of activity. However, the binding activity of ds-Diabody was still 67% after 96 hours of incubation in PBS containing 0.2% BSA at 37°C, indicating good stability.

[0299] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A high-affinity human anti-PD-L1 antibody, characterized in that: including light chain variable regions and heavy chain variable regions; The light chain variable region contains three CDR regions, which are: amino acid sequences such as SLRSYY The LCDR1 shown in the amino acid sequence is as follows GKN The LCDR2 and amino acid sequences shown are as follows NSRDVRLKWV LCDR3 shown; The heavy chain variable region contains three CDR regions, which are: amino acid sequences such as GGSISSYY The HCDR1 and amino acid sequences shown are IKQDESTK The HCDR2 and amino acid sequences shown are as follows ARVWWHAEIASNDY HCDR3 shown.

2. The high-affinity anti-PD-L1 human antibody according to claim 1, characterized in that: The light chain variable region is shown in SEQ ID NO.11; The heavy chain variable region is shown in SEQ ID NO.

12.

3. The nucleic acid encoding the high-affinity anti-PD-L1 human antibody according to claim 1.

4. The nucleic acid encoding the high-affinity anti-PD-L1 human antibody according to claim 3, characterized in that: The sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO.9; The sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO.

10.

5. A disulfide-stabilized human anti-PD-L1 double-chain antibody, characterized in that: The amino acid sequence is shown in SEQ ID NO.

7.

6. A gene encoding the disulfide-stabilized anti-PD-L1 humanized double-chain antibody according to claim 5.

7. The coding gene according to claim 6, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

8.

8. The method for preparing the disulfide-stabilized anti-PD-L1 humanized double-chain antibody according to claim 5, characterized in that The following steps are involved: (1) Using the nucleic acid encoding the light chain variable region as shown in SEQ ID NO.9 and the nucleic acid encoding the heavy chain variable region as shown in SEQ ID NO.10 as templates, the 100th amino acid Gly in the light chain variable region and the 44th amino acid Gly in the heavy chain variable region were site-directedly mutated to Cys by overlapping PCR. The light chain variable region and the heavy chain variable region genes were connected by a connecting peptide gene fragment to construct a V L -GGGGS-V H gene fragments; (2) V obtained in step (1) L -GGGGS-V H The gene fragment was cloned into an expression vector, transferred into host cells for expression, and purified to obtain a disulfide-bond-stabilized humanized anti-PD-L1 double-chain antibody.

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