CN110981958B - A PD-L1 antibody - Google Patents

Abstract

The invention provides a novel anti-PD-L1 monoclonal recombinant single domain antibody, which belongs to the field of tumor immunotherapy drugs. The sequence of the PD-L1 antibody of the present invention is shown in SEQ ID NO. 1, the sequence is short, and the binding ability to PD-L1 is strong. The PD-L1 of the present invention can theoretically be used for the preparation of tumor immunotherapy drugs, and has a good application prospect.

Description

A PD-L1 antibody

technical field

The invention belongs to the field of tumor immunotherapy drugs, in particular to an anti-PD-L1 monoclonal recombinant single domain antibody.

Background technique

Tumor is one of the major diseases endangering human health in the 21st century. Although the current treatment of tumors includes surgery, radiotherapy, chemotherapy, and targeted therapy, the five-year survival rate of patients with advanced lung cancer is only 5%. In recent years, with the development of tumor immunotherapy drugs, immunotherapy has become a This new and more promising treatment is recognized as the fourth pillar of the tumor treatment model in the 21st century. At present, in immunotherapy, immune checkpoint inhibitors and chimeric antigen receptor T cells (CAR-T) and T cell receptor T cells (TCR-T) represented by chimeric antigen receptor T cells (CAR-T) Cellular immunotherapy has achieved good clinical efficacy.

Immune checkpoints are protein molecules and signaling pathways that inhibit the over-activation of immune cells in the immune response; immune checkpoint inhibitors are molecules that can inhibit the function of immune checkpoints. function to promote the body's anti-tumor immunity and kill tumor cells.

Programmed death-1 (PD-1) is a well-known immune checkpoint molecule expressed on the surface of activated T cells. Programmed cell death-Ligand 1 (PD-L1) is a ligand of PD-1, and its binding to PD-1 will promote the latter to play an immunosuppressive role and initiate antigen-specific T Apoptosis, reducing apoptosis of regulatory T cells. PD-L1 is expressed on the surface of autologous cells, and it exists to prevent autologous cells from being arbitrarily cleared by immune cells. And a variety of tumors (such as lung cancer, melanoma, bladder cancer, kidney cancer and other malignant tumors, etc.) also express PD-L1 on the cell surface, which can prevent tumor cells from being cleared by the immune system.

PD-L1 antibody is a class of drugs in tumor immunotherapy. It binds PD-L1 and blocks the combination of PD-1 and PD-L1 to reduce the immunosuppressive effect and enable T cells to play an anti-cancer effect. Based on a similar principle, PD-L1 can also be used in the preparation of anti-infective drugs, especially against pathogens for which there is currently no effective vaccine (such as HIV, hepatitis virus (A, B, C), influenza virus, herpes virus, Giardia, Anti-infective drugs for malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa, etc.).

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel anti-PD-L1 monoclonal recombinant single domain antibody.

The technical scheme of the present invention is as follows:

An anti-PD-L1 monoclonal recombinant single-domain antibody, the amino acid sequence of CDR1 is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is shown in SEQ ID NO.9 shown.

The aforementioned anti-PD-L1 monoclonal recombinant single-domain antibody has the amino acid sequence shown in SEQ ID NO.10.

An anti-tumor biological immune drug, which is a pharmaceutical preparation prepared by using the aforementioned PD-L1 antibody as an active ingredient, and adding pharmaceutically acceptable auxiliary ingredients and/or carriers.

Use of the aforementioned recombinant single-domain antibody in the preparation of PD-L1 inhibitor.

For the aforementioned purposes, the PD-L1 inhibitor is a drug that inhibits the binding of PD-L1 to PD-1.

Use of the aforementioned recombinant single-domain antibody in the preparation of anti-tumor drugs.

Further, the tumor is a malignant tumor.

Further, the malignant tumor is lung cancer, melanoma, bladder cancer, kidney cancer, liver cancer, nasopharyngeal cancer, esophageal cancer, cervical cancer, breast cancer or gastric cancer.

The present invention has the following beneficial effects:

The present invention provides a brand-new anti-PD-L1 monoclonal recombinant single-domain antibody, which has only 368 amino acids, a small molecular weight, and is easy for industrial production.

The PD-L1 antibody of the present invention can fully bind to PD-L1 on the surface of tumor cells and block the combination of PD-L1 and PD-1, and its blocking effect is biologically similar to that of the positive control Tecentriq (an anti-PD-L1 antibody). Medicine is quite.

The PD-L1 antibody of the present invention can be used for preparing anti-tumor immune cell drugs, and has a good application prospect.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1SEC-HPLC diagram

Figure 2 is an RNA electropherogram. M: DNA marker; Lane 1: NB034; Lane 2: NB035.

Figure 3 is a diagram of colony PCR identification. Top: NB034; bottom: NB035; the first lane on the left is a DNA marker, and the marker size is the same as that in Figure 2.

Figure 4 is a statistical graph of PD-1/PD-L1 Bioassay fluorescence signal.

Detailed ways

Example 1 Preparation of PD-L1 antibody B132205 of the present invention

The main steps of PD-L1 monoclonal recombinant single domain antibody preparation are as follows:

1. Library construction to obtain immune antibody protein

III First-StrandSynthesis System for RT-PCR说明书将提取的RNA反转录为cDNA。用巢式PCR扩增编码重链抗体的可变区的核酸片段：To ensure reliable immunization results, two alpacas (number: NB034 and NB035) were immunized. After 4 times of immunization, 50 mL of peripheral blood was collected, and PBMCs (peripheral blood mononuclear cells) were isolated. PBMC total RNA was then extracted with TRIzol reagent. 1 μg of RNA was taken for electrophoresis, and the purity of RNA was detected. The results showed that the purity of RNA was good (Figure 1). see

The instructions for III First-StrandSynthesis System for RT-PCR reverse-transcribe the extracted RNA to cDNA. Amplification of nucleic acid fragments encoding variable regions of heavy chain antibodies by nested PCR:

The first round of PCR:

Upstream primer F1:

Downstream primer R1:

Second round of PCR:

Using the first round PCR product as a template,

Upstream primer F2:

Downstream primer R2:

Downstream primer R3:

The first round of PCR amplification was performed using primers F1 and R1 to obtain a product with a size of about 750bp, purified, and primers F2 and R2, R3 (R2 and R3 were mixed together, because the antibody has 2 subtypes, so 2 were used Reverse primer) for the second round of PCR amplification to obtain the VHH target fragment with a size of about 450 bp.

The vector pocomb3Xss (NBbiolab, P001) and the target fragment were digested with SfiI respectively, and digested overnight at 50°C. After digestion, T4 ligase was used to connect the vector and the target fragment, electrophoresis, and then the target fragment was recovered by gel tapping. The molar ratio of linkage was pocomb3Xss:VHH=1:3.

The aforementioned ligation product was then electroporated into E. coli TG1 competent cells to construct a heavy chain single domain antibody phage library display library against PD-L1 and identify the library. By serial dilution plating, the size of the library volume was calculated to be 1.21×10 ⁹ . In order to detect the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate was 100% (Fig. 2).

2. PD-L1 heavy chain single domain antibody screening

The B132205 antibody was diluted with a carbonate buffer with a pH value of 9.6 to a final concentration of 5 μg/mL, added to the enzyme-labeled well at 100 μL/well, and coated overnight at 4°C. Discard the coating solution, add 300 μL of 3% BSA-PBS blocking solution to each well, block at 37°C for 1 hour, add 100 μL of phage library, incubate at 37°C for 1 hour, add 100 μL of Gly-HCl eluate, and transfer the eluate to In a 1.5mL sterile centrifuge tube, quickly neutralize with 15μL Tris-HCl neutralization buffer. Take 10 μL for gradient dilution, measure the titer, and calculate the recovery rate of panning. The remaining eluates are mixed for amplification and purification for the next round of affinity panning, and the panning conditions are changed. Each round of panning conditions is as follows: Table 1, a total of three rounds.

Table 1 Affinity panning conditions

The panning eluate was mixed with 20 mL of the E. coliTG1 culture in the early stage of logarithmic growth, and allowed to stand at 37°C for 30 min. Add 1 mL of 20% glucose, 37 ° C, 180 r/min shaking culture for 30 min. M13K07 phage was added at the ratio of cell:phage=1:20, and 4 μL of Amp+ (100 mg/mL) was added. After standing at 37°C for 30 min, the cells were incubated with shaking at 180 r/min for 30 min. The culture was divided into centrifuge tubes, and the cell pellet was resuspended overnight. The pellet was suspended in 200 μL of PBS to obtain phage with positive vector, and the titer was determined.

3. Identification and analysis of specific phage clones

On the plate with the titer of the panning eluate, 48 monoclones were randomly picked with a sterilized toothpick and inoculated into 1 mL of 2×YT-A medium, and cultured with shaking at 37°C for 8 hours. Then, 200 μL of the above culture was taken, and M13K07 phage was added at a ratio of cell:phage=1:20, at 37° C., let stand for 15 min, and cultured with shaking at 220 r/min for 45 min. The supernatant was collected by centrifugation the next day and used to identify positive phage clones by ELISA. The B132205 recombinant single-domain antibody was obtained according to the sequence analysis of the positive phage clones. Its amino acid sequence (SEQ ID NO.6) is as follows:

In the sequence of SEQ ID NO.6, the fragment that plays a key role is shown in Table 2,

Table 2 Key fragments of recombinant single domain antibody of the present invention

The beneficial effects of the present invention will be further described below by means of experimental examples.

Experimental Example 1 Antibody B132205 Blocking the Effect of PD-1 and PD-L1 Interaction Verification

1 Experimental principle

PD-1/PD-L1 Bioassay is a functional activity evaluation system for cell-dependent bioluminescence detection, consisting of two genetically engineered cell lines. PD-1 effector cells: Jurkat T cells express human PD-1 and IL2 promoter-induced luciferase reporter genes. PD-L1aAPC/CHO-K1 cells: CHO-K1 cells express human PD-L1 and cell surface proteins that activate TCR. When these two cells were co-cultured, PD-L1 interacted with the ligand PD-1 and inhibited T cell activation signaling and inhibited IL2 promoter-regulated luciferase expression. When the corresponding PD-1 antibody was added, it blocked the interaction between PD-1 and the ligand PD-L1, released the inhibitory signal, thereby activated T cells, and activated IL2 promoter-induced luciferase expression. The blocking functional activity of anti-PD-1 or PD-L1 antibodies was evaluated by adding Bio-Glo reagent (including buffer and substrate) to evaluate the expression of luciferase.

In this experimental example, Jurkat T cells expressing human PD-1 and NFAT-RE-induced luciferase reporter genes were used as effector cells, and CHO-K1 cells expressing human PD-L1 and TCR activating proteins were used as antigen-presenting cells. . A biosimilar of Tecentriq (an anti-PD-L1 antibody) was used as a positive control to evaluate the PD1/PDL1 blocking functional activity of antibody B132205 in vitro.

Similar drug sequence for Tecentriq:

Heavy Chain Sequence (heavy chain sequence, SEQ ID NO. 11):

Light Chain Sequence (light chain sequence, SEQ ID NO. 12)

2 Experimental steps

1) Collect the target cells by centrifugation, resuspend the cells in F12K Nutrient Mixture medium containing 10% FBS, adjust the cell density, and inoculate in a 96-well experimental plate.

2) Transfer the experimental plate to a cell incubator (37°C/5% CO2) and incubate for 16-20 hours.

3) Use Assay Buffer to prepare 2× concentration of positive control and test substance (B132205 prepared by the method of Example 1).

4) Collect the effector cells by centrifugation, resuspend the cells in Assay Buffer and adjust the cell density.

5) Take the incubated 96-well experimental plate out of the cell incubator and remove the medium, and transfer the reference substance, the test substance working solution and the effector cell suspension to the 96-well experimental plate in turn.

6) Transfer the assay plate to a cell incubator (37°C/5% CO ₂ ) and incubate for about 6 hours.

7) Prepare luciferase substrate working solution.

8) Take out the incubated 96-well experimental plate from the cell incubator, and transfer the luciferase substrate working solution to the 96-well experimental plate.

9) Read the chemiluminescence value on the PHERA Star FSX and record the data.

10) Establish a corresponding dose-effect curve according to the corresponding relationship between the relative chemiluminescence signal value (Relative Luminescence Unit) and the final detection concentration.

3 results

As shown in Figure 4, antibody B132205 can effectively block the binding of PD-1 and PD-L1, and activate downstream pathways to generate signals. The EC50 of its dose-response curve was 0.1080μg/ml, which was comparable to the positive drug.

To sum up, the PD-L1 antibody of the present invention can well recognize PD-L1 and effectively block the combination of PD-1 and PD-L1; it can theoretically play a good role in the preparation of tumor immunotherapy drugs. antitumor effect.

SEQUENCE LISTING

<110> West China Hospital of Sichuan University

<120> A PD-L1 antibody

<130> GYKH1352-2019P017256CC

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<170> PatentIn version 3.5

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cttggtggtc ctggctgc 18

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ggtacgtgct gttgaactgt tcc 23

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gatgccatga ctgtggccca ggcggcccag ktgcagctcg tggagtc 47

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catgccatga ctcgcggccg gcctggccat gggggtcttc gctgtggtgc g 51

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catgccatga ctcgcggccg gcctggccgt cttgtggttt tggtgtcttg gg 52

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Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

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Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly

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Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly

35 40 45

Ser Ile Ser Ser Phe Asp Ala Val Leu Trp Tyr Arg Arg Ala Pro Gly

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Lys Gln Arg Asp Trp Val Ala Thr Ser Phe Thr Ala Gly His Thr Ile

65 70 75 80

Tyr Glu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr

100 105 110

Gly Asp Tyr Tyr Cys Asn Ala Arg Arg Leu Gly Ala His Tyr Trp Gly

115 120 125

Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro

130 135 140

Gln Asp Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp

145 150 155 160

Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu

165 170 175

Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu

180 185 190

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

195 200 205

Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His

210 215 220

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

225 230 235 240

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

245 250 255

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

260 265 270

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

275 280 285

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

290 295 300

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

305 310 315 320

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

325 330 335

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

340 345 350

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

355 360 365

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Gly Ser Ile Ser Ser Phe Asp Ala

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Ser Phe Thr Ala Gly His Thr

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Asn Ala Arg Arg Leu Gly Ala His Tyr

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

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20 25 30

Ala Val Leu Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Asp Trp Val

35 40 45

Ala Thr Ser Phe Thr Ala Gly His Thr Ile Tyr Glu Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Gly Asp Tyr Tyr Cys Asn

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Ala Arg Arg Leu Gly Ala His Tyr Trp Gly Gln Gly Thr Gln Val Thr

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Val Ser Ser

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

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Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims (5)

1. An anti-PD-L1 monoclonal recombinant single-domain antibody, characterized in that the amino acid sequence of its CDR1 is as shown in SEQ ID NO.7, the amino acid sequence of CDR2 is as shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.8. shown in SEQ ID NO.9. 2. The anti-PD-L1 monoclonal recombinant single domain antibody according to claim 1, characterized in that it has the amino acid sequence shown in SEQ ID NO.10. 3. An anti-tumor bioimmune drug, characterized in that it is a pharmaceutical preparation prepared by using the PD-L1 antibody of claim 1 as an active ingredient, and adding pharmaceutically acceptable auxiliary ingredients. 4. Use of the recombinant single-domain antibody according to claim 1 or 2 in the preparation of a PD-L1 inhibitor; The PD-L1 inhibitor is a drug for treating lung cancer, melanoma, bladder cancer, kidney cancer, liver cancer, nasopharyngeal cancer, esophageal cancer, cervical cancer, breast cancer or gastric cancer. 5. The use of claim 4, wherein the PD-L1 inhibitor is a drug that inhibits the binding of PD-L1 to PD-1.

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