Long-acting PD-L1 specific affinity as well as preparation method and application thereof

Abstract

The invention relates to the technical field of anti-tumor targeted drugs, in particular to a long-acting PD-L1 specific affinity as well as a preparation method and application thereof. The PD-L1 specific affinity is a dimer of IgBD-ZM1, and the amino acid sequence of the IgBD-ZM1 is as shown in a sequence 8; the preparation method comprises the following steps: fusing IgBD onto an affinity ZM1, forming a disulfide bond through cysteine residues to construct an IgBD-ZM1 dimer, introducing an HE tag, constructing an expression plasmid, transforming the expression plasmid into a bacterial competent state, constructing an expression strain and culturing, adding IPTG to induce expression, collecting a bacteria-broken supernatant, and purifying to obtain a target protein. The fusion structural domain IgBD disclosed by the invention can play a role in FcRn receptor mediated recycling by coupling IgG, so that the enrichment time of effective substances at a tumor part is prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor targeted drugs, and in particular to a long-acting PD-L1 specific affibody and a preparation method and application thereof. Background Art

[0002] During tumor occurrence and progression, the body's immune system can eliminate abnormally proliferating tumor cells and inhibit tumor growth through the "Cancer-Immunity Cycle". However, tumors upregulate the expression of immune checkpoint molecules, establish a complex anti-immune microenvironment, escape immune system surveillance, and promote tumor progression and metastasis. Immune checkpoint blockade therapy can significantly relieve the immunosuppressive effects of tumor cells, reactivate immune cell functions, kill and eliminate tumor cells, and achieve anti-tumor therapeutic effects. Among them, antibody tumor immunotherapy targeting PD-1 / PD-L1 immune checkpoints has been widely used in the late-line treatment of clinical tumor patients, but due to the large molecular weight of antibody drugs and poor tumor penetration, the effective enrichment and penetration into the tumor site are affected, and the half-life of antibody drugs in the body is too long, which is easy to induce a series of immune-related adverse reactions.

[0003] Affibody is a non-immunoglobulin scaffold protein, derived from the high-affinity ligand of the B segment of the staphylococcal A immunoglobulin binding domain. It is composed of 58 amino acids and its skeleton structure is formed by three α-helices. It has no sequence or structural homology with antibodies, low immunogenicity, and good antigen specificity and affinity. In addition, the structure and physicochemical properties of the affibody are stable, which is convenient for subsequent modification. Therefore, the development of endogenous long-acting affibody targeted drugs to block immune checkpoints is a new idea to expand the application of tumor immunotherapy. Summary of the invention

[0004] In view of the technical problem that the anti-tumor effect of existing antibody drugs is not ideal, the present invention provides a long-acting PD-L1 specific affibody and its preparation method and application. The present invention designs a long-acting PD-L1 specific affibody with stronger stability and affinity. The fusion domain IgBD of the present invention can exert the recycling effect mediated by FcRn receptor by coupling IgG, so that the enrichment time of effective substances in the tumor site is prolonged.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a long-acting PD-L1-specific affinity body, which is a dimer of IgBD-ZM1, wherein the amino acid sequence of IgBD-ZM1 is shown in Sequence 8.

[0006] In a second aspect, the present invention provides a method for preparing the above-mentioned PD-L1-specific affinity body, comprising the following steps: The C-terminus of IgBD was fused to the N-terminus of the affinity body ZM1 through a connecting peptide, and a disulfide bond was formed through the cysteine ​​residue at the C-terminus of ZM1 to construct an IgBD-ZM1 dimer. At the same time, in order to separate and purify the protein by using nickel gel, an HE tag was introduced into the N-terminus of IgBD-ZM1 (the nucleotide and amino acid sequences are shown in Sequence 9 and Sequence 10, respectively). The optimized fusion expression gene sequence was cloned into a plasmid to construct an expression plasmid, and then the expression plasmid was transformed into a bacterial competent state, an expression strain was constructed and cultured, IPTG was added to induce expression, and the supernatant after bacterial rupture was collected and purified to obtain the target protein, which was named IgBD-Z PD-L1 .

[0007] Furthermore, the amino acid sequence of IgBD is shown in SEQ ID NO: 2, and the amino acid sequence of ZM1 is shown in SEQ ID NO: 6.

[0008] Furthermore, the connecting peptide is (G4S)3, and the amino acid sequence of (G4S)3 is shown in Sequence 4.

[0009] Furthermore, the plasmid used was pQE30 plasmid, and the constructed expression plasmid was pQE30-IgBD-Z PD-L1 .

[0010] Furthermore, the competent bacteria were M15 Escherichia coli ( Escherichia coli ), the constructed expression strain is M15-pQE30-IgBD-Z PD-L1 .

[0011] Further, the expression strain is cultured and the expression induction method is as follows: The expression strain was inoculated into LB medium containing ampicillin (Amp) and kanamycin (Kan) for expansion culture at 37°C and 220 rpm. 600nm When the value reached 0.6-0.8, 0.1 mM isopropylthiogalactoside (IPTG) was added and the expression was induced at 28°C and 150 rpm for 14-16 h.

[0012] Furthermore, the target protein IgBD-Z PD-L1 The purification and collection method is as follows: After the induction of expression, the cells were collected by centrifugation and resuspended in lysis buffer. Phenylmethylsulfonyl fluoride (PMSF) was added to a working concentration of 1 mM and the cells were ruptured by ultrasonication. After completion, the supernatant was collected by high-speed centrifugation and the target protein was purified by adding Ni-NTA resin gel.

[0013] In a third aspect, the present invention provides an application of the above-mentioned PD-L1 specific affinity body in the preparation of anti-tumor drugs, for example: used as a new endogenous long-acting immune combined anti-tumor therapeutic antibody drug delivery platform.

[0014] Furthermore, the application is to prepare an antibody-drug conjugate by combining a PD-L1-specific affinity with IgG.

[0015] The beneficial effects of the present invention are: First, the present invention incorporates an IgG binding domain IgBD at the Fab end, which has good affinity for IgG and is easy to form a stable complex. Therefore, the affibody provided by the present invention can be coupled with a variety of antibody drugs to form a multifunctional antibody-drug conjugate, which can be targeted at a wider range of disease treatments and has the application prospect of becoming a sustainable drug platform.

[0016] At the same time, plasma protein IgG is used as an endogenous long-acting carrier for drug modification. It is not only rich in sources, but also can reduce the limitations of biocompatibility and immunogenicity. Its Fc receptor (FcRn)-mediated recycling effect can prolong the half-life of the recombinant protein in vivo, effectively increase its action time in the body, improve the anti-tumor biological activity in the body, and exert a more lasting effect in inhibiting tumor growth.

[0017] Secondly, the dimeric affinity body described in the present invention has a tumor targeting effect, can effectively enrich in the tumor tissue site, and recognize PD-L1 on the surface of tumor cells, competitively inhibit the binding of PD-1 and PD-L1 on the surface of T cells, thereby restoring the recognition ability of T cells, blocking the PD-1 / PD-L1 signaling pathway, and exerting a more efficient anti-tumor immunotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 IgBD-Z PD-L1 Preparation; wherein, Figure 1 A in the middle is the dimeric affinity body IgBD-Z PD-L1 Schematic diagram; Figure 1 B is the SDS-PAGE image of different protein samples, and lane 6 is the purified IgBD-Z PD-L1 ; Figure 1 Center C is the SDS-PAGE image of the protein under non-reducing (-2ME) and reducing (+2ME) conditions.

[0020] Figure 2 IgBD-Z PD-L1 Specifically binds to PD-L1 positive tumor cells; Figure 2A in the middle is the PD-L1 expression of cells detected by flow cytometry; Figure 2 B is flow cytometry detection of IgBD-Z PD-L1 Binding to cells; Figure 2 C in the middle is IgBD-Z PD-L1 Fluorescence imaging of cell binding.

[0021] Figure 3 IgBD-Z PD-L1 In vitro binding activity verification with IgG and PD-L1; Figure 3 A in the middle is IgBD-Z PD-L1 IgG and IgBD-Z PD-L1 and the ELISA binding results of its conjugate with mIgG and PD-L1; Figure 3 B is mIgG-IgBD-Z PD-L1 Surface ion resonance results of the conjugate binding to PD-L1; Figure 3 C in the middle is IgBD-Z PD-L1 Surface ion resonance results for binding to mIgG and PD-L1.

[0022] Figure 4 The figure shows the immunofluorescence staining of recombinant protein tissue level.

[0023] Figure 5 The long-term effect of the modified protein on inhibiting tumor growth in vivo; Figure 5 A in the middle shows the tumor growth curves of MC38 tumor-bearing mice in different treatment groups; Figure 5 Middle B is the weight change curve of mice during treatment; Figure 5 Middle C is the mass weight; Figure 5 D in the middle is a photo of the tumor after peeling. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0025] The nucleotide sequence and amino acid sequence information used in the specific implementation of the present invention are as follows: (1) IgBD nucleotide sequence (SEQ ID NO: 1) 5-acgacctataaattggttattaacggcaaaaccctgaaaggtgaaacgactacgaaggcggttgatgcggaaaccgcagctgcggcgtttgcgcagtatgcgaacgacaacggtgtggacggcgtttggacctacgacgacgccaccaagaccttcaccgtcaccgaa-3 (2) IgBD amino acid sequence (sequence 2) NH2-TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATKTFTVTE-COOH (3) (G4S)3 nucleotide sequence (sequence 3) 5-ggtggtggcggctccggtggtggtggctctggtggcggaggcagc-3 (4) (G4S)3 amino acid sequence (SEQ ID NO: 4) NH2-GGGGSGGGGSGGGGS-COOH (5) Nucleotide sequence of ZM1 (SEQ ID NO: 5) 3 (6) Amino acid sequence of ZM1 (SEQ ID NO: 6) NH2-AEAKYAKEHMMAASEILQLPNLTFIQKFVFISKLSDDPSQSSELLSEAKKLNDSQAPKGSC-COOH (7) Nucleotide sequence of IgBD-ZM1 (SEQ ID NO: 7) 5-atgagggggtcacatgaacacgagcacgaaggatctacgacctataaattggttattaacggcaaaaccctgaaaggtgaaacgactacgaaggcggttgatgcgga aaccgcagctgcggcgtttgcgcagtatgcgaacgacaacggtgtggacggcgtttggacctacgacgacgccaccaagaccttcaccgtcaccgaaggtggtggcggc tccggtggtggtggctctggtggcggaggcagcgcagaggccaagtacgcgaaggagcacatgatggctgcttccgaaattctgcagctgccgaatctgaccttcatcc aaaaattcgtgtttatcagcaaactctcggatgatccgagccaaagcagcgagctgctgtccgaggcgaagaagttgaatgatagccaggcaccgaaaggtagttgc-3 (8) Amino acid sequence of IgBD-ZM1 (SEQ ID NO: 8) NH2-MRGSHEHEHEGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSAEAKYAKEHMMAASEILQLPNLTFIQKFVFISKLSDDPSQSSELLSEAKKLNDSQAPKGSC-COOH (9) Nucleotide sequence of HE tag (Sequence 9) 5-atgagggggtcacatgaacacgagcacgaaggatct-3 (10) Amino acid sequence of HE tag (SEQ ID 10) NH2-MRGSHEHEHEGS-COOH The competent M15 Escherichia coli used in the specific implementation of the present invention was purchased from Beina Biotechnology.

[0026] The lysate used in the specific embodiment of the present invention contains 50 mM phosphate, 300 mM sodium chloride and 20 mM imidazole, with a pH of 8.0.

[0027] The high-affinity Ni-NTA resin gel used in the specific embodiment of the present invention was purchased from Nanjing GenScript Biotech Co., Ltd.

[0028] Example 1 IgBD-Z PD-L1 Expression preparation and identification Expression and preparation of PD-L1-specific dimerization affinity body IgBD-Z by genetic engineering technology PD-L1 The C-terminus of IgBD was designed to be fused to the N-terminus of the affinity body ZM1 through a connecting peptide (G4S)3, and a disulfide bond was formed through the cysteine ​​residue at the C-terminus of ZM1 to construct the IgBD-ZM1 dimer. At the same time, in order to separate and purify the protein by using nickel gel, an HE tag was introduced at the N-terminus of IgBD-ZM1. The optimized fusion expression gene sequence was cloned into the pQE30 plasmid through the BamH I and Sal I restriction sites to construct the expression plasmid pQE30-IgBD-Z PD-L1 The expression plasmid was then transformed into competent M15 E. coli to construct M15-pQE30-IgBD-Z PD-L1 The expression strain was inoculated into LB medium containing Amp and Kan for expansion culture (37°C, 220 rpm). 600nm When the value reaches 0.6-0.8, add 0.1 mM isopropylthiogalactoside (IPTG) and induce expression overnight (14-16 h) at 28°C and 150 rpm. After the induction expression is completed on the second day, the bacteria are collected by centrifugation and resuspended with lysis buffer (50 mM phosphate, pH 8.0, 300 mM sodium chloride, 20 mM imidazole), and phenylmethylsulfonyl fluoride (PMSF) is added to a working concentration of 1 mM. Ultrasonic bacteria are broken, and after completion, the supernatant is collected by high-speed centrifugation, and the target protein IgBD-Z is purified by adding high-affinity Ni-NTA resin gel. PD-L1 .

[0029] like Figure 1 As shown in B, compared with before induction (lane 1), a specific protein band appeared at about 15 KDa after induced expression (lane 2). After ultrasonic disruption and centrifugation, the protein was mainly present in the supernatant (lane 3), while the protein was not found in the lower precipitate (lane 4), indicating that the protein was a soluble protein. Finally, the target protein obtained after purification was a single band (lane 6), while the protein was not found in the flow-through peak (lane 5). Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze IgBD-Z under non-reducing conditions (without β-mercaptoethanol, -2ME) and reducing conditions (with β-mercaptoethanol, +2ME). PD-L1 The molecular weights are 30KDa and 15KDa respectively ( Figure 1 C), with IgBD-Z PD-L1 The theoretical molecular weight is consistent with that of PD-L1 .

[0030] Example 2 IgBD-Z PD-L1 In vitro activity studies Mouse MC38 colon cancer and MLE-12 lung epithelial cells were incubated with FITC-labeled PD-L1 antibodies, and flow cytometry was used to verify that MC38 was PD-L1 positive and highly expressed, while MLE-12 was PD-L1 negative and low expressed ( Figure 2 IgBD-Z was subsequently labeled with 6-carboxyfluorescein (6-FAM). PD-L1 , incubated with MC38 and MLE-12 cells for 1 h, washed with PBS and analyzed by flow cytometry. PD-L1 Can specifically bind to PD-L1 positive MC38 cells ( Figure 2 To verify the in vitro expression of IgBD-Z PD-L1 To block the PD-L1, MC38 cells were incubated with free IgBD-Z PD-L1 Pre-incubate with blocked PD-L1 receptor for 30 min, then incubate with 6-FAM labeled IgBD-Z PD-L1 Incubate for 1 hour. Finally, flow cytometry was used to analyze FAM-IgBD-Z before and after PD-L1 receptor blocking. PD-L1 Positive binding rate, found that the binding ratio decreased, reflecting IgBD-Z PD-L1 Blockade of PD-L1 receptors ( Figure 2 At the same time, fluorescence imaging was used to verify the expression of PD-L1 positive cells MC38 and IgBD-Z under the same flow cytometry incubation conditions as above. PD-L1 The binding and blocking conditions of Figure 2 Middle C) shows IgBD-Z PD-L1 It has good binding and blocking ability to PD-L1 receptor.

[0031] The binding of the present invention to IgG and PD-L1 was detected by ELISA, and the ELISA plate was coated with mouse IgG, human IgG, and PD-L1 protein, respectively. PD-L1 and mIgG-IgBD-Z mixed at a molar ratio of 1:2 at room temperature PD-L1 Conjugate, incubate at 37℃ for 1 h. After washing three times with PBST, add horseradish peroxidase-labeled avidin protein and incubate at 37℃ for 30 min. Finally, wash 5 times with PBST and add TMB colorimetric solution, and measure the absorbance at 450nm with an enzyme reader. Figure 3As shown in A, consistent with the flow cytometry and fluorescence results, the present invention can bind to PD-L1; and the binding to PD-L1 is not affected by mIgG coupling. At the same time, ELISA results show that the binding of the present invention to hIgG and mIgG increases in a concentration-dependent manner, indicating that the present invention can bind to IgG from different species and has application prospects in the preparation of multifunctional antibody drug conjugates ( Figure 3 Middle A).

[0032] Surface plasmon resonance technology was further used to verify the binding of the present invention to mIgG and PD-L1 receptor proteins in vitro. mIgG and PD-L1 proteins were coated on a CM5 chip, and then IgBD-Z with different gradient concentrations (5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM) was added to the chip. PD-L1 and (5 nM, 10 nM, 25 nM, 50 nM, 100 nM) mIgG-IgBD-Z PD-L1 The conjugate solution was used as the mobile phase to flow through the chip, and the binding and dissociation processes were monitored and analyzed on the OpenSPR system (Nicoya Lifesciences Inc., Kitchener, Canada). Figure 3 C) shows that the present invention can form a stable conjugate with mIgG. SPR results also prove that IgBD-Z PD-L1 ( Figure 3 C) and mIgG-IgBD-Z PD-L1 ( Figure 3 (B) Both can bind to the PD-L1 protein coated on the CM5 chip.

[0033] Example 3 IgBD-Z PD-L1 In vivo activity studies To explore the in vivo tumor targeting of the present invention, female C57BL / 6 mice (14-16 g) were subcutaneously inoculated with MC38 tumor cells (1×10 6 The tumor-bearing mouse model was established by recording the length (L) and width (W) of the tumor every day. 2 The tumor volume (V) was calculated using the formula / 2.

[0034] Volume up to about 200 mm 3 When the tumor-bearing mice were directly anesthetized and killed, tumor tissues were obtained, frozen sections were incubated with PD-L1 antibodies for immunofluorescence staining, and the expression of PD-L1 in tumor tissues was analyzed to verify the high expression of PD-L1 in MC38 at the tissue level ( Figure 4 DAPI / PD-L1 / Isotype in the middle); another part of tumor-bearing mice were injected with FAM-IgBD-Z by tail vein PD-L1After administrated 5 mg / kg, tumor tissues were taken for immunofluorescence staining to explore the targeted enrichment distribution of the present invention in tumor tissues. The results showed that the tumor tissues had obvious PD-L1 red fluorescence signals, which were consistent with FAM-IgBD-Z PD-L1 The green fluorescence signal of Figure 4 Medium DAPI / PD-L1 / lgBD-Z PD-L1 ), which shows that the present invention can effectively reach the tumor tissue site and bind to the membrane ligand PD-L1 to play a tumor-killing role.

[0035] According to the same model construction method as above, a group of tumor-bearing mouse models were established, divided into IgBD-Z PD-L1 Treatment group, Z PD-L1 Treatment group and PBS treatment group (n=6). When the tumor volume of MC38 tumor-bearing mice subcutaneously transplanted reached about 70 mm 3 When IgBD-Z PD-L1 The treatment group was injected with IgBD-Z into the tail vein every three days. PD-L1 (5 mg / kg), Z PD-L1 The treatment group was injected with an equal molar amount of Z every three days through the tail vein. PD-L1 The PBS-treated group was injected with the same volume of PBS into the tail vein every three days. PD-L1 Preparation method of IgBD-Z PD-L1 Similarly, ZM1 used disulfide bonds to form dimers. The optimized fusion expression gene sequence was cloned into the pQE30 plasmid through the BamH I and Sal I restriction sites. The expression plasmid was then transformed into competent M15 Escherichia coli and inoculated into LB medium containing Amp and Kan for expansion culture (37°C, 220 rpm). 600nm When the value reaches 0.6-0.8, add 0.1 mM IPTG, 28°C, 150 rpm, induce expression overnight, collect the bacteria by centrifugation, resuspend them in lysis buffer, add PMSF to a working concentration of 1 mM, ultrasonically break the bacteria, collect the supernatant by high-speed centrifugation, add high-affinity Ni-NTA resin gel for purification and collection.

[0036] The tumor size and mouse weight were monitored and recorded every day to explore the in vivo anti-tumor immunotherapy effect of the present invention. PD-L1 , the tumor inhibition effect of the present invention is better ( Figure 5 A in the middle), and after the tumor is peeled off, it is also proved that the present invention can effectively treat tumors and reduce tumor weight faster ( Figure 5 Middle C), the tumor volume decreased more significantly ( Figure 5 D), and the weight of mice did not decrease significantly during the treatment, indicating that the present invention has good short-term biosafety ( Figure 5Middle B).

[0037] In summary, this example establishes the MC38 colon cancer tumor model to verify the long-acting dimerization affinity body IgBD-Z PD-L1 The results showed that IgBD-Z PD-L1 As an anti-tumor drug, it can effectively inhibit tumor growth, and the short-acting dimeric affinity body Z PD-L1 Although immunotherapy also has a certain anti-tumor effect, its anti-tumor effect is not as good as that of IgBD-Z provided by the present invention. PD-L1 of excellence.

[0038] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A long-acting PD-L1-specific affinity body, characterized in that: It is a dimer of IgBD-ZM1, wherein the amino acid sequence of IgBD-ZM1 is shown in SEQ ID NO:

8.

2. A method for preparing a PD-L1-specific affinity body according to claim 1, characterized in that: The steps include: The C-terminus of IgBD was fused to the N-terminus of the affinity body ZM1 through a connecting peptide, and a disulfide bond was formed through the cysteine ​​residue at the C-terminus of ZM1 to construct an IgBD-ZM1 dimer. At the same time, an HE tag was introduced into the N-terminus of IgBD-ZM1, and the optimized fusion expression gene sequence was cloned into a plasmid to construct an expression plasmid. The expression plasmid was then transformed into a bacterial competent state, an expression strain was constructed and cultured, IPTG was added to induce expression, the ruptured supernatant was collected, and the target protein was purified.

3. The preparation method according to claim 2, characterized in that: The amino acid sequence of IgBD is shown in SEQ ID NO: 2, and the amino acid sequence of ZM1 is shown in SEQ ID NO:

6.

4. The preparation method according to claim 2, characterized in that: The connecting peptide is (G4S)3, and the amino acid sequence of (G4S)3 is shown in Sequence 4.

5. The preparation method according to claim 2, characterized in that: As the plasmid, pQE30 plasmid was used.

6. The preparation method according to claim 2, characterized in that: The competent bacteria were M15 Escherichia coli ( Escherichia coli ).

7. The preparation method according to claim 2, characterized in that: The culture and induction expression methods of the expression strain are as follows: The expression strain was inoculated into LB medium containing Amp and Kan for expansion culture at 37°C and 220 rpm. 600nm When the value reached 0.6-0.8, 0.1 mM IPTG was added and the expression was induced at 28°C and 150 rpm for 14-16 h.

8. The preparation method according to claim 2, characterized in that: Target protein IgBD-Z PD-L1 The purification and collection method is as follows: After the induction of expression is completed, the bacteria are collected by centrifugation and resuspended in lysis buffer, and PMSF is added to a working concentration of 1 mM for ultrasonic lysis of the bacteria. After completion, the supernatant is collected by high-speed centrifugation and added to Ni-NTA resin gel to purify and collect the target protein.

9. Use of the PD-L1 specific affinity body as claimed in claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that Antibody-drug conjugates were prepared by combining PD-L1-specific affinity with IgG.

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