A long-lasting PD-L1 specific affinity compound, its preparation method and application

Abstract

This invention relates to the field of antitumor targeted drug technology, specifically to a long-acting PD-L1 specific affinity monomer, its preparation method, and its application. The PD-L1 specific affinity monomer is a dimer of IgBD-ZM1, wherein the amino acid sequence of IgBD-ZM1 is shown in Sequence 8. The preparation method includes fusing IgBD to the affinity monomer ZM1, constructing the IgBD-ZM1 dimer by forming disulfide bonds through cysteine ​​residues, introducing an HE tag, constructing an expression plasmid, transforming the expression plasmid into bacterial competent cells, constructing and culturing the expression strain, adding IPTG to induce expression, collecting the bacterial supernatant, and purifying to obtain the target protein. The fused IgBD domain of this invention can couple with IgG to exert an FcRn receptor-mediated recycling effect, thereby prolonging the accumulation time of the effective substance at the tumor site.

Description

Technical Field

[0001] This invention relates to the field of antitumor targeted drug technology, specifically to a long-acting PD-L1 specific affinity compound, its preparation method, and its application. Background Technology

[0002] During tumor development and progression, the body's immune system can eliminate abnormally proliferating tumor cells and inhibit tumor growth through the "Cancer-Immunity Cycle." However, tumors can evade immune system surveillance and promote tumor progression and metastasis by upregulating the expression of immune checkpoint molecules and establishing a complex anti-immune microenvironment. Immune checkpoint blockade therapy can significantly relieve the immunosuppression of tumor cells, reactivate immune cell function, kill and eliminate tumor cells, and achieve anti-tumor therapeutic effects. Among these, antibody-based tumor immunotherapy targeting PD-1 / PD-L1 immune checkpoints has been widely used in the later-line treatment of clinical cancer patients. However, due to the large molecular weight of antibody drugs, their poor tumor penetration affects their effective enrichment and penetration into the tumor site. Furthermore, the long half-life of antibody drugs in vivo can easily induce a series of immune-related adverse reactions.

[0003] Affibody, a non-immunoglobulin scaffold protein, is a high-affinity ligand derived from the B segment of the Staphylococcus A immunoglobulin-binding domain. Composed of 58 amino acids, its backbone structure is formed by three α-helices. It exhibits no sequence or structural homology with antibodies, low immunogenicity, and good antigen specificity and affinity. Furthermore, affibody structures and physicochemical properties are stable, facilitating subsequent modification. Therefore, developing endogenous, long-acting affibody-targeting drugs to block immune checkpoints represents a new approach to expanding the application of tumor immunotherapy. Summary of the Invention

[0004] To address the technical problem of unsatisfactory antitumor effects of existing antibody drugs, this invention provides a long-acting PD-L1 specific affinity polymer, its preparation method, and its applications. This invention designs a long-acting PD-L1 specific affinity polymer with enhanced stability and affinity. The fused IgBD domain of this invention can be coupled with IgG to exert FcRn receptor-mediated recycling effects, thereby prolonging the accumulation time of the effective substance at the tumor site.

[0005] The technical solution of this invention is as follows:

[0006] In a first aspect, the present invention provides a long-lasting PD-L1 specific affinity polymer, which is a dimer of IgBD-ZM1, wherein the amino acid sequence of IgBD-ZM1 is shown in Sequence 8.

[0007] Secondly, the present invention provides a method for preparing the above-mentioned PD-L1 specific affinity polymer, comprising the following steps:

[0008] The C-terminus of IgBD was fused to the N-terminus of its affinity variant ZM1 via a linker peptide, and a disulfide bond was formed through a cysteine ​​residue at the C-terminus of ZM1 to construct the IgBD-ZM1 dimer. Simultaneously, to facilitate protein purification using nickel gel, an HE tag (nucleotide and amino acid sequences shown in sequences 9 and 10, respectively) was introduced to the N-terminus of IgBD-ZM1. The optimized fusion expression gene sequence was cloned into a plasmid to construct the expression plasmid. The expression plasmid was then transformed into bacterial competent cells to construct and culture the expression strain. IPTG was added to induce expression, and the supernatant was collected and purified to obtain the target protein, named IgBD-Z. PD-L1 .

[0009] Furthermore, the amino acid sequence of IgBD is shown in Sequence 2, and the amino acid sequence of ZM1 is shown in Sequence 6.

[0010] Furthermore, the linker peptide is (G4S)3, and the amino acid sequence of (G4S)3 is shown in Sequence 4.

[0011] Furthermore, the pQE30 plasmid was used, and the constructed expression plasmid was pQE30-IgBD-Z. PD-L1 .

[0012] Furthermore, the competent bacterial cells were M15 Escherichia coli (E. coli) Escherichia coli The constructed expression strain was M15-pQE30-IgBD-Z. PD-L1 .

[0013] Furthermore, the methods for culturing and inducing expression of the expression strain are as follows:

[0014] The expression strain was inoculated into LB medium containing ampicillin (Amp) and kanamycin (Kan) for expansion culture at 37°C and 220 rpm until bacterial culture A... 600nm When the value reaches 0.6-0.8, add 0.1 mM isopropyl galactothioglycoside (IPTG) and induce expression at 28℃ and 150 rpm for 14-16 h.

[0015] Furthermore, the target protein IgBD-Z PD-L1 The purification and collection methods are as follows:

[0016] After the induction expression was completed, the bacterial cells were collected by centrifugation and resuspended with lysis buffer. Benzyl sulfonyl fluoride (PMSF) was added to a working concentration of 1 mM and the cells were sonicated to break down the bacteria. After the breakdown was completed, the supernatant was collected by high-speed centrifugation and purified by Ni-NTA resin gel to collect the target protein.

[0017] Thirdly, the present invention provides an application of the above-mentioned PD-L1 specific affinity in the preparation of antitumor drugs, for example, as a novel endogenous long-acting immune-combined antitumor therapy antibody drug delivery platform.

[0018] Further applications include the preparation of antibody-drug conjugates by utilizing the PD-L1 specific affinity monomer to bind with IgG.

[0019] The beneficial effects of this invention are as follows:

[0020] First, this invention incorporates a Fab-terminal IgG-binding domain, IgBD, which exhibits good affinity for IgG and readily forms stable complexes. Therefore, the affinity compound provided by this invention can be conjugated with various antibody drugs to form multifunctional antibody-drug conjugates, targeting a broader range of diseases and demonstrating promising potential as a sustainable drug platform.

[0021] Meanwhile, plasma protein IgG, as an endogenous long-acting carrier for drug modification, is not only abundant in source but also reduces limitations in biocompatibility and immunogenicity. Its Fc receptor (FcRn)-mediated recycling effect can prolong the in vivo half-life of recombinant proteins, effectively increasing their duration of action in vivo, enhancing their anti-tumor biological activity, and exerting a more sustained inhibitory effect on tumor growth.

[0022] Secondly, the dimer affinity of the present invention has a tumor-targeting effect, can be effectively enriched in tumor tissue sites, and recognize PD-L1 on the surface of tumor cells. It competitively inhibits 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. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 IgBD-Z is a dimer affinity variant. PD-L1 Preparation of; wherein, Figure 1 In the middle, A is the dimer affinity IgBD-Z PD-L1 Schematic diagram; Figure 1 Lane B shows SDS-PAGE images of different protein samples, and lane 6 shows purified IgBD-Z. PD-L1 ; Figure 1 In the middle C, the SDS-PAGE images of the protein under non-reducing (-2ME) and reducing (+2ME) conditions are shown.

[0025] Figure 2 For IgBD-Z PD-L1 It specifically binds to PD-L1-positive tumor cells; among which, Figure 2 In section A, PD-L1 expression in cells was detected by flow cytometry. Figure 2 B in the text refers to the flow cytometry detection of IgBD-Z. PD-L1 Binding to cells; Figure 2 C is IgBD-Z PD-L1 Fluorescence imaging of cell binding.

[0026] Figure 3 For IgBD-Z PD-L1 In vitro binding activity against IgG and PD-L1 was validated; among which, Figure 3 In the middle, A is IgBD-Z PD-L1 With IgG and IgBD-Z PD-L1 The results of ELISA binding of the conjugate with mIgG and PD-L1; Figure 3 B is mIgG-IgBD-Z PD-L1 Surface ion resonance results of the coupling compound binding to PD-L1; Figure 3 C is IgBD-Z PD-L1 Surface ion resonance results of binding to mIgG and PD-L1.

[0027] Figure 4 This is an immunofluorescence staining image of recombinant protein at the tissue level.

[0028] Figure 5 To assess the in vivo tumor growth inhibitory effect of the protein before and after long-term modification; among which, Figure 5 In Figure A, the tumor growth curves of MC38 tumor-bearing mice in different treatment groups are shown. Figure 5 Figure B shows the body weight change curve of mice during treatment; Figure 5 In the middle, C represents the weight of the mass; Figure 5 Photograph of the tumor after dissection is shown in D. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0030] The nucleotide and amino acid sequences used in the specific embodiments of this invention are as follows:

[0031] (1) IgBD nucleotide sequence (Sequence 1)

[0032] 5-acgacctataaattggttattaacggcaaaaccctgaaaggtgaaacgactacgaaggcggttgatgcggaaaccgcagctgcggcgtttgcgcagtatgcgaacgacaacggtgtggacggcgtttggacctacgacgacgccaccaagaccttcaccgtcaccgaa-3

[0033] (2) IgBD amino acid sequence (Sequence 2)

[0034] NH2-TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATKTFTVTE-COOH

[0035] (3) (G4S)3 nucleotide sequence (Sequence 3)

[0036] 5-ggtggtggcggctccggtggtggtggctctggtggcggaggcagc-3

[0037] (4) (G4S)3 amino acid sequence (Sequence 4)

[0038] NH2-GGGGSGGGGSGGGGS-COOH

[0039] (5) The nucleotide sequence of ZM1 (Sequence 5)

[0040] 3

[0041] (6) The amino acid sequence of ZM1 (Sequence 6)

[0042] NH2-AEAKYAKEHMMAASEILQLPNLTFIQKFVFISKLSDDPSQSSELLSEAKKLNDSQAPKGSC-COOH

[0043] (7) Nucleotide sequence of IgBD-ZM1 (Sequence 7)

[0044] 5-atgagggggtcacatgaacacgagcacgaaggatctacgacctataaattggttattaacggcaaaaccctgaaaggtgaaacgactacgaaggcggttgatgcgga aaccgcagctgcggcgtttgcgcagtatgcgaacgacaacggtgtggacggcgtttggacctacgacgacgccaccaagaccttcaccgtcaccgaaggtggtggcggc tccggtggtggtggctctggtggcggaggcagcgcagaggccaagtacgcgaaggagcacatgatggctgcttccgaaattctgcagctgccgaatctgaccttcatcc aaaaattcgtgtttatcagcaaactctcggatgatccgagccaaagcagcgagctgctgtccgaggcgaagaagttgaatgatagccaggcaccgaaaggtagttgc-3

[0045] (8) Amino acid sequence of IgBD-ZM1 (Sequence 8)

[0046] NH2-MRGSHEHEHEGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSAEAKYAKEHMMAASEILQLPNLTFIQKFVFISKLSDDPSQSSELLSEAKKLNDSQAPKGSC-COOH

[0047] (9) Nucleotide sequence of the HE tag (Sequence 9)

[0048] 5-atgagggggtcacatgaacacgagcacgaaggatct-3

[0049] (10) The amino acid sequence of the HE tag (Sequence 10)

[0050] NH2-MRGSHEHEHEGS-COOH

[0051] The competent M15 Escherichia coli used in the specific embodiments of this invention was purchased from Beina Biotechnology.

[0052] The lysis buffer used in the specific embodiments of the present invention contains 50 mM phosphate, 300 mM sodium chloride and 20 mM imidazole, and has a pH of 8.0.

[0053] The high-affinity Ni-NTA resin gel used in the specific embodiments of this invention was purchased from Nanjing Genscript Biotech Co., Ltd.

[0054] Example 1 IgBD-Z PD-L1 Expression preparation and identification

[0055] IgBD-Z, a PD-L1-specific dimer affinity variant, was prepared by expression using genetic engineering techniques. PD-L1 The design involved fusing the C-terminus of IgBD to the N-terminus of its affinity variant ZM1 via a linker peptide (G4S)3, and constructing an IgBD-ZM1 dimer by forming a disulfide bond through a cysteine ​​residue at the C-terminus of ZM1. To facilitate protein purification using nickel gel, an HE tag was introduced at the N-terminus of IgBD-ZM1. The optimized fusion expression gene sequence was then cloned into the pQE30 plasmid using BamHI and SalI restriction sites, constructing the expression plasmid pQE30-IgBD-ZM1. 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℃, 220 rpm), and the bacterial culture A was ready. 600nm When the pH reached 0.6-0.8, 0.1 mM isopropyl thiogalactoside (IPTG) was added, and expression was induced overnight (14-16 h) at 28°C and 150 rpm. After the second day of induction, the bacterial cells were collected by centrifugation and resuspended in lysis buffer (50 mM phosphate, pH 8.0, 300 mM sodium chloride, 20 mM imidazole). Benzyl sulfonyl fluoride (PMSF) was added to a working concentration of 1 mM, followed by ultrasonic lysis. The supernatant was collected by high-speed centrifugation and purified using a high-affinity Ni-NTA resin gel to collect the target protein IgBD-Z. PD-L1 .

[0056] like Figure 1As shown in Figure B, compared to before induction (lane 1), induced expression (lane 2) showed a specific protein band at approximately 15 kDa. After sonication and centrifugation, the protein was mainly present in the supernatant (lane 3), while it was not found in the lower precipitate (lane 4), indicating that the protein is soluble. The final purified protein was a single band (lane 6), and it was not found in the flow-through peak (lane 5). Subsequently, IgBD-Z was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under both non-reducing conditions (without β-mercaptoethanol, -2 ME) and reducing conditions (with β-mercaptoethanol, +2 ME). PD-L1 The molecular weights are 30 kDa and 15 kDa, respectively. Figure 1 (C), and IgBD-Z PD-L1 The molecular weight is consistent with the theoretical molecular weight, indicating that IgBD-Z has been successfully expressed and prepared. PD-L1 .

[0057] Example 2 IgBD-Z PD-L1 In vitro activity study

[0058] Mouse MC38 colon cancer cells and MLE-12 lung epithelial cells were incubated with FITC-labeled PD-L1 antibody. Flow cytometry confirmed that MC38 cells showed high PD-L1 expression, while MLE-12 cells showed low PD-L1 expression. Figure 2 IgBD-Z was then labeled with 6-carboxyfluorescein (6-FAM). PD-L1 Incubated with MC38 and MLE-12 for 1 hour respectively, followed by washing with PBS and analysis by flow cytometry, IgBD-Z was found. PD-L1 It can specifically bind to PD-L1 positive MC38 cells ( Figure 2 (B). To verify the in vitro IgBD-Z PD-L1 The blocking effect on PD-L1, MC38 cells were treated with free IgBD-Z. PD-L1 Pre-incubate with PD-L1 receptor blocker for 30 min, then react with 6-FAM-labeled IgBD-Z. PD-L1 Incubation for 1 hour. Finally, flow cytometry was used to analyze FAM-IgBD-Z before and after PD-L1 receptor blockade. PD-L1 Positive binding rate was observed, with a decrease in the binding ratio, indicating a decrease in IgBD-Z. PD-L1 It has the ability to block PD-L1 receptors ( Figure 2 (B). Simultaneously, under the same flow cytometry incubation conditions as described above, fluorescence imaging was used to verify the interaction between PD-L1 positive cells MC38 and IgBD-Z. PD-L1 The binding and blocking situation, results ( Figure 2 (C) Display IgBD-Z PD-L1It has good binding and blocking ability to PD-L1 receptor.

[0059] The binding of this invention to IgG and PD-L1 was detected by ELISA. ELISA plates were coated with mouse IgG, human IgG, and PD-L1 protein, respectively. Subsequently, biotin-labeled IgBD-Z at different concentration gradients was used. PD-L1 and mIgG-IgBD-Z mixed at a molar ratio of 1:2 at room temperature PD-L1 The conjugate was incubated at 37°C for 1 h. After washing three times with PBST, horseradish peroxidase-labeled avidin protein was added, and the mixture was incubated at 37°C for 30 min. Finally, after washing five times with PBST, TMB chromogenic buffer was added, and the absorbance at 450 nm was measured using a microplate reader. Figure 3 As shown in Figure A, consistent with the flow cytometry and fluorescence results, this invention can bind to PD-L1; and the binding to PD-L1 is not affected by mIgG conjugation. Simultaneously, ELISA results show that the binding of this invention to hIgG and mIgG increases in a concentration-dependent manner, indicating that this invention can bind to IgG from different species, and has the potential for application in preparing multifunctional antibody-drug conjugates. Figure 3 (A)

[0060] The surface plasmon resonance (SPR) technique was further used to verify the in vitro binding of the present invention to mIgG and PD-L1 receptor proteins. mIgG and PD-L1 proteins were coated onto a CM5 chip, respectively, and then different gradient concentrations (5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM) of IgBD-Z were applied. PD-L1 and (5 nM, 10 nM, 25 nM, 50 nM, 100 nM) mIgG-IgBD-Z PD-L1 The coupling solution was used as the mobile phase and flowed through the chip. The binding and dissociation processes were monitored and analyzed on an OpenSPR system (Nicoya Lifesciences Inc., Kitchener, Canada). Results ( Figure 3 As shown in Figure C), this invention can form a stable conjugate with mIgG. SPR results also demonstrate that IgBD-Z... PD-L1 ( Figure 3 (C) and mIgG-IgBD-Z PD-L1 ( Figure 3 Both B and C can bind to the PD-L1 protein coated on the CM5 chip.

[0061] Example 3 IgBD-Z PD-L1 In vivo activity study

[0062] To investigate the in vivo tumor targeting of this invention, female C57BL / 6 mice (14-16 g) were subcutaneously inoculated with MC38 tumor cells (1×10⁻⁶). 6 Establish a tumor-bearing mouse model (number of mice per mouse), and record the length (L) and width (W) of the tumor daily, according to V=L×W. 2 The / 2 formula is used to calculate tumor volume (V).

[0063] The volume reaches approximately 200 mm 3 At that time, some tumor-bearing mice were directly anesthetized and sacrificed to obtain tumor tissue. Frozen sections were incubated with PD-L1 antibody and immunofluorescence staining was performed to analyze the expression of PD-L1 in the tumor tissue, verifying the high positive expression of PD-L1 in MC38 at the tissue level. Figure 4 DAPI / PD-L1 / Isotype); another group of tumor-bearing mice were injected with FAM-IgBD-Z via the tail vein. PD-L1 After administering (5 mg / kg), tumor tissue was collected for immunofluorescence staining to investigate the targeted enrichment and distribution of the present invention within the tumor tissue. The results showed that the tumor tissue exhibited a significant PD-L1 red fluorescence signal, which was similar to that of FAM-IgBD-Z. PD-L1 The green fluorescent signal overlaps ( Figure 4 DAPI / PD-L1 / lgBD-Z PD-L1 This indicates that the present invention can effectively reach tumor tissue sites and bind to the membrane ligand PD-L1 to exert a tumor-killing effect.

[0064] Following the same model construction method described above, a batch of tumor-bearing mouse models were established, divided into IgBD-Z. PD-L1 Processing group, Z PD-L1 Treatment group and PBS treatment group (n=6). The tumor volume of MC38 tumor-bearing mice to be subcutaneously transplanted reached approximately 70 mm. 3 At that time, for IgBD-Z PD-L1 The treatment group received IgBD-Z via tail vein injection every three days. PD-L1 (5 mg / kg), Z PD-L1 The treatment group received an equimolar dose of Z via tail vein injection every three days. PD-L1 The PBS-treated group received the same volume of PBS via tail vein injection every three days. PD-L1 The preparation method of IgBD-Z PD-L1 Similarly, ZM1 utilizes disulfide bonds to form a dimer. The optimized fusion expression gene sequence was cloned into the pQE30 plasmid via BamHI and SalI restriction sites. The expression plasmid was then transformed into competent M15 E. coli and inoculated into LB medium containing Amp and Kan for expansion culture (37°C, 220 rpm). Once the bacterial culture A... 600nmWhen the value reaches 0.6-0.8, add 0.1 mM IPTG, induce expression overnight at 28℃ and 150 rpm. After the induction, centrifuge to collect the bacterial cells, resuspend them in lysis buffer, add PMSF to the working concentration of 1 mM, and sonicate to break down the bacteria. After the induction is complete, centrifuge at high speed to collect the supernatant, add high affinity Ni-NTA resin gel to purify and collect the product.

[0065] Tumor size and mouse weight were monitored and recorded daily to investigate the in vivo antitumor immunotherapy effect of this invention. Results showed that, compared to Z... PD-L1 The tumor-suppressing effect of the present invention is better. Figure 5 (A), and after tumor removal, it was also proven that this invention can effectively treat tumors, reducing tumor weight more quickly. Figure 5 In the middle C, the tumor volume reduction trend was more significant ( Figure 5 (D), and the mice did not experience a significant decrease in body weight during treatment, indicating that the present invention has good short-term biocompatibility. Figure 5 (B)

[0066] In summary, this embodiment established an MC38 colon cancer tumor model to validate the long-acting dimer affinity derivative IgBD-Z. PD-L1 The anti-tumor effect of IgBD-Z was observed. PD-L1 As an anti-tumor drug, it can effectively inhibit tumor growth, while the short-acting dimer affinity Z PD-L1 While immunotherapy also has some tumor-suppressing effects, its anti-tumor efficacy is not as strong as that of IgBD-Z provided in this invention. PD-L1 Its excellence.

[0067] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A long-acting PD-L1 specific affibody, characterized in that, A dimer of IgBD-ZM1, wherein the amino acid sequence of IgBD-ZM1 is shown as SEQ ID NO. 8; The preparation method of the PD-L1 specific affimer comprises the following steps: The C terminal of IgBD is fused to the N terminal of the affimer ZM1 through a connecting peptide, and the IgBD-ZM1 dimer is constructed through the disulfide bond formed by the cysteine residue at the C terminal of ZM1, while the HE tag is introduced at the N terminal of IgBD-ZM1, the optimized fusion expression gene sequence is cloned into a plasmid, an expression plasmid is constructed, the expression plasmid is transformed into bacterial competence, an expression strain is constructed and cultured, IPTG is added to induce expression, the supernatant is collected after the bacteria are broken, and the target protein is obtained after purification; The amino acid sequence of IgBD is shown as SEQ ID NO. 2, and the amino acid sequence of ZM1 is shown as SEQ ID NO.

6. The connecting peptide is (G4S)3, and the amino acid sequence of (G4S)3 is shown as SEQ ID NO.

4.

2. The use of the PD-L1 specific affimer of claim 1 in the preparation of an antitumor drug, characterized in that, The antitumor drug is an anticancer drug.

3. Use according to claim 2, wherein the compound is ###0002### The antibody drug conjugate is prepared by using the PD-L1 specific affimer to combine with IgG.

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