A nanobody against PD-L1 protein and its application

Abstract

This invention discloses an anti-PD-L1 protein nanobody and its applications. The inventors obtained an anti-PD-L1 protein nanobody with good binding affinity to the PD-L1 molecule through screening. The CDR region of this nanobody includes GYTISRNS, IENSDGST, AAPKVGQFSDIALGHLAFMTLPALNY, or their conserved substitution sequences. Due to the stronger antigen-antibody binding affinity, it exhibits higher clearance efficiency for specific PD-L1 proteins compared to immunoadsorbents synthesized from the receptor PD-1, reducing immune escape and enhancing the targeted killing effect of NK cells, thus providing a new therapeutic approach for cancer immunotherapy. It can also be used for the detection of PD-L1 proteins.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nanobody against PD-L1 protein and its application. Background Technology

[0002] In 1993, Hamers' research report mentioned the first discovery of a special type of antibody in alpacas, called heavy chain antibody or VHH antibody. These antibodies lack the common light chain and rely solely on variable regions (VHHs) of the heavy chain for antigen binding. VHH antibodies are the smallest functionally complete antibody molecule fragments to date, composed of four framework regions (FR1–FR4) and three complementarity-determining regions (CDR1–CDR3). Their relative molecular mass is approximately 15 kDa, only one-tenth that of conventional antibodies. This antibody molecule is 4.8 nm high and approximately 2.2 nm in diameter, hence the name nanobody. Nanobodies possess many advantages, such as small molecular weight, high physical stability, and ease of expression. Furthermore, nanobodies do not easily aggregate or precipitate and retain their ability to bind to antigens.

[0003] PD-1 (programmed death molecule 1) is an immunosuppressive receptor belonging to the CD28 superfamily of T-cell regulatory receptors. The PD-1 gene is located on chromosome 2q37.3 and encodes a 288-amino acid transmembrane protein. Its structure includes an extracellular region, a transmembrane region, and a cytoplasmic region, with the extracellular region containing an important IgV-like domain and four N-linked glycosylation sites. PD-1 differs in its amino acid sequence from CD28, CTLA-4, and inducible T-cell costimulatory factors, thus exhibiting highly specific ligand binding. PD-1 expression is widely distributed in immune cells such as T lymphocytes, natural killer cells, B lymphocytes, macrophages, dendritic cells, and monocytes, with particularly high expression on tumor-specific T cells. PD-1 transcription is regulated by various transcription factors, such as NFAT, NOTCH, FOXM1, and IRF9. Overexpression of PD-1 plays an immunosuppressive role in tumors, chronic infections, and other immune-related diseases.

[0004] PD-L1 (programmed death-ligand 1) and PD-L2 (programmed death-ligand 2) are two ligands of PD-1, with PD-L1 being the major ligand encoded by the PD-L1 gene. PD-L1, a type 1 transmembrane protein, is encoded by seven exons. PD-L1 expression can be constitutive or inducible. Constitutive low-level expression of PD-L1 is present in resting lymphocytes, antigen-presenting cells, and certain tissues, and helps maintain tissue homeostasis. In cases of inflammation or infection, PD-L1 can be induced to express on immune cells such as hematopoietic cells, endothelial cells, and epithelial cells. PD-L1 expression is influenced by various factors, such as signaling pathways like TLR and IFN-γ. Overexpression of PD-L1 plays a role in evading the immune response in tumors. PD-L1 promotes tumorigenesis in cancer cells by binding to its receptor and activating proliferative and survival signaling pathways.

[0005] After PD-L1 on the surface of tumor cells binds to PD-1 receptors on the surface of T cells, it can inhibit T cell activity, arresting them in the G0 / G1 phase, thereby inhibiting T cell proliferation and inducing T cell apoptosis, leading to tumor immune escape. PD-1 / PD-L1 inhibitors can block the binding of PD-1 and PD-L1, restoring T cell activity and achieving immunosuppression of tumor cells. Although PD-1 / PD-L1 inhibitors have been used clinically to treat various tumors, they still face problems such as low efficacy and the induction of autoimmune diseases.

[0006] In recent years, an increasing number of studies have found high concentrations of soluble PD-L1 in the plasma of patients with various cancers, and that this PD-L1 can exert the same immunosuppressive function as PD-L1 on the cell membrane surface. This may be one reason for the low response rate of PD-1 / PD-L1 therapy. Immune cells exposed to tumor cells that do not contain PD-L1 can be reactivated and can travel to distant sites to attack PD-L1-secreting tumor cells. They can also induce a strong memory response in T cells, inhibiting the growth of PD-L1-secreting tumor cells.

[0007] CN115433281A discloses a humanized nanobody against PD-L1. The results show that the affinities of nanobodies NB43, M2, M5 and M7 are 6.772 nM, 2.667 nM, 1.047 nM and 19.84 nM, respectively.

[0008] Developing a nanobody that combats PD-L1 is of great significance. Summary of the Invention

[0009] The purpose of this invention is to overcome at least one deficiency of the prior art and provide a nanobody of PD-L1 protein and its application.

[0010] The technical solution adopted in this invention is: The first aspect of the present invention provides: A nanobody against PD-L1 protein includes a framework region and a complementation-determining region, wherein the amino acid sequence of the complementation-determining region CDR1–CDR3 is as follows: GYTISRNS CDR2: IENSDGST CDR3: AAPKVGQVSRTVLGHLAFMTLPALNY .

[0011] In some examples of nanobodies, the amino acid sequences of their framework regions FR1–FR4 are as follows: FR1: VQLVESGGGQVQAAGGSLKLSCQAS, FR2: MGWFRQAPGKQREGVVA, FR3: TYADSVKGRFTISLGNAAKNTLYLEMNSLKPEDTAMYC, FR4: WGQGTQVTVSS.

[0012] In some examples of nanobodies, the amino acid sequence is as follows: MDVQLVESGGGQVQAAGGSLKLSCQAS GYTISRNS MGWFRQAPGKQREGVVA IENSDGST TYADSVKGRFTISLGNAAKNTLYLEMNSLKPEDTAMYC AAPKVGQVSRTVLGHLAFMTLPALNY WGQGTQVTVSSHHHHHH.

[0013] A second aspect of the present invention provides: A gene encoding the nanobody described in the first aspect of the present invention.

[0014] In some gene instances, codon optimization is performed based on the different expression systems.

[0015] In some examples of genes, the nucleotide sequence is as follows: CCATGGATGTGCAGCTGGTGGAAAGCGGCGGCGGCCAGGTGCAGGCGGCGGGCGGCAGCCTGAAACTGAGCTGCCAGGCGAGCGGCTATACCATTAGCCGCAACAGCATGGGCTGGTTTCGCCAGGCGCCGGGCAAACAGCGCGAAGGCGTGGTGGCGATTGAAAACAGCGATGGCAGCACCACCTATGCGGATAGCGTGAAAGGCCGCTTTACCAT TAGCCTGGGCAACGCGGCGAAAAACACCCTGTATCTGGAAATGAACAGCCTGAAACCGGAAGATACCGCGATGTATTGCGCGGCCGCGAAAGTGGGCCAGGTGAGCCGCACCGTGCTGGGCCATCTGGCGTTTATGACCCTGCCGGCGCTGAACTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCCACCACCACCACCACCACTGACTCGAG.

[0016] A third aspect of the present invention provides: An expression system in which the gene described in the second aspect of the present invention is inserted.

[0017] In some instances, the expression system is either a prokaryotic or eukaryotic expression system.

[0018] In some examples of expression systems, the prokaryotic expression system is Escherichia coli.

[0019] In some examples of expression systems, the eukaryotic expression system is CHO cells.

[0020] A fourth aspect of the present invention provides: The application of the nanobody described in the first aspect of the present invention in the preparation of PD-1 ligand immunosorbent.

[0021] In some application examples, the PD-1 ligand includes, but is not limited to, the PD-L1 molecule.

[0022] In some application examples, the immunoadsorbent is used to treat tumor suppression caused by soluble PD-1 ligands.

[0023] In some application examples, the tumors include, but are not limited to, clear cell renal cell carcinoma, multiple myeloma, melanoma, non-small cell lung cancer, gastric cancer, head and neck cancer, non-small cell lung cancer, pancreatic ductal adenocarcinoma, and glioblastoma.

[0024] A fifth aspect of the present invention provides: An immunoadsorbent comprising a solid support on which the nanobody described in the first aspect of the present invention is coupled.

[0025] In some examples of immunoadsorbents, the solid support is selected from at least one of chitosan, agarose, cellulose, dextran, resin, and cellulose.

[0026] In some examples of immunoadsorbents, other immunoadsorbent proteins are also coupled to the solid support.

[0027] In some examples of immunoadsorbents, NHS activation is used for coupling.

[0028] In some examples of immunoadsorbents, the coupling step is as follows: 1) The nanobody solution was dialyzed for 2 h using a coupling buffer B solution with pH=8.0-8.5 containing 0.1M NaHCO3 and 0.5M NaCl; 2) Rinse the NHS pre-activated packing material with pre-cooled 1mM HCl coupling solution A. After rinsing, add 0.5 times the packing material volume of coupling solution A, then add 1.5 times the packing material volume of nanobody solution. After shaking and incubation, add 2 times the packing material volume of sealing solution (0.1M Tris-HCl, pH=8.3), shake and seal, and then dry. 3) After drying, add 0.1M Tris-HCl (pH=8-9) and 0.2M acetic acid (pH=3-6) to the packing material and wash alternately. Then add PBS solution to wash the packing material, dry it, and store it in 20% ethanol.

[0029] A sixth aspect of the present invention provides: A blood purification device comprising the immunoadsorbent described in the fifth aspect of this invention.

[0030] The seventh aspect of the present invention provides: A PD-L1 protein detection reagent, comprising the nanobody described in the first aspect of the present invention.

[0031] The beneficial effects of this invention are: The anti-PD-L1 protein nanobodies of some examples of this invention exhibit good binding affinity to free soluble PD-L1 molecules and can be used to adsorb and remove free soluble PD-L1 molecules from the blood of cancer patients. Due to the stronger binding affinity between the antigen and antibody, they demonstrate higher clearance efficiency for specific PD-L1 proteins compared to immunoadsorbents synthesized from receptor PD-1, providing a new therapeutic approach for cancer immunotherapy. They can also be used for the detection of PD-L1 proteins.

[0032] The immunosorbents and blood purification devices of some examples of the present invention can simultaneously remove free soluble PD-L1 molecules from the serum of cancer patients through blood purification, thereby improving the clearance rate, reducing immune escape, and enhancing the targeted killing effect of NK cells. Attached Figure Description

[0033] Figure 1 This is an electrophoresis image of the purified anti-PD-L1 protein nanobody.

[0034] Figure 2 This is the ELISA result of the anti-PD-L1 protein nanobody. Detailed Implementation

[0035] The technical solution of the present invention will be further illustrated below with examples.

[0036] Example 1: Establishment of an anti-PD-L1 protein alpaca phage antibody library

[0037] The PD-L1 protein with a His tag was expressed in eukaryotes and transfected into C1R cells (human B lymphoblasts). Peripheral blood mononuclear cells (PBMCs) were obtained using six immunized alpacas. RNA was then extracted from these cells and reverse transcribed into cDNA. After PCR amplification, the product was cleaved using HindIII and NotI enzymes and ligated into the phage vector pHIAT-1. This recombinant vector was then transformed into *E. coli* TG1 to construct the initial phage library.

[0038] After bacterial strain TG1 reached the logarithmic growth phase, helper phage M13KO7 was introduced into the culture system. After overnight incubation, the supernatant was collected by centrifugation. Subsequently, phage particles were precipitated using polyethylene glycol (PEG), washed with PBS, and filtered through a 0.45 μm filter for sterilization, thereby obtaining the VHH antibody library. The antibody library had a capacity of 4.5 × 10⁻⁶. 12 This provides abundant antibody resources for subsequent experiments.

[0039] Example 2: Screening of monoclonal phage antibodies binding to anti-PD-L1 protein

[0040] The titer of helper phage M13KO7 was determined to be 1 × 10⁻⁶. 12 pfu / ml. The TG1 strain was infected using this helper phage M13KO7.

[0041] First round of screening: First, anti-PD-L1 protein was coated onto ELISA plates at a concentration of 2 µg per well and incubated overnight at 4°C. The plates were then washed and blocked with 3% BSA. 100 µL of phage library solution was added to each well, and the plates were incubated at 37°C for 2 hours, followed by 6 washes. Elution was then performed with glycine-buffered saline (gly-HCl) followed by neutralization with Tris-HCl buffer. 10 μL of the eluent was used for titer determination, and the remaining eluent was added to 5 mL of TG1 bacteria in logarithmic growth phase. These bacteria were then infected and incubated at 37°C for 30 min, followed by the addition of preheated 2×YT medium to a total volume of 10 mL. The plates were incubated at 37°C and 250 rpm for another 30 min, then ampicillin was added to a final concentration of 100 μg / mL, and the plates were incubated for another 2 hours. Subsequently, kanamycin was added to achieve a final concentration of 70 μg / mL, and the mixture was incubated overnight. After centrifugation at 4000 rpm for 15 minutes at 4°C, the supernatant was collected. The supernatant was mixed with 5 mL of PEG / NaCl and incubated on ice for 1 h. Then, it was centrifuged at 9000 rpm for 20 minutes at 4°C, and the supernatant was discarded. Finally, the mixture was resuspended in PBS buffer and centrifuged to remove cell debris, yielding phage antibody particles.

[0042] Second round of screening: Anti-PD-L1 protein was coated onto each ELISA plate, 2µg / well; the remaining procedures were the same as in the first round of screening.

[0043] Third round of screening: Coat the ELISA plate with anti-PD-L1 protein, 2µg / well; the remaining operations are the same as the first round of screening.

[0044] After screening, the selected bacteriophages were used to infect TG1 bacteria, and these bacteria were plated in culture dishes. Single clones were randomly selected from the cultured plates, using the original library as a negative control. Preliminary positive clones with an OD450 value more than twice that of the original library were screened using ELISA. After sequencing, the sequencing results were deduplicated to obtain a series of different sequences. Six of the best-performing anti-PD-L1 protein nanobody sequences were selected and named No. 1 to No. 4.

[0045] The amino acid sequence is as follows: No.1 MD VQLVESGGGQVQAAGGSLKLSCQASGYTISRNSMGWFRQAPGKQREGVVAIENSDGSTTYADSVKG RFTISLGNAAKNTLYLEMNSLKPEDTAMYCAAPKVGQVSRTVLGHLAFMTLPALNYWGQGTQVTVSS HHHHHH (SEQ ID NO.: 18) No.2 MD QVQLVESGGGQVQAAGGSLKLSCQASGYTISRNSMGWFRQAPGKQREGVVAIENSDSTTYADSVKG RFTISLGNAAKNTLYLEMNSLKPEDTAMYCAAPKVGQFSDIALGHLAFMTLPALNYWGQGTQVTVSSHHHHHH (SEQ ID NO.: 19) No. 3 M VQLVESGGGQVQAAGGSLKLSCQASGYTASMGWFRQAPGKEREAVAIENSDSTTYADSVRGRFTISL GNAAKNTLYLQINSLKPEDTAVYYCAARDDDYAFLSRGARDFRYWGQGTQVTVSS HHHHHH (SEQ ID NO.: 20) No. 4 M VQLQESGGGSVQAGGSLRLSCAASRFTASMGWFRQAPGKEREGIATVSGAASTNYADSVRGRFTISK DNAKNTLYLQINSLKPEDTAVYYCAASSTSTYAYYCAARDNYYAFLSRGARDFRYWGQGTQVTVSS HHHHHH (SEQ ID NO.: 21) In the above amino acid sequences, the underlined parts are complementation-determining regions (CDRs) and framework regions (FRs), and the amino acid sequences of each CDR and FR are shown in Table 1.

[0046] Table 1. Structural analysis of different nanobodies 1-FR1 VQLVESGGGQVQAAGGSLKLSCQAS 1 1-CDR1 GYTISRNS 2 1-FR2 MGWFRQAPGKQREGVVA 3 1-CDR2 IENSDGST 4 1-FR3 TYADSVKGRFTISLGNAAKNTLYLEMNSLKPEDTAMYC 5 1-CDR3 AAPKVGQVSRTVLGHLAFMTLPALNY 6 1-FR4 WGQGTQVTVSS 7 2-FR1 VQLVESGGGQVQAAGGSLKLSCQAS 1 2-CDR1 GYTISRNS 2 2-FR2 MGWFRQAPGKQREGVVA 3 2-CDR2 IENSDST 8 2-FR3 TYADSVKGRFTISLGNAAKNTLYLEMNSLKPEDTAMYC 5 2-CDR3 AAPKVGQFSDIALGHLAFMTLPALNY 9 2-FR4 WGQGTQVTVSS 7 3-FR1 VQLQESGGGSVQAGGSLRLSCAAS 10 3-CDR1 GYTAS 11 3-FR2 MGWFRQAPGKEREGIAT 12 3-CDR2 IENSDST 8 3-FR3 NYADSVRGRFTISKDNAKNTLYLQINSLKPEDTAVYYC 13 3-CDR3 AARDDDYAFLSRGARDFRY 14 3-FR4 WGQGTQVTVSS 7 4-FR1 VQLQESGGGSVQAGGSLRLSCAAS 10 4-CDR1 RFTAS 15 4-FR2 MGWFRQAPGKEREGIAT 12 4-CDR2 VSGAAST 16 4-FR3 NYADSVRGRFTISKDNAKNTLYLQINSLKPEDTAVYYC 13 4-CDR3 AASSTSTYAYYCAARDNYYAFLSRGARDFRY 17 4-FR4 WGQGTQVTVSS 7 Its nucleotide sequence is as follows: No.1-DNA sequence CCATGGATGTGCAGCTGGTGGAAAGCGGCGGCGGCCAGGTGCAGGCGGCGGGCGGCAGCCTGAAACTGAGCTGCCAGGCGAGCGGCTATACCATTAGCCGCAACAGCATGGGCTGGTTTCGCCAGGCGCCGGGCAAACAGCGCGAAGGCGTGGTGGCGATTGAAAACAGCGATGGCAGCACCACCTATGCGGATAGCGTGAAAGGCCGCTTTACCAT TAGCCTGGGCAACGCGGCGAAAAACACCCTGTATCTGGAAATGAACAGCCTGAAACCGGAAGATACCGCGATGTATTGCGCGGCCGCGAAAGTGGGCCAGGTGAGCCGCACCGTGCTGGGCCATCTGGCGTTTATGACCCTGCCGGCGCTGAACTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCCACCACCACCACCACCACTGACTCGAG (SEQ ID NO.: 22) No.2-DNA sequence CCATGGATCAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCAGGTGCAGGCGGCGGGCGGCAGCCTGAAACTGAGCTGCCAGGCGAGCGGCTATACCATTAGCCGCAACAGCATGGGCTGGTTTCGCCAGGCGCCGGGCAAACAGCGCGAAGGCGTGGTGGCGATTGAAAACAGCGATAGCACCACCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCTGGGCAACGCGGCGAAAAACACCCTGTATCTGGAAATGAACAGCCTGAAACCGGAAGATACCGCGATGTATTGCGCGGCGCCGAAAGTGGGCCAGTTTAGCGATATTGCGCTGGGCCATCTGGCGTTTATGACCCTGCCGGCGCTGAACTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCCACCACCACCACCACCACTGACTCGAG (SEQ ID NO.: 23) No.3 DNA Sequence CCATGGTGCAGCTGGTGGAAAGCGGCGGCGGCCAGGTGCAGGCGGCGGGCGGCAGCCTGAAACTGAGCTGCCAGGCGAGCGGCTATACCGCGAGCATGGGCTGGTTTCGCCAGGCGCCGGGCAAAGAACGCGAAGCGGTGGCGATTGAAAACAGCGATAGCACCACCTATGCGGATAGCGTGCGCGGCCGCTTTACCATTAGCCTGGGCAACGCGGCGAAAAACACCCTGTATCTGCAGATTAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCGCGCGATGATGATTATGCGTTTCTGAGCCGCGGCGCGCGCGATTTTCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCCACCACCACCACCACCACTGACTCGAG (SEQ ID NO.: 24) No.4 DNA Sequence CCATGGTGCAGCTGCAGGAAAGCGGCGGCGGCAGCGTGCAGGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCCGCTTTACCGCGAGCATGGGCTGGTTTCGCCAGGCGCCGGGCAAAGAACGCGAAGGCATTGCGACCGTGAGCGGCGCGGCGAGCACCAACTATGCGGATAGCGTGCGCGGCCGCTTTACCATTAGCAAAGATAACGCG AAAAACACCCTGTATCTGCAGATTAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCGAGCAGCACCAGCACCTATGCGTATTATTGCGCGGCGCGCGATAACTATTATGCGTTTCTGAGCCGCGGCGCGCGATTTTCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCCACCACCACCACCACCACTGACTCGAG (SEQ ID NO.: 25) The above nucleotide sequence is translated starting from ATG in the CCATGG restriction site, and the last six nucleotides are the restriction sites required to construct the circular plasmid vector.

[0047] Example 3: In vitro expression and purification of nanobodies

[0048] The gene sequences of anti-PD-L1 protein nanobodies No. 1–No. 4 were transformed into the PET28 plasmid via NcoI and XhoI restriction sites, respectively, and expressed in *E. coli* BL21(DE3). After expansion culture in LB medium containing 70 µg / mL kanamycin, the cells were collected, sonicated (5 s on, 10 s off, working time 20 min), and centrifuged at 10,000 rpm for 10 min. The supernatant was collected. Purification was performed using a His-tagged nickel ion chelate packing material, and the elution peak was collected to obtain the anti-PD-L1 protein alpaca nanobodies. The expression level of the nanobodies was determined to be 358 mg / L.

[0049] See purified protein Figure 1 SDS-PAGE analysis revealed that the molecular weight of anti-PD-L1 protein nanobody No. 1 was 15 kDa. The amino acid sequences of the remaining three anti-PD-L1 protein nanobodies were expressed and purified using the same method.

[0050] Example 4: Detection of the binding activity of nanobodies to PD-L1 protein

[0051] S1) Coat PD-L1 protein onto an ELISA plate, dilute the protein concentration to 5 μg / ml with coating buffer (0.1M PBS, pH=7.4), and then add 100 μL / well. Incubate overnight at 4°C. S2) The next day, discard the buffer solution in the wells, pat dry with absorbent paper, and add 250 μL of ELISA washing buffer (0.05% Tween 20 / 0.1M PBS) to each well again, and wash the plate 3 times; S3) Block with 5% BSA, incubate at 37°C for 2 h, and wash the plate 3 times; S4) After washing the plate, add four anti-PD-L1 nanobody proteins to each well. Each nanobody is diluted at different concentrations (2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL..., 3.9 pg / mL). Incubate at 37°C for 2 h and wash the plate 3 times. S5) After washing the plate, add the anti-His tag HRP-labeled antibody at a ratio of 1:5000, add 100 μL / well, and then incubate at 37℃ for 1 h. Wash the plate 6 times. S6) After washing the plate, add 100 μL of TMB colorimetric solution to each well and react at 37°C in the dark for 10-15 min. Terminate the reaction when the wells of the experimental group turn blue and there is no obvious color change in the wells of the blank and negative control groups. S7) Add 100 μL of 1M H2SO4 stop solution to terminate the colorimetric reaction, and use an ELISA reader to detect the absorbance at a wavelength of 450 nm.

[0052] ELISA results showed that all four anti-PD-L1 protein nanobodies had binding activity to the PD-L1 protein. Figure 2 The anti-PD-L1 protein nanobody with the highest binding activity was No. 4.

[0053] Example 5: Synthesis and Adsorption Performance Determination of Immunosorbents

[0054] S1) Take 5 mL of iodoacetyl preactivated agarose packing, rinse the packing with about 30 mL of coupling solution (50 mM Tris, 5 mM EDTA-Na, pH 8.5), and then dry it. S2) Add 5 mL of antibody solution (No.1, No.2, No.3 and No.4) with a concentration of 10 mg / mL respectively, mix thoroughly, transfer to centrifuge tubes, and incubate at 28℃ and 120 rpm for 1 h with shaking. S3) After the reaction is complete, drain the solution and then rinse the packing with 3 column volumes of coupling solution (50 mM Tris, 5 mM EDTA-Na, pH 8.5) and then dry it. S4) Add 5 mL of blocking buffer (50 mM Tris, 5 mM EDTA-Na, 50 mM L-cysteine, pH 8.5), mix thoroughly, transfer to a centrifuge tube, and incubate at 28°C with shaking for 1 h; finally, drain the blocking buffer, wash the packing material with 3 column volumes of pH 8.0, 20 mM PBS, and store with 20% ethanol. S5) Load 1 mL of the above-mentioned synthetic packing material and 2 mL of PD-L1 protein solution (self-made) into a 10 mL EP tube, respectively, and contact at 28℃ and 100 rpm for 1-2 h. Then place the packing material in a disposable affinity chromatography column and remove the packing material from the vacuum. S6) The change in PD-L1 content in the supernatant before and after adsorption was detected using the PD-L1 Human ProcartaPlex™ Simplex Kit. Blank agarose GE Sepharose 6FF was used as a control.

[0055] S7) Calculation Adsorption performance = (content before adsorption - content after adsorption) / packing volume Scavenging rate = 1 - (content before adsorption - content after adsorption) / content before adsorption The adsorption results are shown in Table 2.

[0056] Table 2. Performance determination of the synthetic adsorbent for PD-L1. No.1 100 21.6 78.4 78.4% No.2 100 31.3 68.7 68.7% No.3 100 15.2 84.8 84.8% No.4 100 13.6 86.4 86.4% The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A nanobody against PD-L1 protein, comprising a framework region and a complementarity-determining region, characterized in that, The amino acid sequences of its complementarity-determining regions CDR1 to CDR3 are as follows: CDR1: GYTISRNS, CDR2: IENSDGST, CDR3: AAPKVGQVSRTVLGHLAFMTLPALNY.

2. The nanobody according to claim 1, characterized in that, The amino acid sequences of its framework regions FR1 to FR4 are as follows: FR1: VQLVESGGGQVQAAGGSLKLSCQAS, FR2: MGWFRQAPGKQREGVVA, FR3: TYADSVKGRFTISLGNAAKNTLYLEMNSLKPEDTAMYC, FR4: WGQGTQVTVSS.

3. A gene encoding the nanobody as described in claim 1 or 2.

4. An expression system, characterized in that, The gene described in claim 3 is inserted.

5. The application of the nanobody according to claim 1 or 2 in the preparation of PD-1 ligand immunoadsorbent, wherein the PD-1 ligand is a PD-L1 molecule.

6. An immunoadsorbent comprising a solid-phase support, characterized in that, The solid-phase support is coupled with the nanobody as described in claim 1 or 2.

7. The immunoadsorbent according to claim 6, characterized in that, The solid support is selected from at least one of chitosan, agarose, cellulose, dextran, and resin.

8. The immunoadsorbent according to claim 6, characterized in that, Other immunoadsorption proteins are also coupled to the solid support.

9. A blood purification device, characterized in that, It includes the immunoadsorbent as described in claim 6.

10. A PD-L1 protein detection reagent, characterized in that, Including the nanobody as described in claim 1 or 2.

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