Method for enhancing in-vivo half-life period of PD-L1 nano antibody

Abstract

The invention discloses a C3Fab-fused anti-PD-L1 nano antibody, belongs to the field of nano antibody tumor immunotherapy, and relates to a protein engineering technology. The preparation method comprises the following steps: constructing a PD-L1 nano antibody (NbP3C8-C3Fab) fused with C3Fab; carrying out prokaryotic expression and purification on the NbP3C8-C3Fab (NbP3C8-C3Fab); the combination of the NbP3C8-C3Fab and the antigen PD-L1 is detected by enzyme-linked immunosorbent assay (ELISA); the capacity of NbP3C8-C3Fab for collecting mouse IgG (immunoglobulin G) is detected by ELISA (enzyme-linked immunosorbent assay); carrying out cell detection on the combination of the NbP3C8-C3Fab and the cells for expressing the PD-L1; and determining the plasma half-life period of the NbP3C8-C3Fab in the mouse. The fusion nano-antibody can maintain the advantages of the nano-antibody and restore the anti-tumor effect of the nano-antibody at the same time; moreover, the plasma half-life period of the nano antibody is prolonged, so that the nano antibody has more application possibilities in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of nano-antibody tumor immunotherapy, is a protein engineering technology, and particularly relates to a method capable of enhancing the in vivo half-life of PD-L1 nano-antibody. Background Art

[0002] As a new type of biological molecule for tumor treatment, nanoantibodies have attracted much attention in recent years. Nanoantibodies, also known as single-domain antibodies, are single-domain antibodies (VHH) derived from the heavy chain variable region of camelid IgG. They are the smallest unit molecules known to bind to target antigens. Although their molecular weight is only one-tenth of that of traditional antibodies, nanoantibodies still retain complete antigen-binding ability. Compared with traditional mAbs, nanobodies have many unique advantages: high thermal stability and high tolerance to acid and alkali; the CDR3 of nanobodies often has 6-7 more amino acids than the CDR3 of human VH, which makes the CDR3 of nanobodies have a more expansive convex loop structure, which can target antigenic epitopes that traditional antibodies cannot reach, and even some hidden epitopes; in addition, the four amino acids V37, C44, L45 and W47 on the human VH are mutated to F37, E44, R45 and G47 in nanobodies, which increases the number of hydrophilic amino acids, making nanobodies more water-soluble. In addition to the above properties, nanobodies are also superior to traditional antibodies in some other aspects, including: easy to achieve efficient expression in prokaryotic or eukaryotic systems; easy to modify and transform the structure; stronger tumor and tissue penetration; better stability; can tolerate extreme conditions and have lower immunogenicity; a variety of screening methods, in addition to traditional immunization methods, use natural antibody libraries and in vitro synthesized antibody libraries to prepare nanobodies. These advantages make nanobodies a promising alternative to traditional mAbs.

[0003] Endogenous antibodies are natural antibodies produced by humans when they come into contact with non-self antigens in life, including IgM and IgG, which have a long half-life in human blood. Recent studies have shown that recruiting endogenous antibodies to the surface of tumor cells can activate the immune system to recognize tumor cells and kill tumor cells through the mechanism mediated by their Fc end. By recruiting endogenous antibodies, not only can the anti-tumor effect be exerted, but also the plasma half-life of small molecule protein drugs can be extended.

[0004] In recent years, research on immune checkpoint blockade has become a hot topic in tumor immunity. Immune checkpoint molecules are receptors on the surface of immune cells. After binding to ligands, they can forward inhibitory or stimulatory signals. In recent years, the most studied immune checkpoints are cytotoxic T lymphocyte-associated protein 4 (cTLA-4), programmed death receptor 1 (PD-1), programmed death ligand 1 (PD-L1), T cell immunoglobulin and mucin domain-containing-3 (TIM-3), and lymphocyte activation gene 3 (LAG-3). Related drugs have been developed for these immune checkpoints and have been approved by the FDA for the treatment of renal cell carcinoma, melanoma, lymphoma, typical Hodgkin lymphoma, pancreatic ductal adenocarcinoma, cervical cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. For example, mAbs targeting PD-1, such as nivolumab Pembrolizumab cemiplimab (Sanofi, Regeneron). mAbs targeting PD-L1, such as atezolizumab Avelumab and durvalumab The mAb targeting cTLA-4 is represented by ipilimumab. In recent years, new immunotherapy targets can enrich more treatment options.

[0005] Some small molecule protein drugs show advantages in treatment, but most small molecule protein drugs, namely therapeutic proteins, peptides and nanobodies. In order to maintain therapeutic levels, protein and peptide drugs usually require frequent injections, which may encounter patient compliance and tolerance issues. Improving pharmacokinetics is key to developing biopharmaceuticals. In particular, different proteins may have different physicochemical and biological properties; therefore, protein-targeted strategies may be needed to improve pharmacokinetics. Nanobodies have a small molecular weight, resulting in a very short terminal half-life, which is usually cleared from the blood very quickly by the kidneys within a few minutes to a few hours. Therefore, in order to maximize its effect, continuous or repeated administration is required at short intervals. Recruiting IgG domains for nanoantibody fusion is an effective way to extend the half-life of nanoantibodies.

[0006] The BL21 Escherichia coli prokaryotic expression system is a commonly used protein expression system. The BL21 strain has a high transcription and translation rate and can efficiently express the target protein in a short time. In addition, the BL21 strain lacks intrinsic cytoplasmic proteases, which is beneficial to improve the stability of the target protein. The BL21 strain has a good expression effect on soluble proteins. The commonly used vectors in its expression system, such as the pET system, provide a series of expression vectors and induction methods to achieve efficient expression of soluble proteins. The BL21 strain can also conveniently express target proteins containing in vitro binding structures (inclusion bodies). These structures can obtain their biological activity through protein resolubilization and refolding, and are suitable for the expression of some poorly soluble or complex proteins. The BL21 Escherichia coli expression system has extensive customizability and flexibility. Different expression vectors, promoters, selection markers and other expression regulatory elements can be selected according to different experimental purposes and needs to achieve the best expression effect and convenience of subsequent operations. BL21 Escherichia coli is a common laboratory strain that is relatively easy and economical to cultivate and maintain, without the need for expensive culture media and equipment. This makes the BL21 expression system a widely used protein expression tool.

[0007] At present, monoclonal antibodies are the main anti-tumor antibody drugs. Monoclonal antibodies are complex biological macromolecules that require post-translational modification, so they need to be expressed in eukaryotic cells and are relatively expensive to produce. In addition, monoclonal antibodies have poor penetration into tumor tissues. Therefore, nanobodies can be developed and applied as a substitute for monoclonal antibodies. The lack of Fc fragments and poor pharmacokinetics of ordinary nanobodies limit their clinical application. Summary of the invention

[0008] In view of some problems existing in the prior art, the present invention has developed a method for enhancing the in vivo half-life of PD-L1 nanobodies, providing new ideas for the application of nanobodies and a new optimization scheme for nanobodies as an anti-tumor method.

[0009] The method for enhancing the in vivo half-life of PD-L1 nanoantibodies is to add a modified protein to ordinary nanoantibodies. This domain can be used to enable the PD-L1 nanoantibody to acquire the ability to recruit endogenous IgG. After the nanoantibody binds to IgG, the in vivo half-life can be extended. The modification method is simple, the cost is relatively low, and the nanoantibody has high activity.

[0010] Preferably, the amino acid sequence of the nanobody modified by the related method involves three parts, the amino acid sequence of the nanobody targeting a specific site on the tumor cell between the BamHI and EcoRI restriction sites, the GS linker amino acid sequence part between the EcoRI and XhoI restriction sites, and the IgG Binding Domain amino acid sequence. According to the original PD-L1 nanobody sequence, the relevant primers are designed to amplify the base sequence of the anti-PD-L1 nanobody, and the restriction sites of the nanobody PD-L1 are changed to BamHI and EcoRI, and the target sequence is inserted into the pET21a plasmid carrying the IgBD C3Fab, and then the relevant protein is expressed and purified using the Escherichia coli BL21 prokaryotic expression system.

[0011] The specific steps include: 1. Determine the base fragment of the nanobody and the pET21a vector with C3Fab The base sequence of the PD-L1 nanobody was determined using the PD-L1 nanobody expression plasmid, and the restriction sites were designed to be BamHI, EcoRI, and XhoI, and a His tag was added to the C-terminus of the IgG Binding Domain. The base sequence of the PD-L1 nanobody was amplified using PCR amplification technology after the relevant primers were designed. The fragment was provided with BamHI and EcoRI restriction sites, and the PCR conditions were: The sequences in BamHI and EcoRI in the pET21a plasmid carrying the C3Fab fragment were removed by double digestion with BamHI and EcoRI to obtain the pET21a vector carrying the C3Fab fragment. 2. Insert the PD-L1 nanobody sequence into the pET21a vector with the C3Fab sequence. Use the BamHI and EcoRI restriction sites contained in the pET21a plasmid and the target sequence, and connect the two using restriction endonucleases, DNA ligases, and buffers. After double digestion with BamHI and EcoRI in the previous step, the pET21a plasmid has sticky ends with BamHI and EcoRI sites, and the PCR product after double digestion with BamHI and EcoRI, that is, the target sequence, obtains sticky ends. The digestion conditions are: 37°C, 2h (performed in a water bath). Both need to be purified using a PCR purification kit and quantified. After correct identification using agarose gel electrophoresis, use T4 ligase at a ratio of 3:1 between the target gene and the vector to connect the vector and the PCR product at 4°C. Obtain the recombinant plasmid, and verify the double digestion of BamHI and EcoRI and the double digestion of BamHI and XhoI. 3. Transform the recombinant plasmid into E. coli BL21 competent cells for prokaryotic expression The verified plasmid was transformed into BL21 competent cells by heat shock, and after expansion culture, it was inoculated into 2YT medium containing Amp antibiotics. After 4-5 hours, the inducer was added and the culture was shaken overnight; the induced bacterial liquid was collected and centrifuged to prepare EIRNbs crude protein; centrifuged at 8000rpm for 15 minutes, the supernatant was removed, frozen at -80℃ for 15 minutes, and thawed at room temperature for 15 minutes, and repeated freezing and thawing was repeated 3 times; TES sucrose solution was added to resuspend the bacteria, and placed at 4℃ for 4-6 hours; 4-fold diluted TES sucrose solution was added, mixed and incubated at 4℃ overnight; 2mL of pre-cooled PBS was added to every 100mL of bacterial liquid, and the prepared lysis solution was added. After ice bathing for 30 minutes, the bacteria were lysed by ultrasound; 12000rpm, centrifuged for 15 minutes, and the supernatant was collected; the supernatant after centrifugation was used to purify the nanoantibody carrying the His tag using a nickel ion metal chelate affinity chromatography medium (Ni-NTA) pre-loaded column. 20 mM imidazole was used to wash away impurities and 500 mM imidazole was used to elute the target protein. 15% SDS-PAGE gel electrophoresis was used to identify the target band. 4. Verify the ability of PD-L1 nanoantibodies to recruit IgG after modification using this method Indirect ELISA was used to detect the ability of linker protein to recruit IgG. The protein sample was diluted to 1 μg / mL and coated on a 96-well plate, 100 μL / well, overnight at 4°C; washed 4 times with PBST, added 200 μL of blocking solution and incubated at 37°C for 2 hours; washed 4 times with PBST, mouse IgG was diluted to different concentrations (20 μg / mL to 0.002 μg / mL) and mouse serum (1:10 to 1:1000) was diluted to 100 / well, and incubated at 37°C for 2 hours. Washed 4 times with PBST, patted dry, added 100 μL / well of 1:3000 diluted GoatAnti Mouse IgG HRP, incubated at 37°C for 1 hour; TMB 100 μL / well, stood in the dark for 8 minutes for color development, 50 μL of stop solution per well, and measured OD 450 nm. 5. Verify the ability of the modified PD-L1 nanobody to target PD-L1 antigen and PD-L1 positive cells The PD-L1 nanoantibodies modified by this method can be verified by indirect ELISA and cell ELISA using recombinant PD-L1 surface protein antigens and tumor cells positive for PD-L1 target sites. For the indirect ELISA detection of the binding ability of the modified PD-L1 nanobody with the PD-L1 antigen using this method, the antigen protein was diluted to 1 μg / mL and coated on a 96-well plate, 100 μL / well, overnight at 4°C; washed 4 times with PBST, added 200 μL of blocking solution and incubated at 37°C for 2 hours; washed 4 times with PBST, the purified nanobody was diluted to different concentrations (0.1 ng / mL to 10000 ng / mL) to 100 / well, incubated at 37°C for 1.5 hours; washed 4 times with PBST, patted dry, added 100 μL / well of 1:3000 diluted Anti-His Mouse mAb, incubated at 37°C for 2 hours; washed 4 times with PBST, patted dry, added 100 μL / well of 1:3000 diluted Goat Anti Mouse IgG HRP, incubated at 37°C for 1 hour; TMB 100 μL / well, stood in the dark for 8 minutes for color development, 50 μL of stop solution per well, and measured OD 450 nm. For the cell ELISA test, the modified PD-L1 nanoantibody was used to analyze the binding ability of the PD-L1 positive A549 tumor cells. The cell ELISA method was used to detect the binding affinity of NbP3C8-C3Fab with PD-L1+ cells. It was reported that the A569 lung cancer cell line expressed PD-L1 on the cell surface, so A569 cells were used to detect the binding of NbP3C8-C3Fab. The detection method is as follows: A569 cells were subcultured to the third to fourth generation and then digested, and the cells were plated and counted using a cell counting plate. 5×10 3 -1×10 4 Cells were plated in 96-well plates and cultured overnight in a 37°C incubator containing CO2. The next day, the plates were washed once with PBST, 100 μL of 5% paraformaldehyde was added to each well to fix the cells, and the formaldehyde was discarded after 15 minutes. The plates were washed with PBST, 3% BSA was added, and the plates were blocked at 37°C for 2 hours. After blocking, the plates were washed 5 times with PBST, and NbP3C8 and NbP3C8-C3Fab purified proteins with gradient concentrations (10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL) were added as primary antibodies, and incubated at 37°C for 1 hour. The diluent was 1% BSA. After washing the plates 5 times with PBST (3 minutes / time), anti-His mouse monoclonal antibody (1:1000 dilution) was added as secondary antibody and incubated at 37°C for 1 hour. After washing the plate 5 times with PBST, add goat anti-mouse IgG HRP tertiary antibody (1:4500 dilution) and incubate at 37°C for 1 hour. After washing the plate 5 times with PBST, add 100 μL TMB colorimetric solution and color for 5 to 15 minutes in the dark. Add 50 μL 2 mol sulfuric acid to terminate the reaction and detect OD with an enzyme reader. 450 The absorbance value is in nm. 6. Verify the tumor killing effect of PD-L1 nanoantibodies modified by this method The CC8K method was used to detect the CDC effector function of the modified PD-L1 nanobody. 4 × 10 4 Antigen-positive tumor cells of the second to third generations were cultured overnight; the supernatant was removed, and the cells were washed once with PBS, followed by the addition of 100 μL of nanoantibody linker protein of different concentrations (μg / mL) or PBS, and cultured at 37°C for 2 h; the cells were washed once with PBS, and 100 μL of 9% healthy human serum was added, and TritonX-100 added to a final concentration of 1% was used to achieve maximum cell killing, and the cells were treated at 37°C for 2 h; 10 μL of CCK8 was added, and the cells were cultured at 37°C for 1 h, and the absorbance at 450 nm was measured (each group of experiments was repeated three times, and the average value was taken); the cytotoxicity was calculated using the following formula: %CDC=100×((1-(absorbance of nanoantibody group-absorbance of Triton X-100 group) / (absorbance of PBS group-absorbance of Triton X-100 group)). 7. To verify the half-life extension effect of the PD-L1 nanobody modified by this method, the modified PD-L1 nanobody protein (2 mg / kg, 100 μL) was first injected into mice through the tail vein. The mice were clean-grade ICR mice (6-8 weeks old), and the mouse blood was obtained by orbital blood sampling; the whole blood of the mice was immediately placed at 37°C for 30 min; -4°C for 30 min, and centrifuged at 3500 rpm for 5 min; the supernatant was aspirated and stored at -20°C. The double antibody sandwich ELISA experiment can be used to make a protein half-life standard curve, and the standard curve can be used to calculate the concentration at different times: dilute Coating with Mice anti-His mAb at (1:1000) and coat overnight at 4°C; after washing with PBST, dilute the protein sample to a concentration of (0ng / ml-16384ng / ml) and incubate at 37°C for 2h; incubate rabbit anti-VHH monoclonal antibody (1:1000) and goat anti-rabbit IgG HRP (1:3000) at 37°C for 1h; after washing with PBST, add TMB as a substrate to react with horseradish peroxidase, add 50μL of 2M H2SO4 to terminate the reaction, and detect its absorbance at 450nm; obtain the standard curve by calculation. After the serum was diluted 100 with PBS, its absorbance was measured according to the method of establishing a standard curve, and the plasma half-life and clearance rate were obtained using PKsolver. GraphPad Prism 8.0 was used for statistical analysis and graph drawing, and all curves were fitted with 4-parameter Logistic fitting; one-way analysis of variance was used to compare multiple groups.

[0012] Compared with traditional tumor treatment schemes and traditional nano-antibodies, the beneficial effects of the present invention are as follows: (1) Using PD-L1 nanobodies with the ability to recruit IgG, it still maintains the unique properties of PD-L1 nanobodies, can bind to hidden special epitopes, highly bind to PD-L1 sites, and has good hydrophilicity; it is easy to achieve efficient expression in prokaryotic or eukaryotic systems; it is easy to modify and restructure; it has stronger tumor and tissue penetration ability; it has better stability; it can tolerate extreme conditions and has lower immunogenicity; (2) PD-L1 nanobodies with the ability to recruit endogenous IgG can recruit endogenous IgG in vivo. IgG is one of the most abundant proteins in blood plasma and maintains a long half-life in plasma. The modified PD-L1 nanobodies can extend the half-life of the modified nanobodies by recruiting IgG to bind to it; (3) By utilizing the relevant Fc fragments on IgG recruited by the PD-L1 nanoantibody with the ability to recruit endogenous IgG, the PD-L1 nanoantibody can mediate the Fc-dependent immune killing effect, so that the nanoantibody that can specifically bind to the relevant tumor cell targets can target-directedly bind to the relevant tumor cells and have an anti-tumor effect, making tumor immunotherapy precise. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 3D structural model of anti-PD-L1 nanoantibody linker protein fused with C3Fab

[0014] Figure 2 pET21a expression plasmid map

[0015] Figure 3 Schematic diagram for NbP3C8-C3Fab molecular design

[0016] Figure 4 Agarose gel electrophoresis was used to identify the pET21a-3B6-C3Fab plasmid after double digestion with BamHⅠ / EcoRⅠ and then purified and recovered the pET21a-C3Fab vector

[0017] Figure 5 Agarose gel electrophoresis of P3C8 gene fragment digestion and purification

[0018] Figure 6 Agarose gel electrophoresis diagram for double enzyme digestion verification of the recombinant plasmid pET21a-P3C8-C3Fab

[0019] Figure 7 SDS-PAGE verification of successful purification of nanoantibodies

[0020] Figure 8 Indirect ELISA detection of the binding analysis of NbP3C8-C3Fab, P3C8, B3-C3Fab and antigen PD-L1

[0021] Fig. 9 Indirect ELISA analysis of the recruitment of mouse IgG by NbP3C8-C3Fab and NbP3C8

[0022] Fig.10 Analysis of the binding of NbP3C8-C3Fab to PD-L1 positive cells A569

[0023] Fig.11 Analysis of the results of the plasma half-life determination of NbP3C8-C3Fab and NbP3C8 in mice DETAILED DESCRIPTION

[0024] The present invention is further described below.

[0025] Example 1: Preparation of anti-PD-L1 nanoantibodies with IgG recruitment function by enhancing the in vivo half-life of PD-L1 nanoantibodies 1. Determine the amino acid sequence of the target protein Anti-PD-L1 nanobody sequence: Use the PD-L1 nanobody expression plasmid to determine the base sequence of the PD-L1 nanobody, design the restriction sites as BamHI, EcoRI, and XhoI, and add a His tag to the C-terminus of the IgG Binding Domain. Design relevant primers and use PCR amplification technology to amplify the base sequence of the PD-L1 nanobody. Make the fragment have BamHI and EcoRI restriction sites and bind to the C3Fab sequence with G8S2. The sequence of P3C8-C3Fab is as follows: ggatccQVQLQESGGGSAQAGGSLRLSCEVSGDTSSLNILGWFRQAPGKEREGVAGIYTDAGTPTYTYADSVKGRFAFSRDNGQNTVYLEMSSLKPED TAMYYCAAGTMPVRAATLRPARYIYWGQGTQVTVSSgaattcGGGGSGGGGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATKTFTVTEctcgag The underlined lowercase letters indicate restriction endonuclease cleavage sites: ggatcc is a BamHI cleavage site; gaattc is an EcoRI cleavage site; ctcgag is an XhoI cleavage site. Insert the above sequence into pET21a plasmid The verified plasmid was transformed into BL21 competent cells by electroporation. After expansion culture, it was inoculated into 2YT medium containing Amp antibiotics. After 4-5 hours, the inducer was added and the culture was shaken overnight. The induced bacterial liquid was collected and centrifuged to prepare EIRNbs crude protein. The supernatant was removed after centrifugation at 8000rpm for 15 minutes, and the supernatant was frozen at -80℃ for 15 minutes, and the supernatant was thawed at room temperature for 15 minutes and repeated freezing and thawing for 3 times. The bacteria were resuspended in TES sucrose solution and placed at 4℃ for 4-6 hours. A 4-fold diluted TES sucrose solution was added, mixed and incubated at 4℃ overnight. 2 ml of pre-cooled PBS was added to every 100 mL of bacterial liquid, and the prepared lysis solution was added. After ice bathing for 30 minutes, the bacteria were lysed by ultrasound. The supernatant was centrifuged at 12000rpm for 15 minutes and the supernatant was collected. The supernatant after centrifugation was purified using a nickel ion metal chelate affinity chromatography medium (Ni-NTA) pre-loaded column to purify the nanoantibody carrying the His tag. 20 mM imidazole was used to wash away impurities and 500 mM imidazole was used to elute the target protein. 15% SDS-PAGE gel electrophoresis was used to identify the target band. 3. Transform the recombinant plasmid into E. coli BL21 competent cells for prokaryotic expression The verified plasmid was transformed into BL21 competent cells by heat shock, and after expansion culture, it was inoculated into 2YT medium containing Amp antibiotics. After 4-5 hours, the inducer was added and the culture was shaken overnight; the induced bacterial liquid was collected and centrifuged to prepare crude protein; centrifuged at 8000rpm for 15 minutes, the supernatant was removed, frozen at -80℃ for 15 minutes, and thawed at room temperature for 15 minutes, and repeated freezing and thawing was repeated 3 times; TES sucrose solution was added to resuspend the bacteria, and placed at 4C℃ for 4-6 hours; 4-fold diluted TES sucrose solution was added, mixed and incubated at 4℃ overnight; 2ml of pre-cooled PBS was added to every 100mL of bacterial liquid, and the prepared lysis solution was added. After ice bathing for 30 minutes, the bacteria were lysed by ultrasound; 12000rpm, centrifuged for 15 minutes, and the supernatant was collected; the supernatant after centrifugation was used to purify the nanoantibody carrying the His tag using a nickel ion metal chelate affinity chromatography medium (Ni-NTA) pre-loaded column. 20 mM imidazole was used to wash away impurities and 500 mM imidazole was used to elute the target protein. 15% SDS-PAGE gel electrophoresis was used to identify the target band. SOC medium required for heat shock transformation: Preparation method: Weigh 1 g of yeast extract, 4 g of peptone, and 0.1 g of NaCl, adjust the pH to about 7.0, make up to 200 mL with distilled water, sterilize in a high-pressure steam sterilizer, and store in a refrigerator at 4°C for later use. The culture medium required for BL21 bacterial culture: 2YT medium, LB medium Preparation method: 2YT medium: 16g peptone, 10g yeast extract, 5g NaCl, mix with a magnetic stirrer, adjust the pH to about 7.0 with NaOH, make up to 1L with distilled water, sterilize in a high-pressure steam sterilizer, and store in a refrigerator at 4°C for later use. LB medium: MOPs 10g, peptone 30g, yeast extract 20g, adjust the pH to about 7.0 with NaOH, make up to 1L with distilled water, sterilize in a high-pressure steam sterilizer and store in a refrigerator at 4°C for later use. Required instruments and equipment: For the subsequent PD-L1 nanobody with the ability to recruit IgG, the applicability verification steps 5, 6, 7, and 8: verifying the IgG-raising ability of the PD-L1 nanobody modified by this method, verifying the tumor targeting ability of the PD-L1 nanobody modified by this method, verifying the tumor killing effect of the PD-L1 nanobody modified by this method, and verifying the half-life extension effect of the PD-L1 nanobody modified by this method are consistent with 4, 5, 6, and 7 in the specific operation

[0011] of the invention content. The relevant tumor cells use A549 cells, which are positive for PD-L1 on their cell surface.

[0026] The above examples are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Persons skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for enhancing the in vivo half-life of a PD-L1 nanobody, characterized in that: The PD-L1 nanobody has a significantly improved in vivo plasma half-life after chemical modification. It has an endogenous IgG binding domain (IgBD) and an anti-PD-L1 nanobody constructed and expressed to recruit endogenous IgG, restore the immunological effect mediated by the Fc fragment, and extend the plasma half-life of the nanobody by recruiting endogenous IgG.

2. The IgG Binding Domain with the function of recruiting endogenous IgG according to claim 1, wherein the region is the C3Fab domain, which is derived from Streptococcus G protein. Streptococcus G protein is divided into three parts (SpGC1, SpGC2, SpGC3), CH1 is responsible for binding to the Fab fragment of immunoglobulin, and CH2 and CH3 can bind to the Fc fragment. The SpGC3Fab fragment is obtained by performing site-directed mutation of amino acids on SpGC3, and replacing the three residues (Glu27, Lys28, Lys31) in the SpGC3 helical structure that bind to the Fc region with alanine (Ala). And it has been verified that it has a higher affinity for immunoglobulins and can specifically bind to immunoglobulins of different species (human, mouse, and rat IgG). The C3Fab protein fragment has the ability to serve as an antibody recruitment domain.

3. The nanobody according to claim 1 is a single domain antibody (VHH) derived from the variable region of the heavy chain of camel IgG. Nanobodies are the smallest unit molecules known to bind to target antigens. Although the molecular weight is only one-tenth of that of traditional antibodies, nanobodies still retain complete antigen binding ability. Compared with traditional mAbs, nanobodies have many unique advantages: small molecular weight, ability to bind to specific epitopes, extremely high stability and water solubility, easy structural modification, low immunogenicity, convenient production and preparation, and low cost.

4. The PD-L1 nanobody according to claim 1 is an immune checkpoint inhibitor targeting programmed cell death protein-1 (PD-1) and its ligand (PD-1ligand, PD-L1).

5. The endogenous IgG according to claim 2 is a natural antibody produced by humans when they come into contact with non-self antigens in life. Endogenous antibodies are recruited to the surface of tumor cells, which can activate the immune system to recognize tumor cells and kill tumor cells through the mechanism mediated by their Fc end. Antibody recruiting molecules (ARMs) are a new type of molecule developed based on this concept. ARMs are covalently linked bifunctional molecules with a target binding end (TBT) at one end to interact with tumor-specific markers; the other end is an antibody binding end (ABT) for binding endogenous antibodies. ARMs first form a ternary complex with endogenous antibodies and tumor target cells, and are then recognized by the complement protein system or different immune effector cells, mediating tumor cell apoptosis through a variety of mechanisms. By recruiting endogenous antibodies, not only can anti-tumor effects be exerted, but also the plasma half-life of small molecule protein drugs can be extended.

6. According to the immune checkpoint PD-1 / PD-L1 of claim 4, PD-1 is a membrane protein containing 268 amino acids and is a member of the CD28 family. It is mostly expressed on the surface of activated T cells, B cells, macrophages and other cells. One of the ligands of PD-1, PD-L1, is a 40kDa transmembrane protein belonging to the B7 family. Studies have shown that PD-L1 is not only expressed in a variety of normal cells such as B cells, macrophages, and natural killer cells, but also overexpressed on the surface of most tumor cells such as breast cancer, melanoma, colorectal cancer and non-small cell lung cancer (NSCLC).

7. According to the immune checkpoint of claim 6, the targets of common immune checkpoint therapy are CTLA-4 and PD-1 / PD-L1. CTLA-4 is the first immune checkpoint receptor discovered. Unlike CTLA-4, the expression of the PD-1 / PD-L1 pair of immune checkpoints is more selective. The PD-1 checkpoint only regulates the activity of related cells such as T cells to metastasize to tumors, and the PD-L1 ligand is not overexpressed in normal inflammatory tissues. PD-L1 blockers can improve macrophage immunosuppression by activating NK cells that express PD-L1, and have a certain therapeutic effect on PD-L1 negative tumor cells.

8. According to the PD-L1 nanobody that can recruit endogenous IgG as described in claims 5-7, this method is through the recombinant expression of IgBD and anti-PD-L1 nanobody, and the results show that after fusion of the C3Fab fragment, the plasma half-life of the PD-L1 nanobody in vivo is significantly improved compared with the unfused nanobody.

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