An anti-serum albumin nanobody 1B7 and its use in prolonging the half-life of a polypeptide drug

Abstract

The application discloses an anti-serum albumin nanobody 1B7 and application thereof in prolonging the half-life of a polypeptide drug, and belongs to the technical field of antibody engineering. The nanobody has the following complementarity determining regions (CDRs): CDR-H1 shown in SEQ ID NO. 2 (YTCMG), CDR-H2 shown in SEQ ID NO. 3 (TIDNSGGRTYYADSVKG), and CDR-H3 shown in SEQ ID NO. 4 (QSSGYCLSDNFFRH). The nanobody is coupled with a therapeutic polypeptide, protein or antibody drug by gene fusion technology to construct a long-acting complex. The complex can significantly delay the kidney clearance rate of the drug, resist protease degradation, prolong the half-life of the drug, avoid frequent administration, maintain the curative effect while reducing the administration frequency, and improve the patient compliance and treatment safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody engineering, and in particular to an anti-serum albumin nanobody 1B7 and its application in prolonging the half-life of polypeptide drugs. BACKGROUND

[0002] Biological products play an increasingly important role in the field of disease treatment, among which polypeptide drugs have become a research hotspot due to their high specificity and low toxicity. However, such drugs generally face the serious challenge of short serum half-life. Due to the molecular weight usually less than 10 kDa, polypeptides are easily filtered by the glomerulus (molecular cutoff threshold about 60 kDa) and subjected to extensive degradation by proteases in the body, resulting in a blood circulation time of only a few minutes to a few hours. In order to achieve therapeutic effect, high-dose or frequent injection of drugs has to be adopted in clinic, which not only increases the risk of immunogenicity and toxicity, but also significantly reduces patient compliance, seriously restricting the clinical application value of polypeptide drugs.

[0003] In view of this bottleneck, traditional half-life extension technologies such as PEGylation, Fc fragment fusion and albumin direct fusion have certain effects, but all have fundamental defects. PEGylation can delay kidney clearance by increasing the hydrated radius of the drug, but the PEG chain can shield the active site of the drug, leading to decreased efficacy, and may induce anti-PEG antibodies to accelerate immune clearance; Fc fusion technology can extend the half-life by using the FcRn-mediated recycling mechanism, but the molecular weight after fusion exceeds 100 kDa, which will hinder tissue penetration and may have the risk of activating complement-dependent cytotoxicity; the human serum albumin (HSA) direct fusion strategy can rely on the natural long-circulating properties of HSA (half-life about 19 days), but the introduction of a 66.5 kDa carrier protein will cause the molecular weight of the drug to increase sharply, change the original pharmacokinetic behavior, and may hinder the delivery efficiency of the drug to the target tissue.

[0004] In recent years, the rise of nanobody (VHH) technology has provided a new path to break through the above limitations. These variable domains derived from camelid heavy chain antibodies have unique advantages such as small molecular weight (about 15 kDa), strong tissue penetration, high stability, and easy humanization. Anti-serum albumin nanobodies can obtain long-circulating properties by specific binding to endogenous HSA, while avoiding excessive molecular weight increases. The core mechanism is that the nanobody-HSA complex can effectively reduce kidney clearance and lysosomal degradation through the FcRn-mediated circulation pathway. In the evolution of technology, Jiangnan University developed a rhamnolipid modification strategy to recruit endogenous antibodies to increase the hydration radius, but the efficacy is limited by the abundance of antibodies in the body; Kangzhong Biotechnology screened a broad-spectrum nanobody Nb3 that can bind to human / bovine / mouse serum albumin across species, significantly improving preclinical model adaptability (DOI:10.1016 / j.jconrel.2024.11.080); Nanjing University designed a "black hat" bispecific antibody (NbCD4-NbHSA-NbCD4) that confirmed that HSA binding can extend the half-life by 5-8 times (CN120173100A).

[0005] Current research focuses on three key technological breakthroughs: first, high-affinity humanization, such as the HSA-32 nanobody developed by the Kim team, which maintains sub-nanomolar affinity (KD~10-9 M) after humanization; second, achieving non-pH-dependent binding to ensure stable binding to HSA at physiological pH (7.4) and endosomal acidic environment (pH 5.5-6.0), avoiding dissociation during FcRn recycling; third, establishing an efficient expression system, such as the successful soluble expression of anti-HSA nanobody-therapeutic peptide fusion proteins in E. coli by the Zhou team, significantly reducing production costs (CN119708221B). These advances have pushed anti-HSA nanobodies towards long-acting and intelligent directions, such as in drug fusion with interferon, GLP-1 analogs, etc., extending the half-life from hours to days; the "nanoadapter" developed by South China University of Technology significantly enhances tumor killing efficiency through FcγR1-HSA fusion loading of multi-specific antibodies (DOI:10.1038 / s41551-025-01425-5); Kangzhong Biotechnology has screened a broad-spectrum nanobody Nb3 that can bind to multiple serum albumins (human, bovine, mouse), which can adapt to different animal models and improve drug targeting (CN116023487A, 2023.04.28).

[0006] Despite the promising prospect, the field still faces challenges such as steric hindrance of the conjugate, consistency of the binding across species, and large-scale production. Future trends will focus on the development of intelligent delivery systems (such as the integration of pH / temperature-responsive elements) and the synergistic design of multiple mechanisms (such as the simultaneous targeting of therapeutic targets and HSA by trispecific antibodies). Anti-serum albumin nanobodies provide an efficient, safe, and programmable technical platform for the long-acting of polypeptide drugs by precisely utilizing the human body's natural transport system. With the deep integration of gene editing, artificial intelligence-assisted design, and new expression technologies, this field is expected to completely break through the limitations of traditional pharmacokinetics and lead the biomedicine industry into a new era of "one-time administration, long-term treatment".

[0007] The present application adopts the programmed immunized camel strategy to induce the production of high specificity anti-human serum albumin (HSA) heavy chain antibodies, and combines with the high-throughput panning technology of phage display library to screen candidate single domain antibodies (sdAb) targeting the natural conformation of HSA from the immune library. Further soluble production is realized through yeast and E. coli expression systems, and finally anti-HSA nanobodies with nanomolar to sub-nanomolar binding affinity are obtained. The present application aims to break through the development limitations caused by antigen epitope masking, insufficient antibody affinity or structural heterogeneity in traditional methods, and provides a universal carrier basis for constructing long-acting biological drugs. By fusing the nanobodies with therapeutic polypeptide, protein or antibody drugs, the in vivo half-life of the drugs can be significantly prolonged, the administration convenience can be improved, and the practicality of the clinical treatment window can be enhanced. SUMMARY

[0008] To solve the above problems, the present application provides an anti-serum albumin nanobody and its application. The anti-serum albumin nanobody disclosed by the present application has high affinity with albumin, and can be used to prolong the half-life of biological drugs, and is beneficial to improve the beneficial effect of biological drugs in treatment. The albumin includes serum albumin and recombinant albumin, and the serum albumin is optionally human serum albumin.

[0009] In a first aspect, the present application provides an anti-serum albumin nanobody, which can specifically bind to serum albumin, and the nanobody has the following complementarity determining regions (CDRs): CDR-H1 as shown in SEQ ID NO. 2 (YTCMG), CDR-H2 as shown in SEQ ID NO. 3 (TIDNSGGRTYYADSVKG), and CDR-H3 as shown in SEQ ID NO. 4 (QSSGYCLSDNFFRH). Optionally, the serum albumin is human serum albumin.

[0010] Further, the amino acid sequence of the nanobody includes at least one of the following:

[0011] A1) the amino acid sequence comprises the amino acid sequence as shown in SEQ ID NO. 1;

[0012] A2) the amino acid sequence is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence of the nanobody according to A1), and has more than 95% identity to the amino acid sequence of the nanobody according to A1);

[0013] A3) the amino acid sequence of a fusion protein having the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the amino acid sequence according to A1) or A2).

[0014] In a second aspect, the present application provides a biological material, which comprises at least any one of the following:

[0015] B1) a nucleic acid molecule encoding the nanobody;

[0016] B2) an expression cassette comprising the nucleic acid molecule according to B1);

[0017] B3) a recombinant vector comprising the nucleic acid molecule according to B1), or a recombinant vector comprising the expression cassette according to B2);

[0018] B4) a recombinant microorganism comprising the nucleic acid molecule according to B1), or a recombinant microorganism comprising the recombinant vector according to B2), or a recombinant microorganism comprising the recombinant vector according to B3);

[0019] B5) a recombinant cell comprising the nucleic acid molecule according to B1), or a recombinant cell comprising the recombinant vector according to B2), or a recombinant cell comprising the recombinant vector according to B3), or a recombinant cell comprising the recombinant microorganism according to B4).

[0020] Further, the nucleic acid molecule according to B1) comprises a nucleic acid molecule encoding the nanobody which has been disclosed and / or optimized according to actual needs, and optionally, the nucleic acid molecule according to B1) comprises at least one of the nucleotide sequences as shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0021] It should be understood that SEQ ID NO. 5 and SEQ ID NO. 6 are respectively codon bias optimized sequences for Pichia pastoris and Escherichia coli; one skilled in the art can optimize the nucleic acid encoding SEQ ID NO: 1 according to different expression systems (such as other engineering bacteria), and such variants are within the scope of the present application.

[0022] Further, B3) the recombinant vector comprises at least one of pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, pYES2 vectors.

[0023] Further, B4) the recombinant microorganism or B5) the recombinant cell comprises at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis.

[0024] Optionally, the recombinant microorganism comprises at least one of Escherichia coli, Pichia pastoris.

[0025] It should be noted that the present application does not limit the recombinant microorganism or the recombinant cell, and any cell that can express exogenous genes through recombinant engineering technology is within the protection scope of the present application.

[0026] In a third aspect of the present application, an anti-serum albumin antibody comprising the nanobody amino acid sequence as a VHH chain is provided.

[0027] In a fourth aspect of the present application, a derivative of the nanobody or the anti-serum albumin antibody is provided, wherein the derivative comprises a modifier covalently or non-covalently combined with the nanobody or the antibody.

[0028] Further, the modifier can optionally comprise:

[0029] a particle-like substance combined through non-covalent bond, such as colloidal gold, colloidal silver or colloidal carbon;

[0030] a modifier combined through covalent bond, including but not limited to:

[0031] microspheres (such as colored microspheres, fluorescent microspheres, magnetic microspheres), chromatographic fillers, chemical small molecules (such as biotin, dye molecules, fluorescent molecules).

[0032] In a fifth aspect of the present application, a product is provided, wherein the product comprises at least one of:

[0033] C1) a fusion protein constructed by fusing the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody with a polypeptide or a protein;

[0034] C2) a conjugate constructed by linking the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody with a therapeutic agent;

[0035] C3) a pharmaceutical composition of the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody;

[0036] C4) a kit comprising the nanobody, and / or the biomaterial, and / or the anti-serum albumin antibody, and / or the derivative of the nanobody or the anti-serum albumin antibody, and / or C1) the fusion protein, and / or C2) the conjugate, and / or C3) the pharmaceutical composition.

[0037] Further, the fusion protein is prepared by connecting a polypeptide or a protein at the N-terminus or C-terminus of the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody, and optionally, the polypeptide or the protein and the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody are connected by a chemical bond.

[0038] In the sixth aspect of the present application, a method for preparing the nanobody and / or the fusion protein is provided, and the method comprises the steps of culturing the B4) recombinant microorganism or the B5) recombinant cell in the biomaterial under suitable conditions for culture, and isolating the nanobody or the fusion protein.

[0039] In the seventh aspect of the present application, the nanobody, or the biomaterial, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody, or the product, or the method is applied in any of the following aspects:

[0040] D1) application in preparing a product for prolonging the half-life of a drug;

[0041] D2) application in preparing a product for immunodetection or diagnosis;

[0042] D3) application in preparing a product for promoting the purification or enrichment of serum albumin;

[0043] D4) application in preparing a product for qualitatively or quantitatively detecting serum albumin.

[0044] Further, the drug for prolonging the half-life of the drug comprises a protein drug, an antibody fragment, a small molecule chemotherapeutic drug, and other biological agents; preferably, the protein drug comprises at least one of an IL-6R targeted drug, a TNF-α targeted drug, and an IL-17A / IL-17F targeted drug; the small molecule chemotherapeutic drug comprises at least one of doxorubicin, sorafenib, and sirolimus.

[0045] Further, the product for prolonging the half-life of the drug in A1 includes at least one of a fusion protein of an anti-serum albumin nanobody and a protein drug, a conjugate of an anti-serum albumin nanobody and a therapeutic agent, and a pharmaceutical composition of the anti-serum albumin nanobody.

[0046] Further, the protein drug includes at least one of a polypeptide, an antibody, an antibody fragment, a cytokine, and a tumor marker capable of playing a therapeutic role. In a specific embodiment of the present application, the drug is fibroblast growth factor 21 (FGF21).

[0047] Further, the product further includes an additive acceptable in the medical field. Alternatively, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or an excipient.

[0048] The beneficial effects of the present application include, but are not limited to:

[0049] The present application screens an anti-serum albumin nanobody capable of highly specifically recognizing serum albumin. The nanobody has high affinity to serum albumin and can be widely applied to enhancing the half-life of a drug, tumor targeted therapy and imaging, immune detection or diagnosis, purification or enrichment of recombinant albumin, and the like, and has a wide application prospect in the medical field.

[0050] The present application further provides two expression preparation methods of the nanobody using Pichia pastoris and Escherichia coli as host cells, which are beneficial to commercial production. BRIEF DESCRIPTION OF DRAWINGS

[0051] The accompanying drawings, which are included to provide a further understanding of the present application and are incorporated in and constitute a part of this application, illustrate embodiments of the present application and together with the description serve to explain the present application. In the drawings:

[0052] Figure 1 SDS-PAGE electrophoretograms of each stage of purification of Pichia pastoris-expressed 1B7 in an embodiment of the present application, M: marker; 1: desalination; 2: flow-through; 3: before loading.

[0053] Figure 2 SDS-PAGE electrophoretograms of each stage of purification of Escherichia coli-expressed 1B7 in an embodiment of the present application, M: marker; 1: elution; 2: flow-through; 3: before loading.

[0054] Figure 3 ELISA binding verification analysis graph of Pichia pastoris-expressed 1B7 in an embodiment of the present application.

[0055] Figure 4Figure 1 shows the results of affinity detection of human serum albumin-nanobody 1B7 in the embodiments of the present application. DETAILED DESCRIPTION

[0056] The present application is described in detail below with reference to examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are all purchased through commercial channels.

[0057] The E. coli TG1 competent cells were purchased from Shanghai Moikang Biotechnology Co., Ltd., item number MF2384.

[0058] The E. coli BL-21 competent cells were purchased from Shanghai Moikang Biotechnology Co., Ltd., item number MF2391.

[0059] The X-33 yeast strain was purchased from Thermo Fisher scientific, item number C18000.

[0060] The adult male rats were purchased from Jiangsu Jizhuo Pharmaceutical Biotechnology Co., Ltd., item number SN0159897.

[0061] The fibroblast growth factor 21 (FGF21) was purchased from Abbkine, item number PRP1030.

[0062] The recombinant human serum albumin was self-prepared by Tonghua Anruit Biological Pharmaceutical Co., Ltd.

[0063] The enzyme-hydrolyzed casein was purchased from Solarbio, item number C8210-100.

[0064] PBST: diluted from 10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) to 1X, and then added with 0.1% Tween-20.

[0065] Loading buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and the pH was adjusted to 8.0 with NaOH.

[0066] The application periodically immunizes a camel to make the camel produce specific antibodies against human serum albumin, uses phage display technology to perform panning, and obtains multiple nanobody sequences through three rounds of panning, one of which is named 1B7, and the amino acid sequence is shown as SEQ ID NO. 1. According to the Kabat definition scheme, the CDR is defined, the CDR-H1 amino acid sequence is shown as SEQ ID NO. 2, the CDR-H2 amino acid sequence is shown as SEQ ID NO. 3, and the CDR-H3 amino acid sequence is shown as SEQ ID NO. 4. In addition, the application provides two expression modes of Pichia pastoris and Escherichia coli as host cells, and the high-affinity anti-serum albumin nanobody 1B7 is obtained through purification. The nanobody can be used to prolong the half-life of biological drugs, and is conducive to improving the beneficial effect of biological drugs in treatment.

[0067] The application scheme is described below through specific examples.

[0068] It should be noted that the various biological and chemical reagents used in the embodiments of the application are obtained through conventional commercial channels unless otherwise specified, and the experimental methods not specified are generally performed according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0069] Example 1 Camel immunization and peripheral blood lymphocyte separation

[0070] The camel was immunized by subcutaneous injection of recombinant human albumin mixed with Freund's adjuvant through multiple points on the neck, and a total of 5 immunizations were performed. Blood was collected before immunization, before the fourth immunization, before the fifth immunization, and two weeks after the fifth immunization, and serum was obtained after standing and centrifugation. The ELISA (enzyme linked immunosorbent assay) method was used to detect the titer of the immune antibody. Blood was collected after the fifth immunization, and peripheral blood mononuclear cells (PBMC) were obtained by centrifugation.

[0071] Example 2 Phage library construction

[0072] Trizol method was used to extract total RNA from PBMC, and cDNA was synthesized by reverse transcription. The single-domain antibody fragment was amplified by two PCR, and was connected into the phage plasmid after restriction enzyme digestion. The electroporation method was used to transform into E. coli TG1 competent cells. Gradient dilution method was used to determine the library capacity of 5.15×10 9Forty single clones were randomly selected for PCR identification, and the positive rate was 39 / 40.

[0073] Example 3 Nanobody screening against human albumin

[0074] In the EP tube, magnetic beads and enzyme-hydrolyzed casein (manufacturer: Solarbio, product number: C8210-100) were added, and blocked for 1 h. Recombinant human albumin was added, and incubated for 1 h for coating. After blocking with enzyme-hydrolyzed casein again, the phage library was added for binding, and PBST (10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) was diluted to 1X, and 0.1% Tween-20 was further added. After washing 9 times with PBS (pH 7.4), trypsin was added for elution, and enzyme-hydrolyzed casein was added for termination, and the elution product was obtained. The elution product was used to infect TG1 E. coli, and plated, and cultured at 37°C overnight. The M13K07 was used to infect the E. coli cultured from all the colonies in the logarithmic phase, and the culture was expanded. The library was enriched by 3 cycles of screening. The strains after the last screening were plated on ampicillin-resistant plates, and single colonies were picked on sterile cell culture plates, and subjected to ELISA detection. Qualified positive single colonies were selected and subjected to gene sequencing, and a plurality of nanobody sequences were obtained, one of which was named 1B7, and the amino acid sequence is shown as SEQ ID NO. 1.

[0075] Example 4 Expression and purification of nanobody in Pichia pastoris

[0076] (1) Expression of nanobody 1B7 in Pichia pastoris

[0077] The 1B7 antibody gene was codon-optimized after Pichia pastoris, and was cloned into the yeast vector pPICZαA, and the optimized nucleotide sequence is shown as SEQ ID NO. 5, Sac I After enzyme digestion and linearization, it was electroporated into the X-33 yeast strain, and single colonies were screened on a Zeocin-resistant plate. Routine inoculation was used for induction of secretory expression, and the final concentration of methanol was 0.5% every 24 h. After 72 h of induction and expression, the sample was collected, and the supernatant was collected by centrifugation. The expression of nanobody 1B7 was determined by SDS-PAGE, and it was confirmed that the nanobody 1B7 was expressed correctly.

[0078] (2) Purification of nanobody 1B7 expressed in Pichia pastoris

[0079] The yeast-expressed fermentation supernatant was added to a final concentration of 25 mM imidazole, dissolved, and filtered with a 0.45 μm filter for loading; the nickel column was equilibrated with loading buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 adjusted with NaOH), and the baseline was leveled, the sample was loaded, and the flow-through was collected; the column was washed with loading buffer until the baseline was leveled again, and elution buffer was used for elution, and the eluate was collected; the desalting column was equilibrated with desalting buffer for 4 column volumes, and the eluate was loaded onto the desalting column, and the first peak that appeared was collected as the final antibody solution. If the protein concentration was low after desalting, the solution was concentrated with an ultrafiltration tube. The collected solutions at each stage of protein refolding and purification were detected by SDS-PAGE, and the results are shown in FIG. 2. After purification, the main band of the target protein was clear, and no obvious bands were observed. Figure 1

[0080] Example 5. Expression and purification of nanobodies in E. coli

[0081] (1) Expression of nanobody 1B7 in E. coli

[0082] The 1B7 antibody gene was synthesized after codon optimization of the E. coli gene and was connected to the pET-28A(+) expression vector, and the optimized nucleotide sequence is shown in SEQ ID NO. 6. The transformed BL-21 competent cells were inoculated into LB medium, and the culture was incubated to OD 600 =0.6, and the final concentration of IPTG was 1 mM. The induction expression was performed at 20°C and 250 rpm for 48 h. The bacterial cells were collected by centrifugation, ultrasonically treated, and centrifuged to discard the supernatant. The inclusion bodies were refolded. The expression of nanobody 1B7 was determined by SDS-PAGE, and the protein expression was good after IPTG induction.

[0083] (2) Purification of nanobody 1B7 expressed in E. coli

[0084] The E. coli-expressed refolded solution was added to a final concentration of 25 mM imidazole, dissolved, and filtered with a 0.45 μm filter for loading; the nickel column was equilibrated with loading buffer, and the baseline was leveled, the sample was loaded, and the flow-through was collected; the column was washed with loading buffer until the baseline was leveled again, and elution buffer was used for elution, and the eluate was collected; the desalting column was equilibrated with desalting buffer for 4 column volumes, and the eluate was loaded onto the desalting column, and the first peak that appeared was collected as the final antibody solution. If the protein concentration was low after desalting, the solution was concentrated with an ultrafiltration tube. The collected solutions at each stage of protein refolding and purification were detected by SDS-PAGE, and the results are shown in FIG. 2. After purification, the main band of the target protein was clear, and no obvious bands were observed. Figure 2

[0085] ​​Example 6 Nanobody ELISA binding verification

[0086] Take 100 μL of recombinant human albumin with a concentration of 0.05 μg / mL and add to a 96-well plate, coat overnight at 4°C, and pat dry. Wash three times with 300 μL of PBST, add 100 μL of 5% skimmed milk powder to 37°C for 1 h for blocking, discard the solution and pat dry. Dissolve the nanobodies in 5% skimmed milk powder in a 3-fold serial dilution (initial concentration 1 μg / mL, a total of 12 concentration gradients), add to the 96-well plate, 100 μL per well, 37°C for 1 h, discard the solution and pat dry. Take 1 μL of HRP-labeled secondary antibody (anti-his) and add to 10 mL of 5% skimmed milk powder, mix well, add to the 96-well plate, 100 μL per well, 4°C for overnight incubation. Wash 5 times with 300 μL of PBST, add 100 μL of color developing solution (TMB), avoid light for 10 min. Add 100 μL of stop solution, detect absorbance at 450 nm. Calculate the absorbance OD of each concentration of nanobodies 450 Mean, OD 450 Take the mean as the ordinate and the antibody concentration as the abscissa, draw the reaction curve of human albumin-nanobody at different concentrations, and take the nanobody concentration corresponding to half the absorbance of the flat section of each curve as the EC 50 , as shown in Figure 3 , the detection result is: the yeast expression EC 50 = 8.95E-03 μg / mL.

[0087] Example 7 Human serum albumin-nanobody affinity detection

[0088] The biofilm interference technology (BLI method) was used for affinity detection of human serum albumin-nanobody 1B7. NTA biosensor was used, first immerse the sensor in the analysis buffer for 10 min, immerse the equilibrated sensor in EDC-NHS mixed reagent for 5 min; immerse the activated sensor in nanobody diluent (100 nM) for 10 min and block with ethanolamine (1 M, pH 8.5); immerse the blocked sensor in buffer for baseline zeroing; then immerse the sensor in gradient concentration human serum albumin solution (50-0.78 nM) for 5 min, run the complete binding curve; then transfer the sensor to PBS buffer for 5 min. Use 1:1 binding model for kinetic analysis, as shown in Figure 4 , the dissociation equilibrium constant KD= 6.84E-010 M, indicating that the nanobody 1B7 has strong binding with human serum albumin.

[0089] Example 8 Half-life extension test

[0090] 1) Expression and purification of FGF21-nanobody fusion protein

[0091] The nanobody 1B7 can be expressed in fusion with other polypeptide molecules, and through the characteristics of antigen-antibody specific binding, it can be combined with endogenous albumin to improve the half-life of the drug. In this embodiment, fibroblast growth factor 21 (FGF21) is taken as an example to construct FGF21-1B7 fusion protein (SEQ ID NO. 7). FGF21 is connected with nanobody 1B7 through (G4S)3 flexible linker, and a fusion protein gene fragment (SEQ ID NO. 8) is synthesized and cloned into the yeast vector pPICZαA. After linearization by enzyme digestion, it is electroporated into X-33 yeast strain, and single clone colonies are screened on Zeocin-resistant plates. Routine inoculation is used for secretory expression, and the final concentration of methanol is 0.5% every 24 h. After 72 h of induction expression, the sample is collected by centrifugation. The fermentation supernatant is added to a final concentration of 25 mM imidazole, and after dissolution, it is filtered with a 0.45 μm filter membrane for loading; the nickel column is equilibrated with loading buffer, and after the baseline level, it is loaded and the flow-through is collected; the column is further washed with loading buffer until the baseline level is reached again, and elution buffer is used for elution, and the eluate is collected; the desalting column is equilibrated with desalting buffer for 4 column volumes, and the eluate is loaded onto the desalting column, and the first peak that appears is the target protein solution. If the protein concentration is low after desalting, use ultrafiltration tube for concentration to obtain high-purity FGF21-1B7 fusion protein. Sac I

[0092] 2) Detection of protein half-life

[0093] 6 adult male rats were randomly divided into 2 groups;

[0094] Experimental group: subcutaneous injection of 0.3 mg / kg FGF21-1B7

[0095] Control group: subcutaneous injection of 0.3 mg / kg free FGF21

[0096] Blood was collected from the tail vein at 1, 2, 4, 8, 12, 24, 48, 72, and 96 hours after administration, and plasma was separated;

[0097] The concentrations of FGF21-1B7 and free FGF21 in serum were quantitatively detected by ELISA, and the half-life of each sample in vivo was calculated, and the results are shown in Table 1.

[0098] Table 1 Half-life extension test results

[0099]

[0100] ​The half-life of the FGF21-1B7 fusion protein (20.0 h) is significantly prolonged to 10 times that of free FGF21 (2.0 h), confirming that the anti-serum albumin nanobody 1B7 can effectively improve the retention time of FGF21 in vivo, providing a technical basis for the development of long-acting drugs.

[0101] The above merely describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An anti-serum albumin nanobody, characterized in that, The nanobody can specifically bind to serum albumin, and the nanobody has the following complementarity-determining regions (CDRs): CDR-H1 shown in SEQ ID NO.2 (YTCMG), CDR-H2 shown in SEQ ID NO.3 (TIDNSGGRTYYADSVKG), and CDR-H3 shown in SEQ ID NO.4 (QSSGYCLSDNFFRH).

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody includes at least one of the following: A1) The amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1; A2) An amino acid sequence of a fusion protein with the same function obtained by linking a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

3. A biomaterial, characterized in that, The biomaterial includes at least one of the following: B1) A nucleic acid molecule encoding the nanobody of claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Recombinant cells, wherein the recombinant cells are recombinant cells containing the nucleic acid molecules of B1), or recombinant cells containing the expression cassette of B2), or recombinant cells containing the recombinant vector of B3).

4. The biomaterial according to claim 3, characterized in that, The nucleic acid molecule described in B1) includes at least one of the nucleotide sequences shown in SEQ ID NO. 5 or SEQ ID NO.

6.

5. The biomaterial according to claim 3, characterized in that, The recombinant microorganisms described in B4) or the recombinant cells described in B5) include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

6. A product characterized in that, The product includes at least one of the following: C1) A kit comprising the nanobody of claim 1 or 2, and / or the biomaterial of any one of claims 3-5.

7. A method for preparing the nanobody according to claim 1 or 2, characterized in that, The method includes the steps of culturing the recombinant microorganism (B4) or the recombinant cell (B5) in the biomaterial of claim 3 under suitable culture conditions, and isolating the nanobody.

8. The use of the nanobody of claim 1 or 2, the biomaterial of any one of claims 3-5, the product of claim 6, or the method of claim 7 in any of the following aspects: D1) Applications in the preparation of products with extended FGF21 half-life; D2) Applications in the preparation of serum albumin immunoassay or diagnostic products; D3) Applications in the preparation of products that promote the purification or enrichment of serum albumin; D4) Applications in the preparation of products for qualitative or quantitative detection of serum albumin.

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