Anti-serum albumin nano antibody 1B7 and application thereof in prolonging half-life period of polypeptide drug

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of antibody engineering technology, specifically to an anti-serum albumin nanobody 1B7 and its application in prolonging the half-life of polypeptide drugs. Background Technology

[0002] Biologics are playing an increasingly important role in disease treatment, with peptide drugs becoming a hot research topic due to their high specificity and low toxicity. However, these drugs generally face the serious challenge of a short serum half-life. Because their molecular weight is typically below 10 kDa, peptides are rapidly filtered by the glomeruli (molecular cutoff threshold of approximately 60 kDa) and are extensively degraded by proteases in the body, resulting in their circulation time in the bloodstream being only a few minutes to a few hours. To achieve therapeutic effects, clinicians have to adopt high-dose or frequent injection strategies, which not only increases the risk of immunogenicity and toxic side effects but also significantly reduces patient compliance, severely limiting the clinical application value of peptide drugs.

[0003] While traditional half-life extension techniques such as PEGylation, Fc fragment fusion, and direct albumin fusion have shown some effectiveness in addressing this bottleneck, they all suffer from fundamental drawbacks. PEGylation delays renal clearance by increasing the drug's hydration radius, but the PEG chain can mask the drug's active site, leading to decreased efficacy and potentially inducing anti-PEG antibodies that accelerate immune clearance. Fc fusion technology, while utilizing FcRn-mediated regeneration to extend half-life, results in a molecular weight exceeding 100 kDa, hindering tissue penetration and posing a risk of complement-dependent cytotoxicity. Direct fusion with human serum albumin (HSA), while leveraging HSA's naturally long circulating properties (approximately 19 days half-life), introduces a 66.5 kDa carrier protein, causing a sharp increase in drug molecular weight, altering the original pharmacokinetic behavior, and potentially hindering drug delivery efficiency to target tissues.

[0004] In recent years, the rise of nanobody (VHH) technology has provided a new pathway to overcome the aforementioned limitations. These variable domains, derived from camel heavy chain antibodies, possess unique advantages such as small molecular weight (approximately 15 kDa), strong tissue penetration, high stability, and ease of humanization. Anti-serum albumin nanobodies, by specifically binding to endogenous HSA, allow peptide drugs to "hitch a ride" and acquire long-circulating properties, while avoiding excessive molecular weight increases. The core mechanism lies in the fact that the nanobody-HSA complex can effectively reduce renal clearance and lysosomal degradation through the FcRn-mediated circulation pathway. In the course of technological evolution, Jiangnan University developed a rhamnolipin modification strategy to recruit endogenous antibodies and increase the hydration radius, but the efficacy was limited by the abundance of antibodies in patients; Kangzhong Biotechnology screened out the broad-spectrum nanobody Nb3, which can bind to human / bovine / mouse serum albumin across species, significantly improving the preclinical model suitability (DOI:10.1016 / j.jconrel.2024.11.080); Nanjing University designed a "staff hat type" bispecific antibody (NbCD4-NbHSA-NbCD4), which proved that HSA binding can prolong the half-life by 5-8 times (CN120173100A).

[0005] Current research focuses on three key technological breakthroughs: First, high-affinity humanization modification, such as the HSA-32 nanobody developed by Jin Wanzhu's team, which maintains sub-nanomolar affinity (KD~10-9 M) after humanization; second, achieving pH-independent binding, ensuring stable binding of HSA under both physiological pH (7.4) and endosomal acidic environments (pH 5.5-6.0), avoiding dissociation during FcRn recovery; and third, establishing efficient expression systems, such as the successful soluble expression of anti-HSA nanobody-therapeutic peptide fusion protein in E. coli by Zhou Baisong's team, which significantly reduces production costs (CN119708221B). These advancements have propelled the development of anti-HSA nanobodies towards longer-lasting and more intelligent applications. For instance, in the fusion of drugs such as interferon and GLP-1 analogs, the half-life has been extended from hours to days. The "nanoaptor" developed by South China University of Technology, which loads multi-specific antibodies through FcγR1-HSA fusion, significantly enhances tumor-killing efficacy (DOI:10.1038 / s41551-025-01425-5). Kangzhong Bio has screened out the Nb3 nanobody, which broadly binds to various serum albumins (human, bovine, and mouse), and can be adapted to different animal models and improve drug targeting (CN116023487A, 2023.04.28).

[0006] Despite its promising prospects, this field still faces challenges such as steric hindrance of complexes, cross-species binding consistency, and large-scale production. Future development trends will focus on the development of intelligent delivery systems (such as integrating pH / temperature-responsive elements) and multi-mechanism synergistic design (such as simultaneous targeting of therapeutic targets and HSA with trispecific antibodies). Anti-serum albumin nanobodies, by precisely utilizing the human body's natural transport system, provide an efficient, safe, and programmable technological platform for the long-acting formulation of peptide drugs. With the deep integration of gene editing, AI-aided design, and novel expression technologies, this field is expected to completely break through the limitations of traditional pharmacokinetics, leading biomedicine into a new era of "single-dose, long-acting treatment."

[0007] This invention employs a programmed immune camel strategy to induce the production of highly specific anti-human serum albumin (HSA) heavy chain antibodies. Combined with high-throughput panning technology using phage display libraries, candidate single-domain antibodies (sdAbs) targeting the native conformation of HSA are screened from an immune library. Further soluble production is achieved using yeast and *E. coli* expression systems, ultimately yielding anti-HSA nanobodies with nanomolar to sub-nanomolar binding affinity. This invention aims to overcome the development limitations of traditional methods caused by antigen epitope masking, insufficient antibody affinity, or structural heterogeneity, providing a universal carrier basis for constructing long-acting biopharmaceuticals. By fusing this nanobodies with therapeutic peptides, proteins, or antibody drugs, their in vivo half-life can be significantly extended, dosing convenience improved, and the practicality of the clinical therapeutic window enhanced. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides an anti-serum albumin nanobody and its application. The disclosed anti-serum albumin nanobody exhibits high affinity for albumin and can be used to prolong the half-life of biological drugs, thereby enhancing their therapeutic effects. The albumin includes serum albumin and recombinant albumin; optionally, the serum albumin is human serum albumin.

[0009] In a first aspect, the present invention provides an anti-serum albumin nanobody, wherein the nanobody is capable of specifically binding to serum albumin, and wherein 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). Optionally, the serum albumin is human serum albumin.

[0010] Furthermore, 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) is an amino acid sequence obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the nanobody described in A1), which has more than 95% identity with the amino acid sequence of the nanobody described in A1. A3) An amino acid sequence of a fusion protein with the same function obtained by attaching a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1) or A2).

[0011] In a second aspect, the present invention provides a biomaterial comprising at least any one of the following: B1) Contains a nucleic acid molecule encoding the nanobody described above; 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 nucleic acid molecules of B1), or recombinant microorganisms containing recombinant vectors of expression cassettes of B2), or recombinant microorganisms containing recombinant vectors of B3); B5) Recombinant cells, wherein the recombinant cells contain B1) nucleic acid molecules, or B2) the expression cassette of the recombinant vector, or B3) the recombinant vector, or B4) recombinant microorganisms.

[0012] Further, the nucleic acid molecule described in B1) includes a publicly disclosed nucleic acid molecule encoding the nanobody and / or an optimized nucleic acid molecule as needed. Optionally, 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.

[0013] It should be understood that SEQ ID NO. 5 and SEQ ID NO. 6 are sequences optimized for codon preferences of Pichia pastoris and Escherichia coli, respectively; those skilled in the art can optimize the nucleic acid encoding SEQ ID NO: 1 according to different expression systems (such as other engineered bacteria), and such variants are all within the scope of protection of this application.

[0014] Further, the recombinant vector described in B3) includes at least one of the following: pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, and pYES2 vectors.

[0015] Furthermore, 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.

[0016] Optionally, the recombinant microorganism includes at least one strain of Escherichia coli and Pichia pastoris.

[0017] It should be noted that the present invention does not limit the recombinant microorganisms or recombinant cells mentioned above. Any cell that can express exogenous genes through recombinant engineering technology is protected by the present invention.

[0018] In a third aspect, the present invention comprises an antiserum albumin antibody containing the aforementioned nanobody amino acid sequence as a VHH chain.

[0019] A fourth aspect of the present invention includes the nanobody or a derivative of the antiserum albumin antibody, the derivative comprising a modifier that is covalently or non-covalently bound to the nanobody or the antibody.

[0020] Further, the modification may optionally include: Particulate matter bonded by non-covalent bonds, such as colloidal gold, colloidal silver, or colloidal carbon; Modifiers that are covalently bonded include, but are not limited to: Microspheres (such as colored microspheres, fluorescent microspheres, and magnetic microspheres), chromatography packing materials, and small chemical molecules (such as biotin, dye molecules, and fluorescent molecules).

[0021] In a fifth aspect, the present invention provides a product comprising at least one of the following: C1) A fusion protein constructed by fusing the nanobody, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody with a polypeptide or protein; C2) A conjugate formed by linking the nanobody, or the antiserum albumin antibody, or a derivative of the nanobody or the antiserum albumin antibody, to a therapeutic agent; C3) A pharmaceutical composition comprising the nanobody, or the antiserum albumin antibody, or a derivative thereof; C4) A kit comprising the nanobody, and / or the biomaterial, and / or the anti-serum albumin antibody, and / or the nanobody or a derivative of the anti-serum albumin antibody, and / or the fusion protein of C1), and / or the conjugate of C2), and / or the pharmaceutical composition of C3).

[0022] Furthermore, the fusion protein is prepared by linking a polypeptide or protein to the N-terminus or C-terminus of the nanobody, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody. Optionally, the polypeptide or protein and the nanobody, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody are linked by chemical bonds.

[0023] In a sixth aspect, the present invention provides a method for preparing the nanobody and / or the fusion protein, characterized in that the method includes the steps of culturing the recombinant microorganism (B4) or the recombinant cell (B5) in the biological material under suitable culture conditions, and isolating the nanobody or the fusion protein.

[0024] In a seventh aspect, the present invention provides the nanobody, or the biomaterial, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody, or the product, or the method thereof, and its use in any of the following aspects: D1) Applications in the preparation of products with extended drug half-life; D2) Applications in the preparation of 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.

[0025] Furthermore, the drugs used to extend the half-life of the drugs include protein drugs, antibody fragments, small molecule chemotherapeutic drugs, and other biological agents; preferably, the protein drugs include at least one of IL-6R targeted drugs, TNF-α targeted drugs, and IL-17A / IL-17F targeted drugs; the small molecule chemotherapeutic drugs include at least one of doxorubicin, solefenib, and sirolimus.

[0026] Furthermore, the extended drug half-life product described in A1 includes at least one of the following: a fusion protein composed of an antiserum albumin nanobody and a protein drug, a conjugate formed by linking an antiserum albumin nanobody and a therapeutic agent, and a pharmaceutical composition of the antiserum albumin nanobody.

[0027] Furthermore, the protein-based drug includes at least one of the following: polypeptides, antibodies, antibody fragments, cytokines, and tumor marker molecules capable of therapeutic effects. In one specific embodiment of the present invention, the drug is fibroblast growth factor 21 (FGF21).

[0028] Furthermore, the product also includes pharmaceutically acceptable additives. Optionally, the pharmaceutical composition further includes pharmaceutically acceptable carriers and / or excipients.

[0029] The beneficial effects of the present invention include, but are not limited to: This invention obtains an anti-serum albumin nanobody that can specifically recognize serum albumin through screening. This nanobody has a high affinity for serum albumin and can be widely used in enhancing drug half-life, tumor targeted therapy and imaging, immune detection or diagnosis, purification or enrichment of recombinant albumin, etc., and has broad application prospects in the pharmaceutical field.

[0030] This invention also provides two methods for preparing the nanobody using Pichia pastoris and Escherichia coli as host cells, which is beneficial for commercial production. Attached Figure Description

[0031] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 The images shown are SDS-PAGE electrophoresis images of each stage of purification of Pichia pastoris expression 1B7 in this embodiment of the invention. M: marker; 1: desalting; 2: flow-through; 3: before loading.

[0032] Figure 2 The images shown are SDS-PAGE electrophoresis images of each stage of purification of Escherichia coli expressing 1B7 in this embodiment of the invention. M: marker; 1: elution; 2: flow-through; 3: before loading.

[0033] Figure 3 This is an ELISA binding verification analysis diagram of Pichia pastoris expression 1B7 in an embodiment of the present invention.

[0034] Figure 4 This is a graph showing the affinity detection results of human serum albumin-nanobody 1B7 in an embodiment of the present invention. Detailed Implementation

[0035] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0036] Escherichia coli TG1 competent cells were purchased from Shanghai Maokang Biotechnology Co., Ltd., catalog number MF2384.

[0037] Escherichia coli BL-21 competent cells were purchased from Shanghai Maokang Biotechnology Co., Ltd., catalog number MF2391.

[0038] The X-33 yeast strain was purchased from Thermo Fisher Scientific, catalog number C18000.

[0039] Adult male rats were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., product number SN0159897.

[0040] Fibroblast growth factor 21 (FGF21) was purchased from Abbkine (product number: PRP1030).

[0041] Recombinant human serum albumin: self-supplied by Tonghua Anruit Biopharmaceutical Co., Ltd.

[0042] Enzymatically hydrolyzed casein: purchased from Solarbio, product number: C8210-100.

[0043] PBST: Prepared by diluting 10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) to 1X and then adding 0.1% Tween-20.

[0044] Sample loading buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with NaOH.

[0045] This invention involves periodically immunizing camels to induce the production of specific antibodies against human serum albumin. Phage display technology is used for panning, and after three rounds of panning, multiple nanobody sequences are obtained, one of which is named 1B7, and its amino acid sequence is shown in SEQ ID NO. 1. CDRs are defined according to the Kabat protocol: CDR-H1 amino acid sequence is shown in SEQ ID NO. 2, CDR-H2 amino acid sequence is shown in SEQ ID NO. 3, and CDR-H3 amino acid sequence is shown in SEQ ID NO. 4. Furthermore, this invention provides two expression methods using Pichia pastoris and Escherichia coli as host cells. After purification, a high-affinity anti-serum albumin nanobody 1B7 is obtained. This nanobody can be used to prolong the half-life of biological drugs, which is beneficial for improving the therapeutic effects of biological drugs.

[0046] The present application solution will be described below through specific embodiments.

[0047] It should be noted that, unless otherwise specified, all biological and chemical reagents used in the embodiments of this invention are obtained through conventional commercial channels. Experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0048] Example 1: Camel Immunization and Peripheral Blood Lymphocyte Isolation Camels were immunized via subcutaneous injection at multiple sites in the neck after a mixture of recombinant human albumin and Freund's adjuvant. A total of five immunizations were administered. Blood samples were collected before each immunization, before the fourth immunization, before the fifth immunization, and two weeks after the fifth immunization. Serum was obtained after centrifugation, and antibody titers were detected using ELISA (enzyme-linked immunosorbent assay). Peripheral blood mononuclear cells (PBMCs) were obtained after centrifugation following the fifth immunization.

[0049] Example 2: Phage Library Construction Total RNA was extracted from PBMCs using the Trizol method, and cDNA was synthesized via reverse transcription. Single-domain antibody fragments were amplified by two PCR assays, digested with restriction endonucleases, and ligated into phage plasmids. The phages were then transformed into *E. coli* TG1 competent cells using electroporation. The library volume was determined to be 5.15 × 10⁻⁶ cells using a serial dilution method. 9 Forty single clones were randomly selected for PCR identification, and the positive rate was 39 / 40.

[0050] Example 3: Screening of anti-human albumin nanobodies Magnetic beads and enzymatically hydrolyzed casein (Solarbio, catalog number C8210-100) were added to EP tubes and blocked for 1 h. Recombinant human albumin was added and incubated for 1 h for coating. After blocking again with enzymatically hydrolyzed casein, a phage library was added for binding. The tubes were washed 9 times with PBST (10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) diluted to 1X, then 0.1% Tween-20 was added. The tubes were then washed once with PBS (pH 7.4). Trypsin was added for elution, and the elution was terminated with enzymatically hydrolyzed casein to obtain the elution product. The elution product was used to infect TG1 *E. coli*, plated, and incubated overnight at 37°C. All colonies of *E. coli* in the logarithmic growth phase were infected with M13K07 to expand the culture. The library was enriched by three rounds of cyclic screening. The final screening strains were plated on ampicillin-resistant plates, and single colonies were picked and placed on sterile cell culture plates for ELISA detection. Qualified positive single colonies were selected and their genes were sequenced to obtain multiple nanobody sequences, one of which was named 1B7, and its amino acid sequence is shown in SEQ ID NO. 1.

[0051] Example 4 Expression and purification of nanobodies in Pichia pastoris (1) Pichia pastoris expression of nanobody 1B7 The 1B7 antibody gene was cloned into the yeast vector pPICZαA after codon optimization using Pichia pastoris. The optimized nucleotide sequence is shown in SEQ ID NO. 5. Sac I After linearization by enzyme digestion, the nanobody 1B7 was electroporated into the X-33 yeast strain, and single colonies were screened on Zeocin-resistant plates. Secretory expression was induced by routine inoculation, with methanol added every 24 h to a final concentration of 0.5%. After 72 h of induction, the sample was collected, and the supernatant was collected by centrifugation. The expression of nanobody 1B7 was confirmed by SDS-PAGE.

[0052] (2) Purification of Pichia pastoris expression nanobody 1B7 The yeast fermentation supernatant was added to a final concentration of 25 mM imidazole. After dissolution, it was filtered through a 0.45 μm filter membrane for loading. The nickel column was equilibrated with loading buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with NaOH). Once the column reached baseline, the sample was loaded, and flow-through was collected. The column was further washed with loading buffer until the baseline returned to level. Elution buffer was then used, and the eluent was collected. The desalting column was equilibrated to four column volumes with desalting buffer. All the eluent was loaded onto the desalting column, and the first peak observed was collected as the final antibody solution. If the protein concentration was low after desalting, it was concentrated using an ultrafiltration tube. The purity of the collected solutions at each purification stage was tested, and the SDS-PAGE results are shown below. Figure 1 As shown in the figure, the main band of the purified target protein was clear, and no obvious impurities were observed.

[0053] Example 5 Expression and purification of nanobodies in Escherichia coli (1) Escherichia coli expression of nanobody 1B7 The 1B7 antibody gene fragment, synthesized after codon optimization in *E. coli*, was ligated into the pET-28A(+) expression vector. The optimized nucleotide sequence is shown in SEQ ID NO. 6. The vector was then transformed into *E. coli* BL-21 competent cells. The transformed BL-21 positive colonies were inoculated into LB medium and cultured until OD200. 600 The concentration of the nanobody 1B7 was approximately 0.6 mg / L. IPTG was added to a final concentration of 1 mM and the expression was induced at 20°C and 250 rpm for 48 h. The bacterial cells were collected by centrifugation, sonicated, and the supernatant was discarded after centrifugation. The inclusion bodies were then renatured. SDS-PAGE analysis showed that the expression of the 1B7 nanobody was good after IPTG induction.

[0054] (2) Purification of Escherichia coli expressing nanobody 1B7 The renatured E. coli expression solution was added to a final concentration of 25 mM imidazole. After dissolution, it was filtered through a 0.45 μm filter membrane for sample loading. The nickel column was equilibrated with loading buffer until the baseline was reached, then the sample was loaded and flow-through was collected. The column was washed again with loading buffer until the baseline was reached, then eluted with elution buffer, and the eluent was collected. The desalting column was equilibrated to four column volumes with desalting buffer, and all the eluent was loaded onto the desalting column. The first peak that appeared was collected as the final antibody solution. If the protein concentration was low after desalting, it was concentrated using an ultrafiltration tube. The collected solutions from each stage of protein renaturation and purification were analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown, the main band of the purified target protein is clear, with no obvious impurities.

[0055] Example 6: Validation of Nanobody ELISA Binding Add 100 μL of 0.05 μg / mL recombinant human albumin to a 96-well plate, coat overnight at 4°C, and blot dry. Wash three times with 300 μL PBST, add 100 μL of 5% skim milk powder, and incubate at 37°C for 1 h for blocking. Discard the solution and blot dry. Add 100 μL of serially diluted nanobodies (initial concentration 1 μg / mL, 12 concentration gradients) dissolved in 5% skim milk powder to each well of a 96-well plate, incubate at 37°C for 1 h, and discard the solution and blot dry. Add 1 μL of HRP-labeled secondary antibody (anti-his) to 10 mL of 5% skim milk powder, mix well, add 100 μL to each well of a 96-well plate, and incubate overnight at 4°C. Wash five times with 300 μL PBST, add 100 μL of chromogenic buffer (TMB), and react in the dark for 10 min. Add 100 μL of stop solution and measure the absorbance at 450 nm. Calculate the absorbance OD corresponding to each concentration of nanobody. 450 Mean, in terms of OD 450 The mean was plotted on the ordinate and antibody concentration on the abscissa. Reaction curves of human albumin-nanobodies at different concentrations were plotted. The concentration of nanobodies corresponding to half the absorbance of the flat segment on each curve was defined as EC50. 50 The result is as follows Figure 3 As shown, the detection results are: yeast expresses EC 50 =8.95E-03 μg / mL.

[0056] Example 7: Human serum albumin-nanobody affinity detection The affinity of human serum albumin-nanobody 1B7 was detected using biomembrane interferometry (BLI). Using an NTA biosensor, the sensor was first equilibrated in analytical buffer for 10 min, then activated by immersion in an EDC-NHS mixture for 5 min. The activated sensor was then incubated in nanobody dilution buffer (100 nM) for 10 min and blocked with ethanolamine (1 M, pH 8.5). The blocked sensor was then zeroed by immersion in buffer for baseline adjustment. Next, the sensor was sequentially immersed in gradient concentrations of human serum albumin solution (50–0.78 nM) for 5 min to bind, and a complete binding curve was generated. Finally, the sensor was transferred to PBS buffer for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model, and the results are as follows: Figure 4 As shown, the dissociation equilibrium constant KD = 6.84E-010 M indicates that the nanobody 1B7 has a strong binding to human serum albumin.

[0057] Example 8 Half-life extension test 1) Expression and purification of FGF21-nanobody fusion protein Nanobody 1B7 can be fused with other peptide molecules for expression. Leveraging the antigen-antibody specific binding characteristic, it binds to endogenous albumin, thereby increasing the drug's half-life. This example uses fibroblast growth factor 21 (FGF21) as an example to construct the FGF21-1B7 fusion protein (SEQ ID NO.7). FGF21 and nanobody 1B7 were linked using a (G4S)3 flexible linker to synthesize the fusion protein gene fragment (SEQ ID NO.8), which was then cloned into the yeast vector pPICZαA. Sac I After linearization by enzyme digestion, the enzyme was electroporated into the X-33 yeast strain, and single colonies were screened on Zeocin-resistant plates. Secretory expression was induced by routine inoculation, with methanol added every 24 h to a final concentration of 0.5%. After 72 h of induction, the supernatant was collected by centrifugation. The fermentation supernatant was added to imidazole to a final concentration of 25 mM, and after dissolution, filtered through a 0.45 μm filter membrane for loading. The nickel column was equilibrated with loading buffer until the baseline was reached, then the sample was loaded, and flow-through was collected. The column was further washed with loading buffer until the baseline was again reached, then eluted with elution buffer, and the eluent was collected. The desalting column was equilibrated to four column volumes with desalting buffer, and all the eluent was loaded onto the desalting column. The first peak observed was collected as the target protein solution. If the protein concentration was too low after desalting, it was concentrated using ultrafiltration to obtain high-purity FGF21-1B7 fusion protein.

[0058] 2) Protein half-life detection Six adult male rats were randomly divided into two groups. Experimental group: subcutaneous injection of 0.3 mg / kg FGF21-1B7 Control group: Subcutaneous injection of 0.3 mg / kg free FGF21 Blood was collected via tail vein at 1, 2, 4, 8, 12, 24, 48, 72, and 96 hours after drug administration, and plasma was separated. 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. The results are shown in Table 1 below.

[0059] Table 1 Results of Half-Life Extension Test

[0060] The half-life of the FGF21-1B7 fusion protein (20.0 h) was significantly extended by 10 times compared to that of free FGF21 (2.0 h), confirming that the anti-serum albumin nanobody 1B7 can effectively increase the retention time of FGF21 in vivo, providing a technical basis for the development of long-acting drugs.

[0061] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

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) Contains 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 nucleic acid molecules of B1), or recombinant microorganisms containing recombinant vectors of expression cassettes of B2), or recombinant microorganisms containing recombinant vectors of B3); B5) Recombinant cells, wherein the recombinant cells contain B1) nucleic acid molecules, or B2) the expression cassette of the recombinant vector, or B3) the recombinant vector, or B4) recombinant microorganisms.

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. An antiserum albumin antibody comprising the amino acid sequence of the nanobody as described in claim 1 or 2 as a VHH chain.

7. A derivative comprising the nanobody of claim 1 or 2 or the antiserum albumin antibody of claim 6, wherein the derivative comprises a modifier that is covalently or non-covalently bound to the nanobody or the antibody.

8. A product characterized in that, The product includes at least one of the following: C1) A fusion protein constructed by fusing the nanobody of claim 1 or 2, the anti-serum albumin antibody of claim 6, or the nanobody of claim 7 or a derivative of the anti-serum albumin antibody with a polypeptide or protein; C2) A conjugate formed by linking the nanobody of claim 1 or 2, the anti-serum albumin antibody of claim 6, or the nanobody of claim 7 or a derivative of the anti-serum albumin antibody with a therapeutic agent; C3) A pharmaceutical composition comprising the nanobody of claim 1 or 2, or the anti-serum albumin antibody of claim 6, or the nanobody of claim 7 or a derivative thereof; C4) A kit comprising the nanobody of claim 1 or 2, and / or the biomaterial of any one of claims 3-5, and / or the anti-serum albumin antibody of claim 6, and / or the nanobody of claim 7 or a derivative of the anti-serum albumin antibody, and / or the fusion protein of C1), and / or the conjugate of C2), and / or the pharmaceutical composition of C3).

9. A method for preparing the nanobody of claim 1 or 2 or the fusion protein of C1) in the product of claim 8, 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 or the fusion protein.

10. The use of the nanobody of claim 1 or 2, the biomaterial of any one of claims 3-5, the anti-serum albumin antibody of claim 6, the nanobody of claim 7 or a derivative of the anti-serum albumin antibody, the product of claim 8, or the method of claim 9 in any of the following aspects: D1) Applications in the preparation of products with extended drug half-life; D2) Applications in the preparation of 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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